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Biomedical subjects

T Hunt

Publications and source records attributed to T Hunt.

At least 55 records · Page 3Linked to original sources
PubMed1998

Evaluation of mechanism of increased intracranial pressure with insufflation.

BACKGROUND: Previous studies have documented an increase in intracranial pressure with abdominal insufflation, but the mechanism has not been explained. METHODS: Nine 30-35-kg domestic pigs underwent carbon dioxide insufflation at 1.5 l/min. Intracranial pressure (ICP), lumbar spinal pressure (LP), central venous pressure (CVP), inferior vena cava pressure (IVCP), heart rate, systemic arterial blood pressure, pulmonary arterial pressure, cardiac output, heart rate, respiratory rate, temperature, and end-tidal CO2 were continuously measured. Mechanical ventilation was used to maintain a constant pCO2. Measurements were recorded at 0, 5, 10, and 15 mmHg of abdominal pressure with animals in supine, Trendelenburg (T), and reverse Trendelenburg (RT) positions. Prior to recording measurements, the animals were allowed to stabilize for 40 min after each increase in abdominal pressure and for 20 min after each position change. RESULTS: The animals showed a significant increase in ICP (mmHg) with each 5-mmHg increase in abdominal pressure (0 mmHg: 14 +/- 1.7; 5 mmHg: 19.8 +/- 2.3, p < 0.001; 10 mmHg: 24.8 +/- 2.5, p < 0.001; 15 mmHg: 29.8 +/- 4.7, p < 0.01). The ICP at 15 mmHg abdominal pressure increased further in the T position (39 +/- 4, p < 0.01). Insufflating in the RT position did not significantly reduce the increase in ICP. The IVCP (mmHg) increased with increased abdominal pressure (0 mmHg: 11.5 +/- 6.2, 15 mmHg: 22.1 +/- 3.5, p < 0.01). This increase correlated with the increase in ICP and LP (r of mean pressures >/=0.95). There was no significant change in CVP. CONCLUSIONS: This study suggests that care may be needed with laparoscopy in patients at risk for increased ICP due to head injury or a space occupying lesion. The mechanism of increased ICP associated with insufflation is most likely impaired venous drainage of the lumbar venous plexus at increased intraabdominal pressure. Further studies of cerebral spinal fluid movement during insufflation are currently underway to confirm this hypothesis.

Animals↗
PubMed1998

The pharmacokinetic effects of coadministration of morphine and trovafloxacin in healthy subjects.

BACKGROUND: Morphine and antibiotics are frequently coadministered in the surgical setting. These agents may interact, reducing the efficacy of the antibiotic or increasing the toxicity of morphine. It is therefore important to determine whether antibiotics that might be used for surgical prophylaxis have the potential to change the pharmacokinetics of morphine. It is equally important to learn whether morphine affects the plasma levels of antibiotics and thus may potentially influence their efficacy or tolerability. METHODS: This open, randomized, placebo-controlled, three-treatment, three-period cross-over study enrolled 19 healthy volunteers. Oral trovafloxacin (200 mg), a novel fluoroquinolone antibiotic, and intravenous morphine (0.15 mg/kg) were coadministered, and the effects on the pharmacokinetics of each drug and on changes in the pharmacologic action of morphine, estimated from its effects on respiratory rate and level of sedation, were examined. RESULTS: When trovafloxacin was coadministered with morphine, the half-life of trovafloxacin was unchanged; however, the ratio of the area under the serum concentration versus time curve (AUC(0-infinity)) estimates for trovafloxacin/morphine versus trovafloxacin/placebo was 63.8% (95% confidence interval [CI], 40.7% to 100.3%), indicating a 36% reduction in the bioavailability of trovafloxacin. The ratio of the mean maximum serum concentration (Cmax) estimates of trovafloxacin for the two treatments was 53.8% (95% CI: 36.1% to 80.1%), indicating a 46% reduction in Cmax. The time to Cmax was delayed by 4 hours. With trovafloxacin coadministration, there were no statistically significant changes in either the mean relative bioavailability of morphine or that of its metabolite, 6beta-glucuronide-morphine. Coadministration of trovafloxacin did not exacerbate the reduction in respiratory rate or increase the number of side effects associated with morphine administration. CONCLUSIONS: Coadministration of trovafloxacin and morphine reduces the bioavailability and maximum serum concentrations of trovafloxacin. However, elimination of oral trovafloxacin is not impaired, suggesting that the efficacy of trovafloxacin could be maintained in many patients who receive concomitant morphine. Morphine plasma levels and pharmacologic effects are not significantly altered by coadministration of trovafloxacin. Despite their similar metabolic pathways, the trovafloxacin/morphine combination neither exacerbates the respiratory depressant effects of morphine nor increases the frequency of side effects when compared with placebo/morphine treatment. These results suggest that the efficacy of trovafloxacin may be maintained when coadministered with morphine. Concurrent administration of trovafloxacin and morphine is unlikely to alter the pharmacologic effects of morphine.

Adolescent↗
PubMed1998

A measure of the mitotic index: studies of the abundance and half-life of p34cdc2 in cultured cells and normal and neoplastic tissues.

BACKGROUND: The cdc2 gene encodes a protein kinase, p34cdc2, that is essential for mitosis, and is present at high levels in dividing cells. Classical studies of the levels of this protein in dividing and resting cells used antibodies that cross-react with other members of the CDK family, in particular with CDK2. We have therefore re-examined the abundance of p34cdc2 in a variety of tissues and cell lines, using a highly specific, epitope-mapped monoclonal antibody that does not react with CDK2. RESULTS: We observed high levels of p34cdc2 in proliferating cells, especially those in neoplastic tissues. Cells that have withdrawn from the cell cycle have low or undetectable levels. At the end of mitosis, the level of p34cdc2 declines, with simple first-order kinetics, with a half-life which is never less than 6h and is more typically about 18h. The persistence of p34cdc2 after the last cell division is comparable to that of PCNA, a commonly used marker of proliferation. CONCLUSIONS: The immunochemical detection of p34cdc2 provides an accurate, reliable and meaningful measure of the proliferative activity of cells in tissues. We suggest that p34cdc2 should be considered as the most authentic molecular marker of the mitotic index.

3T3 Cells↗
PubMed1998

Endnote 3.0.

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Computer Communication Networks↗
PubMed1997

Ondansetron modulates GABA(A) current of rat central nervous system neurons.

We examined the effect of ondansetron, a 5-HT3 receptor antagonist, on the whole cell current response of freshly isolated hypothalamic and hippocampal neurons of rats to gamma-aminobutyric acid (GABA). The nystatin perforated patch technique was used to minimize run-down of the GABA current. While 1-150 microM ondansetron had no effect on membrane conductance, co-application with agonist reversibly depressed the maximal end GABA current. The concentration-response relation of GABA reveals a non-competitive mechanism. However, the inhibitory effect was more potent when ondansetron was co-applied with lower concentrations of GABA: i.e., the ondansetron concentration needed to depress the current induced by 5 microM GABA to half amplitude was 7 microM compared to 28 microM for the current induced by 10 microM GABA. Analysis of the current-voltage relationship with and without ondansetron indicated that the effect of ondansetron is not voltage dependent. Current-voltage relations also showed that the effect of ondansetron was not due to activation of a GABA-independent current because the reversal potentials were the same with and without ondansetron. The present data suggest that ondansetron's suppression of GABA-activated current may be the molecular basis of ondansetron-induced seizures observed in vivo.

Animals↗
PubMed1997

Fission yeast Cut2 required for anaphase has two destruction boxes.

The fission yeast Schizosaccharomyces pombe cut2(+) gene is essential for sister chromatid separation. Cut2 protein, which locates in the interphase nucleus and along the metaphase spindle, disappears in anaphase with the same timing as mitotic cyclin destruction. This proteolysis depends on the APC (Anaphase-Promoting Complex)-cyclosome which contains ubiquitin ligase activity. The N-terminus of Cut2 contains two stretches similar to the mitotic cyclin destruction box. We show that both sequences (33RAPLGSTKQ and 52RTVLGGKST) serve as destruction boxes and are required for in vitro polyubiquitination and proteolysis. Cut2 with doubly mutated destruction boxes inhibits anaphase, whereas Cut2 with singly mutated boxes can suppress cut2 mutations. Strong expression of the N-terminal 73 residues containing the destruction boxes leads to the accumulation of endogenous cyclin and Cut2, and arrests cells in metaphase, whereas the same fragment with the mutated boxes does not. Cut2 proteolysis occurs in vitro using Xenopus mitotic extracts in the presence of functional destruction boxes. Furthermore, Cut2 is polyubiquitinated in an in vitro system using HeLa extracts, and this polyubiquitination requires the destruction boxes.

Anaphase↗
PubMed1997

Cell cycle regulatory proteins--an overview with relevance to oral cancer.

The cell cycle is controlled by a number of highly conserved proteins, found in species as diverse as yeast and mammals. The study of these proteins is a rapidly advancing field that is increasing our understanding of normal and abnormal cell division. Disruption of the cell cycle has been demonstrated in several different types of neoplasm, and there is increasing evidence that, in head and neck tumours, there is aberrant control of cyclins, cell cycle protein kinases and their inhibitors. Because of the phase specificity of some of the control proteins, antibodies to them are proving to be of value in studying cell kinetics of both normal tissues and malignant tumours.

Animals↗
PubMed1997

Xenopus cyclin A1 can associate with Cdc28 in budding yeast, causing cell-cycle arrest with an abnormal distribution of nuclear DNA.

BACKGROUND: Cyclins play a regulatory role in cell cycle progression, associated with cyclin-dependent kinases. We have investigated the structure-function relationships of cyclin A, mainly using Xenopus egg extracts in vitro. To further analyse the function and structure of cyclin A in vivo, we expressed Xenopus cyclin A1 in the budding yeast Saccharomyces cerevisiae. RESULTS: We herein show that vertebrate cyclin A1 can associate with endogenous Cdc28 to form histone H1 kinase. The growth of the yeast was inhibited by the expression of indestructible cyclin A1, but not by a non-Cdk binding cyclin A1 mutant. The induction of cyclin A1 expression in yeast caused cell cycle arrest with an abnormal distribution of nuclear DNA to the daughter bud. Suppressors of the cyclin A1-mediated growth arrest were identified as new alleles of the cdc28 mutation that reduced the binding of cyclin A1 and possessed different affinities for the cyclin-Cdc28 complexes. The temperature-sensitivity of the cdc28 mutation was thus preferentially suppressed by the endogenous cyclins CLN2 and CLB2. CONCLUSIONS: These results suggest that the Cdc28 protein kinase activity mediated by vertebrate cyclin A1 may be involved in the process of nuclear movement in the yeast, and thereby affect the dependence of the M phase on the completion of the S phase through a preferential binding affinity of the cyclin-Cdc28 complex.

Amino Acid Sequence↗
PubMed1996

The role of proteolysis in cell cycle progression in Schizosaccharomyces pombe.

A cell-free system derived from Xenopus eggs was used to identify the 'destruction box' of the Schizosaccharomyces pombe B-type cyclin, Cdc13, as residues 59-67: RHALDDVSN. Expression of indestructible Cdc13 from a regulated promoter in S.pombe blocked cells in anaphase and inhibited septation, showing that destruction of Cdc13 is necessary for exit from mitosis, but not for sister chromatid separation. In contrast, strong expression of a polypeptide comprising the N-terminal 70 residues of Cdc13, which acts as a competitive inhibitor of destruction box-mediated proteolysis, inhibited both sister chromatid separation and the destruction of Cdc13, whereas an equivalent construct with a mutated destruction box did not. Appropriately timed expression of this N-terminal fragment of Cdc13 overcame the G1 arrest seen in cdc10 mutant strains, suggesting that proteins required for the initiation of S phase are subject to destruction by the same proteolytic machinery as cyclin.

Amino Acid Sequence↗
PubMed1996

The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase.

We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse-chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis. The destruction box-specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G0 and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells. Extracts of G1 cells degraded cyclin B1 whereas p27Kip1 was stable, in contrast, cyclin B1 remained stable and p27Kip1 was degraded in extracts of S phase cells.

3T3 Cells↗
PubMed1996

The 'destruction box' of cyclin A allows B-type cyclins to be ubiquitinated, but not efficiently destroyed.

The destruction of mitotic cyclins by programmed proteolysis at the end of mitosis is an important element in cell cycle control. This proteolysis depends on a conserved motif of nine residues known as the 'destruction box', which is located 40-50 residues from the N-terminus. The sequences of the A- and B-type destruction boxes are slightly different, which might account for the differences in timing of their destruction. When the cyclin A-type destruction box was substituted for the normal one in cyclin B1 or B2, however, the resulting constructs were unexpectedly stable, although the converse substitution of B-type destruction boxes in cyclin A permitted normal degradation. We compared the ubiquitination of various cyclin constructs, and found that whereas mutation of the highly conserved residues in the destruction box strongly reduced the level of ubiquitinated intermediates, the stable destruction box 'swap' constructs did form such adducts. Thus, while ubiquitination is probably necessary for cyclin destruction, it is not sufficient. We also found that poly-ubiquitinated cyclin derivatives are still bound to p34cdc2, which is not detectably ubiquitinated itself, raising the questions of how cyclin and cdc2 dissociate from one another, and at what stage, in the process of degradation.

Amino Acid Sequence↗
PubMed1996

Cut2 proteolysis required for sister-chromatid seperation in fission yeast.

Although mitotic cyclins are well-known substrates for ubiquitin-mediated proteolysis at the metaphase-anaphase transition, their degradation is not essential for separation of sister chromatids; several lines of evidence suggest that proteolysis of other protein(s) is required, however. Here we report the anaphase-specific proteolysis of the Schizosaccharomyces pombe Cut2 protein, which is essential for sister-chromatid separation. Cut2 is located in the nucleus, where it is concentrated along the short metaphase spindle. The rapid degradation of Cut2 at anaphase requires its amino-terminal region and the activity of Cut9 (ref. 14), a component of the 20S cyclosome/anaphase-promoting complex (APC), which is necessary for cyclin destruction. Expression of non-degradable Cut2 blocks sister-chromatid separation but not cell-cycle progression. This defect can be overcome by grafting the N terminus of cyclin B onto the truncated Cut2, demonstrating that the regulated proteolysis of Cut2 is essential for sister-chromatid separation.

Amino Acid Sequence↗
PubMed1996

The localization of p34cdc2 in the cells of normal, hyperplastic, and malignant epithelial and lymphoid tissues of the oral cavity.

The expression of p34cdc2 has been examined in normal, hyperplastic, and malignant oral epithelia and lymphoid tissues. Four monoclonal antibodies were prepared against Xenopus p34cdc2, three of which react specifically with human p34cdc2 and not with p33cdk2. These produced similar patterns of staining in both fixed and frozen sections of human material. Staining occurred mainly in the proliferative compartments of normal and hyperplastic tissues. In normal oral epithelia, parabasal and basal cells were the most strongly stained, with lighter cytoplasmic staining in lower prickle cells. In tonsillar germinal centres, a high proportion of cells was stained, with fewer positive cells in interfollicular zones, a distribution in keeping with the known pattern of cell proliferation. In normal cells, the intracellular location of p34cdc2 was cytoplasmic until early prophase, but in oral squamous cell carcinomas and lymphomas, it was located in both cytoplasm and nucleus during interphase and a larger fraction of cells was positive than in the equivalent normal tissues. Higher-grade neoplasms showed both a higher intensity of staining and a higher proportion of p34cdc2-positive cells.

Antibodies, Monoclonal↗
PubMed1996

Expression and subcellular localization of CDK2 and cdc2 kinases and their common partner cyclin A in thyroid epithelial cells: comparison of cyclic AMP-dependent and -independent cell cycles.

Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cyclic AMP as a second messenger for TSH. In tis early steps, this mitogenic control is quite distinct from cyclic AMP-independent mitogenic cascades elicited by growth factors. We demonstrate here that TSH (cyclic AMP) and EGF+serum (cyclic AMP-independent) stimulations cooperate and finally converge on proteins that control the cell cycle machinery. This convergence included a common induction of the expression of cyclin A and p34cdc2, and to a lesser extent of p33/38cdk2, which was already expressed in quiescent thyroid cells, and common changes of cdc2 and CDK2 phosphorylations as evidenced by electrophoretic mobility shifts. Kinetic differences in these processes after stimulation by TSH or EGF+serum or by these factors in combination correlated with differences in cell cycle kinetics. Moreover, an immunofluorescence analysis of these proteins using the double labeling of PCNA as a marker of each cell cycle phase shows: (1) a previously undescribed nuclear translocation of CDK2 before S phase initiation; (2) a sudden increase of cdc2 nuclear immunoreactivity at G2/mitosis transition. These data support the roles of CDK2 and cdc2 at G1/S and G2/mitosis transitions, respectively. (3) We were unable to demonstrate in individual cells a strict association between the nuclear appearance of cyclin A and G1/S transition, and an association of cyclin A and CDK2 with PCNA-stained DNA replication sites. On the other hand, the lengthening of G2 phase in the TSH/cyclic AMP-dependent thyroid cell cycle was associated with a stabilization of Tyr15 inhibitory phosphorylation of cdc2 and an especially high nuclear concentration of cyclin A and CDK2. We hypothesize that high nuclear accumulation of cyclin A and CDK2 during G2 phase could be causative in the cyclic AMP-dependent delay of mitosis onset.

Animals↗
PubMed1996

Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts.

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define 'S-phase promoting factor' (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

Animals↗