Expansion of a recurrent V beta 5.3+ T-cell population in newly diagnosed and untreated HLA-DR2 multiple sclerosis patients.

We have used a PCR-based technology to study the V beta 5 and V beta 17 repertoire of T-cell populations in HLA-DR2 multiple sclerosis (MS) patients. We have found that the five MS DR2 patients studied present, at the moment of diagnosis and prior to any treatment, a marked expansion of a CD4+ T-cell population bearing V beta 5-J beta 1.4 beta chains. The sequences of the complementarity-determining region 3 of the expanded T cells are highly homologous. One shares structural features with that of the T cells infiltrating the central nervous system and of myelin basic protein-reactive T cells found in HLA-DR2 MS patients. An homologous sequence was not detectable in MS patients expressing DR alleles other than DR2. However, it is detectable but not expanded in healthy DR2 individuals. The possible mechanisms leading to its in vivo proliferation at the onset of MS are discussed.

Altered surfactant function and structure in SP-A gene targeted mice.

The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs of SP-A(-/-) mice, and perinatal survival of SP-A(-/-) mice was not altered compared with wild-type mice. Lung morphology, surfactant proteins B-D, lung tissue, alveolar phospholipid pool sizes and composition, and lung compliance in SP-A(-/-) mice were unaltered. At the highest concentration tested, surfactant from SP-A(-/-) mice produced the same surface tension as (+/+) mice. At lower concentrations, minimum surface tensions were higher for SP-A(-/-) mice. At the ultrastructural level, type II cell morphology was the same in SP-A(+/+) and (-/-) mice. While alveolar phospholipid pool sizes were unperturbed, tubular myelin figures were decreased in the lungs of SP-A(-/-) mice. A null mutation of the murine SP-A gene interferes with the formation of tubular myelin without detectably altering postnatal survival or pulmonary function.

Cell death in the Schwann cell lineage and its regulation by neuregulin.

The development of Schwann cells, the myelin-forming glial cells of the vertebrate peripheral nervous system, involves a neonatal phase of proliferation in which cells migrate along and segregate newly formed axons. Withdrawal from the cell cycle, around postnatal days 2-4 in rodents, initiates terminal differentiation to the myelinating state. During this time, Schwann cell number is subject to stringent regulation such that within the first postnatal week, axons and myelinating Schwann cells attain the one-to-one relationship characteristic of the mature nerve. The mechanisms that underly this developmental control remain largely undefined. In this report, we examine the role of apoptosis in the determination of postnatal Schwann cell number. We find that Schwann cells isolated from postnatal day 3 rat sciatic nerve undergo apoptosis in vitro upon serum withdrawal and that Schwann cell death can be prevented by beta forms of neuregulin (NRG-beta) but not by fibroblast growth factor 2 or platelet-derived growth factors AA and BB. This NRG-beta-mediated Schwann cell survival is apparently transduced through an ErbB2/ErbB3 receptor heterodimer. We also provide evidence that postnatal Schwann cells undergo developmentally regulated apoptosis in vivo. Together with other recent findings, these results suggest that Schwann cell apoptosis may play an important role in peripheral nerve development and that Schwann cell survival may be regulated by access to axonally derived NRG.

1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease believed to be a model for the human disease multiple sclerosis (MS). Induced by immunizing B10.PL mice with myelin basic protein (MBP), EAE was completely prevented by the administration of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. 1,25-(OH)2D3 could also prevent the progression of EAE when administered at the appearance of the first disability symptoms. Withdrawal of 1,25-(OH)2D3 resulted in a resumption of the progression of EAE. Thus, the block by 1,25-(OH)2D3 is reversible. A deficiency of vitamin D resulted in an increased susceptibility to EAE. Thus, 1,25-(OH)2D3 or its analogs are potentially important for treatment of MS.

How cellular slime molds evade nematodes.

We have found a predator-prey association between the social amoeba Dictyostelium discoideum and the free soil living nematode Caenorhabditis elegans. C. elegans feeds on the amoebae and multiplies indefinitely when amoebae are the sole food source. In an environment created from soil, D. discoideum grows and develops, but not in the presence of C. elegans. During development, C. elegans feeds on amoebae until they aggregate and synthesize an extracellular matrix called the slime sheath. After the sheath forms, the aggregate and slug are protected. Adult nematodes ingest Dictyostelium spores, which pass through the gut of the worm without loss of structure and remain viable. Nematodes kill the amoebae but disperse the spores. The sheath that is constructed when the social amoebae aggregate and the spore coats of the individual cells may protect against this predator. Individual amoebae may also protect themselves by secreting compounds that repel nematodes.

In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase.

Rab8 is a small GTP-binding protein that plays a role in vesicular transport from the trans-Golgi network to the basolateral plasma membrane in polarized epithelial cells (MDCK), and to the dendritic surface in hippocampal neurons. As is the case for most other rab proteins, the precise molecular interactions by which rab8 carries out its function remain to be elucidated. Here we report the identification and the complete cDNA-derived amino acid sequence of a murine rab8-interacting protein (rab8ip) that specifically interacts with rab8 in a GTP-dependent manner. Rab8ip displays 93% identity with the GC kinase, a serine/threonine protein kinase recently identified in human lymphoid tissue that is activated in the stress response. Like the GC kinase, rab8ip has protein kinase activity manifested by autophosphorylation and phosphorylation of the classical serine/threonine protein kinase substrates, myelin basic protein and casein. When coexpressed in transfected 293T cells, rab8 and the rab8ip/GC kinase formed a complex that could be recovered by immunoprecipitation with antibodies to rab8. Cell fractionation and immunofluorescence analyses indicate that in MDCK cells endogenous rab8ip is present both in the cytosol and as a peripheral membrane protein concentrated in the Golgi region and basolateral plasma membrane domains, sites where rab8 itself is also located. In light of recent evidence that rab proteins may act by promoting the stabilization of SNARE complexes, the specific GTP-dependent association of rab8 with the rab8ip/GC kinase raises the possibility that rab-regulated protein phosphorylation is important for vesicle targeting or fusion. Moreover, the rab8ip/GC kinase may serve to modulate secretion in response to stress stimuli.

ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation.

ERK6, a mitogen-activated protein (MAP) kinase-related serine/threonine kinase, is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of myoblasts to myotubes. In transfected 293 cells, activation of the 45-kDa enzyme results in tyrosine-phosphorylated 46- and 56-kDa forms, which phosphorylate myelin basic protein. Overexpression of wild-type ERK6 or the inactive mutant Y185F has no effect on fibroblast and myoblast proliferation, but it enhances or inhibits C2C12 cell differentiation to myotubes, respectively. Our findings suggest ERK6 to be a tissue-specific, differentiation signal-transducing factor that is connected to phosphotyrosine-mediated signaling pathways distinct from those activating other members of the MAP kinase family such as LRK1 and ERK2.

Unimpaired autoreactive T-cell traffic within the central nervous system during tumor necrosis factor receptor-mediated inhibition of experimental autoimmune encephalomyelitis.

The critical role of tumor necrosis factor (TNF) as a mediator in autoimmune inflammatory processes is evident from in vivo studies with TNF-blocking agents. However, the mechanisms by which TNF, and possibly also its homologue lymphotoxin alpha, contributes to development of pathology in rheumatoid arthritis and Crohn disease and in animal models like experimental autoimmune encephalomyelitis is unclear. Possibilities include regulation of vascular adhesion molecules enabling leukocyte movement into tissues or direct cytokine-mediated effector functions such as mediation of tissue damage. Here we show that administration of a TNF receptor (55 kDa)-IgG fusion protein prevented clinical signs of actively induced experimental autoimmune encephalomyelitis. Significantly, the total number of CD4+ T lymphocytes isolated from the central nervous system of clinically healthy treated versus diseased control animals was comparable. By using a CD45 congenic model of passively transferred experimental autoimmune encephalomyelitis to enable tracking of myelin basic protein-specific effector T lymphocytes, prevention of clinical signs of disease was again demonstrated in treated animals but without quantitative or qualitative impediment to the movement of autoreactive T lymphocytes to and within the central nervous system. Thus, despite the uninterrupted movement of specific T lymphocytes into the target tissue, subsequent disease development was blocked. This provides compelling evidence for a direct effector role of TNF/lymphotoxin alpha in autoimmune tissue damage.

Functional implications for the microtubule-associated protein tau: localization in oligodendrocytes.

We present evidence that the microtubule-associated protein tau is present in oligodendrocytes (OLGs), the central nervous system cells that make myelin. By showing that tau is distributed in a pattern similar to that of myelin basic protein, our results suggest a possible involvement of tau in some aspect of myelination. Tau protein has been identified in OLGs in situ and in vitro. In interfascicular OLGs, tau localization, revealed by monoclonal antibody Tau-5, was confined to the cell somata. However, in cultured ovine OLGs with an exuberant network of processes, tau was detected in cell somata, cellular processes, and membrane expansions at the tips of these processes. Moreover, in such cultures, tau appeared localized adjacent to or coincident with myelin basic protein in membrane expansions along and at the ends of the cellular processes. The presence of tau mRNA was documented using fluorescence in situ hybridization. The distribution of the tau mRNA was similar to that of the tau protein. Western blot analysis of cultured OLGs showed the presence of many tau isoforms. Together, these results demonstrate that tau is a genuine oligodendrocyte protein and pave the way for determining its functional role in these cells.

Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules.

We have previously identified a testicular phosphoprotein that binds to highly conserved sequences (Y and H elements) in the 3' untranslated regions (UTRs) of testicular mRNAs and suppresses in vitro translation of mRNA constructs that contain these sequences. This protein, testis/brain RNA-binding protein (TB-RBP) also is abundant in brain and binds to brain mRNAs whose 3' UTRs contain similar sequences. Here we show that TB-RBP binds specific mRNAs to microtubules (MTs) in vitro. When TB-RBP is added to MTs reassembled from either crude brain extracts or from purified tubulin, most of the TB-RBP binds to MTs. The association of TB-RBP with MTs requires the assembly of MTs and is diminished by colcemid, cytochalasin D, and high levels of salt. Transcripts from the 3' UTRs of three mRNAs that contain the conserved sequence elements (transcripts for protamine 2, tau protein, and myelin basic protein) are linked by TB-RBP to MTs, whereas transcripts that lack the conserved sequences do not bind TB-RBP. We conclude that TB-RBP serves as an attachment protein for the MT association of specific mRNAs. Considering its ability to arrest translation in vitro, we propose that TB-RBP functions in the storage and transportation of mRNAs to specific intracellular sites where they are translated.

Tyrosine phosphorylation of protein kinase C-delta in response to the activation of the high-affinity receptor for immunoglobulin E modifies its substrate recognition.

The delta isoform of protein kinase C is phosphorylated on tyrosine in response to antigen activation of the high-affinity receptor for immunoglobulin E. While protein kinase C-delta associates with and phosphorylates this receptor, immunoprecipitation of the receptor revealed that little, if any, tyrosine-phosphorylated protein kinase C-delta is receptor associated. In vitro kinase assays with immunoprecipitated tyrosine-phosphorylated protein kinase C-delta showed that the modified enzyme had diminished activity toward the receptor gamma-chain peptide as a substrate but not toward histones or myelin basic protein peptide. We propose a model in which the tyrosine phosphorylation of protein kinase C-delta regulates the kinase specificity toward a given substrate. This may represent a general mechanism by which in vivo protein kinase activities are regulated in response to external stimuli.

Isolation of cDNA clones encoding RICH: a protein induced during goldfish optic nerve regeneration with homology to mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases.

Using data derived from peptide sequencing of p68/70, a protein doublet induced during optic nerve regeneration in goldfish, we have isolated cDNAs that encode RICH (regeneration-induced CNPase homolog) from a goldfish regenerating retina cDNA library. The predicted RICH protein comprises 411 amino acids, possesses a pI of 4.48, and shows significant homology to the mammalian myelin marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase; EC 3.1.4.37). The mRNA encoding RICH was demonstrated, by both Northern blot analysis and RNase protection assays, to be induced as much as 8-fold in regenerating goldfish retinas at 20 days after nerve crush. Analysis of total RNA samples from various tissues showed a broad distribution of RICH mRNA, with the highest levels observed in gravid ovary. The data obtained strongly suggest that RICH is identical or very similar to p68/70. The molecular cloning of RICH provides the means for a more detailed analysis of its function in nerve regeneration. Additionally, the homology of RICH and CNPase suggests that further investigation may provide additional insight into the role of these proteins in the nervous system.

Cutting activates a 46-kilodalton protein kinase in plants.

Using SDS/polyacrylamide gels that contained myelin basic protein, we identified a 46-kDa protein kinase in tobacco that is transiently activated by cutting. Although the activity of the kinase was rarely detectable in mature leaves, marked activity became apparent within several minutes after isolation of leaf discs and subsided within 30 min. In the presence of cycloheximide (CHX), the kinase activity did not diminish after the isolation over the course of 2 hr, suggesting that protein synthesis was not required for the activation of the kinase. A second cutting of leaf discs between 30 min and 60 min after the isolation failed to activate the kinase, whereas a second cutting given 3 hr after isolation apparently activated the kinase. These results suggest that the 46-kDa protein kinase is desensitized immediately after the first activation, which can be blocked by CHX, but the response ability recovers with time. When protein extracts containing the active kinase were treated with serine/threonine-specific or tyrosine-specific protein phosphatase, the kinase activity was abolished. After immunoprecipitation with antibody against phosphotyrosine, activity of the kinase was recovered in the immunoprecipitate. These results suggest that the active form of the kinase is phosphorylated at both serine/threonine and tyrosine residues. It seems likely that the 46-kDa protein kinase can be activated by dual phosphorylation. The activity of a 46-kDa protein kinase was also detected in leaves of a wide variety of plant species including dicotyledonous and monocotyledonous plants. We propose the name PMSAP (plant multisignal-activated protein) kinase for this kinase because the kinase was also activated by various signals other than cutting.

Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice.

Surfactant protein B (SP-B) is an 8.7-kDa, hydrophobic protein that enhances the spreading and stability of surfactant phospholipids in the alveolus. To further assess the role of SP-B in lung function, the SP-B gene was disrupted by homologous recombination in murine mouse embryonic stem cells. Mice with a single mutated SP-B allele (+/-) were unaffected, whereas homozygous SP-B -/- offspring died of respiratory failure immediately after birth. Lungs of SP-B -/- mice developed normally but remained atelectatic in spite of postnatal respiratory efforts. SP-B protein and mRNA were undetectable and tubular myelin figures were lacking in SP-B -/- mice. Type II cells of SP-B -/- mice contained no fully formed lamellar bodies. While the abundance of SP-A and SP-C mRNAs was not altered, an aberrant form of pro-SP-C, 8.5 kDa, was detected, and fully processed SP-C peptide was markedly decreased in lung homogenates of SP-B -/- mice. Ablation of the SP-B gene disrupts the routing, storage, and function of surfactant phospholipids and proteins, causing respiratory failure at birth.

T-cell receptor (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR beta-chain-variable-region V beta 8.2-positive T cells are not essential for induction and course of disease.

Predominant usage of V beta 8.2 gene segments, encoding a T-cell receptor (TCR) beta chain variable region, has been reported for pathogenic Lewis rat T cells reactive to myelin basic protein (MBP). However, up to 75% of the alpha/beta T cells in a panel of MBP-specific T-cell lines did not display TCR V beta 8.2, V beta 8.5, V beta 10, or V beta 16 elements. To further investigate TCR usage, we sorted the T-cell lines for V beta 8.2- and V beta 10-positive T cells or depleted the lines of cells with these TCRs. V beta 8.2-positive T cells and one of the depleted T-cell lines strongly reacted against the MBP peptide MBP-(68-88). The depleted T-cell line caused marked experimental autoimmune encephalomyelitis (EAE) even in Lewis rats in which endogenous V beta 8.2-positive T cells had been eliminated by neonatal treatment with anti-V beta 8.2 monoclonal antibodies. T-cell hybridomas generated from this line predominantly used V beta 3 TCR genes coexpressed with TCR V alpha 2 transcripts, which were also used by V beta 8.2-positive T cells. Furthermore, V beta 10-positive T cells reactive to MBP-(44-67) were encephalitogenic when injected immediately after positive selection. After induction of EAE by sorted V beta 8.2- or V beta 10-positive T-cell lines, immunocytochemical analysis of the spinal cord tissue showed a predominance of the injected TCR or of nontypable alpha/beta T cells after injection of the depleted line. Our results demonstrate heterogeneity of TCR beta-chain usage even for a single autoantigen in an inbred strain. Moreover, V beta 8.2-positive T cells are not essential for the induction and progression of adoptive-transfer EAE.