Myelin splitting, Schwann cell injury and demyelination in feline diabetic neuropathy

This paper presents a theoretical framework to predict the effects that may arise from mergers in the rapidly-growing Medicare HMO market. We argue that mergers of large Medicare HMOs should be targeted for antitrust investigation because there are significant barriers into this market. The recent merger of PacifiCare and FHP is used to illustrate the potential antitrust issues raised by Medicare HMO mergers.     

Motherhood and nursing stimulate c-FOS expression in the rabbit forebrain.

Mother rabbits nurse once daily with circadian periodicity. The authors investigated brain structures involved in regulating this activity by quantifying c-FOS-immunoreactive (IR) cells in the forebrain of: (1) mothers killed on postpartum Day 1 (PPD 1) after nursing (Group 1) or not given pups (Group 2); (2) mothers killed on PPD 7 after nursing (Group 3) or not given pups on such day (Group 4); (3) unmated virgins (Group 5). Groups 1 through 4 showed similar numbers of c-FOS-IR cells in the preoptic area, an amount around three to fourfold larger than that found in virgins. Nursing increased, on PPD 1 and 7, c-FOS-IR cell number in the lateral septum and paraventricular and supraoptic nuclei. No differences were seen among Groups 1 through 5 in the suprachiasmatic nucleus. In the ventromedial hypothalamus virgins had more c-FOS-IR cells compared with Groups 1 and 2. Results suggest that specific forebrain structures participate in regulating particular aspects of rabbit maternal behavior: the POA and LS seem associated with the establishment of motherhood and the magnocellular nuclei with the occurrence of milk letdown. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Axons modulate the expression of transforming growth factor-betas in Schwann cells

We have investigated the expression of transforming growth factor (TGF)-beta 1,-beta 2, and -beta 3 in developing, degenerating, and regenerating rat peripheral nerve by immunohistochemistry and Northern blot analysis. In normal adult sciatic nerve, TGF-beta 1, -beta 2, and -beta 3 are detected in the cytoplasm of Schwann cells, and the levels of TGF-beta 1 and -beta 3 mRNAs are constant during post-natal development. When sciatic nerves are transected to cause axonal degeneration and prevent axonal regeneration, the level of TGF-beta 1 mRNA in the distal nerve-stump increases markedly and remains elevated, whereas the level of TGF-beta 3 mRNA falls modestly and remains depressed. When sciatic nerves are crushed to cause axonal degeneration and allow axonal regeneration, the level of TGF-beta 1 mRNA initially increases as axons degenerate, and then falls as axons regenerate. TGF-beta 2 mRNA was not detected in developing or lesioned sciatic nerves at any time. Cultured Schwann cells have high levels of TGF-beta 1 mRNA, the amount of which is reduced by forskolin, which mimics the effect of axonal contact. These data demonstrate that Schwann cells express TGF-beta 1, -beta 2, and -beta 3, and that TGF-beta 1 and -beta 3 mRNA predominate over TGF-beta 2 mRNA in peripheral nerve. Axonal contact and forskolin decrease the expression of TGF-beta 1 in Schwann cells.     

Inflammatory cytokines inhibit upregulation of glycolipid expression by Schwann cells in vitro

OBJECTIVE: To determine whether products of inflammatory cells can inhibit differentiation and synthesis of myelin glycolipids by Schwann cells. BACKGROUND: Infiltration of the peripheral nervous system by inflammatory cells is a feature of acquired demyelinating neuropathies. It is not clear what role these cells have in causing demyelination or inhibiting myelin synthesis. METHODS: Nonmyelinating rat Schwann cells were incubated with 1) different concentrations of activated supernatants (AS) from mitogen-activated inflammatory cells; 2) 8-bromo cyclic adenosine monophosphate (8Br cAMP), known to induce Schwann cell differentiation and synthesis of glycolipids; 3) 8Br cAMP and varying concentrations of AS; 4) 8Br cAMP and cytosine arabinoside (Ara C), which inhibits Schwann cell proliferation; 5) 8Br cAMP, AS, and Ara C; or 6) additional medium. RESULTS: AS inhibits the capacity of cAMP to induce Schwann cell expression of myelin-associated glycolipids. Inhibition of glycolipid expression was independent of the capacity of these AS to induce Schwann cell proliferation. CONCLUSIONS: These data suggest that inflammatory mediators are capable of inhibition of Schwann cell differentiation and synthesis of myelin.     

1,25-Dihydroxyvitamin D3 regulates the expression of VDR and NGF gene in Schwann cells in vitro

OBJECTIVES: In mild Alzheimer's disease, SPECT imaging of regional cerebral blood flow has highlighted deficits in the posterior association cortex, and later in the disease process, the deficit spreads to involve the frontal cortex. The sigma4 allele of apolipoprotein E is a risk factor for Alzheimer's disease. The effect of apolipoprotein E polymorphism on cerebral perfusion was studied. The hypothesis was that those patients with Alzheimer's disease who carry the sigma4 allele would have more severe cerebral hypoperfusion. METHODS: Thirty one patients with Alzheimer's disease and eight age and sex matched control subjects were examined in a three year longitudinal study. Patients with Alzheimer's disease were divided into subgroups according to their number of sigma4 alleles. Regional cerebral blood flow ratios referred to the cerebellum were examined by 99mTc-HMPAO SPECT. Apolipoprotein E genotypes were determined by digestion of polymerase chain reaction products with the restriction enzyme Hha1. RESULTS: All patients with Alzheimer's disease had bilateral temporoparietal hypoperfusion compared with control subjects. The two sigma4 allele subgroups had the lowest ratios at the baseline assessment in the parietal and occipital cortices, and at the follow up in the temporal, parietal, and occipital cortices. They had the highest reduction in percentage terms in the temporal and occipital cortices compared with the other subgroups. However, the global clinical severity did not differ at the baseline or follow up examinations between the subgroups. CONCLUSION: Apolipoprotein E polymorphism is involved in the pathogenesis and heterogeneity of Alzheimer's disease as the most severe cerebral hypoperfusion was found in the sigma4 allele subgroups. This might have implications for therapeutic approaches in Alzheimer's disease.     

Coronavirus-induced demyelination occurs in the presence of virus-specific cytotoxic T cells

C57BI/6, but not BALB/c, mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a late onset, symptomatic demyelinating encephalomyelitis. In this report, we characterized anti-viral cytotoxic T cells in the central nervous system and spleen during the acute and chronic stages of the MHV infection. The data show that C57BI/6 mice display a cytotoxic T cell (CTL) response to the surface (S) glycoprotein and this response can be demonstrated in lymphocytes isolated from the brains and spinal cords of mice both acutely and persistently infected with MHV-JHM. Thus, the anti-S CTL activity present in the central nervous system of chronically infected animals is not sufficient to prevent the demyelinating process. BALB/c mice have been shown previously to mount a CTL response against the nucleocapsid (N) protein (Stohlman et al., 1992). Since C57BI/6 mice do not mount a response to the N protein, the role of the N-specific response in preventing the late onset disease was assessed using B10.A(18R) mice, recombinant in the H-2 locus. These mice contain the d alleles of the D and L loci and exhibit a CTL response against the N protein. However, unlike the BALB/c mice, these animals develop the late onset symptomatic disease. These results suggest that the N-specific response is partially protective against the development of the demyelinating disease, but that additional factors are also likely to be involved.     

Activation of the Xmrk proto-oncogene of Xiphophorus by overexpression and mutational alterations

Xmrk is a receptor tyrosine kinase closely related to the human EGF receptor. In the teleost fish Xiphophorus two versions of the Xmrk gene exist, an oncogene (ONC) and a proto-oncogene (INV). While ONC-Xmrk is the melanoma-inducing gene, INV-Xmrk appears not to be involved in transformation of pigment cells. To elucidate the mechanism that converts the proto-oncogene into a transforming oncogene a comparative analysis of the structure, expression and function of both versions of the gene was performed. In contrast to ONC-Xmrk which is expressed at high levels in melanoma cells, the proto-oncogene INV-Xmrk is ubiquitously expressed at very low levels indicating overexpression as one possible reason for tumorigenicity by ONC-Xmrk. As sequence comparison of the proto-oncogene and the oncogene revealed a number of amino acid changes, a possible effect of these mutations on the activation of the ONC-Xmrk receptor was determined. A constitutive activation of the oncogenic receptor was found and ectopic expression of INV-Xmrk after microinjection into medakafish embryos did not lead to the high tumour rate in transgenic fish as observed for the oncogene. Our data therefore suggest that overexpression of the receptor alone is not sufficient for melanoma induction, but that in addition activating mutations in ONC-Xmrk are responsible for its full tumorigenic potential.     

Expression of ubiquitin, actin, and actin-like genes in African swine fever virus infected cells

The distribution, density and histochemical subtype of mast cells (mucosal and connective tissue) were studied in the ileum, trachea and skin of rats treated with IFN alpha (70.000 IU/kg) treated rats. Light and electron microscopic procedures were utilized. The total number of mucosal mast cells in the sections of ileum and trachea were markedly increased in the IFN-alpha treated group (ileum: 31.9 +/- 2.2 cells/villuscrypt unit; trachea: 10,355 +/- 264 cells/mm3). However, the number of connective tissue mast cells did not show any significant change in the skin between IFN-alpha treated (1,472 +/- 125 cells/mm3) and saline-treated (1,757 +/- 264 cells/mm3) groups. We conclude that mast cell proliferation does exist in the rat ileum and trachea but no in the skin response to IFN-alpha. We suggest that this model provides a powerful tool to study differential effects of IFN-alpha on mast cell subtypes and to identify their role in the immunoregulatory and inflammatory reactions.     

Increased ubiquitin expression suppresses the cell cycle defect associated with the yeast ubiquitin conjugating enzyme, CDC34 (UBC3). Evidence for a noncovalent interaction between CDC34 and ubiquitin

The yeast ubiquitin (Ub) conjugating enzyme CDC34 plays a crucial role in the progression of the cell cycle from the G1 to S phase. In an effort to identify proteins that interact with CDC34 we undertook a genetic screen to isolate genes whose increased expression suppressed the cell cycle defect associated with the cdc34-2 temperature-sensitive allele. From this screen, the poly-Ub gene UBI4 was identified as a moderately strong suppressor. The fact that the overexpression of a gene encoding a single Ub protein could also suppress the cdc34-2 allele indicated that suppression was related to the increased abundance of Ub. Ub overexpression was found to suppress two other structurally unrelated cdc34 mutations, in addition to the cdc34-2 allele. In all three cases, suppression depended on the expression of Ub with an intact carboxyl terminus. Only the cdc34-2 allele, however, could be suppressed by Ub with an amino acid substitution at lysine 48 which is known to be involved in multi-Ub chain assembly. Genetic results showing allele specific suppression of cdc34 mutations by various Ub derivatives suggested a specific noncovalent interaction between Ub and CDC34. Consistent with this prediction, we have shown by chemical cross-linking the existence of a specific noncovalent Ub binding site on CDC34. Together, these genetic and biochemical experiments indicate that Ub suppression of these cdc34 mutations results from the combined contributions of Ub-CDC34 thiol ester formation and a noncovalent interaction between Ub and CDC34 and therefore suggest that the correct positioning of Ub on a surface of the ubiquitin conjugating enzyme is a requirement of enzyme function.     

The transplantation of syngeneic Schwann cells in medullary lesions: results, limitations and perspectives

After a central nervous system (CNS) injury, there is only an "abortive regeneration" of axons, while injured axons regenerate vividly in the peripheral nervous system (PNS). This difference is due, at least in part, to the existence in the periphery of Schwann cells and of growth promoting proteins they synthetize. One strategy to promote regrowth of central axons can be therefore, to modify (i.e. "peripheralize") the microenvironment by transplanting biologically active Schwann cells into the lesion site. In a rat model of traumatic paraplegia by inflation of a subdural microballoon, we performed syngeneic transplants of Schwann cells. These cells are cultured from adult dorsal root ganglia and can be kept in vitro for several months. They are transplanted in the injured spinal cord. The grafted Schwann cells are well integrated in the host tissue without detectable inflammatory reaction. Cystic cavitation and astrogliosis are reduced in grafted animals as compared to injured, non-grafted animals. The transplant is invaded by abundant, mainly unmyelinated axons which are immunoreactive for substance P, VIP or CGRP, i.e. transmitters known to be present in DRG afferents. Supraspinal afferents containing 5HT, TH or CCK accumulate at the rostral margin of the graft. Experimental procedures trying to stimulate the invasion of the graft by descending fibers, i.e. by inducing a chemoattraction are therefore of crucial importance for functional recovery.     

Activation of tissue-factor gene expression in breast carcinoma cells by stimulation of the RAF-ERK signaling pathway

Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation. The overexpression of TF in human malignancy has been correlated with the angiogenic phenotype, poor prognosis, and thromboembolic complications. The mechanisms underlying constitutive expression of TF in cancer cells are poorly defined. We cloned TF cDNA on the basis of its strong expression in metastatic MDA-MB-231 breast carcinoma cells in contrast to its weak expression in non-metastatic MCF-7 cells. Transient transfection analysis showed that TF promoter activity in MCF-7 cells could be stimulated by expression of a membrane-targeted raf kinase (raf-CAAX). raf-induced activity was dependent on the presence of an AP-1/NF-kappaB motif in the TF promoter and was inhibited by dominant-negative mutants of jun and by I-kappaB alpha. MDA-MB-231 cells were found to contain higher levels of ERK1/2 kinase activity than did MCF-7 cells. Electrophoretic mobility shift assays showed that MDA-MB-231 nuclear proteins bound strongly to an oligonucleotide corresponding to the AP-1/NF-kappaB sequence, whereas MCF-7 nuclear extracts showed weak binding to this element. Finally, we showed that TF mRNA levels in MDA-MB-231 cells declined after addition of the mitogen-activated protein kinase kinase inhibitor PD98059. Our data showed that activation of the raf-ERK pathway led to activation of TF expression in breast carcinoma cells and suggested that constitutive activation of this pathway leads to high TF expression in MDA-MB-231 cells.     

Regional differences in bromodeoxyuridine uptake, expression of Ki-67 protein, and nucleolar organizer region counts in glioblastoma multiforme

To investigate intratumoral differences in indices of tumor cell proliferation, we measured the bromodeoxyuridine labeling index (BrdUrd LI), the Ki-67 protein proliferating cell indices (PCIs) determined by monoclonal antibody MIB 1 in microwave-processed paraffin sections (MIB 1 PCI) and in some cases by monoclonal antibody in frozen sections (Ki-67 PCI), and counts of argyrophilic nucleolar organizer regions (AgNORs) in 20 glioblastomas. In the most actively proliferating areas, MIB 1 and Ki-67 PCIs correlated well with the BrdUrd LI and with each other, while AgNOR counts correlated less strongly with these indices. In less active areas, the MIB 1 PCI and BrdUrd LI changed concomitantly from one area to another within a tumor except in areas of pseudopalisading with necrosis; in these areas the BrdUrd LI decreased significantly compared with neighboring tumor tissue, while the MIB 1 PCI did not. There was very little staining of gemistocytic nuclei with either anti-BrdUrd or MIB 1 monoclonal antibodies; this supports the concept that gemistocytes are mainly quiescent cells. AgNORs in all of the above-mentioned areas varied from tumor to tumor, which suggests that they may indicate some cellular activity other than proliferation. The close correlation between the BrdUrd LI and Ki-67 protein PCIs in corresponding regions of glioblastomas suggests that MIB 1 staining of microwave-processed paraffin sections can be used to evaluate the growth potential of individual glioblastomas and possibly of other gliomas as well.     

Merlin, the neurofibromatosis type 2 gene product, and beta1 integrin associate in isolated and differentiating Schwann cells

Neurofibromatosis type 2, a disease characterized by the formation of multiple nervous system tumors, especially schwannomas, is caused by mutation in the gene-encoding merlin/schwannomin. The molecular mechanism by which merlin functions as a tumor suppressor is unknown, but is hypothesized to involve plasma membrane and cytoskeleton interaction. Several merlin antibodies were used to study merlin expression, localization, and protein association in primary cultures of rat sensory neurons, Schwann cells (SCs), and SCs grown with neurons (SC/N cultures) before and during differentiation into myelinating cells. Western blot analysis revealed that neurons predominantly expressed a 68-kD protein, but SCs expressed two additional 88- and 120-kD related proteins. Extensive immunological characterization demonstrated that the 88-kD protein shared three domains with the 68-kD merlin protein. Western blot analysis of soluble and insoluble culture fractions demonstrated that the majority of merlin and related proteins were soluble in isolated SCs and undifferentiated SC/N cultures, but became insoluble in myelinating SC/N cultures. Double immunofluorescence staining suggested that merlin translocated from the perinuclear cytoplasm in undifferentiated SCs to the subplasmalemma in differentiating SCs and partially colocalized with beta1 integrin. Finally, beta1 integrin antibody coimmunoprecipitated 68-kD merlin from isolated SC and undifferentiated SC/N cultures, but predominantly the 88-kD protein from differentiating SC/N cultures. Together, these results provide evidence that merlin interacts with beta1 integrin and that merlin localization changes from a cytosolic to cytoskeletal compartment during SC differentiation.     

Osmolality and potassium cause alterations in the volume of glomerulosa cells

Alterations in extracellular osmolality have a powerful inverse effect on aldosterone secretion and potassium- and angiotensin-stimulated aldosterone secretion. Whether alterations in extracellular osmolality produced sustained changes in cell volume that may contribute to the regulation of aldosterone secretion is not known. Using dispersed bovine glomerulosa cells grown in primary culture, the effect of alterations in osmolality on cell volume, measured by the distribution of [14C]urea and [3H]inulin and videometric analysis of the surface area of glomerulosa cells, was determined. Alterations in osmolality had an inverse effect on cell volume and surface area. Changes in cell volume induced by exposure to anisotonic medium were 52% greater (P > 0.02) than that predicted by the changes in osmolality. Increases in potassium concentration also caused sustained (1-h) concentration-dependent increases in cell volume and surface area. Angiotensin-II did not increase glomerulosa cell volume, but did produce a small dose-dependent transient increase in cell surface area. The results demonstrate that alterations in osmolality do cause sustained changes in cell volume, and thus, membrane stretch could be an important part of the cellular mechanism responsible for causing osmolality-induced changes in the cytosolic calcium concentration and subsequent alterations in aldosterone secretion. Alterations in membrane stretch may also be an important component of potassium-induced, but not angiotensin II-induced, aldosterone secretion.     

Hyperplastic neuroretinopathy and disorder of pigment epithelial cells precede accelerated retinal degeneration in the SJL/N mouse

We have found a complex eye disease in the SJL/N mouse. This animal is closely related to the SJL/J mouse, which is homozygous for retinal degeneration (rd) and which also suffers from extraocular reticulum cell sarcomas at around 200 days of age. In the SJL/N animal, a high incidence of subretinal tumor is present at 9 days after birth. Furthermore, we have observed an extensive neuroretinal hyperplasia, a phenomenon that is termed "hyperplastic neuroretinopathy", and that is probably the consequence of elevated levels of cytokines in the animals. In addition to these anomalies, the SJL/N mouse shows progressive dystrophy of the retinal pigment epithelium (RPE) from day 4 onwards, and accelerated photoreceptor cell degeneration is completed by day 16. The early RPE dystrophy appears to be a secondary autoimmune disease, since cells in this structure and in the choroid develop MHC class II antigens, whereas we suspect that the accelerated photoreceptor cell loss is induced by a soluble toxic agent. The F1 progeny derived from cross-breeding the SJL/N and Balb/c +/+ strains also shows a high incidence of subretinal tumor and hyperplastic neuroretinopathy, but neither the RPE dystrophy nor retinal degeneration.     

Pathways of migration of transplanted Schwann cells in the demyelinated mouse spinal cord

We have studied the behavior of Schwann cells transplanted at a distance from an induced myelin lesion of the adult mouse spinal cord. These transplanted cells were mouse Schwann cells arising from an immortalized cell line (MSC80) which expresses several Schwann cell phenotypes including the ability to produce myelin. The behavior of MSC80 cells was compared to that of purified rat Schwann cells transplanted in the same conditions. Schwann cells were labeled in vitro with the nuclear fluorochrome Hoechst 33342 and were transplanted at distances of 2-8 mm from a lysolecithin-induced myelin lesion in the spinal cord of shiverer and normal mice. Our results show that transplanted MSC80 cells migrated toward the lesion, in both shiverer and normal mouse spinal cord, preferentially along the ependyma, meninges, and blood vessels. They also migrated along white matter tracts but traveled a longer distance in shiverer (8 mm) than in normal (2-3 mm) white matter. Using these different pathways, MSC80 cells arrived within the lesion of shiverer and normal mouse spinal cord at the average speed of 166 microns/hr (8 mm/48 hr). Migration was most efficient along the ependyma and the meninges where it attained up to 250 microns/hr. Migration was much slower in white matter tracts (95 microns/hr +/- 54 in the shiverer and only 38 microns/hr +/- 3 in the normal mouse). We also provide evidence for the specific attraction of MSC80 cells by the lysolecithin-induced lesion since 1) their number increased progressively with time in the lesion, and 2) MSC80 cells left their preferential pathways of migration specifically at the level of the lesion. Finally, combining the Hoechst Schwann cell labeling method with the immunohistochemical detection of the peripheral myelin protein, P0, we show that some of the MSC80 cells which have reached the lesion participate in myelin repair in both shiverer and normal lesioned mouse spinal cord. A series of control experiments performed with rat Schwann cells indicate that the migrating behavior of transplanted MSC80 cells was identical to that of purified but non-immortalized rat Schwann cells.     

Activation of the beta-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3

We screened 75 primary hepatocellular carcinomas for somatic mutations in the entire coding region of the beta-catenin gene. We detected somatic mutations in 14 tumors; 12 were considered to cause amino acid substitutions and 2 were interstitial deletions of 51 or 195 nucleotides of genomic DNA, corresponding to exon 3. Among the 12 point mutations, 6 occurred at potential serine/threonine phosphorylation residues of codons 33, 41, or 45. The remaining six tumors contained a mutation at codon 32 (aspartic acid) or 34 (glycine), flanking to the serine residue at codon 33. By Western blot analysis, we confirmed accumulation of beta-catenin in five tumors for which frozen tissues were available; the five included tumors in which amino acid alterations had occurred at codons 32, 34, or 45, and one with a 17-amino acid deletion. Our results suggested that accumulation of beta-catenin due to amino acid substitutions at potential serine/threonine phosphorylation residues or at their neighboring codons or interstitial deletions involving exon 3 could contribute to hepatocellular carcinogenesis.