The role of the coiled-coil motif in interactions mediated by TPD52.

TPD52 (D52)-like proteins are small coiled-coil motif-bearing proteins first identified through their expression in human breast carcinoma that mutually interact in hetero- and homomeric fashions. However, it has been unclear whether the coiled-coil motif is sufficient, or even necessary, for these interactions to occur. We have therefore examined the binding activities of a panel of C-terminally deleted D52 proteins in both the yeast two-hybrid system and pull-down assays. In the yeast two-hybrid system, interactions were only detected when regions C-terminal to the coiled-coil motif were also present. However, using pull-down assays, interactions were detected for all deletion mutants which included the coiled-coil motif. This suggests that the coiled-coil motif is indeed necessary for interactions mediated by D52 proteins, but that C-terminal protein regions facilitate and/or stabilize these interactions.

Caffeine ingestion elevates plasma insulin response in humans during an oral glucose tolerance test.

We tested the hypothesis that caffeine ingestion results in an exaggerated response in blood glucose and (or) insulin during an oral glucose tolerance test (OGTT). Young, fit adult males (n = 18) underwent 2 OGTT. The subjects ingested caffeine (5 mg/kg) or placebo (double blind) and 1 h later ingested 75 g of dextrose. There were no differences between the fasted levels of serum insulin, C peptide, blood glucose, or lactate and there were no differences within or between trials in these measures prior to the OGTT. Following the OGTT, all of these parameters increased (P < or = 0.05) for the duration of the OGTT. Caffeine ingestion resulted in an increase (P < or = 0.05) in serum fatty acids, glycerol, and plasma epinephrine prior to the OGTT. During the OGTT, these parameters decreased to match those of the placebo trial. In the caffeine trial the serum insulin and C peptide concentrations were significantly greater (P < or = 0.001) than for placebo for the last 90 min of the OGTT and the area under the curve (AUC) for both measures were 60 and 37% greater (P < or = 0.001), respectively. This prolonged, increased elevation in insulin did not result in a lower blood glucose level; in fact, the AUC for blood glucose was 24% greater (P = 0.20) in the caffeine treatment group. The data support our hypothesis that caffeine ingestion results in a greater increase in insulin concentration during an OGTT. This, together with a trend towards a greater rather than a more modest response in blood glucose, suggests that caffeine ingestion may have resulted in insulin resistance.

The hD52 (TPD52) gene is a candidate target gene for events resulting in increased 8q21 copy number in human breast carcinoma.

Chromosome band 8q21 is frequently overrepresented in human cancer, but to date no 8q21 target gene has been proposed. The hD52 (TPD52) gene is of potential significance in breast and other cancers due to its location and expression pattern. Fine mapping of hD52 placed this locus within the peak of the 8q21 amplicon delineated in the SK-BR-3 breast carcinoma cell line, and a positive association between hD52 gene dosage and transcript levels was subsequently demonstrated in four breast carcinoma cell lines, including SK-BR-3. Increased copy number (ICN) was measured using Southern blot analyses in 3/32 human breast carcinomas at hD52, and the related hD54 gene in 20q13.2-q13.3. Subsequent immunohistochemical analysis of hD52 expression in 19 breast carcinomas with varying hD52 gene dosages demonstrated a significant positive association between hD52 dosage and hD52 expression using a Spearman rank correlation coefficient (r(s) = 0.573, alpha = 0.01) and a Wilcoxon rank-sum test (alpha = 0.05). On the basis of its map location and expression pattern in breast carcinoma, we therefore propose hD52 as a candidate target gene at chromosome band 8q21.

Glycogenin activity in human skeletal muscle is proportional to muscle glycogen concentration.

The de novo biosynthesis of glycogen is catalyzed by glycogenin, a self-glucosylating protein primer. To date, the role of glycogenin in regulating glycogen metabolism and the attainment of maximal glycogen levels in skeletal muscle are unknown. We measured glycogenin activity after enzymatic removal of glucose by alpha-amylase, an indirect measure of glycogenin amount. Seven male subjects performed an exercise and dietary protocol that resulted in one high-carbohydrate leg (HL) and one low-carbohydrate leg (LL) before testing. Resting muscle biopsies were obtained and analyzed for total glycogen, proglycogen (PG), macroglycogen (MG), and glycogenin activity. Results showed differences (P < 0.05) between HL and LL for total glycogen (438.0 +/- 69.5 vs. 305.7 +/- 57.4 mmol glucosyl units/kg dry wt) and PG (311.4 +/- 38.1 vs. 227.3 +/- 33.1 mmol glucosyl units/kg dry wt). A positive correlation between total muscle glycogen content and glycogenin activity (r = 0.84, P < 0.001) was observed. Similar positive correlations (P < 0.05) were also evident between both PG and MG concentration and glycogenin activity (PG, r = 0.82; MG, r = 0.84). It can be concluded that glycogenin does display activity in human skeletal muscle and is proportional to glycogen concentration. Thus it must be considered as a potential regulator of glycogen synthesis in human skeletal muscle.

Metabolic and exercise endurance effects of coffee and caffeine ingestion.

Caffeine (Caf) ingestion increases plasma epinephrine (Epi) and exercise endurance; these results are frequently transferred to coffee (Cof) consumption. We examined the impact of ingestion of the same dose of Caf in Cof or in water. Nine healthy, fit, young adults performed five trials after ingesting (double blind) either a capsule (Caf or placebo) with water or Cof (decaffeinated Cof, decaffeinated with Caf added, or regular Cof). In all three Caf trials, the Caf dose was 4.45 mg/kg body wt and the volume of liquid was 7.15 ml/kg. After 1 h of rest, the subject ran at 85% of maximal O2 consumption until voluntary exhaustion (approximately 32 min in the placebo and decaffeinated Cof tests). In the three Caf trials, the plasma Caf and paraxanthine concentrations were very similar. After 1 h of rest, the plasma Epi was increased (P < 0.05) by Caf ingestion, but the increase was greater (P < 0.05) with Caf capsules than with Cof. During the exercise there were no differences in Epi among the three Caf trials, and the Epi values were all greater (P < 0.05) than in the other tests. Endurance was only increased (P < 0. 05) in the Caf capsule trial; there were no differences among the other four tests. One cannot extrapolate the effects of Caf to Cof; there must be a component(s) of Cof that moderates the actions of Caf.

Caffeine metabolism and epinephrine responses during exercise in users and nonusers.

This study compared the caffeine (CAF) metabolism and the catecholamine and metabolic responses of users and nonusers of caffeine after acute ingestion of caffeine (5 mg/kg) during 1 h of steady-state exercise (50% maximal oxygen consumption). Nonusers (n = 7) completed two exercise trials after ingesting either CAF (5 mg/kg) or placebo (PL). Users (n = 7) underwent three trials designed to control caffeine use and abstained from voluntary CAF intake for 18 days. After 4 days they had a PL trial and in the following 14 days they were given random 6 days of CAF (2 x 2.5 mg.kg-1 x day-1) or PL ingestion followed in each case on the 7th day by a CAF exercise trial identical to that of the nonusers. In nonusers CAF increased (P < 0.05) plasma epinephrine (EPI) concentration above PL values during exercise. Users did not exhibit any increased EPI with CAF, but the EPI response to exercise in all three trials was twofold greater than that of the nonusers' PL trial (P < 0.05). In all trials both groups had identical norepinephrine responses. The groups had similar plasma and urinary caffeine concentration, but plasma dimethylxanthines varied; the users had greater (P < 0.05) theophylline concentration, and the nonusers had a greater (P < 0.05) rise in paraxanthine (PX) concentration. The users and nonusers' plasma free fatty acids (FFA), glycerol and respiratory exchange ratio were similar after ingestion of CAF. Although PX may increase FFA in resting subjects, in this study PX concentrations in nonusers varied from that of the users, yet FFA data were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular and norepinephrine responses of men and women to two cold pressor tests.

Two types of cold pressor tests were used to study gender differences in cardiovascular and plasma catecholamine responses. Ten male and ten female, young, healthy Caucasian subjects participated. The tests consisted of (1) 5 degrees C air blown at 3.5-4 m/s onto part of the face for 4 min and (2) the open right hand immersed to the wrist in water at 5 degrees C for 4 min. Heart rate, blood pressure (BP), and venous plasma norepinephrine were collected before, during, and 5 min after the 4 min of cold exposures. Test order was decided by a Latin square design, and the subjects rested in a quiet room for 30 min between the two tests. All parameters demonstrated significant (p less than 0.01) increases from rest during the cold tests. Gender differences were significant (p less than 0.01) in diastolic and systolic BP in each test with the males having a greater response, but gender differences were not found in heart rate or norepinephrine concentration. The study demonstrated that gender differences exist in the blood pressure responses to local cold, but that the mechanisms involved do not include a parallel difference in heart rate or venous plasma norepinephrine concentration.

Influence of cold, exercise, and caffeine on catecholamines and metabolism in men.

Recently we found that caffeine ingestion did not enhance either thermal or fat metabolic responses to resting in cold air, despite an increase in plasma epinephrine and free fatty acids. Theophylline, another methylxanthine, has been shown to be effective during exercise but not at rest during cold stress. Therefore we hypothesized that caffeine ingestion before exercise in cold air would have a thermal-metabolic impact by increasing fat metabolism and increasing oxygen consumption. Young adult men (n = 6) who did not normally have caffeine in their diet performed four double-blind trials. Thirty minutes after ingesting placebo (dextrose, 5 mg/kg) or caffeine (5 mg/kg) they either exercised (60 W) or rested for 2 h in 5 degrees C air. Cold increased (P less than 0.05) plasma norepinephrine while both caffeine and exercise increased (P less than 0.05) epinephrine. Serum free fatty acids and glycerol were increased, but there were no differences between rest and exercise or placebo and caffeine. Caffeine had no influence on either respiratory exchange ratio or oxygen consumption either at rest or during exercise. The exercise trials did not significantly warm the body, and they resulted in higher plasma norepinephrine concentrations and lower mean skin temperatures for the first 30 min. The data suggest that skin temperature stimulates plasma norepinephrine while caffeine has little effect. In contrast, caffeine and exercise stimulate plasma epinephrine while cold has minimal effect. Within the limits of this study caffeine gave no thermal or metabolic advantage during a cold stress.

Influence of caffeine on metabolic responses of men at rest in 28 and 5 degrees C.

Cold stress and caffeine ingestion are each reported to increase plasma catecholamines, free fatty acid (FFA) concentrations, and energy metabolism. This study examined the possible interaction of these two metabolic challenges in four double-blind counterbalanced trials. Young adult men (n = 6) ingested caffeine (5 mg/kg) or placebo (dextrose, 5 mg/kg) and rested for 2 h in 28 or 5 degrees C air. Cold stress alone elevated (P less than 0.05) plasma norepinephrine, metabolism (O2 consumption, VO2), and respiratory exchange ratio (RER). Caffeine alone increased (P less than 0.05) plasma epinephrine and FFA but not RER. When the two challenges were combined (caffeine plus 5 degrees C for 2 h) norepinephrine and epinephrine were increased (P less than 0.05) as was FFA. However, VO2, RER, and skin and rectal temperatures were not different from the responses observed at 5 degrees C after placebo ingestion. The data suggest that caffeine selectively increases plasma epinephrine, whereas cold air increases norepinephrine. During the cold exposure, increasing epinephrine and FFA above normal levels did not appear to influence the metabolic or thermal responses to the cold stress. In fact the increase in RER suggested a greater carbohydrate oxidation.

Immunoglobulin IgM detected by ELISA in bovine pituitary intraglandular colloid, non-vascular holocrine secretion of intermediate lobe cells.

ELISA was employed to determine the presence of immunoglobulin IgM in crude bovine pituitary intraglandular colloid (IGC). The percentage weight of IgM was 1.15% per total weight of lyophilized colloid. The colloid from 32 glands (sufficient amount to perform the ELISA) was pooled and placed in 20 ml cold phosphate buffered saline (pH 7.6) and lyophilized. The total weight of the lyophilized material was 2.05 g IGC, housed in the intraglandular lumen (residual lumen). This is the holocrine secretion of the marginal half of bovine pituitary intermediate lobe (IL) cells, known to produce the ACTH/LPH family of peptides. The discovery of immunoglobulin IgM in IGC is a unique phenomenon. Since it has been established that IL cells process common precursor molecules to different final secretory products, it can be envisioned that continuing studies will help to elucidate the relationship between immunoglobulins and the ACTH/LPH family of peptides.

Immunoglobulin IgG detected by ELISA in bovine pituitary intraglandular colloid, a non-vascular holocrine secretion of intermediate lobe cells.

The purpose of this study was to determine the presence of immunoglobulin G in bovine pituitary intraglandular colloid (IGC). Intraglandular colloid, housed in the intraglandular lumen (residual lumen), is the holocrine secretion of the marginal layers of intermediate lobe (IL) cells. Immunoglobulin G was detected and quantitated in bovine IGC using an Enzyme Linked Immunosorbent Assay (ELISA). This study establishes the presence of immunoglobulin G in the intraglandular colloid at levels that are of the order of 10 micrograms per mg of colloid. The existence of this molecule in material scrupulously prepared to exclude the possibility of serum contamination in this normally avascular compartment is a unique phenomenon.