Simulated surface-induced thrombin generation in a flow field.

A computational model of blood coagulation is presented with particular emphasis on the regulatory effects of blood flow, spatial distribution of tissue factor (TF), and the importance of the thrombomodulin-activated protein C inhibitory pathway. We define an effective prothrombotic zone that extends well beyond the dimensions of injury. The size of this zone is dependent on the concentrations of all reactive species, the dimensions of TF expression, the densities of surface molecules, and the characteristics of the flow field. In the case of tandem sites of TF, the relationship between the magnitude of the effective prothrombotic zone and the interval distance between TF sites dictate the net response of the system. Multiple TF sites, which individually failed to activate the coagulation pathway, are shown to interact in an additive manner to yield a prothrombotic system. Furthermore, activation of the thrombomodulin-activated protein C pathway in the regions between sites of TF downregulate the thrombin response at subsequent TF sites. The implications of prothrombotic effects, which extend downstream beyond the discrete site of injury to interact with subsequent lesions are critical given the systemic nature of atherosclerotic disease.

Lipoic acid metabolism in Arabidopsis thaliana: cloning and characterization of a cDNA encoding lipoyltransferase.

Lipoic acid is an essential coenzyme required for activity of several key enzyme complexes, such as the pyruvate dehydrogenase complex, in the central metabolism. In these complexes, lipoic acid must be covalently attached to one of the component proteins for it to have biological activity. We report the cloning and characterization of Arabidopsis thaliana LIP2 cDNA for lipoyltransferase that catalyzes the transfer of the lipoyl group from lipoyl-acyl carrier protein to lipoate-dependent enzymes. This cDNA was shown to code for lipoyltransferase by its ability to complement an Escherichia coli lipB null mutant lacking lipoyltransferase activity. The expressed enzyme in the E. coli mutant efficiently complemented the activity of pyruvate dehydrogenase complex, but less efficiently than that of 2-oxoglutarate dehydrogenase complex. Comparison of the deduced amino acid sequence of LIP2 with those of E. coli and yeast lipoyltransferases showed a marked sequence similarity and the presence of a leader sequence presumably required for import into mitochondria. Southern and northern hybridization analyses suggest that LIP2 is a single-copy gene and is expressed as an mRNA of 860 nt in leaves. Western blot analysis with an antibody against lipoyltransferase demonstrated that a 29 kDa form of lipoyltransferase is located in the mitochondrial compartment of A. thaliana.

Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein.

The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been observed. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein containing a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry.

A new metabolic link. The acyl carrier protein of lipid synthesis donates lipoic acid to the pyruvate dehydrogenase complex in Escherichia coli and mitochondria.

Lipoic acid is an essential enzyme cofactor that requires covalent attachment to its cognate proteins to confer biological activity. The major lipoylated proteins are highly conserved enzymes of central metabolism, the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The classical lipoate ligase uses ATP to activate the lipoate carboxyl group followed by attachment of the cofactor to a specific subunit of each dehydrogenase complex, and it was assumed that all lipoate attachment proceeded by this mechanism. However, our previous work indicated that Escherichia coli could form lipoylated proteins in the absence of detectable ATP-dependent ligase activity raising the possibility of a class of enzyme that attaches lipoate to the dehydrogenase complexes by a different mechanism. We now report that E. coli and mitochondria contain lipoate transferases that use lipoyl-acyl carrier protein as the lipoate donor. This finding demonstrates a direct link between fatty acid synthesis and lipoate attachment and also provides the first direct demonstration of a role for the enigmatic acyl carrier proteins of mitochondria.

Detection of trisomy 7 in nonmalignant bronchial epithelium from lung cancer patients and individuals at risk for lung cancer.

Early identification and subsequent intervention are needed to decrease the high mortality rate associated with lung cancer. The examination of bronchial epithelium for genetic changes could be a valuable approach to identify individuals at greatest risk. The purpose of this investigation was to assay cells recovered from nonmalignant bronchial epithelium by fluorescence in situ hybridization for trisomy of chromosome 7, an alteration common in non-small cell lung cancer. Bronchial epithelium was collected during bronchoscopy from 16 cigarette smokers undergoing clinical evaluation for possible lung cancer and from seven individuals with a prior history of underground uranium mining. Normal bronchial epithelium was obtained from individuals without a prior history of smoking (never smokers). Bronchial cells were collected from a segmental bronchus in up to four different lung lobes for cytology and tissue culture. Twelve of 16 smokers were diagnosed with lung cancer. Cytological changes found in bronchial epithelium included squamous metaplasia, hyperplasia, and atypical glandular cells. These changes were present in 33, 12, and 47% of sites from lung cancer patients, smokers, and former uranium miners, respectively. Less than 10% of cells recovered from the diagnostic brush had cytological changes, and in several cases, these changes were present within different lobes from the same patient. Background frequencies for trisomy 7 were 1.4 +/- 0.3% in bronchial epithelial cells from never smokers. Eighteen of 42 bronchial sites from lung cancer patients showed significantly elevated frequencies of trisomy 7 compared to never smoker controls. Six of the sites positive for trisomy 7 also contained cytological abnormalities. Trisomy 7 was found in six of seven patients diagnosed with squamous cell carcinoma, one of one patient with adenosquamous cell carcinoma, but in only one of four patients with adenocarcinoma. A significant increase in trisomy 7 frequency was detected in cytologically normal bronchial epithelium collected from four sites in one cancer-free smoker, whereas epithelium from the other smokers did not contain this chromosome abnormality. Finally, trisomy 7 was observed in almost half of the former uranium miners; three of seven sites positive for trisomy 7 also exhibited hyperplasia. Two of the former uranium miners who were positive for trisomy 7 developed squamous cell carcinoma 2 years after collection of bronchial cells. To determine whether the increased frequency of trisomy 7 reflects generalized aneuploidy or specific chromosomal duplication, a subgroup of samples was evaluated for trisomy of chromosome 2; the frequency was not elevated in any of the cases as compared with controls. The studies described in this report are the first to detect and quantify the presence of trisomy 7 in subjects at risk for lung cancer. These results also demonstrate the ability to detect genetic changes in cytologically normal cells, suggesting that molecular analyses may enhance the power for detecting premalignant changes in bronchial epithelium in high-risk individuals.

Decreasing rates of cervical cancer among American Indians and Hispanics in New Mexico (United States).

Minority women in New Mexico (United States)--including American Indian and Hispanic women--have shown disproportionately high incidence rates of invasive cervical cancer during the 1960s and 1970s. Several public health programs in New Mexico were directed toward early detection of cervical cellular abnormalities, particularly targeting the state's minority women. To evaluate the effectiveness of these programs, we examined the New Mexico Surveillance, Epidemiology, and End Results (SEER) data collected from 1969-92, and calculated average annual, age-specific, and age-adjusted incidence rates by ethnic group (American Indian, Hispanic, and non-Hispanic White) for five-year time intervals. We also calculated age-adjusted mortality rates for cervical cancer in the same ethnic groups using state vital records. Age-adjusted incidence rates for invasive cervical cancer show substantial temporal decreases, especially for minority women in the state. The age-adjusted incidence rate decreased by 66 percent, from 30.3 to 10.3 per 100,000 for American Indian women, and by 61 percent, from 26.1 to 10.2 per 100,000 for Hispanic women. A stage shift to earlier stages of cervical neoplasia occurred over the study period, with a substantially higher proportion of in situ compared with invasive cancers diagnosed in the most recent cf the most remote time period. The ratio of incidence rates of in situ to invasive cancers changed dramatically for both American Indian and Hispanic women. Cervical cancer mortality rates decreased steadily among Hispanic women from 1958 to 1992; the decrease among American Indian women was less stable and fluctuated due to small numbers. Ongoing targeted screening programs should help to reduce cervical cancer incidence and mortality further in New Mexico.

Contraceptive and reproductive risks for cervical dysplasia in southwestern Hispanic and non-Hispanic white women.

BACKGROUND: Various contraceptive practices and reproductive factors have been associated with cervical neoplasia in case-control studies worldwide. METHODS: To investigate contraceptive and reproductive risk factors associated with high-grade cervical dysplasia in southwestern Hispanic and non-Hispanic white women, we carried out a clinic-based case-control study among university-affiliated clinic attendees. RESULTS: Oral contraceptive use ever (odds ratio [OR] = 0.4, 95% confidence interval [CI]: 0.2-0.9) and past diaphragm use (OR = 0.3, 95% CI: 0.1-0.8) were protective for dysplasia in analyses adjusted for age, ethnicity, sexual behaviour, and for cervical papillomavirus (HPV) infection. After further adjustment for Pap smear screening interval, oral contraceptive use ever remained protective for dysplasia. Vaginal deliveries were strongly associated with dysplasia with > 2 vaginal deliveries associated with a 3.9-fold increase in risk after adjustment for age, ethnicity, sexual behaviour, and HPV infection. Using logistic regression models to simultaneously control for effects of multiple factors as potentially related to cervical dysplasia, we found low educational attainment, cervical HPV infection, cigarette smoking, history of any sexually transmitted disease, and having one or more vaginal deliveries to be associated with dysplasia; oral contraceptive use and past diaphragm use also remained protective for high-grade cervical dysplasia in these regression analyses. CONCLUSIONS: The data suggest that use of oral contraceptives (ever) and past diaphragm use are protective for high-grade cervical dysplasia among Hispanic and non-Hispanic white women in New Mexico. The clinic-based perspective of this research (versus population-based studies) may help explain some of these findings.

Sexually transmitted diseases and other risk factors for cervical dysplasia among southwestern Hispanic and non-Hispanic white women.

OBJECTIVE: To assess risk factors for high-grade cervical dysplasia among southwestern Hispanic and non-Hispanic white women. DESIGN: Clinic-based case-control study. SETTING: University-affiliated gynecology clinics. SUBJECTS: Cases were Hispanic and non-Hispanic white women with biopsy-proven high-grade cervical dysplasia (n = 201). Controls were Hispanic and non-Hispanic white women from the same clinics with normal cervical epithelium (n = 337). METHODS: Study design included interviews focused on histories of sexually transmitted diseases, sexual behavior, reproductive histories, hygienic practices, contraceptive use, cigarette smoking, and diet. Laboratory studies included bacterial and protozoal cultures of the cervix; hybridization tests to identify human papillomavirus (HPV) genome with commercial (ViraPap and ViraType) and polymerase chain reaction-based assays; and serum antibody tests for herpes simplex virus, Chlamydia trachomatis, syphilis, hepatitis B, and hepatitis C. RESULTS: For both ethnic groups combined, after adjustment for ethnicity, age, and sexual behavior, the strongest risks for cervical dysplasia were associated with cervical HPV infection as identified by ViraPap (odds ratio [OR], 12.8; 95% confidence interval [CI], 8.2 to 20.0) or with polymerase chain reaction (OR, 20.8; 95% CI, 10.8 to 40.2). Other factors associated with dysplasia included cigarette smoking at the time of diagnosis (OR, 1.8; 95% CI, 1.2 to 2.8); low income (OR, 2.2; 95% CI, 1.2 to 4.0); low educational level (OR, 6.2; 95% CI, 3.4 to 11.1); history of any sexually transmitted disease (OR, 1.9; 95% CI, 1.3 to 2.7); and seroprevalence of antibodies to hepatitis B (OR, 1.8; 95% CI, 0.9 to 3.5). For Hispanic women, HPV 16/18 identified by ViraType was strongly associated with cervical dysplasia (OR, 171.0; 95% CI, 22.8 to 1280.5). Antibodies to herpes simplex virus type 2 were not associated with dysplasia in Hispanic women but were significantly associated with dysplasia among non-Hispanic whites. Risks associated with cigarette smoking also varied by ethnic group. CONCLUSIONS: The strongest risk factor associated with high-grade cervical dysplasia among clinic attendees was HPV infection. Although most of the risk factors we examined showed similar associations for dysplasia for both ethnic groups, our data suggest that several different risk factors may be relevant to the development of cervical dysplasia in Hispanics compared with non-Hispanic whites who attend the same clinics.

Cigarette smoking and other risk factors for cervical dysplasia in southwestern Hispanic and non-Hispanic white women.

To assess smoking-related and other risk factors for high-grade cervical dysplasia in southwestern Hispanic and non-Hispanic white women in New Mexico, we conducted a clinic-based case-control study among attendees at university-affiliated gynecology clinics. We collected data on cigarette use, sexual behavior, past and current sexually transmitted diseases, hygienic practices, contraception, and diet. For both ethnic groups combined, after adjustment for the effects of human papillomavirus, sexual behavior, and other risk factors, cigarette smoking at the time of diagnosis was associated with high-grade dysplasia (odds ratio, 1.7; 95% confidence limits, 1.0-2.8). In contrast, former smoking was not associated with cervical dysplasia (odds ratio 0.9; 95% confidence limits, 0.5-1.5). Analyses showed dose-response relationships for the amount of cigarettes smoked per day and for cumulative exposure (pack-years of use) in association with cervical dysplasia. Although our study lacked the power to show statistically significant ethnic differences in smoking-related risks for dysplasia, smoking at the time of diagnosis, high pack-years of use, and smoking at the time of menarche were associated with dysplasia only for non-Hispanic white versus Hispanic women. Our data support hypotheses that implicate cigarette use as an etiological factor in the development of high-grade cervical dysplasia and suggest ethnic differences in risks for dysplasia among women attending the same clinics.

Obesity decreases hepatic glutathione concentrations and markedly potentiates allyl alcohol-induced periportal necrosis in the overfed rat.

Liver biopsies from 9 out of every 10 obese individuals exhibit pathological changes of unknown aetiology and 3 out of every 10 reflect severe injury in the form of periportal fibrosis. To examine the hypothesis that excessive fibrosis in obesity arises in part from a predisposition to injury of the liver by drugs and xenobiotics, we administered 5, 10 and 25 mg/kg doses of the model periportal hepatotoxin, allyl alcohol, to obese Sprague-Dawley rats and age-matched non-obese controls. Alanine aminotransferase activity (ALT) in plasma was ten-fold more elevated in obese animals than in non-obese animals given the 25 mg/kg dose (P < 0.05). On fitting the ALT results to a non-linear, parametric model by iterative non-linear least squares regression, we found that the slope of the log dose ALT curve was similar for obese and non-obese rats. However, the minimum dose required to produce elevated ALT (DMIN) was 50% lower for obese animals (DMIN 6.47 +/- 2.75 vs. 13.3 +/- 0.96 mg allyl alcohol; P < 0.05). In a subsequent experiment, allyl alcohol was administered to obese rats based on ideal body weight, which is defined as the mean total body weight of an age-matched non-obese animal. With this dosing normalization, the 25 mg/kg ideal body weight doses translated to administration of a fixed dose of 13.5 mg allyl alcohol to obese rats. Obese rats treated in this fashion exhibited more severe necrosis in the periportal zone (median necrosis score 2 versus 0-1, P < 0.05) and increased mortality over controls (44% versus 0%; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Cervical papillomavirus infection and cervical dysplasia in Hispanic, Native American, and non-Hispanic white women in New Mexico.

BACKGROUND: Human papillomavirus infections of the cervix are found with varying frequencies in different populations worldwide, and have been associated with cervical cytologic abnormalities. METHODS: We studied 1,603 randomly selected Hispanic, Native American, and non-Hispanic White women in New Mexico to determine the prevalence of cervical HPV infection in these ethnic groups, and its association with Pap smear abnormalities, using a new commercial dot-blot hybridization assay. RESULTS: Nine percent of all women screened had evidence of cervical HPV infection (13.7% of non-Hispanic White women, 9.7% of Hispanics, and 6.6% of Native American women). Prevalence was higher in younger women ages 14-19 years than in older age groups. Over half of women with cervical HPV infection (n = 145) had normal Pap smears. The proportion of infected women increased among those with more advanced cytopathologic abnormalities; 5.6 percent with normal Pap smears had cervical HPV vs 66.7 percent with moderate-severe dysplasia. CONCLUSIONS: Cervical HPV infection is common among New Mexico clinic attendees, varies in prevalence among the three major ethnic groups, and is strongly associated with cervical cytopathologic abnormalities.

Cytologic detection of penile malignant melanoma of soft parts in pleural effusion using monoclonal antibody HMB-45.

A case of penile malignant melanoma of soft parts ("clear cell sarcoma") with pulmonary metastases and malignant effusions is reported. The tumor cells in the pleural effusion were scattered singly and admixed with reactive mesothelial cells. They had abundant granular cytoplasm and round nuclei with prominent nucleoli. Although staining for S-100 protein was positive in sections from the penile lesion, it was negative in sections of a cell block prepared from the effusion; however, the effusion tumor cells demonstrated immunoreactivity with HMB-45, an antimelanoma monoclonal antibody.

Fine needle aspiration cytology of malignant spiradenoma arising in congenital eccrine spiradenoma.

The fine needle aspiration (FNA) cytology of malignant eccrine spiradenoma arising in a large benign congenital eccrine spiradenoma is described. The malignant tumor was characterized by cohesive, pleomorphic cells with malignant nuclear features and numerous mitoses. FNA of the adjacent benign eccrine spiradenoma revealed prominent basement membrane deposition with an irregular tubular and nesting growth pattern and bland basaloid cells. To our knowledge, the FNA of malignant eccrine spiradenoma has not been previously described. In this case, FNA cytology was influential in directing patient management.

Quantitative morphology of late renal radiation injury.

Late radiation injury manifests itself in all morphologic compartments of the kidney, but loss of cell mass is most significant in the proximal convoluted tubules. Development of an end stage or nonfunctional kidney requires 12 or more months after single fraction X ray exposures of about 12 Gy (2, 5, 13) and is associated with marked morphologic alterations of renal tubules. Radiation induced changes were studied at 6 months after irradiation, a time interval when histological alterations appear minor, but in previous studies were shown to correlate with the later end stage alterations (5, 8, 9). Renal alterations were graded objectively based on renal weight, variation in size of tubule cell nuclei, and glomerular nuclear volume fraction. Irradiation was associated with loss of renal weight, increased variability of tubule nuclear size, and previously unappreciated changes in glomerular nuclear volume fraction. A classification index derived from a weighted combination of renal weight ratio and tubule cell nuclear variability correlates with radiation dose and with previously established subjective histologic grading of renal damage, and allows objective comparisons of various fractionation schedules.

The cytogenetic and hepatotoxic effects of dioxin on mouse liver cells.

The cytogenetic and hepatotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on mouse liver cells were investigated. Male C57BL/6J strain mice, which have TCDD receptors, were given single intraperitoneal injections of 25, 37.5, 75 and 150 micrograms of TCDD/kg body weight or corn oil carrier alone. Two-thirds hepatectomies were carried out at 1 or 7 days after injection and chromosomal aberrations and mitotic indexes of the regenerating hepatocytes were scored 54 hr after hepatectomy. Liver sections from additional intact mice were studied for TCDD-hepatotoxicity at 1, 7 and 30 days after injection. The three high doses of TCDD caused hepatotoxicity with necrosis of liver cells and focal architectural collapse by 30 days after injection. No evidence was obtained of an increase in the frequency of chromosomal structural aberrations at doses that allowed sufficient mitotic activity for cytogenetic evaluation. We conclude that TCDD is not a clastogen for mouse hepatocytes, although high doses cause marked hepatocellular necrosis.

Video-based image collection for quantitative histopathology.

Obtaining histologic images for computer-based morphometric analysis is associated with a number of standardization problems, which must be solved if reproducible data collection is expected. These problems include tissue processing, sectioning and staining, standardizing and calibrating the video camera and determining the appropriate sampling rate (pixels/micron). Suggested solutions for these problems are presented for a specific image analysis system, but are applicable to other systems with similar capabilities. Biologic variability is not eliminated by computer-assisted analysis, so it is important to minimize data-collection artifacts by parallel processing of experimental and control material, as in other investigative work.

Association of human papillomavirus and Chlamydia infections with incidence cervical neoplasia.

Incidence cervical neoplasia is defined as disease that becomes manifest during a given period of observation. Association with preceding genital infections having characteristic cytologic findings would seem to be more likely for incidence than for prevalence cases since the usual long latency period of carcinoma in situ (CIS) could allow resolution of infectious processes. For this reason, it was elected to examine the preceding Papanicolaou smears from patients with tissue-confirmed incidence CIS or invasive epidermoid carcinoma. There were 67 women with biopsy-proven CIS or invasive carcinoma of the uterine cervix identified in the files of the University of New Mexico Cytopathology Laboratory from 1966 to 1982 who had two initial negative smears as well as smears at intervals of three years or less. All cytologic smears prior to tissue diagnosis were rescreened for confirmation of cytologic atypia or its absence as well as for morphologic evidence of human papillomavirus (HPV) or chlamydial infections. Control cases matched for age, gravidity, ethnicity and number of smears were reviewed in an identical manner. Koilocytes indicative of HPV infection were found in 17 index cases (25%) and 5 controls (7%) (p = 0.005). Chlamydial infections were identified in 18 index cases (27%) and in 4 controls (6%) (p = 0.001). The times required for conversion from smear negativity to malignancy were determined for each incidence case. The results showed great variability but suggest that the progression to malignancy is not hastened in women with antecedent HPV or chlamydial infections. Our results indicate that the presence of koilocytes and/or chlamydial inclusions in cervical smears serves to identify a group of women with a significantly increased risk of developing cervical carcinoma, even in the absence of concurrent dysplasia.