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Biomedical subjects

C Ford

Publications and source records attributed to C Ford.

At least 109 records · Page 6Linked to original sources
PubMed1989

Site-directed mutagenesis at the active site Trp120 of Aspergillus awamori glucoamylase.

Trp120 of Aspergillus awamori glucoamylase has previously been shown by chemical modification to be essential for activity and tentatively to be located near subsite 4 of the active site. To further test its role, restriction sites were inserted in the cloned A.awamori gene around the Trp120 coding region, and cassette mutagenesis was used to replace it with His, Leu, Phe and Tyr. All four mutants displayed 2% or less of the maximal activity (kcat) of wild-type glucoamylase towards maltose and maltoheptaose. Michaelis constants (KM) of mutants decreased 2- to 3-fold for maltose and were essentially unchanged for maltoheptaose compared with the wild type, except for a greater than 3-fold decrease for maltoheptaose with the Trp120----Tyr mutant. This mutant also bound isomaltose more strongly and had more selectivity for its hydrolysis than wild-type glucoamylase. A subsite map generated from malto-oligosaccharide substrates having 2-7 D-glucosyl residues indicated that subsites 1 and 2 had greater affinity for D-glucosyl residues in the Trp120----Tyr mutant than in wild-type glucoamylase. These results suggest that Trp120 from a distant subsite is crucial for the stabilization of the transition-state complex in subsites 1 and 2.

Amino Acid Sequence↗
PubMed1989

Failure of insulin infusion to stimulate fractional muscle protein synthesis in type I diabetic patients. Anabolic effect of insulin and decreased proteolysis.

We evaluated the influence of insulin on fractional mixed skeletal muscle protein synthesis (FMPS) in eight type I (insulin-dependent) diabetic patients in the postabsorptive state. FMPS was calculated from the increment in [13C]leucine in mixed skeletal muscle protein obtained by serial percutaneous needle biopsy during a continuous 8-h intravenous infusion of L-[13C]leucine. We used the plasma [13C]-alpha-ketoisocaproate (representing intracellular leucine labeling) as the precursor pool of protein synthesis for our calculations. FMPS during the insulin treatment (0.0472 +/- 0.0046%/h; plasma glucose 4.6 +/- 1.0 mM) was not different from FMPS during insulin deprivation (0.0499 +/- 0.0046%/h; plasma glucose 16.4 +/- 0.5 mM). Using plasma [13C]-alpha-ketoisocaproate at isotopic plateau for calculation of leucine flux and as the precursor for leucine oxidation, we further confirmed the findings of our group and others that insulin treatment decreases leucine flux, leucine oxidation, and the nonoxidative portion of leucine flux. Our data on direct measurement of FMPS provide further evidence that the anabolic effect of insulin in the postabsorptive type I diabetic patient is mediated via reduction of proteolysis rather than by increasing protein synthesis.

Adult↗
PubMed1988

Comparison of cell cultures for rapid isolation of enteroviruses.

Cell culture isolation is still the most reliable method for the detection of enteroviruses from clinical specimens. Rapid diagnosis of enterovirus infection affects patient management. To increase yield and enhance the rapidity of enterovirus isolation in cell cultures, we used Buffalo green monkey kidney (BGM) cells and subpassages of primary human embryonic kidney (HEK) cells in addition to the human diploid fibroblast (MRC-5) cells and primary cynomolgus or rhesus monkey kidney (MK) cells routinely used for enterovirus culturing. Growth characteristics of enteroviruses from 421 specimens were studied. All specimens were cultured in MRC-5, MK, and BGM cells, and 204 of these specimens were also cultured in HEK cells. Forty-two percent of the enteroviruses became positive within 3 days, and 85% did so within 7 days. MRC-5 cells provided the highest yield of enteroviruses overall and were the best cell type for the recovery of poliovirus and echovirus. MK cells provided the second best yield but were more useful than MRC-5 cells for coxsackievirus. BGM cells supported the growth of additional isolates of coxsackievirus and enhanced the speed of isolation. HEK cells supported the growth of additional isolates of both coxsackievirus and echovirus, but subculturing was always required for definite enterovirus cytopathic effects. The recovery rate increased 11% when two additional cell lines were used. The use of two tubes of MK cells significantly increased the yield of all enterovirus types. We conclude that the use of multiple appropriate cell lines increases yield and enhances the rapidity of enterovirus isolation.

Animals↗
PubMed1987

Intracellular pH changes during neutrophil activation: Na+/H+ antiport.

Activation of the Na+/H+ antiport mechanism was studied in human neutrophils by monitoring intracellular pH with a carboxyfluorescein derivative. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phospholipase C (PLC) induced biphasic pH changes. Amiloride, which inhibits the antiport, completely blocked alkalinization but enhanced acidification. Polymyxin B, which inhibits protein kinase C, only blocked alkalinization. Activation with phorbol 12-myristate 13-acetate (PMA) led to alkalinization only; this was inhibited by amiloride or polymyxin B. Thus, during polymorphonuclear leukocyte (PMN) activation, intracellular alkalinization appears to be mediated by an amiloride-sensitive Na+/H+ antiport. Antiport activity can also be blocked indirectly by inhibition of protein kinase C activity. Early intracellular acidification does not appear to require kinase activity but is observed when phospholipids are remodeled with PLC. The antiport was also activatable by hypertonic buffered media. This response did not appear to be mediated by protein kinase C because it was unaffected by polymyxin B. Finally, superoxide generation was investigated. It is affected by, but not soley controlled by, either antiport or protein kinase C activity.

Amiloride↗
PubMed1987

Failure to reduce cholesterol as explanation for the limited efficacy of antihypertensive treatment in the reduction of coronary heart disease. Evidence from the Hypertension Detection and Follow-up program (1973-1979).

The recent experience of six large trials of antihypertensive therapy has not clearly demonstrated any beneficial effect on the prevention of coronary heart disease (CHD). The data from the HDFP study have been analyzed by three cholesterol strata at baseline. The higher the baseline cholesterol levels, the greater the risk for CHD. In hypertensive patients, the slope of the relationship between cholesterol and CHD event rate was examined. There is indication of an increase of about 6 CHD events per 1,000 patients for each 50 mg/mdl increase in cholesterol (p less than 0.05). This population was further divided into those with major end organ damage (EOD) and those without EOD at baseline. In patients who had no EOD, examination of baseline cholesterol level and 5-year CHD death rates indicates a similar relationship. In contrast, the lack of correlation between baseline cholesterol level and CHD death rates in those hypertensives with EOD, suggests the need to reduce hypercholesterolemia before EOD occurs.

Adult↗
PubMed1986

Cell alkalinization is not necessary and increased sodium influx is not sufficient for stimulated superoxide production.

Preincubation of rabbit neutrophils for 5 min with the protein kinase C inhibitor H7 causes inhibition of the rise in intracellular pH but not the increase in Na+ influx or stimulated oxidative burst produced by the chemotactic factor formyl-methionyl-leucyl-phenylalanine. On the other hand, the stimulated superoxide production, but not the increase in Na+ influx produced by phorbol 12-myristate 13-acetate, is inhibited by H7. The effect is more pronounced on the rate than the extent of the stimulated superoxide release. Furthermore, cell acidification produced by the phorbol ester but not by the chemotactic factor is decreased in the presence of H7. These results suggest that most of the stimulated Na+ influx is not coupled to H+ efflux, in the case of the chemoattractant, the rise in intracellular pH is not necessary for stimulated superoxide production, the increase in Na+ influx, in the case of the phorbol ester, is not sufficient for the stimulation of the oxidative burst, and the sources of the H+ responsible for the stimulated pH drop are the various metabolic activities of the cell, including NADPH oxidation and activation of the hexose monophosphate shunt.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1986

Depth of penetration in periodontal pockets with oral irrigation.

The purpose of this study was to determine the effectiveness of the Water Pik oral irrigator as a vehicle for delivering an aqueous solution into periodontal pockets. Plaque-disclosing dye diluted with sterile saline solution was applied with the irrigator toward the gingival margins of teeth at 90 degrees and at 45 degrees prior to their extraction. The mean % penetration measured between a reference notch at the gingival crest and the periodontal ligament at the bottom of the pocket showed no statistical difference between the two angles of application. Penetration ranged from 44% to 71%, the lowest being into pockets 4-7 mm; higher mean penetration was noted in both subgroups 0-3 and greater than 7 mm. No statistical difference was found between proximal and facial or lingual surfaces, maxilla and mandible, existence of tooth contact, and proximal tissue contour or consistency. The mean % penetration was independent of pocket depth (chi 2 analysis). Correlation between pocket depth and mean penetration was low for all but one subgroup (90 degrees application and pockets greater than 7 mm). The results suggest that the oral irrigator will deliver an aqueous solution into periodontal pockets and will penetrate on average to approximately half the depth of the pockets.

Dental Devices, Home Care↗
PubMed1983

Preliminary characterisation of inhibitors of DNA polymerase isolated from Xenopus laevis early embryos.

Inhibitors of DNA polymerase have been detected in Xenopus laevis ovary and egg extracts. The characteristics of the inhibitors differ between the two extracts. In ovary preparations, the inhibitor is retained by dialysis tubing and is heat sensitive, whereas in egg extracts it is diffusable and heat stable. In both extracts, the activity co-elutes with DNA polymerase after ion exchange chromatography. Chromatography of ovary extracts renders the inhibitor diffusable and heat stable. Preliminary characterisation of inhibitory activity from eggs shows that the substance is sensitive to pronase digestion and has an approx. 300-500 molecular weight. Kinetic studies demonstrate that the inhibitor is uncompetitive with the DNA template and show mixed inhibitory kinetics with respect to the deoxynucleotides.

Animals↗
PubMed1983

Effect of poor diabetic control and obesity on whole body protein metabolism in man.

We have investigated whole body protein turnover in the fasted state in five normal men, five male Type 1 diabetic patients off insulin therapy, and five obese women, using IV 13C-leucine as a tracer. In diabetic patients, there was, as expected, a greater net loss of protein in the fasted state than in normal subjects. However, contrary to animal and studies in vitro, our diabetic patients in the fasted state showed a greater rate of protein synthesis than normal subjects (p less than 0.01). The increased net loss of protein in diabetic patients compared with normal subjects arose because, in the diabetic patients, protein breakdown was increased even more than protein synthesis under the conditions of this study. Plasma leucine concentration was higher in diabetic and in insulin-insensitive obese patients than in normal subjects (p less than 0.01), and higher in diabetic than in obese patients (p less than 0.05). The rate of protein synthesis per kg lean body mass was also higher in diabetic patients than in obese or normal subjects (p less than 0.01), and higher in obese than normal subjects (p less than 0.05). We conclude that, in human subjects, whole body leucine and protein metabolism are very sensitive to the action of insulin.

Adult↗
PubMed1983

A coordinating center in a clinical trial: the Hypertension Detection and Followup Program.

Multicenter clinical trials are often larger and more complex than other methods of clinical inquiry. They tend to involve a number of research or clinical centers and several formal committees. In many of these trials a coordinating center is one of the participating organizational units. This article describes one such coordinating center, that of the Hypertension Detection and Follow-up Program (HDFP). In 1971 the HDFP Coordinating Center was established to assist in planning and implementing this National Heart, Lung, and Blood Institute (NHLBI)-sponsored, multicenter, community-based, randomized, controlled clinical trial. The HDFP Coordinating Center is a large, intricate organization comprised of personnel who perform a wide variety of functions. From 1972 to 1979 it supervised the adherence to a common protocol among the cooperating centers and reported the Program's progress to the various monitoring and review committees, the Steering Committee, and the NHLBI Program Office. The Program screened approximately 159,000 persons ages 30 to 69 years, identifying and following 10,940 hypertensive participants. It has been the responsibility of this Coordinating Center to institute, coordinate, and monitor the data-gathering activities of the study as a whole and to process, store, and analyze the large, multifaceted set of data that were collected.

Clinical Trials as Topic↗
PubMed1983

Further analysis of the effect of ultra-violet irradiation on the formation of the germ line in Xenopus laevis.

Ultra-violet (u.v.) irradiation of the vegetal pole of newly fertilized eggs has three documented effects: reduction of primordial germ cells (PGCs), cytological damage to the vegetal hemisphere and disruption of the normal mechanism by which the vegetal yolk mass induces the formation of the dorsal axis of the embryo. In this study, we find that 90 degrees rotation of the egg for various periods after irradiation rescues the dorsal axial structures but does not restore the number of PGCs found in the dorsal mesentery of the gut; neither is there any correlation between reduced numbers of PGCs and disruption of cleavage at the vegetal pole. We therefore conclude that the effect on the germ line is separate from the other two phenomena. Secondly, 90 degrees rotation of non-irradiated eggs was found to significantly reduce germ cell numbers migrating in the dorsal mesentery of the gut.

Animals↗
PubMed1982

Use of monoclonal antibodies for the histopathological diagnosis of human malignancy.

This paper describes the use of a panel of seven monoclonal antibodies (selected so as to include reagents reactive with both epithelial and lymphoid cells) for distinguishing between anaplastic carcinoma and high grade lymphoma. Details are given of the immunohistological reactions of these antibodies against a wide range of both normal and malignant tissues and of a number of practical instances in which use of the antibody panel enabled a diagnosis to be made when routine histological examination had been inconclusive.

Adult↗
PubMed1980

Three new yellow loci in Chlamydomonas reinhardtii.

Three phenotypically 'yellow', mendelian mutants of Chlamydomonas reinhardtii have been isolated and tested for allelism with the yellow mutant y-1 a1 and with each other. The three mutants represent three new yellow loci, two of which are located on linkage group I. Like y-1a, the mutants accumulate protochlorophyllide when grown under dim light, but have a wildtype phenotype when grown in the light. We conclude that the control of light-independent protochlorophyllide reduction is more complex than has been thought previously.

Alleles↗