Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1)

BACKGROUND: Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop. RESULTS: This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC betaII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC alpha and betaII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme. CONCLUSIONS: We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.     

Predicting adaptation to presymptomatic DNA testing for late onset disorders: who will experience distress? Rotterdam Leiden Genetics Workgroup

The first comparative study on predicting post-test distress (conceptualised by intrusion and avoidance, measured with the Impact of Event Scale) after presymptomatic genetic testing for Huntington's disease (HD, n=25), cancer syndromes (familial adenomatous polyposis (FAP, n=23)), and hereditary breast and ovarian cancer (HBOC, n=10) is reported. The variables with the highest predictive potential of post-test distress are presented. Participants who were depressed before the test were more distressed after testing, but we found that those who were anxious before the test were less distressed, that is, had less intrusive thoughts post-test. Other factors associated with a higher level of post-test intrusion were gender (being a woman), having children, and pre-test intrusion. Religion and being at risk for HBOC were associated with less post-test intrusion. Participants who showed avoidance behaviour before the test and those who had many people available for support showed more avoidance behaviour post-test. The test result did not additionally contribute to post-test distress. The prima facie simple notion that the test result, as such, determines the distress experienced seems to be a misrepresentation of the complex reality.     

Preweaning growth traits for Senepol, Hereford, and reciprocal crossbred calves and feedlot performance and carcass characteristics of steers

We conducted a multiyear study in two phases to determine preweaning performance traits of Senepol (S x S), Hereford (H x H), and reciprocal (S x H and H x S) F1 crossbred calves and feedlot performance and carcass characteristics of steers. In Phase I, from 1985 to 1989, data from S x S (n = 194), H x H (n = 383), and S x H (n = 120) calves were used. Numbers of S x S cows were increased during Phase I so that data from H x S (n = 74) calves could be included in Phase II (1990 to 1992) in addition to S x S (n = 118), H x H (n = 130), and S x H (n = 56) calves. Also during Phase II, feedlot performance and carcass characteristics were determined for S x S (n = 30), H x H (n = 26), H x S (n = 36), and S x H (n = 26) steers. In Phase I, S x S calves had heavier (P < .01) birth weights and heavier (P < .01) 205-d adjusted weaning weights than H x H calves. Birth weights of S x H calves were heavier (P < .01) than the mean of the purebred calves, but 205-d adjusted weaning weights did not differ (P > .10). In phase II, direct heterosis was 3.5% for birth weight (P < .05) and 5.1% for 205-d adjusted weaning weight (P < .01). Senepol maternal breed effects were 1.9 kg for birth weight (P < .10) and 37.9 kg for 205-d adjusted weaning weight (P < .01). Levels of direct heterosis, Senepol maternal breed effects, and Hereford direct breed effects were significant for most feedlot performance traits of steer calves that were fed to a common end point. Breeds did not differ (P > .10) for USDA yield and quality grades, and direct heterosis was not significant for Warner-Bratzler shear force. These results demonstrate significant levels of heterosis in preweaning performance between S x S and H x H calves and in feedlot performance of steers. Levels of heterosis were smaller and nonsignificant for most carcass traits including meat tenderness, which did not differ between S x S and H x H steers in this study.     

Analytical issues in the evaluation of food deprivation and sucrose concentration effects on the microstructure of licking behavior in the rat

A microstructural analysis of licking behavior in nondeprived and 23-hr food-deprived rats (n = 15) presented with various sucrose solutions (0.03-1.0 M) in daily single-bottle, 1-hr sessions was conducted. Food deprivation and concentration interacted to increase total licks. The effects of food deprivation and concentration on burst size (BS), burst number (BN), and other parameters varied as a function of the pause criterion (PC; 0.3-100 s) used to define licking bursts. A rationale for selecting a 1-s PC for further analysis is presented. Despite the lack of correlations between temporally contiguous burst and pause combinations, mean BS decreased and pause duration increased as meals progressed. At the 1-s PC, BS increased linearly with concentration, implying that this microstructural parameter is influenced in part by taste. Food deprivation did not affect BS but rather increased BN and proportionally extended the meal duration.     

Influence of adrenergic agonists on the release of amino acids from rat skeletal muscle studied by microdialysis

Epidermal growth factor (EGF) has been considered to be a candidate for neurotrophic factors on the basis of the results of several in vitro studies. However, the in vivo effect of EGF on ischemic neurons as well as its mechanism of action have not been fully understood. In the present in vivo study using a gerbil ischemia-model, we examined the effects of EGF on ischemia-induced learning disability and hippocampal CA1 neuron damage. Cerebroventricular infusion of EGF (24 or 120 ng/d) for 7 days to gerbils starting 2 hours before or immediately after transient forebrain ischemia caused a significant prolongation of response latency time in a passive avoidance task in comparison with the response latency of vehicle-treated ischemic animals. Subsequent histologic examinations showed that EGF effectively prevented delayed neuronal death of CA1 neurons in the stratum pyramidale and preserved synapses intact within the strata moleculare, radiatum, and oriens of the hippocampal CA1 region. In situ detection of DNA fragmentation (TUNEL staining) revealed that ischemic animals infused with EGF contained fewer TUNEL-positive neurons in the hippocampal CA1 field than those infused with vehicle alone at the seventh day after ischemia. In primary hippocampal cultures, EGF (0.048 to 6.0 ng/mL) extended the survival of cultured neurons, facilitated neurite outgrowth, and prevented neuronal damage caused by the hydroxyl radical-producing agent FeSO4 and by the peroxynitrite-producing agent 3-morpholinosydnonimine in a dose-dependent manner. Moreover, EGF significantly attenuated FeSO4-induced lipid peroxidation of cultured neurons. These findings suggest that EGF has a neuroprotective effect on ischemic hippocampal neurons in vivo possibly through inhibition of free radical neurotoxicity and lipid peroxidation.     

Pre-incision infiltration with lidocaine reduces pain and opioid consumption after reduction mammoplasty

BACKGROUND: The main purpose of the reconstruction of the cranium is the protection of the brain. Besides we have to consider important functional and aesthetic necessities in order to achieve satisfactory results. METHODS: Thirty-six clinical cases, operated from November 1991 to June 1996, in which the reconstruction of the cranial vault is carried out by a polymethylmethacrylate acrylic resin are analysed. The causes and locations of the most common bone defects and the main indications for reconstruction are examined. While the repair of the osseous gaps caused by neoplasms is immediate, in the traumatic occurrences, in order to reduce the probability of infectious complications, an average time of 11 months elapsed from the first operation. The surgical technique, with slightest alterations, is the same in all the presented cases, preparing the acrylic resin straight on the operating table. The resin, moulded and adapted to the defect until its complete hardening, presents, thanks to its properties, manifold advantages (and few real disadvantages). RESULTS: The results, in terms of complications, are very satisfactory, with an infectious rate of 2.7%. Besides, in one third of the patients, a considerable clinical improvement after the repair has been observed. CONCLUSIONS: According to personal experience, it is possible to affirm that polymethylmethacrylate, with its remarkable plasticity and stability in time, can always guarantee a satisfactory functional and aesthetic result.     

Synthesis and analgesic properties of new 5-thioaryl pyrazole derivatives

A new series 5-thio aryl pyrazole derivatives were proposed aiming analgesic activity. In this work, 8 new compounds of this class were synthesized using usual synthetic methodology, having as key intermediate the 3-methyl-4-nitro-5-chloropyrazole-1-phenyl derivative and subsequent reaction with several nucleophiles sulfides. Pharmacological evaluation of this series showed analgesic activity in the some extent in especially for 5-(4-bromophenyl)-thio-3-methyl-4-nitro-1-phenylpyrazole which was the most potent in this series, presenting an analgesic action comparable to that show by dipyrone.     

Trends in mortality of childhood-onset insulin-dependent diabetes mellitus in Leicestershire: 1940-1991

The relative risk of death by calendar date of diagnosis was investigated in a population-based incident cohort of 845 (463 males:382 females) IDDM diagnosed in Leicestershire before the age of 17 years between 1940 and 1989. The mortality status of 844 (99.9%) patients was determined as of the 31 December 1991, representing 14,346 person-years of risk. Trends in relative risk of death were investigated using Cox proportional hazards modelling for within cohort comparisons and age/sex and calendar time adjusted standardized mortality ratios (SMR) using generalized linear modelling for external comparisons. Median age at diagnosis was 10 years (range 3 months to 16 years); median duration of diabetes 15 years (range 1-51 years). Forty-four patients had died (5.2%; median age at death 31 years, range 11-51 years). A further four patients died at presentation (within 24 h) from ketoacidosis and are excluded from all analyses. Calendar date of diagnosis was found to be an important predictor of mortality. Adjusting for attained age there was evidence of a decline in relative risk of death with calendar date of diagnosis of 3.4% (95% CI, 0.005-6.9%) per annum, equivalent to a 32% fall per decade (95% CI, 5-51%), or 84% (95% CI, 21-97) from 1940 to 1989. The data are consistent with a large fall in mortality between the 1940s and 1950s representing over 50% of the total reduction in mortality between 1940 and 1991. Neither sex nor age at diagnosis were significant predictors of mortality. Over the study period 1940-89 the SMR (male and female combined) fell from 981 (541-1556) to 238 (60-953) relative to the general population. This population-based study shows that the prognosis for Type 1 (insulin-dependent) diabetes mellitus has improved markedly over the period 1940-1991.     

EAE TCR motifs and antigen recognition in myelin basic protein-induced anterior uveitis in Lewis rats

T cells infiltrating the iris/ciliary body of Lewis rats with anterior uveitis (AU) that had been induced by myelin basic protein (MBP) immunization were previously found to share surface markers common to the T cells that cause experimental autoimmune encephalomyelitis (EAE). To determine whether these AU-associated T cells are in fact the same as those that infiltrate the central nervous system to cause EAE, we examined TCR V gene expression in T cells infiltrating the anterior chamber in rats with AU. As with EAE, we found a biased expression of Vbeta8.2 and Valpha2 in the iris/ciliary body and, although one would expect an influx of nonspecific inflammatory T cells, these biases were still evident at the peak of AU. An analysis of the TCR Vbeta8.2 and Valpha2 sequences derived from the iris/ciliary body demonstrated the presence of the same complementarity determining region 3 motifs found in MBP-specific T cells that are pathogenic for EAE and found in T cells derived from the central nervous system of rats with EAE. Finally, T cells isolated from the iris/ciliary body of rats with AU were found to proliferate in a specific fashion to MBP Ags. Thus, it appears that MBP-specific T cells are pathogenic for AU as well as EAE in the Lewis rat. In addition, the long-term presence of this highly restricted MBP response in the iris/ciliary body indicates that distinct immunoregulatory mechanisms exist in the environment of the eye. This provides an interesting model with which to address questions pertaining to the nature of T cells infiltrating the eye and their regulation during EAE and other systemic diseases.