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Biomedical subjects

S W James

Publications and source records attributed to S W James.

13 recordsLinked to original sources
PubMed2001

[Investigation of the effects of various factors affecting apoptosis of chronic lymphocytic leukemic cells mediated by P2Z receptors].

OBJECTIVE: To explore the effects of various factors affecting apoptosis of chronic lymphocytic leukemia (CLL) cells mediated by P2Z purinoceptor. METHODS: In vitro exposure of CLL cells with P2Z receptors to 1 mmol/L ATP or 0.1 mmol/L benzoylbenzoic ATP (BzATP) for 8 h in the presence of 1.0 mmol/L Mg2+ or other bivalent cations, 150 mmol/L choline in media, 1, 2, 4 mmol/L EDTA or EGTA, or under different temperature. ATP-induced apoptosis was detected by agarose gel electrophoresis, TdT assay and flow cytometric analysis (FCA). RESULTS: The results showed that 1. Extracellular Mg2+ or Ca2+ stimulated ATP-induced DNA fragmentation in a dose-dependent manner, and the compatible evidence was provided by the inhibition of ATP-induced DNA fragmentation in the presence of EGTA or EDTA; 2. ATP-induced DNA fragmentation was completely inhibited by 1.0 mmol/L Zn2+; 3. ATP-induced DNA breaks were not affected by Ba2+, Sr2+, Co2+ when they were substituted for extracellular Mg2+ or Ca2+; 4. Choline, an inhibitor of phospholipase D (PLD) stimulated by ATP through P2Z receptor in human lymphocytes, was also a partial inhibitor of ATP-induced DNA fragmentation, and the results were confirmed by flow cytometric analysis (FCA); 5. ATP-induced DNA fragmentation was completely obliterated when the temperature was lower than 10 degrees C. CONCLUSION: These results suggest that the endonuclease and PLD may be involved in ATP-induced apoptosis in human lymphocytes via P2Z receptor.

Adenosine Triphosphate↗
PubMed1999

Delays in the detection of hypoxemia due to site of pulse oximetry probe placement.

STUDY OBJECTIVES: To determine if there were any differences in the time to detect hypoxemia related to the site of peripheral pulse oximetry (ear, hand, and foot) during the rapid induction of hypoxemia in healthy volunteers. DESIGN: Repeated-measures, longitudinal, observational study. SETTING: Anesthesia clinical research area of the Department of Anesthesiology. PATIENTS: 13 healthy volunteers, aged 18 to 44 years. INTERVENTIONS: Nellcor N-200 (Nellcor, Inc., Pleasanton, CA) oximeter probes were placed at the ear, hand, and foot. All units were turned on simultaneously with averaging times set for 5 seconds and signals sampled at 2 Hz. A computer-controlled anesthesia circuit was employed to induce mild hypercapnia and hyperoxia (end-tidal gas partial pressures: PETCO2 = 42 +/- 2 mmHg and PETO2 = 130 mmHg) for 5 minutes. PETO2 was then decreased to 45 +/- 2 mmHg over 60 seconds and held at that value for 5 minutes. MEASUREMENTS AND MAIN RESULTS: The mean differences in time (sec) for pulse oximeters to detect hypoxemia (read less than 90%) between probe sites were determined and compared. The following mean differences in time (sec) for pulse oximeters to detect hypoxemia (read less than 90%) between probe sites were found: ear-hand = 6; hand-foot = 57; ear-foot = 63. Paired t-tests revealed statistically significant mean time delay differences of 51 seconds (p < 0.005) and 57 seconds (p < 0.005) for ear-hand versus hand-foot and for ear-hand versus ear-foot, respectively. CONCLUSIONS: In healthy volunteers, significant delays in the detection of acute hypoxemia by pulse oximetry occur when pulse oximeters are placed at the toe as compared with probes at either the ear or hand.

Adolescent↗
PubMed1999

nimO, an Aspergillus gene related to budding yeast Dbf4, is required for DNA synthesis and mitotic checkpoint control.

The nimO predicted protein of Aspergillus nidulans is related structurally and functionally to Dbf4p, the regulatory subunit of Cdc7p kinase in budding yeast. nimOp and Dbf4p are most similar in their C-termini, which contain a PEST motif and a novel, short-looped Cys2-His2 zinc finger-like motif. DNA labelling and reciprocal shift assays using ts-lethal nimO18 mutants showed that nimO is required for initiation of DNA synthesis and for efficient progression through S phase. nimO18 mutants abrogated a cell cycle checkpoint linking S and M phases by segregating their unreplicated chromatin. This checkpoint defect did not interfere with other checkpoints monitoring spindle assembly and DNA damage (dimer lesions), but did prevent activation of a DNA replication checkpoint. The division of unreplicated chromatin was accelerated in cells lacking a component of the anaphase-promoting complex (bimEAPC1), consistent with the involvement of nimO and APC/C in separate checkpoint pathways. A nimO deletion conferred DNA synthesis and checkpoint defects similar to nimO18. Inducible nimO alleles lacking as many as 244 C-terminal amino acids supported hyphal growth, but not asexual development, when overexpressed in a ts-lethal nimO18 strain. However, the truncated alleles could not rescue a nimO deletion, indicating that the C terminus is essential and suggesting some type of interaction among nimO polypeptides.

Amino Acid Sequence↗
PubMed1998

BIMAAPC3, a component of the Aspergillus anaphase promoting complex/cyclosome, is required for a G2 checkpoint blocking entry into mitosis in the absence of NIMA function.

Temperature sensitive (ts) nimA mutants of Aspergillus nidulans arrest at a unique point in G2 which is post activation of CDC2. Here we show that this G2 arrest is due to loss of nimA function and that it is dependent on BIMAAPC3, a component of the anaphase promoting complex/cyclosome (APC/C). Whereas nimA single mutants arrested in G2 with decondensed chromatin and interphase microtubule arrays, nimA, bimAAPC3 double mutants arrested growth with condensed chromatin and aster-like microtubule arrays. nimA, bimAAPC3 double mutants entered mitosis with kinetics similar to bimAAPC3 single mutants and wild-type cells, indicating a checkpoint-like role for BIMAAPC3 in G2. Even cells which had been depleted for NIMA protein and which contained insignificant levels of NIMA kinase activity entered mitosis on inactivation of bimAAPC3. BIMAAPC3 was present in a >25S complex containing BIMEAPC1, and bimAAPC3 mutants were sensitive to elevated CYCLIN B expression, consistent with BIMAAPC3 being a component of the APC/C. Inactivation of bimAAPC3 had little affect on the steady state levels of the B-type cyclin, NIMECyclin B. Our results indicate that BIMAAPC3, and most likely the APC/C itself, is activated in G2 in nimA mutants. We propose that APC/C activation is part of a novel, late G2 checkpoint, which responds to a defective process or structure in nimA mutants, and which prevents inappropriate entry into mitosis.

Alleles↗
PubMed1997

Proteolysis and tyrosine phosphorylation of p34cdc2/cyclin B. The role of MCM2 and initiation of DNA replication to allow tyrosine phosphorylation of p34cdc2.

Previously, it has been shown that Aspergillus cells lacking the function of nimQ and the anaphase-promoting complex (APC) component bimEAPC1 enter mitosis without replicating DNA. Here nimQ is shown to encode an MCM2 homologue. Although mutation of nimQMCM2 inhibits initiation of DNA replication, a few cells do enter mitosis. Cells arrested at G1/S by lack of nimQMCM2 contain p34(cdc2)/cyclin B, but p34(cdc2) remains tyrosine dephosphorylated, even after DNA damage. However, arrest of DNA replication using hydroxyurea followed by inactivation of nimQMCM2 and bimEAPC1 does not abrogate the S phase arrest checkpoint over mitosis. nimQMCM2, likely via initiation of DNA replication, is therefore required to trigger tyrosine phosphorylation of p34(cdc2) during the G1 to S transition, which may occur by inactivation of nimTcdc25. Cells lacking both nimQMCM2 and bimEAPC1 are deficient in the S phase arrest checkpoint over mitosis because they lack both tyrosine phosphorylation of p34(cdc2) and the function of bimEAPC1. Initiation of DNA replication, which requires nimQMCM2, is apparently critical to switch mitotic regulation from the APC to include tyrosine phosphorylation of p34(cdc2) at G1/S. We also show that cells arrested at G1/S due to lack of nimQMCM2 continue to replicate spindle pole bodies in the absence of DNA replication and can undergo anaphase in the absence of APC function.

Amino Acid Sequence↗
PubMed1995

The Aspergillus nidulans bimE (blocked-in-mitosis) gene encodes multiple cell cycle functions involved in mitotic checkpoint control and mitosis.

The bimE (blocked-in-mitosis) gene appears to function as a negative mitotic regulator because the recessive bimE7 mutation can override certain interphase-arresting treatments and mutations, causing abnormal induction of mitosis. We have further investigated the role of bimE in cell cycle checkpoint control by: (1) coordinately measuring mitotic induction and DNA content of bimE7 mutant cells; and (2) analyzing epistasis relationships between bimE7 and 16 different nim mutations. A combination of cytological and flow cytometric techniques was used to show that bimE7 cells at restrictive temperature (44 degrees C) undergo a normal, although somewhat slower cell cycle prior to mitotic arrest. Most bimE7 cells were fully reversible from restrictive temperature arrest, indicating that they are able to enter mitosis normally, and therefore require bimE function in order to finish mitosis. Furthermore, epistasis studies between bimE7 and mutations in cdc2 pathway components revealed that the induction of mitosis caused by inactivation of bimE requires functional p34cdc2 kinase, and that mitotic induction by bimE7 depends upon several other nim genes whose functions are not yet known. The involvement of bimE in S phase function and mitotic checkpoint control was suggested by three lines of evidence. First, at restrictive temperature the bimE7 mutation slowed the cell cycle by delaying the onset or execution of S phase. Second, at permissive temperature (30 degrees C) the bimE7 mutation conferred enhanced sensitivity to the DNA synthesis inhibitor hydroxyurea. Finally, the checkpoint linking M phase to the completion of S phase was abolished when bimE7 was combined with two nim mutations that cause arrest in G1 or S phase. A model for bimE function based on these findings is presented.

Aspergillus nidulans↗
PubMed1993

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii confers resistance to anti-microtubule herbicides.

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii was isolated by using the amiprophos-methyl-resistant mutation apm1-18 as a background to select new mutants that showed increased resistance to the drug. The upA12 mutation caused twofold resistance to amiprophos-methyl and oryzalin, and twofold hypersensitivity to the microtubule-stabilizing drug taxol, suggesting that the mutation enhanced microtubule stability. The resistance mutation was semi-dominant and mapped to the same interval on linkage group III as the alpha 1-tubulin gene. Two-dimensional gel immunoblots of proteins in the mutant cells revealed two electrophoretically altered alpha-tubulin isoforms, one of which was acetylated and incorporated into microtubules in the axoneme. The mutant isoforms co-segregated with the drug-resistance phenotypes when mutant upA12 was backcrossed to wild-type cells. Two-dimensional gel analysis of in vitro translation products showed that the non-acetylated variant alpha-tubulin was a primary gene product. DNA sequence analysis of the alpha 1-tubulin genes from mutant and wild-type cells revealed a single missense mutation, which predicted a change in codon 24 from tyrosine in wild type to histidine in mutant upA12. This alteration in the predicted amino acid sequence corroborated the approximately +1 basic charge shift observed for the variant alpha-tubulins. The mutant allele of the alpha 1-tubulin gene was designated tua1-1.

Acetylation↗
PubMed1992

Mitotic regulation in Aspergillus nidulans.

The nimA and bimE genes of Aspergillus nidulans respectively encode a 79 kDa protein kinase that is a positive regulator of mitosis and a 229 kDa protein that is a negative regulator of mitosis. Either overproduction of nimA or inactivation of bimE can induce mitosis and override the checkpoint associated with incomplete DNA replication. Double mutants between temperature-sensitive nimA and bimE alleles undergo chromatin condensation and spindle polymerization at restrictive temperature, suggesting that the p79nimA kinase is not required for chromatin condensation and spindle polymerization when bimE function is defective. In contrast double mutants carrying ts bimE and nimEcyclinB mutations or bimE and nimTcdc25 mutations are blocked in interphase at restrictive temperature. These results indicate that the mitotic block caused by inactivation of bimE requires activation of the p34cdc2/MPF kinase for chromatin condensation and spindle polymerization to occur. Antibodies against bimE fusion proteins have been used to study p229bimE in wild-type cells and cells overexpressing the bimE gene.

Aspergillus nidulans↗
PubMed1992

Genetic interactions among Chlamydomonas reinhardtii mutations that confer resistance to anti-microtubule herbicides.

We previously described two types of genetic interactions among recessive mutations in the APM1 and APM2 loci of Chlamydomonas reinhardtii that may reflect a physical association of the gene products or their involvement in a common structure/process: (1) allele-specific synthetic lethality, and (2) unlinked noncomplementation, or dominant enhancement. To further investigate these interactions, we isolated revertants in which the heat sensitivity caused by the apm2-1 mutation is lost. The heat-insensitive revertants were either fully or partially suppressed for the drug-resistance caused by the apm2-1 allele. In recombination tests the revertants behaved as if the suppressing mutation mapped within the APM2 locus; the partial suppressors of apm2-1 herbicide resistance failed to complement apm2-1, leading to the conclusion that they were likely to be intragenic pseudorevertants. The apm2-1 partial suppressor mutations reversed apm1-apm2-1 synthetic lethality in an allele-specific manner with respect both to apm1- alleles and apm2-1 suppressor mutations. Those apm1- apm2-1rev strains that regained viability also regained heat sensitivity characteristic of the original apm2-1 mutation, even though the apm2-1 suppressor strains were fully heat-insensitive. The Hs+ phenotypes of apm2-1 partial suppressors were also reversed by treatment with the microtubule-stabilizing agent deuterium oxide (D2O). In addition to the above interactions, we observed interallelic complementation and phenotypic enhancement of temperature conditionality among apm1- alleles. Evidence of a role for the products of the two genes in microtubule-based processes was obtained from studying flagellar assembly in apm1- and apm2- mutants.

Alleles↗
PubMed1989

Isolation and characterization of dominant, pleiotropic drug-resistance mutants in Chlamydomonas reinhardtii.

Three independent pleiotropic drug-resistance (pdr) mutants were isolated by selecting for resistance to the anti-microtubule herbicides amiprophos-methyl (APM) and oryzalin (ORY). These three mutants and a previously isolated mutant, ani1 (anisomycin resistance), were semi-dominant in heterozygous diploids, and they displayed varying degrees of resistance to structurally and functionally unrelated inhibitors such as cycloheximide, cryptopleurine, emetine, atrazine, and nonidet P-40. Linkage analysis and genetic mapping suggested that three of the four mutants, including ani1, define a single locus, here named pdr1. The fourth mutant defined a new locus, pdr2, which is located on the left arm of linkage group VI. One pdr1 mutant exhibited unusual genetic interactions, including enhanced ts-lethality and synergistic increases in drug resistance, when combined with pdr2-1 and with herbicide-resistant alleles of three other genes.

Anisomycin↗
PubMed1989

Extragenic suppression and synthetic lethality among Chlamydomonas reinhardtii mutants resistant to anti-microtubule drugs.

The antimicrotubule agents oryzalin (ORY), colchicine (COL) and taxol (TAX) were used to select three recessive, conditional lethal (ts-) mutants which defined two new essential loci, ory1 and cor1. The two ory1 mutants conferred resistance to ORY, TAX, and COL; the cor1 mutant conferred resistance only to COL. Each of the mutants displayed wild-type sensitivity to a number of unrelated inhibitors. Assembly and disassembly of flagellar microtubules in the ory1 mutants displayed wild-type sensitivity to ORY and COL, suggesting that the ory1 gene product either does not participate in these processes or the ory1 gene product alone is not sufficient to confer resistance. The ory1 locus mapped to linkage group X; cor1 was mapped to the left arm of linkage group XII. A synthetic lethal interaction was observed between ory1 and cor1 mutations, i.e., inferred ory1 cor1 double mutants were inviable at the permissive temperature. The conditional lethal phenotype of ory1-1 was used to select many spontaneous TS+ revertants, which arose at high frequencies. Genetic and phenotypic characterization of the revertants demonstrated that (1) the revertants fell into four phenotypic classes, including some which conferred supersensitivity to ORY and others which conferred cold-sensitive lethality, (2) reversion was caused in most or all cases by extragenic suppressors, (3) suppressor mutations displayed complex behavior in heterozygous (sup/+) diploids, (4) many different loci may be capable of suppressing ory1 mutants, and (5) suppressors of ory1-1 efficiently suppressed an independently isolated allele, ory1-2. Taken together the ory1, cor1, and suppressor mutations identify a number of interacting loci involved in essential cellular processes which are specifically susceptible to antimicrotubule agents.

Alkaloids↗
PubMed1988

Mutants resistant to anti-microtubule herbicides map to a locus on the uni linkage group in Chlamydomonas reinhardtii.

We have used genetic analysis to study the mode of action of two anti-microtubule herbicides, amiprophos-methyl (APM) and oryzalin (ORY). Over 200 resistant mutants were selected by growth on APM- or ORY-containing plates. The 21 independently isolated mutants examined in this study are 3- to 8-fold resistant to APM and are strongly cross-resistant to ORY and butamiphos, a close analog of APM. Two Mendelian genes, apm1 and apm2, are defined by linkage and complementation analysis. There are 20 alleles of apm1 and one temperature-sensitive lethal (33 degrees) allele of apm2. Mapping by two-factor crosses places apm1 6.5 cM centromere proximal to uni1 and within 4 cM of pf7 on the uni linkage group, a genetically circular linkage group comprising genes which affect flagellar assembly or function; apm2 maps near the centromere of linkage group VIII. Allele-specific synthetic lethality is observed in crosses between apm2 and alleles of apm1. Also, self crosses of apm2 are zygotic lethal, whereas crosses of nine apm1 alleles inter se result in normal germination and tetrad viability. The mutants are recessive to their wild-type alleles but doubly heterozygous diploids (apm1 +/+ apm2) made with apm2 and any of 15 apm1 alleles display partial intergenic noncomplementation, expressed as intermediate resistance. Diploids homozygous for mutant alleles of apm1 are 4-6-fold resistant to APM and ORY; diploids homozygous for apm2 are ts- and 2-fold resistant to the herbicides. Doubly heterozygous diploids complement the ts- phenotype of apM2, but they are typically 1.5-2-fold resistant to APM and ORY. From the results described we suggest that the gene products of apm1 and apm2 may interact directly or function in the same structure or process.

Animals↗