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Biomedical subjects

S A Little

Publications and source records attributed to S A Little.

At least 55 records · Page 3Linked to original sources
PubMed1991

Developmental regulation of heat shock protein synthesis and HSP 70 RNA accumulation during postimplantation rat embryogenesis.

Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to an in vitro heat shock of 43 degrees C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage at the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit an increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43 degrees C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages).

Animals↗
PubMed1991

Organization of rat mesenteric artery after removal of cells of extracellular matrix components.

Rat mesenteric arteries, perfusion fixed in relaxed or contracted conditions, were digested with acid and elastase, bleach (sodium hypochlorite), or alkali to selectively remove collagen, elastin, or cells. Scanning electron microscopy was used to study the three-dimensional organization of the remaining cells or extracellular components. Smooth muscle cells of the tunica media were elongated and circumferentially oriented. Superior mesenteric artery cells had an irregular surface with numerous projections and some ends were forked. Small mesenteric artery cells were spindle shaped with longitudinal surface ridges, and showed extensive corrugations upon contraction. Elastin was present both as laminae and as an interconnected fibrous meshwork. Collagen was arranged in an irregular network of individual fibrils and small bundles of fibrils that formed nests around the cells in both arteries. This irregular arrangement persisted, with no apparent reordering or loss of order, upon contraction. The lack of an ordered arrangement or specialized organization at the cell ends suggests mechanical coupling of the cells to elastin or collagen throughout the length of the cell, allowing for force transmission in a number of directions. The tunica media is thus a "composite" material consisting of cells, elastin, and collagen. The isotropic network of fibers is well suited for transmitting the shearing forces placed on it by contraction of smooth muscle cells and by pressure-induced loading.

Animals↗
PubMed1990

Measurement of airborne mite antigen in homes of asthmatic children.

The airborne concentration of major house dust mite antigen Der p1 was measured by low volume sampling (2 litres/min) in the homes of 68 allergic, asthmatic children. The presence of detectable airborne antigen was strongly associated with sensitivity to the mite, whereas there was no significant relation between sensitivity and the previously recommended threshold level of 2 micrograms Der p1 per g carpet dust. There was a significant association with lower threshold levels in carpet dust (0.5 microgram/g) but at no level was the association as strong as that with air measurements. Concentrations of airborne antigen were higher in rooms with wool carpets than in those with synthetic carpets or hard floors, but there was no significant difference between the dust levels of Der p1 in the two carpet types. Air sampling is a more appropriate method of assessing antigen exposure than dust sampling for asthmatic patients.

Adolescent↗
PubMed1990

Effects of temperature and buffer composition on calcium sequestration by sarcoplasmic reticulum and plasma membrane of rabbit renal artery.

45Ca electron microscopic autoradiography was used to examine the effects of buffer composition and temperature on the distribution of calcium in rabbit renal artery smooth muscle cells. The results show that the relative distribution of calcium is dependent on both the buffer used (Tris or Krebs) and the temperature of the bathing solution (25 degrees C or 34 degrees C). Krebs buffer at 34 degrees C gave the highest relative activity in the plasma membrane, sarcoplasmic reticulum, and mitochondria. Buffer and temperature had little effect on the relative activity of the nucleus or cytoplasm. Next, we identified the cellular sites of calcium accumulation after 5, 15, 30, or 60 min exposure to 45Ca in Krebs buffer at 34 degrees C. The results show that sarcoplasmic reticulum and plasma membrane are the primary sites of calcium accumulation during influx into these cells. Although the amount of 45Ca in the cell continues to increase with longer exposure, the relative distribution of calcium is essentially the same after 5 or 60 min. The data also indicate that the relative activity of plasma membrane + sarcoplasmic reticulum (a combination site that includes sarcoplasmic reticulum within a mean distance of 275 nm of the plasma membrane) is similar to the membrane alone and is lower than the sarcoplasmic reticulum alone.

Animals↗
PubMed1990

Binding of (chloroethyl 3H) cyclophosphamide to rat embryo proteins: two-dimensional gel analysis.

Cells of the heart of the day 10 rat embryo are resistant to the cytotoxic effects of cyclophosphamide (CP) while cells of neuroepithelium (head) are sensitive. Using radiolabeled CP we have investigated the hypothesis that this difference in sensitivity is related to the binding (alkylation) of drug to specific proteins in heart and head cells. Two-dimensional gel analysis reveals that CP alkylates a variety of proteins in both heart and head cells. Although minor differences in the profiles of alkylated proteins exist between heart and head cells, the major proteins alkylated by CP in the two populations are identical. These data, therefore, do not support the hypothesis that sensitivity to the cytotoxic effects of CP is related to drug binding to specific proteins.

Alkylating Agents↗
PubMed1990

Relationship of DNA damage and embryotoxicity induced by 4-hydroperoxydechlorocyclophosphamide in postimplantation rat embryos.

4-Hydroperoxydechlorocyclophosphamide (4-OOHdeCl-CP) is a preactivated analogue of cyclophosphamide (CP) that undergoes an elimination reaction to yield acrolein and the nonalkylating derivative of phosphoramide mustard (PM), i.e., dechlorophosphoramide mustard. We used this analogue to assess the role of acrolein in CP-induced embryotoxicity. Embryotoxicity was assessed using day 10 rat embryos cultured in vitro. 4-OOHdeC1-CP was embryotoxic over a concentration range of approximately 75-150 microM and produced complete embryolethality at concentrations of 175 microM and above. This analogue induced abnormal development characterized by tail defects at low drug concentrations and microencephaly or prosencephalic hypoplasia at high concentrations. Using the technique of alkaline elution, we also assessed DNA damage induced by embryotoxic concentrations of drug. When embryos were cultured in serum-containing medium during drug exposure, no DNA damage was detected, even at embryolethal drug concentrations. However, if cellular glutathione (GSH) was depleted with buthionine sulfoximine (BSO) before drug exposure and embryos were cultured in serum-free medium during drug exposure, DNA damage, primarily DNA single-strand breaks, was detected, but only at embryolethal concentrations. Using radiolabeled CP, we showed that acrolein does reach the embryo; however, more acrolein is incorporated into the yolk sac. Binding studies revealed that acrolein binds preferentially to cellular protein, whereas PM binds preferentially to DNA. These results suggest that, unlike the case with PM, the embryotoxic target for acrolein is protein and not DNA. Furthermore, our results indicate that acrolein may mediate its effects on the embryo via the yolk sac.

Acrolein↗
PubMed1988

Effect of calcium channel antagonists on calcium uptake and release by isolated rat cardiac mitochondria.

The effects of calcium channel antagonists on Ca2+ uptake and Na+-induced Ca2+ release were studied in isolated rat cardiac mitochondria. Diltiazem, nitrendipine and nimodipine were more effective inhibitors of Na+-induced Ca2+ release (IC50 = 19-100 microM) than of Ca2+ uptake (IC50 = 0.2-1 mM). Nitrendipine and nimodipine had virtually identical IC50 values for inhibiting Ca2+ uptake, but nitrendipine was 3-4 times more potent than nimodipine at inhibiting Na+-induced Ca2+ release. If these calcium channel antagonists achieve intracellular concentrations in the range of 10(-5)-10(-4) M, our results suggest that calcium channel antagonists would preferentially inhibit mitochondrial calcium release more than mitochondrial calcium uptake.

Animals↗
PubMed1987

DNA cross-linking and single-strand breaks induced by teratogenic concentrations of 4-hydroperoxycyclophosphamide and phosphoramide mustard in postimplantation rat embryos.

Postimplantation rat embryos (Day 10) were exposed in vitro to teratogenic concentrations of 4-hydroperoxycyclophosphamide, an activated form of cyclophosphamide, and phosphoramide mustard, the major teratogenic metabolite of cyclophosphamide. Following a 5-h exposure to these agents, drug-induced DNA damage was assessed by alkaline elution. Both drugs induced detectable DNA cross-linking at teratogenic concentrations. Alkaline elution combined with proteinase K digestion indicated that approximately half of the DNA cross-linking was DNA-DNA cross-linking and the other half was DNA-protein cross-linking. In addition to DNA cross-linking, phosphoramide mustard produced DNA strand breaks and/or alkaline labile sites. However, 4-hydroperoxycyclophosphamide did not produce detectable DNA strand breaks or alkaline labile sites. Our data also indicate that the induction of abnormal morphogenesis by 4-hydroperoxycyclophosphamide and phosphoramide mustard is correlated with drug-induced DNA cross-linking.

Animals↗
PubMed1987

Localization of calcium in vas deferens using 45Ca EM autoradiography: relationship to species and the effect of 45Ca removal.

The distribution of calcium was determined in the vas deferens of the guinea pig using 45Ca electron microscopic autoradiography of rapidly frozen, freeze-dried, and embedded tissue. A selective accumulation of calcium at the plasma membrane and SR was observed in vas deferens that had been incubated in 45Ca for 65-85 min prior to rapid freezing. Rinsing the tissue in nonradioactive calcium for 6 min prior to rapid freezing significantly altered the distribution of calcium among the plasma membrane, mitochondria, and cytoplasmic matrix. The influence of species on the observed distribution of calcium was also examined. The distribution of calcium in the guinea pig vas deferens was not significantly different from that in the rabbit vas deferens when the tissues were prepared under identical conditions.

Animals↗
PubMed1987

Receptor-mediated adenylate cyclase-coupled mechanism for PGE2 inhibition of insulin secretion in HIT cells.

Prostaglandin E2 (PGE2) inhibits glucose-induced insulin secretion, and inhibitors of PGE2 synthesis augment this event. However, there has been confusion regarding prostaglandin regulation of insulin secretion, partly because no mechanism has been demonstrated for the inhibitory action of PGE2 on beta-cell function. These studies were performed with a clonal cell line of glucose-responsive beta-cells (HIT cells) to determine whether PGE2 effects on insulin secretion are receptor mediated and, if so, whether the postreceptor effects are mediated by inhibitory regulatory components (Ni) of adenylate cyclase. Saturable [3H]PGE2 binding to HIT cells was demonstrated. This binding was dissociable and specific for prostaglandins of the E series. Scatchard analyses of binding data indicated a single class of sites with a Kd of approximately 1 X 10(-9) M. Guinea pig islets were also demonstrated to have a single class of binding sites with a similar Kd but only 22% as many binding sites (0.060 vs. 0.013 pmol/mg protein, HIT cells vs. guinea pig islet). HIT cells were demonstrated to synthesize PGE2, and this synthesis was inhibitable by acetylsalicylic acid. Accumulation of cAMP by HIT cells was inhibited in a concentration-dependent manner by PGE2 with an IC50 of approximately 1 X 10(-9) M. Insulin secretion by HIT cells during static incubations with 11.1 mM glucose was also inhibited by PGE2 in a concentration-dependent manner with an IC50 of 1 X 10(-9) M. PGE2 was more potent than epinephrine but less potent than somatostatin in this regard. Maximum inhibition of glucose-induced insulin secretion was 26, 37, and 29% of control values for somatostatin, PGE2, and epinephrine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗
PubMed1985

The cellular distribution of calcium in freeze-dried rabbit vas deferens using EM autoradiography.

The role of calcium in initiating smooth muscle contraction is widely accepted. The sources of this calcium are thought to be located both intracellularly and extracellularly. We have recently developed a method by which the cellular localization of calcium in smooth muscle can be determined. This method involves exposing the tissue to 45Ca, rapidly freezing and vacuum dehydrating the tissue, and preparing the tissue for electron microscopic autoradiography (EM ARG). In the present study the distribution of calcium in control and potassium-contracted tissue of the rabbit vas deferens was compared. No significant differences in distribution were observed in the two treatments. This finding provides morphological support for the hypothesis that the calcium used in potassium-induced contraction is primarily of extracellular origin. In addition, significant sequestration by intracellular organelles does not occur during a potassium contraction. In other experiments, the effect of rinsing tissue in cold calcium prior to freezing was investigated. From these data is appears that calcium is removed from the cytoplasmic matrix, plasma membrane, and organelles in a nonuniform manner. Further investigation into these findings should enable us to characterize more precisely the intracellular redistribution of calcium that occurs as a result of a variety of physiological manipulations.

Animals↗
PubMed1985

Characterization of prostaglandin E2 binding to isolated human adipocytes.

Prostaglandin (PG) E2 binding to fat cells and its consequent antilipolytic effect have been studied in experiments using laboratory animals, but no binding studies have yet been reported using adipocytes from humans. Consequently, we have characterized PGE2 binding to human isolated fat cells to compare the apparent binding constant to the IC50 for the antilipolytic effect of PGE2. Our data indicate that human fat cells contain binding sites that specifically recognize prostaglandins of the E series and demonstrate stereospecific recognition of the more potent of two 15-methyl-PGE2 analogues. There was no evidence for rapid metabolism of PGE2 by isolated adipocytes such as occurs in lung and liver tissue. A double-reciprocal plot of binding data obtained at saturation using [3H]PGE2 and increasing concentrations of PGE2 indicated a single class of binding sites with an apparent binding constant (0.54 nM) that agreed well with the IC50 (0.26 nM) for the antilipolytic effect of PGE2 we observed in human fat cells. The findings from these binding and lipolysis studies are in general agreement with published observations using adipocytes from rodents and provide evidence that the conclusions reached from previous studies of laboratory animals are relevant to adipocyte physiology in humans.

Adipose Tissue↗
PubMed1985

Cobalt-sensitive biphasic uptake of calcium ions in potassium-depolarized smooth muscle.

Isolated longitudinal muscle from guinea-pig ileum was immersed in a calcium-depleted, high-potassium medium for 10 min and then induced to contract by adding 1.8 mM CaCl2. It exhibited a biphasic response that consisted of an initial, rapid, transitory contraction--the phasic response--followed by a slower more sustained contraction--the tonic response. 45Ca uptake was also measured. Only that fraction of the accumulated calcium that could be inhibited by cobalt ion was considered to be intracellular calcium. The data showed that the phasic contraction was accompanied by a brief, rapid uptake of calcium ions and the tonic response was accompanied by a slower, more prolonged uptake of calcium ions. When the muscle, after undergoing an initial biphasic contraction, was reincubated in the calcium-depleted, high-potassium medium for 70 to 80 min, the phasic contraction that it exhibited upon the readdition of 1.8 mM CaCl2 remained unaltered, but the tonic contraction was delayed and substantially diminished. Under these conditions, the rate of the initial brief, rapid uptake of calcium was reduced by 20%, whereas the subsequent slow, prolonged uptake of calcium was measurably delayed and its rate was reduced by an average of 31.4%. Calcium pulse experiments showed that variations in the rate of calcium uptake were due primarily to variations in the rate of the inward movement of calcium ions. The results suggest that the biphasic mechanical response is a reflection of a biphasic influx of calcium ions in the potassium-depolarized longitudinal muscle.

Animals↗
PubMed1983

Down-regulation of prostaglandin E receptors and homologous desensitization of isolated adipocytes.

Adipocytes are known to contain prostaglandin E (PGE) binding sites and PGE is known to be antilipolytic. These studies were performed to ascertain whether PGE binding sites in isolated adipocytes can be down-regulated and whether down-regulation (DR) decreases the sensitivity of the fat cell to the antilipolytic effects of PGE2, adenosine, and insulin. Treatment in vivo of Sprague-Dawley rats with 16,16-dimethyl-PGE2, a PGE analog, induced DR of PGE-specific binding site density in both intact fat cells (175 vs. 307 fmol/mg protein) and triglyceride-free broken fat cell preparations (148 vs. 360 fmol/mg protein). There were no changes in binding affinities. DR was associated with diminished antilipolytic potency of PGE on basal glycerol production by intact fat cells in the presence of adenosine deaminase (IC50/control = 0.31 +/- 0.03 X 10(-9) M vs. DR = 1.6 +/- 0.03 X 10(-9) M; P less than 0.01). In contrast, there was no desensitization of the adipocytes to the antilipolytic effects of phenylisopropyladenosine (IC50/control = 4.65 +/- 0.96 X 10(-10) M vs. DR = 4.90 +/- 0.82 X 10(-10) M; P = NS) or insulin (IC50/control = 4.40 +/- 0.57 X 10(-11) M vs. DR = 2.70 +/- 0.24 X 10(-11); P = NS). PGE desensitization was also observed during studies of isoproterenol-stimulated lipolysis. These data uniquely demonstrate that the adipocyte PGE receptor can be down-regulated and that this decrease in PGE receptor density is associated with homologous desensitization of the fat cell to the antilipolytic effect of PGE and not adenosine or insulin. These findings suggest that a PGE-specific receptor may be involved in regulation of lipolysis by PGE.

Adipose Tissue↗