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J Rosenblatt

Publications and source records attributed to J Rosenblatt.

At least 19 recordsLinked to original sources
PubMed1997

Actin dynamics in vivo.

Actin dynamics in lamellipodia are driven by continuous cycles of actin polymerization, retrograde flow, and depolymerization. In the past year, advances have been made in identifying signaling pathways that regulate actin-filament uncapping and polymerization, in determining the role of myosin motor proteins in retrograde flow, and in evaluating the role of severing proteins in actin depolymerization. Both Listeria monocytogenes and Saccharomyces cerevisiae have emerged as powerful model organisms for studying actin dynamics in cells.

Actins↗
PubMed1996

A purified Drosophila septin complex forms filaments and exhibits GTPase activity.

Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells.

Actin Cytoskeleton↗
PubMed1996

Response of phosphoenolpyruvate cycle activity to fasting and to hyperinsulinemia in human subjects.

We have used a new isotopic tracer technique to investigate the physiological role of the phosphoenolpyruvate (PEP) cycle in metabolic adaptation to fasting and to hyperinsulinemia. The forward direction of the PEP cycle is the conversion of oxaloacetate (OAA) to PEP, and the net flux of the cycle is the rate at which PEP from OAA goes on to form glucose or glycogen, as opposed to being recycled to pyruvate and then OAA. Normal volunteers (n = 6) were studied after an overnight fast and then again after 3 days of fasting, and five additional subjects were studied during a hyperinsulinemic clamp (insulin concentration = 568 +/- 25 microU/ml, glucose infusion = 14.2 +/- 0.55 mg.kg-1.min-1). After an overnight fast, 35.4 +/- 6.7% of PEP from OAA was recycled to pyruvate-lactate. Short-term fasting caused a significant increase in the conversion of OAA to PEP and also a drop in the percentage of PEP from OAA that went to pyruvate-lactate to 15.2 +/- 4.0%. The principal response to hyperinsulinemia was a decrease in the recycling of OAA to lactate, because there was no significant change in the conversion of OAA to PEP. We conclude that changes in both directions of the PEP cycle are important in regulating gluconeogenic-glyconeogenic flux.

Fasting↗
PubMed1996

Comparison of mass isotopomer dilution methods used to compute VLDL production in vivo.

The recent development of mass isotopomer distribution methods represents an important new tool for quantifying synthetic rates. These methods allow precursor enrichment to be determined indirectly from the enrichment of the product, thus sidestepping the often difficult problem of measuring the precursor enrichment. Two different methods have been described to compute synthetic rates by this general approach in the laboratories of M. K. Hellerstein and J. K. Kelleher, and variations of these basic approaches have also been presented by W. N. Lee and by ourselves. A comparison between the different methods has never been reported. In this paper, we take a specific application, calculation of the fractional rate of incorporation of acetyl-CoA into very low density lipoprotein-bound palmitate, and compare the results obtained from all of the mass isotopomer methods using the same data set obtained in vivo in human subjects. We found that it is critical that the measured background isotopomer distribution of palmitate is used rather than the theoretical background isotopomer distribution. We also found that the different methods give comparable precursor enrichments and comparable fractional synthesis rates, provided that the enrichments in Kelleher's method are properly weighted. Thus the choice of method to use is a matter of personal preference.

Humans↗
PubMed1995

The effect of electrostatic charge interactions on release rates of gentamicin from collagen matrices.

PURPOSE: This work studied the effect of changes in the magnitude of electrostatic charge interactions on the release kinetics of gentamicin from collagen matrices. METHODS: The charge distribution on collagen was altered by specific charge chemistries to yield net negative charges which exhibited binding interactions with positively charged gentamicin. The adsorption isotherms were measured to characterize binding interactions and release of gentamicin from modified matrices were measured. The release rates were compared to a mathematical model based on an instantaneous desorption coupled with diffusion mechanism. RESULTS: Ninety percent of the gentamicin loaded was released from native collagen matrices in 2.5 days (one-sided slab geometry in-vitro). Succinylated collagen matrices released 70% in 2.5 days and phosphonylated collagen matrices released 50% in 2.5 days. Excellent agreement between model predictions and experiment results were obtained. CONCLUSIONS: Modified collagen can be much more effective in antibiotic therapy in sustaining release rates compared to native collagen for charged antibiotics like gentamicin.

Adsorption↗
PubMed1995

The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.

Actins↗
PubMed1995

Evaluation of enzyme-linked immunoassay for serological diagnosis of cysticercosis.

We evaluated a commercially available enzyme-linked immunoassay (ELISA) from LMD Laboratories, Inc., Carlsbad, Calif., for the detection of antibodies in serum to the cysticercus of Taenia solium. The ELISA was performed on 308 serum samples; 198 from a pool of healthy individuals, 42 from patients who had antibodies against a variety of parasites other than T. solium, and 68 from patients suspected of having cysticercosis. All of these 68 specimens were tested both by the ELISA and by an immunoblot method (enzyme-linked immunoelectrotransfer blot assay [EITB]) developed at the Parasitic Serology Laboratory of the Centers for Disease Control and Prevention. Twenty-seven of the 68 serum samples from patients suspected of having cysticercosis were positive by both EITB and ELISA, while 31 were negative by both assays. ELISA results for three and two samples were considered false positive and false negative, respectively, when compared with the results of EITB. Results for an additional five samples were considered equivocal but were technically positive because their optical density readings were slightly above the cutoff value. Three of the 198 serum samples from the bank of serum samples from healthy individuals were also false positive by ELISA (the EITB result for the samples was negative). Six other serum samples from healthy individuals which had equivocal results and the five serum samples from patients with equivocal results were EITB negative. Serum samples containing antibodies against Echinococcus spp. frequently cross-reacted with the cysticercus ELISA antigen (13 of 16 specimens), but serum samples with antibodies against other parasites did not (2 of 26 specimens); all of these serum samples were EITB negative. The commercially available ELISA that we describe is a simple and rapid test. Considering all 308 specimens, the ELISA had a specificity of 93% (when samples with equivocal results were considered negative) or 89% (when samples with equivocal results were considered positive); the sensitivity was 93%.

Animals↗
PubMed1994

PITALRE, a nuclear CDC2-related protein kinase that phosphorylates the retinoblastoma protein in vitro.

Members of the cell division cycle 2 (CDC2) family of kinases play a pivotal role in the regulation of the eukaryotic cell cycle. In this communication, we report the isolation of a cDNA that encodes a CDC2-related human protein kinase temporarily designated PITALRE for the characteristic Pro-Ile-Thr-Ala-Leu-Arg-Glu motif. Its deduced amino acid sequence is 47% identical to that of the human cholinesterase-related cell division controller (CHED) kinase, which is required during hematopoiesis, and 42% identical to the Saccharomyces cerevisiae SGV1 gene product, a putative kinase involved in the response to pheromone via its guanine nucleotide-binding protein alpha subunit. PITALRE expression is ubiquitous, but its expression levels are different in various human tissues. PITALRE is an approximately 43-kDa protein that associates with three cellular polypeptides of 80, 95, and 155 kDa. PITALRE is localized primarily to the nucleus. In addition, we have identified a retinoblastoma protein kinase activity associated with PITALRE immunocomplexes that cannot phosphorylate histone H1, suggesting that the target phosphorylation site of PITALRE differs from that of CDC2 kinase. Interestingly, the retinoblastoma kinase activity associated with PITALRE does not oscillate during the cell cycle.

Amino Acid Sequence↗
PubMed1994

Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts.

Within hours of Listeria monocytogenes infection, host cell actin filaments form a dense cloud around the intracytoplasmic bacteria and then rearrange to form a polarized comet tail that is associated with moving bacteria. We have devised a cell-free extract system capable of faithfully reconstituting L. monocytogenes motility, and we have used this system to demonstrate that profilin, a host actin monomer-binding protein, is necessary for bacterial actin-based motility. We find that extracts from which profilin has been depleted do not support comet tail formation or bacterial motility. In extracts and host cells, profilin is localized to the back half of the surface of motile L. monocytogenes, the site of actin filament assembly in the tail. This association is not observed with L. monocytogenes mutants that do not express the ActA protein, a bacterial gene product necessary for motility and virulence. Profilin also fails to bind L. monocytogenes grown outside of host cytoplasm, suggesting that at least one other host cell factor is required for this association.

Actins↗
PubMed1994

Injectable collagen as a pH-sensitive hydrogel.

Injectable collagen is a concentrated dispersion of phase-separated collagen fibres in aqueous solution. The structure and properties of collagen fibres are defined by the magnitudes of electrostatic and hydrophobic attractive forces between neighbouring collagen molecules within collagen fibres. The structure and mechanical properties of collagen fibre dispersions were studied by dynamic rheological measurements and by polarized microscopy. Rheological measurements were performed over pHs ranging from 6 to 9 and over temperatures ranging from 283 to 298 K. At higher pHs the fibre dispersions were found to possess more rigid fibres and stronger inter-fibre attractive forces. This response is argued to result from changes in the ionization of amino acid side chains, which result in larger net-electrostatic attractive forces. Raising the temperature caused fibres to rigidify through enhanced hydrophobic attractive forces. Gels formed by lower pH-higher temperature fibre dispersions possess different properties than gels formed at higher pHs and lower temperatures.

Animals↗
PubMed1994

Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes.

We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Crystal structure of cyclin-dependent kinase 2.

Cyclin-dependent kinase 2 (CDK2) is a member of a highly conserved family of protein kinases that regulate the eukaryotic cell cycle. The crystal structures of the human CDK2 apoenzyme and its Mg2+ ATP complex have been determined to 2.4 A resolution. The structure is bi-lobate, like that of the cyclic AMP-dependent protein kinase, but contains a unique helix-loop segment that interferes with ATP and protein substrate binding and probably plays a key part in the regulation of all cyclin-dependent kinases.

Adenosine Triphosphate↗
PubMed1993

Purification and crystallization of human cyclin-dependent kinase 2.

The major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent protein kinases. We report the purification and crystallization of the catalytic subunit of human cyclin-dependent kinase 2 (CDK2), which has been implicated in the control of the G1/S transition. CDK2 was purified in large quantities from insect cells infected with a recombinant baculovirus, and crystals of the protein were prepared and subjected to preliminary X-ray diffraction analysis.

Animals↗
PubMed1993

Assessment of the mathematical issues involved in measuring the fractional synthesis rate of protein using the flooding dose technique.

1. The fractional synthesis rate of protein is commonly measured by either the constant infusion method or the flooding dose method. The two methods often give different results. 2. An underlying assumption of the traditional flooding dose formula is that the protein synthesis rate is not stimulated by the flooding dose. A new formula for calculation of the fractional synthesis rate is derived with the alternative assumption that the protein synthesis rate is stimulated by an amount proportional to the change in the intracellular concentration of the infused amino acid. The alternative formula is: Fractional synthesis rate = [formula: see text] where EB and EF are the enrichments of bound and free amino acid, respectively (atom per cent excess), and C = 1-(EF/EI), where EI is the enrichment of the infusate. This approach defines the lowest possible value for the fractional synthesis rate. The traditional equation gives a maximal value for the fractional synthesis rate. 3. When data from the literature are considered, the fractional synthesis rate of muscle protein as calculated by the constant infusion technique falls between the values of fractional synthesis rate calculated by the two flooding dose formulae when leucine is the tracer, suggesting that a flooding dose of leucine exerts a stimulatory effect on the rate of protein synthesis, but that the increase is not as great as the increase in the intracellular concentration of leucine.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1993

A transcriptional switch between the Pig-1 and Sgs-4 genes of Drosophila melanogaster.

Pig-1 and Sgs-4 are a pair of closely linked and divergently transcribed Drosophila melanogaster genes, which are both expressed in larval salivary glands but at different times during development. While Sgs-4 is expressed at high levels only at the end of the third instar, Pig-1 exhibits a major peak of expression during late second and early third instar. Thus, Pig-1 expression declines as Sgs-4 expression is induced. In this paper, we show that three adjacent elements located within the short region between these genes can account for the switch from Pig-1 to Sgs-4 expression. A 170-bp segment acts as an enhancer to direct Sgs-4 expression in late-third-instar salivary glands. A 64-bp sequence located just upstream from the enhancer can modify its temporal specificity so that it works throughout the third instar. Expression induced at mid-third instar by a combination of these two elements can be repressed by a negative regulatory sequence located still further upstream. We present evidence suggesting that the changing interactions between these regulatory elements and the Sgs-4 and Pig-1 promoters lead to the correct pattern of expression of the two genes.

Age Factors↗