Separation of inhibition and activation of the allosteric yeast chorismate mutase

Yeast chorismate mutase (EC 5.4.99.5) shows homotropic activation by the substrate, allosteric activation by tryptophan, and allosteric inhibition by tyrosine. In this study mutants of chorismate mutase have been found that remain sensitive to one allosteric effector (tryptophan) but insensitive to the other (tyrosine). These mutations are located in the catalytic domain: loop 220s (212-226) and helix 12 (227-251). The first example starts with the Thr-266 --> Ile mutant that had previously been shown to be locked in the activated R state. The additional mutation Ile-225 --> Thr unlocks the R state and restores the activation by tryptophan but not the inhibition by tyrosine. The second example refers to a molecular trigger for the switch between the T and R state: a hydrogen-bonded system, which stabilizes only the T state, from Tyr-234 to Glu-23 to Arg-157. Various mutants of Tyr-234, especially Tyr-234 --> Phe, are unresponsive to tyrosine but are activated by tryptophan. This separation of activation from inhibition may indicate a pathway for activation that is independent of the allosteric transition and may also be consistent with an intermediate structure between T and R states.     

Combinatorial blockade of calcineurin and CD28 signaling facilitates primary and secondary therapeutic gene transfer by adenovirus vectors in dystrophic (mdx) mouse muscles

Recombinant adenovirus vectors (AdV) have been considered a potential vehicle for performing gene therapy in patients suffering from Duchenne muscular dystrophy but are limited by a cellular and humoral immune response that prevents long-term transgene expression as well as effective transduction after AdV readministration. Conventional immunosuppressive agents such as cyclosporine and FK506, which act by interfering with CD3-T-cell receptor-mediated signaling via calcineurin, are only partially effective in reversing these phenomena and may also produce substantial organ toxicity. We hypothesized that activation of redundant T-cell activation pathways could limit the effectiveness of these drugs at clinically tolerable doses. Therefore, we have tested the ability of immunomodulatory immunoglobulins (Ig) with different modes of action to facilitate AdV-mediated gene transfer to adult dystrophic (mdx) mice. When used in isolation, immunomodulatory Ig (anti-intercellular adhesion molecule-1, anti-leukocyte function-associated antigen-1, anti-CD2, and CTLA4Ig) were only mildly effective in mitigating cellular and/or humoral immunity against adenovirus capsid proteins and the therapeutic transgene product, dystrophin. However, the combination of FK506 plus CTLA4Ig abrogated the immune response against adenovirus proteins and dystrophin to a degree not achievable with the use of either agent alone. At 30 days after AdV injection, >90% of myofibers could be found to express dystrophin with little or no evidence of a cellular immune response against transduced fibers. In addition, the humoral immune response was markedly suppressed, and this was associated with increased transduction efficiency following vector readministration. These data suggest that by facilitating both primary and secondary transduction after AdV administration, combined targeting of CD3-T-cell receptor-mediated signaling via calcineurin and the B7:CD28 costimulatory pathway could greatly increase the potential utility of AdV-mediated gene transfer as a therapeutic modality for genetic diseases such as Duchenne muscular dystrophy that will require long-term transgene expression and repeated vector delivery.     

Stimulation of prohormone convertase-1 mRNA expression by second messenger signaling systems

The purpose of this study was to investigate the effects of activation of various second messenger signaling systems on gene expression (i.e. mRNA levels) of a peptide hormone processing enzyme called prohormone convertase-1 (PC-1, also called PC-3) in a human pancreatic carcinoid cell line (BON) that expresses several endocrine peptides (chromogranin A, pancreastatin, neurotensin). Pharmacologic activation of adenylate cyclase-cAMP, protein kinase-C and Ca2+ mobilization pathways increased PC-1 mRNA levels and neurotensin secretion. Elevations in PC-1 mRNA levels were dose and time-related. Secretagogue-induced cellular depletion of neurotensin was followed by a replenishment of cellular neurotensin stores and an upregulation of PC-1 mRNA levels. Together, these data indicate that PC-1 mRNA expression is increased with peptide secretion and coordinated with maintenance of cellular stores of peptides.     

Fetal retinoblastoma

Fetal tumors are sporadic entities and in some cases can be diagnosed prenatally. A team approach, incorporating the skills of obstetricians, radiation therapists, pediatric medical oncologists and hematologists, neonatal surgeons, rehabilitation specialists, and social workers, is imperative to ensure that the patient receives the best treatment. Retinoblastoma is a relatively uncommon tumor of childhood that arises in the retina. The estimated annual incidence is between 1 in 15,000 and 1 in 34,000. Although it may occur at any age, it most often arises in younger children, with 80% of cases diagnosed before the age of 5 years. We report a case of prenatally diagnosed fetal retinoblastoma.     

Ultraviolet-B induced hyperplasia and squamous cell carcinomas in the cornea of XPA-deficient mice

The purpose of this study was to determine whether presweetened breakfast cereals with various fiber contents and a moderate glycemic index optimize glucose availability and improve endurance exercise performance. Six recreationally active women ate 75 g of available carbohydrate in the form of breakfast cereals: sweetened whole-grain rolled oats (SRO, 7 g of dietary fiber) or sweetened whole-oat flour (SOF, 3 g of dietary fiber) and 300 ml of water or water alone (Con). The meals were provided 45 min before semirecumbent cycle ergometer exercise to exhaustion at 60% of peak O2 consumption (VO2peak). Diet and physical activity were controlled by having the subjects reside in the General Clinical Research Center for 2 days before each trial. Blood samples were drawn from an antecubital vein for glucose, free fatty acid (FFA), glycerol, insulin, epinephrine, and norepinephrine determination. Breath samples were obtained at 15-min intervals after meal ingestion and at 30-min intervals during exercise. Muscle glycogen concentration was determined from biopsies taken from the vastus lateralis muscle before the meal and immediately after exercise. Plasma FFA concentrations were lower (P < 0.05) during the SRO and SOF trials for the first 60 and 90 min of exercise, respectively, than during the Con trial. Respiratory exchange ratios were higher (P < 0.05) at 90 and 120 min of exercise for the SRO and SOF trials, respectively, than for the Con trial. At exhaustion, glucose, insulin, FFA, glycerol, epinephrine, and norepinephrine concentrations, respiratory exchange ratio, and muscle glycogen use in the vastus lateralis muscle were similar for all trials. Exercise time to exhaustion was 16% longer (P < 0.05) during the SRO than during the Con trial: 266.5 +/- 13 and 225.1 +/- 8 min, respectively. There was no difference in exercise time for the SOF (250.8 +/- 12) and Con trials. We conclude that eating a meal with a high dietary fiber content and moderate glycemic index 45 min before prolonged moderately intense exercise significantly enhances exercise capacity.     

Growth of yeasts in milk and associated changes to milk composition

The growth of several yeast species in milk containing added sodium chloride (0-15%, w/v) at 25 degrees C and 10 degrees C was examined in conjunction with yeast metabolism of milk constituents. Depending on conditions, all yeasts grew to maximum populations of 10(7)-10(8) cfu/ml. Kluyveromyces marxianus gave strong utilisation of lactose and weak metabolism of citrate, protein and fat with the production of ethanol, glycerol, lactic acid and propionic acid. As measured by the production of free amino acids and free fatty acids, Candida lipolytica and Candida catenulata gave strong proteolytic and lipolytic reactions, the specificities of which appeared to be influenced by temperature and the presence of NaCl. These species also metabolised organic acids. Although giving strong growth responses, Debaryomyces hansenii and Saccharomyces cerevisiae did not metabolise lactose and gave only very weak lipolytic and proteolytic reactions. Citrate was metabolised by D. hansenii but not by S. cerevisiae. Both species produced small amounts of ethanol, glycerol and lactic acid.     

Mechanism and treatment of diarrhoea due to Vibrio cholerae and Escherichia coli: roles of drugs and prostaglandins

The primary objectives of these studies were to determine the clinical efficacy and safety of the potential antisecretory and antimicrobial drugs in the treatment of diarrhoea due to Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC). The drugs evaluated were chlorpromazine (CPZ), nicotinic acid, berberine, indomethacin, chloroquine, tetracycline, furazolidone, and bioflorin. Additionally, the role of prostaglandins (PGs) in the pathogenesis of cholera diarrhoea has been studied. The drug studies were carried out as placebo-controlled, randomized clinical trials in patients with active diarrhoea due to vibrio cholerae and ETEC. All patients received intravenous (i.v.) or oral rehydration solutions (ORS), but no other medications except the study drugs. Results indicate that CPZ (1 mg/kg or 4 mg/kg), berberine (200 mg), and nicotinic acid (2 g) all reduced stool volumes from 30% to more than 50% in diarrhoeal patients without significant side effects. It appeared that berberine was more effective in ETEC diarrhoea than in cholera. However, chloroquine, indomethacin, clonidine, and bioflorin had no clinically useful effects. Among the antimicrobial agents, a single dose of tetracycline was found to be effective in cholera, because the drug significantly (p < 0.05) reduced the total stool volume from 20.9 +/- 15.9 to 10.5 +/- 8.6 (liters in 6-days, mean +/- SD) compared to furazolidone. Drugs other than antimicrobial and antisecretory agents were also evaluated in the treatment of cholera. It has been shown that treatment with bioflorin, which is a bacterial preparation of lyophilized Streptococcus faecium, did not significantly (p > 0.05) reduce fluid-loss in cholera. Additional studies in animals indicated that treatment with short chain glucose polymers, alone or in combination with a chloride blocking agent, anthracene-9-carboxylic acid (A9C), significantly reduced intestinal secretion in a rat model of secretory diarrhoea. For the first time it was demonstrated that jejunal prostaglandin (PG) E2 concentrations were significantly increased during acute cholera and correlated with the volumes of stool and duration of diarrhoea. Furthermore, it was shown that treatment with indomethacin, a potent inhibitor of PG synthesis, significantly reduced jejunal PGE2 output in adults with acute cholera, in addition to net secretion of water and electrolytes. In summarizing the results, it is concluded that: (1) CPZ, berberine, and nicotinic acid are potential antidiarrhoeal agents, (2) PGs are involved in the pathogenesis of cholera, (3) tetracycline and furazolidone are effective antimicrobial agents in cholera, (4) and glucose short-chain polymers (used with the chloride blocking agent, anthracene-9-carboxylic acid) are better sources of carbohydrates in oral rehydration solutions.     

Morphologic and cytochemical characteristics of blood cells from Hawaiian green turtles

Previous studies have shown that hypoglycemia may reduce counterregulatory responses to subsequent hypoglycemia in healthy subjects and in patients with diabetes. The effect of hypoglycemia on the hormonal response to a nonhypoglycemic stimulus is uncertain. To test the hypothesis that the cortisol response to corticotropin (ACTH) infusion is independent of antecedent hypoglycemia, 10 healthy subjects received a standard ACTH infusion (0.25 mg Cosyntropin [Organon, West Orange, NJ] intravenously over 240 minutes) at 8:00 AM on day 1 and day 3 and a hypoglycemic insulin clamp study (1 mU/kg/min) at 8:00 AM on day 2. During the hypoglycemic clamp, plasma glucose decreased from 5.0 mmol/L to 2.8 mmol/L for two periods of 120 minutes (mean glucose, 2.9 +/- 0.03 and 2.8 +/- 0.02 mmol/L, respectively) separated by a 60-minute interval of euglycemia (mean glucose, 4.7 +/- 0.01 mmol/L). Seven subjects also had paired control studies in random order during which a 330-minute euglycemic clamp (mean glucose, 5.0 +/- 0.11 mmol/L) instead of a hypoglycemic clamp was performed on day 2. Basal ACTH (4.6 +/- 0.7 v 2.6 +/- 0.4 pmol/L, P < .02) and basal cortisol (435 +/- 46 v 317 +/- 40 nmol/L, P < .02) both decreased from day 1 to day 3 following intervening hypoglycemia. In contrast, with intervening euglycemia, neither basal ACTH (5.9 +/- 1.5 v 4.5 +/- 1.0 pmol/L) nor basal cortisol (340 +/- 38 v 318 +/- 60 nmol/L) were reduced significantly on day 3 compared with day 1. Following interval hypoglycemia, the area under the curve (AUC) for the cortisol response to successive ACTH infusions was increased (4,734 +/- 428 nmol/L over 240 minutes [day 3] v 3,526 +/- 434 nmol/L over 240 minutes [day 1], P < .01). The maximum incremental cortisol response was also significantly increased (805 +/- 63 nmol/L (day 3) v 583 +/- 58 nmol/L (day 1), P < .05). In contrast, the AUC for the cortisol response to successive ACTH infusions with interval euglycemia (3,402 +/- 345 nmol/L over 240 minutes [day 3] v 3,709 +/- 391 nmol/L over 240 minutes [day 1] and the incremental cortisol response (702 +/- 62 nmol/L [day 3] v 592 +/- 85 nmol/L [day 1] were unchanged. Following exposure to intermittent hypoglycemia in healthy humans, fasting morning ACTH and cortisol levels are reduced and the incremental cortisol response to an infusion of ACTH is enhanced. The enhanced cortisol response to exogenous ACTH infusion after intervening hypoglycemia (but not intervening euglycemia) may reflect priming of the adrenal gland by endogenous ACTH produced during the hypoglycemia. These data suggest that adrenal function testing by exogenous ACTH administration is not impaired by prior exposure to hypoglycemia. Moreover, the reduced cortisol response to recurrent hypoglycemia in patients with well-controlled diabetes is not likely the result of impaired adrenal responsiveness.     

Antigenic structure of soluble herpes simplex virus (HSV) glycoprotein D correlates with inhibition of HSV infection

Soluble forms of herpes simplex virus (HSV) glycoprotein D (gD) block viral penetration. Likewise, most HSV strains are sensitive to gD-mediated interference by cells expressing gD. The mechanism of both forms of gD-mediated inhibition is thought to be at the receptor level. We analyzed the ability of different forms of soluble, truncated gD (gDt) to inhibit infection by different strains of HSV-1 and HSV-2. Strains that were resistant to gD-mediated interference were also resistant to inhibition by gDt, thereby suggesting a link between these two phenomena. Virion gD was the major viral determinant for resistance to inhibition by gDt. An insertion-deletion mutant, gD-1(delta 290-299t), had an enhanced inhibitory activity against most strains tested. The structure and function of gDt proteins derived from the inhibition-resistant viruses rid1 and ANG were analyzed. gD-1(ridlt) and gD-1(ANGt) had a potent inhibitory effect on plaque formation by wild-type strains of HSV but, surprisingly, little or no effect on their parental strains. As measured by quantitative enzyme-linked immunosorbent assay with a diverse panel of monoclonal antibodies, the antigenic structures of gD-1(rid1t) and gD-1(ANGt) were divergent from that of the wild type yet were similar to each other and to that of gD-1 (delta 290-299t). Thus, three different forms of gD have common antigenic changes that correlate with enhanced inhibitory activity against HSV. We conclude that inhibition of HSV infectivity by soluble gD is influenced by the antigenic conformation of the blocking gDt as well as the form of gD in the target virus.