PubMed Health⌕ Search

Biomedical subjects

J Rosenblatt

Publications and source records attributed to J Rosenblatt.

At least 19 recordsLinked to original sources
PubMed1992

Human cyclin-dependent kinase 2 is activated during the S and G2 phases of the cell cycle and associates with cyclin A.

We have analyzed the cell cycle regulation of human cyclin-dependent kinase 2 (CDK2), a protein closely related to the cell cycle-regulatory protein kinase CDC2. We find that CDK2 activity, like that of CDC2, oscillates during the cell cycle in cultured mammalian fibroblasts. Unlike CDC2 activity (which peaks during mitosis), CDK2 activity rises in late G1 or early S phase and declines during mitosis. Active S-phase CDK2 migrates in multiple large complexes on gel filtration, and CDK2 in one of these complexes is associated with cyclin A. These findings suggest that CDK2 and CDC2, in association with distinct cyclins, regulate separate functions in the mammalian cell cycle.

Amino Acid Sequence↗
PubMed1992

Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15.

We have examined the role of phosphorylation in the regulation of human cyclin-dependent kinase-2 (CDK2), a protein closely related to the cell cycle regulatory kinase CDC2. We find that CDK2 from HeLa cells contains three major tryptic phosphopeptides. Analysis of site-directed mutant proteins, expressed by transient transfection of COS cells, demonstrates that the two major phosphorylation sites are Tyr15 (Y15) and Thr160 (T160). Additional phosphorylation probably occurs on Thr14 (T14). Replacement of T160 with alanine abolishes the kinase activity of CDK2, indicating that phosphorylation at this site (as in CDC2) is required for kinase activity. Mutation of Y15 and T14 stimulates kinase activity, demonstrating that phosphorylation at these sites (as in CDC2) is inhibitory. Similarly, CDK2 is activated in vitro by dephosphorylation of Y15 and T14 by the phosphatase CDC25. Analysis of HeLa cells synchronized at various cell cycle stages indicates that CDK2 phosphorylation on T160 increases during S phase and G2, when CDK2 is most active. Phosphorylation on the inhibitory sites T14 and Y15 is also maximal during S phase and G2. Thus, the activity of a subpopulation of CDK2 molecules is inhibited at a time in the cell cycle when overall CDK2 activity is increased.

Amino Acid Sequence↗
PubMed1992

Tissue compatibility of collagen-silicone composites in a rat subcutaneous model.

Collagen-silicone composites were fabricated and tested for biocompatibility by subcutaneous implantation in rats. The silicone component consisted of addition cure or condensation cure sheets. The collagen component was either (a) a sponge layer 2 mm thick, (b) a thin film 12-20 microns thick, or (c) residual collagen bonded to or incorporated in the silicone rubber. Collagen sponges were mechanically bonded to silicone sheets, and collagen thin films and residual collagen were physically and chemically attached to epoxy-derivatized silicone sheets. Analysis of implanted samples showed that reduced capsule formation occurred around collagen sponge-silicone, compared to control silicone sheets. Only where the underlying silicone sheet, or interpenetrating silicone, was exposed to the tissue, did limited capsule formation occur. In contrast, thin capsule developed completely around silicone coated with a thin collagen film and around silicone bonded to residual collagen. Sponge-silicone composites and control silicone sheets were free of acute and chronic inflammation, except for occasional foreign body giant cells in sponge adjacent to silicone. Silicone coated with micron-thick collagen films exhibited some inflammation, but residual collagen-silicone did not. This study suggests that, to prevent capsule formation, a collagen coat must be of minimum thickness and surface coverage sufficient to prevent any contact between silicone and tissue.

Animals↗
PubMed1992

Effect of electrostatic forces on the dynamic rheological properties of injectable collagen biomaterials.

Injectable collagen is a concentrated dispersion of phase-separated collagen fibres in aqueous solution used to correct dermal contour defects through intradermal injection. The effect of electrostatic forces on the rheology of injectable collagen was studied by observation of the birefringence of collagen fibres through a polarizing microscope as well as by oscillatory rheological measurements on dispersions of varying ionic strengths (0.06-0.30). The birefringence of fibres progressively increased as ionic strength was reduced from 0.30 to 0.06. The linear viscoelastic measurements displayed a logarithmic relationship between storage (and loss) moduli and frequency over oscillation frequencies of 0.1-100 rad/s. The associated relaxation time spectra, interpreted using the theory of Kamphuis et al. for concentrated dispersions, show that collagen fibres become more flexible as ionic strength increases. This result was analysed at the molecular level from the perspective that collagen fibres are a liquid-crystalline phase of rigid rod collagen molecules which have phase-separated from solution. Electrostatic forces affect the volume fraction of water present in the collagen fibres which in turn alters the rigidity of the fibres. Flexible collagen fibre dispersions displayed emulsion-like flow properties whereas more rigid collagen fibre dispersions displayed suspension-like flow properties. Changes in fibre rigidity significantly alter the injectability of collagen dispersions which is critical in clinical performance.

Animals↗
PubMed1992

Stable isotope tracer analysis by GC-MS, including quantification of isotopomer effects.

In metabolic tracer studies it is frequently useful to infuse tracers that are differently labeled variants of the same molecule. These tracers are known as isotopomers. Analysis of the enrichment of each isotopic analogue can be accomplished by gas chromatography-mass spectrometry (GC-MS). However, the raw GC-MS data must be corrected to give the information required. This paper addresses how to transform the raw GC-MS data, consisting of relative abundance ratios at specific ion masses, into relative molar ratios of tracer and tracee molecules. Several correction factors are necessary. First, the background must be measured and corrected for, since it is always present in the sample. Second, the abundances in the spectrum of the labeled molecule are different from those in the unlabeled molecule, and this proportionality "skew" is corrected. A third correction factor accounts for the overlapping spectra of two or more isotopomers that cannot be measured independently. The final correction removes the "double vision" effect that may appear in some spectra due to the presence of (M - H)+ species.

Drug Stability↗
PubMed1991

Isotopic determination of fibronectin synthesis in humans.

Fibronectin is an opsonic protein that, among other functions, activates the reticuloendothelial system. Accurate measurement of its rate of synthesis is necessary to more fully understand its physiological role in normal and pathological conditions. We have determined the rate of fibronectin synthesis in three normal volunteers using a primed-constant infusion of 15N-glycine and 1,2-13C-leucine, and measuring the incorporation of the isotopes into the protein over 5 days of infusion. In nine additional subjects, the fractional synthetic rate (FSR) of fibronectin was calculated during a 24-hour infusion using urinary hippurate and plasma alpha-ketoisocaproic acid enrichment to represent the precursors for incorporation of labeled glycine and leucine, respectively, into fibronectin. The FSR using glycine and leucine was 1.56 +/- 0.14 and 1.29 +/- 0.04 (%/h), respectively, in the 5-day infusion study, and 1.56 +/- 0.10 versus 1.83 +/- 0.09 (%/h), respectively, in the 24-hour study. The results of the 5-day infusion of 15N-glycine justify the use of urinary hippurate to reflect the precursor enrichment for the determination of the FSR of fibronectin during a shorter (less than 24 hour) infusion period.

Carbon Isotopes↗
PubMed1991

Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NS1 proteins and the pestivirus envelope glycoproteins.

Based on the flavi- and pestivirus model of genome organization for the hepatitis C virus (HCV) (1-5), the nucleotide and deduced amino acid sequences of the putative envelope (E1) and the junction between the E1 and NS1/envelope 2 (E2) region from six different human isolates of HCV were compared with the nucleotide and predicted amino acid sequences of the prototype hepatitis C virus (HCV-1) (5). The overall percentage of nucleotide and amino acid changes among all six isolates, including HCV-1, from nucleotide 713 to 1630 (amino acid 129 to 437) was between 3 and 7%, which is comparable to that seen in some flaviviruses (6-8). An analysis of the number of nucleotide and deduced amino acid sequence changes among all six isolates and HCV-1 revealed a moderately variable domain of approximately 40 amino acids in the E1 region and a hypervariable domain (Region V) of approximately 28 amino acids, which is directly downstream from a putative signal peptide sequence, in the junction between E1 and NS1/E2. A similar hypervariable domain is not found in the C-terminus of the envelope polypeptide or in the N-terminus of the NS1 polypeptide domain of the flaviviruses. These findings suggest that the mature NS1/E2 polypeptide starts about amino acid 380 and that the NS1/E2 domain may correspond to a second envelope glycoprotein as in the case of the pestivirus. The observed heterogeneity in the putative structural proteins of HCV may have important ramifications for future vaccine development.

Amino Acid Sequence↗
PubMed1990

Detection of hepatitis C viral sequences in non-A, non-B hepatitis.

The role of hepatitis C virus (HCV) in post-transfusion non-A, non-B hepatitis (NANBH) was investigated by analysing clinical samples for both HCV RNA by cDNA/polymerase chain reaction and antibodies against C100-3 by radioimmunoassay. Of fifteen chronic NANBH patients and one patient with chronic cryptogenic liver disease, ten were positive for anti-C100-3 and seven of the ten had viral sequences in their livers. However, two patients negative for anti-C100-3 also had substantial levels of HCV RNA in their livers. In acute post-transfusion NANBH (one surgical patient and two experimentally infected chimpanzees), HCV RNA was detected in the absence of anti-C100-3. In addition, infectious plasma from a seronegative patient with acute post-transfusion NANBH and a seronegative pool of plasma from a chimpanzee with chronic post-transfusion NANBH had high levels of HCV. These findings show that anti-C100-3-positive patients with chronic post-transfusion NANBH are likely to be viraemic; confirm that antibodies to C100-3 are a marker for infectivity; and suggest that the prevalence of HCV infections may be underestimated from the frequency of antibodies to C100-3 alone.

Animals↗
PubMed1990

Early events in hepatitis C virus infection of chimpanzees.

The cytoplasmic antigen and ultrastructural changes we described previously for chimpanzees (Pan troglodytes) infected with hepatitis C virus (HCV) or with hepatitis D virus have recently been shown to be indirect measures of viral replication and appear to represent a host response to the expression or action of interferon. The time of appearance of these changes in hepatocytes during HCV infection, when compared with similar changes in hepatitis D virus infection, suggests a very early replicative phase for HCV. To investigate the early events in HCV infection, we infected two chimpanzees with HCV and obtained blood and liver biopsy samples from them daily during the first 10 days of infection. The early stage of infection with regard to HCV replication, antigen expression, and ultrastructural changes was similar in both chimpanzees. When tested by cDNA/polymerase chain reaction, HCV sequences became detectable in the serum as early as 3 days after inoculation and remained positive through the peak of aminotransferase elevations. In one chimpanzee the peak of virus production appeared to be 7 weeks after inoculation, which was coincident with rising enzyme values. The cytoplasmic antigen, detected by immunofluorescence, and ultrastructural changes, detected by electron microscopy, became positive in hepatocytes 3 and 6 days, respectively, after HCV sequences were first detected in serum. Circulating anti-HCV appeared 13 weeks and 32 weeks after inoculation, respectively, in the chimpanzees. These data indicate a very early replicative phase for HCV and a potentially long period of infectivity before the appearance of anti-HCV.

Animals↗
PubMed1990

Effect of exercise and recovery on muscle protein synthesis in human subjects.

Previous studies using indirect means to assess the response of protein metabolism to exercise have led to conflicting conclusions. Therefore, in this study we have measured the rate of muscle protein synthesis in normal volunteers at rest, at the end of 4 h of aerobic exercise (40% maximal O2 consumption), and after 4 h of recovery by determining directly the rate of incorporation of 1,2-[13C]leucine into muscle. The rate of muscle protein breakdown was assessed by 3-methylhistidine (3-MH) excretion, and total urinary nitrogen excretion was also measured. There was an insignificant increase in 3-MH excretion in exercise of 37% and a significant increase (P less than 0.05) of 85% during 4 h of recovery from exercise (0.079 +/- 0.008 vs. 0.147 +/- 0.0338 mumol.kg-1.min-1 for rest and recovery from exercise, respectively). Nonetheless, there was no effect of exercise on total nitrogen excretion. Muscle fractional synthetic rate was not different in the exercise vs. the control group at the end of exercise (0.0417 +/- 0.004 vs. 0.0477 +/- 0.010%/h for exercise vs. control), but there was a significant increase in fractional synthetic rate in the exercise group during the recovery period (0.0821 +/- 0.006 vs. 0.0654 +/- 0.012%/h for exercise vs. control, P less than 0.05). Thus we conclude that although aerobic exercise may stimulate muscle protein breakdown, this does not result in a significant depletion of muscle mass because muscle protein synthesis is stimulated in recovery.

Adult↗
PubMed1988

Cubic splines.

Explore the source record for details and available documents.

Regression Analysis↗
PubMed1988

Calculation of substrate flux using stable isotopes.

The use of stable isotope tracers to calculate substrate kinetics in humans is favored over the use of radioactive isotopes because of their greater safety and versatility. However, potential complications not met when dealing with radioactive tracers are caused by 1) the natural occurrence of the stable isotope used as a tracer and 2) the necessity to administer the tracer in an amount that cannot be treated as "massless." We therefore found it desirable to derive a theoretically valid equation for calculating the rate of appearance, Ra, of a substrate under steady-state conditions using a stable isotope tracer. This theoretically valid equation yields results that differ from those of the equations conventionally used to calculate (endogenous) Ra in steady state. Quantitative determination of the error in one of these equations revealed that for tracers commonly used in metabolic studies the error is negligible, whereas the error made in the other equation is likely to be quite high in commonly encountered situations. Finally, to allow for proper use of different definitions of isotopic enrichment that have arisen from practical considerations, we use the results derived above to determine valid equations for Ra appropriate to the two prevalent definitions.

Humans↗
PubMed1988

A more direct approach to compartmental modelling.

By first using some simple linear curve fitting method, (such as spline fitting), on data following a compartmental model, direct application of linear regression can then be made to the system of differential equations describing this data. This allows information about the parameters governing the system to be obtained from tracer and tracee measurements. It simplifies both the process of determining which parameters are estimable from various measurements, as well as the estimation process itself. Since it does not rely on knowledge of a simple closed form of the solution it has the potential to make full use of data measured over very long time periods under nonequilibrium conditions. Essentially it allows introduction of sequential estimation methods of statistics. These results can then be used to predict future substrate concentration from known substrate production, or determine substrate production from concentration measurements. A byproduct of these methods is the ability to estimate parameters in data assumed to be a finite linear combination of exponentials or sinusoids of unknown exponents or frequencies, without use of complicated nonlinear regression methods.

Animals↗