Biomedical subjects

R Strom

Publications and source records attributed to R Strom.

At least 37 records · Page 2Linked to original sources

PubMed1998

Human circular colonic smooth muscle cells possess active somatostatin receptors.

BACKGROUND & AIMS: Somatostatin alters in vivo colonic motility in different species including humans. Few data are available on a cellular basis that could explain the effects of somatostatin on human colon motility. To address these issues we studied the effects of somatostatin on isolated human circular colonic smooth muscle cells to establish whether its actions are directly or neurally mediated. METHODS: Circular smooth muscle cells were prepared by enzymatic digestion from surgical specimens of human colon (Sigma) and resuspended in HEPES buffer containing protease inhibitors. RESULTS: Cholecystokinin (1 nM), carbachol (30 nM) and KCl (20 mM) each caused a contraction of 17%, 16.5% and 15%, respectively. 1 microM of either somatostatin-14, somatostatin-28 or SMS 201-995 alone were able to produce a contraction of 5.1%, 5.7%, and 6.8%, respectively. When smooth muscle cells were preincubated with each of the above-mentioned somatostatin analogs, cholecystokinin-mediated contraction was dose-dependently inhibited only in the presence of antiproteases. The half-maximal effective concentration (EC50) for somatostatin-14, somatostatin-28 and SMS 201-995 were similar (3.5, 5.6, 3.2 nM, respectively). CONCLUSIONS: Somatostatin acts directly on human circular colonic smooth muscle cells through specific somatostatin receptors. SMS 201-995, a somatostatin receptor subtype-2 preferring analogue, shows a high affinity in inhibiting cholecystokinin-mediated contraction, suggesting the presence of somatostatin receptor subtype-2 on human circular colonic smooth muscle cells.

Aged↗

PubMed1997

Intracellular metabolism of 3'-azido-3'-deoxythymidine (AZT): a nuclear magnetic resonance study on T-lymphoblastoid cell lines with different resistance to AZT.

This paper reports the results of 31P and 1H nuclear magnetic resonance (NMR) studies on the uptake and phosphorylation of 3'-azido-3'-deoxythymidine (AZT) in the human CD4+ T-lymphoblastoid cell line CCRF-CEM (CEM-1.3) and in its AZT-resistant cell variant MT-500, isolated by prolonged culturing of CEM cells in the presence of increasing AZT concentrations. After 3 hr of incubation in the presence of 0.5 mM AZT, both AZT and its monophosphorylated form (AZT-MP) could be detected in the sensitive cell line in concentrations above the NMR detection levels. In another cell line, MOLT-4, which is less sensitive to AZT effects, the intracellular level of AZT-MP was much lower and was only slightly raised by increasing the concentration of AZT in the extracellular and intracellular compartments. In the AZT-resistant clone MT-500, characterized by a very low thymidine kinase (TK, EC 2.7.1.21) activity with respect to the parental clone, the intracellular AZT-MP concentration was below detection (<0.02 nmol/10(6) cells). Since, however, not only AZT-MP but also AZT signals failed to be detected in MT-500 extracts following cell incubation with AZT, it was concluded that a TK deficiency cannot be the exclusive mechanism of AZT resistance in these cells. The possible effects of additional mechanisms of drug resistance, such as specific AZT cell extrusion and limited permeation, are discussed, together with the new prospects offered by NMR spectroscopy to further evaluate the limiting steps for the utilization of antiretroviral nucleoside analogues.

Anti-HIV Agents↗

PubMed1997

Deltorphin transport across the blood-brain barrier.

In vivo antinociception studies demonstrate that deltorphins are opioid peptides with an unusually high blood-brain barrier penetration rate. In vitro, isolated bovine brain microvessels can take up deltorphins through a saturable nonconcentrative permeation system, which is apparently distinct from previously described systems involved in the transport of neutral amino acids or of enkephalins. Removing Na+ ions from the incubation medium decreases the carrier affinity for deltorphins (-25%), but does not affect the Vmax value of the transport. The nonselective opiate antagonist naloxone inhibits deltorphin uptake by brain microvessels, but neither the selective delta-opioid antagonist naltrindole nor a number of opioid peptides with different affinities for delta- or mu-opioid receptors compete with deltorphins for the transport. Binding studies demonstrate that mu-, delta-, and kappa-opioid receptors are undetectable in the microvessel preparation. Preloading of the microvessels with L-glutamine results in a transient stimulation of deltorphin uptake. Glutamine-accelerated deltorphin uptake correlates to the rate of glutamine efflux from the microvessels and is abolished by naloxone.

Animals↗

PubMed1997

Does poly(ADP-ribosyl)ation regulate the DNA methylation pattern?

The existence of a possible correlation between poly(ADP-ribosyl)ation and DNA methylation processes was investigated. In vivo and in vitro experiments were carried out on L929 mouse fibroblasts preincubated for 24 h with or without 3-aminobenzamide, a well-known inhibitor of poly(ADP-ribose) polymerase. Both experimental approaches evidenced a close relationship between these two important nuclear enzymatic mechanisms, suggesting that the poly(ADP-ribosyl)ated isoform of H1 histone and/or long and branched protein-free ADP-ribose polymers could act as protecting agents against full methylation of the CpG dinucleotides in genomic DNA.

Animals↗

PubMed1997

Expression of receptors for native and chemically modified low-density lipoproteins in brain microvessels.

Despite the importance of cholesterol metabolism in the central nervous system, only relatively few studies have dealt with the cerebral uptake and transport of lipids into the brain compartment. These functions are mediated by the endothelium of brain microvessels, which forms the anatomical basis of the blood-brain barrier. By a reverse transcriptase PCR study of messenger RNA expression we could show, in bovine brain microvessels, the presence of transcripts of native low-density lipoprotein receptor and of both type I and II scavenger receptors. Brain microvessels therefore appear to play an active role in the uptake of native and modified low-density lipoproteins.

Animals↗

PubMed1997

Developmental study of ultrastructural and biochemical changes in isolated chick brain microvessels.

The morphology of the endothelial junctions, the expression of the alkaline phosphatase (ALKP) and gamma-glutamyltranspeptidase (GGT) activities, and the transport systems for neutral amino acids (NAA) and for d-glucose were studied in parallel in isolated microvessels from the brains of 9-, 12-, and 21-day chick embryos and 30-day chickens using freeze-fracturing and biochemical techniques. In the 9-day embryos, the endothelium junctional plasma membranes show alignments of discrete intramembrane particles (Imps) on the replica P-faces. In the 12-day embryos, the junctional membranes show short fibrils of loosely interconnected fused Imps. In the 21-day embryos, the junction is characterized by simple networks, and in the 30-day chickens, by very dense meshworks of continuous, fibrillary strands. In early embryonic life, the ALKP activity is expressed at high levels, more markedly in the microvessels than in the whole brain. ALKP progressively declines in later embryos and even more in adulthood, when the activity is fivefold higher in the microvessels than in the whole brain. No GGT activity is expressed in either embryonic or adult microvessels. The transport systems for NAA and for d-glucose are most active in 12-day embryonic microvessels, and progressively less expressed by the 21st day and in the adult. The results taken as a whole suggest a close relationship between the morphological and the metabolic maturation of the endothelial barrier of cerebral vessels.

Alkaline Phosphatase↗

PubMed1997

Contribution of factor VII genotype to activated FVII levels. Differences in genotype frequencies between northern and southern European populations.

The relationship between coagulation factor VII (FVII) levels in plasma and FVII genotypes, determined by three polymorphisms (5'F7, IVS7, and 353R/Q), were studied in 500 control subjects enrolled in European multicenter study. The selection of particular FVII genotypes and the analysis of variance clearly indicated the independent contribution of a single 5'F7 insertion (A2) or 353Q (M2) allele to lowering plasma levels of activated FVII (FVIIa) (by a mean 25%). The M2 allele alone was found to make a major contribution to the genetically determined component of the FVIIa levels. Genotypes associated with low FVII levels were significantly rarer in the northern part of Europe (Oslo) than in the southern part (Rome, Murcia). The contribution made by the FVII genotype to the total variance of FVIIa levels was higher (30%) than that made to either FVII activity (25%) or FVII antigen (12%). Subjects with different FVII genotypes showed up to fivefold differences in mean FVIIa values, thus allowing attribution of a substantial part of the considerable interindividual variation to genetic variation, which may be of assistance in the interpretation of FVIIa levels on an individual basis. When FVII levels were adjusted by age and by triglyceride levels, the contribution of FVII genotypes to the FVII phenotypic variance was virtually unchanged. Taken together, these data indicate that in healthy control subjects the FVII genotype is a major predictor of plasma FVIIa levels and would support further study on the role of FVII genetic components in the development of cardiovascular disease.

Adult↗

PubMed1997

Building a theory of grandparent development.

A theory of grandparent development is described that includes the formulation of assumptions, goals for guiding instruction, curriculum design, and a measurement tool to assess learning needs and evaluate the effects of educational intervention. Cultural considerations are illustrated by a three generational study of African-American (n = 777), Caucasian (n = 1086), and Hispanic (n = 672) subjects who were administered the Grandparent Strengths and Needs Inventory. Perceptions of each generation about the attitudes and behaviors of grandparents were examined with multivariate analysis of variance and post-hoc comparisons to analyze the scores, confirm results, and facilitate interpretation of findings. Results showed significant differences between generations and within cultures. Each generation identified elements of grandparent success as well as issues that deserve consideration in building educational programs.

Attitude↗

PubMed1996

AZT-induced hypermethylation of human thymidine kinase gene in the absence of total DNA hypermethylation.

Genome-wide DNA hypermethylation induced by 3'-azido-3'-deoxythymidine (AZT) has been suggested to be involved in the development of AZT resistance. We used a CD4 T-lymphoblastoid CEM line and its AZT-resistant MT500 variant with reduced thymidine kinase activity. Evaluation of total DNA methylation, after AZT treatment, failed to show an increase in the 5-methylcytosine level in both parental and AZT-resistant cells. The effect was instead observed at a more specific gene level, on the three HpaII sites present in exon 1 of the human thymidine kinase gene. These results suggest that AZT treatment can induce site-specific hypermethylation, even in the absence of a more general DNA hypermethylating effect.

5-Methylcytosine↗

PubMed1996

H1-H1 cross-linking efficiency depends on genomic DNA methylation.

Oligonucleosomal DNA preparations from condensed-inactive chromatin were examined, before and after artificial methylation by bacterial SssI methylase, for their ability to allow cooperative H1-H1 interactions under conditions of different ionic strength. Our results support the conclusion that, within the highly methylated genomic DNA, there are some CpG's whose unmethylated state is critical for chromatin folding. Circular dichroism spectra indicate that artificial overmethylation of native oligonucleosomal DNA reduces its efficiency in inducing an ordered conformation of H1 histone. Temperature melting profiles confirm on the other hand that the native and the artificially overmethylated forms of oligonucleosomal DNA are both able to bind H1 histone.

Circular Dichroism↗

PubMed1996

Different effects of histone H1 on de novo DNA methylation in vitro depend on both the DNA base composition and the DNA methyltransferase.

We have characterized the inhibition exerted by histone H1 on the activity of human placenta DNA (cytosine-5-)-methyltransferase. Our experiments demonstrate that the extent of inhibition depends on the DNA base composition, AT-rich substrates being more severely affected than GC-rich substrates and CpG-rich islands. With bacterial SssI methylase, the effect is completely reversed since its activity on AT-rich substrates undergoes a 4-5-fold stimulation upon the addition of H1. Poly(L-lysine) mimicks H1 effects, suggesting an essential role of lysine residues in both the inhibitory and stimulatory effects of H1. By comparison of the different behaviors of the two enzymes, the inhibitory effect over the eukaryotic enzyme might be accounted for by hypothesizing a competition between minor groove-binding motifs (SPKK-like) present in placenta methylase as well as in histone H1.

Base Composition↗

PubMed1996

Specific inhibitory effect of H1e histone somatic variant on in vitro DNA-methylation process.

H1e and H1c histone variants were purified from mouse L929 fibroblasts using a reverse phase HPLC, and their effect on in vitro DNA methylation was investigated, together with their ability to bind unmethylated or methylated CpG-rich 44bp oligonucleotides. In a "physiological" range of H1:DNA ratios only H1e, at variance from H1c, was found to cause a marked inhibition of in vitro enzymic DNA methylation. It was also shown that both variants have a similar affinity in binding a methylated CpG-rich oligonucleotide, but that the binding to the same oligonucleotide in the unmethylated form occurs preferentially with H1e rather than with H1c. H1e is therefore likely to be directly involved in maintaining CpG-rich sequences in the unmethylated state.

Animals↗

PubMed1996

Serum kappa and lambda light immunoglobulin chains in cynomolgus macaques (Macaca fascicularis) during the first twenty months of age.

Growth is coupled to physiological modifications of the immune system which reaches the functional capabilities according to age-related milestones. Few data are available on the circulating immunoglobulin levels and no data exist on total immunoglobulin light chains in infant macaques. Therefore we studied by a nephelometric assay, the age-dependent variations of kappa and lambda serum light chains in the experimental animal model Macaca fascicularis during the first 20 months of age. Both kappa and lambda showed a marked increase in their concentrations during the first 7-8 months of life. Infants' light chain levels were anyhow significantly lower than those of the nursing dams and of the control group, never attaining, even at the 20th month, the same concentration as the adult, although the value of the kappa/lambda ratio was apparently the same.

Age Factors↗

PubMed1996

Erythrocyte glutathione determination in the diagnosis of glucose-6-phosphate dehydrogenase deficiency.

The levels of reduced glutathione in severe glucose-6-phosphate dehydrogenase deficient red blood cells, were found, when assayed immediately after blood withdrawing, almost equal to those of normal human erythrocytes. On the contrary, if the blood samples were stored at 4 degrees C for 4 days (or more) either as whole blood or as washed erythrocytes with or without glucose, the glutathione concentration of the enzyme-deficient cells decreased to half the initial value. After a mild t-butylhydroperoxide (t-BHP) treatment, only glucose-6-phosphate dehydrogenase deficient red cells exhibited a drastic decrease of glutathione, the normal ones being almost unaffected. If t-BHP-treated erythrocytes were incubated in the presence of glucose or of oxidized glutathione, a full recovery of the inital glutathione concentration was detected only in normal samples. Glucose could not be replaced by any other sugar. When the oxidative stress was induced by addition of methylene blue (MB), the behaviour was similar but less marked, it was however impossible, in this case, to restore the normal glutathione levels, through a subsequent incubation of the MB-treated erythrocytes with oxidized glutathione. This discrepancy can be explained by the finding that a marked inhibition of glutathione reductase was observed in MB-treated erythrocytes, while t-BHP exposure had no direct effect on this enzyme.

Erythrocytes↗

PubMed1996

Grandparents in Taiwan: a three-generational study.

Grandparents in the Republic of China want to remain influential, but social policy has not provided them with education to fulfill their changing role. The performance of grandparents was examined to determine suitable content for an intervention program. A sample of 751 non-consanguineous participants from urban and rural Taiwan included 234 grandparents, 241 parents, and 276 grandchildren. Each generation was administered a separate version of the Grandparent Strengths and Needs Inventory that was translated into Mandarin. Respondents identified favorable qualities of grandparents as well as aspects of their relationships in which growth was necessary. Multivariate analysis of variance, univariate analysis of variance, Scheffé and t-tests were used to analyze scores, confirm results, and facilitate interpretation. All three generations described aspects of grandparent success and specific realms of learning they should acquire to become more effective. Significant main effects that influenced responses about grandparent performance were generation, gender of grandchild, age of grandchild, frequency of grandchild care by grandparent, generations living together, and amount of time grandparent and grandchild spent together. Considerations were recommended to improve behavior of grandparents and guide the development of educational programs for them.

Aged↗

PubMed1996

Black grandparents: curriculum development.

The performance of Black grandparents was examined to determine the most suitable content for an educational program intended to assist them in their role. A sample of 626 non-consanguineous subjects included 204 grandparents, 128 parents, and 294 grandchildren. Each generation group completed a separate version of the Grandparent Strengths and Needs Inventory to identify the favorable qualities of grandparents and aspects of their relationships in which further growth was necessary. A combination of multivariate analysis of variance procedures, univariate analysis of variance tests, and t-tests were used to analyze scores, confirm results, and assist with interpretation of data. All groups described aspects of grandparents success and made known specific realms of learning that grandparents should acquire in order to become more effective. Analysis of the responses revealed significant main effects for generation, gender of grandchild, age of grandchild, and amount of time that grandparent and grandchild spent together. Considerations were identified for improving grandparent influence and guiding the development of grandparent education programs.

Adolescent↗

PubMed1996

Simultaneous myogenin expression and overall DNA hypomethylation promote in vitro myoblast differentiation.

Two clones of the L5 myoblast line (M6 and the fusion-defective M12) were examined for the expression of myogenin, one of the regulatory genes involved in the regulation of differentiation to myofibers after treatment with 3-deazaadenosine, a metabolic inhibitor of methyl transfer reactions. Cultures treated with 3-deazaadenosine showed, using Northern blot hybridization, a conspicuous increase in myogenin expression, which in clone M6 correlated to the extent of cell differentiation under fusing conditions but was evident also in growth medium, although the drug was unable to start the myogenic program. We also tested the extent of total DNA methylation to verify whether the activation of the regulatory cascade could be correlated to the decrease of the overall number of 5-methylcytosines present in the genome. The results show that the loss of 5-methylcytosine from newly synthesized DNA, but not from preexisting DNA, is evident in fusing conditions and enhanced by 3-deazaadenosine. It appears that there is a positive correlation between the passive demethylation of newly synthesized DNA, the activation of the myogenin gene by demethylation, and the differentiation of myoblasts. However, in fusing conditions, the defective clone M12, although it is able to express myogenin and its DNA is hypomethylated, fuses only in the presence of 3-deazaadenosine, suggesting some alternative way of induction.

Animals↗

PubMed1995

DNA binding and methyl transfer catalysed by mouse DNA methyltransferase.

By using a purified fraction of mouse DNA methyltransferase we have shown, by gel-retardation analysis, that the enzyme forms a low-affinity complex preferentially with hemimethylated DNA; the complexes formed with unmethylated or with fully methylated DNA are of even lower affinity, and only very weak interaction occurs with DNA lacking CG dinucleotides. Interaction is inhibited by N-ethylmaleimide. Methyl transfer from S-adenosyl-methionine is associated with the release of the fully methylated product from the complex. Complexes formed with the intact enzyme are extremely large, but limited trypsin treatment allows a major complex to enter the gel. DNA binding is not inhibited by this limited proteolysis of the native enzyme.

Animals↗