December Science News

Sheep Cloned from adult animal
Significance of milk production drop for BSE
Infectivity by tissue: 5 species
Louping ill and scrapie: science update
Prion protein dimerization based on alpha-helical packing
Protein markers in CSF from BSE-affected cattle
Preclinical detection of TSE in mouse spleen
Allelic frequencies of an ovine scrapie susceptibility gene
Spongiform encephalopathies of cattle, sheep and cats in Hungary
Neuropeptide Y and GFAP after prion infection
Anti-sense mRNA and GABA transmission control by c-fos
Localization of Menkes disease copper protein
Polymorphism in transcriptional control for HTT

Sheep Cloning

12.7.96 BBC
Scientists at the Roslin Institute, Edinburgh, have developed the world's first technique for mass producing cloned sheep. The research also opens up the astonishing possibility of being able to clone entire new animals from a single cell from an adult sheep.

This result offers important prospects for studying scrapie in genetically identical sheep, as well as possibilities for breeding out any propensity to acquire the disease -- webmaster

Significance of milk production drop in BSE

Listserve 12.07.96 ... Roland Heynkes

I am not looking for a disgnostic tool, when I ask for a statistical plot of milk productivity against month before the detection of BSE. I am of course not interested in the daily productivity of single cattle, but in the average monthly decrease of productivity (and of fertility if possible) during the last year of many BSE-cattle.

With this data it would be possible to find out, if a decrease of the monthly milk production as an early consequence of BSE infection can increase the probability of slaughtering of cows in the last months before clinical signs of BSE become obvious. This question is important because such an effect would influence the BSE-statistics, the calculation of maternal transmission and prognoses for the future of BSE.

Milk drop not an early warning for BSE

Milk drop not an early warning for BSE

B. D. Slenning 12.05.96

A great deal of interest lies in coming up with an early warning system for identifying BSE animals. Ideally, we would be able to catch them before they show obvious neurological signs, because we know how long a period that can take. So, what we want (and what the above excerpt describes) are "pre-clinical" signs of BSE that we can use to identify and deal with infected cows. This message is meant to show that these signs are completely useless in that endeavor.

If we take the first paragraph (starting with "The character...") to describe pre-clinical BSE cases, and the second paragraph (starting with "Shipley's cow video...") to describe clinical BSE cases, we can look at the quality of information garnered by the signs displayed. Note, the first few sentences of paragraph 1, however, describe the clinical neurological and behavioral manifestations of the disease, so cannot be considered as "early" signs, but as signs highly suggestive of neurological disorders. So, the remainder of the signs for the pre-clinical cases described are:

          decrease feed intake
                stop ruminating/cudding
                digestive upsets
                loss of condition (i.e., weight loss)
                fall in milk production
The two major signs displayed early in the course of disease, prior to the manifestation of neurological signs, are [1] decrease feed intake (leads to decrease rumination and digestive upsets and loss in weight), and [2] fall in milk production. Though not mentioned specifically, farmers will describe cows that "hang back" or "change their patterns" as dull or depressed. That could be a third sign.

I went to the Cornell College of Vet Med "Consultant" site (a search algorithm that compares described signs and symptoms to known diseases), and entered these signs in a hypothetical bovine:

     anorexia                (decreased intake)
                dullness                (defined above)
                agalactia               (loss of milk production)
The database kicked back 242 different infectious diseases, intoxications, metabolic disorders, and neoplasias that can manifest themselves this way (As an aside, this USA database does not list a spongiform encephalopathy as one of the differential diagnoses). Given this, the listed signs may truly describe the "pre-clinical" BSE case, but they describe very many more-common disorders as well. As such, by themselves they are pretty worthless in helping to screen a population for a spongiform encephalopathy.

Next, I added in Tremors, to account for the described "... shaking movement across her shoulders ...". I did not use the excitatory signs, because, again, in most dairy cattle, anyway, those are not specific enough to be of much value. The database, using the first search criteria plus tremors produced 48 different infectious diseases, intoxications, metabolic disorders, and neoplasias that can manifest themselves this way.

Granted, a list of 48 is easier to work with than one of 242, but I think the point is made: The signs and symptoms attributed to these early cases of BSE are so generally shared by a multitude of other diseases that they do not add any clinical decision making value by their existence.

>From an individual animal perspective (i.e., the vet being called out to look at a sick cow), the results from applying such a BSE screening test are bad. One way to evaluate this is by using the Odds Likelihood ratio of the test. Without going into its derivation, the OL ratio says that the odds of a disease being present, given a positive test result, are equal to the prior odds of the disease (i.e., its prevalence in the selected group divided by 1-prevalence) times the ratio formed by taking the probability of a test positive result given the presence of disease, and dividing that by the probability of a test positive result given no disease is present. This is a spin-off of Bayes' theorem for those of you into probability games.

Suppose our vet and owner understand that the prevalence of BSE in adult cows in this area is 1% or less. Given that the Cornell listing of 242 diseases covers well over 90% of the cases a vet is likely to see in sick cows (my estimate, not theirs), then the probability of a test positive result given no disease is present is at least 90%. The probability of a test positive result given the presence of disease is 100%, as assumed above. The odds likelihood ratio, then, would be:

Probability the animal showing anorexia, agalactia, and dullness is displaying pre-clinical BSE (i.e., it posterior odds of BSE) equals :

 (prior odds) * (likelihood ratio) = 1/99 * 100/90  =  0.010 * 1.11 =  0.011
In plain english, the probability assigned to this animal having BSE was 1% prior to the test (1/99). After running the test, the new likelihood of the animal having BSE was 1.1%. So how much value can we place on these signs? Very, very little. This is true of the 242 diseases as well -- the signs are not helpful in differentiating any of them.

We often are handed diseases to work with where the diagnosis is defined by "manifestational means" (what signs and symptoms is the animal showing?). Unfortunately, when other diseases share those same manifestations, that diagnostic definition fails us in our attempts to help make decisions. We all want to find an early warning system. These manifestations are not capable of giving us that device.

Infectivity in different tissues of living beings with TSE
KEY: Infectivity: + found, - not found, 0 no information

SheepGoat Cow Mink Man Mouse
Spinal cord++++++++++++
Peripheral nerve++000+
Lymph tissue++-+0+
Bone marrow+---0+

Louping ill and scrapie

Listserve 12.01.96

John Lanchester wrote in the 2 Dec New Yorker:
1930's: 18,000 UK sheep were inoculated against louping ill, a brain inflammatory illness, spread by ticks. Despite formalin-treatment of the inoculated agent, the procedure gave rise to 1500 cases of scrapie. Louping is a Scottish word for fleeing or leaping, related to loping. In humans, louping ill is called Russian spring-summer encephalitis, a meningo-encephalitis with muscular trmeors and spasms followed by varying degrees of paralysis. Update on louping illness (the best of Medline):

 1) Sequencing and antigenic studies of a Norwegian virus isolated from
encephalomyelitic sheep confirm the existence of louping ill virus outside Great Britain and
Ireland. J Gen Virol 74 ( Pt 1): 109-14 (1993) 

         2) Nucleotide sequence of the envelope glycoprotein of Negishi virus shows very close
homology to louping ill virus. Virology 190: 515-21 (1992) 

         3) Genomic sequence of the structural proteins of louping ill virus: comparative
analysis with tick-borne encephalitis virus. Virology 180: 411-5 (1991) 

         4) Louping ill virus envelope protein expressed by recombinant baculovirus and
vaccinia virus fails to stimulate protective immunity. Virus Res 26: 213-29 (1992) 

         5) The virus causing encephalomyelitis in sheep in Spain: a new member of the
tick-borne encephalitis group. Res Vet Sci 58: 11-3 (1995) 

         6) Tick-borne flavivirus NS1 gene: identification of conserved peptides and antigenic
analysis of recombinant louping ill virus NS1 protein. Virus Res 31: 245-54 (1994) 

         7) Studies on the glycosylation of flavivirus E proteins and the role of carbohydrate in
antigenic structure. Virology 159: 237-43 (1987) 
There are some intriguing parallels to prion disease, like failure of the envelope protein to stimulate protective immunity, glycosylation, focus in UK, and the association with scrapie. [Guess: brains of sheep with louping ill were homogenized, treated with formaldehyde, and injected intra-muscularly into sheep.]

A model for prion protein dimerization based on alpha-helical packing

Warwicker,J.; Gane,P.J.
Biochemical and Biophysical Research Communications 1996; 226(N3): 777-82 1996

Residues 109-122 of the human prion protein (PrP) are highly conserved across species, and are predicted to be alpha- helical in PrPc, the cellular form. A computational search of the potential for alpha-helical dimerisation has been made for residues 109-122. The conformation which consistently scores highest in terms of burying non-polar surface area is a tight association involving alanine, glycine and valine residues. A model of heterodimerisation for PrPc and PrPSc (the misfolded form) is presented in which species barrier mutations would arise from interaction specificities that would follow, at least in part, the same framework as formation of a putative homodimer.

Protein markers in CSF from BSE-affected cattle

Protein markers in CSF from BSE-affected cattle

Jones,V.; Martin,T.C.; Keyes,P.; Dawson,M
Veterinary Record 1996; 139(N15): 360-3 1996

Two-dimensional gel electrophoresis was used to analyse cerebrospinal fluid (CSF) from 75 suspect cases of bovine spongiform encephalopathy (BSE), 61 of which were confirmed by post mortem brain histopathology, and 38 normal cattle. CSF samples were also examined from cattle killed at periodic intervals through the incubation period following experimental challenge. Consistent changes were recorded in all CSF samples from the confirmed cases of natural BSE and also from cattle showing early signs of experimental disease. The changes consisted of an increased intensity of staining of apolipoprotein E and the presence of two protein spots, as yet unidentified, of molecular weights 35 and 36 kDa, both with a pI of 5.5. These changes were absent in the CSF samples from the normal cattle, from the clinically suspect cattle which were not confirmed as BSE and from the experimentally challenged cattle in the preclinical phase of infection.

Preclinical detection of TSE in mouse spleen

Grathwohl,K.U.D.; Horiuchi,M.; Ishiguro,N.; Shinagawa,M. Archives of Virology 1996; 141(N10): 1863-74 1996

Scrapie in sheep has recently become again a target of control measures and eradication programs. Crucial for the effectiveness of these measures is the detection of infected sheep during the long and potentially hazardous incubation period. However, routine-diagnosis is mostly limited to clinical examinations when disease: becomes apparent, and to postmortem investigations. Through the detection of the scrapie-specific isoform of the prion protein (PrPSc) by Western blot in the spleen and lymph nodes from scrapie- infected mice and sheep, we have shown previously that diagnosis during the preclinical stage is possible. We introduce here an improved method for the diagnosis of mouse scrapie shortly after infection. Through a homogenization procedure that includes a collagenase digestion step, and through extraction and salting-out of PrPSc by Sarkosyl and NaCl, respectively, we were able to detect PrPSc in spleen tissue of intraperitoneally infected mice seven days postinfection. Moreover, the new protocol makes sample- handling easier and reduces the hands-on time. We also successfully enriched PrPSc from spleen tissue through immobilized metal affinity chromatography (IMAC); however, for the diagnosis at the earliest stage of infection, extraction of PrPSc by Sarkosyl and NaCl was more effective.

Allelic frequencies of an ovine scrapie susceptibility gene

Orourke,K.I.; Melco,R.P.; Mickelson,J.R.
Animal Biotechnology 1996; 7(N2): 155-62 1996

The allelic frequencies of an ovine gene associated with susceptibility to scrapie was analyzed in a sample of 30 scrapie affected sheep and 545 clinically normal sheep from 12 flocks. The allele encoding glutamine at codon 171 occurred at a frequency of 0.76 in the overall population. All 30 scrapie affected sheep were homozygous for glutamine at codon 171. This genotype was observed in 56.5% of the clinically normal sheep. None of the 30 scrapie affected sheep carried the allele encoding Valine at codon 136 although this allele was observed in 2/12 flocks sampled.

Spongiform encephalopathies of cattle, sheep and cats in Hungary

Glavits,R.; Ratz,F.
Magyar Allatorvosok Lap1996; 51(N9): 529-31 1996

Between 1989 and 1995, after having excluded rabies, histological examination of brains of 1037 cattle, 1494 sheep and 1436 cats with symptoms of CNS were carried out in the Hungarian diagnostic institutes (Table). None of the cases were found alterations characteristic for spongiform encephalopathies (BSE, scrapie, FSE) considering the diagnostic criteria specified in the European communities and USA. These findings confirm that Hungary remains free of spongiform encephalopathy of cattle, sheep and cats. In cattle and sheep, most frequently listeriosis and other purulent meningitides and/or focal purulent encephalitides caused by other bacteria, Aujeszky's disease, CCN, as well as encephalitis caused by ruminal acidosis, alkalosis, methaemoglobinaemia, ketosis, certain toxic matters and plants, infectious bovine rhinotracheitis virus and chlamydias were found associated with the nervous symptoms observed. In cats, Aujeszky's disease and different poisonings (toxicoses) were most frequently diagnosed. It has been pointed out that besides the histological investigation of the brains directed to the diagnosis of spongiform encephalopathies, the evaluation of clinical symptoms observed in vivo (the relatively long course of the disease, extending weeks or more frequently months and the gradually progressing, characteristic clinical symptoms) is also essential in each suspected case.

Aberrant induction of neuropeptide-y messenger-RNA in hippocampal ca3 pyramidal neurons in scrapie-infected mice

Diez,M.; Koistinaho,J.; DeArmond,S.J.; Camerino,A.P.; Groth,D.; Caytano,J.C.; Prusiner,S.B.; Hokfelt,T.
Neuroreport 1996; 7(N12): 1887-92 1996

The neurochemical alterations preceding neurological dysfunction and neuronal death in prion diseases are not well characterized. Here we examined, using in situ hybridization histochemistry, the expression of neuropeptide Y (NPY), an inducible and abundant neuropeptide in mammalian brain with known neuroregulatory functions, and glial fibrillary acidic a protein (GFAP), a marker for astroglial activation, in the hippocampus at different time points following intracerebral prion inoculation in male CD-1 mice. Between 110 and 140 days postinoculation NPY mRNA expression was specifically up- regulated in CA3 pyramidal neurones, whereas expression of NPY in hilar neurones remained unaltered. Up-regulation of GFAP mRNA was observed in the CA1 stratum radiatum at 60 days, and spread throughout the hippocampus, cortex and thalamus between 110 and 140 days, suggesting early accumulation of scrapie prion protein in these regions. The clinical symptoms were first manifested 120 days postinoculation. Aberrant induction of NPY mRNA in the hippocampal CA3 pyramidal neurones preceded the onset of neurological symptoms, and may be involved in the regulation of glutamate release at the Schaffer collateral-CA1 synapses in scrapie-infected mice.

Intrastriatally injected c-fos antisense oligonucleotide interferes with striatonigral but not striatopallidal gamma-aminobutyric acid transmission in the conscious rat

W. Sommer, R. Rimondini, W. O'Connor, A. C. Hansson, U. Ungerstedt, K. Fuxe
PNAS Volume 93, Number 24; Pages: 14134-14139

Antisense c-fos oligonucleotides injected into the neostriatum of conscious rats selectively inhibited c-fos expression associated with compensatory increases in striatal c-fos mRNA levels and also with increased expression of junB and NGFI-A mRNA, probably as a result of regulatory phenomena. Dual probe in vivo microdialysis was used to investigate gamma-aminobutyric acid (GABA) release in the substantia nigra and the globus pallidus, which represent the terminal sites of the dopamine D1 receptor regulated striatonigral and the dopamine D2 receptor regulated striatopallidal GABA pathways, respectively. Intrastriatal infusion of the c-fos antisense oligonucleotide profoundly decreased dialysate GABA levels in the ipsilateral substantia nigra within 60 min but did not influence the dialysate GABA levels in the globus pallidus compared with the sham and control oligonucleotide treated groups. The site of action of the antisense oligonucleotides was mainly restricted to striatal neurons as shown by the distribution of locally injected fluoresceine isothiocyanate and radiolabeled oligonucleotides. The findings demonstrate a facilitatory role for c-fos mediated gene regulation in striatonigral GABA transmission and strengthen the evidence that the regulation of neurotransmission is different in the striatonigral and striatopallidal GABA pathways.

Biochemical characterization and intracellular localization of the Menkes disease protein

Yukitoshi Yamaguchi, Mark E. Heiny, Mariko Suzuki, Jonathan D. Gitlin
PNAS Volume 93, Number 24; Pages: 14030-14035

Menkes disease is a fatal neurodegenerative disorder of childhood due to the absence or dysfunction of a putative copper-transporting P-type ATPase encoded on the X chromosome. To elucidate the biosynthesis and subcellular localization of this protein, polyclonal antisera were generated against a bacterial fusion protein encoding the 4th to 6th copper-binding domains in the amino terminus of the human Menkes protein. RNA blot analysis revealed abundant Menkes gene expression in several cell lines, and immunoblotting studies utilizing this antiserum readily detected a 178-kDa protein in lysates from these cells. Pulse-chase studies indicate that this protein is synthesized as a single-chain polypeptide which is modified by N-linked glycosylation to a mature endoglycosidase H-resistant form. Sucrose gradient fractionation of HeLa cell lysates followed by immunoblotting of individual fractions with antibodies to proteins of known intracellular location identified the Menkes ATPase in fractions similar to those containing the cation-independent mannose-6-phosphate receptor. Consistent with this observation, confocal immunofluorescence studies of these same cells localized this protein to the trans-Golgi network and a vesicular compartment with no expression in the nucleus or on the plasma membrane. Taken together, these data provide a unique model of copper transport into the secretory pathway of mammalian cells which is compatible with clinical observations in affected patients and with recent data on homologous proteins identified in prokaryotes and yeast.

Polymorphism in transcriptional control for HTT

Science November 1996

Recently, we reported a polymorphism in the transcriptional control region upstream of the 5-HTT coding sequence. Initial experiments demonstrated that the long and short variants of this 5-HTT gene-linked polymorphic region (5-HTTLPR) had different transcriptional efficiencies when fused to a reporter gene and transfected into human placental choriocarcinoma (JAR) cells . The 5-HTTLPR is located 1 kb upstream of the 5-HTT gene transcription initiation site and is composed of 16 repeat elements. The polymorphism consists of a 44-base pair (bp) insertion or deletion involving repeat elements 6 to 8 (Fig. 1A). In the present study, polymerase chain reaction (PCR)-based genotype analysis of 505 subjects revealed allele frequencies of 57% for the long (l) and 43% for the short (s) allele (11). The 5-HTTLPR genotypes were distributed according to Hardy-Weinberg equilibrium: 32% l/l, 49% l/s, and 19% s/s.

Map of the human 5-HT transporter gene promoter (5-HTTP) (EMBL-GenBank accession number X76753). The 5-HTTLPR comprises a repetitive sequence with an insertion-deletion variation indicated by a black box. (B) Basal and PKC- or cAMP-induced transcriptional activity of the long (l) and short (s) 5-HTTP variants in human lymphoblast cell