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Biomedical subjects

J Wiseman

Publications and source records attributed to J Wiseman.

At least 19 recordsLinked to original sources
PubMed1992

Rapid optimization of enzyme substrates using defined substrate mixtures.

A strategy is described for the rapid optimization of kcat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enhanced kcat/Km. High performance liquid chromatography/continuous flow fast atom bombardment mass spectrometry is used to assign structure to each peak in the chromatogram. As an example of the utility and efficiency of "substrate mapping" we describe optimization of the collagenase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (where Dnp is dinitrophenyl) at the P'1 and P'2 positions. Six different mixtures were prepared for evaluation, representing the synthesis of 128 different synthetic substrates. "Substrate mapping" has led to Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2, a substrate that possesses a 10-fold better kcat/Km than Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2.

Amino Acid Sequence↗
PubMed1992

Evaluation of inert markers for the determination of ileal and faecal apparent digestibility values in the pig.

A series of experiments was designed to evaluate inert markers employed in studies of ileal and faecal apparent digestibilities of nitrogen and amino acids in pigs fitted with simple 'T' piece cannulas. Trial 1 assessed the palatability of diets containing (a) 5 g chromic oxide/kg, (b) 1 and (c) 5 g titanium dioxide/kg at two levels of feeding. The results indicated that there were slight problems in terms of period of feed consumption associated with diets (a) and (c), but that these effects were transitory and disappeared following acclimatization, although higher levels of feeding may compound the problem. Trial 2 estimated N balance, faecal apparent digestibility of amino acids as determined by both total collection and ratio of markers, ileal apparent digestibility of amino acids and recovery of markers using diets containing (a) 1 and (b) 5 g Cr2O3/kg, (c) 1 and (d) 5 g TiO2/kg. An additional assessment of acid-insoluble lignin present within the diet was also undertaken. Cr2O3 was associated with the lowest recovery, whilst calculations based on TiO2 gave lower standard errors. Trial 3 examined diurnal variation in digesta sampling and its effect on faecal apparent digestibility of N and amino acids. There were no evident effects. In general, it was concluded that the most appropriate marker to use in studies of this nature was TiO2 at a rate of 1 g/kg.

Amino Acids↗
PubMed1992

TRISS unexpected survivors--a statistical phenomenon or a clinical reality?

Data from patients treated in Pennsylvania-accredited trauma centers during 1989 were analyzed. TRISS expected and unexpected survivors (1.6% of all survivors) differed in many ways. Unexpected survivors were more than twice as likely to have been transferred from a nondesignated trauma center (45.8% vs. 22.8%, p < 0.001). Unexpected survivors had significantly higher frequencies of motor vehicle injuries (56.2% vs. 38.3%, p < 0.001), pedestrian injuries (9.6% vs. 5.4%, p < 0.01), and gunshot wounds (7.3% vs. 4.7%, p < 0.01). Expected survivors were injured more frequently in falls (26.1% vs. 10.8%, p < 0.001) and were less frequently male (64.5% vs. 75%, p < 0.001). Unexpected survivors had significantly longer average hospital stay (29.6 s vs. 9.3 days, p < 0.001) and more frequent (98.8% vs. 36.8%, p < 0.001) and longer average stays in the ICU (13.3 s vs. 4.1 days, p < 0.001). The percentage of unexpected survivors discharged to rehabilitation centers (61.9%) was significantly greater than that for expected survivors (8.7%), (p < 0.001). Unexpected survivors were more frequently judged "completely dependent" in five measures of functional disability than expected survivors. We conclude that unexpected survivors are a seriously injured and clinically relevant patient set, not just a statistical phenomenon.

Activities of Daily Living↗
PubMed1992

Does Graves' disease or thyrotoxicosis affect the prognosis of thyroid cancer.

Twenty-one patients who underwent surgical treatment for thyrotoxicosis and who were found at operation to have thyroid cancer are presented. Sixteen had Graves' disease and 5 had toxic nodular goiter. The group with Graves' is compared with 110 euthyroid patients with thyroid cancer who underwent their initial surgery in the same time period and who were of the same age (+/- 1 yr) and sex as the patients with Graves' disease. None of the thyrotoxic patients died during follow-up of 2-24 yr or developed subsequent metastases. The 1 patient with a local lymph node metastasis has not shown evidence of recurrence. Hypoparathyroidism appeared as a complication in only 1 patient. The size of tumors in the patients with Graves' disease was significantly smaller than in the euthyroid group. The course of the disease in both the patients with Graves' disease and the thyrotoxic group as a whole was relatively benign. This series does not support the recent suggestions that thyroid cancer in patients with Graves' disease is more aggressive than in either patients with toxic nodular goiter or euthyroid subjects. Patients with Graves' disease and thyroid cancer should be treated identically to other patients with thyroid cancer. Therapy should consist of total thyroidectomy followed by a postoperative 131I scan. Residual tissue or metastases found on the scan should be ablated with 6 GBq 131I. The patient should receive a suppressive dose of T4.

Adult↗
PubMed1991

N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine.

Authentic N omega-hydroxy-L-arginine was synthesized and used to determine whether it is an intermediate in nitric oxide (.NO) synthesis from L-arginine by macrophage .NO synthase. The apparent Km (6.6 microM) and Vmax (99 nmol x min-1 x mg-1) observed with N omega-hydroxy-L-arginine were similar to those observed with L-arginine (Km = 2.3 microM; Vmax = 54 mumol x min-1 x mg-1). N omega-Hydroxy-D-arginine was not a substrate. Stable isotope studies showed that .NO synthase exclusively oxidized the hydroxylated nitrogen of N omega-hydroxy-L-arginine, forming .NO and L-citrulline. As with L-arginine, O2 was the source of the ureido oxygen in L-citrulline from N omega-hydroxy-L-arginine. In the presence of excess N omega-hydroxy-L-arginine, .NO synthase generated a metabolite of L-[14C]arginine that cochromatographed with authentic N omega-hydroxy-L-arginine. The labeled metabolite exhibited identical chromatographic behavior in three solvent systems and generated the same product (L-citrulline) upon alkaline hydrolysis as authentic N omega-hydroxy-L-arginine. Experiments were then run to identify which redox cofactor (NADPH or tetrahydrobiopterin) participated in the enzymatic synthesis of N omega-hydroxy-L-arginine. Both cofactors were required for synthesis of .NO from either N omega-hydroxy-L-arginine or L-arginine. However, with L-arginine, the synthesis of 1 mol of .NO was coupled to the oxidation of 1.52 +/- 0.02 mol of NADPH; whereas with N omega-hydroxy-L-arginine, only 0.53 +/- 0.04 mol of NADPH was oxidized per mol of .NO formed. These results support a mechanism in which N omega-hydroxy-L-arginine is generated as an intermediate in .NO synthesis through an NADPH-dependent hydroxylation of L-arginine.

Amino Acid Oxidoreductases↗
PubMed1991

Determination of enzyme specificity in a complex mixture of peptide substrates by N-terminal sequence analysis.

A method has been developed to determine preferred residue substitutions in the P' position of peptide substrates for proteolytic enzymes. The method has been validated with four different enzymes; the angiotensin I-converting enzyme, atrial dipeptidyl carboxyhydrolase, bacterial dipeptidyl carboxyhydrolase, and meprin A. A mixture of N-acylated potential peptide-substrates for each of the enzymes was prepared in a single synthesis procedure on the same solid-phase synthesis resin. The peptides were identical in all residue positions except the P' position to be studied, into which numerous amino acid residues were incorporated on a theoretical equimolar basis. After cleavage and extraction of the peptides from the resin, no attempt was made to purify them individually; the exact concentration of each peptide in the mixture was determined by quantitative amino acid analysis. Incubation of an enzyme with its peptide-substrate mixture at [S] much less than Km yielded peptide hydrolytic products with newly exposed N-termini. The identity and amount of each hydrolysis product was determined by automated N-terminal sequence analysis. One cycle of sequencing revealed preferred amino acid substitutions in the P'1 position, two cycles the P'2 position, and so forth. Comparison of the rates of production of the various products indicates the preferred substitution in that particular P' position. New information on the substrate specificities of each of the enzymes tested was obtained and it is clear that this approach can be applied to any protease with a defined (or suspected) point of cleavage in a peptide substrate.

Amino Acid Sequence↗
PubMed1991

The influence of free fatty acid content and degree of saturation on the apparent metabolizable energy value of fats fed to broilers.

To determine the influence of free fatty acid (FFA) content on the subsequent nutritive value of fats, the following were studied: tallow (FFA content of 138.4 g/kg) and tallow acid oil (FFA = 952.1 g/kg) in Experiment 1; palm oil (FFA = 57.5 g/kg) and palm acid oil (FFA = 917.5 g/kg) in Experiment 2; and soybean oil (FFA = 14.4 g/kg) and soybean acid oil (FFA = 683.4 g/kg) in Experiment 3. The two fats within each experiment were blended in proportions to give three mixtures of intermediary FFA content. Thus, each experiment investigated five fats by substituting them into a basal diet at the expense of the energy-yielding ingredients at rates of 40, 80, and 120 g/kg. Experimental diets were evaluated for apparent fat availability (AFA) of broiler chicks 1.5 and 7.5 wk of age. Corresponding values for fats were obtained through extrapolation of the linear response, or interpolation of the quadratic response, of AFA of diets. The AME was calculated as the product of fat AFA and gross energy. The degree of saturation had a pronounced effect upon the AME of fats; values decreased in the following order: soybean oil, tallow, and palm oil. Overall AME of fats linearly decreased with increasing FFA content, and the decrease was more pronounced with the younger birds. This rate of reduction appeared to be influenced by degree of saturation of the fat, but only with younger birds. Thus, AME of fats declined from 30.9 to 20.4, 27.7 to 14.8, and 38.5 to 33.1 MJ/kg (1 MJ = .239 Mcal) for young birds in Experiments 1, 2, and 3, respectively. Corresponding data for older birds were from 32.9 to 28.3, 32.3 to 27.5, and 38.5 to 35.1 MJ/kg. Effects of rate of inclusion of fats were minor and, generally, were confined to fats of a higher degree of saturation with greater contents of FFA evaluated with younger birds.

Analysis of Variance↗
PubMed1991

Prediction of the apparent metabolizable energy content of fats fed to broiler chickens.

Soybean oil and tallow acid oil were blended in the ratio 75:25, 50:50, and 25:75 to give 3 blends (A, B, and C) of increasing free fatty acid (FFA) content but decreasing ratio of unsaturated to saturated fatty acids (U:S). Tallow and soybean acid oil were blended in similar proportions to give three blends (D, E, and F) of increasing FFA content and increasing U:S ratio. The six fats were incorporated into a basal diet at rates of 40, 80, and 120 g/kg. Experimental diets were evaluated for apparent available fat (AAF) with broilers aged 1.5 and 7.5 wk of age. The AAF of fats was calculated from extrapolation of the regression of AAF of diets to rate of inclusion of fats. The AME of fats was determined as the product of AAF and gross energy. Increasing FFA content but reducing U:S ratio lowered the AME giving 31.1, 28.7, and 21.7 MJ/kg (1 MJ = .239 Mcal) and 33.2, 31.5, and 28.4 MJ/kg, respectively, for A, B, and C for young and old birds. Corresponding data for D, E, and F (increasing FFA content and U:S ratio) were 27.7, 28.0, and 29.4 MJ/kg and 31.8, 32.9, and 33.3 MJ/kg, respectively, for young and old birds. Data generated, together with results from previous reports, were subject to regression analysis. The AME of fats could be predicted from knowledge of U:S ratio and FFA content with equations accounting for .816 and .925 of the variation in AME values for young and old birds, respectively.

Analysis of Variance↗
PubMed1990

European reference method for the in vivo determination of metabolisable energy with adult cockerels: reproducibility, effect of food intake and comparison with individual laboratory methods.

1. A common reference procedure adopted by several European laboratories to determine apparent metabolisable energy corrected to zero-nitrogen balance (AMEn) is described. 2. Reproducibility has been estimated by comparing results from 4 diets measured in 7 laboratories. The standard deviations between laboratories of dry matter, gross energy, Kjeldahl nitrogen and AMEn were respectively, on average, 11.29 g/kg, 0.242 MJ/kg, 1.56 g/kg and 0.380 MJ/kg dry matter; the corresponding coefficients of variation between laboratories were 1.27, 1.29, 4.39 and 2.92%. 3. Effect of food intake was tested by comparing AMEn from birds fed either 0.90 or 0.45 of ad libitum: the amount of food intake had a negligible effect on AMEn values. Endogenous energy losses corrected (EELn) or not (EEL) to zero nitrogen balance were estimated either by regression between excreted energy and ingested energy or in fasted and glucose-fed cockerels. EELn estimated by regression was, on average, 8.7 kJ/bird/d.; it was independent of diet composition. EEL and EELn determined in fasted or glucose-fed birds were higher than those determined by regression. 4. AMEn values measured by individual laboratory procedures were very close to those obtained by the reference method, except from laboratories using a tube feeding procedure, where deviations were probably a consequence of overestimated EEL.

Analysis of Variance↗
PubMed1989

Influence of age, chemical composition and rate of inclusion on the apparent metabolisable energy of fats fed to broiler chicks.

1. Vegetable oil (VO) and tallow (T) were evaluated at rates of 25, 50, 75, 100 and 125g/kg in a basal diet with broiler chicks aged 1.5, 3.5, 5.5 and 7.5 weeks of age using a total collection period of 72 h. 2. Apparent metabolisable energy, corrected to zero nitrogen retention (AMEN, MJ/kg) and apparent fat availability (AFA, g/kg) of the experimental diets were employed to estimate the dietary energy values of fats, by linear and quadratic regression. 3. AME of fats, determined from AFA, had smaller standard errors than those determined from AMEN. 4. There was a marked increase in the AME of fats from 1.5 to 3.5 weeks of age; thereafter, the improvement was less pronounced. 5. Rate of improvement in fat AME with age was greater with T than VO.

Age Factors↗
PubMed1987

Interactions between fats of differing chemical content: apparent metabolisable energy values and apparent fat availability.

1. Tallow (A) and rape oil (E) were obtained for evaluation. They were blended in the ratios A95:E5, A90:E10 and A80:E20. The three blends together with the two pure fats were each included at 40, 80 and 120 g/kg into a basal diet. 2. The experimental diets were evaluated for apparent metabolisable energy corrected to zero nitrogen retention (AMEN MJ/kg) and apparent fat availability (g/kg) using 6 replicates of a cage of two male broiler chicks 14 d old and 8 replicates of a cage of one Rhode Island cross cockerel approximately 1 year old. Diets were fed for 72 h then removed for 24 h. This was followed by a 48 h period when food was available ad libitum and a further 24 h of starvation. A total collection of excreta was undertaken each 24 h for the latter 72 h period. 3. Evaluation of AMEN and apparent fat availability for the individual fat blends was achieved through both linear and quadratic regression. 4. Synergism, as demonstrated by a higher determined than calculated AMEN value, was detected with fat blends fed to chicks but not cockerels and only at rates of inclusion beyond 80 g/kg. 5. No consistent differences between AMEN determined through calorimetry or AME determined with reference to apparent fat availability multiplied by fat gross energy were detected.

Animals↗
PubMed1987

Interactions between fats of differing chemical content: apparent availability of fatty acids.

1. Tallow (A) and rape oil (E) were obtained for evaluation. They were blended in the ratios A95:E5, A90:E10 and A80:E20. The three blends together with the two pure fats were each included at 40, 80 and 120 g/kg into a basal diet. 2. The experimental diets were evaluated for apparent availability (g/kg) of the fatty acids palmitic (C16:0), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) using 6 replicates of a cage of two male broiler chicks 14 d old and 8 replicates of a cage of one Rhode Island cross cockerel approximately 1 year old. Diets were fed for 72 h then removed for 24 h. This was followed by a 48 h period when food was available ad libitum and a further 24 h starvation. A total collection of excreta was undertaken for the latter 72 h period. 3. Evaluation of apparent available fatty acid (AAFA g/kg fatty acid) was achieved by linear regression. 4. All results indicated a progressive increase in AAFA with both chicks and adults for C16:0 and C18:0 with increasing proportions of the more unsaturated rape oil in the fat blends and for C18:1 and C18:2 with chicks. 5. Evaluation of AAFA by quadratic regression indicated an additional effect of rate of inclusion for C16:0 and C18:0 with chicks and adults and for C18:1 with chicks.

Animals↗
PubMed1987

Reduced life span of the osteoclast in osteopetrotic (mi and midi) mice.

Osteoclasts were enumerated on the parietal bones of mice carrying combinations of alleles (mi, midi and +) at the microphthalmic locus. Homozygous mutants at this locus show varying degrees of osteopetrosis due to defective bone resorption. As the severity of the bone resorption defect increased across the genotypes, there was an increase in the total number of osteoclasts and also an increase in the proportion of uninucleate osteoclasts. In parietal bones incubated for 24 h with parathyroid hormone (PTH) there was no significant difference between osteopetrotic and normal bones in terms of the number of osteoclast nuclei that had taken part in fusion. When bones were incubated for 24 h in the absence of PTH the percentage of osteoclasts remaining was correlated inversely with the severity of the bone resorption defect. Thus, we suggest that the increased proportion of uninucleate osteoclasts in these mutants results from a shortened life span, reducing the time-dependent accumulation of nuclei. This implies a reduced efficiency of uninucleate osteoclasts over multinucleate ones and we speculate on the reason for this.

Alleles↗
PubMed1986

Serum osteocalcin concentrations in Paget's disease of bone.

Total body scintiscans, serum alkaline phosphatase estimations, and serum osteocalcin radioimmunoassays were performed in 49 consecutive patients with Paget's disease of bone. Eleven were receiving calcitonin (salmon synthetic) at the time of the study. The serum alkaline phosphatase activities were elevated in all but one patient, with the highest value almost 50 times the upper limit of the reference range. Serum osteocalcin concentrations were elevated in 53% of patients and normal in the rest. The highest serum osteocalcin value was 4.2 times the upper limit of the reference range. The correlation coefficient between the extent of skeletal involvement and serum osteocalcin level was .70, while that between skeletal involvement and serum alkaline phosphatase level was .55. In spite of the better correlation between bone scintiscans and serum osteocalcin level, osteocalcin measurements are diagnostically less useful than serum alkaline phosphatase estimations in patients with Paget's disease of bone.

Aged↗