PubMed Health⌕ Search

Biomedical subjects

R L Baldwin

Publications and source records attributed to R L Baldwin.

At least 37 records · Page 2Linked to original sources
PubMed2001

Refinements in primary rumen epithelial cell incubation techniques.

The objectives of this study were to 1) determine if the number of rumen epithelial cells in primary cell incubation affects the rate of metabolite production, and 2) determine the optimum mode of data expression to standardize reporting criteria. Sections of rumen epithelial tissue were excised from five Holstein heifers and subjected to serial tryptic digestion to isolate cells. Isolated cells had a mean viability of 86% (+/- 1.29) and were incubated at concentrations of 0.5, 1, 5, 10, 20, and 40 million cells per flask. Oxidation of [1-14C]butyrate to 14CO2 and production of acetoacetate (ACAC), beta-hydroxybutyrate (BHBA), lactate, and pyruvate were measured for cell dilution comparisons. Cell number, cell dry matter, cell crude protein, epithelial wet tissue weight, body weight, and metabolic body weight were measured to generate 12 different forms of data expression. Coefficients of variation were calculated for each type of expression. Expressing data per cell number resulted in the lowest variation. Oxidation of [1-14C]butyrate to 14CO2 and pyruvate production per million cells did not significantly differ between treatments for 90-min incubation. Acetoacetate and lactate concentrations were greatest at 0.5 and 1 million cells/flask, respectively, with no differences between 5 to 40 million cells/flask. Production of BHBA for 1 million cells/flask was greater than 0.5 and 40 million cells/flask, but did not change between cell concentrations 5 to 20 million. The BHBA:ACAC concentration ratios for 0.5 and 1 million cell dilutions were both 1.1 to 1 indicating low mitochondrial redox potentials. Concomitantly, lactate:pyruvate ratios for 0.5 and 1 million cells were greater than other cell dilutions, indicating a high cytosolic redox potential. The suggested range of rumen epithelial cells to include in incubations is 5 to 20 million cells/flask. This will minimize experimental error associated with using low cell numbers and the potential for reduced metabolite production caused by incubating large cell quantities. When rumen tissue taken from animals of the same species, size, and stage of development; data adjusted by cell number is preferred. However, it is recommended that cell protein, cell DM, and animal metabolic weight be included to facilitate future comparison between species and laboratories.

Animals↗
PubMed2001

Effects of brown midrib corn silage on the energy balance of dairy cattle.

Effects of genotype and level of intake on net energy for lactation values of corn silage were evaluated by indirect calorimetry in two experiments using lactating and dry, nonpregnant dairy cows. In experiment 1, six multiparous Holstein cows in early lactation were fed experimental diets containing either brown midrib (bm3) or isogenic normal corn silage. Dietary treatments were isogenic and bm3 diets fed ad libitum, and the bm3 diets restricted-fed. Dry matter (DM) intake was 2.4 kg/d greater for cows fed the bm3 diet ad libitum compared with cows fed the isogenic diet. Apparent digestibilities of DM, organic matter, neutral detergent fiber, and acid detergent fiber were greater for cows restricted-fed bm3 than the isogenic diet. In experiment 2, six dry, nonpregnant Holstein cows were fed maintenance diets containing either bm3 or isogenic corn silage. Apparent digestibilities of DM, organic matter, neutral detergent fiber, and acid detergent fiber were greater for cows fed bm3 compared with isogenic corn silage. Digestible energy and metabolizable energy were greater for maintenance diets containing bm3 compared with isogenic corn silage, respectively. These data indicate increased milk production seen in other studies is a result of increased DMI rather than an increase in energy efficiency. Increased organic matter digestibility of bm3 corn silage resulted in greater digestible energy and metabolizable energy values in cows fed at maintenance energy intake. However, calculated net energy for lactation values of bm3 and isogenic corn silages were similar at both productive and maintenance levels of feeding.

Animals↗
PubMed2001

Ruminal fermentation and intestinal flow of nutrients by lactating cows consuming brown midrib corn silages.

Rumen characteristics and digestive kinetics of brown midrib corn silage were evaluated with five late-lactation (221 DIM +/- 20 d) multiparous cows fitted with ruminal and duodenal cannula. Dietary treatments were applied by using a single reversal design with two 21-d periods where either brown midrib (BM3) or isogenic (ISO) corn silage were included in a total mixed ration formulated to be 40% concentrate and 60% corn silage on a dry matter (DM) basis. Rumen and total tract digestibilities of DM, organic matter, neutral detergent fiber, acid detergent fiber, starch, and N were determined and rumen characteristics evaluated. Apparent rumen DM and organic matter digestibilities were greater for the BM3 corn silage (7.1 and 4.7 percentage units, respectively). Dietary intake and duodenal flow of starch were greater and rumen and total tract starch digestibilities were lower for BM3 corn silage diets than ISO corn silage diets. However, more starch (1.1 kg/d) was apparently digested and absorbed postruminally in cows fed the BM3 corn silage diets. Duodenal flow of neutral detergent fiber was 0.9 kg/d lower, and ruminal (15.9 percentage units) and total tract digestibilities (4.4 percentage units) were higher for BM3 treatment compared with the ISO treatment. Digestive patterns of ADF were similar for the BM3 and ISO treatments. Ruminal pH was lower in the cows fed the BM3 corn silage than those fed the ISO corn silage. As a result of a tendency for decreased N excretion in urine and slight increases in N intake due to increased DM intake, N balance tended to be greater for the BM3 treatment compared with the ISO treatment. These results may partially explain the benefits of feeding BM3 corn silage to cows during early lactation, as the observed increases in fiber component digestibility and improved N economy may combine to enhance DM intake and better support the nutritional demands of milk production for the high producing dairy cow.

Animal Nutritional Physiological Phenomena↗
PubMed2000

Differential gene expression between normal and tumor-derived ovarian epithelial cells.

The majority of ovarian tumors arise from the transformation of the ovarian surface epithelial cells, a single layer of cells surrounding the ovary. To identify genes that may contribute to the malignant phenotype of ovarian cancers, cDNA representational difference analysis was used to compare expressed genes in primary cultures of normal human ovarian surface epithelium (HOSE) and ovarian tumor-derived epithelial cells from the Cedars-Sinai Ovarian Cancer (CSOC) repository. A total of 255 differentially expressed genes were identified, of which 160 and 95 were specifically expressed in HOSE and CSOC cells, respectively. Using cDNA array hybridization, the expression profiles of the genes identified by cDNA-representational difference analysis were examined in an additional 5 HOSE and 10 CSOC lines. The comparison of average signal of each gene revealed 44 HOSE-specific and 16 CSOC-specific genes that exhibited at least a 2.5-fold difference in expression. A large number of genes identified in this study encode membrane-associated or secreted proteins and, hence, may be useful as targets in the development of serum-based diagnostic markers for ovarian cancer. Very few genes associated with protein synthesis or metabolism were identified in this study, reflecting the lack of observable differences in phenotypic or growth characteristics between HOSE and CSOC cells. Northern blot analysis on a subset of these genes demonstrated comparable levels of gene expression as observed in the cDNA array hybridization.

Blotting, Northern↗
PubMed2000

BRCA1 promoter region hypermethylation in ovarian carcinoma: a population-based study.

There is a clear association between germ-line BRCA1 mutations and inherited ovarian cancer; however, the association between BRCA1 mutations and sporadic ovarian cancer remains ambiguous. The frequency of BRCA1 promoter hypermethylation as an epigenetic means of BRCA1 inactivation was determined for a large, population-based cohort of ovarian cancer patients. BRCA1 promoter hypermethylation was determined by methylation-specific restriction digestion of tumor DNA, followed by Southern blot analysis and confirmed by methylation-specific PCR. BRCA1 promoter hypermethylation was observed in 12 of 98 ovarian tumors. BRCA1 methylation status of the primary tumor was conserved in six recurrent tumors after interim chemotherapy. None of the 12 tumors with BRCA1 promoter hypermethylation demonstrated BRCA1 protein expression by immunohistochemistry. BRCA1 methylation was only seen in ovarian cancer patients without a family history suggestive of a breast/ ovarian cancer syndrome. Therefore, the 12 BRCA1 methylated tumors represented 15% (12 of 81) of the sporadic cancers analyzed in this study. Although the clinical significance of BRCA1 promoter hypermethylation is yet to be determined, promoter hypermethylation may be an alternative to mutation in causing the inactivation of the BRCA1 tumor suppressor gene in sporadic ovarian cancer.

Alleles↗
PubMed2000

Energetics of the interaction between water and the helical peptide group and its role in determining helix propensities.

The alanine helix provides a model system for studying the energetics of interaction between water and the helical peptide group, a possible major factor in the energetics of protein folding. Helix formation is enthalpy-driven (-1.0 kcal/mol per residue). Experimental transfer data (vapor phase to aqueous) for amides give the enthalpy of interaction with water of the amide group as approximately -11.5 kcal/mol. The enthalpy of the helical peptide hydrogen bond, computed for the gas phase by quantum mechanics, is -4.9 kcal/mol. These numbers give an enthalpy deficit for helix formation of -7.6 kcal/mol. To study this problem, we calculate the electrostatic solvation free energy (ESF) of the peptide groups in the helical and beta-strand conformations, by using the delphi program and parse parameter set. Experimental data show that the ESF values of amides are almost entirely enthalpic. Two key results are: in the beta-strand conformation, the ESF value of an interior alanine peptide group is -7.9 kcal/mol, substantially less than that of N-methylacetamide (-12.2 kcal/mol), and the helical peptide group is solvated with an ESF of -2.5 kcal/mol. These results reduce the enthalpy deficit to -1.5 kcal/mol, and desolvation of peptide groups through partial burial in the random coil may account for the remainder. Mutant peptides in the helical conformation show ESF differences among nonpolar amino acids that are comparable to observed helix propensity differences, but the ESF differences in the random coil conformation still must be subtracted.

Alanine↗
PubMed2000

A rodent model of protein turnover used to design an experiment for measuring the rates of channeling, recycling and protein synthesis.

We described previously a mechanistic model of whole-body protein turnover in rodents. Channeling was defined as the flow of amino acids from the extracellular compartment to aminoacyl tRNA and protein synthesis. Recycling was defined as the flow of amino acids from protein degradation to aminoacyl tRNA (protein synthesis) without mixing with the intracellular pool of amino acids. In this paper, the model is applied to tissues and whole body and is used to develop an experimental protocol for estimating protein fractional synthesis rate, recycling and channeling. Channeling, recycling and protein synthesis must be estimated simultaneously because changes in specific radioactivities over time are highly dependent on the rate of protein synthesis. Injection-specific radioactivities, body weights and experimental variation were used with the model to generate data at different rates of recycling and channeling. The data generated were then used to determine the best time points and experimental method to estimate percentages of recycling, channeling and protein synthesis rate by the iterative Method of Maximum Likelihood. Specific radioactivity at each time point was based on simulated data from three rodents at each of six time points. Predicted protein synthesis rates were within 5%/d of observed rates for all methods. Predicted rates of recycling and channeling were generally within 15% of observed rates except recycling in muscle at high channeling and high recycling. Standard deviations of the predictions of percentages of channeling and recycling were between 0.148 and 44.5% for the pulse dose method, 0.0655 and 197% for the continuous infusion method and 0.351 and 962% for the flooding dose method. The experimental design that yields the best estimates of channeling, recycling and protein synthesis is the pulse dose. Changes in amino acid specific radioactivities in the extracellular, aminoacyl tRNA and protein pools were greatest and should be measured at 2, 6, 10, 40, 70 and 100 min in the pulse method.

Amino Acids↗
PubMed2000

The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry.

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 degrees C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 degrees C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.

Apoproteins↗
PubMed2000

Performance and carcass merit of growing beef steers with chlortetracycline-modified sensitivity to pituitary releasing hormones and fed two dietary protein levels.

This paper reports the effects of reduced sensitivity to growth hormone-releasing hormone and thyrotropin-releasing hormone through feeding a subtherapeutic level of chlortetracycline (CTC; 350 mg CTC/d) and two levels of dietary CP (10% and 13% of diet DM) on growth performance and carcass merit characteristics. Thirty-two steers (initial average BW, 286 kg) were adapted to a common 13% CP diet consisting primarily of grass hay, corn, and soybean meal fed to gain 1.25 kg/d. The steers were assigned to four treatments (with or without CTC and 10% or 13% dietary CP in a factorial arrangement) and fed ad libitum amounts of diet for 91 d. Feed intake was determined daily and steers were weighed weekly. Steers were killed at the end of the feeding period for carcass merit determinations. Efficiency of BW gain was greater (P < .05) for steers fed the 13% CP diet than for the 10% CP diet and tended to be less for CTC-steers when the 10% CP diet was fed and greater for the CTC-steers when the 13% CP diet was fed (CTC x dietary CP interaction, P < .10). Feeding CTC increased (P < .01) fat over the longissimus muscle and marbling. This study is interpreted to indicate that the sustained effect of subtherapeutic feeding of CTC to cattle appears to increase fat deposition consistent with a reduced growth hormone and thyroid status reported earlier for these same steers. This would tend to increase energy utilization but may not necessarily produce a measurable increase in BW gain.

Administration, Oral↗
PubMed2000

Influence of chlortetracycline and dietary protein level on visceral organ mass of growing beef steers.

Thirty-two beef steers (285 +/- 3 kg BW) were used to determine the effects of chlortetracycline and dietary protein level on visceral tissue mass, chemical composition, intestinal morphology, and proliferation rate indices. Steers were allotted randomly by weight to a factorial arrangement of dietary treatments consisting of either 10 or 13% CP diets top-dressed with a corn meal carrier (500 g/d) containing either 0 or 350 mg of chlortetracycline. After 84 d, steers were slaughtered and visceral organs removed and separated. Rinsed wet tissue mass was recorded; total RNA, total DNA, tissue DM, and tissue N content were determined; and tissue sections were prepared for immunohistochemical analysis. Thin tissue sections were evaluated to determine crypt depth and villus height as well as proliferation rate by immunohistochemical detection of the nuclear antigen Ki67. Rumen and abomasum weights and small intestinal length were greater (P < 0.04) in steers fed the 13% CP diet than in those fed the 10% CP diet on both an absolute weight basis and a percentage of empty BW. Chemical composition of the small intestinal and ruminal segments were largely unaffected by increased dietary protein. Increasing the dietary CP also increased the villus height in duodenal (P = 0.02) and the crypt depth of jejunal (P = 0.03) sections. Dietary administration of chlortetracycline decreased (P < 0.01) small intestinal weight both on absolute and empty BW bases. Nitrogen and RNA concentrations of the small intestinal segments were unaffected (P > 0.1) by dietary administration of subtherapeutic levels of chlortetracycline; however, because of increases (P < 0.05), or tendencies for an increase (P < 0.1), in the tissue content of DNA, the ratio of N to DNA was decreased (P < 0.05) or tended to be decreased (P < 0.1) in the small intestinal segments of the chlortetracycline-treated animals. The observed decrease in small intestinal epithelial mass does not appear to be due to alterations in cell proliferation rate but rather cell size. Consistent with this finding, cell proliferation, as determined by Ki67 antigen staining, was not affected by dietary treatment. Chlortetracycline administration decreased small intestinal mass that may be a result of decreased cell size.

Administration, Oral↗
PubMed2000

Effects of diet forage:concentrate ratio and metabolizable energy intake on visceral organ growth and in vitro oxidative capacity of gut tissues in sheep.

We used 28 crossbred wether lambs to determine the effects of dietary forage:concentrate ratio and metabolizable energy intake on visceral organ growth and oxidative capacity of gut tissues in lambs. Lambs were assigned randomly to a factorial arrangement of dietary treatments consisting of pelleted diets containing either 75% orchardgrass or 75% concentrate fed once daily at either .099 or .181 Mcal ME x (kg BW(.75))(-1) x d(-1). After a 52-d feeding period, lambs were slaughtered to obtain measurements of visceral organ mass and composition and oxidative capacity of isolated epithelial cells. Lamb performance, as measured by DMI, ADG, and efficiency of gain, was greater (P = .0001) for both diets at high ME intake. Likewise, lambs fed 75% concentrate gained faster and more (P < or = .01) efficiently than lambs fed 75% forage. Total digestive tract (TDT; includes rumen, reticulum, omasum, abomasum, and intestines) weight increased (P = .0001) with ME intake and was greater (P = .03) in lambs fed 75% forage than in those fed 75% concentrate. As a percentage of empty body weight (EBW), TDT weight increased with ME intake in lambs fed 75% forage, but it was unaffected by ME intake in lambs fed 75% concentrate (diet x intake, P = .03). Liver weight increased (P = .0001) with ME intake and was greater (P = .005) in lambs fed 75% concentrate vs 75% forage; however, liver weight as a percentage of EBW was increased (P = .0002) with ME intake but was unaffected by diet. Greater ME intake increased (P < or = .02) small intestinal (SI) epithelial and muscle mass of 15-cm sections, whereas jejunal epithelial mass was greater (P = .01) for lambs fed 75% forage vs 75% concentrate. Rumen epithelial concentrations of DNA and RNA increased (P < or = .02) with greater ME intake, whereas SI concentrations of DNA and RNA were largely unaffected by diet or ME intake. The activity of Na(+)-K(+)-ATPase increased in ileal epithelium (P < or = .02) with ME intake and concentrate in the diet, but activity in ruminal epithelium increased (P = .05) with concentrate. Total oxygen consumption by isolated ruminal and intestinal epithelial cells was unaffected by treatment. These data suggest that ME intake and level of dietary forage affect ruminal and intestinal growth via changes in cellular hyperplasia. Additionally, this study supports the concept that ME intake and diet composition alter gut energy expenditure, at least in part, through changes in mass rather than mass specific metabolism.

Animal Feed↗
PubMed2000

Effects of diet forage:concentrate ratio and metabolizable energy intake on isolated rumen epithelial cell metabolism in vitro.

Crossbred wether lambs were used to assess the effect of altered forage:concentrate ratio and metabolizable energy intake on metabolism of substrates by ruminal epithelium using an isolated cell system. Lambs (n = 28; 20.1 +/- 3 kg BW) were assigned randomly to a factorial arrangement of dietary treatments consisting of either 75% forage or 75% concentrate fed once daily at either .099 or .181 Mcal ME x(kg BW(.75))(-1) x d(-1) for 52 d. After a 52-d feeding period, isolated rumen epithelial cells (IREC) were incubated in the presence of an oxidizable substrate with a single 14C label (acetate, propionate, butyrate, glucose, glutamate, and glutamine) at concentrations ranging from .1 to 50 mM, and substrate oxidation to 14CO2 or metabolism to beta-hydroxybutyrate (beta-HBA), acetoacetate, pyruvate, and lactate was determined. For all substrates, oxidation to CO2 was concentration-dependent and saturable within the physiological range. Differences in substrate oxidation to CO2 by IREC at specific substrate concentrations did not affect Vmax (maximal rate of substrate oxidation, nmol oxidized to CO2 x 1 x 10(6) cells(-1) x 90 min(-1)) and K(ox) (concentration of substrate at which half Vmax oxidation rate is achieved, mmoles/L) estimates for the dietary treatments. Production of beta-HBA from butyrate by IREC from the lambs fed 75% forage was not affected by ME intake; however, production was elevated by high ME intake of the 75% concentrate diet (diet x intake interaction; P < .02). Acetoacetate production from butyrate by IREC from lambs fed at high ME intake was greater (P = .001) than from those fed at low ME intake. Lactate and pyruvate production from glucose, glutamate, and propionate were generally unaffected by dietary treatment; however, rate of glutamine metabolism to lactate and pyruvate by IREC was increased with increased ME intake. The observed changes in metabolite production rates across groups did not affect the predicted Vmax and K(ox) parameter estimates. The estimated K(ox) values corroborate that VFA are the primary oxidizable fuels used by ruminal epithelial cells while illustrating that other substrates such as glucose, glutamate, and glutamine would not be expected to be oxidized extensively in vivo due to the high K(ox) relative to substrate concentrations in vivo. In conclusion, the capacity of isolated ruminal epithelial cells to oxidize substrates was largely unaffected by ME intake or dietary forage:concentrate ratio of the diet.

Animal Feed↗
PubMed2000

Sheep rumen metabolic development in response to age and dietary treatments.

This study examined the time course of rumen metabolic development in the absence of solid feed consumption and the effect of delayed solid feed consumption on sheep rumen development. Twenty-seven lambs consumed milk replacer until slaughter at nine ages from 1 to 84 d (milk group). Three additional lambs consumed milk replacer from 1 to 48 d. From 49 d until slaughter at 84 d, these lambs were weaned onto solid feed (fed group). At slaughter, rumen contents were removed for VFA analysis and rumen epithelium was preserved for morphological examination. Rumen epithelial cells were isolated and incubated in media containing 2.5 mM U-[14C]-glucose or 10 mM 1-[14C]-butyrate. Rumen VFA concentrations did not change with age in lambs given milk replacer. At 84 d of age, intraruminal VFA concentrations were elevated in lambs consuming solid feed compared to 84-d-old lambs given milk replacer (P < .05). The number of ruminal papillae per square centimeter decreased (P < .05) while papillae length and width did not change significantly with age in rumen epithelium from lambs given milk replacer. At 84 d of age, rumen epithelium from lambs in the fed group had fewer and larger papillae/per square centimeter than rumen epithelium from lambs given milk replacer (P < .05). Rates of glucose and butyrate oxidation and acetoacetate and lactate production by rumen cells isolated from lambs given milk replacer did not change with age. Beta-hydroxybutyrate (BHBA) production was undetectable before 42 d of age in lambs given milk replacer and increased to levels found in conventionally raised adults by 84 d. At 84 d there were no differences in rates of glucose and butyrate oxidation or acetoacetate and lactate production by rumen cells between the two treatment groups. Thus, the change in substrate oxidation from glucose to butyrate, indicative of rumen metabolic maturation, does not occur in the absence of solid feed consumption. However, the development of rumen ketogenesis, as evidenced by increased BHBA production, does occur in the absence of solid feed consumption. Delaying the initiation of solid feed consumption results in rumen morphological development but does not stimulate rumen metabolic development. Increased intraruminal VFA concentrations, earlier exposure to VFA, or a longer period of exposure to VFA may be required to induce the genes responsible for rumen metabolic development.

Aging↗
PubMed2000

Estimation of parameters describing lipid metabolism in lactation: challenge of existing knowledge described in a model of metabolism.

We have been conducting research to improve quantitative descriptions of metabolism in lactating dairy cows depicted in an existing mechanistic, computer-assisted model. The model is dynamic and deterministic and is based on biochemical equations describing ruminal fermentation and chemical interactions in body tissues. The objective was to challenge this model with data collected in in vivo and in vitro experiments on high producing dairy cattle fed a range of energy. Cows that varied in genetic propensity for milk production (7045 to 12,909 kg of milk/305 d), lactation number (1 to 4), stage of lactation (-30 to 345 d in milk), and rate of intake (14 to 29 kg/d of dry matter) and that were fed various energy-yielding feedstuffs were used. Dietary inputs, milk component outputs, body fat, nutrient concentrations in blood, and maximal velocity and substrate sensitivity of adipose tissue metabolic reactions were observed. Model simulations were conducted; simulated yields of milk components for a 305-d lactation were within 5% of observed means. Simulated lipid metabolism and accumulation of body fat were adequate in many situations; however, the model response to changes in energy intake was too sensitive. This inadequacy was especially noticeable in later lactation because of inadequate representation of dynamic responses over periods more than a few weeks long. The model behaves consistently with biochemical principles, behavior was in the correct direction, and precision was adequate for many variables. Lack of precision in long-term dynamic changes indicates that the parameters describing energy-utilizing reactions are inadequate. This severe challenge of the model supports its functionality. Further experiments must be designed to determine how nutrients in viscera, muscle, and adipose tissue are used; these experiments must encompass sufficient range in genetic ability, nutrient input, and time to adequately describe the dynamic and integrated nature of metabolic reactions.

Adipose Tissue↗
PubMed1999

Comparative hybridization of an array of 21,500 ovarian cDNAs for the discovery of genes overexpressed in ovarian carcinomas.

Comparative hybridization of cDNA arrays is a powerful tool for the measurement of differences in gene expression between two or more tissues. We optimized this technique and employed it to discover genes with potential for the diagnosis of ovarian cancer. This cancer is rarely identified in time for a good prognosis after diagnosis. An array of 21,500 unknown ovarian cDNAs was hybridized with labeled first-strand cDNA from 10 ovarian tumors and six normal tissues. One hundred and thirty-four clones are overexpressed in at least five of the 10 tumors. These cDNAs were sequenced and compared to public sequence databases. One of these, the gene HE4, was found to be expressed primarily in some ovarian cancers, and is thus a potential marker of ovarian carcinoma.

Biomarkers, Tumor↗
PubMed1999

The 28-111 disulfide bond constrains the alpha-lactalbumin molten globule and weakens its cooperativity of folding.

Our aim is to determine whether the disulfide bonds of alpha-lactalbumin account for the lack of cooperative folding behavior reported for some molten globule variants, in contrast to the highly cooperative folding reported for the pH 4 molten globule of apomyoglobin. Two different alpha-lactalbumin genetic constructs are studied: [28-111], which has a single disulfide bond connecting two segments of the alpha-helix domain, and [all-Ala], which has no disulfide bonds. The superposition test used earlier to probe for cooperative folding of the apomyoglobin molten globule is used to determine whether there is an important difference in folding cooperativity between the molten globules of [28-111] and [all-Ala]. The [all-Ala] construct behaves in the same manner as the apomyoglobin molten globule: its folding satisfies the superposition test in the three sets of anion conditions studied, and anions stabilize it against urea unfolding. The [28-111] construct behaves differently in both respects: the folding of its molten globule does not satisfy the superposition test in two of the three sets of anion conditions, and anions barely affect its stability. The 28-111 disulfide bond stabilizes the molten globule substantially, as expected from earlier work. Comparison of the unfolding transition curves monitored by circular dichroism also demonstrates that [28-111] folds in a less cooperative manner than [all-Ala]: the unfolding curve of [28-111] is significantly broader. Moreover, the unfolding curves indicate that [28-111] has a lower helix content than [all-Ala]. Consequently, the 28-111 bond constrains the folding behavior of the molten globule and weakens its cooperativity of folding.

Disulfides↗
PubMed1999

Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate.

Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia equilibrium Ib. In urea-induced unfolding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib-->Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U equilibrium Ia equilibrium Ib equilibrium N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2. 7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.

Animals↗