The Melibiose Transporter of Escherichia coli: CRITICAL CONTRIBUTION OF LYS-377 TO THE STRUCTURAL ORGANIZATION OF THE INTERACTING SUBSTRATE BINDING SITES*

We examine the role of Lys-377, the only charged residue in helix XI, on the functional mechanism of the Na⁺-sugar melibiose symporter from Escherichia coli. Intrinsic fluorescence, FRET, and Fourier transform infrared difference spectroscopy reveal that replacement of Lys-377 with either Cys, Val, Arg, or Asp disables both Na⁺ and melibiose binding. On the other hand, molecular dynamics simulations extending up to 200–330 ns reveal that Lys-377 (helix XI) interacts with the anionic side chains of two of the three putative ligands for cation binding (Asp-55 and Asp-59 in helix II). When Asp-59 is protonated during the simulations, Lys-377 preferentially interacts with Asp-55. Interestingly, when a Na⁺ ion is positioned in the Asp-55-Asp-59 environment, Asp-124 in helix IV (a residue essential for melibiose binding) reorients and approximates the Asp-55-Asp-59 pair, and all three acidic side chains act as Na⁺ ligands. Under these conditions, the side chain of Lys-377 interacts with the carboxylic moiety of these three Asp residues. These data highlight the crucial role of the Lys-377 residue in the spatial organization of the Na⁺ binding site. Finally, the analysis of the second-site revertants of K377C reveals that mutation of Ile-22 (in helix I) preserves Na⁺ binding, whereas that of melibiose is largely abolished according to spectroscopic measurements. This amino acid is located in the border of the sugar-binding site and might participate in sugar binding through apolar interactions.     

Nicotinic acids: liver-targeted SCD inhibitors with preclinical anti-diabetic efficacy

An in vitro screening protocol was used to transform a systemically-distributed SCD inhibitor into a liver-targeted compound. Incorporation of a key nicotinic acid moiety enables molecular recognition by OATP transporters, as demonstrated by uptake studies in transfected cell lines, and likely serves as a critical component of the observed liver-targeted tissue distribution profile. Preclinical anti-diabetic oGTT efficacy is demonstrated with nicotinic acid-based, liver-targeting SCD inhibitor 10, and studies with a close-structural analog devoid of SCD1 activity, suggest this efficacy is a result of on-target activity.     

Azaindoles as potent CRTH2 receptor antagonists

A new class of 7-azaindole analogs of MK-7246 as potent and selective CRTH2 antagonists is reported. The SAR leading to the identification of the optimal azaindole regioisomer as well as the pharmacokinetics and off-target activities of the most potent antagonists are disclosed.     

New indole amide derivatives as potent CRTH2 receptor antagonists

A new series of indole amide acting as hCRTH2 receptor ligands had been explored and are described herein. Several amide derivatives displaying low nanomolar activity in hCRTH2 binding and whole blood assays were identified. They were found to behave as a full antagonists, exhibiting good selectivity over related prostaglandin receptors. Also, prototypical compounds in this novel series which displayed acceptable CYP profiles and were orally bioavailable in rats were identified.     

R5-SHIV induces multiple defects in T cell function during early infection of rhesus macaques including accumulation of T reg cells in lymph nodes

Background

HIV-1 is a pathogen that T cell responses fail to control. HIV-1gp120 is the surface viral envelope glycoprotein that interacts with CD4 T cells and mediates entry. HIV-1gp120 has been implicated in immune dysregulatory functions that may limit anti-HIV antigen-specific T cell responses. We hypothesized that in the context of early SHIV infection, immune dysregulation of antigen-specific T-effector cell and regulatory functions would be detectable and that these would be associated or correlated with measurable concentrations of HIV-1gp120 in lymphoid tissues.

Methods

Rhesus macaques were intravaginally inoculated with a Clade C CCR5-tropic simian-human immunodeficiency virus, SHIV-1157ipd3N4. HIV-1gp120 levels, antigen-specificity, levels of apoptosis/anergy and frequency and function of Tregs were examined in lymph node and blood derived T cells at 5 and 12 weeks post inoculation.

Results/Conclusions

We observed reduced responses to Gag in CD4 and gp120 in CD8 lymph node-derived T cells compared to the peripheral blood at 5 weeks post-inoculation. Reduced antigen-specific responses were associated with higher levels of PD-1 on lymph node-derived CD4 T cells as compared to peripheral blood and uninfected lymph node-derived CD4 T cells. Lymph nodes contained increased numbers of Tregs as compared to peripheral blood, which positively correlated with gp120 levels; T regulatory cell depletion restored CD8 T cell responses to Gag but not to gp120. HIV gp120 was also able to induce T regulatory cell chemotaxis in a dose-dependent, CCR5-mediated manner. These studies contribute to our broader understanding of the ways in which HIV-1 dysregulates T cell function and localization during early infection.     

Brain, spine, and muscle cytochrome Cu-A redox patterns of change during hypothermic circulatory arrest in swine

Past near infrared spectroscopy (NIRS) studies have reported different changes in cytochrome C oxidase (Cyt) redox status during similar interventions that cause tissue ischaemia. We investigated whether there were distinctive differences when NIRS signals were obtained simultaneously from different tissues during total circulatory arrest. Forty-two healthy 10 kg commercial swine (Sus scrofa) on cardiopulmonary bypass, each underwent 2 to 8 sequential periods of hypothermic circulatory arrest for 7.5 min. Prior to each arrest, key physiologic variables were adjusted to 1 of 81 combinations of high, normal, or low levels of core temperature, hematocrit, pH, and serum glucose. Each combination was repeated at least twice. Simultaneous NIRS monitoring yielded 202 brain, 191 spine, and 199 muscle Cyt data sets, which were then classified into 13 distinctive patterns of change. The data sets always differed between tissues in the same arrest trial and subject. Typically, brain Cyt rapidly became more reduced at the start of arrest and changed little thereafter, muscle Cyt behaved comparably to brain Cyt but continued to become reduced throughout the arrest, and spine Cyt either did not change status or gradually became more reduced over the course of arrest. The spine pattern's mean rate of change was 12 times slower than those of the brain or muscle. The Cyt patterns of change were classified into 13 groups which were significantly related to core temperature in the brain and spine, and hematocrit in muscle. The respiratory response in mitochondria during systemic circulatory arrest differs between brain, spine and muscle tissues in the same subject.     

Testosterone-Fatty Acid esterification: a unique target for the endocrine toxicity of tributyltin to gastropods

Over the past thirty years, a global occurrence of sexual aberrationhas occurred whereby females among populations of prosobranchsnails exhibit male sex characteristics. This condition, calledimposex, has been causally associated with exposure to the biocidetributyltin. Tributyltin-exposed, imposex snails typically haveelevated levels of testosterone which have led to the postulatethat this endocrine dysfunction is responsible for imposex.This overview describes recent evidence that supports this postulate.Gastropods maintain circulating testosterone levels and administrationof testosterone to females or castrates stimulates male sexdifferentiation in several snail species. Studies in the mudsnail (Ilyanassa obsoleta) have shown that gastropods utilizea unique strategy for regulating free testosterone levels. Excesstestosterone is converted to fatty acid esters by the actionof a testosterone-inducible, high capacity/low affinity enzyme,acyl-CoA:testosterone acyl transferase, and stored within theorganisms. Free testosterone levels are regulated during thereproductive cycle apparently due to changes in esterification/desterificationsuggesting that testosterone functions in the reproductive cycleof the organisms. Testosterone esterification provides a uniquetarget in the testosterone regulatory machinery of snails thatis altered by tributyltin. Indeed, imposex and free testosteronelevels were elevated in field collected snails containing hightin levels, while testosterone-fatty acid ester pools were reducedin these organisms. These observations indicate that tributyltinelevates free testosterone by reducing the retention of testosteroneas fatty acid-esters. This endocrine effect of tributyltin maybe responsible for imposex.     

Evaluation of medetomidine/ketamine for short-term immobilization of variable flying foxes (Pteropus hypomelanus)

Four medetomidine/ketamine (M/K) doses (30 microg/kg/3 mg/kg; 40/4; 50/5; 60/6), administered by intramuscular injection, were evaluated for short-term immobilization of adult male variable flying foxes (Pteropus hypomelanus). The highest dose (60 microg/kg/6 mg/kg) produced a significantly faster induction (31 +/- 46 sec) than the lowest dose (30/3) (125 +/- 62 sec). The highest dose levels (50/5, 60/6) produced significantly longer immobilization times (52.5 +/- 25.7 min and 60.6 +/- 20.8 min, respectively) than did the lower doses (30/3, 40/4) (18.8 +/- 8.7 min and 31.0 +/- 14.3 min, respectively). The dose at which 50% of the bats were immobilized for > or = 30 min (ED(50)) was approximately 40 microg/kg/4 mg/kg. This dose produced a mean immobilization time of 31 +/- 14 min, bradypnea and bradycardia. In conclusion, a M/K dose of 50 microg/kg/5 mg/kg is recommended for greater than 30 min of relaxed immobilization in free-living variable flying foxes and is sufficient for safe collection of samples.