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Biomedical subjects

A J Siegel

Publications and source records attributed to A J Siegel.

At least 19 recordsLinked to original sources
PubMed2001

Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus.

We have examined the targeting of proliferating cell nuclear antigen (PCNA), an integral component of the mammalian replicative enzyme DNA polymerase delta, with sites of DNA replication by using confocal microscopy and computer image analysis. Labeling (5 min pulse) of DNA replication sites in normal human diploid fibroblast cells (NHF1) with BrdU was followed by immunostaining with PCNA antibodies. A striking degree of colocalization was seen between PCNA and the characteristic patterns of DNA replication sites of early, middle and late S-phase (Nakayasu and Berezney [1989] J. Cell. Biol. 108:1-11). These observations were confirmed by quantitative computer image analysis which revealed that approximately 90% of the PCNA-stained area overlapped with DNA replication sites in early S-phase. Pulse-chase experiments, involving in vivo labeling for replication followed by PCNA staining at later time points, suggested that PCNA disassembles from previously replicated sites and targets to newly active sites of DNA replication. To further study this phenomenon in living cells, stable GFP-PCNA transfectants under the control of a tetracycline-inducible promoter were created in mouse 3T6 cells. Like the endogenous PCNA, GFP-PCNA targeted to sites of replication (approximately 80% colocalization) and demonstrated similar dynamic changes following pulse-chase experiments in fixed cells. Studies of living cells revealed progressive changes in the GFP-PCNA distribution that mimic the replication patterns observed in fixed cells. We conclude that GFP-PCNA targets to DNA replication sites in living cells and is an effective marker for tracking the spatio-temporal dynamics of DNA replication as cells transverse the S-phase.

Animals↗
PubMed2001

Effects of cocaine on luteinizing hormone in women during the follicular and luteal phases of the menstrual cycle and in men.

Cocaine stimulates luteinizing hormone (LH) release in rhesus monkeys and in men, but its effects on LH in women are unknown. Cocaine (0.2 and 0.4 mg/kg i.v.) was administered to groups of follicular and luteal phase women (N = 22) and to men (N = 12) to examine the influence of gender and menstrual cycle phase on cocaine and LH interactions. All subjects met American Psychiatric Association Diagnostic and Statistical Manual IV criteria for cocaine abuse, and menstrual cycle phase was verified by estradiol and progesterone measures. Baseline LH levels were equivalent between groups. Peak cocaine levels did not differ significantly between men and women and averaged between 87 +/- 21 and 124 +/- 18 ng/ml after 0.2 mg/kg cocaine and between 227 +/- 22 and 287 +/- 21 ng/ml after 0.4 mg/kg cocaine. The lower dose of cocaine (0.2 mg/kg) significantly increased LH levels in men (P < 0.001) but not in women at either phase of the menstrual cycle. The higher dose of cocaine (0.4 mg/kg) stimulated significant increases in LH in men (P < 0.001) and in women at both phases of the menstrual cycle (P < 0.004-0.001). Although cocaine's effects on LH in women were dose-dependent, there were no significant differences as a function of menstrual cycle phase. LH remained significantly elevated longer in men (32 min) than in women (8 and 12 min). This gender difference in cocaine's potency in stimulating LH was unexpected.

Cocaine↗
PubMed1999

Cocaine-induced erythrocytosis and increase in von Willebrand factor: evidence for drug-related blood doping and prothrombotic effects.

BACKGROUND: Mechanisms that mediate cocaine-induced cardiovascular events following vasoconstriction are incompletely understood. OBJECTIVE: To examine the effects of cocaine in moderate doses on hematologic and hemostatic parameters that influence blood viscosity and thrombotic potential. METHODS: Changes in hemoglobin concentration, hematocrit, and red blood cell counts were measured in human subjects who met Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria for long-term cocaine abuse, before and sequentially after moderate intranasal and intravenous doses of cocaine. Hemostatic parameters, including von Willebrand factor, fibrinolytic activity, fibrinogen, plasminogen activator inhibitor antigen, and tissue-type plasminogen activator antigen, were sequentially measured after intravenous cocaine or saline placebo with cardiac troponin subunits T and I. RESULTS: Hemoglobin level (P= .002), hematocrit (P =.01), and red blood cell counts (P = .04) significantly increased from 4% to 6% over baseline from 10 to 30 minutes after intranasal (n = 14) and intravenous (n = 7) cocaine administration in doses of 0.9 mg/kg and 0.4 mg/kg, respectively, with no change in white blood cell or platelet counts. There was a significant increase (P =.03) in von Willebrand factor from 30 to 240 minutes, peaking at 40% over baseline following intravenous cocaine administration in a dose of 0.4 mg/kg (n = 12), with no change after 0.2 mg/kg (n = 3) or placebo (n = 6). Other hemostatic factors, creatinine, blood urea nitrogen, and cardiac troponin subunits T and I showed no changes. CONCLUSIONS: Cocaine induced a transient erythrocytosis that may increase blood viscosity while maintaining tissue oxygenation during vasoconstriction. An increase in von Willebrand factor without a compensatory change in endogenous fibrinolysis may trigger platelet adhesion, aggregation, and intravascular thrombosis.

Adult↗
PubMed1999

Association of chromosome territories with the nuclear matrix. Disruption of human chromosome territories correlates with the release of a subset of nuclear matrix proteins.

To study the possible role of the nuclear matrix in chromosome territory organization, normal human fibroblast cells are treated in situ via classic isolation procedures for nuclear matrix in the absence of nuclease (e.g., DNase I) digestion, followed by chromosome painting. We report for the first time that chromosome territories are maintained intact on the nuclear matrix. In contrast, complete extraction of the internal nuclear matrix components with RNase treatment followed by 2 M NaCl results in the disruption of higher order chromosome territory architecture. Correlative with territorial disruption is the formation of a faint DNA halo surrounding the nuclear lamina and a dispersive effect on the characteristically discrete DNA replication sites in the nuclear interior. Identical results were obtained using eight different human chromosome paints. Based on these findings, we developed a fractionation strategy to release the bulk of nuclear matrix proteins under conditions where the chromosome territories are maintained intact. A second treatment results in disruption of the chromosome territories in conjunction with the release of a small subset of acidic proteins. These proteins are distinct from the major nuclear matrix proteins and may be involved in mediating chromosome territory organization.

Ammonium Sulfate↗
PubMed1999

Chromosome movement during meiotic prophase in crane-fly spermatocytes: IV. Actin and the effects of cytochalasin D.

Cytochalasin D (CD) was applied to crane-fly spermatocytes at late diakinesis with the aim of perturbing actin structure and actin function, thereby testing the hypothesis that intranuclear chromosome movement during late diakinesis is actin-based. Isolated tests were incubated in a range of CD concentrations (2-100 microM) for 1 or 2 h. None of those treatments resulted in cessation of prophase movements in living cells. An immediate effect of 10-100 microM CD at late diakinesis was the formation of highly refractile, actin-containing cables within the nonchromosomal nucleoplasm. No such cables were observed in vehicle-treated control cells. CD treatments caused autosomal bivalents in unusually large numbers of spermatocytes to become aggregated into densely-packed clusters; for example, with 40 microM CD about 80% of late diakinesis spermatocytes had clustered autosomes, vs. about 25% clustering in untreated cells. We conclude from these data that the mechanism of chromosome positioning at the nuclear envelope is CD-sensitive. Rhodamine-conjugates of phalloidin and DNase I were used to assess the status of actin in untreated cells as well as the effect of CD on actin distribution. Differences in nucleoplasmic staining with phalloidin and DNase I conjugates suggest that nucleoplasm at late diakinesis contains actin in a nonfilamentous form.

Actins↗
PubMed1999

Cocaine pharmacokinetics in men and in women during the follicular and luteal phases of the menstrual cycle.

Preclinical and clinical studies suggest that females may be less vulnerable to cocaine's toxic effects than males. The pharmacokinetics of intravenous cocaine (0.2 and 0.4 mg/kg) were measured in 12 men and 22 women with a history of cocaine abuse, matched with respect to age and body mass index (BMI). Women were studied during the follicular and the luteal phases of the menstrual cycle. There were no differences between men and women in pharmacokinetic measures [peak plasma cocaine levels (Cmax), elimination half-life (T 1/2 min), area under the curve (AUC)] or cardiovascular or subjective effects "high" measures. Heart rate increases were cocaine dose-related (p < .01-.02) and also did not differ between men and women. Cocaine's pharmacokinetic and pharmacodynamic effects were similar in men and women, and in women during the follicular and mid-luteal phases of the menstrual cycle.

Adult↗
PubMed1998

Segregation of transcription and replication sites into higher order domains.

Microscopy shows that individual sites of DNA replication and transcription of mammalian nuclei segregate into sets of roughly 22 and 16 higher order domains, respectively. Each domain set displayed a distinct network-like appearance, including regions of individual domains and interdigitation of domains between the two networks. These data support a dynamic mosaic model for the higher order arrangement of genomic function inside the cell nuclei.

3T3 Cells↗
PubMed1998

Visualization of kinetochores and analysis of their refractility in crane-fly spermatocytes after aldehyde fixation.

Glutaraldehyde and formaldehyde were used to fix crane-fly spermatocytes for observation with differential interference contrast (DIC) microscopy. In aldehyde-fixed cells, kinetochores exhibit contrast not normally observed in living cells. Although the mechanism underlying this result is not understood, the visualization of kinetochores as distinct refractile objects opens the way for analysis of unstained kinetochores with the light microscope. The analysis of kinetochore refractility reported in this paper is made possible by the finding that the refractility of chromosomes in formaldehyde-fixed cells decreases as the concentration of formaldehyde is increased. In 4% formaldehyde, the refractility of chromosomes is matched with that of its surround, chromosomes appear invisible, and kinetochores may be analyzed as if chromosomes were not present. Kinetochores were imaged with DIC optics, and then digital image analysis was performed. Gray-level scans through the highlight and shadow of an individual kinetochore parallel to the axis of shear resulted in a curve having a slope proportional to the DIC optical path gradient. Curves from autosomal kinetochores imaged in anaphase had slopes approximately one-half those recorded at metaphase under identical optical conditions. By contrast, kinetochore thicknesses (defined as the distance between the peak and the valley of a gray-level scan) at those two stages were not significantly different. These data suggest a loss of dry mass from autosomal kinetochores during anaphase. Neither the refractility nor thickness of lagging sex kinetochores varied as autosomes went through anaphase. The conclusion drawn from these findings is that the decreased refractility of autosomal kinetochores in anaphase is movement-related.

Anaphase↗
PubMed1998

Cocaine administration induces human splenic constriction and altered hematologic parameters.

Cocaine is a potent vasoconstrictor that has been shown to alter hemoglobin, hematocrit, and red blood cell counts in both animals and humans. The present study evaluated whether cocaine administration induces splenic constriction in men and whether spleen-volume changes temporally correlate with altered hematologic parameters. Spleen volume was assessed at baseline and after cocaine administration (0.4 mg/kg) by using magnetic resonance imaging. A group of five healthy men, aged 31 +/- 2 (SE) yr and reporting occasional cocaine use (13 +/- 5 lifetime exposures), participated. Cocaine reduced spleen volume by 20 +/- 4% (P < 0.03) 10 min after drug administration. Spleen volume returned to normal (101 +/- 3% baseline) within 35 min after cocaine administration, indicating that the reduction is a transient phenomenon. In subjects administered cocaine from whom blood samples were obtained (n = 3), cocaine increased hemoglobin levels, hematocrit, and red blood cell count to 104.5 +/- 0.9, 105.6 +/- 1.2, and 106.5 +/- 1.0% of baseline levels, respectively (P < 0.03), but it did not alter white blood cell and platelet counts. Placebo administration (n = 5) did not alter hematologic parameters. These results suggest that cocaine induces splenic constriction in humans, and this may contribute to temporally concordant hematologic parameter changes. These events may help to preserve or increase tissue oxygenation in periods of high oxygen demand and/or increased vascular resistance.

Adult↗
PubMed1998

Concurrent pharmacokinetic analysis of plasma cocaine and adrenocorticotropic hormone in men.

The purpose of this study was to determine the covariance between plasma cocaine and ACTH pharmacokinetics. Twelve healthy male occasional cocaine users participated in a double blind study. Intravenous cocaine (0.2 mg/kg) or placebo was infused over 1 min, and samples for cocaine, ACTH and cortisol analysis were collected at 2, 4, 8, 12, 16, 20, 30, 40, 60, 80, 120, 180, and 240 min. Peak cocaine plasma levels averaged 101.2 +/- 14.6 ng/mL. ACTH increases were significantly correlated (P < 0.0001) with increases in plasma cocaine levels (r = 0.67; r2 = 0.44). Pharmacokinetic analysis showed that the t(max) (observed time to maximum concentration) values for cocaine (6.0 +/- 1.4 min) and ACTH (7.3 +/- 1.2 min) were almost identical. The area under the curve was calculated using the trapezoidal rule. The area under the curve for plasma cocaine was 6463 +/- 1070 ng/min x mL, and the area under the curve for ACTH was 1873 +/- 188 pmol/min x L. The mean half-life for plasma cocaine was 46.7 +/- 4.0 min, and that for ACTH was 35.8 +/- 5.1 min. Cardiovascular and subjective effect measures were correlated with concurrent increases in plasma cocaine and ACTH levels.

Adrenocorticotropic Hormone↗
PubMed1997

Risperidone in the elderly: a pharmacoepidemiologic study.

BACKGROUND: The possibly limited adverse effects of risperidone encourage interest in its use in geriatric patients. METHOD: Medical records of 122 hospitalized psychogeriatric patients (> or = 65 years old) newly treated with risperidone were reviewed and scored for indications, doses, and effects of this novel neuroleptic. RESULTS: Subjects (83 women, 39 men), mean +/- SD age = 76.5 +/- 6.8 years (range, 65-95), were given risperidone for agitation or psychosis associated with dementia (53%), a major mood disorder (29%), or other disorders (18%). Most (77%) were also medically ill and received other psychotropic (76%) or cardiovascular agents (70%). Daily doses of risperidone averaged 1.6 +/- 1.1 mg (range, 0.25-8.0) (0.025 mg/kg body wt.); 78% received 2.0 mg. Risperidone appeared to be effective in 85% of cases, but 18% were discontinued due to intolerability (11%) or inefficacy (7%). Adverse events occurred in 32% of the patients (36% of those discontinued). These adverse events included hypotension (29%) or symptomatic orthostasis (10%), cardiac arrest (1.6%) with fatality (0.8%), and extrapyramidal effects (11%) or delirium (1.6%). Benefits were associated with younger age and male gender, but not risperidone dose. Adverse effects were associated with cardiovascular disease and its treatment, cotreatment with an SRI antidepressant or valproate, and relatively rapid dose increases. CONCLUSION: Risperidone appeared to be effective and may be safe for many elderly psychiatric patients with comorbid medical conditions provided that doses are low and increased slowly. Particular caution is advised in the presence of cardiovascular disease or cotreatment with other psychotropic agents.

Age Factors↗
PubMed1995

Normal post-race antimyosin myocardial scintigraphy in asymptomatic marathon runners with elevated serum creatine kinase MB isoenzyme and troponin T levels. Evidence against silent myocardial cell necrosis.

Recent epidemiologic studies confirm that heavy physical exertion can trigger myocardial infarction. Diagnosis of acute myocardial injury in marathon runners is complicated by elevations of serum creatine kinase MB isoenzyme activity in asymptomatic finishers with normal post-race infarct-avid myocardial scintigraphy. Such isoenzyme elevations can arise from exertional rhabdomyolysis of skeletal muscle biochemically altered by training, from silent injury to the myocardium or from a combined tissue source. To assess silent myocardial cell necrosis in marathon runners, we performed quantitative anti-myosin myocardial scintigraphy after competition with serum immunoassays for creatine kinase MB isoenzyme and troponin T. Therefore, 8 male marathon runners with a mean age of 52 years underwent quantitative antimyosin myocardial scintigraphy immediately following the 1988 and 1993 Boston Marathons. Serum immunoassays for creatine kinase MB isoenzyme by a chemiluminescent method (CLIA) and troponin T by an enzyme-linked immunosorbent assay were performed in 4 runners after the 1993 race. Quantitative antimyosin myocardial scintigraphy was normal in all runners including 3 who participated after both races 5 years apart. Post-race serum creatine kinase MB isoenzyme and/or troponin T levels were in a range otherwise diagnostic of acute myocardial infarction in 3 of 4 subjects. Normal quantitative antimyosin myocardial imaging in asymptomatic marathon runners excludes silent myocardial cell necrosis as the source of elevated serum protein markers. Such imaging may be the optimal diagnostic modality for detection of myocardial cell necrosis in symptomatic athletes when results of conventional testing are inconclusive.

Adult↗