Strain-related effects of fenbendazole treatment on murine experimental autoimmune encephalomyelitis.

Parasitic infections are a concern in animal facilities, in view of their influence on physiological processes and the immune status of animals. Pinworms are effectively controlled with the anthelminthic fenbendazole (FBZ, [5-(phenylthio)-1H-benzamidazol-2-yl]carbamic acid methyl ester; C(15)H(13)N(3)O(2)S); however, questions remain as to whether prolonged FBZ exposure alters the disease course in specific experimental models, such as those pertaining to the immune system. We report that a three-month regimen of FBZ-medicated feed severely affected the onset and disease severity of murine experimental autoimmune encephalomyelitis (EAE), a disease that mimics multiple sclerosis. Differences were recorded between mouse strains used. Our data suggest that where the use of FBZ is mandatory, its full effect should be verified on the particular EAE variant adopted by the laboratory.

Disturbed oligodendrocyte development and recovery from hypomyelination in a c-myc transgenic mouse mutant.

The complexity of interactions underlying the elaboration of myelin has been extensively demonstrated. We provide evidence that signals promoting myelination are not confined to the normal developmental time window for myelination and persist well into adult life. The 2-50 mutant, described previously, carries a c-myc transgene regulated by a myelin basic protein promoter. This mutant is characterised by severe hypomyelination and abnormal oligodendrocytes in early life, followed by loss of the phenotype and normal longevity. We show that c-myc expression in early oligodendrocyte development results in a substantial reduction of cells of this lineage. However, apparent complete recovery, associated with loss of c-myc expression, axonal survival, and gradual myelin accumulation, is observed by 4 months of age. Thus, stimulation of myelination continues during adult life until normal myelin levels are established. We propose that this mutant may contribute to the characterisation of oligodendrocyte responses to myelinating signals.

Axonal degeneration is an early pathological feature in autoimmune-mediated demyelination in mice.

Multiple sclerosis (MS) is an inflammatory disorder of the central nervous system (CNS), characterised by focal destruction of myelin. Although it is evident that the immune system contributes to tissue destruction in MS, it is still unclear as to whether this immune response is a cause or a consequence of the disease process. In addition, there is debate over the contribution of axonal damage to clinical progression. We have described a murine model of relapsing-remitting MS (RR-MS), the most common form of the disease, following immunisation with the myelin component, myelin oligodendrocyte glycoprotein (MOG). We showed that a single injection of a MOG peptide (MOG(35-55)) in NOD/Lt mice induces a paralytic relapsing disease with extensive plaque-like demyelination. This model also mimics many of the immunological features associated with RR-MS. To investigate the relationship between clinical episodes, inflammation, and demyelination/remyelination, we analysed lesions during each attack and remission over the course of the disease, using histological, immunocytochemical, and electron microscopy (EM) techniques. We show that morphological features of lesions in our model resemble those observed in MS. Indeed, severe inflammation and demyelination coincide with the peak of clinical episodes while remissions are characterised by quiescent plaques. Furthermore, axonal damage is evident from the earliest stage of the disease and increases in severity with subsequent relapses. These data establish that in the model of MS-like disease, the peak of clinical episodes coincides with severe inflammation and demyelination and that axonal pathology correlates with clinical progression.

Regulation of myelin oligodendrocyte glycoprotein in different species throughout development.

The assembly and function of central nervous system (CNS) myelin requires the coordinated expression of several myelin-specific proteins, including myelin oligodendrocyte glycoprotein (MOG). Despite the recent cloning of MOG, the function of this molecule is still unknown. Because MOG is a late marker of oligodendrocyte maturation and is exclusively expressed in the CNS on the outermost lamellae of the myelin membrane, it is possible that this molecule plays an important role in the control and maintenance of myelination. Furthermore, as a member of the immunoglobulin superfamily that carries the L2/HNK-1 epitope, it has also been suggested that MOG is involved in cell-cell interaction, perhaps functioning as an adhesive molecule for bundles of nerve fibres. In order to further delineate the role of MOG throughout development we have analysed, by immunoblotting, the developmental appearance and accumulation pattern of MOG in the CNS of three mammalian species. We have also purified MOG to homogeneity from five different species including rat, guinea pig, bovine, monkey and human. Immunoblotting revealed two major MOG bands at 28 and 55 kD in all species. The 55 kD band appears to be a dimer of the lower band although treatment with 2-mercaptoethanol or EDTA failed to abolish it. Purified MOG from all species also displayed faint reactivity with bands at 36, 48 and 78 kD. While the 78 kD band may represent a trimer of MOG, the identity of the other bands remains unknown. Developmental studies in mouse, rat, guinea pig and bovine showed at as for other myelin proteins, MOG displayed a caudorostral gradient of expression, appearing in the spinal cord before the brain. The sensitivity of the detection system used here allowed us to detect MOG protein earlier than in previous reports such that its presence was clearly demonstrated in the CNS of mice and rats at 14 and 10 days after birth, respectively. Analysis of MOG expression in a novel transgenic mouse model that has both delayed and reduced myelination revealed that, like other myelin proteins, MOG expression was delayed compared with normal littermates. These results demonstrate that the expression of MOG is similar in all species and is regulated in a manner consistent with other myelin-specific proteins.

Delayed and incomplete myelination in a transgenic mouse mutant with abnormal oligodendrocytes.

In search of animal models suitable for investigating myelin repair, we have analysed myelinogenesis in a transgenic mouse mutant with delayed myelination, but with a normal life-span. The 2-50 mutant which carries a c-myc gene under the regulation of the myelin basic protein promoter has been described previously (Orian et al.: J Neurosci Res 39:604-612, 1994). Here we show that appropriate mRNA transcripts and their corresponding protein products are generated, but that the accumulation of these products is delayed in transgenic mice with respect to nontransgenic littermates. This phenomenon is associated with aberrant myelin and paucity of normal oligodendrocytes. Myelination appears to be carried out by abnormal, oligodendrocyte-like cells. We propose that the primary defect in the 2-50 mutant is an inability to generate the normal number of mature oligodendrocytes. This mutant represents a novel class of mutant in which oligodendrocyte development and myelination can be studied in the absence of interference with a gene for a structural protein of myelin, in an animal with normal survival. It may also represent a new tool to investigate in vivo gliogenesis and regulatory events bringing about the coordinated regulation of myelin protein synthesis.

Transformation of astrocytes in transgenic mice expressing SV40 T antigen under the transcriptional control of the glial fibrillary acidic protein promoter.

There are many animal models of glioma, but few that represent the biology of low-grade tumors and allow the study of the genetic mechanisms of glial oncogenesis. We report the in vivo transformation of astrocytic cells in transgenic mice by the SV40 T antigen under the control of the 5'-flanking sequence of the murine glial fibrillary acidic protein (GFAP) gene. High levels of T antigen expression were detectable in a tissue distribution that mirrored the normal expression of GFAP. This was associated with a consistent phenotype in the founder mice. Diffuse proliferation occurred in cells of the periventricular subependymal zone with diffuse invasion into the brain parenchyma, leading to death by 19-30 days postnatally. Transformed cells exhibited secondary structuring, a typical histopathological feature of human astrocytomas. Early passage cultures of these cells expressed GFAP in vitro and were transformed on the basis of tumor formation after transplantation into nude mice. These results demonstrate the susceptibility of periventricular astrocytic cells in the immature brain to malignant transformation. Furthermore, this study demonstrates the potential of the transgenic approach for the in vivo determination of genetic events involved in astrocyte transformation and for the development of novel models of astrocytoma.

Aberrant p53 expression does not correlate with the prognosis in anaplastic astrocytoma.

Mutations of the p53 tumor-suppressor gene, as determined by the immunohistochemistry of archival formalin-fixed specimens, have been correlated with the prognosis for patients with many different types of malignancy. Similar correlations have been shown in series including patients with all grades of astrocytoma. We hypothesized that this might be a clinically useful prognostic indicator for patients with a defined grade of astrocytoma, anaplastic astrocytoma. This series comprised 54 consecutive patients with biopsy-proven anaplastic astrocytoma treated at one institution. When the CM-1 antiserum was used for testing, 33 (61%) of the 54 biopsies exhibited positive nuclear staining for p53, indicating an abnormal accumulation of the protein. This staining was graded according to the number of positive cells per high-power field. Positive immunohistochemical staining for p53 in the tumor cell nuclei did not correlate with the patient's outcome. Significant correlates of improved patient survival were the presentation with epilepsy in the absence of a neurological deficit (P = 0.005) and the surgeon's performance of a macroscopically complete surgical resection of the tumor (P = 0.01).

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice.

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomers Msp I and Hpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition of Msp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pair BstN I and EcoR II. A minority of MToGH1 animals in the F1 generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.

Insertional mutagenesis inducing hypomyelination in transgenic mice.

Investigations of myelin disorders, in particular multiple sclerosis (MS), have concentrated on immunemediated damage to formed myelin, while there has been less emphasis on the molecular genetics of myelin formation. We have generated a transgenic mouse mutant (designated 2-50) which carries an insertional mutation in a locus regulating myelination. These mice carry a transgene comprising 1.3 Kb of the mouse myelin basic protein (MBP) promoter conjugated to a fragment containing exons 2 and 3 of the human c-myc gene. Positive mice show a significant reduction in myelin compared to controls and a shivering phenotype. Unlike other myelin mutants, all 2-50 mice lose the shivering phenotype and breed normally. Expression of c-myc is detectable in only 65% of transgene-carrying mice, and when present occurs at extremely low levels. This shows that the phenotype is caused by insertional inactivation of a gene necessary for myelination rather than ectopic expression of the transgene. The transgene was found by in situ hybridization to be inserted into a single site which is very distally located on chromosome 9. The 2-50 mice represent a unique model which will be ideal for investigating the molecular basis of myelin assembly and for developing gene therapy to promote remyelination in conditions such as MS.

Overexpression of multiple oncogenes related to histological grade of astrocytic glioma.

The expression of the c-erbB-1, c-myc, Ha/N-ras and c-fos oncogenes was investigated in 62 astrocytomas of low, intermediate and high grades by immunogold silver histochemistry. Elevated expression of c-erbB-1 was observed in 95%, 48% and 86% of low, intermediate and high grade tumours respectively, c-myc in 5%, 33% and 76% respectively, Ha/N-ras in 0, 43% and 71% respectively and c-fos in 55%, 48% and 52% respectively. Controls included normal brain and tumour sections immunoreacted with pre-immune serum or with antisera absorbed with synthetic peptides. Analysis of co-overexpression revealed that low grade tumours co-overexpressed a maximum of two of these genes, intermediate grade tumours a maximum of three of these genes, while co-overexpression of all four genes was observed in some high grade tumours. Co-overexpression of c-erbB-1 and c-fos was frequently observed in low grade astrocytomas and may be predictive of non-progression. On the other hand, there was a statistically significant increase in the number of tumours overexpressing Ha/N-ras or c-myc with increasing grade of tumour, suggesting that overexpression of these two oncogenes may be indicative of progression.

Elevation of growth hormone (GH) and prolactin receptors in transgenic mice expressing ovine GH.

The effect of elevated serum ovine GH (oGH) concentration on liver somatotrophic and lactogenic receptors was studied in transgenic mice expressing a metallothionein 1(MT)-oGH fusion gene. The mice belonged to three different pedigrees and were killed between 14 and 63 weeks of age. The levels of GH receptor (GH-R) and PRL receptor (PRL-R) determined by competitive binding assays were similar to those observed in late pregnant, nontransgenic mice. This observation was made for all transgenic mice expressing elevated serum oGH levels, irrespective of sex, final size, or age. Cross-linking studies revealed that binding occurred predominantly to a Mr 48,000 polypeptide with a small amount of binding to polypeptides of Mr 60,000, 70,000, and 100,000 in transgenic mice as well as in a late pregnant, nontransgenic mouse. Total cellular RNA was isolated from livers of transgenic and nontransgenic mice and analyzed on Northern blots using probes specific for GH-R and PRL-R. Results showed that the levels of messenger RNA for both GH-R and PRL-R were elevated in transgenic mice expressing high levels of serum oGH. Since levels of PRL in these mice were within the normal range, these results demonstrate that oGH is capable of inducing hepatic GH-R and PRL-R in vivo and that PRL is not required for the induction of its own receptor. These data also demonstrate, for the first time, the suitability of transgenic mice expressing a foreign GH for the study of the regulation of hepatic GH and PRL receptors.

New murine model for hepatocellular carcinoma: transgenic mice expressing metallothionein-ovine growth hormone fusion gene.

Hepatocellular carcinomas developed at a high frequency in the livers of transgenic (C57BL/6 X SJL/J)F1 mice under the influence of growth hormone. Three lines of giant transgenic mice expressing a mouse metallothionein-ovine growth hormone fusion gene were generated. The giant mice weighed twice as much as control littermates. The three lines of giant mice expressing very high levels of growth hormone were bred over several generations. Mice from all three lines developed hepatocellular tumors, including adenoma and carcinoma. The occurrence of tumors was age-dependent, and their incidence increased to 70% of the mice studied after 43 weeks of age. Pathologic changes in the livers resembled those observed in rats in which hepatocellular carcinomas are induced chemically. Transgenic mice carrying the metallothionein-ovine growth hormone fusion gene represent a new model for hepatocellular carcinogenesis. This model exemplifies the oncogenic potential for a sustained proliferative growth stimulus within an organ.

The expression of a metallothionein-ovine growth hormone fusion gene in transgenic mice does not impair fertility but results in pathological lesions in the liver.

The physiological effects of high serum levels of ovine GH (oGH) were studied in three generations of transgenic mice carrying a metallothionein 1-(MT)oGH fusion gene. Livers of mice expressing oGH were enlarged, irrespective of the level of serum oGH detected. In mice expressing high levels of oGH, direct measurements of hepatocytes in liver sections revealed that cell and nuclear size were abnormally large. Hepatocytes of different transgenic mice varied from 1.4-2.2 times normal size and hepatocyte nuclei varied from 1.7-2.4 times normal size. In addition, intranuclear inclusions were observed in hepatocytes of transgenic mice and their presence was always associated with high serum levels of oGH. In contrast to female transgenic mice containing mouse MT-human, rat, or bovine GH fusion genes female mice containing the MT oGH fusion gene were fertile and their pituitary glands showed synthesis of GH.

Biogenesis of mitochondria: defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated.

We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H+-ATPase can proceed in a mtDNA-less (rho degree) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H+-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized beta-subunit of the H+-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H+-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.

The largest mitochondrial translation product copurifying with the mitochondrial adenosine triphosphatase of Saccharomyces cerevisiae is not a subunit of the enzyme complex.

Mitochondrial adenosine triphosphatase isolated from a double mutant of Saccharomyces cerevisiae lacking cytochrome b apoprotein and subunit II of cytochrome oxidase does not contain the mitochondrial translation product (approximate molecular weight, 32,000) previously suggested to be a subunit of the enzyme complex.

Mitochondrially synthesized protein subunits of the yeast mitochondrial adenosine triphosphatase. A reassessment.

Evidence is presented that a mitochondrial translation product (Mr, 32,000) previously thought to be a subunit of the membrane sector of the yeast mitochondrial ATPase is a contaminant, consisting of subunit II of the cytochrome oxidase complex and cytochrome b apoprotein. Our data suggest that only two subunits (Mr, 7600 and 20,000) of the mitochondrial ATPase are synthesized in the mitochondria.