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Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

San Antonio, Texas, was one of the first metropolitan areas where 2009 pandemic influenza A (H1N1) virus (pH1N1) was detected. Identification of laboratory-confirmed pH1N1 in 2 students led to a preemptive 8-day closure of their high school. We assessed transmission of pH1N1 and changes in adoption of nonpharmaceutical interventions (NPIs) within households of students attending the affected school. Household secondary attack rates were 3.7% overall and 9.1% among those 0-4 years of age. Widespread adoption of NPIs was reported among household members. Respondents who viewed pH1N1 as very serious were more likely to adopt certain NPIs than were respondents who viewed pH1N1 as not very serious. NPIs may complement influenza vaccine prevention programs or be the only line of defense when pandemic vaccine is unavailable. The 2009 pandemic provided a unique opportunity to study NPIs, and these real-world experiences provide much-needed data to inform pandemic response policy.

Studies suggest that insulin-signaling molecules are present in the pancreatic islets. For this reason, the effects of insulin glulisine, insulin aspart and regular human insulin (RHI) on the function and molecular features of isolated human pancreatic islets were investigated.

Human pancreatic islets were prepared by collagenase digestion and density-gradient purification of pancreata from multiple organ donors. Islets were then cultured for 48 h in the presence of 5.5 (normal) or 22.2 (high) mmol/L of glucose with and without glulisine, aspart and RHI (10 or 100 nmol/L). Functional (glucose-stimulated insulin secretion) and molecular (quantitative RT-PCR and immunoblot) studies were performed at the end of the different incubation conditions.

Glucose-stimulated insulin secretion was blunted in islets cultured in 22.2 mmol/L of glucose, with no significant effects from the exogenous added insulins. In islets maintained at 5.5 mmol/L of glucose, insulin receptor (IR) expression was reduced by low RHI, while phosphatidylinositol-3 kinase p110-alpha (PI3K) was enhanced by both concentrations of glulisine and aspart, and by high RHI. In islets preexposed to high glucose, IR expression was increased by both concentrations of aspart and RHI, but not by glulisine. Glulisine at high concentration significantly (P<0.05) increased PI3K expression. Glulisine and RHI significantly increased IRS-2 phosphorylation compared with control and aspart (P<0.05).

Insulin analogues have differential effects on the expression of insulin-signaling molecules in human pancreatic islets that are also dependent on the degree of glucose exposure.

In the postprandial state, insulin regulates metabolic and cardiovascular responses. In insulin resistance, the insulin action is impaired at both levels. However, postprandial hemodynamic responses are poorly characterized in this setting.

We investigated fasting and postprandial cardiac and vascular hemodynamic responses in subjects with and without insulin resistance.

Sixty-six atherosclerosis-free, healthy volunteers were studied in a fasted state and ≤180 min after ingestion of a mixed meal. The insulin sensitivity index was determined by using a minimal model analysis; hemodynamic response was monitored by using continuous impedance cardiography that allowed a reliable beat-to-beat noninvasive evaluation of stroke volume, cardiac contractility, and several derived variables.

Subjects were divided into insulin-resistant (IR; n = 33) and insulin-sensitive (IS; n = 33) groups. After fasting, IR subjects had significantly higher values of systolic and diastolic blood pressures and the systemic vascular resistance index (SVRI) than did IS subjects. In the postprandial state, acute vasodilatation was comparable and synchronous (at 30 min) in IR and IS subjects (P = 0.209), but subsequent vascular tone recovery (30-180 min) was significantly impaired in IR subjects (P = 0.018), even after adjustment for age and sex (P = 0.031). Hemodynamic dysregulation was directly correlated with metabolic disturbances in the postprandial state. In basal and postprandial states, hemodynamic variables related to cardiac function were not significantly different in IR and IS subjects.

IR subjects had a worse fasting vascular performance than did IS subjects. In the postprandial phase, insulin resistance was associated with a shorter duration of vasodilatation in the absence of an altered cardiac performance. Peripheral hemodynamic alterations in fasting and postprandial states may have a negative effect on cardiovascular performance in IR patients.

Arg/Arg homozygotes for the Gly16Arg polymorphism in the β₂-adrenoreceptor gene (ADRB2) have a reduced response to short-acting β₂-agonists but no effect has been associated with long-acting β₂-agonists (LABAs). We selected 604 subjects with current asthma from the European Community Respiratory Health Study to evaluate whether asthma control and lung function decline were associated with Gly16Arg polymorphism, and to test whether LABA or inhaled corticosteroid (ICS) use modified these effects. There was an increased risk of noncontrolled asthma (OR 1.33, 95% CI 1.01-1.75; p = 0.046) for each Arg allele. Among nonusers of ICS, the odds ratio of noncontrolled asthma among Arg/Arg versus Gly/Gly subjects was 2.73 (95% CI 1.28-5.82; p = 0.009). No increased risk of noncontrolled asthma associated with the Arg allele was observed among ICS and/or LABA users. For each Arg allele, a mean ± se decrease in decline in forced expiratory volume in 1 s of 7.7 ± 2.5 mL·yr⁻¹ was found (p-value for trend 0.003), irrespective of ICS or LABA use. Arg/Arg subjects had an increased risk of bronchial hyperresponsiveness (BHR) versus Gly/Gly subjects, with an odds ratio of 2.51 (95% CI 1.12-5.63; p = 0.025) if they did not use ICS. The Arg allele was associated with poorer asthma control, a steeper lung function decline and BHR. Absence of genotypic effects on asthma control among ICS users may be due to reversed β₂-adrenoreceptor desensitisation.

Brazil initiated universal immunization of infants with the G1P[8] human rotavirus (RV) vaccine in March 2006. This study evaluated vaccine effectiveness (VE) against severe rotavirus gastroenteritis (RVGE) hospitalizations.

Matched case-control study conducted at 4 hospitals in Belém from May 2008 to May 2009. Cases were children hospitalized with RVGE age-eligible to have received 2 doses of the human RV vaccine (≥ 12 weeks of age and born after March 6, 2006). For each case, 1 neighborhood and 1 hospital control without gastroenteritis was selected, matching by birth date (± 8 and ± 6 weeks, respectively). Matched odds ratio of 2-dose RV vaccination in cases versus controls was used to estimate VE (1 - odds ratio × 100%).

Of 538 RVGE cases, 507 hospital controls and 346 neighborhood controls included, 54%, 61%, and 74% had received both RV vaccine doses. VE against RVGE hospitalization was 75.8% (95% confidence interval [CI]: 58.1-86.0) using neighborhood controls and 40.0% (95% CI: 14.2-58.1) using hospital controls. VE in children 3 to 11 months and ≥ 12 months of age was 95.7% (95% CI: 67.8-99.4) and 65.1% (95% CI: 37.2-80.6) using neighborhood controls, and 55.6% (95% CI: 12.3-77.5) and 32.1% (95% CI: -3.7-55.5) using hospital controls. G2P[4] accounted for 82.0% of RVGE hospitalizations. G2P[4]-specific VE was 75.4% (95% CI: 56.7-86.0) using neighborhood controls and 38.9% (95% CI: 11.1-58.0) using hospital controls.

Although fully heterotypic G2P[4] was the predominant RV strain, good VE was demonstrated. VE was highest in children aged 3 to 11 months. However, protection in children ≥ 12 months of age, important for optimal public health impact, was significantly sustained based on estimates obtained using neighborhood controls.

Post-resuscitation syndrome leads to death in approximately 2 out of every 3 successfully resuscitated victims, and myocardial microcirculatory dysfunction is a major component of this syndrome. The aim of this study was to determine if glucagon-like peptide-1 (GLP-1) improves post-resuscitation myocardial microcirculatory function.

Ventricular fibrillation (VF) was induced electrically in 20 anesthetized domestic swine (30-35 kg). Following 8 min of untreated VF, animals were resuscitated with aggressive advanced cardiac life support (ACLS). Animals were blindly randomized to receive a continuous infusion of either GLP-1 (10 pM/kg/min) or equal volume saline as placebo (PBO) for 4h, beginning 1 min after return of spontaneous circulation (ROSC). Left ventricular (LV) haemodynamics, LV ejection fraction, cardiac output, and coronary flow reserve (CFR) [using a standard technique of intracoronary Doppler flow measurements before and after intracoronary administration of 60 microg adenosine] were performed pre-arrest and at 1 and 4h post-resuscitation. In the present study, CFR is a measure of myocardial microcirculatory function since these swine had no obstructive coronary artery disease. Twenty-four hour post-resuscitation survival and neurological functional scores were also determined.

CFR was significantly increased in GLP-1-treated animals, 1h (1.79+/-0.13 in control animals vs. 2.05+/-0.12 in GLP-1-treated animals, P = <0.05) and 4h (1.82+/-0.16 in control animals vs. 2.31+/-0.13 in GLP-1-treated animals, P = <0.05) after ROSC. In addition, compared to PBO-treated animals, GLP-1 increased cardiac output 1h after ROSC (2.1+/-0.1 in control animals vs. 2.7+/-0.2 in GLP-1-treated animals, P = <0.05). There was no statistically significant difference in survival between GLP-1-treated (100%) and PBO-treated animals (78%).

In this swine model of prolonged VF followed by successful resuscitation, myocardial microcirculatory function was enhanced with administration of GLP-1. However, GLP-1 treatment was not associated with a clinically significant improvement in post-resuscitation myocardial function.

Oligomers incorporating the tetrapeptide MSH4, the minimum active sequence of melanocyte stimulating hormone, were synthesized by an A2 + B2 strategy involving microwave-assisted copper-catalyzed azide-alkyne cycloaddition. A2 contained an MSH4 core while B2 contained a (Pro-Gly)3 spacer. Soluble mixtures containing compounds with up to eight MSH4 units were obtained from oligomerizations at high monomer concentrations. The avidities of several oligomeric mixtures were evaluated by means of a competitive binding assay using HEK293 cells engineered to overexpress the melanocortin 4 receptor. When based on total MSH4 concentrations, avidities were only minimally enhanced compared with a monovalent control. The lack of variation in the effect of ligands on probe binding is consistent with high off rates for MSH4 in both monovalent and oligomeric constructs relative to that of the competing probe.

Vascular smooth muscle is characterized by a high rate of aerobic lactate production, which may be altered independently of oxidative phosphorylation. This finding suggested a cytoplasmic compartmentation of metabolism. Exogenous glucose was found to be the sole precursor of aerobic glycolysis under unstimulated conditions. Although tissue depolarization with high K+ resulted in a substantial reduction of endogenous glycogen, exogenous glucose remained the sole precursor of aerobic lactate production. These data showed unequivocally that carbohydrate metabolism is compartmentalized in vascular smooth muscle.

ATP utilization (delta approximately P) during an isometric contraction has been studied in terms of both measurements of oxygen consumption and lactate production as well as of the tissue nucleotide and metabolite levels. The contribution of breakdown of preformed ATP and phosphocreatine (PCr) pools to delta approximately P during contraction is minor compared to that made by metabolic synthesis of ATP. For tonic vascular smooth muscle (VSM), in fact, no change in ATP or PCr from resting levels can be measured. In contrast to amphibian skeletal muscle, a P:O of 3 can be demonstrated in VSM. In both tonic and phasic VASM, delta approximately P is biphasic with contraction duration, attaining a maximal value before that of isometric force and declining to a steady-state value approximately 60% of the maximal suprabasal rate during the maintenance of constant isometric force. The steady-state rate of ATP utilization per unit force maintained increases with extracellular Ca2+. Both the pre-steady-state temporal dependence and the steady-state dependence on Ca2+ are consistent with the hypothesis that myosin phosphorylation modulates the cross-bridge cycle rates. VSM metabolism, when viewed in terms of ATP synthesis, is primarily oxidative. However, even under fully oxygenated conditions, lactate is the major end product of glucose catabolism. Recent work has shown that aerobic lactate production is specifically coupled to Na-K transport in many, but not all, vascular tissues. Oxidative metabolism, on the other hand, is strongly related to active isometric force. The biochemical basis of this functional compartmentation was investigated at the level of substrate specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

In vascular smooth muscle (VSM), aerobic lactate production can account for as much as 30% of the basal rate of ATP production. Generally, glucose transport is thought to be the rate-limiting step for glycolysis in unstimulated VSM. In this work we provide evidence that the intracellular concentration of glucose is negligible in porcine carotid artery, indicating that glucose transport is rate limiting for its utilization. Since aerobic glycolysis appears to be coupled to active Na+-K+ transport in this tissue, we examined the effects of altering ion transport on glucose transport. Glucose uptake and 3-O-methyl-D-glucose transport were accelerated, though intracellular glucose remained negligible in artery rings that were incubated with 80 mM KCl, which is known to stimulate active Na+-K+ transport, as well as aerobic glycolysis and mechanical activity. On the other hand, inhibitors of active Na+-K+ transport (ouabain, Na+-free media), which also elicit mechanical activity, had little effect on sugar transport but significantly inhibited aerobic glycolysis and caused an intracellular accumulation of glucose. Our results indicate the following: 1) that glucose transport is regulated in VSM; 2) that the intracellular concentration of Ca2+ does not appear to regulate sugar transport, since changes in glucose and 3-O-methyl-D-glucose transport are not always seen in association with increased mechanical activity, and 3) that the decrease in aerobic glycolysis associated with the inhibition of active Na+-K+ transport is not due to a decrease in glucose transport but rather to an inhibition of glucose utilization.

The neuroradiologist and the clinician are partners in the search for pathology; the clinician must help the radiologist tailor the study by indicating where pathology is likely to be. The orbit, the sellar and parasellar regions, and new imaging modalities and interventional techniques are discussed in this light.

Early investigations into the nature of the coupling between energy transduction and metabolism in smooth muscle, particularly from the laboratories of Bülbring and Lundholm, suggested that specific metabolic pathways could independently supply energy for ion transport and actin-myosin interactions. Subsequent work has solidified the concept that oxidative phosphorylation is specifically coupled to tension generation and maintenance, whereas, aerobic glycolysis is not only a vital characteristic of smooth muscle metabolism, but also is likely to be independently coupled to Na-K transport at the plasmalemma. The independence of oxidative and glycolytic metabolism is reflected as a compartmentation of carbohydrate metabolism in the porcine carotid artery. The coupling of these independent metabolic pathways with specific energy utilizing processes, indicates a means by which energy production and transduction can be closely and efficiently regulated. The coupling of glycogenolysis to mitochondrial respiration may have evolved as a direct response to the energetic needs of VSM. That is, the large glycogenolytic response in the initial minutes of stimulation may be necessary to maximize the cellular production of ATP during the presteady state. Likewise, the coupling between aerobic glycolysis and Na-K transport indicates a sensitive and efficient means of coordinating energy metabolism with ion transport at the membrane level. Additionally, the regulation of substrate supply, i.e. glucose transport, also may be closely coordinated with changes in ion transport. One may speculate that alterations in the microenvironment of each compartment can independently regulate intermediary metabolism and therefore allow the cell to quickly and efficiently respond to localized stimuli. Thus, stimulation of Na-K transport could effectively regulate energy production at the membrane level without mobilizing or competing with the energy transduction of other cellular processes. This compartmentation of energy utilization may be highly advantageous, since oxidative metabolism is closely coordinated with mechanical activity and therefore regulation of blood flow. Future investigations will attempt to elucidate which intracellular signals which are responsible for the regulation of these functionally independent compartments of energy metabolism and transduction in VSM. In more general terms, our findings provide a basis from which future questions concerning the regulation of cellular metabolism must be directed. The cellular cytoplasm can no longer be envisioned as a homogeneous compartment, but rather a complex array of functional subcompartments which may be individual

Previous work has indicated that there are at least two functionally independent Embden-Meyerhof pathways within the vascular smooth muscle of porcine carotid artery. We tested this hypothesis by analyzing the isotopic equilibrium between medium glucose and intracellular glucose 6-phosphate under basal conditions and after 30 min of mechanical activation, during which time the rate of glycogenolysis has been found to be substantial. Under basal conditions, the specific activity of glucose 6-phosphate equilibrated to a level which was not in isotopic equilibrium with medium glucose suggesting that there is a significant pool of glucose 6-phosphate which is not readily accessible to medium glucose. After 15 min of mechanical activation, the specific activity of intracellular glucose 6-phosphate was found to decrease significantly from its apparent steady-state distribution, indicating that glycogen was likely to be a significant source for glucose 6-phosphate. Since the specific activity of lactate was unaltered from its equilibrium distribution under similar stimulus conditions, these findings substantiate the existence of at least two independent pools of glucose 6-phosphate.

No abstract given.

The energy metabolism of two continuous cell lines of renal origin, MDCK (Madin-Darby dog kidney) and A6 (toad kidney), was investigated by measuring the oxygen consumption (QO2) and lactate production (Jlac) by cells taken into suspension from monolayer cultures. Cells suspended from fully differentiated monolayers produce approximately 80% of their ATP requirements from oxidative metabolism. The interrelationship between ion transport and metabolism was determined by analyzing the ouabain sensitive components of intermediary metabolism under control conditions and after the stimulation of active Na-K transport with nystatin. In both cell lines, approximately 25% of the net rate of ATP production was inhibited by ouabain. Ouabain inhibited Jlac by 40% in MDCK and 45% in A6 cells, whereas QO2 was decreased by only 20% in both cell lines. In the presence of 0.05 mg nystatin/mg cell protein, ouabain sensitive Jlac increased by 75% in MDCK and was more than doubled in A6, whereas the ouabain-sensitive QO2 was not statistically different than control. This preferential stimulation of glycolysis with nystatin was not due to a limited capacity of mitochondrial oxidative phosphorylation since nystatin treatment of cells incubated without glucose (no glycolysis) significantly elevated the rate of QO2. These data demonstrate that aerobic glycolysis is more sensitive than is QO2 to changes in hydrolytic activity of the Na-K-adenosine triphosphatase (ATPase), in both cell lines.

In vascular smooth muscle, oxidative phosphorylation and glycolysis are independently regulated. Previous studies indicated that the independent regulation of these pathways was related to a compartmentation of carbohydrate metabolism. To further study carbohydrate metabolism, glucose transport and the incorporation of radiolabel from glucose into glycogen and lactate were measured after the oxidative and glycolytic pathways were independently altered. Ouabain stimulated mechanical activity, oxygen consumption, and glycogenolysis, whereas lactate production was decreased. Although glycogenolysis was substantial, glucose was the only substrate for lactate, indicating that intermediates derived from glycogen do not mix with those from glucose uptake. Thus glycogenolysis and glycolysis are carried out by independent enzymatic pathways. Insulin-stimulated lactate production and glucose transport without affecting the other parameters. Again, lactate was produced only from glucose. Phenytoin decreased isometric tension and oxygen consumption, whereas stimulating lactate production and glycogenolysis. Glycogen was the primary substrate for the lactate produced. Our findings indicate that the compartmentation of substrate utilization is ascribable to the coordination of glycogenolysis with increases in oxygen consumption and the coupling of glycolysis to the Na-K-adenosine triphosphatase. The coupling of independent energy providing pathways to specific endergonic processes indicates a mechanism by which cellular energetic efficiency may be optimized.

The relation between the activity of the Na+-K+-ATPase and the metabolic source of ATP was investigated in suspensions of MDCK cells. The pump activity of Na+-K+-ATPase was estimated from the initial rate of ouabain-sensitive K+ uptake into K+-depleted cells. Uptake was initiated by the reintroduction of K+ to the medium in which the cells were suspended. The metabolic source of ATP was varied by changing the substrates supplied to the suspension. Cells respiring on glutamine produced ATP from oxidative metabolism alone, whereas cells incubated with glucose and glutamine produced ATP via glycolysis and oxidative phosphorylation. Over a wide range of extracellular K+ concentrations, the initial rate of K+ uptake was faster in cells incubated with glucose and glutamine when compared with cells incubated with glutamine alone. Kinetic analysis together with ouabain-binding data demonstrated that this increase in K+ uptake was due to an increase in maximal velocity (Vmax) at a constant number of Na+-K+-ATPase transport sites. In addition, steady-state studies revealed that the addition of glucose to K+-depleted cells respiring on glutamine alone resulted in a net ouabain-sensitive influx of K+. These data demonstrate that in MDCK cells the maximal capacity for transport via the Na+-K+-ATPase is greater when ATP is produced from both glycolysis and oxidative phosphorylation than when ATP is produced from oxidative phosphorylation alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicotinamide adenine dinucleotide (NADH) plays a critical role in oxidative phosphorylation as the primary source of reducing equivalents to the respiratory chain. Using a modified fluorescence microscope, we have obtained spectra and images of the blue autofluorescence from single rat cardiac myocytes. The optical setup permitted rapid acquisition of fluorescence emission spectra (390-595 nm) or intensified digital video images of individual myocytes. The spectra showed a broad fluorescence centered at 447 +/- 0.2 nm, consistent with mitochondrial NADH. Addition of cyanide resulted in a 100 +/- 10% increase in fluorescence, while the uncoupler FCCP resulted in a 82 +/- 4% decrease. These two transitions were consistent with mitochondrial NADH and implied that the myocytes were 44 +/- 6% reduced under the resting control conditions. Intracellular fluorescent structures were observed that correlated with the distribution of a mitochondrial selective fluorescent probe (DASPMI), the mitochondrial distribution seen in published electron micrographs, and a metabolic digital subtraction image of the cyanide fluorescence transition. These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originates from mitochondrial NADH.

To study the regulation of glycogen utilization in vascular smooth muscle, we measured the content of glycogen and glucose 6-phosphate and the activity of the glycogen phosphorylase and glycogen debrancher enzymes in porcine carotid artery. During active contraction, the rates of glycogen phosphorylase and glycogenolysis were as high as expected. Despite this, glycogen content did not decrease to less than approximately 50% of control levels even after sustained contractions. The activity of glycogen debrancher enzyme was found to be limiting glycogen utilization at this point. Although glycogenolysis is closely coordinated with increases in oxidative metabolism concomitant with active contraction, the maximal level of tension obtained after stimulation was not substantially reduced under conditions where glycogen debrancher enzyme was limiting glycogen utilization. On the other hand, the rate of tension generation was increased in these tissues. Thus glycogen utilization is not necessary for maximal force generation per se, but may influence other muscle contractile properties. Finally, during steady-state tension maintenance, glycogen utilization is likely to be regulated by the intracellular concentrations of metabolic intermediates (glucose, glucose 6-phosphate), as it is in skeletal muscle.