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Cautionary new CJD epidemiology: meat consumption, glaucoma tonometry
New human amyloid identified
Tubingen meeting of Sept 23, 1999
Differences in proteinase K resistance of BSE and 87V
Argyrophilic grains in late-onset Creutzfeldt-Jakob diseased brain.
Bovine or ovine origin for florid plaque dura mater CJD?
Glycosylation differences by brain region
The genetics of prions--a contradiction in scholarship?
Prion polyA variation -- does it lead to over-production?
Postdoc available at U Mass Med School

Cautionary new CJD epidemiology: meat consumption, glaucoma tonometry

Amer J Epidemiol 1998 Davanipour, Sobel et al.
Comment (webmaster):
Check out these 1998 published meeting abstracts in Amer J Epidemiol. Reading them, one sees that it will be very difficult to get them published because of the awkward ramifications; the better the full text (not yet available), the less likely that they will get past politically sensitive journal gatekeepers. There is no 'problem' with the authors -- they are well-established professors at a medical school, one in neurology, the other in preventative medicine.

Again we see the need for a biology preprint server -- let these referees and others come forward with their secret objections and the authors' reportedly excellent responses. The webmaster does not care to have his reading matter screened by people with one (or both) eyes fixed on beef exports.

As Collinge noted in the July 1999 Lancet review, "...the appearance and rapid evolution to epidemic of BSE, with the legitimate concerns of human transmission and a potentially severe threat to public health, have placed these diseases, and the people who have managed and studied them, under an unprecedented political and inquisitorial spotlight."

1. Dietary risk factors for a neurodegenerative disease: a confirmatory case-control study.

Amer J Epidemiol 1998; 147 S12 (abstract)
Z Davanipour, A. Ziogas, E. Sobel et al.
University of Southern California School of Medicine, Los Angeles 90033, USA. 
CJD is a transmissible fatal neurodegenerative disease of humans. A hypothesis testing case-control studty was conducted to confri or refulte earlier findings suggesting n association between CJD and certain aspects of diet [Amer J Epidemiol 1985: 122: 443-541].

The a priori dietary hypotheses were that eating (i) foods containing organ tissue and (ii) raw meat increases CJD risk.

Information was collected on 110 sporadic, neuropathologically confirmed cases of CJD from 11 US states. Matched controls (114) were obtained using community random digit dialing. Data were obtained from case surrogates, directly from controls, and from a sample of control surrogates.

Consumption of hot dogs, sausage, pepperoni, kielbasa, canned meat, poultry liver, any stomach or intestine, beef stomach or intestine, any organ tissue, and beef organ tissue was associated with increased CJD rise, e.g., odds rations (OR) were 4.4 (95%CI. 1.2-24.3) for sausage consumption; 2.4 (95% CI. 1.2-5.1) for pepperoni consumption, 3.2 (95% CI. 1.4-4.8) for beef organ tissue consumption.

Consumtion of bologna, salami, any liver, beef liver, and pork stomach or intestine was marginally associated with CJD. Consumption of rare or raw meat was associated with CJD, OR = 2.0 (95% CI, 0.9-4.7)/

Increased consumption was associated with increased risk. Analyses using control surrogate date indicate that use of the control self-responses did not bias the results away from the null. The study supports the a priori hypotheses [ie, eating (i) foods containing organ tissue and (ii) raw meat increases CJD risk. -- webmaster]

A case-control study of Creutzfeldt-Jakob disease. Dietary risk factors.

Am J Epidemiol 1985 Sep;122(3):443-51
Davanipour Z, Alter M, Sobel E, Asher DM, Gajdusek DC
The mode of natural transmission of Creutzfeldt-Jakob disease remains unknown. In a case-control study conducted in 1981-1983 to evaluate possible dietary and other sources of the disease, 26 cases were ascertained in the mid-Atlantic region of the United States, 23 of which were obtained from accumulated records of the Laboratory of Central Nervous System Studies of the National Institutes of Health. Controls included 18 family members and 22 hospital-matched individuals (total sample size, 66). An increased consumption among patients was found for roast pork, ham, hot dogs (p less than 0.05), roast lamb, pork chops, smoked pork, and scrapple (p less than 0.1). An excess consumption of rare meat (p less than 0.01) and raw oysters/clams (p less than 0.1) was also reported among the patients. Liver consumption, among organ foods, was greater (p less than 0.1) among the cases. If Creutzfeldt-Jakob disease is acquired through ingestion of foods containing the agent, then the food items identified may be among those which need to be evaluated more intensively. Larger case-control studies with more focused dietary questions are warranted.

2. Ocular tonometry and TSE in humans

Amer J Epidemiol 1998; 147 S76 (abstract)
Z Davanipour, A. Ziogas, E. Sobel et al. 
Departments of Neurology and Preventive Medicine 
USC School of Medicine, Los Angeles
The infectious agent of the spongiform encephalopathies is present in the cornea prior to clinical symptoms. Tonometers are not disinfected sufficiently to kill the infections agent between uses.

The hypothesis that ocular tonometry is a CJD risk factor was suppported by the results of an earlier study [Neurology 1985: 35 1483-1486]. To test the a priori hypothesis that glaucoma testing is a risk factor for CJD, a multi-state confirmatory case-control study was conducted.

110 sporadic, neuropathologically confirmed cases of CJD identified through multiple sources in 11 US states were included in the study. 114 matched controls were included using community random digit dialing.

Histories of glaucoma testing were obtained. The odds ration [OR] of 'ever' vs 'never' having had a tonometry test were statistically significanly greater than 1 for both matched and unmatchd analyses for the period age15 through 3 years prior to disease onset, using control self-responses.

The results using 'ever' vs 'never' control surrogate data support these results. The ORs, using control self-responses, increased as the nlumber of tonometry tesets increased during this age period.

For 5+ tests, the ORs were 5.8, 95% CI 2.2-19.1 (matched) and 3.7, 95% CI, 1.9-7.0 (unmatched), p < 0.00005.

Ocular tonometry appears to be a risk factor for the transmission of CJD. Consideration should be given to developoing an instrument whcih can toloerate adequate autoclaving or adequate disinfectant whcih kills the infectious agend and can be employed after each use. Alternatively, disposable covers on the tonometer can be used.

See also:

Rizzo M, et al 
       Is applanation tonometry a risk factor for transmission of Creutzfeldt-Jakob disease? 
       Arch Ophthalmol. 1987 Mar;105(3):314.  
Davanipour Z, et al.  
       Possible modes of transmission of Creutzfeldt-Jakob disease. 
       N Engl J Med. 1984 Dec 13;311(24):1582-3. 

Davanipour Z, et al. 
       Creutzfeldt-Jakob disease. 
       Neurol Clin. 1986 May;4(2):415-26. Review. 
Creutzfeldt-Jakob disease: possible medical risk factors.
Neurology 1985 Oct;35(10):1483-6
Davanipour Z, Alter M, Sobel E, Asher D, Gajdusek DC
To explore possible risk factors in the past medical history of patients with Creutzfeldt-Jakob disease (CJD), we conducted a case-control study among 26 cases and 40 matched controls. Statistically significant odds ratios were obtained for intraocular pressure testing; injury to or surgery on the head, face or neck; and trauma to other parts of the body. The odds ratios were nearly significant for head trauma and procedures requiring sutures. These data suggest that the CJD agent may be acquired by inoculation through injury or during surgery, and perhaps on certain absorbable sutures of animal origin. The tonometer used for glaucoma testing may also be a vehicle of transmission.

New human amyloid identified

PNAS Vol. 96, Issue 15, 8669-8674, July 20, 1999
Bo H”ggqvist, ..., Per Westermark
Comment (webmaster):
This remarkable paper characterizes a new and very common human amyloid -- said to occuer in 97% of the over-50 population as part of the aging process. (This may also occur in other amyloidoses, in prions, it might mean with the new sensitive methods that just about every older person has some PrpSc (with implications for transmission).

It is too soon to say whether this amyloid is infectious or even particularly pathological (though loss of flexibility to the aorta would hardly be beneficial). Lactadherin, as a glycosylated, multidomain protein with a 23-aa signal peptide, tandem repeats, pre-amyloid beta sheets (known from discoidin homology) and unknown function possibly involving cell adhesion and regulation of blood coagulation in the vessel walls, is presumbably exposed on the outer cell surface, features reminiscent of prion protein.

Highlights from the article:

Aortic medial amyloid is a form of localized amyloid that occurs in virtually all individuals older than 60 years. The importance and impact of the amyloid deposits are unknown. In this study we have purified a 5.5-kDa aortic medial amyloid component from three individuals; the amyloid, medin, is derived from an internal proteolytic fragment (positions 245-294) of lactadherin.

Lactadherin (formerly BA46; bovine, murine, rat, pig homologs known, called MFGM_HUMAN for human milk fat globule at SwissProt) is a 364-aa glycoprotein, previously known to be expressed by mammary epithelial cells as a cell surface protein and secreted as part of the milk fat globule membrane. The multidomain protein has a C-terminal domain showing homology to blood coagulation factors V and VIII.

We found that the main constituent of aortic medial amyloid is a 50-aa-long peptide, here called medin, that is positioned within the coagulation factor-like domain of lactadherin. Our result is supported by the specific labeling of aortic medial amyloid in light and electron microscopy with two rabbit antisera raised against two synthetic peptides corresponding to different parts of medin. By using in situ hybridization we have shown that lactadherin is expressed by aortic medial smooth muscle cells. Furthermore, one of the synthetic peptides forms amyloid-like fibrils in vitro. Lactadherin was not previously known to be an amyloid precursor protein or to be expressed in aortic tissue. The structure of lactadherin may implicate an important regulatory function in the aorta.

The localized amyloid deposits in some of the major age-related diseases have attracted immense interest in recent years. Investigations have focused mainly on Abeta and AIAPP (islet amyloid polypeptide, type 2 diabetes).... Albeit not proven, it may be that all localized amyloidoses reflect primary pathological changes.

One extremely common form of senile localized amyloid is confined to the aortic media. This form of amyloid is biochemically different from that seen in association with atheromatous plaques, which is derived from apolipoprotein A1). In a previous study our group found aortic medial amyloid in 97% of the subjects above the age of 50; it is possible that they contribute to the age-related diminished elasticity of the vessels.

The synthetic peptide with the C-terminal octapeptide NFGSVQFV solubilized in DMSO instantly formed a gel-like aggregate when a small aliquot of water was added (final concentration, 1 mg/ml). This aggregate had typical amyloid properties, was Congo red-positive, and showed intense green birefringence under polarized light. Electron microscopy of negatively stained material confirmed the presence of fibrils with an amyloid-like appearance.

Congo red birefringence and fibrils were also seen in aortic media sections colocalized to antibody stain. In situ hybridization on sections of the aorta of a patient with extensive aortic media amyloid resulted in a strong and specific labeling of smooth muscle cells in the aortic media and in the endothelium of small medial vessels. Lactadherin is known to be expressed by mammary epithelial cells and has been used as a mammary tumor cell marker. To find lactadherin as the precursor protein to localized amyloid in the aortic media therefore was highly unexpected.

Lactadherin is a glycosylated, multidomain protein, expressed as a 387-aa-long, single polypeptide chain from which a 23-aa signal peptide is cleaved. The normal function of lactadherin is not known. Lactadherin has an epidermal growth factor-like N terminus that contains an integrin v3-binding Arg-Gly-Asp motif that promotes cell adhesion.

The C-terminal part of lactadherin contains a tandem repeated domain with 43% and 38% identity, respectively, to the similarly positioned C1 and C2 domains of blood coagulation factors V and VIII). Medin is positioned within the C2-like domain of lactadherin, which comprises amino acid positions 206-364. The C2 domains are necessary for the procoagulant activity of factors V and VIII, and mutations in the C2 domain of factor VIII may cause hemophilia.

Given the strong homology between functionally important parts of factors V and VIII and the C1 C2 domains of lactadherin, it is tempting to speculate that expression of lactadherin in the aortic media is of importance for the regulation of blood coagulation in the vessel walls. However, further investigation is required to establish this protein's normal function in the aortic media.

A multiple-alignment model of a family of proteins with discoidin domains (DS) closely related to the C2 domain suggests three conserved beta-strands present within the medin peptide. One of the conserved beta-strands is positioned in the C-terminal flank of medin, corresponding to the octapeptide susceptible to fibril formation.

>sp|Q08431|MFGM_HUMAN MILK FAT GLOBULE-EGF FACTOR 8 PRECURSOR (MFG-E8) . NxT and NxS are generally glycosylation sites; there are 4 such sites in this protein, however NPS at the start of medin and probably NWT are excluded because of proline imino and tryptophan bulk.


Tubingen meeting of Sept 23, 1999

Tue, 27 Jul 1999 
Comment (webmaster):
The first meeting has come and gone with no report from anyone in attendence 985.

In a recent article in the 24 Jul 99 Lancet Collinge wrote, "The lack of a humoral immune response, presumably because of immune tolerance to what are host proteins widely expressed in the immune system, has precluded a serological assay."

This raises the question, if the 3 dimensional shape of the PrPSc is so different from that of the normal PrP, how can immune tolerance against host proteins explain the lack of a humoral immune response?

It seems that no one has actually looked for endogenous antibody to PrPSc in the blood in nvCJD (or elsewhere, in the last decade). Perhaps MJ Schmerr's capillary electrophoresis method could be adapted to this -- sensitivity is paramount. In dietary transmission acrdoss species, there are inevitable differences in primary sequence compounded by tertiary cross-beta, both seemingly non -self. We know from the Prionics PrpSC-specific monoclonal and other results that the mammalian immune system is perfectly capable of distinguishing both primary and cross beta differences.

So another answer might be is that there is always a certain amount of amyloid around in everyone (perhaps turning over without pathological accumulation; 97% of population had some aortic medial amyloid) and so PrPSc gets recognized as self early on, preventing later immune response. this would fit the implausible levels of contamination of growth hormone and dura mater.

The Immunology of Prion Disease

Monday 12 July 1999 at Barnes Hall, Royal Society of Medicine, London. 
       The immune system in TSEs: historical overview Moira Bruce 
       nvCJD: speaker not listed
       Pathogenesis of scrapie and BSE in sheep: Jim Foster 
       Detection of nvCJD in human lymphoid tissues: James Ironside 
       Inflammation in the CNS in scrapie infection: Hugh Perry 
       B cells in thepathogenesis of scrapie: Adriano Aguzzi 
       Dendritic cells in the peripheral pathogenesis of scrapie: Karen Brown 
       Interspecies transmission of TSEs and the species barrfier effect: Christine Farquar 
       PrPc - the two edged sword on the immune system: Neil Mabbott 
       Infection and autoimmunity in CJD: Alan Ebringer
Sophie Blackham ,Section of Clinical Immunology & Allergy, writes on Mon, 2 Aug 1999 that:

"Unfortunately, there were no abstracts produced for this meeting and the meeting was not taped. However, I have spoken to the meeting organiser who informs me that most of the speakers' work has been published, although there was some new work presented. I cannot give out speakers' details, however if you wanted to contact them, I can pass your details on to them."

The Tubingen meeting, on 23-25 Sep 99 is said by Dealler to be the main European meeting of the year. Further details about talks may emerge on the conference web site.

Registration Deadline: August 15th, 1999 
Abstract Submission Deadline: June 30th, 1999 
 Organized by Martin H. Groschup  via telephone number+49 7071 967-257 or fax 967-305.

Characterisation and diagnosis of prion diseases in animals and man
Preliminary program and list of symposium topics
 Scientific Board:  
 A. Aguzzi, Z¸rich  
 J. Collinge, London  
 D. Dormont, Paris  
 M.H. Groschup, T¸bingen  
 H. Kretzschmar, G–ttingen  
 C. Weissmann, London

 Invited Speakers 
A. Aguzzi (Zurich)
M. Bruce (Edinburgh)
H. Budka (Vienna)
A. Buschmann (T¸bingen)
B. Caughey (Hamilton, MT)
B. Chesebro (Hamilton, MT)
J. Collinge (London)
D. Dormont (Paris)
P. Gambetti (Ohio)
A. Giese (G–ttingen)
J. Grassi (Gif sur Yvette, F)
M. Groschup (T¸bingen)
A. Hill (London)
D. Harris (St. Louis) 
J. Hope (Compton, UK)
N. Hunter (Edinburgh)
J. Ironside (Edinburgh)
R. Jackmann (Weybridge, UK)
M. Jeffrey (Penicuik, UK)
H. Kretzschmar (G–ttingen)
J. Manson (Edinburgh)
B. Oesch (Zurich)
S. Poser (G–ttingen)
D. Riesner (D¸sseldorf)
J. Safar (San Francisco)
R. Sayers (Dublin)
W. Schulze-Sch”fer (G–ttingen) 
M. Scott (San Francisco)
F. Tagliavini (Milano)
G. Telling (London)
L. VanKeulen (Lelystad, NL)
I. Vorberg (T¸bingen)
C. Weissmann (London)
J. Wilesmith (Weybridge, UK)
R. Will (Edinburgh)
Dear Colleague,

You are invited to participate in an international symposium on the 'Characterization and Diagnosis of Prion Diseases in Animals and Man' which will be held in T¸bingen from Thursday, September, 23rd, 1.30 p.m. to Saturday, September 25th, 1.30 p.m.

In the light of the huge BSE epidemic in cattle and the emergence of a new variant form of CJD in humans, this symposium attempts to support the exchange and communication of advances within the scientific community. Moreover, it may serve for industrial and political decision-makers as a forum for gathering first-hand information and advice. The symposium has been made possible by supportive funding from the EU Commission. The following questions will be addressed in particular:

What characterizes the pathogenesis of prion diseases in animals and man? Which molecular mechanisms determine prion transmissibility across species barriers? What are the factors that influence the host species susceptibility to disease? Which animal and in-vitro models are currently available for the detection and quantification of prion infectivity? Which diagnostic techniques allow the reliable investigation and confirmation of suspect cases in animals and humans? What characterizes prion strains?

Oral presentations will be held by invited speakers (to be listed later) and by the members of the Scientific Board. In addition, participants selected on the basis of submitted abstracts will be invited to present their data orally.

Thursday, September 23rd, 1999

13.30-15.30 Keynote lecture and two epidemiological updates

The structure of PrP
15.45-16.45 Poster display session I

16.45-18.15 PrP Conversion

Friday, September 24th, 1999

8.30-9.30 Pathogenesis of prion diseases

9.30-10.30 Poster presentation session I
10.45-12.15 Histo- and cell pathology of prion diseases
13.15-14.00 Poster display session II

14.00-15.00 Poster presentation session II
15.20-17.20 Post-mortem diagnosis of TSEs in preclinical and in clinically diseased victims

17.20-18.00 Roundtable discussion on the chances and limitations of currently available diagnostic techniques

Participants: Grassi, Budka, Oesch, Detwiler, Hope, Bommeli, Riesner

Saturday, September 25th, 1999

8.30-10.30 Characterization of the infectious agent

10.30-11.30 Poster display session III 

11.30-13.00 Conventional and transgenic models for detection of BSE and scrapie infectivity

Argyrophilic grains in late-onset Creutzfeldt-Jakob diseased brain.

Pathol Int 1999 May;49(5):369-73 
Kawashima T, Doh-ura K, Iwaki T
Braak's argyrophilic grains (ArG) are spindle-shaped structures originally described in patients with dementia. Herein, a unique case of sporadic Creutzfeldt-Jakob disease (CJD) accompanied by numerous ArG is presented. The pathological picture was typical of CJD based on the findings of routine hematoxylin-eosin staining. The highest density of ArG was observed throughout the parahippocampal gyrus and the temporal gyri; however, the sector CA1 of the hippocampus showed less ArG. An immunohistochemical analysis for prion protein (PrP) revealed diffuse fine neuropil staining in the cerebral cortex, while the ArG themselves did not demonstrate any immunoreaction for PrP. No correlation was observed between the densities of ArG and either the presence of senile plaques, neurofibrillary tangles or neuropil threads in the present case. To our knowledge, this is the first report of CJD demonstrating numerous ArG.

Comment (webmaster):

Argyrophilic grain disease: frequency of occurrence in different age categories and neuropathological diagnostic criteria.

J Neural Transm 1998;105(8-9):801-19
Braak H, Braak E 
Argyrophilic grain disease is a progressive degenerative disorder of the human brain which becomes increasingly prevalent with advancing age. The disease entails multiple neuronal systems and results from cytoskeletal degeneration in only a few neuronal types and in oligodendrocytes. Immunoreactions for abnormally phosphorylated tau protein permit identification of the changes. Only a fraction of the emerging abnormal fibrillary material shows a pronounced argyrophilia. Essential for neuropathological diagnosis is assessment of the presence of small spindle-shaped argyrophilic grains in neuronal processes. The anteromedial portion of the temporal lobe bears the brunt of the lesions. Grains generally can be found in abundance in the entorhinal region, the first Ammon's horn sector, the subcortical nuclear complex of the amygdala, and the hypothalamic lateral tuberal nucleus. Frequently, the lesions co-exist with those typically found in Alzheimer's disease or other tauopathies. Owing to the characteristic grains, the disorder easily can be differentiated from other tauopathies. 2661 non-selected brains obtained at autopsy included 125 cases of argyrophilic grain disease (5%) from individuals between 51 and 96 years of age (mean 79 years) .

The fact that the same material contained 146 cases of fully developed Alzheimer's disease (6%) supports the view that argyrophilic grain disease is not a rare disorder. Its prevalence with and without concomitant neurofibrillary changes of the Alzheimer type grows with increasing age. Argyrophilic grain disease merits attention because of its frequent occurrence and its potential to cause severe brain dysfunction.

Neuritic conglomerates in the cerebral cortex of a patient with Creutzfeld-Jakob disease.

Shintaku M, Ogura J, Terashima A
Acta Neuropathol (Berl) 1996 Sep;92(3):319-23 
In the cerebral cortex of a patient with Creutzfeld-Jakob disease (CJD), we found peculiar glomeruloid or skein-like structures which have not previously been described. The patient was a 67-year-old man, whose clinical features and neuropathological findings were consistent with CJD. The glomeruloid or skein-like structures were distributed in the deep layers of the cortex and consisted of intricately entangled masses of thick argyrophilic fibers. These structures were immunostained with anti-neurofilament antibodies and were considered to have originated from neuronal cytoplasmic processes, most likely axons. The pathogenesis and pathological significance of these structures, which were tentatively termed "neuritic conglomerates," remains unclear. However they probably represent an overgrowth of the distal portion of axons and indicate the plasticity of the injured neurons.

Experimental induction of beta-amyloid plaques and cerebral angiopathy in primates.

Ann N Y Acad Sci 1993 Sep 24;695:228-31 
Baker HF, Ridley RM, Duchen LW, Crow TJ, Bruton CJ
Moderate numbers of amyloid plaques with associated argyrophilic dystrophic neurites and cerebral amyloid angiopathy (CAA) but no neurofibrillary tangles (NFTs) were found in the brains of 3 middle-aged common marmosets (Callithrix jacchus) inoculated intracerebrally (i.c.) 6-7 years earlier with brain tissue from a patient with early onset Alzheimer's disease. The plaques and vascular amyloid stained positively with antibodies to beta (A4)-protein.

Panencephalopathic type of CJD with neuropathologic features similar to Pick's disease.

Clin Neuropathol 1993 Jan-Feb;12(1):1-6
Pietrini V, Danieli D, Bevilacqua P, Lechi A
The panencephalopathic type of Creutzfeldt-Jakob disease is characterized by a serious degeneration of the white matter in addition to the other pathological features of the classic Creutzfeldt-Jakob disease. The clinical and neuropathological findings of a new case are described in a woman aged 62, who died after a year of illness. The brain appeared seriously affected by atrophy and white matter degeneration. Microscopically, it showed a marked cortical spongiosis, with gemistocytic astrogliosis and degeneration of the white matter of both hemispheres. Although a serious loss of nerve cells was evident, some residual neurons with a ballooned aspect were found in the fronto-temporal cortex. Other neurons presented argyrophilic inclusions similar to Pick bodies.

Argyrophilic plaques in mice inoculated with scrapie from particular sources.

Lancet 1973 Mar 17;1(7803):617-8 
Fraser H, Bruce M

Bovine or ovine pericardium: a superior alternative for repair of dural defects?

29 July 99
Comment (webmaster):
Bovine or ovine pericardium for repair of dural defects -- the choice is yours. From the abstracts below, it is clear that there is no rejection issue with allografts or xenografts of either bovine or ovine dura mater or pericardium. Note also the widespread use of these materials in many types of surgeries -- no wonder the first properly controlled study found such a pronounced CJD effect with multiple surgeries [Lancet 1999; 353:693-7].

The UK Health Ministry reportedly pulled bovine pericardium for human dura mater repairs in November, 1986, indicating that it was used prior to this time. The BSE epidemic was already in full swing by then unfortunately.

In the webmaster's opinion, this makes it quite clear that the sheep dura mater collected by the orthopaedic surgeon for the Lyodura company was intended for human use. Notice both Japan and France are finding florid plaque in some of their dura mater cases [Arch Neurol 1999 Mar;56(3):357-62, Rinsho Shinkeigaku 1997 Sep;37(9):824-8, Lancet 1997 Sep 20;350(9081):865-6, Lancet 1996 Nov 2;348(9036):1239-40 -- abstracts below].

These could very well represent bovine or ovine CJD. These cases may well be strain-type 4, though a different route might cause some differences. If so, a great many more cases may be in the pipeline from popular surgeries.

Not all dura mater CJD could result from scrapie or BSE xenografts. Human cadaveric dura mater is separately implicated -- commerical sources in the US having no connection with Lyodura or animal sources are giving dura mater CJD [Oral Maxillofac Surg 1991 Mar;49(3):272-4; discussion 274-5].

Comparative study of lyophilized human dura mater and lyophilized bovine pericardium as dural substitutes in neurosurgery.

Acta Neurochir (Wien) 1990;107(1-2):16-21
Laun A, Tonn JC, Jerusalem C
In a prospective, controlled randomized study either lyophilized bovine pericardium or lyophilized human dura mater have been used as a patch for the closure of the dura in 102 patients. The aim of this investigation was to compare both materials in terms of immunogenic response of the patients. The rate of post-operative complications was comparably low in both groups (wound infection in 1/51 patients each). In regard of workability, thickness of the material and flexibility the pericardium patches were judged to be by far superior. Neither signs of a cellular nor of an intesified humoral response could be detected in patients who received the pericardium implants. Thus, lyophilized bovine pericardium seems to be a superior alternative for the surgical repair of dural defects.

Ovine pericardium: a new material for duraplasty.

J Neurosurg 1996 Mar;84(3):508-13
Parizek J, Husek Z, Mericka P, Tera J, Nemecek S, Spacek J, Nemeckova J, Suba P 
The authors report on their 2 1/2-year clinical experience using a dural substitute, ovine pericardium, stabilized with 0.3% glutaraldehyde, flat freeze-dried, and sterilized with gamma-irradiation. Packaging of the ovine pericardium in double-plastic transparent bags allows simple storage in operating rooms and the opportunity for the surgeon to choose an ideal graft according to its shape, size, and plasticity. The ovine pericardia were examined histologically and by transmission and scanning electron microscopy in their native, freeze-dried, and irradiated forms. The final product is composed solely of pericardium fibrosum interwoven with artificially formed extracellular microcavities that serve as natural pores for the ingrowth of host tissue. ... As a new dural substitute, ovine pericardium proved to be superior to bovine and allogeneic pericardia because of its workability, flexibility, and reduced thickness. In a study of 120 grafts, all but one healed without complications.

Bovine pericardium for dural grafts: clinical results in 35 patients.

Neurosurgery 1996 Oct;39(4):764-8
Anson JA, Marchand EP .
The United States FDA has recently approved the marketing of bovine pericardium as a dural graft material, but literature reports of this use are limited. Bovine pericardium has been widely used for grafts in cardiac surgery and seems to have suitable properties for use as a dural graft. We report the use of glutaraldehyde-processed bovine pericardium for dural grafts in 35 patients undergoing cranial and craniospinal operations with the objective of providing a clinical assessment of this material and technique..... The material is relatively inexpensive and requires no additional incision. It has low antigenicity and toxicity, good strength, and minimal elasticity. :In this clinical assessment, bovine pericardium was found to be an excellent dural graft material.

The use of Vicryl Collagen as a dura substitute: a clinical review of 78 surgical cases.

Acta Neurochir (Wien) 1997;139(2):120-3 
Van Calenbergh F, Quintens E, Sciot R, Van Loon J, Goffin J, Plets C 
We performed a retrospective review of 78 consecutive neurosurgical procedures using Vicryl Collagen, a resorbable mesh of polyglactin 910 coated with bovine collagen, for dural substitution. The complications we encountered were infrequent and mostly minor.... We conclude that Vicryl Collagen is a valuable alternative to the patient's own fibrous tissues when dural substitution is necessary.

Experimental evaluation of collagen sponge as a dural graft.

Br J Neurosurg 1993;7(6):635-41 
Narotam PK, Van Dellen JR, Bhoola K, Raidoo D
Early collagen products, when used as dural substitutes, promoted severe inflammatory responses and fell into disrepute. A more recent advance, collagen sponge, which is derived from bovine flexor tendons was used in this experimental study. Collagen sponge was surgically implanted as an onlay dural replacement graft following skull trephination and dural excision in 12 primates. Macroscopic, histological and electron-microscopical evaluations were performed at periods of 1, 3 and 9 months. This preliminary animal study indicated that collagen sponge is suitable to use as a graft since it does not induce any inflammatory response or adhesions in the absence of pia arachnoid injury. If forms an ideal scaffold for the early ingrowth of fibroblasts to effect dural repair.

Detailed evaluation of 2,959 allogeneic and xenogeneic dense connective tissue grafts (fascia lata, pericardium, and dura mater) used in the course of 20 years for duraplasty in neurosurgery.

Parizek J, Mericka P, Husek Z, Suba P, Spacek J, Nemecek S, Nemeckova J, Sercl M, Elias P [Czech Republic]
Acta Neurochir (Wien) 1997;139(9):827-38  
Surgical experience with 2959 allogeneic and xenogeneic dense connective tissue grafts (1767 of fascia lata, 909 of pericardium, and 283 of dura mater), used in 2665 neurosurgical operations performed in the course of 20 years (1976 to 1995) is reported. Duraplasty using either allogeneic or xenogeneic grafts has had a similar, and favourable clinical outcome. Nevertheless, the pliable deep frozen fascia lata grafts, which could be used in any location, have been reserved for sella turcica plugging, anterior cranial base plasty, aneurysmal wrapping, and surgery of lipomyelomeningocele. Pericardium and dura mater grafts were in the majority of cases used over the brain convexity and posterior cranial fossa. Ovine pericardium proved to be superior to bovine and allogeneic pericardia because of its workability, flexibility, reduced thickness, and better transparency.

Xenogeneic pericardium as a dural substitute in reconstruction of suboccipital dura mater in children.

J Neurosurg 1989 Jun;70(6):905-9
Parizek J, Mericka P, Spacek J, Nemecek S, Elias P, Sercl M [Czechoslovakia]
A 5-year experience with the glutaraldehyde-stabilized freeze-dried radiation-sterilized calf pericardium used as a dural substitute is reported. The structure of pericardium xenograft is compared with other collagenous materials used for duraplasty (allogeneic fascia lata and dura mater) by light and electron microscopy. The special neurosurgical techniques involved in using pericardium xenografts in the reconstruction of suboccipital dura mater in children are presented in detail.

Dura mater for soft-tissue augmentation. Evaluation in a rabbit model.

Nordstrom MR, Wang TD, Neel HB 3d Irradiated human dura mater, a commercially available preparation of dura mater (Tutoplast), and irradiated rabbit dura mater were implanted in subcutaneous pockets in the pinna and forehead of New Zealand white rabbits

[Xenoplasty of the dura mater by means of formalinized grafts]

Zh Vopr Neirokhir 1985 Jul-Aug;(3):49-54
Beloded VG, Ryvniak VV
The possibility of using as plastic material animal (neat cattle, pig) dura mater stored in 0.5% formaldehyde solution was studied. The structure of the dura mater tissue is maintained for a 3-year storage period, and it therefore can be used during this period. The results of experiments (on 30 dogs) and clinical observations (63 patients) show that dura mater grafts used to repair a defect ensure airtightness of the subdural space and separate the brain from the overlying tissues, thus preventing the development of an adhesive-cicatricial process. On resorption, the graft is replaced by an organ-typical regenerated material performing the function of the lost dura mater.

Porcine small intestinal submucosa as a dural substitute.

Surg Neurol 1999 Jan;51(1):99-104
Cobb MA, Badylak SF, Janas W, Simmons-Byrd A, Boop FA
The continuing search for the ideal dural substitute is currently directed toward collagen preparations. Xenogeneic porcine small intestinal submucosa (SIS), a naturally occurring extracellular matrix rich in collagen, has been successfully used as a soft tissue graft in several body organ systems, including preliminary studies as a dural substitute in the rat. ...There was no clinical or histologic evidence of sensitization or graft rejection. No evidence of adverse effect on the underlying cerebral cortex was observed. Porcine small intestinal submucosa demonstrates a favorable biologic response as a dural substitute in the canine model. It is a promising biomaterial for dural replacement.

Comparative experimental study of esophageal wall regeneration after prosthetic replacement.

J Biomed Mater Res 1999 May;45(2):84-91 
Freud E, Efrati I, Kidron D, Finally R, Mares AJ
This study compares three prosthetic materials for potential use in patching and bridging congenital and acquired esophageal defects. The study was divided into two parts. In the first part, full-thickness, 6-cm2 cervical esophageal defects were induced in three groups of young mongrel dogs and were replaced with patches of lyophilized dura mater (Lyodura)...

Neurosurgery and iatrogenic transmission of Creutzfeldt-Jakob disease.

Med J Aust 1996 May 20;164(10):603-4 
Newcombe RL
The Neurosurgical Society of Australasia has not recommended that recipients of dura mater grafts be actively sought out and informed, but rather that neurosurgeons should attempt to discover for individual patients whether they have received dura mater grafts.

Dura mater (Lyodura) in reconstruction of the abdominal and chest wall defects after radical excision of soft tissue neoplasms--case reports.

Eur J Surg Oncol 1987 Feb;13(1):63-8 
Kulakowski A, Ruka W
Three cases of abdominal and chest wall defects after radical excision of soft tissue sarcomas reconstructed with dura mater are presented. Based on intraoperative findings and early results it is suggested that dura mater (Lyodura) is of great value for the repair of full-thickness integument defects.

[Use of dura matter (Lyodura) in the surgical treatment of complete rectal prolapse.]

Minerva Chir 1986 Sep 30;41(17-18):1403-7  
Latteri M, Fricano S, Bajardi G, Pantuso G, Spinnato G

Sling operation for urinary incontinence.

Acta Obstet Gynecol Scand 1985;64(2):187-90 
Iosif CS
105 women with urinary stress incontinence were treated over an 8-year period. The operations carried out were: 44 Zoedler slings using mersilene and 61 slings using lyodura...

[Critical review of 10 years' use of the surgical method of closing the cleft palate with lyodura].

Chir Maxillofac Plast 1982;12(1):37-40 
Barlovic M, Bagatin M

[Direct capping of human teeth with Lyodura].

Dtsch Zahnarztl Z 1981 Jun;36(6):354-6 
Albers HK

Use of lyophilized dura in aural surgery.

ORL J Otorhinolaryngol Relat Spec 1978;40(3):129-38
Palva T, Ylikoski J, Makinen J
Lyophilized dura was utilized in chronic ear surgery to cover raw promontory surfaces, to stabilize the reconstructed ossicles, to raise the posterior insertion point of the grafted drum after canal wall removal, and to fortify the soft posterior canal wall. In 3 guinea pigs, lyodura was also placed in the bulla and studied 6, 12 and 16 months later.... Lyophilized dura contains no irritating material and is considered to be a versatile and useful material in chronic ear surgery.

Creutzfeldt-Jakob disease from allogeneic dura: a review of risks and safety.

J Oral Maxillofac Surg 1991 Mar;49(3):272-4; discussion 274-5 
Marx RE, Carlson ER 
Surgeons and the lay public have recently expressed concern over the safety of allogeneic dura as it relates to the transmission of Creutzfeldt-Jakob Disease. Indeed, two cases have resulted from use of tissue procured from a commercial agency that did not adhere to criteria accepted by the American Association of Tissue Banks or the Southeast Organ Procurement Foundation. This review discusses the risks and safety of allogeneic dura. The findings should reassure the surgeon of the safety of allogeneic dura when it is properly processed and catalogued by a bona fide, reputable tissue bank. To date, there have been no documented cases reported to the Center for Disease Control in which Creutzfeldt-Jakob Disease was transmitted from allogeneic dura obtained from a registered tissue bank.

Creutzfeldt-Jakob disease with florid-type plaques after cadaveric dura mater grafting.

Arch Neurol 1999 Mar;56(3):357-62 
Shimizu S, Hoshi K, Muramoto T, Homma M, Ironside JW, Kuzuhara S, Sato T, Yamamoto T, Kitamoto T
...We report herein 2 autopsy cases of dura-associated CJD wit atypical clinicopathological features.... These 2 cases are clinicopathologically distinct from typical dura-associated cases of CJD. They may be a subtype with florid-type plaques in dura-associated CJD.

[Creutzfeldt-Jakob disease with a widespread presence of kuru-type plaques after cadaveric dural graft replacement. An autopsy case]

Rinsho Shinkeigaku 1997 Sep;37(9):824-8
Takashima S, Tateishi J, Taguchi Y, Hirade S, Inoue H, Matsui Y, Furukawa H
A case of Creutzfeldt-Jakob disease (CJD) is reported in a 48-year-old woman who had received a cadaveric dural graft after a clipping procedure of a cerebral artery aneurysm in September 1985. In November 1994, she noticed unsteady gait and blurred vision at first...Immunohistochemically, PrP plaques, so called kuru-type plaques, were extensively distributed throughout the cerebrum and cerebellum. Moreover, some of these plaques resembled "florid" plaques, which were surrounded by a zone of spongiform change. The PrP gene analysis of her blood and brain tissue revealed no mutations with homozygosity, Met/Met, at codon 129. The unusual features of this case, that is the absence of PSD on EEG and the widespread presence of kuru-type plaques including "florid" plaques, may be similar to the features of "new variant" CJD.

Creutzfeldt-Jakob disease in a 52-year-old woman with florid plaques.

Lancet 1996 Nov 2;348(9036):1239-40 
Kopp N, Streichenberger N, Deslys JP, Laplanche JL, Chazot G

Panencephalopathic type of Creutzfeldt-Jakob disease associated with cadaveric dura mater graft.

J Neurol Neurosurg Psychiatry 1997 Oct;63(4):524-7 
Yamada M, Itoh Y, Suematsu N, Matsushita M, Otomo E

Creutzfeldt-Jakob disease with florid plaques after cadaveric dural graft in a Japanese woman.

Lancet 1997 Sep 20;350(9081):865-6 
Takashima S, Tateishi J, Taguchi Y, Inoue H
A 52 year old man with Creutzfeldt-Jakob disease who received a cadaveric dura mater graft 99 months before the onset is reported. The prion protein gene was homozygous for methionine at the polymorphic codon 129. Neuropathological examination disclosed a panencephalopathic type of Creutzfeldt-Jakob disease which was characterised by severe involvement of the cerebral white matter and cerebellum, as well as of the cerebral cortical and deep grey matter. Thus the panencephalopathic type of Creutzfeldt-Jakob disease may occur in association with cadaveric dura mater grafts.

Creutzfeldt-Jakob disease after an embolization of intercostal arteries with cadaveric dura mater suggesting a systemic transmission of the prion agent.

Neurology 1997 May;48(5):1470-1 
Defebvre L, Destee A, Caron J, Ruchoux MM, Wurtz A, Remy J

Host and transmissible spongiform encephalopathy agent strain control glycosylation of PrP.

J Gen Virol 1999 Jul;80 (Pt 7):1865-72
Somerville RA
PrP is a host-encoded glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs) or 'prion' diseases. The normal form of the protein (PrP(C)) is heavily but incompletely glycosylated; it shows structural diversity in three neuroanatomically distinct regions of the brain. No effect of TSE infection on PrP(C) glycosylation has been detected.

TSE-specific forms of PrP (PrP(Sc)) vary in their degree of glycosylation according to strain of TSE infectious agent. PrP(Sc) also varies independently in the amount and pattern of glycosylation according to brain region. This diversity shows that the glycosylation of PrP is under both host- and TSE agent-specified control, probably within the biosynthetic pathway for protein N-glycosylation. These findings challenge assumptions that PrP(Sc) is formed from the normal, mature form of PrP(Sc) but are compatible with a model in which the glycosylation phenotype of PrP(Sc) is under the control of both host cellular factors and TSE agent-specified information.

Comment (webmaster): Here is a paper with careful experimentation, excellent literature awareness, and thoughtful discussion. There was however lukewarm characterization of glycosylation -- it seems that the sialic acids could be counted from pH and size given the Birlingame paper. It is unsatisfactory to speak of non-, mono-, and di- substituted protein when there are two distinct glycosylation sites and over 60 known partial glycosylation products. Here again we see how the prion literature suffers from authors not being aware of simultaneous complimentary papers.

The authors point out but cannot resolve a seemingly conflicting result from another study, Kuczius et al. J. Infectious Diseases 178, 693-99 1998. The differences by brain region in Somerville's Fig 1 may be reproducible but are ultimately ±5%. Medulla, cortex, and cerebellum pool many cell types.

The webmaster was not persuaded that the deglycosylation or pool selection models were ruled out. It is basically absence of evidence taken as evidence of absence. Looking at amyloid fibril, it seem self-evident that the growing points of the fibrils will exercise some restraints on what can be added. Physical strain types are observed in quite a few of the other 25-30 amyloidoses yet no one frets over extra factors in these diseases. An obscure and diminished role is left for proposed virino nucleic acid, at best. However good data, as here, is model-independent and useful to all.

The discussion addresses the possibility of getting at whether an unoccupied glycosylation site had ever been occupied, by looking at coded asparagine becoming aspartate during catabolic cleavage. That is, in strain-typing, does partial glycosylation represent partially biosynthesized or partially catabolized?

However, there is no evidence linking PNGase, the enzyme discussed in the paper, to catabolism of prion protein in vivo or in vitro and the presence of fucose could rule out its relevence; PNGase is non-lysosomal as well. By microscopic reversibility, there is no reason why some other enzyme could not regenerate asparagine. Finding DxS/T instead of NxS/T could be in part artifactual deamidation -- few glycoproteins are sequenced directly with some extreme measure.

The webmaster would be very curious where Suzuki's database of experimentally sequenced glycoproteins is to be found online -- each would have to be annotated as experimental method. This area of mammalian brain metabolism is very poorly studied as is the biosynthetic side. So we are left with an inconclusive situation with prion protein except where non-artifactual aspartate is found by mass spec. Recall direct amino acid sequencing of aglycosyl hamster PrP showed asparagine at the second site (Stahl et al., 1993), not aspartate; status of the first site was not determinable.

Purification and enzymatic properties of peptide:N-glycanas: possible widespread occurrence of post-translational remodification of proteins by N-deglycosylation.

Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S 
J Biol Chem 1994 Jul 1;269(26):17611-8
Recently, we found the occurrence of N-deglycosylating enzyme, peptide:N-glycanase (PNGase), in mammalian cells and observed that PNGase is a rather common enzyme involved in post-translational remodification of proteins. Although this enzyme was capable of hydrolyzing structurally diverse natural glycopeptide substrates bearing high mannose, hybrid, and complex-type glycan units, the activity was completely inhibited by the presence of the fucose residue either alpha-1-->3- or alpha-1-->6-linked to the proximal GlcNAc residue. The enzyme showed maximal activity at pH near 7. This and the inability to act on glycoasparagine strongly support our view that this enzyme would not be involved in lysosomal degradation pathway.

Possible wide occurrence of N-deglycosylation of glycoproteins was shown by a data bank survey of the protein sequences showing discrepancies between those determined directly (-D-X-(S/T)-) and those deduced from cDNA sequencing (-N-X-(S/T)-). We propose here that PNGase-catalyzed N-deglycosylation is a functionally important universal feature in living cells.

Identification of peptide:N-glycanase activity in mammalian-derived cultured cells.

Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S
Biochem Biophys Res Commun 1993 Aug 16;194(3):1124-30 
The recent finding of peptide:N-glycanase (PNGase) raised the question of how widespread is the occurrence of this type of de-N-glycosylating enzyme....HPLC, amino acid and carbohydrate composition analyses, and peptide sequence analysis unequivocally established the reaction products to be the free glycan having di-N-acetylchitobiosyl sequence at its reducing end and free peptide in which the originally glycan-linked Asn residue was converted to the Asp residue. This represents the first demonstration of PNGase in mammalian cells and thus PNGase appears to be a very common enzyme expressed in not only plants and bacteria but also a wide range of animals although its functional significance remains to be clarified.

Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing.

Stahl N, Baldwin MA, Teplow DB, Hood L, Gibson BW, Burlingame AL, Prusiner SB
Biochemistry 1993 Mar 2;32(8):1991-2002
The only component of the infectious scrapie prion identified to date is a protein designated PrPSc. A posttranslational process converts the cellular PrP isoform (PrPC) into PrPSc. Denatured PrPSc was digested with endoproteases, and the resulting fragments were isolated by HPLC. By both mass spectrometry and Edman sequencing, the primary structure of PrPSc was found to be the same as that deduced from the PrP gene sequence, arguing that neither RNA editing nor protein splicing feature in the synthesis of PrPSc. Mass spectrometry also was used to search for posttranslational chemical modifications other than the glycosylinositol phospholipid anchor attached to the C-terminus and two Asn-linked oligosaccharides already known to occur on both PrPSc and PrPC. These results contend that PrPSc molecules do not differ from PrPC at the level of an amino acid substitution or a posttranslational chemical modification.

The genetics of prions--a contradiction in scholarship?

Lancet 1999; 354 (suppl l): 22-25
Adriano Aguzzi, Sebastian Brandner
" Many variants of the protein-only hypothesis have been formulated, three of which are the most hotly debated (figure 1). "

Comment (webmaster): This debate has been going on for decades with all the amyloid diseases. Exasperatingly, figure 1 fails to show cross-beta structure which was proven long ago from xray and synchrotron radiation studies. GG Glenner is not cited -- he characterized prion amyloid correctly in 1980. The turnover of fibril and covalent heterogeneity of polymerizing material also needs to be addressed, especially in the case like here of N- and C- truncated fragments with variable glycosylation occupancy and completeion.

"Even a stop codon (at position 145) has been identified,20 but the lack of transmissibility in experiments in animals prevents a fully confident assignment of this particular disease to the TSE group.

Comment (webmaster): The high priests can't stand this mutation because (a) it implies the downstream part of the molecule is irrelevent and (b) it was discovered by the Japanese (1, 2, 3, 4). The histopathology and symptoms fit perfectly well with GSS which is extremely variable in its presentation -- see the P105L siblings below.. Nobody ever had a problem with many of the other point mutations transmitting poorly if at all (or never being tested).

For example, P105L, A117V, Y145stop, V180I, M232R have no or inconclusive transmission to mice at this point. However transmission failure of Y145stop means little unless the recipient mice were knock-outs with human Y145stop additionally knocked in -- otherwise there could be interference, too much of a species barrier, and too short a lifespan. Pity -- Y145stop makes a nice mini-prion. The 106-126 region is key to cross-beta formation and would be available in Y145stop -- the rest, as in other amyloidoses, is probably window-dressing. An August 1999 JBC paper provides further information on this mutation.

Note that the beautifully drawn figure 2 has V180I incorrectly shown as V180L, H187R omitted, D202N omitted, V210I incorrectly shown as V210L, Q212P omitted, N171S omitted as a polymorphism and the silent mutations are either misplaced or missing. This is a completely unacceptable number of errors. The correct set of point mutations has been posted here for many months and is also available in various prominent review articles.

"To date, all familial cases of CJD are inherited as autosomal dominant traits. Although penetrance varies from low >to almost 100% depending on the underlying mutation..."

'All' is a single cite to P102L because in point of fact, the vast majority of known mutations have never been shown to be familial and indeed showed up instead in screens of sporadic CJD. 'Dominant inheritance with low penetrance' is going to be a really tough one to prove with LOD scores, a single kindred, and a disease with late onset first recognized in the 1920's. Situations like N171S are resolvable as polymorphisms, despite the Brazilian study, based on its occurrence in great apes and the general equivalence of these two amino acids at this position throughout mammalian history.

Not addressed are promoter, splice, and polyA mutations. These are found in most of the other amyloidoses where they again need only be present in one allele. Since few of the known prion mutations were found through screenings of familial dementia, it seems likely that some fraction sporadic CJD will be attributable to these. Sporadic CJD is never sequenced outside of the coding region, even though polyA polymorphims have shown up in sheep. (The issue of course is over-production of prion protein and driven equilibrium.)

An inherited prion disease with a PrP P105L mutation: clinicopathologic and PrP heterogeneity.

Neurology 1999 Jul 13;53(1):181-8 
Yamada M, Itoh Y, Inaba A, Wada Y, Takashima M, Satoh S, Kamata T, Okeda R, Kayano T,
Suematsu N, Kitamoto T, Otomo E, Matsushita M, Mizusawa H 
To clarify a clinical and neuropathologic phenotype of an inherited prion disease associated with a missense mutation at codon 105 in the prion protein (PrP) gene that was originally described as a variant of Gerstmann-Straussler-Scheinker disease demonstrating spastic paraparesis. Two siblings from a Japanese family are described. PrP gene analyses, neuropathologic studies with immunohistochemistry, and Western blot analysis of the PrP were performed. RESULTS: Both patients showed a missense (proline-->leucine) mutation at codon 105 and a methionine/valine polymorphism at codon 129 of the PrP gene.

Clinically, Patient 1 presented with progressive spastic paraparesis, ataxia, and dementia. Patient 2, the sister of Patient 1, showed prominent action myoclonus and dementia. Neuropathologically, multiple PrP-positive amyloid plaques and diffuse PrP deposition in the deep cortical layers were found in the cerebral cortex with primarily frontal dominant atrophy in both patients.

Tau-positive pathologic structures including neurofibrillary tangles, neuropil threads, and dystrophic neurites around the plaques were abundant in the brain of Patient 2. In contrast, the tau pathology was scarce in Patient 1. Western blot analysis of the brain showed different patterns of detergent-insoluble PrP fragments between the patients.

Despite the identical codon 105 mutation and codon 129 polymorphism of the PrP gene, remarkable clinical and neuropathologic differences, and PrP heterogeneity were present between the affected siblings. The phenotypic variability might be related to PrP heterogeneity.

PrP (prion) gene expression in sheep may be modulated by alternative polyadenylation of its messenger RNA

J Gen Virol 1999 80: 2275 [to appear: August 99]
W. Goldmann, G. O'Neill, F. Cheung, F. Charleson, P. Ford and N. Hunter 
This paper has broad relevence to amyloid diseases where over-production of protein is an issue (nearly all of them). In sheep, it means that tweaking the open reading frame is probably insufficient for breeding out scrapie -- a low susceptibility allele could be offset by a high production polyA. The same issue arises upstream around exon 1 and 2.

This all arises in sporadic CJD and nvCJD. Unbelievably, no one has looked at either promoter or polyA regions in affected humans, even though they remain a totally puzzling subset of the exposed met/met population. (Indeed, met/met may be irrelevent -- met could be simply tightly linked to some polyA allele.). Prion researchers are going to look extremely stupid if a polyA or promoter allele in humans turns out to be a good predictor of CJD susceptibility. Sequencing is a simple, quick, sure, and cheap experiment -- the golden rule is always do the easy experiments first.

The paper looks at two length of mRNA found in various sheep tissues, the 2.1kb (from AATAAA at 1523) and 4.6kd which differ only in polyadenylation site. The 4.6 is highest in brain; the 2.1 highest in spleen. 2.1 is found in sheep and goat, marginally in cow, not seen in human or mouse. [This statement is not backed by homology alignment.]

Sheep also show a minor 3.3kb band (amounting to 1-5% of mRNA) and a 2.6kb species seen in kidney. No tissues are 2.1 only, some is in brain heart and liver, contrary to earlier findings. 2.1 in brain ils possibly from specific regions, this was not tested. Goldmann et al. also claim unpublished alternative 5' splicing sites in cow but not sheep.

M131313 and AJ223072 said to have 6 allelic differences and 30 differences relative to Lee's heroic sequence U67922 and in several positions to Cheviot (Goldmann, unpublished), showing high mutation rate or (very likely, for reasons given by Lee) sequencing error. This could have been tested by alignment with cow and mink 3' UTR but was not.

Sheep 3'UTR have 3 retrotransposons, poorly described in this paper and claimed to have high GC and high GT said to function as RNA polymerase stop signals. The mRNA species are not breed or allele or scrapie related. Regions ABCDEFG were previously defined by Goldmann [PNAS 87 2476-2480 1990] -- it is unclear what significance these have. Goldmann Brit Med Bulletin 49: 839-8601 1993 says retrotransposons are regions D and F, lacking in 2.1. Sheep 2.1 is found in domains ABC, said to be non-homologous to human.

Protein expression was measured in a heterologous system , mouse neuroblastoma. The best protein expression correlated with shortest 3' UTR in region G; short 3' UTR in region C was distinctly less efficient. Poor translation of full length mRNA intransfected cells, unlike sheep brain, suggests murine neuroblasomat cells were unsuitable for characterizing relative translation efficiencies in vivo in sheep.

Bovine ovary, uterus, and brain also low 2.1 but still 0.5-2% of total mRNA. Horiuchi 1995 compared ovine kidney and brain: 20% less mRNA in kidney, yet 2.5% the protein, factor of 8x less use. Here ovine kidney was 75% 4.6 and 25% 2.1 or 15% and 5% of brain, consistent with but hardly proving that only 2.1 is translated in kidney. Sheep express 4.6 kb mRNA two-thirds through gestation in brain, as does mouse. Various fetal tissues and young lambs and placenta also express the prion gene. Fetal only tonsil had both mRNA forms. Prion mRNA is easily detected at 98 days gestation, is 100 x higher at day 134, and 200x higher in early lamb. 4.6/2.1 ratio was 1 at fetal 98, 3 at fetal 138 and early lamb and held steady. Thymus had a ratio of 4, drops during early lamb by factor of 8.

Calf had 4.6 in brain, kidney, and spleen, liver, ovary, and uterus (which both had some 2.1). Goat spleens had both; brains were 4.6. Humans have 2.5k mRNA; brain 4x as abundantly as liver and heart, 8x that of lung, placenta, muscle, kidney, and pancreas. Humans are said to have 3 additional consensus sites at 1765, 1978, and 2098 that could give shorter mRNAs. This 2.5k must be adjusted for exon 1 and the leader and coding portion of exon 3 to give numbering in terms of the 3' UTR; exon 1 is 134 bp, exon 2 is 97bp, exon 3 leader is 11 bp, coding + stop is 738 totalling 980 bp.

Sheep said to have 3220 polyA signal versus 3246 given at GenBank annotation of Lee's UTR. Human 3' UTR consists of 1606 bp. 9 Suffolk polyA signals said to be at 1a AATAAA 1523, 1b TATAAA 1523, AATAAA 2222, AATAAA 2285, AATAAA 2667, ATTAAA 4063. Not in table: 1253, 4038, 4678. Cheviot unpublished have 1253, 1523, 4038, and 4063.

The majority of mRNA is polyadenylated about 20 bp downstream of the polyA signal at 4063 in Cheviot. 2.1 is polyadenylated 23 bp downstream of ATTAAA 1523.

Comment (webmaster): Recall that messenger RNA for the prion protein in all known species contains a long untranslated 3' sequence. This has signal regions (typically AATAAA) for polyadenylation (necessary for mRNA stability and use) and the actual polyadenylation site itself.

In all species studied, there are alternative signals and alternative sites (even for a single signal). The question comes up, especially when alleles are found, is does this have anything to do with sporadic TSE or susceptibility to infection or regulation of prion protein production by tissue or stage of development?

The region has never been considered in sporadic CJD -- in any other human disease they would have looked at this ten years ago to see how much sporadic was actually cryptic familial CJD.

Ruminants such as sheep and cow (but not ferungulates such as mink or primates or rodents) have three separate retrotransposons interupting their 3' UTR messenger RNA:

Bov-B    =   387 bp [alt: BOV2; LINE-like element]
Bov-tA3  =   159 bp [art-2 SINE]
OaMAR1   = 1,220 bp [mariner element]

The mariner pseudogene and its terminal inverted repeats is described as a 'fossil mariner' element with transposase protein homology to Mellifera (bee) subfamily. It is probably an old insertion probably shared by all ruminants since it has 7-8 frameshifts and 5 stop condons -- figure 3 of the Lee paper shows a guided translation and the correct flanking human gene alignment: The insertion occured between 27587 and 27588 in terms of human 3' UTR numbering, just prior to the Bov-tA3. At a page in progress, the webmaster has collected relevent GenBank references and polyA web analytic tools, reconstructed the ancestral ruminant 3'UTR by masking retrotransposons and using mink for outgroup arbitration, characterized the type of mutations fixed in this region, found the homologous regions in human and rodent, compared the mariner pseudogene rate of evolution with those of more conservative functional regions, annotated full texts of relevent cites, and so on.

Then there is the matter of what information lies in the EST database for mouse and human -- these RT PCR from bulk mRNA with a polyT primer and so pick up tissue variation in use of polyA signal and site. These sequences can be of extremely high fidelity for several hundred nucleotides.

Post Doctoral position, available immediately.

 A post doctoral position is available immediately for the
investigation of prion protein expression in yeast.  Transmembrane forms of
epitope-tagged fusions predominate and the orientation of insertion appears
to follow the pattern established in mammalian microsomes by Hegde, Lingappa
et al.  We are investigating the influences on topology of mutations in both
charged residues adjacent to the conserved central hydrophobic segment and
in disease-associated conformational determinants (e.g., p105L, A117V).  Our
aims are to better understand the effects of such mutations on prion protein
translocation at the ER and to provide further tests of the correlation of
transmembrane insertion with pathogenesis proposed by Lingappa.  

This is a recently initiated project that will form the basis of a new grant application. Funds are available for 12-15 months. Interested individuals should send a CV and a list of referees, or contact DJT for additional information.

Donald Tipper
Dept of Molecular Genetics and Microbiology
Room S5-121, U Mass Med Shool
Worcester MA 01655
Phone    508 856 2308
FAX        508 856 5920
Transmembrane protein insertion orientation in yeast depends on the charge difference across transmembrane segments, their total hydrophobicity, and its distribution.
J Biol Chem 1998 Sep 18;273(38):24963-71 
Harley CA, Holt JA, Turner R, Tipper DJ
The determinants of transmembrane protein insertion orientation at the endoplasmic reticulum have been investigated in Saccharomyces cerevisiae using variants of a Type III (naturally exofacial N terminus (Nexo)) transmembrane fusion protein derived from the N terminus of Ste2p, the alpha-factor receptor. Small positive and negative charges adjacent to the transmembrane segment had equal and opposite effects on orientation, and this effect was independent of N- or C-terminal location, consistent with a purely electrostatic interaction with response mechanisms. A 3:1 bias toward Nexo insertion, observed in the absence of a charge difference, was shown to reflect the Nexo bias conferred by longer transmembrane segments. Orientation correlated best with total hydrophobicity rather than length, but it was also strongly affected by the distribution of hydrophobicity within the transmembrane segment. The most hydrophobic terminus was preferentially translocated. Insertion orientation thus depends on integration of responses to at least three parameters: charge difference across a transmembrane segment, its total hydrophobicity, and its hydrophobicity gradient. Relative signal strengths were estimated, and consequences for topology prediction are discussed. Responses to transmembrane sequence may depend on protein-translocon interactions, but responses to charge difference may be mediated by the electrostatic field provided by anionic phospholipids.

The role of charged residues in determining transmembrane protein insertion orientation in yeast.

J Biol Chem 1996 Oct 4;271(40):24625-33 
Harley CA, Tipper DJ
The first 79 residues of the yeast Ste2p G protein-coupled pheromone receptor, including the negatively charged N-terminal domain, the first transmembrane segment, and the following positively charged cytoplasmic loop, has been fused to a Kex2p-cleavable beta-lactamase reporter. Insertion orientation was determined by analysis of cell-associated and secreted beta-lactamase activities and independently corroborated by analysis of membrane association and glycosylation patterns. This fusion inserts with exclusively N terminus exofacial (Nexo) topology, serving as a model type III membrane protein. Orientation is unaffected by removal of all three positively charged residues in the cytoplasmic loop or by deletion of all but 12 residues from the N-terminal domain. The residual -2 N-terminal charge apparently provides a signal sufficient to determine Nexo topology. This is entirely consistent with the statistically derived rule in which the charge difference, Delta(C-N), counted for the 15 immediately flanking residues, is the primary topology determinant. Mutations altering Delta(C-N) to zero favors Nexo insertion by 3 to 1, whereas increasingly negative values cause increasing inversion of orientation. All results are consistent with the charge difference rule and indicate that whereas positive charges promote cytoplasmic retention, negative charges promote translocation.

Differences in proteinase K resistance and neuronal deposition of abnormal prion proteins

Mol Med 1999 Jun;5(6):406-418
Kuczius T, Groschup MH
A number of prion strains can be distinguished by "glycotyping" analysis of the respective deposited PrP(Sc) compound. In this study, the long-term proteinase K resistance, the molecular mass, and the localization of PrP(Sc) deposits derived from conventional and transgenic mice inoculated with 11 different BSE and scrapie strains or isolates were examined. Differences were found in the long-term proteinase K resistance (50 mug/ml at 37 degrees C) of PrP(Sc). For example, scrapie strain Chandler or PrP(Sc) derived from field BSE isolates were destroyed after 6 hr of exposure, whereas PrP(Sc) of strains 87V and ME7 and of the Hessen1 isolate were extremely resistant to proteolytic cleavage. Nonglycosylated, proteinase K-treated PrP(Sc) of BSE isolates and of scrapie strain 87V exhibited a 1-2 kD lower molecular mass than PrP(Sc) derived from all other scrapie strains and isolates.

With the exception of strain 87V, PrP(Sc) was generally deposited in the cerebrum, cerebellum, and brain stem of different mouse lines at comparable levels. Long-term proteinase resistance, molecular mass, and the analysis of PrP(Sc) deposition therefore provide useful criteria in discriminating prion strains and isolates (e.g., BSE and 87V) that are otherwise indistinguishable by the PrP(Sc) "glycotyping" technique.

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