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Biomedical subjects

L O'Brien

Publications and source records attributed to L O'Brien.

At least 19 recordsLinked to original sources
PubMed1995

Interaction of ceramides, sphingosine, and sphingosine 1-phosphate in regulating DNA synthesis and phospholipase D activity.

C2- and C6-ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) abolished the stimulation of DNA synthesis by sphingosine 1-phosphate in rat fibroblasts. This inhibition by ceramide was partially prevented by insulin. C2-ceramide did not alter the stimulation of DNA synthesis by insulin and decreased the sphingosine-induced stimulation by only 16%. The ceramides did not significantly modify the actions of sphingosine or sphingosine 1-phosphate in decreasing cAMP concentrations. C2- and C6-ceramides blocked the activation of phospholipase D by sphingosine 1-phosphate, and this inhibition was not affected by insulin. Okadaic acid decreased the activation of phospholipase D by sphingosine 1-phosphate and did not reverse the inhibitory effect of C2-ceramide on this activation. Therefore, this effect of C2-ceramide is unlikely to involve the stimulation of phosphoprotein phosphatase activity. Sphingosine did not activate phospholipase D activity significantly after 10 min. C2-ceramide stimulated the conversion of exogenous [3H]sphingosine 1-phosphate to sphingosine and ceramide in fibroblasts. Ceramides can inhibit some effects of sphingosine 1-phosphate by stimulating its degradation via a phosphohydrolase that also hydrolyzes phosphatidate. Furthermore, C2- and C6-ceramides stimulated ceramide production from endogenous lipids, and this could propagate the intracellular signal. This work demonstrates that controlling the production of ceramide versus sphingosine and sphingosine 1-phosphate after sphingomyelinase activation could have profound effects on signal transduction.

Animals↗
PubMed1995

Short-chain ceramide-1-phosphates are novel stimulators of DNA synthesis and cell division: antagonism by cell-permeable ceramides.

Ceramide and ceramide-1-phosphate are sphingolipid analogues of diacylglycerol and phosphatidate, respectively, and they are putative second messengers of agonist-stimulated sphingomyelin metabolism. The interactions of exogenous cell-permeable ceramides and ceramide-1-phosphates in modifying DNA synthesis and signal transduction were investigated in Rat-1 fibroblasts. C2- and C8-Ceramide-1-phosphates (N-acetylsphingosine-1-phosphate and N-octanoylsphingosine-1-phosphate, respectively) at 1-10 microM stimulated DNA synthesis and cell division. This effect was blocked by cell-permeable ceramides. C2-Ceramide stimulated the conversion of exogenous C8-ceramide-1-phosphate to C8-ceramide, with very little production of sphingosine or sphingosine-1-phosphate. This mechanism may be partly responsible for preventing the stimulation of DNA synthesis. Unlike phosphatidate or lyso-phosphatidate, concentrations of C8-ceramide-1-phosphate that stimulated DNA synthesis did not inhibit adenylate cyclase activity, nor did they increase the activities of phospholipase D or mitogen-activated protein kinases (42- and 44 kDa isoforms). Although ceramide-1-phosphate can be considered as an analogue of phosphatidate, the effects of this compound on signal transduction differ considerably from those of phosphatidate. This work demonstrates that short-chain ceramide-1-phosphates can be used as novel external agonists that can stimulate DNA synthesis. This effect can be counteracted by short-chain ceramides.

Animals↗
PubMed1994

Cell-permeable ceramides inhibit the stimulation of DNA synthesis and phospholipase D activity by phosphatidate and lysophosphatidate in rat fibroblasts.

The interactions of ceramides with phosphatidate and lysophosphatidate in the regulation of signal transduction in rat fibroblasts were examined. C2- and C6-ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) at 10 microM inhibited the stimulation of DNA synthesis that was produced by 50-100 microM phosphatidate, or lysophosphatidate, or by exogenous phospholipase D. Sphingosine (10 microM) had the opposite effect to the ceramides on DNA synthesis. C2- or C6-ceramides failed to inhibit the stimulation of DNA synthesis by insulin or serum. The ceramides did not modify the actions of phosphatidate, or lysophosphatidate, in decreasing the forskolin-induced increase in cAMP. C2- and C6-ceramides inhibited the stimulation of phospholipase D activity by: (a) phosphatidate, lysophosphatidate, phorbol ester, thrombin, or serum in intact fibroblasts and (b) phorbol ester or guanosine 5'-3-O-(thio)triphosphate in permeabilized fibroblasts. The ceramides can therefore modify cell signaling via phospholipase D, but this effect alone could not explain the decreased DNA synthesis. Incubation of fibroblasts with C2- or C6-ceramides or sphingomyelinase inhibited the interaction of exogenous phosphatidate or lysophosphatidate with the fibroblasts by 42 and 53%, respectively. Furthermore, a greater proportion of the phosphatidate, or lysophosphatidate, that was associated with the fibroblasts was metabolized further when the cells were pretreated with ceramides or sphingomyelinase. This effect was accompanied by an increased activity of N-ethylmaleimide-insensitive phosphatidate phosphohydrolase. Ceramides may therefore produce part of their growth inhibitory effects by blocking some of the signal transducing effects of phosphatidate and lysophosphatidate.

Animals↗
PubMed1994

Apparent killing of Mycobacterium tuberculosis by cytokine-activated human monocytes can be an artefact of a cytotoxic effect on the monocytes.

A protocol using combined exposure to interferon-gamma (IFN-gamma), calcitriol and tumour necrosis factor-alpha (TNF-alpha) has been reported to activate human monocytes in vitro to kill Mycobacterium tuberculosis. We have attempted to repeat the findings in two laboratories, with negative results; treated cells were no different from untreated cells in this respect. However, the treated cells were more sensitive to a toxic effect of the bacteria. We suggest that the reported dramatic mycobacterial killing may have been an illusory consequence of the toxicity leading to cell lysis and loss of the liberated bacteria from the assay.

Artifacts↗
PubMed1994

Strains of Mycobacterium tuberculosis differ in susceptibility to reactive nitrogen intermediates in vitro.

The effects on the viability of Mycobacterium tuberculosis strains and one Mycobacterium bovis strain from exposure to sodium nitrite for 24 h, in both neutral and acidic media, were tested. The in vitro resistance of mycobacteria to reactive nitrogen intermediates, generated at an acidic pH, was found to have a significant (P < 0.05) positive correlation to the virulence of strains in guinea pigs.

Hydrogen-Ion Concentration↗
PubMed1993

Purification and characterization of eukaryotic initiation factor (eIF)-2 alpha kinases from Ehrlich ascites tumor cells.

A major mechanism of regulation of mammalian protein synthesis initiation is accomplished by the phosphorylation of the alpha subunit of eukaryotic initiation factor (eIF) 2. This modification inhibits the activity of another initiation factor, guanine nucleotide exchange factor, preventing conversion of eIF-2.GDP to eIF-2.GTP and hence binding of initiator tRNA and formation of ternary complex (eIF-2.GTP.Met-tRNAf). Inhibition of protein synthesis and phosphorylation of eIF-2 occurs in Ehrlich cells when they are amino acid- or serum-deprived or heat-shocked as well as in other nucleated cells under similar conditions. This paper describes the purification of two eIF-2 alpha kinases from Ehrlich cells. Unlike the two well characterized eIF-2 alpha kinases, HRI (heme-regulated inhibitor from reticulocytes) and P68 (double-stranded RNA-dependent kinase found in interferon-treated cells), the Ehrlich cell kinases do not appear to autophosphorylate. The two kinases chromatograph differently on DEAE-cellulose and Mono Q. Furthermore, their Michaelis constants (Km values) for ATP are different. Both kinases can inhibit purified guanine nucleotide exchange factor (GEF) from stimulating ternary complex formation. However, only one kinase inhibits reticulocyte lysate cell free protein synthesis. The other kinase co-purifies with a factor that suppresses inhibition of protein synthesis in reticulocyte lysates by eIF-2 alpha kinases. This suppressing activity is probably guanine nucleotide exchange factor.

Animals↗
PubMed1993

Inhibition of Plasmodium falciparum phospholipase A2 by chloroquine, quinine, and arteether.

Phospholipase A2 activity was detected in Plasmodium falciparum-infected human erythrocytes but not in uninfected red cells. The activity was similar both for trophozoite- and schizont-infected cells. Enzyme activity was Ca(2+)-independent and had a broad pH optimum, with a maximum at pH 8. No detectable phospholipase A1 activity was found either in infected or uninfected erythrocytes. Phospholipase A2 was inhibited by the anti-malarials chloroquine, quinine, and arteether with concentrations that cause 50% inhibition (IC50) of 1.3, 1.0, and 1.8 mM, respectively. The IC50 value for chloroquine is within the range of concentrations found in the food vacuoles of the malaria parasite. Inhibition of the plasmodial phospholipase A2 may, therefore, be relevant for the therapeutic action of this drug.

Animals↗
PubMed1992

Trichosanthin treatment of HIV-induced immune dysregulation.

Twenty patients with the acquired immunodeficiency syndrome (AIDS), AIDS-related complex (ARC) or asymptomatic HIV infection (HIV+) were given 20 mcg kg-3 trichosanthin (TCS; 'Compound Q'), a ribosome-inactivating protein with in vitro antiviral activity against human immunodeficiency virus (HIV) once every four weeks for up to 12 weeks. With the concurrent administration of prostaglandin inhibitors, the drug was moderately well tolerated, with most subjects experiencing mild arthralgia, hives and malaise. Additionally, four patients experienced neurological complications which resolved spontaneously without intervention. Four of 20 subjects in this open label pilot study showed progressive although transient reductions in viral activity as measured by p-24 antigen level decreases. Subjects also experienced decreases in levels of beta 2-microglobulin. Ten HIV+ and healthy ARC subjects demonstrated improved immunological status as measured by significant increases in percentage of CD4+ cells and augmentations in delayed hypersensitivity reactions. Eight of 20 subjects reported improved appetites and increased energy levels. The group as a whole had a weight gain of 3.2 kg. Eight of 20 subjects who presented with persistent generalized lymphadenopathy exhibited a marked diminution in the size of their lymph nodes after the first treatment. No subject who presented with oral candidiasis experienced an improvement in that condition. We conclude that, in the short term, TCS seems to have the ability to reduce viral activity and improve certain symptoms in healthy ARC patients and HIV + asymptomatics although it may not be able to restore immune competence in persons with advanced AIDS or poor prognosis ARC. Additionally, the drug may pose a special risk for patients with HIV-related dementia.

AIDS-Related Complex↗
PubMed1992

Intracellular pH on protein kinase C and ionomycin potentiation of isoproterenol-stimulated cyclic AMP and cyclic GMP production in rat pinealocytes.

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.

Ammonium Chloride↗
PubMed1992

Intracellular pH and growth hormone-releasing factor-stimulated adenosine 3'5'-monophosphate, intracellular calcium and growth hormone release from rat anterior pituitary cells.

In this study, we examined the effect of changes in intracellular pH (pHi) on basal and GH-releasing factor (GRF)-stimulated cyclic AMP (cAMP), intracellular Ca2+ and GH release using a static monolayer culture prepared from dispersed rat anterior pituitary cells. To modulate pHi, two approaches were used: variation of extracellular pH (pHo) and addition of sodium propionate and ammonium chloride which alter pHi directly. Direct pHi measurement with 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein showed that for pHo values between 6.9 and 7.6, a change in pHo of 0.1 units resulted in a change in pHi of 0.045 units. Sodium propionate (30 mmol/l) reduced pHi by 0.06 units whereas ammonium chloride (30 mmol/l) increased pHi by 0.1 units. Increasing pHo from 6.6 to 7.8 enhanced the maximal GRF-stimulated cAMP and GH responses by 80% and 300% respectively, indicating that the GRF-stimulated cAMP and GH release were both pH-dependent. Acute elevation of pHo from 6.6 to 7.8 also increased basal GH release by sixfold. Reduction of pHi by sodium propionate, however, had no significant effect on GRF-stimulated cAMP levels while the corresponding GRF-stimulated GH release was reduced by up to 40%. In comparison, elevation of pHi by ammonium chloride enhanced the GRF-stimulated cAMP release by up to 75% and the corresponding increase in GH was less than 20%. When the relationship between pHi and intracellular Ca2+ was determined with the fluorescent Ca2+ indicator, Fura-2, it was found that increasing pHo and treatment with ammonium chloride increased intracellular Ca2+, while sodium propionate and reducing pHi had no effect on intracellular Ca2+. These results indicate that activation of adenylate cyclase and mobilization of intracellular Ca2+, two intracellular signalling pathways of importance to GH secretion, are both sensitive to changes in pHi.

Ammonium Chloride↗
PubMed1991

Behavioral thermoregulation and immobilization: conflicting demands for survival.

In three studies, core temperatures of immature chicks rose during immobilization induced by simulated predation and fell rapidly when immobilization terminated. Immobilization termination was predicted by proximity of the core temperature to the daily resting core temperature of adult fowl. Although immobilization duration increased with age and was shorter in cold and hot environments, immobilization terminated at the same core temperature at all ages and ambient temperatures. The common termination temperature and pattern of temperature change across all Ss suggests that immobilization is terminated in response to the demand that the rising core temperature be behaviorally maintained within a vital range. The generality of this finding was confirmed in a fourth study with preweanling kittens during exhibition of the transport response--an immobilization reaction with a different evolutionary history. These findings reveal that when immobilization and behavioral thermoregulation present conflicting survival demands for avian and mammalian young, the control of core temperature assumes behavioral priority.

Adaptation, Physiological↗
PubMed1990

The effect of peritoneal dialysate on pulmonary function and blood gasses in C.A.P.D. patients.

There have been many reports showing diminished vital capacity (VC), total lung capacity (TLC), and functional residual capacity (FRC), after the infusion of peritoneal dialysate in patients on continuous ambulatory peritoneal dialysis (C.A.P.D.) for chronic renal failure. We also examined the effects of the infusion of two litres of dialysate on airways resistance (Raw) using total body plethysmography and on arterial blood gasses. Ten patients on C.A.P.D. were selected. The mean results of dialysate infused (in) and dialysate drained (out) are as follows: FVC 3.66 l (in) and 3.73 l (out) (not significant); VC 3.81 l (in) and 3.99 l (out) (p less than 0.05); FEV1 3.02 l (in) and 2.94 (out) (n.s.); TLC 5.89 l (in) and 6.33 l (out) (p less than 0.05); FRC 3.56 l (in) and 3.78 l (out) (p less than 0.05); Raw 4.79 cmsH21/l/s (in) and 4.72 cmsH20/l/s (out) (n.s.); Pa02 11.03 kPA (in) and 11.35 kPA (out) (p less than 0.001). We conclude that two litre dialysate causes significant reduction of TLC, VC and FRC, and a reduction in Pa02 and A-a02 but has no effect on airways resistance.

Airway Resistance↗
PubMed1990

Interferon-alpha and 3'-azido-3'-deoxythymidine are highly synergistic in mice and prevent viremia after acute retrovirus exposure.

This study was undertaken to calculate the in vivo drug interactions between recombinant human interferon-alpha A/D (rHuIFN-alpha A/D) and 3'-azido-3'-deoxythymidine (AZT) in a quantitative model for retroviral viremia. When given as single agents, both AZT and rHuIFN-alpha A/D suppressed virus-induced splenomegaly in a dose-dependent fashion in mice inoculated with Rauscher murine leukemia virus (RLV). However, suppressive doses of single-agent AZT caused anemia after 20 days of therapy. Combining rHuIFN-alpha A/D with AZT allowed drastic dose reductions for each agent while maintaining greater than or equal to 93% inhibition of splenomegaly. No clinically significant toxicity was seen. Computer analysis with the isobologram technique and combination index method showed that these combination regimens were highly synergistic. A 20-day course of AZT + rHuIFN-alpha A/D started 4 h after virus exposure was protective against RLV viremia and disease. After cessation of therapy, the animals were resistant to rechallenge with fully infectious RLV. We conclude that prompt initiation of effective combination therapy after retroviral exposure prevented viremia and disease and led to protective immunity.

Animals↗
PubMed1988

Totally implantable system for venous access in children with cystic fibrosis.

Thirteen children with cystic fibrosis had a totally implantable reservoir surgically inserted for repeated courses of intravenous antibiotics. The youngest patient was 17 months at the time of implantation. The catheters have been in place for an average of 463 +/- 200 days. Forty seven courses of intravenous antibiotics have been given over a total of 832 treatment days. Three patients had complications. One experienced catheter separation from the portal, one catheter became occluded, and a third catheter fractured and migrated to the right atrium but was successfully retrieved percutaneously. The catheters were accepted well by patients and parents. We conclude that implantable reservoir systems, although moderately expensive and not totally free of complications, offer another modality in the management of cystic fibrosis patients.

Anti-Bacterial Agents↗
PubMed1985

Internal fixation of acute scaphoid fractures: a new approach to treatment.

Thirty-five patients with acute, unstable fractures of the scaphoid were treated by primary internal fixation using the Herbert Bone Screw System. The use of plaster was avoided whenever possible, and operation was carried out as soon as practicable after injury. The average time for return to work after operation was 3.7 weeks. Only one fracture failed to unite, and this was due to avascular necrosis of the proximal fragment of the scaphoid. Internal fixation of the acute scaphoid fracture successfully avoids the morbidity associated with standard methods of treatment in plaster.

Adult↗
PubMed1980

Crystalline ribosomes are present in brains from senile humans.

Paracrystalline inclusions known as Hirano bodies characteristically appear in the hippocampal region of the brains of humans exhibiting senile and presenile dementias as well as several other neurodegenerative diseases. We present evidence that the currently accepted model for those structures based on alternating filament sheets is not correct, but that Hirano bodies are stacked sheets of membrane-bound ribosomal particles derived from partially degraded rough endoplasmic reticulum or Nissl substance. Using fluorescence staining with acridine orange and ethidium bromide, were have shown that the bodies contain RNA. Spatial filtering of electron micrographs by Fourier techniques shows that the individual particles that make up the arrays have a characteristic shape previously reported for the large subunit of eukaryotic ribosomes. The storage of these ribosomal particles in inclusion bodies may indicate a quiescent state of protein synthesis in the cells. This withdrawal of synthetic mechanisms in the hippocampus may have significant consequences in the loss of ability to consolidate short-term to long-term memory.

Alzheimer Disease↗