Mesencephalic astrocyte-derived neurotropic factor is an important factor in chondrocyte ER homeostasis.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) resident protein that can be secreted due to an imperfect KDEL motif. MANF plays a cytoprotective role in several soft tissues and is upregulated in conditions resulting from intracellular retention of mutant protein, including two skeletal diseases, metaphyseal chondrodysplasia, Schmid type (MCDS) and multiple epiphyseal dysplasia (MED). The role of MANF in skeletal tissue homeostasis is currently unknown. Interestingly, cartilage-specific deletion of Manf in a mouse model of MED resulted in increased disease severity, suggesting its upregulation may be chondroprotective. Treatment of MED chondrocytes with exogenous MANF led to a decrease in the cellular levels of BiP (GRP78), confirming MANF's potential to modulate ER stress responses. However, it did not alleviate the intracellular retention of mutant matrilin-3, suggesting that it is the intracellular MANF that is of importance in the pathobiology of skeletal dysplasias. The Col2Cre-driven deletion of Manf from mouse cartilage resulted in a chondrodysplasia-like phenotype. Interestingly, ablation of MANF in cartilage did not have extracellular consequences but led to an upregulation of several ER-resident chaperones including BiP. This apparent induction of ER stress in turn led to dysregulated chondrocyte apoptosis and decreased proliferation, resulting in reduced long bone growth. We have previously shown that ER stress is an underlying disease mechanism for several skeletal dysplasias. The cartilage-specific deletion of Manf described in this study phenocopies our previously published chondrodysplasia models, further confirming that ER stress itself is sufficient to disrupt skeletal growth and thus represents a potential therapeutic target.

Long-term consequences of the Alzheimer's caregiver role: a qualitative analysis.

A qualitative analysis was performed on responses of 51 participants to open-ended questions about the experience of being family caregivers for a loved one affected by Alzheimer's disease. Participants had been in the caregiver role for at least eight years. Results indicated a pattern of adapting successfully or unsuccessfully to the caregiver role, experiencing initial caregiver burden, finding relief in social support when available, and long-term distress or long-term positive change. Memories and feelings were strong about individuals and institutions that had been helpful or indifferent many years earlier as the caregivers struggled to cope with their unplanned predicament.

Insulin continues to induce plasminogen activator inhibitor 1 gene expression in insulin-resistant mice and adipocytes.

BACKGROUND: Although the association between insulin resistance and cardiovascular risk is well established, the underlying molecular mechanisms are poorly understood. The antifibrinolytic molecule plasminogen activator inhibitor 1 (PAI-1) is a cardiovascular risk factor that is consistently elevated in insulin-resistant states such as obesity and non-insulin-dependent diabetes mellitus (NIDDM). The strong positive correlation between this elevated PAI-1 and the degree of hyperinsulinemia not only implicates insulin itself in this increase, but also suggests that PAI-1 is regulated by a pathway that does not become insulin resistant. The data in this report supports this hypothesis. MATERIALS AND METHODS: We show that insulin stimulates PAI-1 gene expression in metabolically insulin-resistant ob/ob mice and in insulin-resistant 3T3-L1 adipocytes. Moreover, we provide evidence that glucose transport and PAI-1 gene expression are mediated by different insulin signaling pathways. These observations suggest that the compensatory hyperinsulinemia that is frequently associated with insulin-resistant states, directly contribute to the elevated PAI-1. CONCLUSIONS: These results provide a potential mechanism for the abnormal increases in cardiovascular risk genes in obesity, NIDDM, and polycystic ovary disease.

Diverse mechanisms of inhibition of pyruvate dehydrogenase kinase by structurally distinct inhibitors.

The mechanism of action of structurally distinct pyruvate dehydrogenase kinase (PDK) inhibitors was examined in assays with experimental contexts ranging from an intact pyruvate dehydrogenase complex (PDC) with and without supplemental ATP or ADP to a synthetic peptide substrate to PDK autophosphorylation. Some compounds directly inhibited the catalytic activity of PDKs. Some of the inhibitor classes tested inhibited autophosphorylation of recombinant PDK1 and PDK2. During these studies, PDC was shown to be directly inhibited by a novel mechanism; the addition of supplemental recombinant PDKs, an effect that is ADP-dependent and partly alleviated by members of each of the compound classes tested. Overall, these data demonstrate that small molecules acting at diverse sites can inhibit PDK activity.

Pancreatic beta-cell K(ATP) channel activity and membrane-binding studies with nateglinide: A comparison with sulfonylureas and repaglinide.

Nateglinide (A-4166) is an amino acid derivative with insulinotrophic action in clinical development for treatment of type 2 diabetes. The aim of this study was to determine whether nateglinide's interaction at the K(ATP) channel/sulfonylurea receptor underlies its more rapid onset and shorter duration of action in animal models. Binding studies were carried out with membranes prepared from RIN-m5F cells and HEK-293 cells expressing recombinant human sulfonylurea receptor 1 (SUR1). The relative order for displacement of [(3)H]glibenclamide in competitive binding experiments with RIN-m5F cell membranes was glibenclamide > glimepiride > repaglinide > glipizide > nateglinide > L-nateglinide > tolbutamide. The results with HEK-293/recombinant human SUR1 cells were similar with the exception that glipizide was more potent than repaglinide. Neither nateglinide nor repaglinide had any effect on the dissociation kinetics for [(3)H]glibenclamide, consistent with both compounds competitively binding to the glibenclamide-binding site on SUR1. Finally, the inability to measure [(3)H]nateglinide binding suggests that nateglinide dissociates rapidly from SUR1. Direct interaction of nateglinide with K(ATP) channels in rat pancreatic beta-cells was investigated with the patch-clamp method. The relative potency for inhibition of the K(ATP) channel was repaglinide > glibenclamide > nateglinide. Kinetics of the inhibitory effect on K(ATP) current showed that the onset of inhibition by nateglinide was comparable to glibenclamide but more rapid than that of repaglinide. The time for reversal of channel inhibition by nateglinide was also faster than with glibenclamide and repaglinide. These results suggest that the unique characteristics of nateglinide are largely the result of its interaction at the K(ATP) channel.

Hypoglycemic prodrugs of 4-(2,2-dimethyl-1-oxopropyl)benzoic acid.

SAH 51-641 (1) is a potent hypoglycemic agent, which acts by inhibiting hepatic gluconeogenesis. It is a prodrug of 4-(2, 2-dimethyl-1-oxopropyl)benzoic acid (2) and 4-(2, 2-dimethyl-1-hydroxypropyl)benzoic acid (3), which sequester coenzyme A (CoA) in the mitochondria, and inhibits medium-chain acyltransferase. 1-3 and 4-tert-butylbenzoic acid all cause testicular degeneration in rats at pharmacologically active doses. 14b (FOX 988) is a prodrug of 3, which is metabolized in the liver at a rate sufficient enough to have hypoglycemic potency (an ED50 of 65 micromol/kg, 28 mg/kg/day, for glucose lowering), yet by avoiding significant escape of the metabolite 3 to the systemic circulation, it avoids the testicular toxicity at doses up to 1500 micromol/kg/day. 14b was selected for clinical studies.

Substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones as hypoglycemic agents.

A series of substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones was synthesized from amino alcohols or amino thiols and keto acids. A pharmacological model based on the results obtained with these compounds led to the synthesis and evaluation of a series of isoxazoles and other monocyclic compounds. These were evaluated for their ability to enhance glucose utilization in cultured L6 myocytes. The in vivo hypoglycemic efficacy and potency of these compounds were evaluated in a model of type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus), the ob/ob mouse. 25a(2S) (SDZ PGU 693) was selected for further pharmacological studies.

Heterologously expressed inner lipoyl domain of dihydrolipoyl acetyltransferase inhibits ATP-dependent inactivation of pyruvate dehydrogenase complex. Identification of important amino acid residues.

The activity of the pyruvate dehydrogenase multienzyme complex (PDC), which catalyses the oxidation of pyruvate to acetyl-CoA within the mitochondrion, is diminished in animal models of diabetes. Studies with purified PDC components have suggested that the kinases responsible for inactivating the decarboxylase catalytic subunits of the complex are most efficient in their regulatory role when they are bound to dihydrolipoyl acetyltransferase (E2) subunits, which form the structural core of the complex. We report that the addition of an exogenous E2 subdomain (inner lipoyl domain) to an intact PDC inhibits ATP-dependent inactivation of the complex. By combining molecular modelling, site-directed mutagenesis and biophysical characterizations, we have also identified two amino acid residues in this subdomain (Ile229 and Phe231) that largely determine the magnitude of this effect.

Sexual harassment: the "reasonable person" vs "reasonable woman" standards have not been resolved.

Previous research suggests that women are more likely than men to perceive a hostile environment of sexual harassment in job-related scenarios. Such findings raise questions about whether a "reasonable woman" standard might be preferable to a "reasonable person" standard for adjudication of some sexual harassment cases. There are sound arguments for both positions, and there is no basis at the present time for unequivocal and categorical support for one position over the other.

Environmental psychology 1989-1994.

A review of research and theory on transactions between people and physical environments emphasizes new contributions to theory and empirical research published in major journals of environmental psychology, 1989-1994. Theories focused on arousal, load, stress, privacy-regulation, behavior settings, and transactional analysis; new theory increasingly incorporated situational and contextual variables. Empirical research emphasized field settings over the laboratory and employed increasingly diverse methods, populations, and cultures. Environmental design studies integrated scientific and applied goals through post-occupancy evaluation. New findings concerned features of residences, work places, hospitals, schools, prisons, and larger community environments. New studies also addressed environmental stressors (e.g., temperature, noise); effects of attitudes and behaviors on conservation, crime, pollution, and hazards; and issues for neighborhoods, public places, and natural environments. Directions for the future include integrated theory to guide research, more design experiments, and development of conventions for case studies.

Effects of substrate binding and pH on the secondary structure of carnitine acetyltransferase.

Carnitine acetyltransferase (CAT) exists as a monomer in solution as demonstrated by dynamic light scattering measurements. Under these conditions, interactions between CAT and its substrates, L-carnitine and acetyl-CoA, were studied by circular dichroism (CD) and fluorescence spectroscopy over a wide range of substrate concentrations. CD data indicated that the binding of L-carnitine and acetyl-CoA caused changes in the secondary structure of the protein. Quenching of the intrinsic protein fluorescence upon binding of either substrate corroborated these findings. Analysis of the binding data suggests that binding of both substrates to CAT is specific and saturable, and that there is a single binding site (or multiple identical and independent binding sites) on CAT for each substrate. Estimated L-carnitine/CAT dissociation constants were 506 +/- 58 microM and 236 +/- 27 microM in the absence or presence of acetyl-CoA, respectively. The dissociation constant for acetyl-CoA/CAT was estimated at 19 +/- 7 microM. The effect of pH on the secondary structure of the protein was determined in order to investigate the structural cause for the pH-dependent enzymatic activity of CAT. Loss of alpha-helices and a reduction of thermal stability in CAT was detected at both acidic and basic pH. Thus, the reduced catalytic activity of CAT at acidic or basic pH may be due to pH-induced protein unfolding.

Gender influence on perceptions of hostile environment sexual harassment.

Perceptions of sexual harassment were investigated as a function of perpetrators' and recipients' gender. Undergraduate students (100 women, 98 men) were presented 34 scenarios of men and women interacting at work. Participants were asked to read carefully each scenario and indicate on a scale anchored by 1 (strongly disagree) and 7 (strongly agree) their opinions as to whether the scenario represented an incident of sexual harassment. Analysis indicated that women rated "hostile environment" scenarios as more harassing than men, and male perpetrators were rated as more harassing than female perpetrators. Even though some scenarios were rated as more harassing than others, the full range of the 7-point scale was used on every scenario, indicating a lack of agreement on what constitutes harassment. This lack of agreement highlights the debate within the legal community about whether the "reasonable person" or the "reasonable woman" standard should be used to judge sexual harassment in the workplace.

Over-expression and characterization of active recombinant rat liver carnitine palmitoyltransferase II using baculovirus.

The cDNA encoding rat liver carnitine palmitoyltransferase II (CPT-II) was heterologously expressed using a recombinant baculovirus/insect cell system. Unlike Escherichia coli, the baculovirus-infected insect cells expressed mostly soluble active recombinant CPT-II (rCPT-II). CPT activity from crude lysates of recombinant baculovirus-infected insect cells was maximal between 50 and 72 h post-infection, with a peak specific activity of 100-200 times that found in the mock- or wild-type-infected control lysates. Milligram quantities (up to 1.8 mg/l of culture) of active rCPT-II were chromatographically purified from large-scale cultures of insect cells infected with the recombinant baculovirus. The rCPT-II was found to be: (1) similar in size to the native rat liver enzyme (approximately 70 kDa) as judged by SDS/PAGE; (2) immunoreactive with a polyclonal serum raised against rat liver CPT-II; and (3) not glycosylated. Kinetic analysis of soluble rCPT-II revealed Km values for carnitine and palmitoyl-CoA of 950 +/- 27 microM and 34 +/- 5.6 microM respectively.

Place attachment, isolation, and the power of a window in a hospital environment: a case study.

This paper describes the relevance of the literature on environmental psychology to the coping strategies a leukemia patient used in adapting to psychological and physical isolation on a hospital bone marrow transplant unit and oncology unit. The case study describes the difficulty of place attachment on the isolation unit and its evolution on the oncology unit. The power of a window with a natural view--including a view of a cemetery--was especially evident even as the disease became terminal.

The effects of wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, on insulin-stimulated glucose transport, GLUT4 translocation, antilipolysis, and DNA synthesis.

PI 3-kinase, an enzyme that selectively phosphorylates the 3-position of the inositol ring, is acutely activated by insulin and other growth factors. The physiological significance of PI 3-kinase activation and, more specifically, its role in insulin action is an area under intense investigation. In this study, we have examined the role of PI 3-kinase activation in mediating selected metabolic and mitogenic effects of insulin employing the fungal metabolite wortmannin, a potent inhibitor of PI 3-kinase activity. In isolated rat and cultured 3T3-L1 adipocytes, wortmannin inhibited insulin-stimulated glucose transport (IC50 = 9 nM) without a significant effect on basal transport. Insulin-stimulated translocation of GLUT4 in isolated rat adipocytes was markedly inhibited by wortmannin. Wortmannin had no effect on either basal or insulin-stimulated glucose utilization in L6 myocytes, a skeletal muscle cell line in which GLUT1 is the predominant transporter isoform. Wortmannin also partially antagonized the antilipolytic effect of insulin on adenosine deaminase-stimulated lipolysis in isolated rat adipocytes. Furthermore, wortmannin caused a significant reduction in insulin-stimulated DNA synthesis in Fao rat hepatoma cells. We conclude that PI 3-kinase activation is necessary for maximum insulin-stimulated glucose transport, translocation of GLUT4, antilipolysis and DNA synthesis.

Kinetic, circular dichroism and fluorescence studies on heterologously expressed carnitine palmitoyltransferase II.

Km estimates for carnitine and palmitoyl-CoA of heterologously expressed rat liver carnitine palmitoyl-transferase-II (rCPT-II) were 950 +/- 27 microM and 34 +/- 6 microM, respectively. Vmax for the enzyme was 1.8 mumol/min/mg purified protein. Consistent with an ordered reaction mechanism in which palmitoyl-CoA binds first, SDZ CPI 975, a reversible carnitine palmitoyltransferase inhibitor containing both carnitine and alkyl moieties, inhibited rCPT-II competitively with carnitine and uncompetitively with palmitoyl-CoA. Substrate-enzyme interactions were examined by circular dichroism (CD) and fluorescence. Both carnitine and palmitoyl-CoA alone induced conformational changes in the enzyme; dissociation constant estimates by CD for carnitine and palmitoyl-CoA were 41 +/- 5 microM and 7 +/- 2 microM, respectively.

Expression of rat microsomal epoxide hydrolase in Escherichia coli. Identification of a histidyl residue essential for catalysis.

The cDNA containing the complete coding region for rat microsomal epoxide hydrolase (EC 3.3.2.3) was cloned into the expression/secretion vector pIN-III-OmpA3 and expressed in Escherichia coli strain TG1. Recombinant epoxide hydrolase was found to represent 4-9% of total bacterial protein and catalyzed the hydrolysis of styrene oxide and benzo[a]pyrene 4,5-oxide with specific activities of 421 and 734 nmol min-1 mg of epoxide hydrolase-1, respectively. Previous work implicated a histidyl residue at or near the active site of the enzyme (DuBois, G. C., Appella, E., Levin, W., Lu, A. Y. H., and Jerina, D. M. (1978) J. Biol. Chem. 253, 2932-2939). Comparison of the amino acid sequences of rat, human, and rabbit epoxide hydrolases revealed the presence of 14 conserved histidyl residues. To investigate the role of these residues in epoxide hydrolysis, site-specific mutants were generated and expressed in E. coli. Mutants H64L, H82L, H115N, H126N, H129L, H148N, H170L, H176L, H242L, H247L, H301L, H385L, K386M-H387L, delta 385-391, and H407L catalyzed the hydrolysis of benzo[a]pyrene 4,5-oxide with specific activities between 115 and 830 nmol min-1 mg-1. Mutants H431L, H431N, and H431R were all found to have activities of < 5 nmol min-1 mg-1, which is at least 150-fold less than the activity of the wild type enzyme. A Vm versus pH profile for the recombinant wild type epoxide hydrolase revealed a broad pH optimum of 6.5 to 8.5 and the presence of three ionizable groups with pKa values of 5.8 +/- 0.2, 9.2 +/- 0.1, and 9.7 +/- 0.4. The group with a pKa of 5.8 is preferentially unprotonated, while the other two groups are preferentially protonated for catalysis. We propose that histidine 431 corresponds to the group with a pKa of 5.8, while the others, with pKa values of 9.2 and 9.7 likely represent lysyl, cysteinyl, or tyrosyl residues. Thus, the data are consistent with a model where His-431 acts as a general base, abstracting a proton from water, while another residue(s), perhaps lysine, act as a general acid protonating the alkoxide anion that forms upon cleavage of the carbon-oxygen bond.

Spondylolysis in fast bowlers: principles of prevention and a survey of awareness among cricket coaches.

Back injury in general, and spondylolysis in particular, represents a serious threat to the fast bowler. Hereditary factors, poor technique, overuse, and poor preparation for fast bowling may combine to produce the 'at risk' bowler. Three first-class county coaches were interviewed to establish a level of awareness of the lesion, with an emphasis on preparation of fast bowlers and the roles of technique alteration and bowling prescription in reducing the risk of back injury.