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Abstract: The objective of this study was to study was to compare chromium (Cr), nickel-chromium (Ni-Cr), and control groups for sister chromatid exchange (SCE) in lymphocytes to obtain an understanding of the mutagenic effect of Cr(VI) in humans. Subjects totaled 91 persons from the 3 groups, including 14 Cr and 34 Ni-Cr electroplating workers and 43 control group members. Results showed that blood and urine Cr concentrations were highest among Cr workers (11.39 μg/l, 14.7 μg/g creatinine), next highest among Ni-Cr workers (5.28 μg/l, 6.2 μg/g creatinine), and lowest among the control group (2.36 μg/l, NA). After adjustment for smoking habits, SCE/cell values were 10.6, 9.4, and 8.3 for Cr workers, Ni-Cr workers, and controls, respectively. A synergetic effect was shown on HFC (high-frequency cells) percentages for Cr workers who also smoked. Odds ratios were 31.78 and 3.66 that Cr and Ni-Cr workers would have higher HFC percentages than the control group, respectively. The authors conclude that SCE in lymphocytes is useful for evaluation of the biological effects of environmental mutagens.

We have evaluated the antiemetic effect of i.v. dexamethasone compared with saline in the prevention of nausea and vomiting after laparoscopic cholecystectomy. We studied 90 patients requiring general anaesthesia for laparoscopic cholecystectomy, in a randomized, double-blind, placebo-controlled study. The dexamethasone group (n = 45) received dexamethasone 8 mg i.v. and the saline group received saline 2 ml i.v. at induction of anaesthesia. Anaesthesia was maintained with isoflurane in oxygen. We found that 10% of patients in the dexamethasone group compared with 34% in the saline group reported vomiting (P < 0.05). Of note, the total incidence of nausea and vomiting was 23% in the dexamethasone group and 63% in the saline group (P < 0.001). We conclude that dexamethasone 8 mg significantly decreased the incidence of nausea and vomiting after laparoscopic cholecystectomy.

Members of the JAK family of protein tyrosine kinase (PTK) proteins are required for the transmission of signals from a variety of cell surface receptors, particularly those of the cytokine receptor family. JAK function has been implicated in hematopoiesis and regulation of the immune system, and recent data suggest that the vertebrate JAK2gene may play a role in leukemia. We have isolated and characterizedjak cDNAs from the zebrafish Danio rerio. The zebrafish genome possesses 2 jak2 genes that occupy paralogous chromosome segments in the zebrafish genome, and these segments conserve syntenic relationships with orthologous genes in mammalian genomes, suggesting an ancient duplication in the zebrafish lineage. The jak2a gene is expressed at high levels in erythroid precursors of primitive and definitive waves and at a lower level in early central nervous system and developing fin buds. jak2b is expressed in the developing lens and nephritic ducts, but not in hematopoietic tissue. The expression of jak2a was examined in hematopoietic mutants and found to be disrupted in clocheand spadetail, suggesting an early role in hematopoiesis. Taken together with recent gene knockout data in the mouse, we suggest that jak2a may be functionally equivalent to mammalianJak2, with a role in early erythropoiesis.

DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2'deoxyuridine, which produces DNA dsb, signaled de novo ceramide synthesis by post-translational activation of ceramide synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.

Abstract: In this paper the Fourier series method is introduced to obtain the solution for the displacement response of rectangular plates subjected to a blast loading. Applying the Fourier series method, the von Karman nonlinear equations of plates are transformed into Duffing type nonlinear ordinary differential equations and then solved by adopting the Lindstedt-Poincar'e perturbation method. The complete analysis of square plates was carried out for all-clamped edge conditions. The results of deflection history and stress resultants which uses one perturbation term sufficiently correlated with results coming from both related literature as well as the finite element method.

Silicon carbon nitride films have been successfully synthesized at a temperature below 100°C from an adenine(C5N5H5)-silicon-mixed target sputtered by Ar ion beam. The effects of Ar ion sputtering voltage, area ratio of Si to adenine in the mixed target and nitrogen atom addition during deposition on the film growth are investigated. XPS, XRD, and ellipsometry were employed to characterize the composition, chemical bonding, structure, and optical property of the films. The growth characteristic and film properties of the silicon carbon nitride films are also compared with those of the carbon nitride films deposited from an adenine target to elucidate the effect of silicon incorporation. The silicon content of the resultant films increased significantly as the area ratio of Si to adenine increased, whereas a higher Ar ion voltage led to a lower level of silicon incorporation, presumably due to differential sputtering yield of Si and adenine. XPS chemical state analysis revealed multiple bonding structures for every element in the SiCN films, of which possible implications are given. XRD studies showed that only amorphous films for Si-rich SiCN were obtained, while the films with low Si incorporation and deposited at high Ar ion beam voltage contained nanocrystallites. Furthermore, the refractive index of the SiCN films increased with increasing silicon content. The appearance of the nanocrystalline structure in the films led to a reduction in the refractive index.

Predicting metabolic fluxes of a genetically engineered organism is an important step toward rational pathway design. However, because of various regulatory mechanisms, which are complex, often ill-characterized, and sometimes undiscovered, predicting metabolic fluxes using kinetic simulation is difficult. We propose to incorporate regulatory constraints in flux calculation to allow prediction of the steady-state fluxes without complete kinetics. The regulatory constraint, in its linear form, is derived from the dynamic metabolic control theory and involves the flux control coefficients. It is shown that with these constraints, the responses to metabolic perturbation can be predicted. Conversely, the regulatory constraints and the control coefficients can be determined by comparing the experimental data with the prediction. Therefore, this approach may offer a practical direction toward prediction of fluxes for metabolically engineered organisms.

Human centromeres contain large arrays of alpha-satellite DNA that are thought to provide centromere function. The arrays show size and sequence variation, but the extent to which extremely low levels of this DNA can occur on normal centromeres is unclear. Using a set of chromosome-specific alpha-satellite probes for each of the human chromosomes, we performed interphase fluorescence in situ hybridization (FISH) in a population-screening study. Our results demonstrate that extreme reduction of chromosome-specific alpha satellite is unusually common in chromosome 21 (screened with the alphaRI probe), with a prevalence of 3.70%, compared to < or =0.12% for each of chromosomes 13 and 17, and 0% for the other chromosomes. No analphoid centromere was identified in >17,000 morphologically normal chromosomes studied. All of the low-alphoid centromeres are fully functional as indicated by their mitotic stability and binding to centromere proteins CENP-B, CENP-C, and CENP-E. Sensitive metaphase FISH analysis of the low-alphoid chromosome 21 centromeres established the presence of residual alphaRI as well as other non-alphaRI alpha-satellite DNA suggesting that centromere function may be provided by (1) the residual alphaRI DNA, (2) other non-alphaRI alpha-satellite sequences, (3) a combination of 1 and 2, or (4) an activated neocentromere DNA. The low-alphoid centromeres, in particular those of chromosome 21, should provide unique opportunities for the study of the evolution and the minimal DNA requirement of the human centromere.

AbstractVoronoi polygons (VP) has been successfully employed for providing microstructural information to many multi-particle assemblies. This work has, however, highlighted the difficulties of employing Voronoi polygons (VP) to identify channels in a two-dimensional, multi-particle assembly. We noted that the VP area distribution is a good channel identifier as the constituent particles on the 2-D surface is only slightly perturbed from a crystal alignment. The asphericity parameter, which has been proposed as a superior index for microstructural descriptor, fails to detect the channel existing in a staggered lattice. Furthermore, the capability of VP in detecting channels decays markedly as the packing order decreases. Limitations of using VP had been addressed.

We have deduced the entire 1,370-amino-acid sequence of the human insulin receptor precursor from a single complementary DNA clone. The precursor starts with a 27-amino-acid signal sequence, followed by the receptor α-subunit, a precursor processing enzyme cleavage site, then the β-subunit containing a single 23-amino-acid transmembrane sequence. There are sequence homologies to human epidermal growth factor receptor and the members of the src family of oncogene products.

Antibody genes are assembled from a series of germ-line gene segments that are juxtaposed during the maturation of B lymphocytes. Although diversification of the adult antibody repertoire results in large part from the combinatorial joining of these gene segments, a restricted set of antibody heavy chain variable (VH), diversity (DH), and joining (JH) region gene segments appears preferentially in the human fetal repertoire. We report here that one of these early-expressed VH elements (termed VH6) is the most 3' VH gene segment, positioned 77 kilobases on the 5' side of the JH locus and immediately adjacent to a set of previously described DH sequences. In addition to providing a physical map linking human VH, DH, and JH elements, these results support the view that the programmed development of the antibody VH repertoire is determined in part by the chromosomal position of these gene segments.

Lipopolysaccharide (LPS), a causal agent of sepsis, has been shown to induce systemic nitric oxide (NO) synthesis through complex mechanisms. However, the effect of LPS on endothelial cells is incompletely understood. To investigate the mechanism by which LPS influences the release of NO from endothelial cells, the effect of this compound on endothelial constitutive nitric oxide synthase (ecNOS) was studied in cultured bovine coronary venular endothelial cells. Western and Northern analyses showed that LPS decreased ecNOS expression at the protein and mRNA levels in a time-dependent and dose-responsive manner. Concurrent treatment of the endothelial cells with LPS and a transcription inhibitor, actinomycin D, resulted in decreased ecNOS mRNA within 8 hours. In contrast, treatment with actinomycin D had only a relatively insignificant effect on the ecNOS transcript level. This result suggests that the reduction of ecNOS by LPS resulted from an increased degradation rate of its transcript.

Chromophore-assisted laser inactivation (CALI) is a technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions and an understanding of its mechanism is required for its judicious application. We report here that CALI is not mediated by photoinduced thermal denaturation but by photogenerated free radicals. Thermal diffusion calculations suggest that the temperature changes resulting from CALI are too small to cause thermal denaturation, and Arrhenius plots of CALI are inconsistent with a photothermal mechanism. CALI shows an energy dose reciprocity above a threshold and can be inhibited by free-radical quenchers, thus demonstrating a photochemical mechanism of protein inactivation. The type of quenchers that are effective in inhibiting CALI indicates that the active species is a hydrogen abstractor which is not derived from molecular oxygen. We suggest that the active free-radical species is the hydroxyl radical and its very short lifetime explains the spatial specificity of CALI such that half-maximal damage is effected within 15 A from the dye moiety and no significant damage occurs at 34 A. The data are consistent with free-radical formation resulting from a sequential two-photon process.

Critical to policy planning for a program of national health insurance or a National Health Service is an understanding of the strengths and weaknesses of current federally-supported health programs. One program which has been subjected to criticism is Medicaid. A major problem facing Medicaid is the high proportion of physicians who refuse to participate in the program, thus preventing the target population from obtaining access to medical care. A telephone survey was conducted to assess the attitudes and behavior of a stratified random sample of physicians regarding their participation in a Medicaid program. Physicians were asked to identify major advantages and disadvantages of the program to individual patients, to society and to their medical practice. Non-participants were asked to identify the major reasons why they did not participate in the program. Medical specialty was the only significant demographic determinant of participation. Inadequate reimbursement, excessive paperwork, patient abuses of the program and bureaucratic complexity were among the most prominent factors contributing to nonparticipation. Implications of these findings are discussed with respect to the role of primary care providers' perceptions in the planning of future national health programs.

Phosphorylation of CD45, a transmembrane protein-tyrosine phosphatase (PTPase), has been proposed to mediate docking of signaling proteins and to modulate PTPase activity. To study the role of phosphorylation in CD45, in vivo phosphorylation sites of CD45 from 70Z/3.12 cells were identified using 32P labeling, trypsin digestion, two-dimensional peptide mapping, high performance liquid chromatography, phosphoamino acid analysis, matrix-assisted laser desorption/ionization mass spectrometry, and specific enzymatic degradation. Eight phosphopeptides, a through h, were isolated and four phosphorylation sites were identified. All four phosphorylation sites were in the membrane-distal PTPase domain (D2) and the C-terminal tail and none were in the membrane-proximal PTPase domain (D1). One site, Ser(P)939 peptide h, was in the D2 domain and, by comparison to the three-dimensional structure of PTP1B, is predicted to lie at the apex of the substrate binding loop. Ser939 was the only in vitro phosphorylation site for protein kinase C among the phosphorylation sites identified. Four of the C-terminal peptides identified (d, e, f, and g) spanned the same sequence and were derived from the same phosphorylation site in the C-terminal tail, Ser1204. Peptide a was derived from the intact C terminus and comprised a mixture of monophosphorylated peptides containing either Ser(P)1248 or Thr(P)1246. Knowledge of the precise phosphorylation sites of CD45 will lead to the design of experiments to define the role of phosphorylation in PTPase activity and in signaling.

This paper, a continuation of our previous work, is a presentation of the effects of slight deformations of both the coating film and the drug matrix on the concentration distributions and the slow release characteristics of a coated drug particle. The coated particle shape, in practice, is not perfectly spherical. With restriction of the deformation shape functions considered, it is noted that if the deformation is slight or if only the average release rate is of interest, the effects of shape deformation are secondary. Otherwise, the shape deformation can have a significant effect on the concentration profiles and/or the release rate from different parts of the particle surface. The deformations of both coating film and the drug matrix could introduce a greater deviation from the spherical case than those considering only coating film or drug matrix shape deformation.

A transient model considering the emission rate of volatile and semivolatile organic compounds from an intermittently aerated bioreactor is developed and solved analytically. If the aeration time is relatively short, and/or the overall mass transfer resistance is high, a large deviation can occur from the pseudo-steady-state mass transfer model by Chern and Yu (Chern, J.-M.; Yu, C.-F. Ind. Eng. Chem. Res. 1995, 34, 2634) or from the conventional ASCE standard model by Matter-Müller et al. (Matter-Müller, C.; Gujer, W.; Giger, W. Water Res. 1981, 15, 1271).

Flux Control Coefficients have been used in the analysis of metabolic regulation for quantifying the effect of an enzyme on the overall steady-state flux. However, the experimental determination of these coefficients is very time-consuming, involving either determining the individual enzyme kinetics or perturbing the enzyme activity by genetic or other means. We developed a methodology that enables the determination of the Flux Control Coefficients from transient metabolite concentrations without knowing kinetic parameters. The transient states can be generated by changing the incubation conditions or adding the initial substrate. This approach is suitable for investigating metabolic regulation in vivo or multiple enzyme systems in vitro. It is particularly helpful if used in conjunction with n.m.r. measurements. The approach is based on a relationship between transient metabolite concentrations and the Flux Control Coefficients. The methodology has been improved from our previous results, and it is illustrated by three examples with simple pathway topologies.

The safety and biochemical effects of AL 1576 (HOE 483), a recently developed aldose reductase inhibitor, were evaluated. In a double‐blind, placebo‐controlled, clinical trial, AL 1576 (HOE 483) was administered to diabetic patients for the first time. Four single, orally administered dose levels were tested, (2, 5,10, and 20 mg). No clinically important adverse effects were seen in any of the patients. AL 1576 (HOE 433) suppressed red blood cell (RBC) sorbitol concentrations in a dose‐related fashion. Also found were statistically significant inverse correlations between the plasma drug concentration and both RBC sorbitol concentrations as well as RBC sorbitol/serum glucose ratios. In single doses up to 20 mg, AL 1576 (HOE 433) is well tolerated and decreases BBC sorbitol, a biochemical marker of pharmacologic activity, in diabetic patients.

SCL/Tal-1 is a transcription factor necessary for hematopoietic stem cell differentiation. Although SCL is also expressed in endothelial and neural progenitors, SCL function in these cells remains unknown. In the zebrafish mutant cloche (clo), SCL expression is nearly abolished in hematopoietic and vascular tissues. Correspondingly, it was shown previously that clo fails to differentiate blood and angioblasts. Genetic analysis demonstrates that the clo mutation is not linked to the SCL locus. Forced expression of SCL in clo embryos rescues the blood and vascular defects, suggesting that SCL acts downstream of clo to specify hematopoietic and vascular differentiation.

The swelling equilibrium and diffusion kinetics in various solvents of the maleimide-terminated polyurethanes (UBMIs) and of the triol and tetraol-crosslinked polyurethanes (PU) were studied. The polymer volume fraction of the UBMIs at swelling equilibrium is much higher than that of the tetraol-crosslinked PU networks for the same type of polyol used in the PU. It was explained by the high functionality of the UBMIs produced in the network structure. Furthermore, the molecular weight between crosslinks (Mc) has been calculated from the swelling model and the results exhibit good agreement with the proposed network structure. The early time sorption kinetic data were obtained to investigate the diffusion mechanism of the solvent in the networks. The solubility, diffusion coefficients, and permeability of the solvent in UBMI networks were found to be lower than in the multiol-crosslinked PU networks. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35: 1747–1755, 1997

Aromatic metabolites in Escherichia coli and other microorganisms are derived from two common precursors: phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). During growth on glucose, the levels of both E4P and PEP are insufficient for high throughput of aromatics because of the low carbon flux through the pentose pathway and the use of PEP in the phosphotransferase system. It has been shown that transketolase and PEP synthase are effective in relieving this limitation and promoting high throughput of aromatics. To determine whether transaldolase, another E4P-producing enzyme, is also a limiting factor in directing carbon flux to the aromatic pathway, E. coli transaldolase gene (tal) was cloned and overexpressed in an aroB strain which excretes 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), the first intermediate in the aromatic pathway. We found that overexpression of transaldolase did significantly increase the production of DAHP from glucose. This result further supports the contention that the supply of E4P is limiting when glucose is the carbon source. However, overexpression of transaldolase in strains which already overexpress transketolase did not show a further increase in production of aromatics. This result was attributed to the saturation of E4P supply when TktA was overexpressed. The flux control of DAHP production was discussed on the basis of Metabolic Control Analysis. © 1997 John Wiley & Sons, Inc.

It is well-known that Escherichia coli grows more slowly on gluconeogenic carbon sources than on glucose. This phenomenon has been attributed to either energy or monomer limitation. To investigate this problem further, we varied the expression levels of pck, encoding phosphoenolpyruvate carboxykinase (Pck), and pps, encoding phosphoenolpyruvate synthase (Pps). We found that the growth rates of E. coli in minimal medium supplemented with succinate and with pyruvate are limited by the levels of Pck and Pps, respectively. Optimal overexpression of pck or pps increases the unrestricted growth rates on succinate and on pyruvate, respectively, to the same level attained by the wild-type growth rate on glycerol. Since Pps is needed to supply precursors for biosyntheses, we conclude that E. coli growing on pyruvate is limited by monomer supply. However, because pck is required both for biosyntheses and catabolism for cells growing on succinate, it is possible that growth on succinate is limited by both monomer and energy supplies. The growth yield with respect to oxygen remains approximately constant, even though the overproduction of these enzymes enhances gluconeogenic growth. It appears that the constant yield for oxygen is characteristic of efficient growth on a particular substrate and that the yield is already optimal for wild-type strains. Further increases in either Pck or Pps above the optimal levels become growth inhibitory, and the growth yield for oxygen is reduced, indicating less efficient growth.

The determination of intracellular fluxes from the measurement of extracellular rates, a technique known as flux analysis, has been used successfully on several systems important to biotechnology. However, calculation of intracellular fluxes does not directly suggest ways to improve the performance of the cell. Here, we introduce a variation of this analysis, termed inverse flux analysis, which allows the prediction of the flux distribution as a function of manipulable internal fluxes. This approach is applied to analyze the acetate excretion problem commonly observed in aerobic Escherichia colicultures. The effect of manipulating each flux is quantified by a flux distribution sensitivity coefficient, which is dependent on the stoichiometry. For the acetate excretion problem, our results suggest that the anaplerotic pathways, including the reactions catalyzed by phosphoenolpyruvate carboxylase and the glyoxylate bypass are the best choices for manipulating acetate excretion in E.coli. Increasing the anaplerotic flux will decrease acetate production while increasing the growth yield.

Determination of the control coefficients allows the identification of rate‐controlling steps in a reaction system. However, the measurement of the flux control coefficients in a biochemical system is not a trivial task, except for some special cases. We have developed a theoretical basis for the direct determination of these coefficients from dynamic responses. In order to show the validity of this methodology experimentally, the dynamic approach is applied to an in vitro reconstituted partial glycolytic pathway to determine the flux control coefficients of hexokinase and phosphofructokinase. It is shown that the dynamic approach gives consistent results, which agree well with values obtained by the direct enzyme titration method. The detailed procedure and potential applications to other systems, such as immobilized enzyme or cell reactors, are discussed. © 1993 Wiley & Sons, Inc.

Vascular endothelial cell injury or activation by lipopolysaccharide (LPS) plays an important role in the pathogenesis of endotoxin shock. However, the effect of LPS on NO production from vascular endothelial cells (ECs) is incompletely understood. In this study, bovine coronary venular ECs were treated with LPS and the release of NO and expression of the endothelial NO synthase (ecNOS) were examined. We found that the ecNOS activity is transiently enhanced by LPS within the time scale of about 10 h due to the interplay between two LPS-induced mechanisms. Within the first 10 h of LPS treatment, the specific activity of ecNOS is increased by a post-translational modification mediated through a protein tyrosine kinase cascade. After about 10 h of treatment, however, LPS destabilizes the transcript of ecNOS and thus decreased the expression level and total activity.

The methodology previously developed for determining the Flux Control Coefficients [Delgado & Liao (1992) Biochem. J. 282, 919-927] is extended to the calculation of metabolite Concentration Control Coefficients. It is shown that the transient metabolite concentrations are related by a few algebraic equations, attributed to mass balance, stoichiometric constraints, quasi-equilibrium or quasi-steady states, and kinetic regulations. The coefficients in these relations can be estimated using linear regression, and can be used to calculate the Control Coefficients. The theoretical basis and two examples are discussed. Although the methodology is derived based on the linear approximation of enzyme kinetics, it yields reasonably good estimates of the Control Coefficients for systems with non-linear kinetics.