Biomedical subjects

G Weeks

Publications and source records attributed to G Weeks.

At least 55 records · Page 3Linked to original sources

PubMed1990

Regulation of DdrasG gene expression during Dictyostelium development.

DdrasG gene expression during the early development of Dictyostelium discoideum has been examined in detail. The amount of DdrasG-specific mRNA increased approximately twofold during the first 2 to 3 h of development and then declined rapidly, reaching negligible levels by the aggregation stage. The increase in mRNA levels that occurred during the first 2 to 3 h of development also occurred during differentiation in cell suspensions and was enhanced when cells were shaken rapidly. This initial increase was unaffected by cell density. When cells were set up to differentiate on filters, the addition of a glucose-amino acid mixture slightly delayed differentiation and had a similar effect on the expression of the gene. The decline in DdrasG expression during development did not occur when cells were treated with cycloheximide, suggesting that the expression of a developmentally regulated gene product is essential for the reduction of DdrasG gene mRNA. There was no decrease in DdrasG mRNA level during differentiation in shake suspension, but the decrease did occur upon application of pulses of cyclic AMP to shaking cultures. The application of a continuously high level of cyclic AMP delayed the increase in expression of the gene and did not result in the subsequent decline. These results suggest that the induction of a functional cyclic AMP relay system is important in reducing DdrasG gene mRNA levels.

Amino Acids↗

PubMed1990

Perioperative myocardial infarction after coronary artery bypass surgery. Clinical significance and approach to risk stratification.

The clinical significance of perioperative myocardial infarction (MI) after coronary artery bypass surgery is not known. Therefore, strategies for the risk stratification of these patients do not exist. This study was undertaken to define the effect of perioperative MI on prognosis after discharge from the hospital and to develop an approach to the risk stratification of these patients. Fifty-nine patients with and 115 patients without perioperative MI were observed for 30 months for the development of cardiac events (death, nonfatal MI, and admission to hospital for unstable angina or congestive heart failure). Patients with perioperative MI were significantly more likely than patients without to have a cardiac event (31% versus 12%, p less than 0.01) and multiple events (19% versus 1%, p less than 0.001). Cox regression analysis identified two independent predictors of cardiac events other than perioperative MI (relative risk, 2.7): inadequate revascularization (relative risk, 3.5) and depressed (less than 40%) postoperative ejection fraction (EF) (relative risk, 2.1). Event-free survival rate of patients with perioperative MI varied markedly depending on the number of other negative prognostic variables present. Patients with perioperative MI who were adequately revascularized and had a postoperative EF greater than 40% had an event-free survival rate similar to patients without a perioperative MI (92% versus 87%, p = NS). Patients with perioperative MI who were inadequately revascularized and had depressed postoperative EF had an event-free survival rate of 13% (p less than 0.001 versus all other subsets). Event-free survival rate was intermediate (68%) in patients with perioperative MI and with only one of the other two variables (p less than 0.001 versus other subsets). In conclusion, perioperative MI adversely affects prognosis. Patients can be stratified into low, high, and intermediate risk subsets based on a simple assessment of the adequacy of revascularization and a determination of residual left ventricular function.

Coronary Artery Bypass↗

PubMed1990

A Dictyostelium morphogen that is essential for stalk cell formation is generated by a subpopulation of prestalk cells.

The stalk cell differentiation inducing factor (DIF) has the properties required of a morphogen responsible for pattern regulation during the pseudoplasmodial stage of Dictyostelium development. It induces prestalk cell formation and inhibits prespore cell formation, but there is as yet no strong evidence for a morphogenetic gradient of DIF. We have measured DIF accumulation by monolayers of isolated prestalk and prespore cells in an attempt to provide evidence for such a gradient. DIF is accumulated in the largest quantities by a subpopulation of prestalk cells that specifically express the DIF-inducible genes pDd56 and pDd26. Since it has been shown recently that cells that express pDd56 are localized in the central core of the prestalk cell region of the pseudoplasmodia, our current results suggest a morphogenetic gradient generated by this region.

Blotting, Northern↗

PubMed1989

Studies on the mechanism of action of the alkaline phosphatase from Dictyostelium discoideum.

The Dictyostelium discoideum alkaline phosphatase was investigated kinetically in an attempt to elucidate its mechanism of action. Analysis of the hydrolysis of p-nitrophenyl phosphate by stopped-flow spectrophotometry revealed biphasic kinetics, suggesting a double displacement enzyme mechanism. Furthermore, Tris stimulated activity in an uncompetitive manner, a result that was consistent with this interpretation. The enzyme was inhibited reversibly by phosphate at low ionic strength, but the inhibition was irreversible at high ionic strength and the latter effect was enhanced at alkaline pH values. These results indicate that high ionic strength and alkaline pH conditions bring about a conformational change that renders the enzyme susceptible to irreversible inhibition by phosphate.

Alkaline Phosphatase↗

PubMed1989

Studies on the accumulation of the differentiation-inducing factor (DIF) in high-cell-density monolayers of Dictyostelium discoideum.

A number of factors that have been shown to influence cell type determination in Dictyostelium discoideum were assessed for their effects on the accumulation of the stalk cell differentiation-inducing factor (DIF) in high-cell-density monolayers of strain V12-M2. DIF accumulation is markedly enhanced by low pH, butyrate, and the proton pump inhibitor diethylstilbestrol (DES), conditions that induce stalk cell formation in low-cell-density monolayers in the absence of added DIF. These results are discussed in relation to a model for cell type determination recently proposed by (J.D. Gross, M.J. Peacey, and R. Pogge Von Strandmann (1988, Differentiation, 38: 91-98). DIF accumulates in high-cell-density monolayers after the cells have become independent of cyclic AMP for stalk cell formation. This accumulation is greatly enhanced by the addition of cyclic AMP. This result may explain why cyclic AMP stimulates stalk cell formation in low-density monolayers in the presence of suboptimal levels of DIF, following preincubation in the presence of saturating levels of cyclic AMP (L. Kwong, A. Sobolewski, and G. Weeks, 1988, Differentiation 37, 1-6). Adenosine has no effect on DIF accumulation in high-cell-density monolayers.

Butyrates↗

PubMed1989

Growing and developing Dictyostelium cells express different ras genes.

The expression of ras-related protein in the cellular slime mold Dictyostelium discoideum is developmentally regulated. It was previously reported that Dictyostelium possesses a single ras gene (Ddras) that is maximally expressed during the pseudoplasmodial stage of development. We have isolated a series of cDNA clones derived from a second ras gene, DdrasG. It encodes a protein that is very similar to the protein encoded by Ddras, but in contrast to Ddras, DdrasG is only expressed during growth and early development. Although other eukaryotic organisms possess more than one ras gene, Dictyostelium is thus far unique in expressing different ras genes at different stages of development. In Dictyostelium the two ras proteins may fulfill different functions, with the DdrasG protein playing a role during cell growth and the Ddras protein playing a role in signal transduction during multicellular development.

Amino Acid Sequence↗

PubMed1989

Tissue acidosis: role in sustained arteriolar dilatation distal to a coronary stenosis.

This study tested the hypothesis that myocardial tissue acidosis is responsible for maintenance of reduced arteriolar tone distal to a severe coronary arterial stenosis. Domestic swine (n = 10) were instrumented with a coronary arterial stenosis that reduced vessel diameter 80%. Measurements of hemodynamic indexes were made 1) before stenosis, 2) at 5, 20, and 60 minutes after stenosis placement, and 3) after each of three, 20-minute NaOH infusions (0.05 M, 0.1 M, and 0.5 M) distal to the stenosis (group 1). Intracellular pH at the end of 30 minutes of 0.5 M NaOH infusion distal to the stenosis was measured in a second group (n = 6) of swine (group 2). After stenosis placement in group 1, endocardial blood flow declined significantly, and evidence of regional acidosis (increased coronary venous Pco2 and decreased coronary venous pH) and ischemia (lactate production) developed. One hour later, evidence of acidosis persisted, though to a lesser extent. Myocardial oxygen and lactate metabolism exhibited similar patterns. Infusion of 0.5 M NaOH (0.38 ml/min) reduced (p less than 0.01) distal zone epicardial blood flow but did not change endocardial flow. Regional myocardial oxygen extraction (75 +/- 8%, mean +/- SD) and consumption (8.2 +/- 2.3 ml/min/100 g) also declined significantly (p less than 0.01) in response to 0.5 M NaOH infusion compared with 60 minutes after stenosis (86 +/- 4 and 12.4 +/- 2.8 ml/min/100 g respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗

PubMed1988

Purification and characterization of a Ca2+- or Mg2+-stimulated ATPase from plasma membrane enriched fractions of Dictyostelium discoideum.

Evidence is presented for the presence of both diethylstilbestrol (DES)-sensitive and DES-insensitive Mg2+-ATPase activities in plasma membrane enriched fractions of Dictyostelium discoideum. When removed from the membrane, the DES-sensitive activity is markedly less stable than the DES-insensitive activity, and the two activities display a number of quite distinct properties. The DES-sensitive enzyme has a decided preference for Mg2+ over Ca2+, displays saturation kinetics in response to ATP as substrate (Km = 0.2 mM) and has a narrow pH optimum range. In contrast, the DES-insensitive activity is stimulated equally by Mg2+ or Ca2+, is not saturable by ATP within the mM concentration range and has a much broader pH optimum. The DES-insensitive activity has been purified extensively. The purified enzyme is inhibited by vanadate and fluoride, but is insensitive to N,N'-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide and thimerosal. In the absence of divalent cations, the enzyme displays a sigmoidal activity curve in response to substrate concentration, which is abolished by addition of either Mg2+ or Ca2+, suggesting a binding site for a divalent cation and a positive cooperative interaction. The enzyme is capable of hydrolyzing other nucleotide triphosphates and ADP, but is without activity on AMP, p-nitrophenyl phosphate and pyrophosphate. The enzyme has an apparent molecular weight of approximately 64,000.

Adenosine Triphosphate↗

PubMed1988

Evidence for a membrane-bound pyrophosphatase in Dictyostelium discoideum.

Plasma membrane enriched fractions of Dictyostelium discoideum contain a Des-insensitive ATPase activity that can be fractionated by DEAE-Sephacel into a major vanadate-sensitive activity and a minor vanadate-insensitive activity. The vanadate-insensitive activity hydrolyzed pyrophosphate considerably more rapidly than ATP or any other substrate tested, and the enzyme was therefore designated a pyrophosphatase. The enzyme had no activity on AMP or p-nitrophenyl phosphate. The pyrophosphatase activity was maximal at alkaline pH values and stimulated by Mg2+ but not by Ca2+, properties of the enzyme that are very similar to those of the previously characterized pyrophosphatases of the plant tonoplast membrane. The pyrophosphatase activity of total membrane extracts changed very little during Dictyostelium differentiation.

Cell Membrane↗

PubMed1988

Stalk cell formation in monolayers of Dictyostelium discoideum V12-M2.

Stalk cell formation in low-cell-density monolayers of Dictyostelium discoideum, strain V12-M2, occurs following the sequential addition of cyclic AMP and the differentiation-inducing factor (DIF). Both cyclic AMP and DIF are essential for the appearance of the prestalk-specific isozyme alkaline phosphatase-II, which suggests that both factors are necessary for prestalk cell formation. The available evidence suggests that the cyclic AMP requirement for stalk cell formation is mediated through the cell surface cyclic AMP receptor. However, stalk cell formation is inhibited by caffeine and this inhibition is reversed by the cell-permeable analogue 8-Br-cyclic AMP, which suggests in addition a possible involvement for elevated intracellular cyclic AMP concentrations in stalk cell formation. During in vivo development cells first become independent of cyclic AMP at the tipped aggregate stage, but the acquisition of cyclic AMP independence is advanced by several hours when cells are incubated in the presence of cyclic AMP for 2 hours. Cells do not become independent of DIF until the culmination stage of development, which suggests the possibility that DIF is required for the conversion of prestalk cells to stalk cells. There is an absolute requirement for DIF for stalk cell formation in low-density monolayers of prestalk cells but only part of population exhibits a requirement for cyclic AMP, which suggests that the prestalk cell population consists of two distinct cell types. Stalk cell formation from prespore cells is totally dependent on both cyclic AMP and DIF.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differentiation↗

PubMed1988

Purification and characterization of PCPP-260: a Purkinje cell-enriched cyclic AMP-regulated membrane phosphoprotein of Mr 260,000.

PCPP-260 (Purkinje cell phosphoprotein of Mr 260,000), a substrate for cAMP-dependent protein kinase, appears to be an integral membrane protein highly enriched in Purkinje cells of the mammalian cerebellum (Walaas et al.: J. Neurosci., 3:291-301, 1983; Walaas et al.: J. Neurosci., 6:954-961, 1986). PCPP-260 has now been purified from a crude particulate fraction of bovine cerebellum, using the ionic detergent N-lauryl sarcosine (NLS) as solubilizing agent, and monitoring the purification by silver stain and autoradiography of 32P-phosphorylated samples, after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Concanavalin A was found to bind to PCPP-260, suggesting that it is a glycoprotein. PCPP-260 was therefore extracted, retained on a column of concanavalin A-agarose, and eluted by alpha-methyl mannoside. Further chromatography on Sephacryl S-400 yielded a preparation that was purified approximately 250-fold relative to the initial particulate fraction and that was at least 95% pure. The protein was estimated to represent approximately 0.4% of total membrane protein in the cerebellum. Peptide mapping and phosphoamino acid analysis following phosphorylation of the protein by cAMP-dependent protein kinase showed one major tryptic phosphopeptide containing phosphoserine. A similar, less prominent protein was also found in membranes from other brain regions but could not be detected in liver membranes. The availability of highly purified PCPP-260 should facilitate the investigation of its possible functional roles in the nervous system.

Animals↗

PubMed1988

The requirement for DIF for prestalk and stalk cell formation in Dictyostelium discoideum: a comparison of in vivo and in vitro differentiation conditions.

In Dictyostelium discoideum stalk cell formation is induced by cyclic AMP and differentiation-inducing factor (DIF) when cells are plated in in vitro monolayers (Kay et al., 1979, Differentiation 13: 7-14). The in vivo developmental stages at which cells became independent of these factors were determined. Independence was defined as the stage at which dispersed cells no longer required the factors for stalk cell formation in low density monolayers. Cyclic AMP independent cells were first detected at around 12 hr of development, a time that corresponds to the transition between the tipped aggregate and the first finger stages. In contrast cells did not become independent of DIF until late culmination. The prestalk cell-specific isozyme acid phosphatase II and a stalk cell-specific 41,000 Mr antigen (ST 41) were expressed during differentiation in low density monolayers in the presence of both cyclic AMP and DIF, but neither component was expressed in the presence of cyclic AMP alone. This result implies that DIF is essential for both prestalk and stalk cell formation. The two components were expressed within 2 hr of each other during differentiation in vitro, whereas during development in vivo acid phosphatase II was first detected at the first finger stage and ST 41 was first detected during late culmination, 8-12 hr later. These contrasting results suggest that the conversion of prestalk cells to stalk cells is unrestrained in monolayers, following directly after prestalk cell induction, but restrained in vivo until the culmination stage. This interpretation is consistent with the finding that cells become independent of DIF early during in vitro differentiation (A. Sobolewski, N. Neave, and G. Weeks, 1983, Differentiation 25, 93-100), but do not become independent of DIF until the culmination stage when differentiating in vivo.

Acid Phosphatase↗

PubMed1988

Stalk cell formation in monolayers from isolated prestalk and prespore cells of Dictyostelium discoideum: evidence for two populations of prestalk cells.

Cells from the pseudoplasmodial stage of Dictyostelium discoideum differentiation were dispersed and separated on Percoll gradients into prestalk and prespore cells. The requirements for stalk cell formation in low-density monolayers from the two cell types were determined. The isolated prespore cells required both the Differentiation Inducing Factor (DIF) and cyclic AMP for stalk cell formation. In contrast, only part of the isolated prestalk cell population required both cyclic AMP and DIF, the remainder requiring DIF alone, suggesting the possibility that there were two populations of prestalk cells, one independent of cyclic AMP and one dependent on cyclic AMP for stalk cell formation. The finding that part of the prestalk cell population required only a brief incubation in the presence of DIF to induce stalk cell formation, whilst the remainder required a considerably longer incubation in the presence of both DIF and cyclic AMP was consistent with this idea. In addition, stalk cell formation from cyclic-AMP-dependent prestalk cells was relatively more sensitive to caffeine inhibition than stalk cell formation from cyclic-AMP-independent prestalk cells. The latter cells were enriched in the most anterior portion of the migrating pseudoplasmodium, indicating that there is spatial segregation of the two prestalk cell populations. The conversion of prespore cells to stalk cells took longer and was more sensitive to caffeine when compared to stalk cell formation from cyclic-AMP-dependent prestalk cells.

Caffeine↗

PubMed1987

A ras-encoded protein in Dictyostelium discoideum is acylated and membrane-associated.

Dictyostelium discoideum synthesizes a 23,000 Mr protein, p23dd-ras, closely related to the mammalian oncogene-encoded protein p21ras. To investigate the subcellular localization of p23dd-ras, conditions were optimized to reduce protein degradation following cell breakage. Subcellular fractionation of D. discoideum showed that p23dd-ras was associated predominantly with the membrane fraction during both vegetative growth and differentiation. In the absence of suitable protease inhibitors considerable amounts of a truncated form of p23dd-ras were recovered in the cytosol fraction, suggesting that intact p23dd-ras is attached to the membrane by a short terminal peptide sequence. Radio-isotope labelling of D. discoideum with myristic acid or palmitic acid in the presence of excess unlabelled acetate resulted in radio-isotope incorporation into a select group of proteins including p23dd-ras. No acyl label appeared in the truncated cytoplasmic form of p23dd-ras when cell breakage was performed in the absence of suitable protease inhibitors, indicating that the acyl group is associated with the short terminal peptide that is cleaved. These data suggest that p23dd-ras, like its mammalian counterpart, is acylated and associated with the plasma membrane. There was no evidence during a 30-minute pulse of methionine for a cytoplasmic precursor to the membrane-bound p23dd-ras, suggesting that the turnover of the presumptive precursor must be much more rapid in D. discoideum than for pro-p21ras in mammalian cells.

Acetates↗

PubMed1987

The synthesis and degradation of ras-related gene products during growth and differentiation in Dictyostelium discoideum.

A Dictyostelium discoideum protein with an Mr of 23,000 (p23dd-ras) is structurally related to the mammalian proto-oncogene ras-gene product, p21ras, and is specifically precipitated from cell-free extracts of D. discoideum by the Y13-259 monoclonal antibody against p21ras. p23dd-ras was degraded at rates that were very similar to those observed for total protein during both growth and differentiation, suggesting that the previously reported decline in p23dd-ras levels during differentiation is due to a change in the rate of synthesis rather than a change in the rate of degradation. p23dd-ras synthesis did not decrease immediately after the initiation of differentiation, but rather its rate of synthesis increased for the first 1-2 h, suggesting that p23dd-ras is not rapidly down-regulated in response to nutrient deprivation. There were differences in the extent of p23dd-ras turnover during the differentiation of the three tested strains, A-3, NC4, and V12-M2. The relative level of p23dd-ras dropped most rapidly in V12-M2, which may reflect the slightly faster differentiation process exhibited by this strain. In all three strains, very little p23dd-ras was present by the end of the differentiation process. A second protein with an Mr of 24,000 (p24dd-ras) was also immunoprecipitated using the Y13-259 antibody. The amount of p24dd-ras was small or undetectable in vegetative cells, but relatively larger amounts of p24dd-ras were synthesized in pseudoplasmodial cells. We found no evidence to suggest that p24dd-ras is a precursor of p23dd-ras.(ABSTRACT TRUNCATED AT 250 WORDS)

Dictyostelium↗

PubMed1985

The biosynthesis and turnover of lipid during the differentiation of Dictyostelium discoideum.

Despite the fact that there are only relatively slight changes in lipid composition during the differentiation of Dictyostelium discoideum, the rates of lipid synthesis were found to vary considerably. Polar lipid synthesis declined markedly during aggregation and pseudoplasmodium formation and then increased during the terminal stages of differentiation. Several neutral lipid classes (sterol, the diacylglycerols and triacylglycerol) exhibited similar changes in synthetic rates, although the effects were somewhat less pronounced. In contrast, the rates of synthesis of steryl ester and free fatty acid increased slightly throughout the differentiation period, so that, by the end of the later stages of fruiting body culmination, the rates were essentially doubled. Finally, the synthesis of an unknown component increases at least 10-fold during differentiation. Of the newly synthesized lipid, only triacylglycerol and polar lipid exhibited marked turnover. Accumulation of radioactivity in steryl ester and free fatty acid continued after the removal of radioactive acetate, presumably due to the incorporation of fatty acid produced by polar lipid degradation.

Acetates↗

PubMed1985

The membrane-bound alkaline phosphatase and 5'-nucleotidase activities of vegetative cells of Dictyostelium discoideum.

Earlier reports suggested that the adenosine monophosphate (AMP)- and the p-nitrophenyl phosphate (pNPP)-hydrolyzing activities of Dictyostelium discoideum membrane preparations are due to different proteins. These results have been apparently contradicted by the recent purification to homogeneity of the two activities from culmination phase cells as a single protein [D. R. Armant and C. L. Rutherford (1981) J. Biol. Chem. 256, 12710-12718]. Results presented here from studies on the activities of vegetative cells support the concept of a single protein. Nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Triton X-100 extracts of cell membrane preparations of D. discoideum showed identical migration of pNPPase and AMPase activities. Furthermore, the previously reported different pH optima of the two activities was due to the fact that pH optima are dependent upon the substrate concentration, and the selective solubilization of AMPase from membrane preparations by phospholipase C can probably be accounted for by the finding that phospholipase C preparations from the same commercial source contain 5'-nucleotidase activity. Moreover, there are alterations in the Km and the stability of both AMPase and pNPPase in a strain with a mutationally altered alkaline phosphatase, further supporting the concept that the two activities are due to a single protein. Both substrates serve as transphosphorylation donors demonstrating that the enzyme activity is mechanistically an alkaline phosphatase.

5'-Nucleotidase↗

PubMed1985

Regulation of a ras-related protein during development of Dictyostelium discoideum.

Recent work has shown that DNA sequences related to the mammalian ras proto-oncogenes are highly conserved in eucaryotic evolution. A monoclonal antibody (Y13-259) to mammalian p21ras specifically precipitated a 23,000-molecular-weight protein (p23) from lysates of Dictyostelium discoideum amoebae. Tryptic peptide analysis indicated that D. discoideum p23 was closely related in its primary structure to mammalian p21ras. p23 was apparently derived by post-translational modification of a 24,000-molecular-weight primary gene product. The amount of p23 was highest in growing amoebae, but declined markedly with the onset of differentiation such that by fruiting body formation there was less than 10% of the amoeboid level. The rate of p23 synthesis dropped rapidly during aggregation, rose transiently during pseudoplasmodial formation, and then declined during the terminal stages of differentiation. There was, therefore, a strong correlation between the expression of the ras-related protein p23 and cell proliferation of D. discoideum.

Cell Differentiation↗