Biomedical subjects
Publications and source records attributed to G King.
The structure of the Tus-Ter DNA replication fork arrest complex of Escherichia coli reveals a novel architecture for the bound Tus protein and a new type of DNA-binding motif. The structure of the complex may explain how Tus can block movement of a replication fork approaching from one direction and not the other.
A highly sensitive method for light microscopic immunohistochemistry is described. The increased sensitivity compared with current methodologies is based on the horseradish peroxidase-catalysed deposition of biotinylated tyramine at the sites of immunoreactivity, followed by the detection of the biotin with streptavidin biotin horseradish peroxidase complex. This method is of general applicability in immunohistochemistry and has several important advantages over currently used immunohistochemical detection procedures. The most significant advantage is that several antibodies which to data have been non-reactive, even in antigen-retrieval formalin-fixed, was-embedded sections, now show strong and reproducible immunoreactivity using biotinylated tyramine amplification. In addition, many other antibodies can be used at significantly higher dilutions.
Parvalbumins are a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. It is suspected that these proteins evolved via intragene duplication from a single EF-hand. Silver hake parvalbumin (SHPV) consists of three EF-type helix-loop-helix regions, two of which have the ability to bind calcium. The three helix-loop-helix motifs are designated AB, CD, and EF, respectively. In this study, native silver hake parvalbumin isoform B (SHPV-B) has been sequenced by mass spectrometry. The sequence indicates that this parvalbumin is a beta-lineage parvalbumin. SHPV-B was cleaved into two major fragments, consisting of the ABCD and EF regions of the native protein. The 33-amino acid EF fragment (residues 76-108), containing one of the calcium ion binding sites in native SHPV-B, has been isolated and studied for its structural characteristics, ability to bind divalent and trivalent cations, and for its propensity to undergo metal ion-induced self-association. The presence of Ca2+ does not induce significant secondary structure in the EF fragment. However, NMR and CD results indicate significant secondary structure promotion in the EF fragment in the presence of the higher charge-density trivalent cations. Sedimentation equilibrium analysis results show that the EF fragment exists in a monomer-dimer equilibrium when complexed with La3+.
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The acyl binding site of Rhizopus delemar prolipase and mature lipase was altered through site-directed mutagenesis to improve lipase specificity for short- or medium-chain length fatty acids. Computer-generated structural models of R. delemar lipase were used in mutant protein design and in the interpretation of the catalytic properties of the resulting recombinant enzymes. Molecular dynamics simulations of the double mutant, val209trp + phe112trp, predicted that the introduction of trp112 and trp209 in the acyl binding groove would sterically hinder the docking of fatty acids longer than butyric acid. Assayed against a mixture of triacylglycerol substrates, the val209trp + phe112trp mature lipase mutant showed an 80-fold increase in the hydrolysis of tributyrin relative to the hydrolysis of tricaprylin while no triolein hydrolysis was detected. By comparison, the val94Trp mutant, predicted to pose steric or geometric constraints for docking fatty acids longer than caprylic acid in the acyl binding groove, resulted in a modest 1.4-fold increase in tricaprylin hydrolysis relative to the hydrolysis of tributyrin. Molecular models of the double mutant phe95asp + phe214arg indicated the creation of a salt bridge between asp95 and arg214 across the distal end of the acyl binding groove. When challenged with a mixture of triacylglycerols, the phe95asp + phe214arg substitutions resulted in an enzyme with 3-fold enhanced relative activity for tricaprylin compared to triolein, suggesting that structural determinants for medium-chain length specificity may reside in the distal end of the acyl binding groove. Attempts to introduce a salt bridge within 8 A of the active site by the double mutation leu146lys + ser115asp destroyed catalytic activity entirely. Similarly, the substitution of polar Gln at the rim of the acyl binding groove for phe112 largely eliminated catalytic activity of the lipase.
Multiple parvalbumin isoforms have been detected in the tail (skeletal) muscle of the American alligator (Alligator mississipiensis). One of these isoforms (APV-1) has been highly purified and partially characterized. Protein purification involved mainly gel filtration and anion exchange chromatography, and characterization included gel electrophoresis, amino acid composition analysis, metal ion analysis, MALDI-TOF and ESI mass spectrometry, ultraviolet and fluorescence spectroscopy, and one- and two-dimensional 500 MHz proton NMR spectroscopy. The alligator isoforms are rich in phenylalanine and deficient in the other aromatic residues as is typical for parvalbumins. In fact, the one highly purified isoform that forms the basis of this study has only phenyl-alanine as an aromatic residue. Ion exchange chromatography further indicates that this isoform has a relatively high isoelectric point (pl approximately 5.0), indicating that it is an alpha-lineage parvalbumin. This alligator parvalbumin isoform is unusual in that it has an atypically high Ca2+ content (almost 3.0 mole of Ca2+ per mole of protein) following purification, a fact supported by terbium fluorescence titration experiments. Preliminary comparative analysis of the highly purified alligator parvalbumin isoform (in the Ca2-loaded state) by two-dimensional 1H-NMR (2D 1H TOCSY and 2D 1H NOESY) indicates that there is considerable similarity in structure between the alligator protein and a homologous protein obtained from the silver hake (a saltwater fish species).
This paper presents an analysis of the "race" concept as used by researchers who have studied the smoking behavior of African Americans. Most researchers in the field have failed to address the conceptual dimensions and meanings of "race" and accept uncritically the use of the term. This practice is viewed as an impediment in explaining inter- and intra-racial group differences and intervening effectively to reduce consumption of tobacco products. Adopting the majority-minority intergroup relations paradigm, the conceptual and practical meanings of "race" are reviewed by focusing on the history of relations between blacks and tobacco, conceptions of "race," "biology" and cigarette smoking, and the sociological nucleus (e.g. social class, racism and culture) of "race." Genetic or biologic assumptions and meanings of "race" in research on the smoking behavior of African Americans are critically examined. It is argued that "race" is a dynamic social construct reflecting societal transformations in relations between racially classified social groups (RCSGs).
OBJECTIVE: We analysed nm23, c-erbB-2 and p53 protein expression in lung adenocarcinoma in relation to clinicopathological status and patient survival, to elucidate any potential prognostic value. Published reports suggest that high p53 and c-erbB-2 protein expression and loss of nm23 protein expression are associated with poor prognosis. METHODS: A total of 162 pulmonary adenocarcinomas resected between 1980 and 1991 were stained using monoclonal antibodies to nm23 (NCL-nm23), c-erbB-2 (NCL-CB11) and p53 (DAKO Do7). Antigen retrieval was by microwave heating and bound antibody was visualised using standard immunohistochemical methods. Staining was scored by two observers blinded to tumour status and patient survival. RESULTS: Of the tumours, 101/162 (62.3%) exhibited high-level p53 expression, 30 (18.5%) showed high-level c-erbB-2 membrane staining, and 77 (47.5%) demonstrated loss of nm23 positivity. The influence of T and N status and disease stage on postoperative survival was as expected. The predicted effect on patient survival of nm23, c-erbB-2 or nm23 protein expression suggested by previous studies was not verified by our work. This was true both overall and for groups within the same T or N status or stage. CONCLUSION: Immunohistochemical assessment of the nm23, c-erbB-2 and p53 proteins using the above monoclonal antibodies does not have value as an independent prognostic indicator in pulmonary adenocarcinoma.
Immunohistochemical staining of bcl-2 and p53 proteins was compared with thymidine labelling index (TLI) and cell loss factor (O) in lung cancer. Neither bcl-2 nor p53 overexpression was associated with high cell loss but strong bcl-2 staining was associated with higher TLI. Concomitant strong p53 and bcl-2 expression, not the usual inverse relationship, plus high cell-loss factor was present in three neuroendocrine carcinomas. Other factors presumably have a role in controlling cell death in these tumours.
The purpose of this research was to evaluate the combined effect of intensive neurodevelopmental therapy (NDT) and casting in improving hand function, quality of upper-extremity movement and range of motion in children aged between 18 months and 4 years with cerebral palsy (CP). A randomized crossover design was used to evaluate the difference between intensive NDT plus casting and a less intensive regular occupational therapy (OT) program. Blinded assessments of hand function, quality of upper-extremity movement, and parents' perception of hand-function performance were carried out at baseline, 4 months (end of first intervention period), 6 months (after a 2-month 'washout' period), and 10 months (end of second intervention period). Analysis of the outcomes revealed no significant differences in hand function, quality of upper-extremity movement, or parents' perception of hand-function performance between the two treatment groups-intensive NDT plus casting or regular OT programs. There does not appear to be any beneficial effect of an increased amount of therapy for the children in this study.
AIMS: To determine the usefulness of antibodies HBME-1 and antithrombomodulin in the differential diagnosis of malignant mesothelioma of the pleura. METHODS: Using microwave antigen retrieval and streptavidin-biotin complex horseradish peroxidase immunohistochemistry the above antibodies were used to stain sections of 57 malignant mesotheliomas, 17 reactive pleural hyperplasias, 23 cases of carcinoma metastatic in pleura, 20 primary ovarian cell carcinomas, and 20 primary renal cell carcinomas. RESULTS: Eighty six per cent of mesotheliomas and 82% of reactive mesothelial hyperplasias stained strongly with HBME-1. However, 48% of carcinomas metastatic to pleura also stained, as did all serous ovarian carcinomas. Seventy two per cent of mesotheliomas and 24% of reactive mesothelial hyperplasias stained strongly with the antithrombomodulin antibody; 86% and 88%, respectively, of these cases showed staining of any type. While 26% of metastatic carcinomas showed some staining with antithrombomodulin, only one third of these (9%) showed strong, yet focal, staining. Of 40 ovarian and renal carcinomas only two (5%) showed any staining with antithrombomodulin. CONCLUSIONS: HBME-1, although a sensitive mesothelial marker, is not sufficiently specific to be useful diagnostically, as almost half of carcinomas metastatic to pleura also stained positive. Antithrombomodulin is also a sensitive mesothelial marker and is sufficiently specific to be a useful discriminator, positively identifying, in appropriate circumstances, the mesothelial nature of a cell population.
AIM: To investigate immunocytochemical changes in intestinal tissues from patients with intra-abdominal sepsis, and to relate the changes to the possibility of enhanced bacterial adhesion and translocation. METHODS: Tissues from 17 patients suffering from intra-abdominal sepsis and from controls were sectioned and stained immunocytochemically for IgA, IgM, secretory component, J chain, and HLA-DR. Differences in the distribution and characteristics of positively staining cells between the patient groups were assessed. RESULTS: Patients with intra-abdominal sepsis had noticeable reductions in numbers of IgA and IgM plasma cells, reduced J chain staining, and had little immunoglobulin on the surfaces of enterocytes. In contrast, HLA-DR positive cells were increased in the sepsis compared with the control group. The plasma cells present showed cytological changes suggestive of apoptosis. CONCLUSIONS: Stress associated with sepsis and its immediate causes might result in increased plasma glucocorticoid levels that bring about apoptosis of mucosal plasma cells (or their precursors). The consequent reduction in expression of IgA and IgM may favour bacterial adhesion to the enterocytes and facilitate bacterial translocation into the tissues.
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A paucity of data exists regarding the smoking prevention and cessation practices of African-American physicians. Advice from a physician is an important strategy in helping patients quit smoking. This article presents the results of a national survey conducted in the mid- 1980s of African-American physicians about their attitudes and practices toward smoking cessation and prevention. In this study, an adjusted response rate of 54% was achieved, yielding 188 returned questionnaires. Four specific smoking intervention practices were examined as dependent variables: (1) exploring patients' feelings about smoking; (2) discussing smoking with patients' families; (3) providing educational materials to patients; and (4) recording patients' smoking status in the medical charts. The proportion of African-American physician smokers in this study (16%) was greater than the reported rates (5% to 10%) of other physicians but well below the national average for African Americans (32.9%) in 1987. In bivariate analyses of the professional and demographic predictors, age, medical specialty, practice setting, physician smoking status, and certain attitudinal variables were significantly related to smoking intervention practices. Multivariate logistic regression analysis showed that older physicians were less likely to discuss smoking with a patient's family and to record patient smoking status and that respondents who felt their advice was effective were more likely to explore a patient's feelings about smoking.
Seventy-two healthy males and females serially breathed air and 100% O2 at 1.0 atm abs (1.01 bar; AIR and O2, respectively), then 100% O2 at 2.36 atm abs (2.39 bar, HBO) to establish reference values for chest (CH), leg (LG), and foot (FT) PtcO2. Subjects sequentially a) rested supine with legs extended (baseline); b) elevated their monitored leg; c) returned to supine/extended position; d) assumed a seated, both legs dependent posture; and e) returned to supine/extended position. LG and FT PtcO2 decreased during leg elevation and increased when both legs were dependent during AIR, O2, and HBO (P < 0.0001, respectively). LG PtcO2 of females exceeded that for males in all conditions (P < 0.05 to P < 0.0001). Baseline CH PtcO2 also was greater than LG PtcO2 for all subjects in all conditions (P < 0.01 to P < 0.0001) and greater than FT PtcO2 for all conditions (P < 0.01 to P < 0.0001) except AIR. We conclude that: a) position and hyperoxygenation of an extremity significantly affect PtcO2; b) PtcO2 does not follow a decreasing CH to FT gradient in all conditions; c) a gender difference exists for LG PtcO2; and d) PtcO2 reference data are established for the comparative evaluation and clinical management of problem wounds.
Seasonal effects on post-partum ovarian activity, duration and intensity of sexual behaviour were determined for Holstein dairy cattle imported from a temperate climate into a tropical region. Animals were observed continuously during the cooler (temperature-humidity index (THI) < 25) and hotter (THI > 25) seasons for 2 years. They were restricted to a cement footing in the hotter season observation period in Year 1, but had access to both concrete and dirt footing during all other seasons. Sequential milk progesterone profiles provided an indication of when follicular phases occurred, and recorded sexual behaviour was compared with these to determine if oestrous signs accompanied ovulations. Most cows had normal ovarian cycles and ovulated regularly during both seasons, but quiet ovulations occurred with greater frequency during the hotter times of the year (P < 0.05). Demonstrations of sexual behaviour were affected by choice of footing rather than season. The actual time when cows stood passively and allowed herdmates to complete mounting ranged from 5.1 +/- 0.7 to 5.8 +/- 1 h with access to exercise yards and cement or dirt footing, but declined to only 1.3 +/- 1.1 h when animals were confined to cement (P < 0.05). Similarly, the total duration of oestrus and mean number of interactions were significantly (P < 0.05) reduced during the observation period conducted with cows confined to concrete footing. These findings further emphasize that the duration of oestrus in dairy cows in considerably shorter than the commonly quoted 18 h.
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Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.