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R Hecht

Publications and source records attributed to R Hecht.

At least 19 recordsLinked to original sources
PubMed1997

Autocatalytic networks with intermediates. I: Irreversible reactions.

A class of autocatalytic reaction networks based on template-dependent replication and specific catalysis is considered. Trimolecular "elementary steps" of simple replicator dynamics are resolved into two consecutive irreversible reactions. The extreme cases, competition for common resources and hypercycle-like cooperative feedback, were analyzed in some detail. Although the dynamics of the extended networks resembles corresponding replicator dynamics in general, there are significant differences. Most notably, the interior fixed points in the cooperative model turned out to be asymptotically stable for an arbitrary number of species, whereas simple replicator dynamic predicts an asymptotically stable periodic orbit fixed for four species and fewer and a stable periodic orbit for all other cases.

Bioreactors↗
PubMed1996

Refined crystal structure and mutagenesis of human granulocyte-macrophage colony-stimulating factor.

The crystal structure of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) has been refined against data extending to a resolution of approximately 2.4 A along a* and approximately 1.9 A along b* and c*. Anisotropic scale factors of B11 = -20.8 A2, B22 = 7.4 A2, B33 = 13.3 A2 corrected for the more rapid fall of diffraction in the a* direction. The anisotropy correlates with the weak crystal packing interactions along the a axis. In addition to apolar side chains in the protein core, there are 10 buried hydrogen bonding residues. Those residues involved in intramolecular hydrogen bonding to main chain atoms are better conserved than those hydrogen bonding to other side chain atoms; 24 solvation sites are observed at equivalent positions in the two molecules in the asymmetric unit, and the strongest among these are located in clefts between secondary structural elements. No buried water sites are seen. Two surface clusters of hydrophobic side chains are located near the expected receptor binding regions. Mutagenesis of 11 residues on the helix A/helix C face confirms the importance of Glu-21 and shows that Gly-75 and Gln-86, located on helix C, each cause a greater than fourfold drop in activity. Glu-21 and Gly-75, but not Gln-86, are structurally equivalent to residues involved in the growth hormone binding to its receptor.

Amino Acid Sequence↗
PubMed1995

Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice.

Megakaryocyte growth and development factor (MGDF) is a potent inducer of megakaryopoiesis in vitro and thrombopoiesis in vivo. The effects of MGDF appear to be lineage-selective, making this cytokine an ideal candidate for use in alleviating clinically relevant thrombocytopenias. This report describes a murine model of life-threatening thrombocytopenia that results from the combination treatment of carboplatin and sublethal irradiation. Mortality of this regimen is 94% and is associated with widespread internal bleeding. The daily administration of pegylated recombinant human MGDF (PEG-rMGDF) significantly reduced mortality (to < 15%) and ameliorated the depth and duration of thrombocytopenia. The severity of leucopenia and anemia was also reduced, although it was not clear whether these effects were direct. Platelets generated in response to PEG-rMGDF were morphologically indistinguishable from normal platelets. PEG-rMGDF administered in combination with murine granulocyte colony-stimulating factor completely prevented mortality and further reduced leukopenia and thrombocytopenia. These data support the concept that PEG-rMGDF may be useful to treat iatrogenic thrombocytopenias.

Animals↗
PubMed1995

Megakaryocyte growth and development factor and interleukin-3 induce patterns of protein-tyrosine phosphorylation that correlate with dominant differentiation over proliferation of mpl-transfected 32D cells.

Recently, the ligand for c-mpl, a member of the family of cytokine receptors, was cloned and found to be a physiologic regulator of platelet homeostasis. We report that megakaryocyte growth and development factor (MGDF, thrombopoietin [TPO], c-mpl ligand ) induces differentiation in a majority of mpl-transfected 32D cells, while interleukin (IL)-3 is exclusively mitogenic in this system. MGDF differentiation, as measured by decreased proliferation, changes in cellular morphology, increased adherence, and downregulation of very late antigen (VLA)-4, is dominant over IL-3 proliferation. MGDF induces tyrosine-phosphorylation of mpl, JAK2, SHC, SHPTP-1 (HCP, motheaten) and SHPTP-2 (Syp, PTP-1D) within 30 seconds of stimulation, as well as of vav and MAPK with slightly delayed kinetics. A fraction of mpl and JAK2 is preassociated, and the stoichiometry of this complex is unaltered by cytokine stimulation. After MGDF stimulation, we detect interactions among SHC, grb2, SHPTP-1, SHPTP-2, and the mpl/JAK2 complex. IL-3 induces phosphorylation of the above proteins with the exception of mpl and also causes weak JAK1 phosphorylation. Although similar in composition, the MGDF- and IL-3-induced complexes of signal transducers appear to be assembled in different configurations, especially with respect to SHPTP-2. Both MGDF and IL-3 induce tyrosine phosphorylation of STAT3 (APRF) and STAT5 (MGF), with MGDF favoring STAT3 while IL-3 predominantly causes STAT5 phosphorylation. In addition, some proteins become tyrosine-phosphorylated in response to MGDF only, suggesting that we may have detected differentiation-specific signal transducers. These include a number of high-molecular-weight proteins (140 to 200 kD) and one 28-kD protein that becomes tyrosine-phosphorylated only briefly.

Amino Acid Sequence↗
PubMed1995

Effects of the obese gene product on body weight regulation in ob/ob mice.

C57BL/6J mice with a mutation in the obese (ob) gene are obese, diabetic, and exhibit reduced activity, metabolism, and body temperature. Daily intraperitoneal injection of these mice with recombinant OB protein lowered their body weight, percent body fat, food intake, and serum concentrations of glucose and insulin. In addition, metabolic rate, body temperature, and activity levels were increased by this treatment. None of these parameters was altered beyond the level observed in lean controls, suggesting that the OB protein normalized the metabolic status of the ob/ob mice. Lean animals injected with OB protein maintained a smaller weight loss throughout the 28-day study and showed no changes in any of the metabolic parameters. These data suggest that the OB protein regulates body weight and fat deposition through effects on metabolism and appetite.

Adipose Tissue↗
PubMed1995

Characterization of the cDNA and three processed pseudogenes from the murine protein kinase CK2 alpha subunit.

Seven protein kinase CK2 alpha clones were isolated from a murine genomic DNA library. They were assigned to four different genomic loci (A,B,C,D). Locus D was previously identified as a processed pseudogene (Boldyreff et al. (1992) Biochem. Biophys. Res. Commun. 186, 723-730). Here we present sequences of genomic loci B and C and the murine CK2 alpha cDNA. Loci B and C are like locus D processed pseudogenes, however, with considerable differences among each other and to the cDNA, especially with respect to the lengths of the putative gene products. Genomic locus D would code for a protein of 82 amino acids, locus B for a protein of 132 amino acids and locus C product for the full length product of 391 amino acids as the murine cDNA.

Amino Acid Sequence↗
PubMed1995

[A new method for measuring internal ear circulation using colored microspheres].

The pathogenesis of sudden hearing loss is as yet unknown. Vascular disturbances are thought to play a major role, but they cannot be diagnosed clinically. We have therefore developed an animal model which allows measurements of the cochlear blood flow (CBF) and function. For CBF measurements a new technique based on coloured microspheres was developed. New Zealand rabbits were anaesthetised, ventilated and blood pressure and heart frequency were continuously monitored. 6 x 10(6) microspheres of one colour were injected in the left ventricle; different colours are available and allow multiple measurements. The amount of microspheres trapped in the capillary bed of the cochlea depends on the regional blood flow. An arterial reference blood sample makes it possible to calculate absolute values of the regional blood flow in the cochlea. The amount of microspheres trapped in the cochlea was determined microscopically after dissecting, dissolving and filtrating the cochlea. Measurements of the CBF in a control group (5 rabbits, 10 inner ears) at the beginning and after 120 minutes were performed. Mean CBF was 3.8 +/- 1.1 microliters/min and 4.3 +/- 1.3 microliters/min. The coloured microsphere technique allows repeated measurements of the CBF with good reproducibility and an acceptable standard deviation. CBF can be measured in absolute values, which is of advantage for determining a lower limit of the CBF for the cochlea function.

Animals↗
PubMed1994

Role of Mac-1 and ICAM-1 in ischemia-reperfusion injury in a microcirculation model of BALB/C mice.

The leukocyte beta 2-integrin Mac-1 (CD11b/CD18) and its endothelial ligand intercellular adhesion molecule 1 (ICAM-1) are involved in leukocyte adhesion to and macromolecular leakage from postcapillary venules during inflammatory reactions. Both events are also encountered after ischemia-reperfusion of striated muscle, suggesting a central role of both adhesion proteins in reperfusion injury. Using intravital fluorescence microscopy and a microcirculation model in awake BALB/C mice, we investigated the effects of monoclonal antibodies (MAb) and Fab fragments to Mac-1 and MAb to ICAM-1 on leukocyte-endothelium interaction and macromolecular leakage of fluorescein isothiocyanate-dextran (1.5 x 10(5) mol wt) in striated skin muscle after 3 h of ischemia followed by reperfusion. We demonstrated that administration of MAb and Fab to Mac-1 before reperfusion was as effective as administration of MAb to ICAM-1, which was found to be significantly upregulated in the postischemic tissue by immunohistochemical analysis, in preventing postischemic leukocyte adhesion to and macromolecular leakage from postcapillary venules, whereas postischemic leukocyte rolling was not affected after MAb administration. Postischemic capillary perfusion was efficiently preserved in animals treated with anti-Mac-1 and anti-ICAM-1 MAb compared with animals receiving the isotype-matched control antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Leukocyte rolling in venules of striated muscle and skin is mediated by P-selectin, not by L-selectin.

Leukocyte rolling in post-capillary venules is mediated by adhesion molecules of the selectin family expressed on both leukocytes (L-selectin) and endothelial cells (E- and P-selectin). With the use of intravital fluorescence microscopy, the effects of antibodies against these selectins were analyzed in the skinfold chamber model of BALB/c mice and the ear model of homozygous hairless mice (hr/hr) that permit chronic observation of striated muscle and skin microcirculation in awake animals, respectively. Mice were injected intravenously with monoclonal antibodies (MAb) to murine L-selectin and E-selectin and affinity-purified polyclonal antibodies to P-selectin. The antibodies, which are known to block cell adhesion, were tested by immunoprecipitation to selectively bind to L-, E-, or P-selectin. Leukocyte rolling was a constant finding in both microcirculation models in the absence of inflammatory stimuli. In both models, injection of anti-P-selectin antibodies completely prevented baseline leukocyte rolling over an observation period of 2 h (P < 0.01 vs. baseline), while no effects were seen after administration of either anti-L-selectin or anti-E-selectin MAb. Treatment with the isotype-matched control antibodies did not affect leukocyte rolling in either model. We conclude that leukocyte rolling in postcapillary venules of murine striated muscle and skin is a physiological process mediated via P-selectin, whereas L- and E-selectin appear not to play a significant role under these circumstances.

Animals↗
PubMed1994

Diaspirin crosslinked hemoglobin: evaluation of effects on the microcirculation of striated muscle.

Hemoglobin-based oxygen carriers such as diaspirin-crosslinked hemoglobin (DCLHb) have been proposed for blood substitution due to their plasma expansion and oxygen transport capacity. This study investigates the effects of DCLHb on the microcirculation of striated muscle after moderate topload infusion and isovolemic exchange transfusion in awake hamsters. The skinfold chamber model in hamsters and intravital fluorescence microscopy were used for analysis of vessel diameter, red blood cell velocity (RBCV), leukocyte sticking to the microvascular endothelium, and macromolecular leakage in striated skin muscle. In each animal, arteriolar and postcapillary vessel segments were chosen and sequentially recorded on videotape (baseline). Animals were subjected to either topload infusion (10% of blood volume) or isovolemic exchange transfusion (hct 30%) of DCLHb followed by measurements at 10, 30, and 60 min thereafter. In vivo visualization of plasma and leukocytes was performed using FITC-dextran 150,000 and rhodamine 6G, respectively. No significant changes of vessel diameter and RBCV were observed after topload infusion or isovolemic exchange transfusion with DCLHb, either in postcapillary venules or in arterioles when compared with baseline values. Leukocyte sticking and macromolecular leakage were not found enhanced after administration of DCLHb. We conclude that the introduction of DCLHb-bound oxygen into the tissue does neither stimulate leukocyte adhesion nor impair endothelial integrity.

Animals↗
PubMed1993

Comparison of the biophysical characteristics of human brain-derived neurotrophic factor, neurotrophin-3, and nerve growth factor.

The structural properties of human brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were studied using sedimentation equilibrium and circular dichroism (CD), fluorescence and Fourier-transform infrared spectroscopies, and compared with those of human nerve growth factor (NGF). Both the far UV CD and infrared spectra indicate that these three proteins have similar, but not identical, secondary structures which contain primarily beta-sheet and irregular structures. NGF appears to contain the most beta-sheet while NT-3 contains a small fraction of alpha-helix. The near UV CD spectra appear to indicate that the three proteins contain disulfide bonds in similar environments, suggesting a resemblance in tertiary structure. The fluorescent tryptophans found in the molecules are relatively solvent exposed, while Trp102 found only in NT-3 is possibly quenched. The fluorescent Trp(s) in NGF are significantly quenched relative to those in the other two neurotrophic factors. Both NT-3 and BDNF have very hydrophilic surfaces at neutral pH, as indicated by a low binding affinity to a hydrophobic probe, anilinonaphthalenesulfonate. Sedimentation equilibrium showed that BDNF, NT-3, and NGF exist as strongly associated dimers in phosphate-buffered saline, pH 7.1. Fits of the observed fringe displacements to various association models suggested that the BDNF, NT-3, and NGF samples contain, in addition to the principal dimeric species, some oligomers, and that NT-3 contains a small fraction of incompetent monomer.

Amino Acid Sequence↗
PubMed1992

Identification of four genomic loci highly related to casein-kinase-2-alpha cDNA and characterization of a casein kinase-2-alpha pseudogene within the mouse genome.

Using the coding region of the human CK-2 alpha cDNA as a probe for screening a genomic mouse library, positive clones representing four different genomic loci were isolated. Partial DNA sequences of these loci encompassing the first 120 nucleotides of the putative coding region are reported. One positive clone was further analyzed by sequencing a 3.1 kb XbaI fragment. This clone displays the characteristics of a pseudogene, i.e. lack of introns and several nucleotide insertions and deletions. In its 3' region it contains a 91 bp large CT-rich stretch which consists of (CCTT) and (CT) repeats; in the 5' region three (CCCCCT) repeats.

Animals↗
PubMed1980

Electrophoretic mobility of PM2 DNA treated with ultimate chemical carcinogens or with ultraviolet light.

Superhelical DNA of the Pseudomonas phage PM2 was irradiated with UV-light or reacted with covalently binding carcinogens, such as 7-bromomethyl-benz[a]anthracene, (Ac)2ONFln, K-region epoxides, and alkylating agents. Migration velocity of the DNA products was determined using agarose gel electrophoresis. In gels of more than 1.3%-1.9% agarose, modified PM2 DNA exhibited a dose-(concentration-)dependent decrease of migration velocity. This phenomenon is probably due to a decrease in superhelix density which caused the compact DNA coil to assume eventually an open-circular conformation. Comparison of the extent of DNA modification with the decrease of migration velocity revealed that the superhelical structure sensitively reflected the chemical DNA alterations. DNA species exhibiting, in 1.6% agarose gels, a migration velocity of up to 30% of that of control DNA showed an increase of velocity in 0.4% agarose. Therefore, in 1.3%-1.9% agarose gels, the decrease os superhelix density is accompanied by an increase of the frictional coefficient, whereas in 0.4%-0.9% agarose gels the same decrease of superhelix density apparently led to a higher degree of flexibility of the macromolecule and/or exposure of additional electric charges.

2-Acetylaminofluorene↗
PubMed1978

Purification and characterization of an endonuclease from Micrococcus luteus that acts on depurinated and carcinogen-modified DNA.

An endonuclease which is active with regard to depurinated, alkylated, arylated, and arylamidated DNA has been purified 500-fold from Micrococcus luteus. In this purification, separation from the pyrimidine-dimer-specific ultraviolet-endonuclease has been achieved. The enzyme has a molecular weight of 30000 on the basis of gel filtration; its activity is not absolutely dependent upon the presence of Mg2+, but 5--30 mM Mg2+ produces a five-fold stimulation. Potassium chloride concentrations of less than 100 mM are optimal, while concentrations exceeding 100 mM inhibit. The enzyme has no effect on native DNA, but introduces single-strand breaks into DNA containing apurinic/apyrimidinic sites produced by heating at an acidic pH. DNA treated with such carcinogens as N-alkyl-N-nitrosoureas, alkyl methanesulfonates, alkyl sulfates, nitrogen mustard, beta-propiolactone, 7-bromomethyl-benz[a]anthracene, N-acetoxy-2-acetylaminofluorene, and 7,12-dimethyl-benz[a]anthracene-5,6-oxide also becomes susceptible to enzymic action. The activity of the enzyme has been detected by making use of the difference in mobility between supercoiled closed-circular DNA of Pseudomonas phage PM2 and its nicked form in agarose gel elctrophoresis. Even depurinated or carcinogen-modified supercoiled PM2 DNA migrated faster than the respective relaxed nicked forms. A comparison of the number of enzyme-catalyzed single-strand breaks with the number of alkali-labile (i.e. apurinic) sites in carcinogen-modified PM2 DNA showed that the enzyme preparation introduced approximately twice as many breaks into the substrates as the number of apurinic sites present. We conclude that the enzyme preparation either recognizes both apurinic sites and DNA bases carrying carcinogenic residues or contains DNA glycosidase activity in addition to the endonuclease activity. Exposure of ultraviolet-irradiated PM2 DNA to the endonuclease preparation showed that pyrimidine dimers were not substrates. The yield of enzyme-catalyzed single-strand breaks found in ultraviolet-irradiated DNA was five times the number of alkali-labile sites present suggesting that minor photoproducts, possibly 5,6-saturated pyrimidine residues, were recognized in addition to apurinic sites.

Carcinogens↗