Brain region-dependent effects of dexamethasone on counterregulatory responses to hypoglycemia in conscious rats

The aim of this study was to determine whether activation of central type II glucocorticoid receptors can blunt autonomic nervous system counterregulatory responses to subsequent hypoglycemia. Sixty conscious unrestrained Sprague-Dawley rats were studied during 2-day experiments. Day 1 consisted of either two episodes of clamped 2-h hyperinsulinemic (30 pmol x kg(-1) x min(-1)) hypoglycemia (2.8 +/- 0.1 mM; n = 12), hyperinsulinemic euglycemia (6.2 +/- 0.1 mM; n = 12), hyperinsulinemic euglycemia plus simultaneous lateral cerebroventricular infusion of saline (24 microl/h; n = 8), or hyperinsulinemic euglycemia plus either lateral cerebral ventricular infusion (n = 8; LV-DEX group), fourth cerebral ventricular (n = 10; 4V-DEX group), or peripheral (n = 10; P-DEX group) infusion of dexamethasone (5 microg/h), a specific type II glucocorticoid receptor analog. For all groups, day 2 consisted of a 2-h hyperinsulinemic (30 pmol x kg(-1) x min(-1)) or hypoglycemic (2.9 +/- 0.2 mM) clamp. The hypoglycemic group had blunted epinephrine, glucagon, and endogenous glucose production in response to subsequent hypoglycemia. Consequently, the glucose infusion rate to maintain the glucose levels was significantly greater in this group vs. all other groups. The LV-DEX group did not have blunted counterregulatory responses to subsequent hypoglycemia, but the P-DEX and 4V-DEX groups had significantly lower epinephrine and norepinephrine responses to hypoglycemia compared with all other groups. In summary, peripheral and fourth cerebral ventricular but not lateral cerebral ventricular infusion of dexamethasone led to significant blunting of autonomic counterregulatory responses to subsequent hypoglycemia. These data suggest that prior activation of type II glucocorticoid receptors within the hindbrain plays a major role in blunting autonomic nervous system counterregulatory responses to subsequent hypoglycemia in the conscious rat.     

Regulation of cardiac volume-sensitive chloride channel by focal adhesion kinase and Src kinase

The volume-sensitive chloride current (ICl,swell) is found in the mammalian myocardium and is activated by osmotic swelling. The goal of this study was to examine the importance of the tyrosine kinases focal adhesion kinase (FAK) and Src kinase in cardiac ICl,swell regulation. Neonatal rat ventricular myocytes were cultured on collagen membranes and infected with adenovirus expressing beta-galactosidase (AdLacZ), FAK, or FAK-related nonkinase. FAK-related nonkinase (FRNK) is an endogenous cardiac protein, which functions as an inhibitor of FAK. Whole cell patch-clamp recordings demonstrated that osmotic swelling was associated with the activation of an outward rectifying current in uninfected and AdLacZ-infected cells. Consistent with the properties of ICl,swell, this current displayed a reversal potential close to the equilibrium potential for Cl-; was inhibited by the Cl- channel blockers 4,4'-dinitrostilbene-2,2'-disulfonic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and tamoxifen; and was eliminated in hypertonic solution. In addition to activating ICl,swell, hypotonic swelling enhanced the tyrosine phosphorylation of multiple cardiac proteins including those in the range of 68-70 and 120-130 kDa. Pretreatment of the cells with the drug 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, an inhibitor of FAK and Src, diminished swelling-induced phosphorylation of these proteins but paradoxically increased ICl,swell. Furthermore, overexpression of FRNK but not FAK caused a twofold augmentation in I(Cl,swell) and increased the rate of current activation. Thus the tyrosine kinases FAK and Src contribute to the regulation of ICl,swell.     

Deficiency of iNOS does not attenuate severe congestive heart failure in mice

Inducible nitric oxide synthase (iNOS) has been implicated in the pathophysiology of congestive heart failure (CHF). Given the extensive evidence supporting this concept, we hypothesized that iNOS deficiency (iNOS(-/-)) would attenuate the severity of CHF in mice. Mice were subjected to permanent occlusion [myocardial infarction (MI)] of the proximal left anterior descending coronary artery to produce CHF. Cardiac function was assessed in vivo using echocardiography and ultraminiature ventricular pressure catheters. Sham wild-type (n = 17), sham iNOS(-/-) (n = 8), MI wild-type (n = 56), and MI iNOS(-/-) (n = 48) mice were subjected to MI (or sham MI) and followed for 1 mo. Deficiency of iNOS did not alter survival during CHF compared with wild type (35% vs. 32%, P = not significant). Furthermore, fractional shortening and cardiac output were not significantly different between wild-type (9.6 +/- 2.0% and 441 +/- 20 microl.min(-1).g(-1)) and iNOS(-/-) (9.8 +/- 1.3% and 471 +/- 26 microl.min(-1).g(-1)) mice. The extent of cardiac hypertrophy and pulmonary edema was also similar between wild-type and iNOS(-/-) mice. None of the indexes demonstrated any significant differences between iNOS(-/-) and wild-type mice subjected to MI. These findings indicate that deficiency of iNOS does not significantly affect severe CHF in mice after MI.     

Morphological engineering of Streptomyces hygroscopicus var. geldanus: regulation of pellet morphology through manipulation of broth viscosity

Actinomycetes, especially members of the genus Streptomyces, are responsible for producing the majority of known antibiotics. The production of antibiotics by filamentous organisms is often dependent on the morphology and size distribution of the pellet population within the culture. Particle interaction and subsequent pellet formation are primarily dependent on the rate of collision of particles in culture, which is in turn, a function of fluid turbulence. The microbial polysaccharide xanthan gum was used to artificially regulate the apparent viscosity (_a) of S. hygroscopicus fermentation broths with the aim of controlling particle interaction, aggregation and hence pellet formation. An increase in both pellet count and biomass concentration from approximately 2,000 to 8,000 pellets ml^–1 and 0.9–2.1 g l^–1 dry weight of biomass, as well a decrease in the mean pellet volume from 0.014 to 0.004 mm³ was observed in cultures supplemented with 3 g l^–1 xanthan gum. The addition of xanthan gum significantly alters fluid rheology by increasing the _a. Counter-intuitively, an increase in the _a within the experimental range examined resulted in an increase in the rate of gas–liquid mass transfer. This was attributed to the predominantly diffusive nature of oxygen transfer in shake flask cultures.     

Therapeutic insulins and their large-scale manufacture

Biotechnological innovations over the past 25 years have underpinned the rapid development of a thriving biopharmaceutical sector. Therapeutic insulin remains one of the most commonly used products of pharmaceutical biotechnology and insulin-based products command annual global sales in excess of $4.5 billion. Innovations in its method of production and in particular the advent of engineered insulin analogues provide a fascinating insight into how scientific and technological advances have impacted upon the pharmaceutical biotechnology sector as a whole. Current insulin-based diabetes research is increasingly focused not on the insulin molecule per se, but upon areas such as the development of non-parenteral insulin delivery systems, as well as organ-/cell-based and gene therapy-based approaches to controlling the disease.     

Elucidating the substrate specificity and condensation domain activity of FkbP, the FK520 pipecolate-incorporating enzyme

Rapamycin, FK506, and FK520 are potent immunosuppressant natural product macrocycles generated by hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) systems in streptomycetes. An important functional element within these molecules is an l-pipecolate moiety that is incorporated into the completed polyketide chain by the action of RapP/FkbP, a four-domain NRPS that also putatively serves to cyclize the chain after amino acid insertion. Here we report the expression and purification of recombinant FkbP from the FK520 biosynthetic pathway. Using a combination of radioassays and Fourier transform mass spectrometry, we demonstrate that once FkbP has been phosphopantetheinylated in vitro, its peptidyl carrier protein domain can be successfully loaded with l-pipecolic acid and, to a lesser extent, l-proline. The first condensation domain of FkbP is shown to be active through the successful acetylation of aminoacyl-S-FkbP using the appropriately loaded terminal acyl carrier protein from the PKS array, FkbA, as the chain donor. Site-directed mutagenesis confirmed that the N-terminal condensation domain catalyzes the transfer reaction. Acetylation of prolyl-S-FkbP was more rapid and occurred to a greater extent than that of pipecolyl-S-FkbP, a trend which was also observed with alternative acyl chain donors. These observations suggest that the adenylation domain of FkbP serves as the primary selectivity filter for pipecolate incorporation.     

PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis

MOTIVATION: PSORTb v.1.1 is the most precise bacterial localization prediction tool available. However, the program's predictive coverage and recall are low and the method is only applicable to Gram-negative bacteria. The goals of the present work are as follows: increase PSORTb's coverage while maintaining the existing precision level, expand it to include Gram-positive bacteria and then carry out a comparative analysis of localization. RESULTS: An expanded database of proteins of known localization and new modules using frequent subsequence-based support vector machines was introduced into PSORTb v.2.0. The program attains a precision of 96% for Gram-positive and Gram-negative bacteria and predictive coverage comparable to other tools for whole proteome analysis. We show that the proportion of proteins at each localization is remarkably consistent across species, even in species with varying proteome size. AVAILABILITY: Web-based version: http://www.psort.org/psortb. Standalone version: Available through the website under GNU General Public License. CONTACT: psort-mail@sfu.ca, brinkman@sfu.ca SUPPLEMENTARY INFORMATION: http://www.psort.org/psortb/supplementaryinfo.html.     

Synthesis and inhibitory activity of benzoic acid and pyridine derivatives on influenza neuraminidase

Based upon the activity and X-ray crystallographic studies of tri-substituted benzene derivatives containing carboxylic acid, acetamido and guanidine groups, we investigated the effect of the fourth substituent to fulfill the fourth pocket of neuraminidase enzyme. The groups selected as fourth substituents were hydroxymethyl, hydroxyethyl, oxime and amino. These tetra-substituted benzene derivatives were synthesized and evaluated for neuraminidase inhibitory activity. All these compounds were found to have poorer IC(50) values than the tri-substituted compounds. Further, benzene ring was replaced by pyridine ring and di, tri and tetra-substituted pyridine derivatives were synthesized. The activity of the pyridine derivatives was comparable to benzene derivatives. The fourth substituent seems to disturb the binding of the other three substituents, so the activity is reduced as compared to tri-substituted benzene and pyridine derivatives.     

The role of biphasic shocks for transthoracic cardioversion of atrial fibrillation

The modern generation of transthoracic defibrillators now employ impedance compensated biphasic waveforms. These new devices are superior to those with monophasic waveforms and practice is currently switching to biphasic defibrillators for the treatment of both ventricular and atrial fibrillation. However, there is no universal guideline for the use of biphasic defibrillators in direct current cardioversion of atrial fibrillation. This article reviews the use of biphasic defibrillation waveforms for transthoracic cardioversion of atrial fibrillation.     

Mechanism of eosinophil induced signaling in cholinergic IMR-32 cells

Eosinophils interact with nerve cells, leading to changes in neurotransmitter release, altered nerve growth, and protection from cytokine-induced apoptosis. In part, these interactions occur as a result of activation of neural nuclear factor (NF)-kappaB, which is activated by adhesion of eosinophils to neural intercellular adhesion molecule-1 (ICAM-1). The mechanism and consequence of signaling after eosinophil adhesion to nerve cells were investigated. Eosinophil membranes, which contain eosinophil adhesion molecules but not other eosinophil products, were coincubated with IMR-32 cholinergic nerve cells. The studies showed that there were two mechanisms of activation of NF-kappaB, one of which was dependent on reactive oxygen species, since it was inhibited with diphenyleneiodonium. This occurred at least 30 min after coculture of eosinophils and nerves. An earlier phase of NF-kappaB activation occurred within 2 min of eosinophil adhesion and was mediated by tyrosine kinase-dependent phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Coimmunoprecipitation experiments showed that both extracellular signal-regulated kinase 1/2 and IRAK-1 were recruited to ICAM-1 rapidly after coculture with eosinophil membranes. This was accompanied by an induction of ICAM-1, which was mediated by an IRAK-1-dependent pathway. These data indicate that adhesion of eosinophils to IMR-32 nerves via ICAM-1 leads to important signaling events, mediated via IRAK-1, and these in turn lead to expression of adhesion molecules.