Expression pattern of human P2Y receptor subtypes: a quantitative reverse transcription-polymerase chain reaction study.

The diverse biological actions of extracellular nucleotides in tissues and cells are mediated by two distinct classes of P2 receptor, P2X and P2Y. The G protein-coupled P2Y receptors comprise at least six mammalian subtypes (P2Y(1,2,4,6,11,12)), all of which have been cloned from human tissues, as well as other species. The P2Y receptor subtypes differ in their pharmacological selectivity for various adenosine and uridine nucleotides, which overlap in some cases. Data concerning the mRNA expression patterns of five P2Y receptors (P2Y(1,2,4,6,11)) in different human tissues and cells are currently quite limited, while P2Y mRNA distribution in the human brain has not previously been studied. In this study, we have addressed this deficiency in receptor expression data by using a quantitative reverse transcription-polymerase chain reaction approach to measure the precise mRNA expression pattern of each P2Y receptor subtype in a number of human peripheral tissues and brain regions, from multiple individuals, as well as numerous human cell lines and primary cells. All five P2Y receptors exhibited widespread yet subtype-selective mRNA expression profiles throughout the human tissues, brain regions and cells used. Our extensive expression data indicate the many potentially important roles of P2Y receptors throughout the human body, and will help in elucidating the physiological function of each receptor subtype in a wide variety of human systems.

The influence of supporting the Oxylog instrument on estimated maximal aerobic capacity during a step test and heart rate in a lifting test.

This study investigated the influence of wearing, an Oxylog mask and heart rate monitor while the Oxylog instrument was supported in a stand versus that of wearing the Oxylog mask, heart rate monitor, and the Oxylog instrument, on oxygen consumption (VO2) and heart rate response during a graduated submaximal step test protocol. Also, the predicted maximal aerobic capacities ( VO2max) produced by the two graduated step tests were compared. In addition, differences in the working heart rates in a submaximal lifting test were analyzed in two lifting tests, one with the participant wearing the Oxylog mask, heart rate monitor, and Oxylog instrument and the other required the participant to only wear a heart monitor. Seventeen experienced male manual materials handlers participated in the study, and each treatment was seen by each participant in a randomized Latin Square design. Results from the two investigations indicate that there was no significant difference in the estimated maximal oxygen consumption (p = 0.1384) and no significant difference in the heart rate between the two lift tests. The analysis did show that the 4th stage (participants reaching their physiological limits) of the step tests indicated a statistically significant difference (p = 0.0259 for oxygen consumption, and p = 0.0465 for heart rate).

Identification, cloning, and expression of a functional phenylalanyl-tRNA synthetase (pheRS) from Staphylococcus aureus.

Phenylalanyl-tRNA synthetase (pheRS) is unique among aminoacyl tRNA synthetases in that it is a heterotetrameric enzyme composed of two alpha-subunits and two larger beta-subunits. In prokaryotes, the alpha- and beta-subunits of pheRS are encoded by the genes pheS and pheT, respectively. In this report we describe the isolation of a DNA fragment (3.52 kb) containing the pheS and pheT genes from a Staphylococcus aureus (WCUH29) genomic DNA library. Both genes, found as a part of transcriptional operon, were predicted to encode polypeptides which showed strong primary and structural similarity to prokaryotic phenylalanyl-tRNA synthetase alpha- and beta- subunits. We describe the high-level overexpression and purification of recombinant S. aureus pheRS using pheS and pheT genes as part of an artificial operon in Escherichia coli. For comparative analysis we also report a procedure for the purification of native pheRS from S. aureus (Oxford Strain) and demonstrate that Michaelis-Menten parameters for both recombinant and native enzyme, at least for phenylalanine tRNA aminoacylation are comparable.

Effects of chronic murine and human leptin infusion on plasma leptin and corticosterone levels and energy balance in lean Zucker rats.

AIM: To clarify whether centrally delivered leptin can access the circulation and to determine to what extent the effects of i.c.v. h-leptin and m-leptin on body weight and plasma corticosterone are due to reduced food intake. METHODS: Male lean Zucker rats were infused i.c.v. with recombinant m-leptin or h-leptin (42 microg/day) for 7 days. Terminal plasma leptin levels were measured using selective r-leptin, m-leptin and h-leptin RIA. Plasma h-leptin and corticosterone levels were determined on days 0, 2, 4 and 6 of h-leptin infusion. Interscapular brown adipose tissue weight and UCP-1 mRNA expression (an indicator of thermogenic capacity) were also measured. RESULTS: The terminal plasma leptin level was elevated (from 2.2 +/- 0.4 to 42.7 +/- 20.2 ng/ml) in the h-leptin-treated lean rats to levels similar to those in vehicle i.c.v. infused fa/fa rats (72.2 +/- 4.7 ng/ml), but this was only detectable when the h-leptin radioimmunoabsorbent assay (RIA) was used. Further, both m-leptin and h-leptin infusions in lean rats elevated terminal plasma corticosterone (352 +/- 37 and 389 +/- 55 ng/ml, respectively) to levels similar to those in i.c.v. rats (386 +/- 62 ng/ml), whereas diet-restriction by pair-feeding, with the h-leptin group, in lean rats had no effect (207 +/- 45 ng/ml). The increase in plasma corticosterone level coincided with the maximum hypophagic effects of leptin and preceded the appearance and sustained elevation of exogenous human leptin in the circulation. Both m-leptin and h-leptin i.c.v. infusion reduced body weight gain (3% and 4%, respectively, compared to pair-fed group) and increased UCP-1 expression (11-fold and 16-fold, respectively) in lean rats. However, h-leptin elicited an earlier effect than m-leptin on body weight, manifested as an earlier reduction in food intake and greater increase in UCP-1 expression. h-Leptin also elicited a greater reduction in body weight gain than did pair-feeding. CONCLUSIONS: Intracerebroventricular-infused m-leptin or h-leptin was detected in the circulation. Furthermore, m-leptin and h-leptin elevated plasma corticosterone levels and h-leptin caused some weight loss in lean rats independently of its suppression of food intake. The elevation of corticosterone levels in the lean rats may be a mechanism whereby they resist excessive weight loss in response to leptin.

The human GABA(B1b) and GABA(B2) heterodimeric recombinant receptor shows low sensitivity to phaclofen and saclofen.

1. The aim of this study was to characterize the pharmacological profile of the GABA(B1)/GABA(B2) heterodimeric receptor expressed in Chinese hamster ovary (CHO) cells. We have compared receptor binding affinity and functional activity for a series of agonists and antagonists. 2. The chimeric G-protein, G(qi5), was used to couple receptor activation to increases in intracellular calcium for functional studies on the Fluorimetric Imaging Plate Reader (FLIPR), using a stable GABA(B1)/GABA(B2)/G(qi5) CHO cell line. [(3)H]-CGP-54626 was used in radioligand binding studies in membranes prepared from the same cell line. 3. The pharmacological profile of the recombinant GABA(B1/B2) receptor was consistent with that of native GABA(B) receptors in that it was activated by GABA and baclofen and inhibited by CGP-54626A and SCH 50911. 4. Unlike native receptors, the GABA(B1)/GABA(B2)/G(qi5) response was not inhibited by high microMolar concentration of phaclofen, saclofen or CGP 35348. 5. This raises the possibility that the GABA(B1)/GABA(B2)/G(qi5) recombinant receptor may represent the previously described GABA(B) receptor subtype which is relatively resistant to inhibition by phaclofen.

Expression, purification, and functional analysis of the human serine protease HtrA2.

HumHtrA2 or Omi is a recently described member of a novel family of mammalian serine proteases homologous to the Escherichia coli htrA gene product. Although the physiological function of members of this new family is unclear, the current understanding is that as well as being involved with the degradation aberrantly folded proteins during conditions of cellular stress, they may possess a chaperone-like role under normal conditions. In this report we describe the overexpression of humHtrA2 in two heterologous systems comparing the merits of each. We found that molecular analysis of processing events in Sf9 cells allowed us to revisit E. coli expression systems which were initially unsuccessful. Using E. coli we were able to produce milligram amounts of >90% pure recombinant enzyme as determined by SDS-PAGE gels. By means of fluorescently labeled substrates alpha- and beta-casein and zymography, the proteolytic activity of recombinant HumHtrA2 was also demonstrated.

Leptin expression in colorectal and breast cancer patients.

Leptin is a hormone which controls fat metabolism. Leptin plasma levels and adipose tissue mRNA expression were measured in cancer patients. Plasma levels were correlated with TNM staging, cachexia parameters, tumour markers and hormones. Breast and colorectal cancer patients showed blood plasma levels of insulin, TNF-alpha and tumour markers higher than controls. Breast cancer patients, but not colorectal cancer patients, had plasma levels and adipose tissue expression of leptin significantly higher than controls associated with elevated values of estrogen- and progesterone-receptors. These data suggest the possible use of leptin as a clinical marker.

Identification, molecular cloning, expression, and characterization of a cysteinyl leukotriene receptor.

The cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory disorders, in particular asthma, for which the CysLT receptor antagonists pranlukast, zafirlukast, and montelukast, have been introduced recently as novel therapeutics. Here we report on the molecular cloning, expression, localization, and pharmacological characterization of a CysLT receptor (CysLTR), which was identified by ligand fishing of orphan seven-transmembrane-spanning, G protein-coupled receptors. This receptor, expressed in human embryonic kidney (HEK)-293 cells responded selectively to the individual CysLTs, LTC(4), LTD(4), or LTE(4), with a calcium mobilization response; the rank order potency was LTD(4) (EC(50) = 2.5 nM) > LTC(4) (EC(50) = 24 nM) > LTE(4) (EC(50) = 240 nM). Evidence was provided that LTE(4) is a partial agonist at this receptor. [(3)H]LTD(4) binding and LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor were potently inhibited by the structurally distinct CysLTR antagonists pranlukast, montelukast, zafirlukast, and pobilukast; the rank order potency was pranlukast = zafirlukast > montelukast > pobilukast. LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor was not affected by pertussis toxin, and the signal appears to be the result of the release from intracellular stores. Localization studies indicate the expression of this receptor in several tissues, including human lung, human bronchus, and human peripheral blood leukocytes. The discovery of this receptor, which has characteristics of the purported CysLT(1) receptor subtype, should assist in the elucidation of the pathophysiological roles of the CysLTs and in the identification of additional receptor subtypes.

Factors affecting minimum push and pull forces of manual carts.

The minimum forces needed to manually push or pull a 4-wheel cart of differing weights with similar wheel sizes from a stationary state were measured on four floor materials under different conditions of wheel width, diameter, and orientation. Cart load was increased from 0 to 181.4 kg in increments of 36.3 kg. The floor materials were smooth concrete, tile, asphalt, and industrial carpet. Two wheel widths were tested: 25 and 38 mm. Wheel diameters were 51, 102, and 153 mm. Wheel orientation was tested at four levels: F0R0 (all four wheels aligned in the forward direction), F0R90 (the two front wheels, the wheels furthest from the cart handle, aligned in the forward direction and the two rear wheels, the wheels closest to the cart handle, aligned at 90 degrees to the forward direction), F90R0 (the two front wheels aligned at 90 degrees to the forward direction and the two rear wheels aligned in the forward direction), and F90R90 (all four wheels aligned at 90 degrees to the forward direction). Wheel width did not have a significant effect on the minimum push/pull forces. The minimum push/pull forces were linearly proportional to cart weight, and inversely proportional to wheel diameter. The coefficients of rolling friction were estimated as 2.2, 2.4, 3.3, and 4.5 mm for hard rubber wheels rolling on smooth concrete, tile, asphalt, and industrial carpet floors, respectively. The effect of wheel orientation was not consistent over the tested conditions, but, in general, the smallest minimum push/pull forces were measured with all four wheels aligned in the forward direction, whereas the largest minimum push/pull forces were measured when all four wheels were aligned at 90 degrees to the forward direction. There was no significant difference between the push and pull forces when all four wheels were aligned in the forward direction.

Australasian perfusion incident survey.

An anonymous postal survey about perfusion accidents, incidents and safety procedures was conducted retrospectively in all identifiable cardiac surgery units in Australia and New Zealand over an 18-month period from January 1994 to July 1995. Responses were received from 69% of all eligible perfusionists representing 39 to 42 operating units. The most frequent incidents reported were heater/cooler failure (43% of respondents), urgent return to bypass following circuit disposal (38%), air embolus in a circuit not reaching the patient (24%), accidental cannula displacement (28%), protamine-induced circuit clotting postbypass (20%), hospital power failure (31%) and oxygenator membrane leaks (24%). There were 11 serious injuries and 10 deaths reported, giving an overall rate of serious injury or death of one in 1300 cases. However, the perfusion-related injury rate was lower, at one injury or death per 2500 perfusions. The use of safety equipment was widespread with low-level alarms in use by all respondents, bubble detectors by 74%, arterial line filters by 82% and written or computerized checklists by 80%. While the rate of injury was lower than that reported in surveys done elsewhere, the rate of reporting of incidents was greater and this may reflect a changing attitude of perfusionists to accountability in the delivery of their services. The data suggest that the level of safety in perfusion in Australasia is high but that improved incident reporting may help to further improve practices.

Effects of prenatal stress on the fetal calf.

Twelve pregnant Brahman cows were randomly assigned to one of two treatment groups: 1) transported in a stock trailer for 24.2 km, unloaded at a second farm and penned for 1 hr, and then returned to the original farm (TRANS, n = 6); or 2) walked through the handling facilities (SHAM, n = 6). Treatments were repeated at 60, 80, 100, 120, and 140 d of gestation. Calves were delivered by cesarean section on d 266 of gestation. The male:female ratio was 4:2 and 5:1 for the TRANS and SHAM treatment groups, respectively. Before calf removal and severance of the umbilical blood flow, a blood sample was collected from the calf to determine plasma concentrations of adrenocorticotropin (ACTH) and cortisol. The calf was then sedated and exsanguinated, after which pituitary and adrenal glands were collected. The adrenals were immediately weighed, and a cross-section from the left adrenal was stored in 4% paraformaldehyde until being embedded in paraffin. Eight sections from each adrenal were sliced (5 microns), fixed, and then stained with Harris' hematoxylin and eosin. Areas of the cortex and medulla were calculated with a computerized digitizing unit and tracing of the viewed section. The TRANS calves had heavier body weights (BW) (28.7 vs. 23.9 +/- 1.8 kg; P < 0.07), pituitary glands (12.63 vs. 8.24 +/- 1.10 g/kg BW; P < 0.008), and heart weights (5.58 vs. 5.17 +/- 0.58 g/kg BW; P < 0.05) than did the SHAM calves. Plasma concentrations of ACTH and cortisol did not differ between SHAM and TRANS calves (57 vs. 82 +/- 14 pg/ml and 7.0 vs. 6.7 +/- 0.9 ng/ml, respectively; P > 0.2). Adrenal gland weight and medulla-to-cortex ratio did not differ between SHAM and TRANS calves (0.61 and 0.73 +/- 0.03 g and 0.97 and 0.99 +/- 0.12 g, respectively; P > 0.2). These results suggest that the altered response to stress in prenatally stressed calves is not associated with morphological changes in the adrenal gland but may be due to effects of prenatal stress on the fetal pituitary.

Effects of prenatal stress on suckling calves.

Pregnant Brahman cows (n = 42), bred to either Brahman or Tuli bulls, were randomly assigned to one of three treatments: 1) transported in a stock trailer for 24.2 km, unloaded at a second farm and penned for 1 h, and then returned to the original farm (TRANS); 2) i.v. injection of ACTH, 1 IU/kg BW (ACTH); or 3) walked through the handling facilities (SHAM). Treatments were initiated on d 60 and repeated at 80, 100, 120, and 140 d of gestation. The calves from these cows were subjected to tests to measure their capacity to react to stress. In Test 1, Tuli-sired calves were restrained at 10 and 150 d of age for 3.5 h. In Test 2, Brahman-sired calves were restrained for 3.5 h and given an injection of ACTH (.125 IU ACTH/kg of BW). In Test 3, Test-2 calves were restrained at 180 d of age and hot-iron branded. In Test 4, Test-1 calves were restrained at 180 d of age and given an injection of cortisol (6.7 ng/kg BW) to estimate cortisol clearance rate. During all tests, calves were restrained for 3.5 h, and heart rates were recorded and blood samples were taken at -15, 0, 15, 30, 45, 60, 90, 120, and 180 min. The 10- and 150-d-old TRANS calves maintained greater plasma cortisol in Test 1 (restraint) than the ACTH and SHAM calves (P < .01). The ACTH challenge (Test 2) increased plasma cortisol and ACTH, but cow treatment did not alter the response (P > .4). In response to branding (Test 3), the TRANS, ACTH, and SHAM calves' overall mean plasma cortisol was not affected by treatment (52, 51, and 43 +/- 3 ng/mL, respectively; P > .1), nor was the calves' overall heart rate (91, 94, and 86 +/- 3 beats/min, respectively; P > . 1). In Test 4, TRANS calves cleared plasma of cortisol at a slower rate than did the SHAM calves (P < .01), but not the ACTH calves (261, 374, and 473 +/- 50 mL/min, respectively; P > .1). The TRANS calves had an overall greater heart rate than did the ACTH or the SHAM calves (91, 79, and 77 +/- 2 beats/min, respectively; P < .001). Exposing cows to repeated transportation stress during gestation altered their calf's physiological response to stress, and these alterations could have a profound influence on the calfs ability to adapt to stress, thereby influencing its welfare. Further research should examine the growth, immune function, and reproductive function of prenatally stressed calves to determine whether these changes in plasma cortisol are beneficial or deleterious.

CpG methylation directly inhibits binding of the human papillomavirus type 16 E2 protein to specific DNA sequences.

CpG methylation of the human papillomavirus upstream regulatory region has previously been shown to reduce virus promoter activity. Here, we demonstrate that methylation of the CpG dinucleotides contained within the binding site of the human papillomavirus type 16 E2 protein has a direct effect on the interaction of this protein with DNA. Methylation of both CpG dinucleotides within the E2 site abolishes the binding of E2.

Characterization of the helicase and ATPase activity of human papillomavirus type 6b E1 protein.

Human papillomavirus type 6b (HPV-6b) is one of the most common causes of human genital warts, an important sexually transmitted disease. Discovery of antiviral therapies for this condition has been hampered by the inability to propagate the virus using standard tissue culture techniques and through difficulties in expressing sufficient recombinant viral proteins in vitro. Replication of papillomavirus DNA requires two viral proteins, E1 and E2. In an effort to establish assays to discover compounds active against this virus, we have co-expressed HPV-6b E1 and E2 proteins in insect cells. We demonstrate that the two proteins form a heteromeric complex which can be purified by sequence-specific DNA affinity chromatography. We also demonstrate that the complex has both E1-associated ATPase and ATP-dependent DNA helicase activity and report further characterization of these functions.

Adrenocorticotropic hormone dose response and some physiological effects of transportation on pregnant Brahman cattle.

The appropriate dose and the ability of exogenous ACTH to mimic the physiological effects of a real stressor need to be determined. In Exp. 1, 25 pregnant Brahman heifers were injected i.v. with either 0 (saline), .125, .25, .5, or 1 i.u. of ACTH/kg BW. Plasma cortisol was determined in blood samples collected during a 5-h period, and an integrated cortisol response was calculated for each cow. The greater the dose of ACTH, the greater was the integrated cortisol response (P < .001). However, peak plasma cortisol in response to the four doses of ACTH did not differ (P > .6). The plasma cortisol concentrations returned to baseline more slowly in those cows receiving the greater doses of ACTH, making their integrated areas of response greater. In Exp. 2, pregnant Brahman cows were either transported 48 km (n = 28), injected with 1 i.u. of ACTH/kg BW (n = 21), or served as shams (n = 28). Each treatment was repeated at 60, 80, 100, 120, and 140 d of gestation. Shrink was greater for the transported cows than for either the ACTH-treated or sham cows, 14.3, 6.0, and 5.2 kg (P < .001). Shrink also decreased in response to each subsequent application of treatment for all three treatment groups (P < .001). Transported cows had lower plasma cortisol concentrations after the first two applications of treatments (P < .006). The range of doses of ACTH caused a similar peak cortisol release; however, it took cortisol longer to return to baseline as ACTH dose increased. Repeated administration of exogenous ACTH did not cause the same amount of shrinkage as transportation, and the resultant cortisol concentrations remained consistent for each administration. There was no apparent carryover effect of repeated administration of ACTH at 20-d intervals. Maximal plasma cortisol concentrations in Brahman cattle can be obtained with doses of ACTH much smaller than those traditionally injected. However, larger doses of ACTH maintained plasma cortisol concentrations for a longer duration. Repeated transportation caused a decrease in cortisol release and shrinkage indicative of psychological habituation. Injections of ACTH did not cause the same physiological response as transportation.

Echocardiographic assessment of aortic root dimensions in normal children based on measurement of a new ratio of aortic size independent of growth.

Two-dimensional echocardiography is commonly used as a method of monitoring aortic root dimensions in children with connective tissue disease. Measurements are usually standardized to body surface area (BSA) to account for growth. However, there are several theoretical and practical disadvantages to adopting this approach, and there has been little investigation of alternative methods of standardization. This study of 48 normal children and adolescents (age range 2 weeks to 23 years) was performed to determine the relation of 2-dimensional echocardiographic aortic root dimensions to indexes of body size and growth, and to examine a simple means of internally standardizing aortic root measurements to create an index of aortic root size independent of growth. Maximal diameters in the parasternal long-axis view were recorded at 4 levels: annulus, sinuses of Valsalva (SOV), supraaortic ridge (SAR), and ascending aorta (AAO). Ratios of aortic root size were created by internally standardizing aortic root diameters to aortic annular size. All diameters correlated closely with age, height, weight, and BSA (all r > 0.87). Linear regression in each case showed a significant positive slope (all p < 0.0001). The best predictor of aortic dimensions was height, with r values of 0.93 for annulus, SOV, and AAO, and 0.95 for SAR. Ratios of SOV/annulus, SAR/annulus, and AAO/annulus remained constant, with no correlation with age or any growth parameters. Mean values and 95% confidence limits were: SOV/annulus 1.37 (1.18-1.56); SAR/annulus 1.11 (0.95-1.28); and AAO/annulus 1.16 (0.97-1.35). Standardization to height, or the use of internally standardized aortic root ratios, provides a simple and accurate alternative to standardization to BSA for assessing aortic root dimensions in normal growing children.