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Biomedical subjects

R L Baldwin

Publications and source records attributed to R L Baldwin.

At least 19 recordsLinked to original sources
PubMed2007

Salvage of blood urea nitrogen in sheep is highly dependent on plasma urea concentration and the efficiency of capture within the digestive tract.

The aims of this study were 1) to determine whether transfer of blood urea to the gastrointestinal tract (GIT) or the efficiency of capture of urea N within the GIT is more limiting for urea N salvage, and 2) to establish the relationship between plasma urea concentration and recycling of urea N to the GIT. We used an i.v. urea infusion model in sheep to elevate the urea entry rate and plasma concentrations, thus avoiding direct manipulation of the rumen environment that otherwise occurs when feeding additional N. Four growing sheep (28.1 +/- 0.6 kg of BW) were fed a low-protein (6.8% CP, DM basis) diet and assigned to 4 rates of i.v. urea infusion (0, 3.8, 7.5, or 11.3 g of urea N/d; 10-d periods) in a balanced 4 x 4 Latin square design. Nitrogen retention (d 6 to 9), urea kinetics([(15)N2]urea infusion over 80 h), and plasma AA were determined. Urea infusion increased apparent total tract digestibility of N (29.9 to 41.3%) and DM (47.5 to 58.9%), and N retention (1.45 to 5.46 g/d). The plasma urea N entry rate increased (5.1 to 21.8 g/d) with urea infusion, as did the amount of urea N entering the GIT (4.1 to 13.2 g/d). Urea N transfer to the GIT increased with plasma urea concentration, but the increases were smaller at greater concentrations of plasma urea. Anabolic use of urea N within the GIT also increased with urea infusion (1.43 to 2.98 g/d; P = 0.003), but anabolic use as a proportion of GIT entry was low and decreased (35 to 22%; P = 0.003) with urea infusions. Consequently, much (44 to 67%) of the urea N transferred to the GIT returned to the liver for resynthesis of urea (1.8 to 9.2 g/d; P < 0.05). The present results suggest that transfer of blood urea to the GIT is 1) highly related to blood urea concentration, and 2) less limiting for N retention than is the efficiency of capture of recycled urea N by microbes within the GIT.

Amino Acids↗
PubMed2006

Milk production of dairy cows fed differing concentrations of rumen-degraded protein.

Thirty-two multiparous and 16 primiparous Holstein cows in midlactation averaging 126 d in milk were used to determine the effects of rumen-degraded protein (RDP) concentration on lactation performance. Cows were assigned to diets in a repeated Latin square design with 3-wk experimental periods. Diets were formulated to provide 4 concentrations of dietary RDP [6.8, 8.2, 9.6, and 11.0% of dry matter (DM)] while rumen-undegraded protein remained constant (5.8% of DM). Diets contained 50% corn silage and 50% concentrate (DM basis). Ingredients within diets were equal across treatments except for ground corn, soybean meal, and ruminally protected soybean meal. Dry matter intake was not affected by treatment. Milk yield, fat yield, and protein yield all increased linearly when cows were fed diets with greater RDP. Milk fat and protein concentration each increased by 0.16 percentage units for cows fed 11% RDP compared with 6.8% RDP. Milk protein yield increased by 0.19 g/d for every 1 g/d increase in crude protein supplied mainly as RDP. As RDP increased, the efficiency of N use declined linearly. Milk urea N increased linearly when cows were fed increasing amounts of RDP, indicating increased losses of N via urine. Feeding deficient RDP diets to dairy cows can decrease nitrogen excretion, but it also decreases lactation performance. These data show an environmental benefit from underfeeding RDP to dairy cows according to National Research Council requirements, but at a financial cost to the dairy producer.

Animals↗
PubMed2006

Effect of mastitis on milk perchlorate concentrations in dairy cows.

Recent surveys have identified the presence of perchlorate, a natural compound and environmental contaminant, in forages and dairy milk. The ingestion of perchlorate is of concern because of its ability to competitively inhibit iodide uptake by the thyroid and to impair synthesis of thyroid hormones. A recent study established that milk perchlorate concentrations in cattle highly correlate with perchlorate intake. However, there is evidence that up to 80% of dietary perchlorate is metabolized in clinically healthy cows, thereby restricting the available transfer of ingested perchlorate into milk. The influence of mastitis on milk perchlorate levels, where there is an increase in mammary vascular permeability and an influx of blood-derived components into milk, remains unknown. The present study examined the effect of experimentally induced mastitis on milk perchlorate levels in cows receiving normal and perchlorate-supplemented diets. Over a 12-d period, cows were ruminally infused with 1 L/d of water or water containing 8 mg of perchlorate. Five days after the initiation of ruminal infusions, experimental mastitis was induced by the intramammary infusion of 100 microg of bacterial lipopolysaccharide (LPS). Contralateral quarters infused with phosphate-buffered saline served as controls. A significant reduction in milk perchlorate concentration was observed in the LPS-challenged glands of animals ruminally infused with either water or perchlorate. In control glands, milk perchlorate concentrations remained constant throughout the study. A strong negative correlation was identified between mammary vascular permeability and milk perchlorate concentrations in LPS-infused glands. These findings, in the context of a recently published study, suggest that an active transport process is operative in the establishment of a perchlorate concentration gradient across the blood-mammary gland interface, and that increases in mammary epithelial and vascular endothelial permeability lead to a net outflow of milk perchlorate. The overall finding that mastitis results in lower milk perchlorate concentrations suggests that changes in udder health do not necessitate increased screening of milk for perchlorate.

Animals↗
PubMed2005

Fate of dietary perchlorate in lactating dairy cows: Relevance to animal health and levels in the milk supply.

Perchlorate is a goitrogenic anion that competitively inhibits the sodium iodide transporter and has been detected in forages and in commercial milk throughout the U.S. The fate of perchlorate and its effect on animal health were studied in lactating cows, ruminally infused with perchlorate for 5 weeks. Milk perchlorate levels were highly correlated with perchlorate intake, but milk iodine was unaffected, and there were no demonstrable health effects. We provide evidence that up to 80% of dietary perchlorate was metabolized, most likely in the rumen, which would provide cattle with a degree of refractoriness to perchlorate. Data presented are important for assessing the environmental impact on perchlorate concentrations in milk and potential for relevance to human health.

Animals↗
PubMed2005

Characterization of a normalized cDNA library from bovine intestinal muscle and epithelial tissues.

Tissue-specific cDNA library sequences (expressed sequence tags, or EST) yield a detailed snapshot of gene expression and are useful in developing second-generation molecular resources (i.e., microarrays) for gene expression profiling. The objective of this study was to develop and characterize an intestine-specific cDNA library to examine the transcriptome of the bovine gut and identify expressed genes that influence ruminant nutrition and health. We describe BARC-8BOV, a normalized cDNA library developed from mRNA isolated from four distinct intestinal locations (duodenal, jejunal and ileal small intestine, colon) of Holstein dairy cattle resulting in 19,110 5'-EST deposited into the NCBI GenBank EST database. Assembly and clustering of these 19,110 clone sequences yielded 11,208 unique elements (3,419 contigs and 7,789 singletons) with an average length of 695 base pairs. Analysis strongly suggests normalization and tissue pooling were effective at increasing the discovery rate of new bovine sequence. A total of 1,123 sequence elements not previously identified in cattle, but with similarity to known genes in other animal species, were identified and shown to be involved in numerous critical biological processes. An additional 745 transcripts were not previously represented as EST in nucleotide or protein databases, and further analysis of these could lead to the identification of gut-specific transcript variants of known genes or potentially the discovery of novel bovine genes. Of the 11,208 assembled sequences, 11,034, or 98.4%, match sequences present in the bovine DNA trace archive at NCBI, and add to a bovine EST database previously lacking significant gut tissue representation. Ultimately, these data will also contribute in efforts to annotate the bovine genome.

Animals↗
PubMed2005

Metabolic fates of ammonia-N in ruminal epithelial and duodenal mucosal cells isolated from growing sheep.

The objective of this experiment was to determine the capability of ruminant gut tissues to detoxify ammonia-N using short-term incubations of isolated cells in vitro. Ruminal epithelial cells (REC) and duodenal mucosal cells (DMC) were isolated from growing Texel-Polypay ram lambs (n = 4) fed a pelleted forage:concentrate-based diet. Immediately after isolation, primary cells were incubated for 60 min with glucose (1mM), glutamate (1mM), [15N]ammonium chloride (5, 10, 20, or 40 mM), and 1 of 4 combinations of substrates (1 mM each) that could support urea synthesis [control, N-carbamoylglutamate (NCG); NCG + ornithine (ONCG); and ONCG + aspartate (AONCG)]. Treatments were arranged in a 4 x 4 factorial design. Incorporation of ammonia-15N into alanine, citrulline, arginine, and urea was determined by gas chromatography-mass spectrometry. For both cell types, ammonia-N transfer to alanine was lower when incubation medium contained NCG compared with control, whereas use of ammonia-N for net alanine synthesis increased quadratically with ammonia concentration regardless of substrate treatment. For REC, ammonia-N was not incorporated into citrulline, arginine, or urea, nor into arginine or urea by DMC. Ammonia-N use for net citrulline synthesis exhibited an inverse relationship with ammonia concentration, decreasing linearly as media ammonia concentration increased. Thus, ala-nine synthesis may be a significant metabolic pathway for ruminant gut tissues to detoxify ammonia-N when it is presented luminally at high concentrations as compared with detoxification by the ornithine-urea cycle. Furthermore, DMC do exhibit a metabolic capability to incorporate ammonia-N into citrulline, but low or absent activity of downstream enzymes of the ornithine-urea cycle appears to limit ammonia-N transfers to urea.

Alanine↗
PubMed2004

Influence of abomasal carbohydrates on small intestinal sodium-dependent glucose cotransporter activity and abundance in steers.

Most animals adapt readily to increased supplies of carbohydrate in the intestinal lumen by increasing enzymes for degradation and increasing glucose transporter activity. However, the extent of upregulation of Na+-dependent glucose cotransporter 1 (SGLT1) activity and content in response to increased delivery of carbohydrate to the small intestinal lumen of ruminants is unclear. Therefore, an experiment was conducted to determine the effect of glucose and starch hydrolysate on the activity and abundance of SGLT1 in the small intestine of steers. In a randomized complete block design, 40 crossbred beef steers (243+/-2 kg BW) were fed 0.163 Mcal of ME/(kg BW0.75(d; W), 0.215 Mcal of ME/(kg BW0.75 x d; 2M), or 0.163 Mcal ME/(kg BW0.75 x d) and infused for 35 d into the rumen (R) or abomasum (A) with 12.6 g/(kg BW0.75 x d) of starch hydrolysate (S) or into the abomasum with 14.4 g/(kg BW0.75 x d) of glucose (G). Steers were slaughtered, and brush-border membrane vesicles were prepared from the small intestinal samples obtained from five equidistant sites along the intestine. Maltase activity in vesicles and homogenates differed with intestinal sampling site (quadratic, P < 0.001). Steers on the AG treatment yielded a greater intestinal maltase activity (38 nmol glucose x mg protein(-1) x min(-1)) compared with the AS, RS, W, or 2M treatments (34, 26, 23, and 23 nmol glucose x mg protein(-1) x min(-1) respectively [SEM = 3; P = 0.02]). Sodium-dependent glucose uptake averaged 18.4+/-3.94 pmol glucose/(mg protein x s) and was not affected by treatment, but uptake decreased distally along the intestine (P < 0.001). There was no effect of treatment on SGLT1 protein abundance, but SGLT1 protein abundance increased linearly from the duodenum to the ileum (P = 0.05). The inverse relationship between glucose uptake and SGLT1 abundance suggests that the regulation of brush border Na+-dependent glucose transport capacity is complex, involving factors other than the presence of luminal carbohydrate.

Abomasum↗
PubMed2004

Oxidation of glucose, glutamate, and glutamine by isolated ovine enterocytes in vitro is decreased by the presence of other metabolic fuels.

The objective of this study was to evaluate oxidative metabolism of glucose, glutamate, and glutamine by isolated ovine enterocytes in the presence of other metabolic fuels in vitro. A mixed mucosal primary cell culture containing enterocytes was isolated from crossbred wether sheep (n = 6) fed a mixed forage-concentrate diet and incubated for 90 min with 1 mM U-14C-glucose, -glutamate, or -glutamine and additional substrates (water as negative control, acetate, propionate, butyrate, glucose, glutamate, or glutamine) at concentrations of 0.1, 1.0, and 10.0 mM. Oxidation of labeled substrates to CO2 and net production of lactate and pyruvate in incubation media were measured. Oxidation of glucose and glutamine to CO2 was decreased (P < 0.05) by 5 to 40% in the presence of additional substrates except acetate. Our observation that glutamine oxidation can be decreased by the presence of additional substrates is contrary to observations in the literature using enterocytes from nonruminants, indicating that ruminant enterocytes might rely on glutamine to a lesser extent as an energy source. Net glucose utilization was decreased (P < 0.05) 16% by propionate (10 mM) compared with control but was not affected by the other additional substrates. Glutamate oxidation to CO2 was decreased 28% (P < 0.05) in the presence of propionate (10 mM) or by 17 and 33% in the presence of glutamine (1.0 and 10 mM, respectively), but not by that of the other additional substrates. Acetate did not affect the oxidation of glucose, glutamate, and glutamine. Propionate decreased (P < 0.05) the oxidation of glucose and glutamate only at the highest concentration (10 mM), indicating that the sparing effects of propionate on substrate oxidation are affected by its concentration in the incubation media. These observations indicate that ruminant enterocytes possess metabolic flexibility for oxidative metabolism of glucose, glutamine, and glutamate depending on the type and concentration of available additional substrates.

Acetates↗
PubMed2004

Urea synthesis by ruminal epithelial and duodenal mucosal cells from growing sheep.

The objective was to determine whether ruminant gut tissues have the capability to synthesize urea in a short-term incubation. Mixed primary cell cultures containing ruminal epithelial (REC) or duodenal mucosal cells (DMC) were isolated from growing sheep (n = 4) fed a mixed forage-concentrate diet. Cells were incubated (90 min) in a Krebs salts-based buffer with either acetate (5 mM) or propionate (5 mM) plus a combination of substrate intermediates (5 mM) for urea synthesis: arginine, aspartate + citrulline (AspC), aspartate + ornithine + ammonia (AspON), or AspON + N-carbamoylglutamate (AspONG) in a 2 x 4 factorial arrangement of treatments. Volatile fatty acid, propionate vs. acetate, did not influence net urea synthesis. For REC, net urea synthesis (nmoles x (10(6) cells)(-1) x 90 min(-1)) was greatest with Arg (54.5 +/- 6.3) followed by AspC (4.6 +/- 1.1) and AspONG (3.6 +/- 1.4). For DMC, net urea synthesis for Arg (2.1 +/- 0.7) and AspONG (1.9 +/- 0.7) treatments was greater than for AspC (0.3 +/- 0.7) and AspON (-0.6 +/- 0.7) treatments. Thus, for both REC and DMC, arginase activity appeared to be sufficient for catabolism of arginine to urea. Furthermore, greater urea synthesis from ammonia, ornithine and aspartate in the presence of the N-acetylglutamate analogue suggests that carbamoyl phosphate synthetase is probably rate-limiting for urea synthesis and ammonia detoxification by ruminant gut tissues.

Ammonia↗
PubMed2004

Visceral tissue growth and proliferation during the bovine lactation cycle.

Twenty one multiparous, nonpregnant, lactating dairy cows were used to assess the impact of stage of lactation on visceral tissue mass and small intestinal cell proliferation. Cows were slaughtered at each of 4 stages of lactation: 14, 90, 120, and 240 d of lactation. With stage of lactation, DMI increased through d 90 and thereafter remained similar through d 240 (quadratic). Carcass weight and empty body weight (EBW) declined with stage of lactation through d 120 and increased thereafter (quadratic). As a percentage of EBW, rumen, small intestine, and liver weights increased with increasing stage of lactation (quadratic), increasing from 14 to 120 d and declining through 240 d. Stage of lactation did not have a measurable affect on reticulum, omasum, abomasum, or large intestine weights as a percentage of EBW. Visceral adipose mass as a percentage of EBW declined with stage of lactation to a minimum at 120 d and increased by 240 d (quadratic). Concentrations of RNA and DNA of digestive tract organs were largely unaffected by stage of lactation with the exception of the liver DNA concentration through d 120 (quadratic). The proliferative growth fraction (Ki67) was unaffected by stage of lactation. However, bromo-deoxyuridine labeling of jejunal crypts exhibited a cubic response with stage of lactation and tritiated thymidine incorporation by duodenal epithelium increased with stage of lactation through d 120, declining thereafter (quadratic). Mass of visceral tissues increase to meet the energetic demands of lactation and that increased absorption capacity of the intestines is achieved by hyperplastic growth of the intestinal epithelium.

Animals↗
PubMed2004

Bovine somatotropin and rumen-undegradable protein effects in prepubertal dairy heifers: effects on body composition and organ and tissue weights.

The objectives of this study were to determine the effect of recombinant bovine somatotropin (bST) and added dietary rumen undegradable protein (RUP) on organ and tissue weights and body composition in growing dairy heifers. Thirty-two Holstein heifers were in the experiment, 8 killed initially at 3 mo of age, with the remaining 24 Holstein heifers randomly assigned to treatments (n = 6) consisting of 0.1 mg/kg of body weight per day of bST and 2% added dietary RUP (dry matter basis) applied in a 2 x2 factorial design. A total of 6 heifers per treatment group (3 each at 5 and 10 mo of age), were slaughtered to determine body composition and organ masses. Feed intake measured from group intakes were increased by 0.25 and 0.35 kg/d with bST and RUP, respectively. Administration of bST tended to increase the weights of visceral organs including heart, kidney, and spleen by 16, 16, and 38%, respectively. At 10 mo of age, there was a trend for increased empty body weights (EBW) and non-carcass components for heifers treated with bST, but there were no effects of RUP. Body components and organ weights, expressed as a percentage of BW were not affected by RUP or bST. Somatotropin increased ash weight at 10 mo without affecting amounts of protein, fat, and energy. Rates of ash deposition between 3 and 10 mo of age were increased 7 and 4 g/d by bST and RUP, respectively. There were no treatment effects on rates of body fat, protein, and energy deposition. Bovine somatotropin and RUP altered the metabolism of growing heifers in a manner that was consistent with increased rates of skeletal growth. This suggests that nutritional and endocrine manipulations could increase growth rates of skeletal tissues without increasing fat deposition in prepubertal dairy heifers.

Aging↗
PubMed2002

Developmental changes in ketogenic enzyme gene expression during sheep rumen development.

Ketogenesis is the conversion of acetyl-CoA to the ketone bodies acetoacetate and beta-hydroxybutyrate (BHBA). In hepatic ketogenesis, which occurs during fasting in both nonruminant and ruminant animals, the source of acetyl-CoA is the mitochondrial oxidation of predominantly long-chain fatty acids. In the mature, fed ruminant animal, the ruminal epithelium is also capable of producing ketone bodies. In this case, the source of acetyl-CoA is the mitochondrial oxidation of butyrate produced by the microbial fermentation of feed. The purposes of this study were to determine ontogenic and dietary effects on ketogenic enzyme gene expression in developing lamb ruminal epithelium. Twenty-seven conventionally reared lambs and twenty-seven milk-fed lambs were slaughtered between 1 and 84 d of age. Six additional milk-fed lambs were weaned (the fed group) or maintained on milk replacer with a volatile fatty acid gavage (the VFA group) until 84 d of age. At slaughter, total RNA was extracted from samples of ruminal epithelium. The expression of the genes encoding acetoacetyl-CoA thiolase, the first enzyme in the ketogenic pathway, and 3-hydroxy-3-methylglutaryl-CoA synthase, the rate-limiting enzyme in the ketogenic pathway in nonruminant liver, were examined. Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase mRNA concentrations increased with age independent of diet. 3-Hydroxy-3-methylglutaryl-CoA synthase mRNA levels in ruminal epithelium obtained from milk-fed lambs were low before 42 d of age, but a marked increase occurred by 42 d of age. At 84 d of age, there were no differences in acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase expression due to diet. The pattern of the expression of these genes, in particular, 3-hydroxy-3-methylglutaryl-CoA synthase, parallels the rate of production of BHBA by rumen epithelial cells isolated from the same lambs, which increased to conventionally reared adult levels at 42 d of age and did not differ with diet. In conclusion, development of the ketogenic capacity of the ruminal epithelium occurs as the animal ages, regardless of dietary treatment. Thus, the expression of the genes encoding the ketogenic enzymes are not affected by the presence of VFA in the ruminal lumen.

3-Hydroxybutyric Acid↗
PubMed2002

Effects of ruminal and postruminal infusion of starch hydrolysate or glucose on the microbial ecology of the gastrointestinal tract in growing steers.

Forty crossbred steers were used to determine the effects of carbohydrate supply site on the indigenous bacteria of the gastrointestinal tract. Steers were fitted with ruminal and abomasal infusion catheters and assigned randomly to one of eight groups in a complete randomized block design. The experimental period was 36 d. Treatments included: 1) a pelleted basal diet fed at 0.163 Mcal ME x (kg BW(0.75)) x 1 x d(-1) (LE); 2) the basal diet fed at 0.215 Mcal ME x (kg BW(0.75)) (-1) x d(-1) (HE); 3) the basal diet fed at 0.163 Mcal ME x (kg BW(0.75))(-1) x d(-1) with ruminal infusion of starch hydrolysate (SH) (RSH); 4) the basal diet fed at 0.163 Mcal ME x (kg BW(0.75))(-1) x d(-1) with abomasal infusion of SH (ASH); and 5) the basal diet fed at 0.163 Mcal ME x (kg BW(0.75))(-1) x d(-1) with abomasal infusion of glucose (AG). The total volume ofinfusate (5 kg x site(-1) x d(-1)) was equalized across treatments and infusion sites by infusion of water. Glucose and SH were infused at rates of 14.35 and 12.64 g x (kg BW(0.75)) x d(-1), respectively. Ruminal, cecal, and fecal samples were obtained on d 36. Ruminal pH was low (5.79) in LE steers and unaffected (P > 0.10) by increased energy intake or carbohydrate infusion. Cecal and fecal pH were 6.93 and 7.00, respectively, for LE steers. Increasing energy intake (P < 0.10) and the rate of carbohydrate infusion (P < 0.01) significantly decreased cecal and fecal pH compared with LE. Ruminal counts of anaerobic bacteria in LE steers were 8.99 log10 cells/g and abomasal carbohydrate infusion had no affect (P > 0.10) on these numbers. However, ASH and AG steers had approximately 1.5 log10 cells/g more (P < 0.01) cecal and fecal anaerobic populations. Ruminal, cecal, and fecal aerobic bacterial counts were 40, 22, and 23%, respectively, lower than anaerobic counts. Generally, aerobic counts responded similarly to the anaerobic counts. Less than 1% of the anaerobic bacteria enumerated in the rumen, cecum, and feces were coliforms, and 97% of the coliforms were Escherichia coli. Carbohydrate infusions resulted in only numerical increases in fecal coliform and E. coli concentrations (P > 0.10). Fecal E. coli were highly acid sensitive in all steers, with less than 1% surviving a 1-h exposure to low pH (2.0). This suggests that cecal or fecal pH is not a good indicator of acid resistance, and it supports the concept that there are other factors that may induce acid resistance.

Abomasum↗
PubMed2001

The pKa of His-24 in the folding transition state of apomyoglobin.

In native apomyoglobin, His-24 cannot be protonated, although at pH 4 the native protein forms a molten globule folding intermediate in which the histidine residues are readily protonated. The inability to protonate His-24 in the native protein dramatically affects the unfolding/refolding kinetics, as demonstrated by simulations for a simple model. Kinetic data for wild type and for a mutant lacking His-24 are analyzed. The pK(a) values of histidine residues in native apomyoglobin are known from earlier studies, and the average histidine pK(a) in the molten globule is determined from the pH dependence of the equilibrium between the native and molten globule forms. Analysis of the pH-dependent unfolding/refolding kinetics reveals that the average pK(a) of the histidine residues, including His-24, is closely similar in the folding transition state to the value found in the molten globule intermediate. Consequently, protonation of His-24 is not a barrier to refolding of the molten globule to the native protein. Instead, the normal pK(a) of His-24 in the transition state, coupled with its inaccessibility in the native state, promotes fast unfolding at low pH. The analysis of the wild-type results is confirmed and extended by using the wild-type parameters to fit the unfolding kinetics of a mutant lacking His-24.

Acetates↗
PubMed2001

How Ala-->Gly mutations in different helices affect the stability of the apomyoglobin molten globule.

The apomyoglobin molten globule has a complex, partly folded structure with a folded A[B]GH subdomain; the factors determining its stability are not yet known in detail. Ala-->Gly mutations, made at solvent-exposed positions, are used to probe the role of helix propensity of individual helices in stabilizing the molten globule. Molten globule stability is measured by reversible urea unfolding, monitored both by circular dichroism and by tryptophan fluorescence. Two-state unfolding is tested by superposition of these two unfolding curves, and stability data are reported only for variants which satisfy the superposition test. Results for sites Q8 in the A helix and E109 in the G helix confirm that the helix propensities of the A and G helices both strongly affect molten globule stability, in contrast to results for the G65A/G73A double mutant which show that changing the helix propensity of the E-helix sequence has no significant stabilizing effect. Changing the helix propensity of the B-helix sequence with the G23A/G25A double mutant affects molten globule stability to an intermediate extent, confirming an earlier report that this mutant has increased stability. These results are consistent with the bipartite structure for the molten globule in which the A, G, and H helices are stably folded, while the long E helix is unfolded and the B helix has intermediate stability. Some differences are found in the shapes of the unfolding curves of different mutants even though they satisfy the superposition test for two-state unfolding, and possible explanations are discussed.

Alanine↗
PubMed2001

Allelotype of papillary serous peritoneal carcinomas.

OBJECTIVE: Papillary serous peritoneal carcinoma (PSPC) is histologically indistinguishable from papillary serous ovarian carcinoma (PSOC) with a similar clinical presentation, yet may differ in its carcinogenesis. The purpose of this study was to determine the incidence of allelic loss and the frequency of p53 mutation by p53 overexpression in PSPC compared to PSOC. METHODS: An allelotype analysis of 26 patients with PSPC was performed using 39 microsatellite markers from 25 chromosomal arms. Thirty-seven previously studied patients with PSOC served as the comparison. P53 mutations were detected by immunohistochemical protein overexpression. RESULTS: There was significantly less LOH in PSPC than PSOC. Both the number of chromosomes with LOH and the proportion of tumors with allelic loss were less frequent. Significant LOH, defined as >/=30% of informative tumors having loss at a chromosome locus, was seen on 4 chromosome arms in PSPC: 12p, 17p, 17q, and 18q, compared to 18 arms in PSOC: 4q, 5q, 6p, 6q, 9p, 9q, 12p, 12q, 13q, 15q, 16q, 17p, 17q, 18q, 19p, 19q, 22q, and Xq (P < 0.001). The median LOH frequency was higher in PSOC than PSPC, 43% versus 33%, respectively (P = 0.013), and more PSOC tumors had LOH than PSPC tumors, 91% versus 65% (P = 0.042). P53 overexpression was detected in 80% of PSPC tumors. CONCLUSIONS: LOH occurs less frequently in PSPC compared to PSOC. Chromosomal regions with high frequencies of LOH common to PSPC and PSOC, such as 12p, 17p, 17q, and 18q, may harbor tumor suppressor genes important in the carcinogenesis of both malignancies and likely include p53.

Aged↗
PubMed2001

Impact of separating amino acids between plasma, extracellular and intracellular compartments on estimating protein synthesis in rodents.

Three models representing different separations of amino acid sources were used to simulate experimental specific radioactivity data and to predict protein fractional synthesis rate (FSR). Data were from a pulse dose of 14C-U Leu given to a non-growing 20-g mouse and a flooding dose of 3H Phe given to a non-growing 200 g rat. Protein synthesis rates estimated using the combined extracellular and intracellular (Ec + Ic) source pool and extracellular and plasma (Ec + Pls) source pool mouse models were 78 and 120% d(-1) in liver, 14 and 16% d(-1) in brain and 15 and 14% d(-1) in muscle. Predicted protein synthesis rates using the Ec + Ic, Ec + Ic + Tr (combined extracellular, intracellular and aminoacyl tRNA source pool) and Ec + Pls rat models were 57, 3.4 and 57% d(-1) in gastrocnemius, 58, 71 and 62% d(-1) in gut, 8.3, 8.4 and 7.9% d(-1) in heart, 32, 23 and 25% d(-1) in kidney, 160, 90 and 80% d(-1) in liver, 57, 5.5 and 57% d(-1) in soleus and 56, 3.4 and 57% d(-1) in tibialis. The Ec + Ic + Tr model underestimated protein synthesis rates in mouse tissues (5.0, 27 and 2.5% d(-1) for brain, liver and muscle) and rat muscles (3.4, 5.5 and 3.4% d(-1) for gastrocnemius, soleus and tibialis). The Ec + Pls model predicted the mouse pulse dose data best and the Ec + Ic model predicted the rat flooding dose data best. Model predictions of FSR imply that identification and separation of the source specific radioactivity is critical to accurately estimate FSR.

Amino Acids↗