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A partial amino acid sequence of a serine protease from Dermatophilus congolensis allowed the design of oligonucleotide primers that were complemented with additional ones from previously published partial sequences of the gene encoding the enzyme. The polymerase chain reaction (PCR), using combinations of specific and degenerate oligonucleotide primers, allowed the amplification of a 1738-bp internal fragment of the gene, which was finally characterised by inverse PCR as the first full-length sequenced serine protease gene (nasp) from Dermatophilus congolensis. The deduced amino acid sequence of this enzyme, probably involved in the pathogenesis of dermatophilosis, links it to the subtilisin family of proteases.

Human endogenous retroviruses (HERVs) are a family of viruses within our genome with similarities to present day exogenous retroviruses. HERVs have been inherited by successive generations and it is possible that some have conferred biological benefits. However, several HERVs have been implicated in certain cancers and autoimmune diseases. This article demystifies these retroviruses by providing an insight into HERVs, their means of classification, and a synopsis of HERVs implicated in cancer and autoimmunity. Furthermore, the biological roles of HERVs are explored.

Serum and synovial antibody reactivities of caprine arthritis encephalitis virus (CAEV) infected goats were assessed by Western blotting against purified CAEV antigen and the greatest intensity of reactivity in the serum of arthritic goats was to the gp45 transmembrane protein (TM). The extracytoplasmic domain of the TM gene was cloned into a pGEX vector and expressed in Escherichia coli as a glutathione S transferase fusion protein (GST-TM). This clone was found to be 90.5 and 89.2% homologous to published sequences of CAEV TM gene. Serum of 16 goats naturally infected with CAEV were examined by Western blotting for reactivity to the fusion protein. Antibody reactivity to the GST-TM correlated with clinically detectable arthritis (R = 0.642, P < or = 0.007). The hypothesis that the immune response to the envelope proteins of the CAEV contributes to the severity of arthritis in goats naturally infected with CAEV via epitope mimicry was tested. Antibodies from 5 CAEV infected goats were affinity purified against the GST-TM fusion protein and tested for cross-reactivity with a series of goat synovial extracts and proteogylcans. No serum antibody response or cross-reactivity of affinity purified antibodies could be detected. Peptides of the CAEV SU that were predicted to be linear epitopes and a similar heat shock protein 83 (HSP) peptide identified by database searching, were synthesized and tested for reactivity in CAEV goats using ELISA, in vitro lymphocyte proliferation and delayed type hypersensitivity (DTH) assays. Peripheral blood lymphocytes from 10 of 17 goats with long term natural CAEV infections proliferated in vitro in response to CAEV and in vivo 3 of 7 CAEV infected goats had a DTH reaction to CAEV antigen. However, none of the peptides elicited significant cell mediated immune responses from CAEV infected goats. No antibody reactivity to the SU peptides or HSP peptide was found. We observed that the antibody reactivity to the CAEV TM protein associated with severity of arthritis however epitope mimicry by the envelope proteins of CAEV is unlikely to be involved.

Serine proteases are thought to be involved in the initial attack on sheep skin by Dermatophilus congolensis and are obvious antigens for inclusion in a vaccine to prevent lumpy wool disease (dermatophilosis). Degenerate primers were designed after alignment of seven bacterial serine proteases. Inosine was incorporated into the primers at positions of three- and four-base redundancy, and this reduced the complexity of the primer mixtures from several thousand to sixteen different sequences for each primer. The primers were validated by production and sequencing of amplicons from serine protease genes in Bacillus subtilis and Serratia marcescens. The primers were used with heat-soaked polymerase chain reaction (PCR) to produce amplicons from two D. congolensis strains, AG and MB. In the amplicon codons for arginine, rather than the expected serine, were found where inosine was used for both the first and third positions for a codon in the primer. A search with the deduced amino acid sequences of the amplicons showed significant similarity to a keratinase and other serine proteases from various organisms. Similarity was most apparent around the active site residues and other essential secondary structural elements.

Epitope mimicry is the theory that an infectious agent such as a virus causes pathological effects via mimicry of host proteins and thus elicits a cross-reactive immune response to host tissues. Weise and Carnegie (1988) found a region of sequence similarity between the pol gene of the Maedi Visna virus (MVV), which induces demyelinating encephalitis in sheep, and myelin basic protein (MBP), which is known to induce experimental allergic encephalitis (EAE) in laboratory animals. In this study, cross-reactions between sera raised in sheep against synthetic peptides of MVV (TGKIPWILLPGR) and 21.5 kDa MBP (SGKVPWLKPGR) were demonstrated using enzyme-linked immunosorbant assay (ELISA) and thin layer chromatography (TLC) immunoprobing. The antibody responses of MVV-infected sheep were investigated using ELISA against the peptides, and MBP protein, immunoprobing of the peptides on TLC plates and Western blotting against MBP. Slight significant reactions to the 21.5 kDa MBP peptide (P < 0.001) and to a lesser extent sheep MBP (P < 0.004) were detected in ELISA. The MBP peptide evoked stronger responses from more sera than the MVV peptide on immunoprobed TLC plates. On the Western blots, eight of the 23 sheep with Visna had serum reactivity to MBP. This slight reaction to MBP in MVV-infected sheep is of interest because of the immune responses to MBP evident in multiple sclerosis and EAE, but its relevance in Visna is limited since no correlation with disease severity was observed. The cell-mediated immune responses of MVV-infected sheep against similar peptides was assessed. The peptides did not stimulate proliferation of peripheral blood lymphocytes of MVV-infected sheep. Since the MVV peptide was not recognised by antibodies or T lymphocytes from MVV-infected and encephalic sheep, it was concluded that epitope mimicry of this 21.5 kDa MBP peptide by the similar MVV pol peptide was not contributing to the immunopathogensis of Visna. The slight antibody response to MBP and the MBP peptide can be attributed to by-stander effects of the immunopathology of MVV-induced encephalitis.

Based upon Blalock's complementary recognition approach, a complementary or antisense peptide (CP) was designed to the experimental autoimmune encephalomyelitis (EAE) epitope peptide, rat myelin basic protein (MBP) peptide 72-82. This peptide (EAE CP) was shown to have some sequence similarities to T-cell receptors (TCR) and MHC II molecules in a sequence homology search. Solid-phase binding assays demonstrated specific and high affinity binding (3 and 4 microM) between the EAE CP and the rat and guinea pig EAE epitope peptides (Rt72-82 and Gp69-82), respectively. This EAE CP was also found to be immunogenic in rats in an ear swelling test for delayed type hypersensitivity (DTH) reactions and an ELISA for antibody responses. However, a rabbit antibody generated to EAE CP was shown to be unable to stain the V beta 8+ EAE susceptible T-cells in immunofluorescence analyses. This EAE CP was also used in attempts to down-regulate EAE and the results showed that prior immunization with EAE CP in complete Freund's adjuvant could not prevent the Lewis rats from developing EAE. Although the data on sense-antisense peptide interaction were positive and the EAE CP was immunogenic, the inability of EAE CP to regulate EAE indicates that the CP approach may not be generally applicable.

Gamma inulin and Algammulin, two new adjuvants, were examined and compared with alum and Freund's Complete/Incomplete Adjuvant (FCA/FIA), for potentiation of cell-mediated immunity (CMI) and humoral immunity in sheep to a recombinant Taenia ovis antigen. The ability to protect sheep when challenged with live T. ovis eggs was also assessed. The results showed that gamma inulin and Algammulin induced a CMI response which was comparable to the FCA/FIA and alum groups and significantly higher than the control saline group. While gamma inulin, Algammulin and alum performed similarly and induced a significantly higher humoral immune response than the saline group. FCA/FIA elicited a much higher humoral immune response. Algammulin did not show the synergistic effect seen in mice and performed similarly to gamma inulin and alum alone. All the adjuvant groups induced significantly higher IgG1 and IgG2 levels than the saline group and they all favoured IgG1 production. When the sheep were challenged with live T. ovis eggs, at 25 weeks after primary immunization, the only group to show significant protection was the one which received FCA/FIA.

An important part of quality control in the food industry are tests to determine the origin of materials used in processed products. Because of the stability of DNA, tests which make use of DNA in the product can be used to authenticate the species used in meat pies, tinned fish and even the variety of oats used in porridge. Tests based on hybridization, sequencing of oligonucleotides and amplification of DNA are likely to find increasing application with the only inhibition being the unpredictable nature of the market which makes commercial development difficult.

Proviral DNA from four Australian isolates of feline immunodeficiency virus (FIV) was amplified by PCR and the nucleotide sequence determined for two conserved regions within gag (p15/p24) and pol (RT) genes. Comparison with the nucleotide and deduced amino acid sequence of two previously described U.S. isolates from California (Petaluma and PPR), and a third from Maryland (MD) as well as the Japanese isolate TM2, revealed a close similarity between the Australian and Californian isolates with 95-97% nucleotide and 96-99% amino acid homologies. By contrast, the Maryland and Japanese isolates were more distantly related with only 84-87% nucleotide and 90-94% amino acid homology with either the Australian or Californian isolates. The relationship of the Australian FIV isolates to other domestic isolates as well as eight lentiviral isolates from wild felidae (panthers) published previously, was investigated further by constructing a phylogenetic tree based on the pol sequence. This revealed two subgroups of FIV, an Australian/Californian group and a less tightly clustered Maryland/Japanese group. These results suggest that the genomic variability of FIV is reflected by more than simply geographic distance. Furthermore, the relative genetic homogeneity found between Australian isolates suggest a shorter period of evolution of the virus in Australia than in North America.

In an investigation of the evolution of feline immunodeficiency virus (FIV) in vivo, sequential isolates from a persistently infected cat were examined by direct sequencing following amplification of selected subgenomic regions by polymerase chain reaction (PCR). Three isolates, T90, T91, and T92, obtained over a three-year period revealed no changes to regions known to be conserved within gag and pol genes. Additionally, no change occurred within gag and pol in an isolate recovered from a second cat which was experimentally infected with T90. Changes were detected within an N-terminal region of the envelope glycoprotein gp 120 (env). These consisted of point mutations, some of which would result in amino acid substitutions and the predicted amino acid changes tended to cluster within variable domains. Inoculation of T90 into a second cat resulted in a different pattern of mutations than that observed for the three isolates from the first cat. In all cases, virus isolates derived from the same cat were much more highly related to each other (extent of env variation was 0.5-1.5%) than to isolates from other cats (10-12% env variation). The rate of change of FIV was estimated to be 3.4 x 10(-3) nucleotide substitutions per site per year for the env gene and less than 10(-4) nucleotide substitutions per site per year for the gag and pol genes, values concordant with that found for human immunodeficiency virus 1. Both nucleotide and amino acid changes in the gp 120 region were found to be directional, suggesting that selective pressures influence FIV envelope gene sequences.

Since the discovery of human immunodeficiency virus (HIV) as the cause of AIDS the study of other members of the virus group lentivirinal has intensified. A major outcome of this research have been the development of "state of the art" diagnostic tests. While it has long been realised that these viruses were important causes of slowly developing diseases in sheep and horses other lentiviruses have recently been implicated in similar disease situations in goats, cats and cattle. Membership of the lentivirus group of viruses has increased dramatically over the past fifteen years and it is likely that most if not all mammalian species will be found to be infected with their own lentivirus. This paper will describe the problems caused by these viruses in domestic animals and the steps being taken to prevent their spread. Biotechnology companies have a major role in devising simple and economical tests to detect the viruses and antibodies to these viruses. In addition if a vaccine can be produced against any of the animal viruses this will perhaps enable a similar vaccine to be prepared against HIV.

The reactivity to a peptide from the HTLV-I polyprotein (FKLPGLNSR) and a similar sequence from myelin basic protein (MBP) (FKLGGRDSR) was examined in relation to the proposal that mimicry of MBP by HTLV-I could be involved in autoimmune responses in HTLV-I-associated myelopathy (HAM). It was found that rabbit antibodies raised against the HTLV-I peptide recognised both peptides, with a titre of 1/10240 to the HTLV-I peptide and 1/5220 to the MBP peptide. Human sera from HAM patients and a HTLV-I carrier without HAM showed slightly higher responses to the HTLV-I peptide compared to the responses from uninfected human sera. HAM patients had greater responses to the HTLV-I peptide than to the similar MBP peptide and an unrelated bovine MBP peptide. There was no recognition of the peptides by peripheral blood lymphocytes from HAM patients or a HTLV-I carrier without HAM. It was concluded that although cross-reactivity was demonstrated in rabbits and the HTLV-I peptide was recognised by sera from HAM patients, the epitope does not appear to evoke a mimicking response to the similar region in MBP. Hence it is not likely to be involved in the pathogenesis of HAM through molecular mimicry.

The 21,500 molecular weight (21.5K) variant of myelin basic protein (MBP) was isolated from sheep brain and partially characterized. Digestion with cyanogen bromide and trypsin yielded peptides which showed that approximately 30 additional amino acids were inserted at the equivalent of the amino acid at position 57 in the bovine 18.5K MBP sequence. An unusually hydrophobic peptide Pro, Val, Leu, Trp, Lys was present in this region. Ornithine was present in hydrolyzates of 21.5K MBP, but it was not detected in any of the peptides.

Methylated amino acids are excreted in urine upon degradation of some tissue proteins. The urinary excretion ratios of NG,N'G-dimethylarginine (syn-DMA) and NG,NG-dimethylarginine (unsym-DMA) were studied in healthy adults and in patients with various diseases. The normal ratio of sym- to unsym-DMA in urine was 0.98 and ranged from 0.71 to 1.33; ratios were not significantly different in multiple sclerosis, cerebrovascular accident, cancer, and systemic lupus erythematosus. However, patients with liver, disease, including chronic active hepatitis, were found on average to have a significantly altered ratio of 0.79, range 0.49-1.30, owing to an increase in the excretion of unsym-DMA. Hence measurements of the urinary excretion of dimethylarginine could become a useful aid in assessing recovery of liver cells in patients with chronic liver disease.

A solid phase competitive assay for human myelin basic protein (MBP) has been developed using a monoclonal antibody to MBP. The assay has been applied to the detection of antigenic peptides derived from MBP, the measurement of anti-idiotypic antibody and the detection of MBP in human serum.

A method for the electroimmunoblotting and immunodetection of peptides of less than 50 amino acid residues is described. Excellent resolution of a mixture of myelin basic protein (MBP) peptides was achieved by electrophoresis in a polyacrylamide stacking, urea-dodecyl sulphate minislab gel. Following electrophoresis, the peptides were transferred to various matrices and probed with monoclonal and polyclonal antibodies. Variables such as transfer time, membrane type, fixation and the amount of peptide loaded on the gel have been optimized as a consequence native and synthetic peptides can now be visualized in gels and immunodetected on immobilizing matrices. This procedure is particularly suited to the analysis and identification of small MBP fragments arising in various neuropathological conditions as well as for the rapid characterization of antigenic determinants recognized by monoclonal and polyclonal anti-MBP antibodies.

It was confirmed that experimental autoimmune encephalomyelitis EAE, could be induced in SJL/J mice with mouse spinal cord homogenate. It was shown that induction of EAE in mice was critically dependent on the concentration of pertussis vaccine. The encephalitogen present in mouse brain was the basic protein of myelin. The smaller form of the mouse and rat basic proteins induced EAE; thus the mouse like the rat responds to determinants other than the "tryptophan region," which induced EAE in guinea-pigs. Mice with EAE developed a cell-mediated immune response to myelin basic protein, as judged by inhibition of peritoneal cell migration. However, levels of antibody to mouse basic protein were low, as judged by radioimmunoassay. The establishment of this autoimmune disease model in the mouse will allow the application of well established techniques for the analysis of the immunologic mechanisms leading to disease manifestation.

Hind-limb motor function in adult female Lewis rats with experimental autoimmune encephalomyelitis (EAE) was investigated using an objective behavioral measurement of motor ability. Rats were pretrained to avoid falling from the external surface of a power-driven running wheel. The performance of EAE-group rats on the wheel was then compared with that of saline and adjuvant controls immediately prior to the onset of clinical signs of EAE, and within 3 days of apparent recovery from EAE. Results indicate no apparent hind-limb motor deficit in the absence of overt clinical signs of EAE, despite histological evidence of severe inflammatory lesions persisting in the central nervous system (CNS) at the time of the post-recovery test. The remarkably transient nature of motor impairment is discussed within the context of a continuing search for the underlying cause(s) of clinical signs of EAE.

A rapid method for similarity searches (FASTP program) was used to identify similarities between a protein database and the human basic proteins from myelin [P2 protein and 17.2K, 18.5K, and 21.5K variants of myelin basic protein (MBP)]. From similarity scores, we concluded that none of the presently known proteins are in a family containing the MBPs. No new members were found for the lipid-binding family of which P2 is a member. Sequence similarities deemed relevant to the molecular mimicry hypothesis for virus-induced autoimmunity were identified in FASTP data with the aid of microcomputer programs. Several MBP/viral protein similarities were found that have not been reported previously. Of note because of their association with demyelinating conditions were proteins from visna and vaccinia. Similarity with visna was specific to the 21.5K and 20.2K MBPs. The most interesting new possibility for mimicry involving the P2 protein was between the influenza A NS2 protein and a sequence region of P2 thought to be neuritogenic in animals and mitogenic for lymphocytes from some patients with Guillain-Barré syndrome (GBS). This may have relevance for some cases of GBS associated with the 1976 U.S.A. swine flu vaccination program. Because FASTP reports only the best similarities between proteins, searches with FASTP may not have detected all the examples of mimicry present in the database. Searches might also be more effective if similarities could be scored on immunological rather than structural relatedness.

Attempts were made to transmit possible infectious agents from tissue of MS patients into three animal species, under conditions designed to enhance the development of such an agent. Myelination was monitored by measuring CNPase activity and myelin basic protein. Although no significant effects were observed, the usefulness of a new assay for CNPase was demonstrated. Nude mice were found to have a lower level of CNPase than their heterozygous littermates.

The cell-mediated inflammatory component of experimental autoimmune encephalomyelitis (EAE) in mice is measured by the radioisotopic technique. Mice are challenged with autologous spinal cord homogenate in Freund's complete adjuvant and at various time intervals after such immunization given [125I]5-iodo-2'-deoxyuridine which is incorporated into the mononuclear cell pool. The degree of cell-mediated inflammation is determined by radiometry of the brain and spinal cord tissues. Increased radiolabelling is detected in the brains 2 days prior to the onset of clinical signs of EAE; increased radioactivity of the spinal cord is concomitant with clinical signs. This technique is useful in staging the extent of EAE and may prove to be a powerful tool in studying cell-mediated reactions in other autoimmune diseases.

1. The complete amino acid sequence of the smaller basic protein from rat brain myelin was determined. This protein differs from myelin basic proteins of other species in having a deletion of a polypeptide of 40 amino acid residues from the centre of the molecule. 2. A detailed comparison is made of the constant and variable regions in a group of myelin basic proteins from six species. 3. An arginine residue in the rat protein was found to be partially methylated. The ratio of methylated to unmethylated arginine at this position differed from that found for the human basic protein. 4. Three tryptic peptides were isolated in more than one form. The differences between the two forms of each peptide are discussed in relation to the electrophoretic heterogeneity of myelin basic proteins, which is known to occur at alkaline pH values. 5. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50029 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.

The studies described were designed to examine the question of cross-reactivity between basic protein of myelin and a basic protein common to many human tumours. The lymphocytes from guinea-pigs injected with acid extracts of human cancer tissue showed significant (P less than 0-01) tranformation on exposure to basic protein of human myelin. Conversely lymphocytes from guinea-pigs injected with basic protein of human myelin showed significant (P less than 0-01) transformation on exposure to the acid extracts of human cancer tissue. Lymphocytes from control non-injected guinea-pigs did not transform on exposure to either antigen. These findings demonstrate immunological cross-reactivity in guinea-pigs between basic proteins of human myelin and of human cancer tissue using an assay system other than the macrophage electrophoretic migration hitherto used to show this effect in man.

A previous hypothesis in which myasthenia gravis was explained by an immune response to acetylcholine receptors has been validated, and is here extended to cell receptors in general. Receptors on target cells, being accessible to circulating trophic hormones or transmitters, must also be accessible to antibodies which compete with the natural mediator for access to the site. To detect anti-receptor antibodies, physiological assay systems would be more sensitive than conventional immunological assays. Autoimmune responses to receptor sites would require a genetic predisposition to failure of immunological tolerance such as occurs in various autoimmune diseases. This hypothesis is supported by recent findings in hyperthyroidism and a type of insulin-resistant diabetes mellitus, and is applicable to other endocrinopathies, diseases in which dysfunction at receptor sites can be postulated, and regulatory functions within the immune system itself.

High performance liquid chromatography on a Partisil-10 SCX column was examined as an alternative to conventional ion exchange chromatography as a means of quantitating the histidine dipeptides, anserine, carnosine and balenine in processed pig meats. Because the ratio of these dipeptides in skeletal muscle of the pig is different from that in other species commonly used for meat, it is possible to estimate the proportion of pig meat in processed meats. Several Australian pork products were found to contain a low proportion of lean pig meat., (Copyright © 1984. Published by Elsevier Ltd.)

A developmental study of myelin basic protein (MBP) variants in eight regions of pig nervous system (NS) was performed using a quantitative electroimmunoblotting procedure. Four major MBP forms with apparent molecular weights of 21.5K, 20.2K, 18.5K, and 17.3K were identified in both the CNS and the PNS and were detected as early as 22 days before birth. Quantification of the most abundant forms, the 21.5K and 18.5K MBPs, revealed characteristic profiles of accumulation of these two variants in different regions of the NS. The ratio of 21.5K:18.5K MBP varied with developmental time as well as with the various NS regions, peaking 20 days postnatally. The 17.3K MBP was observed from embryonic stages to adulthood, as were the 21.5K and 18.5K forms. In contrast, the 20.2K variant appeared most abundant from 10 days before to 22 days after birth and thereafter decreased in intensity so as to be no longer detectable in the brain of a 5-year-old pig. A similar pattern was also observed with an anti-MBP-reacting protein with an apparent molecular weight of 23K. Taken together, these results suggest that in the pig NS, the expression of MBP variants may be regulated both regionally and developmentally.

An electroimmunoblotting technique was used with a monoclonal antibody to myelin basic protein (MBP) to assess demyelination in 3 defined regions of the spinal cord in rats with acute experimental autoimmune encephalomyelitis (EAE). A slight loss in MBP was detected only in the sacrococcygeal region of the spinal cord after the onset of clinical signs. In all 3 spinal cord regions studied, significantly elevated levels of albumin and IgG were detected during the course of EAE by the same technique. At the onset of clinical signs, the levels of IgG and albumin were highest in the more caudal regions of the spinal cord. As the clinical signs became more severe, IgG and albumin levels increased in the more cranial regions of the spinal cord. These changes thus correlated with the ascending progression of clinical signs typical of EAE in rats. These results provided added evidence that in rats affected with acute EAE, the clinical signs occur independently of demyelination and coincide with vasogenic edema.

The neurological signs induced by injection of tunicamycin are, in young adult rats, virtually identical to those typical of acute experimental autoimmune encephalomyelitis (EAE). Vasogenic exudation, of which the occurrence in the spinal cord of EAE rats has been shown to coincide with the onset of clinical signs, was investigated by quantitative electroimmunoblotting of central nervous system (CNS) tissue at various times following tunicamycin injection of young adult rats. Highly elevated levels of extravasated plasma proteins were observed in the spinal cord from 48 h after injection and, as in EAE rats, these increases coincided with the onset of neurological impairment. At 72 h post-injection, significant increases were also found in the brain of affected animals, albeit at much reduced levels. This is in contrast to previously reported findings in nursling rats where oedema was shown to be predominantly located in the brain. Quantitative electroimmunoblotting for myelin basic protein (MBP) in the CNS of tunicamycin-treated young adult rats indicated that, as in acute EAE, no extensive demyelination had occurred. These data provided further evidence that in both neurological diseases, vasogenic oedema of the spinal cord may be causally related to the appearance of neurological signs and suggested that its differential localization in the CNS may lead to differential neurological impairment.

A case is described of progressive cerebellar degeneration in association with ovarian carcinoma, with nearly 4 years elapsing between the onset of neurological illness and the discovery of the carcinoma. Neither treatment with prednisolone nor surgical removal of the tumour effected any improvement, CAT scanning late in the illness showed marked cerebellar atrophy. Histology at post-mortem showed complete loss of cerebellar Purkinje cells. The cerebrospinal fluid gave weak binding to multiple neural components in a solid-phase radio-immuno-assay.

Chickens injected with cycloleucine developed vacuolation of myelin similar to that seen in humans with vitamin B12 deficiency. Cycloleucine, an inhibitor of the formation of S-adenosylmethionine, decreased the incorporation of methyl groups into methylarginine in myelin basic protein in vivo. The need for methylcobalamin for the conversion of homocysteine to methionine and the requirement that myelin basic protein may be methylated, offer a rational explanation for the myelin lesions observed in cases of vitamin B12 deficiency where there is increasing evidence that methyl-, rather than adenosylcobalamin is required to prevent dysmyelination.

Degradation of myelin basic protein (MBP) in human man myelin was monitored by electroimmunoblotting. Problems of variation between, as well as within, electroimmunoblots were overcome by the introduction of an internal standard in each sample, thus allowing reproducible quantification of MBP. The Ca2+-dependent protease acting on MBP was active at endogenous levels of Ca2+ (congruent to 300 micrograms/g myelin) and was inhibited in the presence of Ca2+ chelators. Extensive degradation of MBP occurred rapidly in the presence of added Ca2+, reaching a plateau after a 1 h incubation (80-85% degradation). The proteolytic activity was not enhanced in the presence of 2-mercaptoethanol. It was most active at neutral pH and at temperatures approaching physiological conditions. No difference was observed between proteolytic activities of control and multiple sclerotic myelin. It is suggested that fluctuations in the accessibility of free Ca2+ to the protease may lead to the regulation of Ca2+-activated myelinolysis.

Isoelectric focusing was used to characterize proteolytic enzymes in homogenate and excretory-secretory preparations of the larvae of L. cuprina, the sheep blowfly. Zymogram overlays showed that the larvae produce a number of highly active proteases which have a wide range of isoelectric points and molecular weights. The alkaline and neutral pI proteases were inhibited by phenylmethyl-sulfonylfluoride, leupeptin and aprotinin; this indicated the presence of serine in the active site. Pepstatin and the metal chelating agent ethylenediaminetetraacetic acid had no effect on the activity of any of the proteases. Optimal pH for activity of the proteases was between 7 and 8. In addition, the proteases were found to be heat labile. Digestion of collagen fibrils confirmed the existence of collagenolytic activity in the excretory-secretory enzyme preparations. It is suggested that these enzymes may be involved in the nutrition of the larvae and in the pathogenesis of the lesion on the skin.

A solid phase radioimmunoassay (RIA) for the detection of antibodies to myelin basic protein (MBP) in sera has been developed employing MBP-coated flexible polyvinylchloride microtiter trays and [125I]protein-A as the radiolabel. [125I]Protein-A directly binds to the Fc region of serum IgG from several animal species and serves as an excellent reagent for detecting antibody. It can also be used to bind to a second antibody ligand, thereby making it useful even when it does not bind directly to the primary antibody Fc region. The use of one preparation of [125I]protein-A label allows sera from several species to be tested for antibodies to MBP simultaneously, thereby making this RIA technically simple, rapid and economical. This assay has been particularly useful in examining serum samples from animals with experimental autoimmune encephalomyelitis and hypomyelinogenisis congenita. In patients with multiple sclerosis low levels of antibody to MBP can be detected in cerebrospinal fluid (CSF) and acid-eluted extracts from brain plaque material; detection of low levels of antibody in human serum has not been possible due to non-specific binding of human serum Ig in this RIA.