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Biomedical subjects

R Hecht

Publications and source records attributed to R Hecht.

At least 19 recordsLinked to original sources
PubMed2000

Cell death during development of intercalated ducts in the rat submandibular gland.

Programmed cell death, or apoptosis, occurs during the development of many tissues and organs in almost all multicellular organisms. Although apoptosis of salivary gland cells has been demonstrated in several pathological conditions, the role of apoptosis in the postnatal development of the salivary glands is unknown. We have studied the development of the rat submandibular gland (SMG) during its transition from the perinatal stage to the mature adult stage. Terminal tubule or Type I cells, which synthesize the secretory protein SMG-C, are prominent in the perinatal acini and are believed to form the intercalated ducts of the adult gland. Between 25 days and 30 days after birth, the number of Type I cells and their SMG-C immunoreactivity markedly decreased. Apoptotic cells in association with the developing intercalated ducts were labeled with the Terminal Deoxyribonucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) method. Between 25 and 40 days of age, from 50 to 80% of the apoptotic cells in cryostat sections of the SMG were closely associated with the intercalated ducts. Electron microscopy showed that the Type I cells became vacuolated, their secretory granules were reduced in size and number, and the amount of rough endoplasmic reticulum was decreased. Cellular debris resembling apoptotic bodies was phagocytosed by macrophages and adjacent intercalated duct cells. These observations suggest that the loss of Type I cells and reduction of SMG-C immunoreactivity during development of the intercalated ducts of the adult rat SMG is due, at least in part, to apoptosis.

Animals↗
PubMed1998

Efficacy of exogenous recombinant murine leptin in lean and obese 10- to 12-mo-old female CD-1 mice.

Leptin efficacy was compared in obese and lean female CD-1 mice. Body weights in these 10- to 12-mo-old mice ranged from 29.7 to 62.0 g, and leptin levels correlated with body weight. Mice from the lean and obese ends of the weight distribution were treated with daily peripheral leptin injections (1-100 mg/kg) for a 33-day period. The half-maximal effective doses for weight loss and fat reduction were shifted 0.5-0.7 log to the right for obese mice. Leptin was less efficacious at low doses (1-3 mg/kg) in obese mice but equal to or more efficacious in obese than lean mice at high doses (30-100 mg/kg). Leptin's initial effects on weight loss could be explained by appetite suppression in both groups, but its effects on fat reduction were greater in leptin-treated than pair-fed mice, particularly in the lean group. Leptin also prevented the elevations in serum corticosterone and ketones found in pair-fed lean mice. These data allow a quantitative comparison of leptin sensitivity in obese vs. lean CD-1 mice and suggest that in mice where obesity is a function of outbreeding and age, leptin sensitivity is moderately reduced. Furthermore, although appetite suppression has a clear role in leptin's effects on body weight, leptin may also have specific effects on lipid metabolism and mobilization that are different from the metabolic compensations that normally occur with food deprivation.

Adipose Tissue↗
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↗
PubMed1993

Improving the implementation of cost recovery for health: lessons from Zimbabwe.

In the current debate over health financing policy in developing countries, governments are increasingly focusing on cost recovery--having patients pay part or all of their health care costs--as a way to mobilize more resources for health, improve equity by selectively charging the wealthy, and increase efficiency by encouraging reinvestment of fee revenues into cost-effective primary care. Zimbabwe offers an important example of a country with a tradition of levying fees in government health facilities, but where enforcement became lax in the 1980s. In 1991, policymakers resolved to resuscitate and strengthen cost recovery, as part of a broader economic reform program. This paper discusses the strengths and weaknesses of Zimbabwe's cost recovery system, its potential for improvement, and the obstacles to change in revising the fee structure and billing and collection procedures. It argues that cost recovery can help to achieve Zimbabwe's health objectives, but only in conjunction with other measures to redirect public spending to essential public health and clinic care and improve the efficiency of government services. The paper finds that during the 1980s, the fee schedule became badly misaligned with actual medical care costs and created distortions in patient referral patterns. Billing and collection were also weak, because of deficiencies in personnel and information systems and lack of incentives for revenue generation. The paper concludes that if key steps were taken to raise the collections-to-billings ratio, recover fees from privately-insured patients, and adjust fees in line with medical cost inflation, recoveries could increase fourfold, from 5% to 20% of government spending for clinical care. At the same time, access to government health services for the poor could be maintained by improving exemption procedures.

Cost Sharing↗
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↗