Oriented immobilization of Pseudomonas putida putidaredoxin at a gold (111)-buffer interface: a real time scanning tunnelling microscopy study.

A scanning tunnelling microscopy study of adsorption of wild-type Pseudomonas putida putidaredoxin at a gold (111)-buffer interface has been made in real time. Reversible adsorption has been observed reflecting weak interaction of the wild-type protein with a gold (111) electrode. A genetically engineered mutant, C73S-D58C, which contains a surface thiol, has been used for 'immobilization' and 'orientated adsorption' on the gold surface. The implication of such orientated immobilization in development of a bio-electrode surface has been predicted.

Correlates of experiencing life events just before or during pregnancy.

This study evaluates the prevalence of selected life events around the time of pregnancy, examining changes in the prevalence of these events, and identifying maternal characteristics associated with these events. We used data from the Pregnancy Risk Assessment Monitoring System (PRAMS) to examine 18 stressful life events among women who recently gave birth and to identify maternal characteristics associated with these events. PRAMS is a mail sample survey with telephone follow-up for non-respondents. Sixty-four percent of women experienced at least one event. The prevalence of specific events ranged from 0.4 to 30%. Women who experienced events differed from those who did not. Most notably, women of low socioeconomic status (SES) were much more likely to experience stressful life events. These events were also associated with other demographic and behavioral characteristics after controlling for SES. These results have implications for interpreting studies of stressful life events. The strong associations with SES highlight the importance of controlling for SES in studies of life events and health, and of considering differences in SES when interpreting these studies.

High resolution scanning force microscopy of cardiac myocytes.

The advent of scanning probe microscopy has introduced a powerful new method of probing the structural features of biological specimens. In this study, high resolution atomic force microscopy micrographs of single, isolated, cardiac myocytes are presented. Significantly, our images show not only the features to be expected of the external sarcolemma, but also resolve sub-surface features, including the striated pattern of the contractile proteins and their associated sarcoplasmic reticulum and mitochondria.

A voltammetric study of direct electron transfer to cytochrome c using a very large assembly of carbon microelectrodes.

A very large assembly of more than 8000 carbon fibre microdisk electrodes was used to study direct electron transfer to cytochrome c. Near steady-state cyclic voltammograms were observed, which exhibited excellent signal-to-noise ratios despite the low concentrations of cytochrome c employed (1-50 microM). The high resolution of the voltammograms allowed the formal potential of the native form of cytochrome c to be determined over a range of solution pH.

Direct electrochemistry of pentachlorophenol hydroxylase.

The direct electrochemistry of the flavin-containing monooxygenase, pentachlorophenol hydroxylase (PCPH), at an edge plane graphite electrode was observed and a catalytic response, linear with concentration, was found with the substrate pentachlorophenol (PCP).

Ultrastructural organization of amyloid fibrils by atomic force microscopy.

Atomic force microscopy has been employed to investigate the structural organization of amyloid fibrils produced in vitro from three very different polypeptide sequences. The systems investigated are a 10-residue peptide derived from the sequence of transthyretin, the 90-residue SH3 domain of bovine phosphatidylinositol-3'-kinase, and human wild-type lysozyme, a 130-residue protein containing four disulfide bridges. The results demonstrate distinct similarities between the structures formed by the different classes of fibrils despite the contrasting nature of the polypeptide species involved. SH3 and lysozyme fibrils consist typically of four protofilaments, exhibiting a left-handed twist along the fibril axis. The substructure of TTR(10-19) fibrils is not resolved by atomic force microscopy and their uniform appearance is suggestive of a regular self-association of very thin filaments. We propose that the exact number and orientation of protofilaments within amyloid fibrils is dictated by packing of the regions of the polypeptide chains that are not directly involved in formation of the cross-beta core of the fibrils. The results obtained for these proteins, none of which is directly associated with any human disease, are closely similar to those of disease-related amyloid fibrils, supporting the concept that amyloid is a generic structure of polypeptide chains. The detailed architecture of an individual fibril, however, depends on the manner in which the protofilaments assemble into the fibrillar structure, which in turn is dependent on the sequence of the polypeptide and the conditions under which the fibril is formed.

Catalytic reductive dehalogenation of hexachloroethane by molecular variants of cytochrome P450cam (CYP101).

CYP101 (cytochrome P450cam) catalyses the oxidation of camphor but has also been shown to catalyse the reductive dehalogenation of hexachloroethane and pentachloroethane. This reaction has potential applications in the biodegradation of these environmental contaminants. The hexachloroethane dehalogenation activity of CYP101 has been investigated by mutagenesis. The effects of active-site polarity and volume were probed by combinations of active-site mutations. Increasing the active-site hydrophobicity by the Y96A and Y96F mutations strengthened hexachloroethane binding but decreased the rate of reaction. Increasing the polarity with the F87Y mutation drastically weakened hexachloroethane binding but did not affect the rate of reaction. The Y96H mutation had little effect at pH 7.4, but weakened hexachloroethane binding while increasing the rate of dehalogenation by up to 40% at pH 6.5, suggesting that the imidazole side-chain was partially protonated at pH 6.5 but not at pH 7.4. Substitutions by bulkier side-chains at F87, T101 and V247 weakened hexachloroethane binding but increased the dehalogenation rate. The effect of the individual mutations was additive in multiple mutants, and the most active mutant for hexachloroethane reductive dehalogenation at pH 7.4 was F87W-V247L (80 min-1 or 2.5 x the activity of the wild-type). The results suggested that the CYP101 active site shows good match with hexachloroethane, the Y96 side-chain plays an important role in both hexachloroethane binding and dehalogenation, and hexachloroethane binding and dehalogenation places conflicting demands on active-site polarity and compromises were necessary to achieve reasonable values for both.

A scanning tunnelling microscopy study of Clostridium pasteurianum rubredoxin.

Scanning tunnelling microscopy (STM), which can provide 'direct' and 'non-averaged' information on molecular structure in three dimensions, has been used to achieve sub-molecular resolution in a 'single molecule' of rubredoxin, an important iron-sulphur protein, at the gold (111)/water interface. The metal-ligand site [Fe(III)-Cys4] appears distinct because of an enhancement of the tunnelling current over this region compared to the surrounding protein structure.

A scanning tunnelling study of immobilised cytochrome P450cam.

A site-specifically engineered surface cysteine residue, located in a region where the haem moiety is closest to the surface, is used to anchor cytochrome P450cam enzyme molecules covalently to a gold electrode. More reproducibly ordered adsorption, at high coverage, occurs with this K344C mutant than with the wild-type enzyme. The subsequently formed close-packed monolayer arrays have been probed by scanning tunnelling microscopy under ambient conditions and under aqueous (buffered) solution at high resolution. Initial indications suggest that the immobilised enzyme is both electrochemically addressable and catalytically active.

Does inadequate prenatal care contribute to growth retardation among second-born African-American babies?

The authors evaluated the relation between adequacy of prenatal care and risk of delivery of full term small-for-gestational-age (SGA) infants. Data were derived from maternally linked birth certificates for 6,325 African-American women whose first two pregnancies ended in singleton, full term live births in Georgia from 1989 through 1992. The authors used stratified analysis to assess the effect of prenatal care on the risk of having an SGA baby in the second pregnancy among women with and without an SGA baby in their first pregnancy. The group of women with a history of SGA birth may be more likely to include persons for whom SGA delivery is related to factors, such as genetics, that are not amenable to intervention by prenatal care. Inadequate prenatal care was not associated with the risk of SGA delivery among women who had previously delivered an SGA baby. In unadjusted analyses, inadequate prenatal care was associated with an increased risk of delivering a full term SGA baby in the second pregnancy among women whose first baby was not SGA (risk ratio = 1.28; 95% confidence interval: 1.05, 1.55). The association did not persist when data were adjusted for confounding variables (odds ratio = 1.11; 95% confidence interval: 0.89, 1.38). Regardless of outcome in the first pregnancy, adequate prenatal care did not reduce the risk of full term SGA birth among second pregnancies in this population.

Surface-modified mutants of cytochrome P450cam: enzymatic properties and electrochemistry.

We report the electrochemistry of genetic variants of the haem monooxygenase cytochrome P450cam. A surface cysteine-free mutant (abbreviated as SCF) was prepared in which the five surface cysteine residues Cys-58, Cys-85, Cys-136, Cys-148 and Cys-334 were changed to alanines. Four single surface cysteine mutants with an additional mutation, R72C, R112C, K344C or R364C, were also prepared. The haem spin-state equilibria, NADH turnover rates and camphor-hydroxylation properties, as well as the electrochemistry of these mutants are reported. The coupling of a redox-active label, N-ferrocenylmaleimide, to the single surface cysteine mutant SCF-K344C, and the electrochemistry of this modified mutant are also described.

Viral infections among patients with hemophilia in the state of Georgia.

This analysis evaluated the extent to which infections with selected blood-borne viruses, specifically infection with hepatitis B virus, hepatitis C virus, and/or the human immunodeficiency virus (HIV), continue to contribute to the morbidity of persons with hemophilia. The Georgia Hemophilia Surveillance System collected information on 336 state residents with hemophilia A or B who were followed by a physician in 1994. Data abstracted from medical records included information on demographics, sources of hemophilia care, clinical characteristics, joint range of motion measurements, hospitalization, and results of laboratory testing for hepatitis B, hepatitis C, and the human immunodeficiency virus. Prevalence of infection with one or more of these viruses was determined, and relationships with disease severity, bleeding frequency, and amount of clotting factor prescribed were explored. No child under the age of ten was positive for the human immunodeficiency virus; hepatitis infection was also uncommon in this age group, in contrast to the very high frequency of such infections among older subjects. There was a strong association between HIV positive status and infection with one of the hepatitis viruses. The likelihood of all types of viral infection increased with frequency of bleeding and with amount of clotting factor received. Efforts to prevent transmission of lipid-enveloped viruses via clotting factor have been extremely effective. However, currently infected hemophilia patients will likely experience significant morbidity and mortality due to chronic liver disease and AIDS-related complications.

African-American physicians' and dentists' perceptions of smoking cessation: solo versus HMO practices.

This study sought to assess the differences in smoking cessation (SC) patient practices and attitudes of physicians and dentists by type of practice (i.e. solo versus group or health maintenance organization [HMO]). Few studies have compared demographics, attitudes, and practices between solo and group providers. The results from this survey of African-American (AA) physicians and dentists provide insights into the perceptions of SC by AA health-care providers in a Southern metropolitan area. Approximately 373 physicians and 90 dentists were mailed questionnaires along with a letter describing the purpose of the study and elements of confidentiality. Dentists and physicians reported nearly the same percentage of smoke-free offices. Physicians were more likely to report that they record information on patients' smoking status and that they counsel patients to quit. Measuring attitudes and current practices among physicians and dentists with regards to SC is essential if health promotion efforts are to have an impact among the AA population.

Site-specific introduction of an electroactive label into a non-electroactive enzyme (beta-lactamase I).

A cysteine residue was introduced close to the active site of beta-lactamase I by site-directed mutagenesis to replace tyrosine-105 and was subsequently modified with an electroactive SH-specific reagent, N-(2-ferrocene-ethyl)maleimide. The resulting modified enzyme became electroactive, showing good quasireversible electrochemistry which was characteristic of the attached ferrocene moiety while retaining its specific enzymatic activity. In the presence of a suicide substrate, 6beta-iodopenicillanic acid, the redox potential shifted +20 mV suggesting that the label was sensitive to changes in the active site of the enzyme.

Attitudes, beliefs, and practices regarding smoking and smoking cessation among African-American physicians and dentists.

African-American physicians and dentists in metropolitan Atlanta were surveyed to assess smoking cessation practices and perceptions. Questionnaires were mailed to 373 physicians and 90 dentists. A total of 154 questionnaires were returned, for an overall response rate of 33.3%. More physicians than dentists considered smoking a "very serious" threat to patients' health, and physicians were more likely to document smoking status in charts and to counsel smokers to quit. Physicians also were approached more frequently by patients seeking cessation advice. Both types of practitioners considered the nicotine patch, formal cessation programs, and behavior modification/psychotherapy to be among the most effective cessation methods, and nicotine gum and acupuncture to be among the least effective. These results indicate African-American physicians are much more involved than dentists in promoting smoking cessation among patients. Advice of health professionals generally is viewed as a powerful influence for African-American patients. Further work is needed to utilize fully the power of health care providers, especially dentists, in the fight against tobacco-related morbidity and mortality.

Racial differences in endometrial cancer survival: the black/white cancer survival study.

OBJECTIVE: To identify factors that explain a lower survival rate among black women with endometrial cancer when compared to white women. METHODS: Data are from the National Cancer Institute's Black/White Cancer Survival Study, a population-based study of racial differences in cancer survival. Subjects included 329 white and 130 black women, ages 20-79 years, residing in the metropolitan areas of Atlanta, New Orleans, or San Francisco-Oakland, diagnosed with endometrial cancer from 1985 to 1987. Known prognostic factors were assessed as potential explanatory variables for the black-white survival difference using proportional hazards regression. Information was derived from interviews, abstracts of hospital and physicians' records, and a centralized review of biopsy and surgical specimens. RESULTS: Adjusting for age and geographic location, risk of death among black women was 4.0 times (95% confidence interval [CI] 2.8, 5.6) that of white women. Approximately 40% of this difference could be attributed to a more advanced stage at diagnosis among black women, and 23% to tumor characteristics and treatment. Further adjustment for all remaining factors reduced the hazard ratio to 1.6 (95% CI 1.0, 2.6). CONCLUSION: Eighty percent of the excess mortality among black women is explained by racial differences in stage at diagnosis, tumor characteristics, treatment, sociodemographic characteristics, hormonal and reproductive factors, and factors related to comorbidities and health behavior. Difference in stage at diagnosis is prominent in explaining the disparity in endometrial cancer survival rates in black and white women. Potential differences in treatment within stage merit further exploration.

Direct electrochemistry of the hydroxylase of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

The redox properties of the hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been thoroughly investigated. Previous studies used redox indicator titrations and spectroscopic methods for the determination of the concentrations of reduced species. Herein we report, for the first time, direct electrochemistry (i.e. without the use of mediators) of the diiron centers of the hydroxylase from M. capsulatus (Bath) at a modified gold electrode giving rise to two waves at 4(+/- 10) mV and -386(+/- 14) mV versus saturated calomel electrode (SCE). In addition, the effects of proteins B and B' on the redox reactions were determined. The redox potentials of the complex with protein B are -25(+/- 14) mV and -433(+/- 8) mV versus SCE whereas protein B' had no effect though it did alter the effect of protein B on the redox potentials.

Bis-methionine ligation to heme iron in mutants of cytochrome b562. 1. Spectroscopic and electrochemical characterization of the electronic properties.

We have generated mutants of cytochrome b562 in which the histidine ligand to the heme iron (His102) has been replaced by a methionine. The resulting proteins can have bis-methionine coordination to the heme iron, but the stability of this arrangement is dependent on oxidation state and solution pH. We have used optical, MCD, and EPR spectroscopies to study the nature of the heme coordination environment under a variety of conditions. Optical spectra of the reduced state of the single variant, H102M, are consistent with bis-methionine ligation. In its oxidized state, this protein is high-spin under all conditions studied, and the spectroscopic properties are consistent with only one of the methionine ligands being coordinated. We cannot identify what, if anything, provides the other axial ligand. A double variant, R98C/H102M (in which the heme is covalently attached to the protein through a c-type thioether linkage), is also bis-methionine coordinated in the ferrous state, but has significantly different properties in the oxidized state. With a pKa of 7.1 at 20 degrees C, the protein converts from a low-spin, 6-coordinate heme protein at low pH, to a high-spin species, similar to the high-spin species observed for the single variant. Our spectroscopic data prove that the low-spin species is bis-methionine coordinated. The reduction potential of this bis-methionine species has been measured using direct electrochemical techniques and is +440 mV at pH 4.8. The electrochemistry of these proteins is complicated by coupled coordination-state changes. Proof that the ferrous state is bis-methionine coordinated is provided by NMR results presented in the following paper.

Factors associated with inadequate prenatal care during the second pregnancies among African-American women.

A longitudinally linked data set for Georgia was used to identify characteristics, including previous prenatal care use and complications at the first birth, associated with prenatal care use in the second pregnancy among 8,224 African-American women. More than 70% of the women who were < 25 years of age at their first birth (younger women) and almost 40% of women who were > or = 25 years at their first birth received inadequate care with at least one of their first two births. Women who received inadequate care in their first pregnancy were more likely to receive inadequate care in their second pregnancy than women who received adequate care in their first pregnancy. Younger women with a history of a stillbirth, neonatal death, or vacuum extraction were less likely to receive inadequate care in their subsequent pregnancy. Although this study was not able to evaluate the content of prenatal care, it suggested that many African-American women may not receive sufficient care to prevent adverse pregnancy outcomes. Women who receive inadequate care in their first pregnancy must be targeted for interventions that help them overcome economic, situational, or attitudinal barriers to receiving adequate care in their next pregnancy.