AMPK and substrate availability regulate creatine transport in cultured cardiomyocytes

Profound alterations in myocellular creatine and phosphocreatine levels are observed during human heart failure. To maintain its intracellular creatine stores, cardiomyocytes depend upon a cell membrane creatine transporter whose regulation is not clearly understood. Creatine transport capacity in the intact heart is modulated by substrate availability, and it is reduced in the failing myocardium, likely adding to the energy imbalance that characterizes heart failure. AMPK, a key regulator of cellular energy homeostasis, acts by switching off energy-consuming pathways in favor of processes that generate energy. Our objective was to determine the effects of substrate availability and AMPK activation on creatine transport in cardiomyocytes. We studied creatine transport in rat neonatal cardiomyocytes and HL-1 cardiac cells expressing the human creatine transporter cultured in the presence of varying creatine concentrations and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Transport was enhanced in cardiomyocytes following incubation in creatine-depleted medium or AICAR. The changes in transport were due to alterations in V(max) that correlated with changes in total and cell surface creatine transporter protein content. Our results suggest a positive role for AMPK in creatine transport modulation for cardiomyocytes in culture.     

Gut Hormone Pharmacology of a Novel GPR119 Agonist (GSK1292263), Metformin,and Sitagliptin in Type 2 Diabetes Mellitus: Results from Two Randomized Studies

GPR119 receptor agonists improve glucose metabolism and alter gut hormone profiles in animal models and healthy subjects. We therefore investigated the pharmacology of GSK1292263 (GSK263), a selective GPR119 agonist, in two randomized, placebo-controlled studies that enrolled subjects with type 2 diabetes. Study 1 had drug-naive subjects or subjects who had stopped their diabetic medications, and Study 2 had subjects taking metformin. GSK263 was administered as single (25–800 mg; n = 45) or multiple doses (100–600 mg/day for 14 days; n = 96). Placebo and sitagliptin 100 mg/day were administered as comparators. In Study 1, sitagliptin was co-administered with GSK263 or placebo on Day 14 of dosing. Oral glucose and meal challenges were used to assess the effects on plasma glucose, insulin, C-peptide, glucagon, peptide tyrosine-tyrosine (PYY), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). After 13 days of dosing, GSK263 significantly increased plasma total PYY levels by ∼five-fold compared with placebo, reaching peak concentrations of ∼50 pM after each of the three standardized meals with the 300 mg BID dose. Co-dosing of GSK263 and metformin augmented peak concentrations to ∼100 pM at lunchtime. GSK263 had no effect on active or total GLP-1 or GIP, but co-dosing with metformin increased post-prandial total GLP-1, with little effect on active GLP-1. Sitagliptin increased active GLP-1, but caused a profound suppression of total PYY, GLP-1, and GIP when dosed alone or with GSK263. This suppression of peptides was reduced when sitagliptin was co-dosed with metformin. GSK263 had no significant effect on circulating glucose, insulin, C-peptide or glucagon levels. We conclude that GSK263 did not improve glucose control in type 2 diabetics, but it had profound effects on circulating PYY. The gut hormone effects of this GPR119 agonist were modulated when co-dosed with metformin and sitagliptin. Metformin may modulate negative feedback loops controlling the secretion of enteroendocrine peptides.

Trial Registration:

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01119846","term_id":"NCT01119846"}}NCT01119846 Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01128621","term_id":"NCT01128621"}}NCT01128621     

Thermoregulation of weaned northern elephant seal (Mirounga angustirostris) pups in air and water

Fasting weaned northern elephant seal pups (Mirounga angustirostris) experience diverse environmental conditions on land and in water on a daily basis. Each environment undoubtedly induces distinct energetic costs that may vary for pups of differing body condition. To determine the energetic costs associated with different environmental conditions and whether costs vary between individuals, body mass, surface area, volume, body composition, resting metabolic rate, and core body temperature were determined for 17 weaned northern elephant seal pups from A?o Nuevo, California. Metabolic rate and body temperature were measured for pups resting in air (20.9 degrees +/-0.8 degrees C), cold water (3.8 degrees+/-0.4 degrees ;C), and warm water (14.5 degrees+/-0.2 degrees C). Resting metabolic rate increased with body mass (range: 62.0-108.0 kg) and was also correlated with lean mass and lipid mass. Metabolic rates ranged from 293.6 to 512.7 mL O(2) min(-1) and were lowest for pups resting in cold water. Thermal conductance, calculated from metabolic rate and core body temperature, ranged from 3.1 to 15.2 W degrees C(-1), with the highest values in air and the lowest values in cold water. Metabolic responses to the three environmental conditions did not differ with individual variation in body condition. For all elephant seal pups, a consequence of high lipid content is that thermoregulatory costs are greatest on land and lowest in cold water, a pattern that contrasts markedly with terrestrial mammals.     

Broad Neutralization of Human Immunodeficiency Virus Type 1 (HIV-1) Elicited from Human Rhinoviruses That Display the HIV-1 gp41 ELDKWA Epitope

In efforts to develop AIDS vaccine components, we generated combinatorial libraries of recombinant human rhinoviruses that display the well-conserved ELDKWA epitope of the membrane-proximal external region of human immunodeficiency virus type 1 (HIV-1) gp41. The broadly neutralizing human monoclonal antibody 2F5 was used to select for viruses whose ELDKWA conformations resemble those of HIV. Immunization of guinea pigs with different chimeras, some boosted with ELDKWA-based peptides, elicited antibodies capable of neutralizing HIV-1 pseudoviruses of diverse subtypes and coreceptor usages. These recombinant immunogens are the first reported that elicit broad, albeit modest, neutralization of HIV-1 using an ELDKWA-based epitope and are among the few reported that elicit broad neutralization directed against any recombinant HIV epitope, providing a critical advance in developing effective AIDS vaccine components.The development of an AIDS vaccine is an ongoing and urgent challenge. One of the major hurdles is that the specific correlates of protection against human immunodeficiency virus (HIV) are still largely unknown. Nonetheless, most agree that the full complement of cellular and humoral components of the immune system will be needed to combat this virus. This is especially true given that the virus resides permanently in its host, infects the very cells needed to direct effective immune responses, and evades the immune system, either by changing in appearance or hiding in subcellular compartments.A broadly reactive neutralizing antibody response is likely to be critical as a first line of defense upon initial HIV exposure by aiding in the clearance of cell-free virions, targeting infected cells for destruction, and preventing viral spread through cell-to-cell transmission. The presence of inhibitory antibodies in highly exposed persistently seronegative individuals testifies to the importance of the humoral response (9, 37). Additionally, broadly neutralizing serum has been associated with healthier prognoses for infected individuals (27, 65) and may be vital for protecting offspring from their infected mothers (7, 79) and preventing superinfection by heterologous HIV strains (23, 84). Even if complete protection cannot be achieved by vaccine-derived antibodies, an early, well-poised and effective neutralizing antibody repertoire may be able to lower the set point of the viral load following the initial burst of viremia, an outcome that has been reported to translate into improved disease outcomes and reduced transmission of HIV (66, 74). Further benefits of neutralizing antibodies have been seen with passive immunization studies in macaques, in which administration of broadly neutralizing monoclonal antibodies (MAbs) has demonstrated that it is possible to provide protection from—and even sterilizing immunity against—HIV infection (5, 51, 66). There is also evidence that such antibodies may provide therapeutic benefits for chronically infected individuals, analogous to benefits realized with anti-HIV drug treatment regimens (87).Despite the promising potential of broadly neutralizing MAbs, designing immunogens that can elicit such cross-reactive neutralizing responses against HIV has been a surprisingly difficult task. Since the majority of the host''s B-cell response is directed against the envelope (Env) glycoproteins, gp120 and gp41, vaccine efforts have concentrated on these proteins and derivatives thereof in approaches ranging from the use of Env-based peptide cocktails to recombinant proteins and DNAs made with varied or consensus sequences and diverse, heterologous prime/protein boost regimens (reviewed in references 36, 58, and 70). These iterative studies have shown notable improvements in the potency and breadth of neutralizing responses induced. However, concerns exist regarding immunogens containing extraneous epitopes, as is the case with intact subunits of Env, and the nature of the immune responses they may elicit. A polyclonal burst of antibodies against a multitude of nonfunctional epitopes may include a predominance of antibodies that are (i) low affinity and/or nonfunctional (reviewed in reference 72); (ii) isolate specific (25); (iii) able to interfere with the neutralizing capabilities of otherwise-effective antibodies (via steric hindrance or by inducing various forms of B-cell pathology) (67); or (iv) directed against irrelevant epitopes instead of more conserved (and sometimes concealed) epitopes that might be able to elicit more potent and cross-reactive neutralizing responses (28, 71, 91).We have developed a system that can be used to present essentially any chosen epitope in a stable, well-exposed manner on the surface of the cold-causing human rhinovirus (HRV). HRV is itself a powerful immunogen and is able to elicit T-cell as well as serum and mucosal B-cell responses (reviewed by Couch [22]) and has minimal immunologic similarity to HIV (data not shown). Chimeric viruses displaying optimal epitopes should be able to serve as valuable components in an effective vaccine cocktail or as part of a heterologous prime/boost protocol. We have shown previously that HRV chimeric viruses displaying HIV-1 gp120 V3 loop sequences are able to elicit neutralizing responses against HIV-1 (75, 82, 83).In this study, we focused our attention on presenting part of the membrane-proximal external region (MPER) of the transmembrane glycoprotein gp41, a region of approximately 30 amino acids adjacent to the transmembrane domain (reviewed in references 59 and 97). The MPER plays an important role in the process of HIV fusion to the host cell membrane (60, 78). This region is also involved in binding to galactosylceramide, an important component of cell membranes, thus permitting CD4-independent transcytosis of the virus across epithelial cells at mucosal surfaces (1, 2). These functions likely explain this region''s sequence conservation and the efficacy of antibodies directed against the MPER (97), particularly given that an estimated 80% of HIV-1 infections are sexually transmitted at mucosal membranes. In fact, potent responses against the MPER are associated with stronger and broader neutralizing capabilities in infected individuals (68). A conserved, contiguous sequence of the MPER, the ELDKWA epitope (HIV-1 HxB2 gp41 residues 662 to 668), is recognized by the particularly broadly neutralizing human MAb 2F5 (11, 62, 85) and is highly resistant to escape mutation in the presence of 2F5 (49). 2F5 was also used in the MAb cocktails reported to confer passive, protective immunity in macaques (5, 51). In addition, infected individuals producing neutralizing antibodies directed against the ELDKWA epitope have been seen to exhibit better health (16, 29), including persistent seronegativity (8), and reduced transmission of HIV to offspring (89). While none of the vaccine-induced immune responses generated against this region has been effective thus far (19, 24, 26, 33, 35, 38, 40, 42, 44-48, 50, 53, 54, 56, 57, 61, 63, 69, 93, 96) (see Table S1 in the supplemental material), more appropriate presentations of MPER epitopes should produce valuable immunogens that can contribute to a successful vaccine.In this study, we have grafted the ELDKWA epitope onto a surface loop of HRV connected via linkers of variable lengths and sequences and selected for viruses well recognized and neutralized by MAb 2F5. In so doing, we have been able to create immunogens capable of eliciting antibodies whose activities mimic some of those of 2F5. The combinatorial libraries produced were designed to encode a large set of possible sequences and, hence, structures from which we could search for valuable conformations. This work illustrates that HRV chimeras have the potential to present selected HIV epitopes in a focused and immunogenic manner.     

An E. coli expression system optimized for DELLA proteins

The DELLA proteins are involved in regulation of plant growth in response to phytohormonal signals such as GA, ethylene, and auxin. They have become one of most challenging and active area of research due to their fundamental roles in plant biology. Here, we describe the first successful expression of the N-terminal domains of DELLA proteins of Arabidopsis thaliana and Malus domestica in Escherichia coli system which will be used to produce monoclonal antibodies for profiling protein micro-arrays. Optimizations of the cloning, expression, and purification using specific tags have been discussed.     

Reliability of air displacement plethysmography

The purpose of this study was to examine the reliability of an air displacement plethysmography device (BOD POD) over trials performed on 3 different days. Subjects consisted of 24 healthy adults (8 men, 16 women), ages 18-38 years, with body weights 46.8-93.6 kg, body mass indexes of 19.1-30.1 kg x m(-2), and percentage body fats (BF) of 7.9-43.1%. Two estimates of BF were performed on 3 days. Paired t-tests revealed no significant within-day differences in body volume (BV), thoracic gas volume (V(TG)), body density (BD), and BF. Correlations between the two V(TG) measures on a day were r = 0.86 for day 1, r = 0.93 for day 2, and r = 0.96 for day 3. BF estimates within a day had high correlations of r = 0.98. Significant differences were found between days for measures of BV, V(TG), BD, and BF. These results indicate a high reliability for within-day estimates of BF and significant differences in between-day estimates of BF using air displacement plethysmography. Reliability of BF may be increased by requiring subjects to practice the procedure for V(TG) measurement.     

Evolution of amino acid frequencies in proteins over deep time: inferred order of introduction of amino acids into the genetic code

To understand more fully how amino acid composition of proteins has changed over the course of evolution, a method has been developed for estimating the composition of proteins in an ancestral genome. Estimates are based upon the composition of conserved residues in descendant sequences and empirical knowledge of the relative probability of conservation of various amino acids. Simulations are used to model and correct for errors in the estimates. The method was used to infer the amino acid composition of a large protein set in the Last Universal Ancestor (LUA) of all extant species. Relative to the modern protein set, LUA proteins were found to be generally richer in those amino acids that are believed to have been most abundant in the prebiotic environment and poorer in those amino acids that are believed to have been unavailable or scarce. It is proposed that the inferred amino acid composition of proteins in the LUA probably reflects historical events in the establishment of the genetic code.     

What does it take to be a plant pathogen: genomic insights from <Emphasis Type="Italic">Streptomyces</Emphasis> species

Plant pathogenicity is rare in the genus Streptomyces, with only a dozen or so species possessing this trait out of the more than 900 species described. Nevertheless, such species have had a significant impact on agricultural economies throughout the world due to their ability to cause important crop diseases such as potato common scab, which is characterized by lesions that form on the potato tuber surface. All pathogenic species that cause common scab produce a family of phytotoxins called the thaxtomins, which function as cellulose synthesis inhibitors. In addition, the nec1 and tomA genes are conserved in several pathogenic streptomycetes, the former of which is predicted to function in the suppression of plant defense responses. Streptomyces scabies is the oldest plant pathogen described and has a world-wide distribution, whereas species such as S. turgidiscabies and S. acidiscabies are believed to be newly emergent pathogens found in more limited geographical locations. The genome sequence of S. scabies 87-22 was recently completed, and comparative genomic analyses with other sequenced microbial pathogens have revealed the presence of additional genes that may play a role in plant pathogenicity, an idea that is supported by functional analysis of one such putative virulence locus. In addition, the availability of multiple genome sequences for both pathogenic and nonpathogenic streptomycetes has provided an opportunity for comparative genomic analyses to identify the Streptomyces pathogenome. Such genomic analyses will contribute to the fundamental understanding of the mechanisms and evolution of plant pathogenicity and plant-microbe biology within this genus.