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Biomedical subjects

B A Clark

Publications and source records attributed to B A Clark.

At least 73 records · Page 4Linked to original sources
PubMed1989

Human papillomavirus DNA in glandular dysplasia and microglandular hyperplasia: presumed precursors of adenocarcinoma of the uterine cervix.

Presumed precursors of adenocarcinoma of the uterine cervix were investigated with specific techniques to identify human papillomavirus (HPV) DNA. The presence of HPV DNA in 36 lesions of glandular dysplasia and 16 lesions of microglandular hyperplasia of the uterine cervix was studied by in situ hybridization using 3H-labeled HPV 16 and HPV 18 DNA probes. Only two of 36 lesions (6%) of glandular dysplasia contained HPV 18 DNA, although 64% of coexisting adenocarcinoma in situ, microinvasive adenocarcinoma, and cervical squamous intraepithelial neoplasia III lesions contained HPV 18 and/or HPV 16 DNA. Two lesions of HPV 18 DNA-positive glandular dysplasia coexisted with adenocarcinoma in situ that contained the same type of HPV DNA. None of the microglandular hyperplasia lesions contained HPV 16 DNA or HPV 18 DNA. These results suggest that, if HPV infection is an initial step toward carcinogenesis, it is unlikely that glandular dysplasia and microglandular hyperplasia are precursor lesions of adenocarcinoma of the uterine cervix. A large proportion of glandular dysplasia may represent reactive lesions of endocervical columnar epithelium. Two lesions of HPV 18 DNA-positive glandular dysplasia may represent well-differentiated components of adenocarcinoma in situ of the uterine cervix.

Adenocarcinoma↗
PubMed1989

The effects of near-UV radiation on human lens beta-crystallins: protein structural changes and the production of O2- and H2O2.

beta-Crystallins (beta 1-, beta 2- and beta 3-crystallin) comprise nearly half the protein of the human lens. The effect of near-UV radiation, which is one of the possible risk factors in cataract formation, on the beta-crystallins is investigated in this study. Protein intersubunit crosslinking, change in charge of the protein subunits to more acidic species and changes in protein tertiary structure (conformation) by 300 nm irradiation are reported. The fluorescence yield of protein tryptophan residues decreases by 300 nm irradiation. There is an increase in nontryptophan fluorescence (lambda cx 340 nm, lambda cm 400-600 nm), and in protein absorption at 340 nm, due to the formation of tryptophan photooxidation products. Both tryptophan and its oxidation products can be photoexcited by 300 nm irradiation and the latter are known to be good photosensitizers. The results provide evidence for the generation of H2O2 in the irradiated human beta-crystallin solutions by the Type I photosensitizing action of the chromophores absorbing at 300 nm. The H2O2 is generated via the intermediate production of O2 anion; the latter spontaneously dismutates to H2O2, presumably via O2- protein interactions. The amount of H2O2 generated per absorbed photon is compared for various solutions of beta 1-, beta 2- and beta 3-crystallins from human lenses of different age.

Circular Dichroism↗
PubMed1989

Generation of oxidants in the near-UV photooxidation of human lens alpha-crystallin.

In this study we report on the generation of superoxide anion (O2-) and hydrogen peroxide in the near-UV irradiation of human lens alpha-crystallin by monochromatic light at 300 nm. Photolysis of human lens alpha-crystallin at 300 nm, at irradiances similar to those encountered in sunlight causes an alteration of protein tertiary structure, a loss of tryptophan fluorescence and increase of nontryptophan fluorescence. The nontryptophan fluorescence is likely to be due to the photooxidation of tryptophan to N-formylkynurenine (N-FK or related species), which is a good photodynamic sensitizer, has significant absorption at 300 nm, and can thus react via its triplet state with O2 to generate 1O2 or with reducing substrates (amino acids of the protein) to generate free radicals. The latter, in the presence of O2 can lead to the generation of O2- and H2O2. These species have been directly assayed in this study in photolyzed solutions of fetal, young and old human lens alpha-crystallin. The addition of superoxide dismutase (SOD) to the protein solution prior to photolysis increased the amount of H2O2 generated by 3- to 4-fold. This observation not only provides definitive evidence for the photogeneration of O2-, but also indicates that only a fraction of this species is transformed into H2O2 in the absence of SOD. Significant amounts of O2- and H2O2 were formed by 340 nm irradiation of old human lens alpha-crystallin, in which the basal level of N-FK is high. The role of 1O2 in these photoreactions has been studied by investigating the quenching effect of azide and the enhancing effects of D2O on the rate of loss of tryptophan fluorescence yield and the effect of azide on the rate of H2O2 generation.

Adult↗
PubMed1989

Abnormalities of cytoplasmic Ca2+ in platelets from patients with uremia.

Uremic patients have a hemorrhagic tendency, often associated with prolonged bleeding times and decreased platelet function in vitro. Whether these defects result from abnormalities in plasma factors affecting platelet activity, platelet surface receptors, intracellular platelet mediators, or other aspects of platelet behavior is unknown. To examine the possibility that the abnormality in platelet function may result from aberrations in Ca2+ homeostasis, blood was obtained from 29 patients with severe uremia. The platelets were washed, loaded with the Ca2+ -sensitive probes indo-1 and aequorin, gel-filtered, and resuspended in either plasma or buffer. Of the 29 patients, seven had template bleeding times prolonged to 11 minutes or more, but platelet aggregation in plasma was not consistently impaired in these patients. However, in aequorin-loaded platelets from the patients with long bleeding times, the highest elevation of cytoplasmic calcium [( Ca2+]i) in response to the Ca2+ ionophore A23187, arachidonate, adenosine diphosphate (ADP), or epinephrine was lower than that seen in platelets from both uremic patients with less prolonged bleeding times and normal volunteers. The reduced [Ca2+]i response was associated with decreased aggregation of gel-filtered platelets suspended in buffer. Suspending washed aequorin-loaded uremic platelets in normal plasma for 20 minutes did not reverse the decreased agonist-induced rise in [Ca2+]i; platelets from a normal donor resuspended in uremic plasma aggregated and produced a normal increase in [Ca2+]i in response to agonists. We conclude that the platelet defect seen in some patients with uremia is associated with a decreased rise in platelet [Ca2+]i after stimulation and that this is a manifestation of an intrinsic platelet defect.

Aequorin↗
PubMed1988

Human papillomavirus types and localization in adenocarcinoma and adenosquamous carcinoma of the uterine cervix: a study by in situ DNA hybridization.

Formalin-fixed, paraffin-embedded tissues from 108 cases of invasive carcinoma of the uterine cervix, consisting of 40 cases of adenocarcinoma, 44 cases of adenosquamous carcinoma, and, as a control, 24 cases of squamous cell carcinoma were examined for the presence of human papillomavirus (HPV) DNA by in situ hybridization of high sensitivity using tritium-labeled HPV-2, HPV-6, HPV-16, and HPV-18 DNA probes. This method detects five genome copies of homologous HPV DNA per cell. HPV DNA was detected with mixed HPV DNA probes in 17 cases (42.5%) of adenocarcinoma, 16 cases (36.4%) of adenosquamous carcinoma, and in 13 cases (54.2%) of squamous cell carcinoma. The types of HPV DNA in the HPV-positive tissues were also analyzed with each individual probe under high stringency conditions. HPV-18 DNA was detected in all but one case of the HPV DNA-positive adenocarcinoma and one-half of the HPV DNA-positive adenosquamous carcinoma. HPV-16 DNA was detected in one case of the HPV DNA-positive adenocarcinoma, one-half of the HPV DNA-positive adenosquamous carcinoma, and all cases of the HPV DNA-positive squamous cell carcinoma. HPV DNA was confined to the areas of carcinoma and squamous cervical intraepithelial neoplasia (CIN) associated with carcinoma. Among 36 cases in which CIN was associated with adenocarcinoma (9 cases), adenosquamous carcinoma (19 cases), and squamous cell carcinoma (8 cases), the same type of HPV DNA was present in the carcinoma and the associated CIN that constituted 12 cases (3 adenocarcinoma, 5 adenosquamous carcinoma, and 4 squamous cell carcinoma). Two cases (one adenocarcinoma and one adenosquamous carcinoma) contained HPV DNA in the carcinoma but not in the associated CIN. The incidence of HPV DNA did not show a significant correlation with the existence of CIN or histological differentiation of carcinoma.

Adenocarcinoma↗
PubMed1988

Accessibilities of the sulfhydryl groups of native and photooxidized lens crystallins: a fluorescence lifetime and quenching study.

Fluorescence lifetime and acrylamide quenching studies on the N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS)-labeled sulfhydryl groups of bovine lens alpha-, beta H-, and gamma-crystallins were carried out to characterize the microenvironment of the sulfhydryls and changes produced by singlet oxygen mediated photooxidation. For the untreated proteins, the lifetimes of the major decay component of the fluorescence-labeled crystallins were 15.2, 14.4, and 13.0 ns, and the quenching rate constant, kq, values were 16.6 x 10(7), 26.9 x 10(7), and 32.7 x 10(7) M-1 s-1 for alpha-, beta H-, and gamma-crystallins, respectively. The results indicate that as the polarity of the sulfhydryl site increased (i.e., its lifetime decreased), its accessibility to collisional quenching by acrylamide also increased. The minor decay component of the fluorescence label was not significantly quenched by acrylamide for all three classes of crystallins. When the proteins were irradiated in the presence of methylene blue, in a system generating singlet oxygen, the kq value for acrylamide quenching of the major decay component of alpha-crystallin decreased to zero, while its lifetime decreased to 6 ns. Neither the lifetime nor the kq of alpha-crystallin recovered completely in the presence of the singlet oxygen quencher sodium azide. Light-induced binding of the photosensitizer methylene blue to the crystallins was observed by absorption spectroscopy. The bound photosensitizer partially quenches the fluorescence lifetime of the N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) label in irradiated alpha-crystallin. Further decrease in the lifetime occurs as a result of the singlet oxygen mediated conformational change. The results suggest that the fluorescence lifetime of the AEDANS is fully quenched in the irradiated alpha-crystallin and there is no further quenching by acrylamide. An increase in the fraction of the minor component of beta H-crystallin which was inaccessible to acrylamide quenching was observed after irradiation. There was no effect of irradiation on the kq for acrylamide quenching of the major component of the decay of AEDANS bound to beta H- or gamma-crystallins. Static quenching was found to contribute significantly to the steady-state quenching plots of the polar sulfhydryl sites of irradiated alpha-crystallin and of untreated and irradiated beta H- and gamma-crystallins, but it had no detectable role in the case of untreated alpha-crystallin. Fluorescence anisotropy of the AEDANS label bound to the crystallins was higher in the irradiated crystallins as compared with the controls.

Acrylamide↗
PubMed1988

Conformational changes of beta H-crystallin in riboflavin-sensitized photooxidation.

The effect of riboflavin-sensitized photooxidation on calf lens beta H-crystallin has been investigated by using fluorescence and circular dichroism techniques. beta H-Crystallin showed a pronounced change in its tertiary structure (conformation) as manifested in the near-u.v. circular dichroism spectra and fluorescence yield of tryptophan residues. The rate of tryptophan photolysis was significantly diminished under anaerobic conditions, but was not affected appreciably when D2O was used in the reaction mixture instead of H2O. Ferricyanide and ferricytochrome c added to the solution prior to irradiation inhibited the rate of photolysis of tryptophan, suggesting the involvement of O2- anion in the photoreactions. Quantitative assays of O2- and H2O2 in the irradiated protein solution strongly suggest that the Type I photosensitization pathway is involved in the RF-sensitized photooxidation of beta H-crystallin. The effect of photolysis on the cysteine residues of the protein was also studied. The sulfhydryl specific fluorophore N-iodoacetyl-N'-(5-sulfonaphthyl) ethylenediamine (1,5-IAEDANS) was used to study the change in the microenvironment of the cysteine (sulfhydryl) residues of the protein by photolysis. The results indicate that there is a quantitative loss of IAEDANS labeling sites due to photooxidation as well as structural changes of the protein. Fluorescence lifetime measurements indicate that the probe is bound in two environments--the major one (95%) is exposed and the minor one (5%) hydrophobic. A decrease in the lifetimes of the bound label occurs after photooxidation. However, the relative proportion of the hydrophobic IAEDANS-labeling sites increases in the photooxidized beta H-crystallin, probably due to the formation of supra-aggregated protein by photolysis.

Animals↗
PubMed1988

The analysis of carcinomas of the vagina for human papillomavirus DNA.

Invasive cancers of the vagina are relatively rare and often resistant to effective treatment. While studies on the more abundant premalignant lesions of the vagina and premalignant and malignant tumors of the vulva and cervix have shown a frequent association with human papillomavirus (HPV) DNA infection, lack of fresh tissue samples has precluded similar studies on malignant tumors of the vagina. Using mostly in situ hybridization, we have retroactively examined 14 formalin-fixed, paraffin-embedded biopsies of invasive squamous cell carcinomas of the vagina. We have found 21% of the samples to have HPV DNA. These findings confirm a role for HPV in malignancies of the entire female lower genital tract.

Biopsy↗
PubMed1988

Treadmill protocols for determination of maximum oxygen uptake in runners.

Four testing protocols were completed by each of 10 runners using a common speed for protocols 1 and 2 (P1 and P2), each runner's training pace for protocol 3 (P3) and a speed selected manually by the runner for protocol 4 (P4). Stages were increased by 2.5% grade every 2 min for each protocol except for P1, which had 1 min stages. There were no significant differences in maximum oxygen uptake (VO2 max) between protocols (P1, 65.0 +/- 5.6 ml.kg-1 min-1; P2, 64.5 +/- 5.3 ml.kg-1 min-1; P3, 66.2 +/- 3.9 ml.kg-1 min-1; P4, 64.7 +/- 5.8 ml.kg-1 min-1). Treadmill time was significantly less for P1 than for the other protocols. The rate of perceived exertion obtained at maximal exercise during P1 was less than that obtained during the other three protocols. Heart rate was significantly lower (P less than 0.05) at any level of submaximal VO2 during P3 than during the other protocols. We recommend a testing protocol using speeds approximating the runner's training pace and 1 min stages. This may result in lower perception of difficulty and HR throughout the test and shorter testing times.

Adult↗
PubMed1988

Spectroscopic studies on the photooxidation of calf-lens gamma-crystallin.

The photooxidation of calf gamma-crystallin has been investigated in (a) riboflavin-sensitized reaction and (b) direct photolysis of the tryptophan residues of the protein at 300 nm. Partial insolubilization of the protein is evident by the increase in turbidity of the photolyzed protein solution. The turbidity is diminished when anaerobic conditions are used for irradiation. Spectroscopic studies on the soluble phase of the photolyzed protein reveal significant changes in the near-UV circular dichroism spectrum, suggesting that changes in the tertiary structure of the protein precede insolubilization. Isoelectric focusing analyses reveal that the gamma-crystallin polypeptides have more acidic pI's after photooxidation under both conditions. The insoluble protein is highly crosslinked into dimers and higher oligomers via covalent, non-disulfide crosslinks, but inter-subunit crosslinking is negligible in the soluble phase of the photolyzed protein. The mechanism of photooxidation involves the generation of H2O2 in the riboflavin-sensitized reaction. In the direct photolysis of tryptophan residues by 300 nm irradiation, there is a rapid disruption of the protein structure, apparently by the oxidation of the hydrophobic tryptophan residues.

Animals↗
PubMed1988

Human papillomavirus DNA in adenosquamous carcinoma and squamous cell carcinoma of the vulva.

The tissues from 16 cases of adenosquamous carcinoma (pseudoglandular squamous cell carcinoma or adenoacanthoma of the sweat glands of Lever) and 26 cases of invasive squamous cell carcinoma of the vulva were studied for the presence of human papillomavirus (HPV) genomes using Southern blot hybridization on fresh tissues. Types 1, 2, 3, 4, 5, 6, 16, and 18 HPV DNA probes and in situ hybridization were used on formalin-fixed paraffin sections using type 2, 6, 16, and 18 HPV DNA probes. Only one case of adenosquamous carcinoma contained an undetermined type of HPV DNA, whereas five cases of squamous cell carcinoma contained HPV DNA. Three of these five cases contained type 16, one type 6 HPV, and two an undetermined type. These results demonstrate HPV DNA associations with malignancy of the vulva that are similar to those observed elsewhere in the genital tract.

Adenocarcinoma↗
PubMed1988

Condylomatous carcinoma of the vulva with special reference to human papillomavirus DNA.

Nine cases of condylomatous carcinoma (squamous cell carcinoma arising in condyloma acuminatum) of the vulva were studied for their clinical history, histopathology, and presence of human papillomavirus (HPV) DNA. Condylomatous carcinoma occurred primarily in an elderly population with a mean age of 70 years. There was an antecedent history of vulvar condyloma in 77%, with a median of nine months before the documentation of an invasive lesion. The disease had a good prognosis, with few recurrences and no metastasis or deaths from the disease. Human papillomavirus DNA was demonstrated to be present in 55% of these tumors by either filter or in situ hybridization techniques. Both HPV 6 and HPV 16 DNA were identified in an equal number of cases.

Aged↗
PubMed1987

Detection of human papillomavirus DNA in invasive carcinomas of the cervix by in situ hybridization.

An examination of 27 invasive cancers of the cervix was performed using the technique of in situ hybridization using human papillomavirus DNA probes. Four tissues, previously found to harbor papillomavirus DNA by filter hybridization, were confirmed by in situ analysis. One further tissue never previously studied was also found to be positive by in situ hybridization. Overall, we found 33% of invasive cancers of the cervix to contain human papillomavirus DNA. In contrast, 55% of carcinoma in situ and severe dysplasia of the cervix were found to be positive for human papillomavirus DNA. These results confirmed that the sample population of patients in our studies have a relatively low association of human papillomavirus DNA with invasive cancers of the cervix and that in situ hybridization provides an effective complementation to filter hybridization for human papillomavirus-infected tumors.

Carcinoma↗
PubMed1986

Sensitivity of koilocytosis, immunocytochemistry, and electron microscopy as compared to DNA hybridization in detecting human papillomavirus in cervical and vaginal condyloma and intraepithelial neoplasia.

The sensitivity in detecting human papillomavirus (HPV) by histological observation of koilocytosis, immunocytochemistry, and electron microscopy with reference to the results of Southern blot deoxyribonucleic acid (DNA) hybridization were reviewed in 41 lesions (37 patients) of cervical and vaginal condylomata acuminata and intraepithelial neoplasia. Human papillomavirus DNA was demonstrated in fresh tissues by Southern blot DNA hybridization in all but one lesion of moderate dysplasia (98%). The rate of koilocytosis observed in tissue sections was 80% in condyloma, and ranged from 89-20% in cervical intraepithelial neoplasia (CIN), with steady reduction as the grade of CIN or vaginal intraepithelial neoplasia (VaIN) was higher. The immunocytochemistry for HPV capsid antigens was positive in 80% of condylomata and ranged from 61-0% in CIN or VaIN. The rate declined in inverse proportion to the grade of CIN or VaIN. Electron microscopy of preselected areas containing intranuclear inclusions in paraffin sections of 10 lesions demonstrated HPV-like particles in 90% of the lesions. Although immunocytochemistry and observation of koilocytosis may be useful in detecting HPV in condylomata acuminata and mild dysplasia, their sensitivity was poor in CIN or VaIN of higher grades. Electron microscopy on preselected areas in paraffin blocks showed better sensitivity, presumably due to its ability to detect immature virions.

Condylomata Acuminata↗
PubMed1985

Intraoperative serum and tissue activity of cefazolin and cefoxitin.

We determined the intraoperative serum and wound-muscle concentrations of cefazolin and cefoxitin in 40 patients who were undergoing cholecystectomies. The study employed an open-label design in which all of the patients randomly received cefazolin sodium (20 mg/kg) or cefoxitin sodium (30 mg/kg) intravenously while the patient was in the ward ("on call") or with the induction of anesthesia. Multiple blood and wound-muscle samples were collected intraoperatively and assayed for their cephalosporin concentrations. Considerable differences in intraoperative serum and tissue concentrations between antibiotics were apparent; there were usually higher levels of cefazolin. In all of the patients who received cefazolin sodium, the antimicrobial was detectable in wound tissue at wound closure, while it was detectable in 86% and 38% of patients who received cefoxitin sodium with anesthesia and on call, respectively. Because cefoxitin has a much shorter elimination half-life than cefazolin it seems prudent to administer the agent as close to the start of the operation as possible, and readminister the agent every two to three hours until the wound is closed. For cefazolin, on-call administration appears to be acceptable, with readministration not required for at least four hours.

Abdominal Muscles↗