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Biomedical subjects

M L Smith

Publications and source records attributed to M L Smith.

At least 145 records · Page 8Linked to original sources
PubMed1996

Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to u.v.-irradiation or cisplatin.

Loss of p53 function in cancer cells commonly results in a condition of genomic instability. This is believed to emanate from a loss of the G1 checkpoint response to DNA damage. While the role of p53 in the induction of a G1 arrest is well-accepted, additional p53 functions are being discovered. Cell cycle checkpoints presumably function to allow additional time for DNA repair after damage is incurred, however, genetic studies in yeast suggest that components of the checkpoint pathway may also be involved in DNA lesion processing (Lydall and Weinert, 1995). Recent evidence suggests that this may also be the case for p53, as suggested by numerous reports linking p53 function to DNA repair. Thus, loss of p53 function might contribute to genomic instability independent of G1-arrest. In the present study, we explored the effect of p53 disruption and consequences of antisense GADD45 expression on the DNA repair capacity of human colon carcinoma RKO cells. DNA repair was assayed using host-cell reactivation of u.v.-damaged reporter plasmids and unscheduled DNA synthesis experiments in transiently-transfected cells. We show that a number of transfected genes that suppress p53 function reduce the ability of cells to repair u.v.-induced DNA damage. Moreover, cells in which expression of the p53-regulated gene GADD45 was blocked by antisense vectors, also showed altered levels of DNA repair. Blocking Gadd45 expression by constitutive antisense expression sensitized cells to killing by u.v.-radiation or by cis-platinum (II) diamine-dichloride (CDDP, or cisplatin), a cancer chemotherapy drug which produces DNA cross-links. These findings suggest the involvement of downstream effectors of the p53 pathway in the coordination of cell cycle arrest and DNA repair.

Antineoplastic Agents↗
PubMed1996

Characterization of the GADD45 response to ionizing radiation in WI-L2-NS cells, a p53 mutant cell line.

We have previously reported that WI-L2-NS, a human lymphoblastoid cell line, has very high basal levels of GADD45 mRNA and protein in spite of a p53 mutation at amino acid 237. Regardless of the amount of Gadd45 in this cell line, no growth suppression activity was detected. We report here that in WI-L2-NS, the mutated p53 protein adopts predominantly a wild type (wt) conformation and binds to the p53 binding site in the GADD45 third intron. In this cell line, the already high levels of mutated p53 protein can be induced further by ionizing radiation (IR) but the response of the p53 downstream effector genes is altered. Induction of GADD45 and CIP1/WAF1 is reduced compared to p53 wt cell lines but is still substantially higher than the average fold induction obtained from 39 p53 mutant cell lines. Induction of the MDM2 gene was not detected in WI-L2-NS following IR. The induction pattern of the three p53 effector genes by the alkylating agent methylmethane sulfonate (MMS) was also attenuated in WI-L2-NS cells. In TK6 cells, a WI-L2-NS sister cell line having a p53 wt genotype, the induction of the p53 downstream effectors is normal, i.e. induced, both at the protein and the mRNA levels. These results indicate that the DNA binding activity of the mutated p53 protein in WI-L2-NS might be responsible, at least in part, for the high basal levels of GADD45 but can not mediate the full induction of the p53 downstream effector genes. The reason(s) for the inability of Gadd45 to suppress growth in this cell line remains however unknown.

Alkylating Agents↗
PubMed1996

Reflex control of sympathetic activity during ventricular tachycardia in dogs: primary role of arterial baroreflexes.

BACKGROUND: The determinants of hemodynamic outcome during ventricular tachycardia (VT) are not well understood. In the present study, we addressed the relative contributions of arterial and cardiopulmonary baroreflexes to the sympathetic and arterial pressure responses to VT or ventricular pacing (VP) in dogs with inducible VT. METHODS AND RESULTS: Responses of renal sympathetic nerve activity (RSNA), pulmonary capillary wedge pressure (PCWP), and mean arterial pressure (MAP) to induced VT or VP (220 to 280 beats per minute) were determined in 12 dogs with a healed anteroapical infarction and inducible VT and in 8 control dogs. The responses were determined with all reflexes intact, after selective denervation of either arterial or cardiopulmonary baroreflexes, and after combined denervation. Differences between intact and denervated conditions were used to assess the relative effects of each baroreflex. In the infarct group, responses during VT were comparable to those during VP. RSNA and PCWP increased significantly (P<.01), whereas MAP decreased significantly (P<.001) during VT or VP with baroreflexes intact in both groups. The increase in RSNA and the recovery of MAP during sustained VP were greater in the infarct group (P<.05); in addition, the increase in PCWP was greater in the infarct group (P<.05). Arterial baroreflex denervation abolished the increased RSNA and recovery of MAP during VP in both groups. After cardiopulmonary baroreflex denervation, the increase in RSNA was augmented in both groups (control group more than infarct group), but recovery of MAP was increased further only in the control group. CONCLUSIONS: These results suggest that arterial baroreflex mediated sympathoexcitation plays an important role in determining the hemodynamic outcome during VT, whereas cardiopulmonary baroreflexes play only a modest modulatory role.

Animals↗
PubMed1996

An abnormality in the p53 pathway following gamma-irradiation in many wild-type p53 human melanoma lines.

DNA-damaging agents such as ionizing radiation (IR) activate the tumor suppressor p53, and, in turn, p53 transactivates a number of downstream effector genes such as GADD45, CIP1/WAF1, and MDM2. The induction of these downstream genes following IR appears to be strictly dependent upon the presence of wild-type functional p53 known to evoke G1 arrest. In this study, we characterized 56 cell lines from 9 different tumor types with predetermined p53 genotype by measuring the induction of GADD45, CIP1/WAF1, and MDM2 relative mRNA levels after IR. A higher fraction of melanoma lines had wild-type (wt) p53 (5/8, or 63%) compared to the nonmelanoma lines (11/48, or 23%). Most wt p53 (nonmelanoma) cell lines (11/12, or 92%) showed clear induction of both GADD45 and CIP1/WAF1. On the other hand, many wt p53 melanoma lines (4/5, or 80%) showed normal induction of CIP1/WAF1, but little or no induction of GADD45. Despite this defect in GADD45 induction, we found that all wt p53 melanoma lines exhibited strong G1 arrest and increased levels of p53 protein after IR. The results demonstrated that radiation-induced G1 arrest could occur by the p53-CIP1/WAF1 pathway without appreciable induction of GADD45 in melanoma lines. Time course experiments demonstrated prolonged induced expression of CIP1/WAF1 mRNA transcripts in melanoma lines in which GADD45 induction was lacking, suggesting some sort of compensatory mechanism involving CIP1/WAF1, in cell lines with defective GADD45 induction. We could reproduce this compensatory effect in RKO colon carcinoma cells in which GADD45 expression was blocked by constitutive antisense vectors. These findings reveal that defective induction of GADD45 following IR is common in human melanoma cell lines.

Base Sequence↗
PubMed1996

Characterization of p21Cip1/Waf1 peptide domains required for cyclin E/Cdk2 and PCNA interaction.

The cyclin-dependent kinase inhibitor p21Cip1/Waf1 is responsible for the p53-dependent growth arrest of cells in G1 phase following DNA damage. In the present study we investigated regions of p21 involved in inhibition of the G1/S phase cyclin-dependent kinase, cyclin E/Cdk2, as well as regions of p21 important for binding to this kinase and recombinant PCNA. To perform these studies we synthesized a series of overlapping peptides spanning the entire p21 sequence and used them in in vitro assays with cyclin E/Cdk2-immune complexes and with recombinant p21 and PCNA proteins. One amino-terminal p21 peptide spanning amino acids 15-40, antagonized p21 binding and inhibition of cyclin E/Cdk2 kinase. Antagonism of p21 binding was, however, lost in a similar peptide lacking amino acids 15-20, or in a peptide in which cysteine-18 was substituted for a serine. These results suggest that this peptide region is important for p21 interaction with cyclin E/Cdk2. A second peptide (amino acids 58-77) also antagonized p21-activity, but this peptide did not affect the ability of p21 to interact with cyclin E/Cdk2. A region of p21 larger than 26 amino acids is presumably required for Cdk-inhibition because none of the peptides we tested inhibited cyclin E/Cdk2. We also found that a peptide spanning amino acids 21-45 bound recombinant p21 in ELISA assays, and additional studies revealed a requirement for amino acids 26 through 45 for this interaction. A p21 peptide spanning amino acids 139-164 was found to bind PCNA in a filter binding assay and this peptide suppressed recombinant p21-PCNA interaction. Conformational analysis revealed that peptides spanning amino acids 21-45 and 139-164 tended towards an alpha-helical conformation in trifluoroethanol buffer, indicating that these regions are probably in a coiled conformation in the native protein. Taken together, our results provide an insight into domains of p21 that are involved in cyclin E/Cdk2 and PCNA interaction. Our results also suggest that a potential p21 dimerization domain may lie in the amino-terminus of p21. Continued exploration of these domains could prove useful in assessing p21-mimetic strategies for cancer treatment.

Amino Acid Sequence↗
PubMed1996

Role of hypoxemia in sleep apnea-induced sympathoexcitation.

The importance of hypoxemia in determining sympathoexcitation during obstructive sleep apnea was examined by comparing changes in efferent sympathetic nerve activity (SNA) during spontaneous obstructive apneas with hypoxemia alone of similar magnitude and duration induced by 1-4 breaths of 100% nitrogen in six patients with obstructive sleep apnea and with spontaneous apneas while breathing 100% oxygen (apnea without hypoxemia) in three patients. In addition, eight control subjects were studied during induced hypoxemia. The magnitude of sympathoexcitation during spontaneous apneas (103 +/- 15%) was more than twice that observed during induced hypoxemia (47 +/- 14%) during episodes in which the nadir of oxygen desaturation (78 +/- 2 and 75 +/- 2%, respectively) and duration of hypoxemia (27 +/- 3 and 33 +/- 3 s, respectively) were the same (P > 0.20). Similarly, in three patients SNA increased 115% during normoxic spontaneous obstructive apneas, but increased only 43% during hyperoxic spontaneous obstructive apneas in which oxygen saturation did not decrease significantly. Sympathetic neural responses to induced hypoxemia in control subjects (17 +/- 7%) were significantly less than that of the sleep apnea patients. We conclude that hypoxemia contributes importantly, but is not the sole determinant of the sympathoexcitation provoked during episodes of obstructive sleep apnea.

Adult↗
PubMed1996

Microsurgery in a rat lung transplant model: analysis of benefit.

With the use of microsurgery, we have developed a method of measuring hemodynamic parameters in a rat not possible with previous technology. Three groups of rats were studied: a chemically induced pulmonary hypertensive group (PH); a chemically induced pulmonary hypertensive group treated with single lung transplantation (LT); and an untreated, control group (C). Cardiac output, heart rate, and pulmonary vascular resistance were then calculated in each group from data obtained by 1 mm high fidelity micromanometers and an ultrasonic flow probe. The results show that the data collected from the rodent model are reproducible within each group, and data quality is comparable to large animal models. With this new method, data can be collected in a small animal model at a fraction of the time and cost of large animal studies. Additionally, the complications of graft rejection in large animal studies are eliminated in an isogenic rodent model.

Animals↗
PubMed1996

Immunodetection of squamous cell carcinoma-associated variant of nuclear protein.

A new monoclonal antibody, MAb C63.3, was developed by immunizing mice with the high molecular weight fraction of the cytoplasmic proteins from the cervical squamous cell carcinoma. In Western blotting, MAb C63.3 reacted with cytoplasmic and chromatin antigens expressed by cervical and vulvar squamous cell carcinomas and much weaker with normal cervical tissue and skin cells. Different molecular variants of C63.3 chromatin antigens were found in normal tissues (heavy variants, M(r) 65,000 and 59,000) compared to squamous cell carcinomas (light variant, M(r) 48,000). Diagnostic value of the cancer-associated M(r) 48,000 antigen is discussed. MAb C63.3 was internalized by cells and bound to the chromatin which suggests that antigen(s) C63.3 might represent a receptor for a yet unknown ligand (growth factor). It is suggested that the high molecular weight C63.3 antigens play a role in maintaining the nonmalignant phenotype.

Animals↗
PubMed1996

Mapping translocation breakpoints by orthogonal field agarose-gel electrophoresis.

Orthogonal field agarose-gel electrophoresis (OFAGE) of chromosomes from translocation-bearing and normal Neurospora crassa strains was utilized, first, to recover cosmids from a translocated region, and second, to map translocation breakpoints. Surprisingly, the right breakpoints in two independently derived, interstitial translocations, T(II-->III) AR18 and T(II-->VI)P2869, are within about 5.6 kbp of each other suggesting that this region of linkage group (LG) II may be fragile or otherwise subject to chromosome breakage. Mapping translocation breakpoints through OFAGE, or other similar methods, should allow for DNA sequencing across breakpoints that are not associated with mutant phenotypes or that are not within walking distance of cloned markers.

Centromere↗
PubMed1996

Recall of frequency of occurrence of self-generated and examiner-provided words after frontal or temporal lobectomy.

Patients with unilateral excisions from the frontal or temporal lobe and normal control subjects were given two tasks requiring the recall of the frequency of occurrence of words that differed in the number of times they occurred in study lists. In one task, subjects were required to generate words in response to cues, whilst in the other they merely had to read responses that satisfied the cues. All subject groups gave higher frequency estimates to words they had generated than to words they had read. Across both tasks, the frontal-lobe groups (left more so than right) underestimated frequency of occurrence.

Adolescent↗
PubMed1996

Regional brain-derived neurotrophic factor mRNA and protein levels following transient forebrain ischemia in the rat.

Levels of BDNF mRNA and protein were measured in the rat brain using in situ hybridization and a two-site enzyme immunoassay. Under basal conditions, the highest BDNF concentration was found in the dentate gyrus (88 ng/g), while the levels in CA3 (50 ng/g), CA1 (18 ng/g) and parietal cortex (8 ng/g) were markedly lower. Following 10 min of forebrain ischemia, BDNF protein increased transiently in the dentate gyrus (to 124% of control at 6 h after the insult) and CA3 region (to 131% of control, at 1 week after the insult). In CA1 and parietal cortex, BDNF protein decreased to 73-75% of control at 24 h. In contrast, BDNF mRNA expression in dentate granule cells and CA3 pyramidal layer was transiently elevated to 287 and 293% of control, respectively, at 2 h, whereas no change was detected in CA1 or neocortex. The regional BDNF protein levels shown here correlate at least partly with regional differences in cellular resistance to ischemic damage, which is consistent with the hypothesis of a neuroprotective role of BDNF.

Animals↗
PubMed1996

Enalaprilat augments arterial and cardiopulmonary baroreflex control of sympathetic nerve activity in patients with heart failure.

OBJECTIVES: This study sought to determine the effects of enalaprilat on reflex control of sympathetic nerve activity. BACKGROUND: Angiotensin-converting enzyme inhibitors decrease mortality in patients with congestive heart failure. Their efficacy appears to be related importantly to antiadrenergic effects, the mechanism for which has not been determined. Because baroreflexes tonically inhibit sympathetic outflow, and baroreflexes are blunted in heart failure, we hypothesized that these agents reduce sympathetic activity by augmenting baroreflexes. METHODS: We assessed baroreflex control of sympathetic nerve activity and heart rate in patients with congestive heart failure and in control subjects before and after enalaprilat (0.02 mg/kg body weight intravenously). Arterial baroreflexes were perturbed by bolus administration of sodium nitroprusside and phenylephrine. Cardiopulmonary baroreflexes were perturbed by lower body negative pressure and head-down tilt. Muscle sympathetic nerve activity was recorded by microneurography. RESULTS: Enalaprilat decreased systolic blood pressure in patients with heart failure and control subjects. Sympathetic nerve activity increased in control subjects but decreased in patients with heart failure after enalaprilat despite reductions in central venous pressure in this group. Baroreflex control of sympathetic nerve activity was unchanged by enalaprilat in control subjects. In patients with heart failure, both arterial and cardiopulmonary baroreflex control of sympathetic nerve activity was enhanced by enalaprilat. Baroreflex control of heart rate was unchanged by enalaprilat in either group. CONCLUSIONS: Enalaprilat augments both arterial and cardiopulmonary baroreflex control of sympathetic activity in heart failure. These augmented inhibitory influences are associated with a reduction in sympathetic outflow and may contribute to the beneficial effects of angiotensin-converting enzyme inhibitors in heart failure.

Adult↗
PubMed1996

Determination of 207Pb2+ chemical shift tensors from precise powder lineshape analysis.

207Pb solid state NMR powder spectra at 296 K are presented for PbSO4, PbMoO4, PbCrO4, PbCO3, PbTiO3, PbZrO3, Pb(NO3)2, Pb(SCN)2, and PbS. Analysis for principal values of the anisotropic chemical shift tensors of the generally very broad spectra included the frequency dependent excitation of the pulse sequence used. Commonly used solid and liquid secondary shift standards for lead were studied with high precision as a function of temperature between 295 K and 315 K to establish a clean 207Pb shift scale. Errors in the existing literature are discussed.

Chemical Phenomena↗
PubMed1996

Mammalian DNA damage-inducible genes associated with growth arrest and apoptosis.

Mammalian cells are exposed to a wide variety of genotoxic stresses from both endogenous and exogenous sources. Cells typically exhibit cell cycle delays, or checkpoints, in response to acute genotoxic stress. Other types of cellular responses to DNA damage include apoptosis and probably increases in DNA repair levels. These response pathways are altered in cancer cells, by genetic alterations such as overexpression or mutation of oncogenes, or loss of tumor suppressor gene functions. As cancer chemotherapy relies primarily on the selective killing of cancer cells by DNA-damaging agents, genetic alterations affecting cellular stress response pathways may affect the outcome of cancer treatment.

Animals↗