PubMed Health⌕ Search

Biomedical subjects

N Moskowitz

Publications and source records attributed to N Moskowitz.

At least 19 recordsLinked to original sources
PubMed2000

Rapid purification of protein complexes from mammalian cells.

The evaluation of the protein binding partner(s) of biologically important proteins is currently an area of intense research, especially since the development of the yeast two-hybrid assay. However, not all protein-protein interactions uncovered by this assay are biologically relevant and another confirmatory assay must be performed. Ideally, this assay should be rapid, versatile and performed under conditions which mimic the 'normal' physiological state as closely as possible. Towards this goal, we have constructed two eukaryotic expression vectors that facilitate the purification of a protein of interest, along with any associated proteins, from mammalian cells. These vectors incorporate the following features: (i) a tetracycline-responsive promoter so that the level of protein production can be regulated; (ii) an N-terminal glutathione S-transferase tag or a triple repeat of the HA1 epitope, to facilitate purification of the protein either by glutathione affinity chromatography or immunoprecipitation, respectively, followed by a multiple cloning site; (iii) the gene for the enhanced green fluorescent protein (for detection of the presence of the fusion protein and subcellular localization); (iv) a puromycin marker for the selection of stable transformants; (v) a truncated EBNA protein and oriP sequence for episomal replication of the vector. These latter two features permit expansion of small cultures of transfected cells under puromycin selection, thereby increasing the amount of tagged protein that can be purified. We show that these vectors can be used to direct the doxycycline-inducible expression of tagged proteins and to recover tagged CIP1-p21 protein complexes from HeLa cells. Furthermore, we show that these tagged p21-purified complexes contain both cyclin A and Cdk2, which are known to interact with p21, but not beta-actin.

Animals↗
PubMed1998

Humanizing the yeast telomerase template.

Saccharomyces cerevisiae contains an irregular telomere sequence (TG1-3)n, which differs from the regular repeat (TTAGGG)n found at the telomeres of higher organisms including humans. We have modified the entire 16-nt template region of the S. cerevisiae telomerase RNA gene (TLC1) to produce (TTAGGG)n repeats, the human telomere sequence. Haploid yeast strains with the tlc1-human allele are viable with no growth retardation and express the humanized gene at a level comparable to wild type. Southern hybridization demonstrates that (TTAGGG)n repeats are added onto the yeast chromosome ends in haploid strains with the tlc1-human allele, and sequencing of rescued yeast artificial chromosome ends has verified the addition of human telomeric repeats at the molecular level. These data suggest that the irregularity of the yeast telomere sequence is because of the template sequence of the yeast telomerase RNA. Haploid strains with the tlc1-human allele will provide an important tool for studying the function of telomerase and its regulation by telomere-binding proteins, and these strains will serve as good hosts for human artificial chromosome assembly and propagation.

Blotting, Southern↗
PubMed1996

Effect of Mycobacterium tuberculosis and its components on macrophages and the release of matrix metalloproteinases.

BACKGROUND: Pulmonary tuberculosis is associated with caseating necrosis, parenchymal lung destruction, and cavity formation. It was hypothesised that tuberculous lung destruction is mediated, at least in part, by the participation of matrix metalloproteinases released by mononuclear phagocytes. METHODS: Cells of the myelomonocytic leukaemia cell line THP-1 were incubated with lipoarabinomannan (LAM), the major antigenic cell wall component, and with Mycobacterium tuberculosis and analysed by Northern blot analysis. Two patients with active cavitary tuberculosis also underwent bronchoalveolar lavage and the cells were analysed by Northern blotting. RESULTS: Incubation of THP-1 cells with LAM resulted in the stimulated release of matrix metalloproteinase-9 (MMP-9), a 92 kDa gelatinase, by 24 hours in a dose-dependent fashion. In addition, Northern analysis revealed that LAM upregulated the gene for MMP-9 by 24 hours, but not the gene for the 72 kDa gelatinase MMP-2. Heat killed M tuberculosis H37Ra also upregulated the MMP-9 gene. Bronchoalveolar lavage of the two patients with active cavitary tuberculosis showed striking upregulation of the MMP-9 gene compared with a normal control using Northern analysis. LAM also upregulated the type I interstitial collagenase (MMP-1) gene by 24 hours in both THP-1 cells and peripheral blood monocytes. CONCLUSIONS: These data suggest that M tuberculosis and its major cell antigenic component, LAM, stimulate the release of MMP-9 and upregulate the expression of genes for MMP-1 and MMP-9. It is possible that M tuberculosis and its components contribute directly to cavity formation by their ability to stimulate macrophages to release matrix metallo-proteinases that digest collagens I-IV, and indirectly by stimulating the release of the cytokines interleukin 1 beta and tumour necrosis factor alpha that induce fibroblasts to amplify the release of matrix metalloproteinases.

Antigens, Bacterial↗
PubMed1990

Theories on the promotion of CNS transplant integration by selective activation of presynaptic enzyme cascades: prospects for future clinical applications.

It is hypothesized that multiple parallel presynaptic enzyme cascades act concertedly to regulate synaptic plasticity. These enzyme cascades include phospholipases A2 and C, protein kinase C, calcium/calmodulin and cAMP-dependent protein kinases and adenylate cyclase. New putative neurotrophic agents are postulated based on their ability to activate these enzymes. The artificial induction and amplification of multiple presynaptic enzymes in the fetal graft-mature host CNS tissue complex should maximally augment axon growth and synaptogenesis. In turn this would lead to enhanced transplant integration and hence maximal functional neurologic restoration. These enzyme cascades could be stimulated in-vivo by the intraventricular infusion of receptor-specific and/or lipophilic neurotrophic agents as well as by the application of external electric fields. A theoretical construct is formulated for developing future grafting experimentation with the hope of ultimately applying these concepts to the amelioration of human neurodegenerative diseases.

Animals↗
PubMed1989

Foramen magnum decompression in an infant with homozygous achondroplasia. Case report.

Homozygous achondroplasia is a rare yet distinct clinical entity. Most infants succumb to an early death as a result of respiratory compromise due to upper airway obstruction, thoracic cage deformity, and/or cervicomedullary compression. The successful cervicomedullary decompression of a 16-week-old infant with homozygous achondroplasia is described. This report suggests that homozygous achondroplasia is not universally fatal and that these infants are potentially viable if managed by aggressive respiratory and surgical measures.

Achondroplasia↗
PubMed1989

Systemic lupus erythematosus of the central nervous system: 2. Molecular theories and models for mental disease.

A molecular theory attempting to account for the complexity and diversity of the manifestations of systemic lupus erythematosus (SLE) central nervous system (CNS) disease is presented. The clinical manifestations of seizures, pareses, and movement and cranial nerve disorders can be accounted for primarily by immune-complex-mediated small and medium-sized cerebral vasculopathy. The disorders of thought and affect can be explained predominantly by systemic and intrathecal synthesis of antineuronal autoantibodies and brain-enzyme-inhibiting factors leading to alterations in the quantity of mesolimbic neurotransmitter release. It is possible that a multitude of different molecular pathologic derangements can lead to the same or different CNS dysfunctions. The final clinical expression of CNS disease in patients with SLE is dictated by the permutations and combinations of the individuals' underlying genetic predisposition, environmental triggers, specific systemic and intrathecal antineuronal autoantibodies, and the diversity and extent of immune-complex-mediated cerebral vasculopathy. Ways in which SLE-CNS disease may be viewed as a theoretical model for the nature and etiology of mental disease are discussed.

Autoantibodies↗
PubMed1986

Brain protein kinase in synaptic vesicles is inhibited by a factor in sera from systemic lupus erythematosus patients with central nervous system manifestations.

Sera from systemic lupus erythematosus patients with clinical central nervous system manifestations had a high mean percent of brain synaptic vesicle protein kinase inhibition (62 +/- 9.3, P less than 0.001). This conclusion was derived from screening sera of a total of 287 patients with heterogeneous diseases and from 12 healthy controls. Low levels of synaptic vesicle protein kinase inhibition also were detected in the sera of patients with certain autoimmune, inflammatory, neurological and/or psychiatric symptoms. Because 87.5% of the patients whose sera showed strong synaptic vesicle protein kinase inhibition (over 50%) had neuropsychiatric manifestations, we postulate this relationship may be due to the presence of an inhibitor factor, of which the etiology and molecular characteristics were investigated.

Adult↗
PubMed1985

A unified theory of presynaptic chemical neurotransmission.

The mechanism of neurotransmission and its modulation involves the direct role of calcium on membranes, and calcium's ability to activate synergistically and simultaneously a host of interdependent enzymatic cascades in synaptic and coated vesicles and the presynaptic plasma membrane. Enzymatic products formed can either amplify or depress synaptic vesicle exocytosis and synaptic vesicle regeneration via the coated pit/vesicle system. Rate amplification produced by a series of parallel, multistepped, interconnected enzymatic cascades as well as the optimal geometric spatial orientation of synaptic vesicles induced by presynaptic structures is hypothesized to explain how neurotransmitter is released within 200 musec upon calcium entry into the axon terminal.

Calcium↗
PubMed1985

Calcium-dependent binding of calmodulin to phospholipase A2 subunits induces enzymatic activation.

Calmodulin interacted with phospholipase A2 from two different sources, as established by affinity chromatography, dimethylsuberimidate protein crosslinking, and phospholipase A2 assays. Calmodulin was covalently crosslinked to pancreatic and bee venom phospholipases A2 in a calcium-dependent manner, and enhanced the enzymatic activities of these phospholipases. Pancreatic phospholipase A2 was separated into two species of identical molecular weight by calmodulin affinity chromatography; the species that bound to immobilized calmodulin in a calcium-dependent manner was stimulated by calmodulin. This presents further evidence that phospholipase A2 is directly activated by calmodulin.

Animals↗
PubMed1984

Regulation of endogenous calcium-dependent synaptic membrane phospholipase A2.

Synaptic plasma membrane preparations from brain tissue have endogenous Ca2+-dependent phospholipase A2 activity. Characterization of this activity revealed that it was maximally active at 10(-7)-10(-5) M Ca2+ and pH 7.0. The enzyme had a Km of 62.0 microM and a Vmax of 98.0 nmol/mg/h. Calmodulin and prostaglandin F2 alpha stimulated phospholipase A2 activity, whereas prostaglandin E2, cyclic AMP and ATP were inhibitory. Addition of exogenous phospholipase A2 to synaptic plasma membrane and synaptic vesicle preparations led to their disruption and/or lysis. We suggest that Ca2+-dependent regulation of phospholipase A2 activity may be required for synaptic vesicle and synaptic plasma membrane interaction.

Adenosine Triphosphate↗
PubMed1983

Phospholipase A2 modulation by calmodulin, prostaglandins and cyclic nucleotides.

Phospholipase A2 in the presence of Ca2+ was stimulated by calmodulin and by prostaglandin F2 alpha. Prostaglandin E2, cyclic-AMP and cyclic-GMP inhibited phospholipase A2 in the presence or absence of calmodulin. Dimethylsuberimidate cross-linking of phospholipase A2 with calmodulin was found to be Ca2+ dependent. These results indicate that phospholipase A2 is directly regulated by a host of key intracellular regulators and is one of the calmodulin-regulated enzymes.

Animals↗
PubMed1983

Preliminary characterization of synaptic vesicle/calmodulin interaction.

The association of calmodulin with brain synaptic vesicle proteins was analyzed. Scatchard analysis of [125I]calmodulin binding to brain synaptic vesicles revealed one high-affinity, low-binding-capacity, Kd = 1.0 (+/- 0.15) nM, Bmax = 4.1 (+/- 0.6) pmol/mg, and one low-affinity high-binding-capacity site, Kd = 177. (+/- 12.0) nM and Bmax = 202 (+/- 15.0) pmol/mg. Triton X-100 solubilization of synaptic vesicle proteins and subsequent elution on a Sepharose-4B-CNBr-calmodulin affinity column demonstrated that two protein doublets of approximate MrS 55 K and 30 K were the major synaptic vesicle calmodulin binding proteins. In addition there were two minor calmodulin binding singlet polypeptides with MrS 62 K and 40 K. Calmodulin stimulated endogenous synaptic vesicle protein kinase, Ca2+, Mg2+-ATPase and Ca2+ uptake activities. Phosphorylation assays coupled with immunological studies using affinity-purified antibodies suggested that the synaptic vesicle Ca2+/calmodulin-dependent protein kinase migrated in the 30 K Mr region.

Animals↗
PubMed1983

Characterization of brain synaptic vesicle phospholipase A2 activity and its modulation by calmodulin, prostaglandin E2, prostaglandin F2 alpha, cyclic AMP, and ATP.

Brain synaptic vesicle phospholipase A2 (PLA2) activity was characterized. It is Ca2+-dependent and has a pH optimum of 9.0. The enzyme has a Km of 60 microM and a Vmax of 2.0 nmol/mg/h. Calmodulin, prostaglandin F2 alpha, and cAMP, and ATP all increased the Vmax of the enzyme. Prostaglandin E2 inhibited the Vmax in the presence or absence of calmodulin. Light-scattering techniques in conjunction with phase-contrast and electron microscopy demonstrated that an increase in Vmax of PLA2 was correlated with synaptic vesicle aggregation, lysis, and possible fusion. In vitro synaptic vesicle-vesicle association that was stimulated by conditions that increased PLA2 activity could be diminished when synaptic vesicles were preincubated with PLA2 inhibitors. It is suggested that endogenous synaptic vesicle PLA2 activity may be an important mechanism underlying Ca2+-mediated neurotransmitter release.

Adenosine Triphosphate↗
PubMed1983

Phosphorylation of brain synaptic and coated vesicle proteins by endogenous Ca2+/calmodulin- and cAMP-dependent protein kinases.

Phosphorylation of brain synaptic and coated vesicle proteins was stimulated by Ca2+ and calmodulin. As determined by 5-15% sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE), molecular weights (Mr) of the major phosphorylated proteins were 55,000 and 53,000 in synaptic vesicles and 175,000 and 55,000 in coated vesicles. In synaptic vesicles, phosphorylation was inhibited by affinity-purified antibodies raised against a 30,000 Mr protein doublet endogenous to synaptic and coated vesicles. When this doublet, along with clathrin, was extracted from coated vesicles, phosphorylation did not take place, implying that the protein doublet may be closely associated with Ca2+/calmodulin-dependent protein kinase. Affinity-purified antibodies, raised against clathrin used as a control antibody, failed to inhibit Ca2+/calmodulin-dependent phosphorylation in either synaptic or coated vesicles. Immunoelectron cytochemistry revealed that this protein doublet was present in axon terminal synaptic and coated vesicles. Synaptic vesicles also displayed cAMP-dependent kinase activity; coated vesicles did not. The molecular weights of phosphorylated synaptic vesicle proteins in the presence of Mg2+ and cAMP were: 175,000, 100,000, 80,000, 57,000, 55,000, 53,000, 40,000, and 30,000. Based on the different phosphorylation patterns observed in synaptic and coated vesicles, we propose that brain vesicle protein kinase activities may be involved in the regulation of exocytosis and in retrieval of synaptic membrane in presynaptic axon terminals.

Animals↗
PubMed1982

Comparison of calmodulin binding to brain synaptic and coated vesicles.

Several characteristics of calmodulin association with brain synaptic and coated vesicles wer analyzed and compared. Radioimmunoassay revealed that both classes of vesicles contain approx. 1 microgram of calmodulin per mg of vesicle protein. Discontinuous sucrose gradients revealed that coated and synaptic vesicles preparations were homogeneous and had different sedimentation properties. Binding of 125I-labeled calmodulin to sympatic and coated vesicles was Ca2+ dependent and displaced by unlabeled calmodulin but not by troponin-C. Scatchard analysis revealed the presence of two binding sites. In both vesicle types there was one high-affinity, low-binding-capacity site (Kd = 1-39 nM and Bmax = 4-16 pmol/mg) and one low-affinity, high-binding-capacity site (Kd = 102-177 nM and Bmax = 151-202 pmol/mg). (Ca2+ +Mg2+)-ATPase activity was stimulated in both synaptic and coated vesicles by calmodulin. Thus synaptic and coated vesicles may possess similar calmodulin binding sites.

Animals↗