Dana L. Madison

Differential expression of rab3 isoforms in oligodendrocytes and astrocytes.

The small GTP‐binding protein Rab3a is involved in regulated secretory pathways and is enriched in synaptic and neuroendocrine secretory vesicles. We have reported previously the developmental regulation of Rab3a in oligodendrocytes in culture and purified central nervous system myelin (Huber et al.: FEBS Lett 347: 273–278, 1994). Since multiple rab3 isoforms exist in the brain and may be associated with different secretory pathways, we have investigated the differential expression of the rab3 isoforms in oligodendrocytes, astrocytes, and Schwann cell line RT4‐D6P2T. The expression of specific rab3 isoforms (rab3a‐c) was detected by polymerase chain reaction (PCR) amplification and confirmed by sequence analyses. These data show that in addition to the previously reported expression in neurons, the two macroglial populations, astrocytes and oligodendrocytes, also express rab3 isoforms. Rab3b was preferentially amplified from purified, cultured astrocytes, while rab3a and rab3c were preferentially amplified from highly enriched populations of both cultured oligodendrocytes and those isolated directly from the brain by immunopanning. No novel rab3 isoform was detected in glia. These results indicate that glial cells in the brain express specific isoforms of the vesicular trafficking Rab3 protein family.

SNARE complex proteins, including the cognate pair VAMP-2 and syntaxin-4, are expressed in cultured oligodendrocytes

Abstract : Myelin membrane synthesis in the CNS by oligodendrocytes (OLs) involves directed intracellular transport and targeting of copious amounts of specialized lipids and proteins over a relatively short time span. As in other plasma membrane‐directed fusion, this process is expected to use specific trafficking and vesicle fusion proteins characteristic of the SNARE model. We have investigated the developmental expression of SNARE proteins in highly enriched primary cultures of OLs at discrete stages of differentiation. VAMP‐2/synaptobrevin‐2, syntaxin‐2 and ‐4, nsec‐1/munc‐18‐1, Rab3a, synaptophysin, and synapsin were expressed. During differentiation, expression of the vesicular SNARE VAMP‐2, the small GTP‐binding protein Rab3a, and the target SNARE syntaxin‐4 were up‐regulated. VAMP‐2 and Rab3 proteins detected immunocytochemically in cultured OLs were localized within the developing process network ; in situ anti‐VAMP‐2 antibody stained the perikarya of rows of cells with the distribution and appearance of OLs. We discuss the potential involvement of SNARE complex proteins in a plasma membrane‐directed transport mechanism targeting nascent myelin vesicles to the forming myelin sheath.

Myelin membrane biogenesis by oligodendrocytes Developmental regulation of low molecular weight GTP-binding proteins

Oligodendrocytes synthesize dramatic amounts of myelin membrane. We hypothesized that this requires unique aspects of vesicular trafficking. Specific stages of the oligodendrocyte lineage were assayed for low molecular weight GTP‐binding proteins implicated in the regulation of vesicular transport pathway (two dimensional gel electrophoresis, [α‐32P]GTP overlay). Consistent with the hypothesis, as oligodendrocytes differentiate from early progenitors to mature myelin‐producing cells, ≥≥ 12 small GTP‐binding proteins become up‐regulated. Myelin membrane also has a complex pattern of GTP‐binding proteins. Several of these proteins may be specific to oligodendrocytes, suggesting that oligodendrocytes may utilize cell‐type specific GTP‐binding proteins for biogenesis and maintenance of the myelin membrane.

The PARP inhibitor PJ34 causes a PARP1-independent, p21 dependent mitotic arrest.

Poly(ADP)-ribose polymerase (PARP) inhibitors modify the enzymatic activity of PARP1/2. When certain PARP inhibitors are used either alone or in combination with DNA damage agents they may cause a G2/M mitotic arrest and/or apoptosis in a susceptible genetic context. PARP1 interacts with the cell cycle checkpoint proteins Ataxia Telangectasia Mutated (ATM) and ATM and Rad3-related (ATR) and therefore may influence growth arrest cascades. The PARP inhibitor PJ34 causes a mitotic arrest by an unknown mechanism in certain cell lines, therefore we asked whether PJ34 conditionally activated the checkpoint pathways and which downstream targets were necessary for mitotic arrest. We found that PJ34 produced a concentration dependent G2/M mitotic arrest and differentially affected cell survival in cells with diverse genetic backgrounds. p53 was activated and phosphorylated at Serine15 followed by p21 gene activation through both p53-dependent and -independent pathways. The mitotic arrest was caffeine sensitive and UCN01 insensitive and did not absolutely require p53, ATM or Chk1, while p21 was necessary for maintaining the growth arrest. Significantly, by using stable knockdown cell lines, we found that neither PARP1 nor PARP2 was required for any of these effects produced by PJ34. These results raise questions and cautions for evaluating PARP inhibitor effectiveness, suggesting whether effects should be considered not only on PARPs diverse ADP-ribosylation independent protein interactions but also on homologous proteins that may be producing either overlapping or distinct effect.

C-terminal binding protein and poly(ADP)ribose polymerase 1 contribute to repression of the p21 waf1/cip1 promoter

Transcriptional repression by the C-terminal binding protein (CtBP) is proposed to require nicotinamide adenine dinucleotide dehydrogenase (NAD(H). Previous studies have implicated CtBP in transcriptional repression of the p21waf1/cip1 gene. Similarly, the NAD-dependent poly(adenosine diphosphate)ribose polymerase 1 (PARP1) may affect p21 expression via its NAD-dependent enzymatic activity; we therefore asked if PARP1 and CtBP were functionally linked in regulating p21 transcription. We found that restraint of basal p21 transcription requires both CtBP and PARP1. PARP inhibition attenuated activation of p21 transcription by both p53-independent and p53-dependent processes, in a CtBP-dependent manner. CtBP1+2 or PARP1+2 knockdown partially activated p21 gene expression, suggesting relief of a corepressor function dependent on both proteins. We localized CtBP-responsive repression elements to the proximal promoter region, and found ZBRK1 overexpression could also overcome DNA damage-dependent, but not p53-dependent activation through this region. By chromatin immunoprecipitation we find dismissal of CtBP from the proximal promoter following DNA-damage, and that PARP1 associates with a CtBP corepressor complex in nuclear extracts. We propose a model in which both CtBP and PARP functionally interact in a corepressor complex as components of a molecular switch necessary for p21 repression, and following DNA damage signals activation of p21 transcription by corepressor dismissal and co-activator recruitment.

Henoch-Schönlein purpura: a possible complication of hepatitis C related liver cirrhosis

Henoch-Schonlein purpura (HSP) is a systemic small vessel vasculitis predominantly affecting children and, less commonly, adults. Classical HSP includes a tetrad of palpable purpura, arthritis, abdominal pain, and glomerulonephritis. Adults may present with any two of the four criteria in the tetrad (87% sensitivity and specificity). Gastrointestinal disease has been recorded in up to 82% of adult patients in one series1 and is usually self limiting with colicky abdominal pain, but may progress to ischaemic bowel perforation.2 We present the case of a 63 year old man with IgA vasculitis, probably HSP, confounded by undiagnosed hepatitis C related cirrhosis. He was admitted with a two week history of dyspnoea, malaise, cough, fevers, and chills, myalgias, one day of a non-blanching erythematous rash on his legs, and an ileus. His hepatitis …

Purification and partial characterization of an ice nucleator protein from the intertidal gastropod, Melampus bidentatus

An ice nucleator protein has been purified from the intertidal gastropod Melampus bidentatus by a combination of gel filtration and ion exchange chromatography. Partial characterization of this ice nucleator protein indicates that in the native form, it exists as an aggregate with a molecular weight in excess of 600 kDa. SDS-PAGE analysis indicates that this aggregate is composed of two low molecular weight protein species of approximately 16.6 and 17.4 kDa. Biochemical analysis indicates that it is neither a lipoprotein nor a glycoprotein. The amino acid composition indicates that it contains a high percentage of polar amino acids with Asx and Glx representing over 20 mol% of the protein. The characteristics of this molluskan ice nucleator protein are compared with those of purified ice nucleator proteins obtained from bacteria and various species of insects.

Transient transfection of oligodendrocyte progenitors by electroporation

The transient transfection of transgenes into oligodendrocytes offers an important tool for studying the function of proteins during myelin formation. Currently established procedures, however, have generally resulted in low survival rates and low levels of uptake of the transgene into primary oligodendrocyte progenitors. We describe an electroporation method which yields transient transfection of oligodendrocyte progenitors of up to 10–15% of the surviving cells, and provides approximately 104 surviving, transfected cells per electroporation reaction. In recent applications transgene expression persisted as the transfected progenitors progressed through subsequent stages of the oligodendrocyte lineage. This technique is expected to facilitate the study of the function of key proteins and lipids during the development of primary cultured oligodendrocytes.

Nicotinamide Adenine Dinucleotide Induced Multimerization of the Co-repressor CtBP1 relies on a Switching Tryptophan

Background: C-terminal binding protein 1 (CtBP1) assembles into a tetrameric transcriptional co-repressor but how it directs gene expression is not clear. Results: CtBP1 requires NAD(H) for transition into multimers. Its biochemical activities are separable from transcriptional repression. Conclusion: Tryptophan 318 permits CtBP1 to first dimerize and then tetramerize after the binding of NAD(H). Significance: Clarification of how CtBP1 tetramerizes will permit development of CtBP inhibitors to target oncogenesis. The transcriptional co-repressor C-terminal binding protein (CtBP) interacts with a number of repressor proteins and chromatin modifying enzymes. How the biochemical properties including binding of dinucleotide, oligomerization, and dehydrogenase domains of CtBP1 direct the assembly of a functional co-repressor to influence gene expression is not well understood. In the current study we demonstrate that CtBP1 assembles into a tetramer in a NAD(H)-dependent manner, proceeding through a dimeric intermediate. We find that NAD-dependent oligomerization correlates with NAD+ binding affinity and that the carboxyl terminus is required for assembly of a dimer of dimers. Mutant CtBP1 proteins that abrogate dinucleotide-binding retain wild type affinity for the PXDLS motif, but do not self-associate either in vitro or in vivo. CtBP1 proteins with mutations in the dehydrogenase domain still retain the ability to self-associate and bind target proteins. Both co-immunoprecipitation and mammalian two-hybrid experiments demonstrate that CtBP1 self-association occurs within the nucleus, and depends on dinucleotide binding. Repression of transcription does not depend on dinucleotide binding or an intact dehydrogenase domain, but rather depends on the amino-terminal domain that recruits PXDLS containing targets. We show that tryptophan 318 (Trp318) is a critical residue for tetramer assembly and likely functions as a switch for effective dimerization following NAD+ binding. These results suggest that dinucleotide binding permits CtBP1 to form an intranuclear homodimer through a Trp318 switch, creating a nucleation site for multimerization through the C-terminal domain for tetramerization to form an effective repression complex.

Do Secretory Pathway Snare Proteins Mediate Myelinogenesis

Oligodendrocyte development proceeds along a well defined developmental lineage both in vivo and in vitro (Fig. 1). The differentiated oligodendrocyte synthesizes a vast amount of myelin membrane, in the range of 5,000–50,000 mm2 per cell per day (Pfeiffer et al., 1993). In the neo-natal rat brain, or example, the mg of dry weight or myelin increases 600% from P0 to P30 and 1800% by P180; over this same time period total brain weight increases by only 50–60% (Norton and Cammer, 1984). How do oligodendrocytes accurately target and assemble myelin lipids and proteins into mature myelin membrane?