Specificity of RSG-1.2 peptide binding to RRE-IIB RNA element of HIV-1 over Rev peptide is mainly enthalpic in origin

Rev is an essential HIV-1 regulatory protein which binds to the Rev responsive element (RRE) present within the env gene of HIV-1 RNA genome. This binding facilitates the transport of the RNA to the cytoplasm, which in turn triggers the switch between viral latency and active viral replication. Essential components of this complex have been localized to a minimal arginine rich Rev peptide and stem IIB region of RRE. A synthetic peptide known as RSG-1.2 binds with high binding affinity and specificity to the RRE-IIB than the Rev peptide, however the thermodynamic basis of this specificity has not yet been addressed. The present study aims to probe the thermodynamic origin of this specificity of RSG-1.2 over Rev Peptide for RRE-IIB. The temperature dependent melting studies show that RSG-1.2 binding stabilizes the RRE structure significantly (ΔT _m = 4.3°C), in contrast to Rev binding. Interestingly the thermodynamic signatures of the binding have also been found to be different for both the peptides. At pH 7.5, RSG-1.2 binds RRE-IIB with a K_a = 16.2±0.6×10⁷ M⁻¹ where enthalpic change ΔH = −13.9±0.1 kcal/mol is the main driving force with limited unfavorable contribution from entropic change TΔS = −2.8±0.1 kcal/mol. A large part of ΔH may be due to specific stacking between U72 and Arg15. In contrast binding of Rev (K_a = 3.1±0.4×10⁷ M⁻¹) is driven mainly by entropy (ΔH = 0 kcal/mol and TΔS = 10.2±0.2 kcal/mol) which arises from major conformational changes in the RNA upon binding.     

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.     

Recruitment Kinetics of DNA Repair Proteins Mdc1 and Rad52 but Not 53BP1 Depend on Damage Complexity

The recruitment kinetics of double-strand break (DSB) signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/µm) to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET  = 2.6 keV/µm) to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T₀ after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ₁. Mdc1 accumulates faster (T₀ = 17±2 s, τ₁ = 98±11 s) than 53BP1 (T₀ = 77±7 s, τ₁ = 310±60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T₀ = 73±16 s, τ₁ = 1050±270 s) after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ₁ of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies.     

Genetic Modulation of Lipid Profiles following Lifestyle Modification or Metformin Treatment: The Diabetes Prevention Program

Weight-loss interventions generally improve lipid profiles and reduce cardiovascular disease risk, but effects are variable and may depend on genetic factors. We performed a genetic association analysis of data from 2,993 participants in the Diabetes Prevention Program to test the hypotheses that a genetic risk score (GRS) based on deleterious alleles at 32 lipid-associated single-nucleotide polymorphisms modifies the effects of lifestyle and/or metformin interventions on lipid levels and nuclear magnetic resonance (NMR) lipoprotein subfraction size and number. Twenty-three loci previously associated with fasting LDL-C, HDL-C, or triglycerides replicated (P = 0.04–1×10⁻¹⁷). Except for total HDL particles (r = −0.03, P = 0.26), all components of the lipid profile correlated with the GRS (partial |r| = 0.07–0.17, P = 5×10⁻⁵–1×10⁻¹⁹). The GRS was associated with higher baseline-adjusted 1-year LDL cholesterol levels (β = +0.87, SEE±0.22 mg/dl/allele, P = 8×10⁻⁵, P _interaction = 0.02) in the lifestyle intervention group, but not in the placebo (β = +0.20, SEE±0.22 mg/dl/allele, P = 0.35) or metformin (β = −0.03, SEE±0.22 mg/dl/allele, P = 0.90; P _interaction = 0.64) groups. Similarly, a higher GRS predicted a greater number of baseline-adjusted small LDL particles at 1 year in the lifestyle intervention arm (β = +0.30, SEE±0.012 ln nmol/L/allele, P = 0.01, P _interaction = 0.01) but not in the placebo (β = −0.002, SEE±0.008 ln nmol/L/allele, P = 0.74) or metformin (β = +0.013, SEE±0.008 nmol/L/allele, P = 0.12; P _interaction = 0.24) groups. Our findings suggest that a high genetic burden confers an adverse lipid profile and predicts attenuated response in LDL-C levels and small LDL particle number to dietary and physical activity interventions aimed at weight loss.     

The slow-releasing hydrogen sulfide donor, GYY4137, exhibits novel anti-cancer effects in vitro and in vivo

The slow-releasing hydrogen sulfide (H₂S) donor, GYY4137, caused concentration-dependent killing of seven different human cancer cell lines (HeLa, HCT-116, Hep G2, HL-60, MCF-7, MV4-11 and U2OS) but did not affect survival of normal human lung fibroblasts (IMR90, WI-38) as determined by trypan blue exclusion. Sodium hydrosulfide (NaHS) was less potent and not active in all cell lines. A structural analogue of GYY4137 (ZYJ1122) lacking sulfur and thence not able to release H₂S was inactive. Similar results were obtained using a clonogenic assay. Incubation of GYY4137 (400 µM) in culture medium led to the generation of low (<20 µM) concentrations of H₂S sustained over 7 days. In contrast, incubation of NaHS (400 µM) in the same way led to much higher (up to 400 µM) concentrations of H₂S which persisted for only 1 hour. Mechanistic studies revealed that GYY4137 (400 µM) incubated for 5 days with MCF-7 but not IMR90 cells caused the generation of cleaved PARP and cleaved caspase 9, indicative of a pro-apoptotic effect. GYY4137 (but not ZYJ1122) also caused partial G₂/M arrest of these cells. Mice xenograft studies using HL-60 and MV4-11 cells showed that GYY4137 (100–300 mg/kg/day for 14 days) significantly reduced tumor growth. We conclude that GYY4137 exhibits anti-cancer activity by releasing H₂S over a period of days. We also propose that a combination of apoptosis and cell cycle arrest contributes to this effect and that H₂S donors should be investigated further as potential anti-cancer agents.     

A novel, "double-clamp" binding mode for human heme oxygenase-1 inhibition

The development of heme oxygenase (HO) inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308). Using a carbon monoxide (CO) formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC₅₀ = 0.27±0.07 µM) than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC₅₀ = 4.0±1.8 µM). The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This “double-clamp” binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors.     

The missing part of seed dispersal networks: structure and robustness of bat-fruit interactions

Mutualistic networks are crucial to the maintenance of ecosystem services. Unfortunately, what we know about seed dispersal networks is based only on bird-fruit interactions. Therefore, we aimed at filling part of this gap by investigating bat-fruit networks. It is known from population studies that: (i) some bat species depend more on fruits than others, and (ii) that some specialized frugivorous bats prefer particular plant genera. We tested whether those preferences affected the structure and robustness of the whole network and the functional roles of species. Nine bat-fruit datasets from the literature were analyzed and all networks showed lower complementary specialization (H₂'' = 0.37±0.10, mean ± SD) and similar nestedness (NODF = 0.56±0.12) than pollination networks. All networks were modular (M = 0.32±0.07), and had on average four cohesive subgroups (modules) of tightly connected bats and plants. The composition of those modules followed the genus-genus associations observed at population level (Artibeus-Ficus, Carollia-Piper, and Sturnira-Solanum), although a few of those plant genera were dispersed also by other bats. Bat-fruit networks showed high robustness to simulated cumulative removals of both bats (R = 0.55±0.10) and plants (R = 0.68±0.09). Primary frugivores interacted with a larger proportion of the plants available and also occupied more central positions; furthermore, their extinction caused larger changes in network structure. We conclude that bat-fruit networks are highly cohesive and robust mutualistic systems, in which redundancy is high within modules, although modules are complementary to each other. Dietary specialization seems to be an important structuring factor that affects the topology, the guild structure and functional roles in bat-fruit networks.     

S100B protein, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in human milk

Background

Human milk contains a wide variety of nutrients that contribute to the fulfillment of its functions, which include the regulation of newborn development. However, few studies have investigated the concentrations of S100B protein, brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF) in human milk. The associations of the concentrations of S100B protein, BDNF, and GDNF with maternal factors are not well explored.

Methodology/Principal Findings

To investigate the concentrations of S100B protein, BDNF, and GDNF in human milk and characterize the maternal factors associated with their levels in human milk, human milk samples were collected at days 3, 10, 30, and 90 after parturition. Levels of S100B protein, BDNF, and GDNF, and their mRNAs in the samples were detected. Then, these concentrations were compared with lactation and other maternal factors. S100B protein levels in human milk samples collected at 3, 10, 30, and 90 d after parturition were 1249.79±398.10, 1345.05±539.16, 1481.83±573.30, and 1414.39±621.31 ng/L, respectively. On the other hand, the BDNF concentrations in human milk samples were 10.99±4.55, 13.01±5.88, 13.35±6.43, and 2.83±5.47 µg/L, while those of GDNF were 10.90±1.65, 11.38±1., 11.29±3.10, and 11.40±2.21 g/L for the same time periods. Maternal post-pregnancy body mass index was positively associated with S100B levels in human milk (r = 0.335, P = 0.030<0.05). In addition, there was a significant correlation between the levels of S100B protein and BDNF (z = 2.09, P = 0.037<0.05). Delivery modes were negatively associated with the concentration of GDNF in human milk.

Conclusions

S100B protein, BDNF, and GDNF are present in all samples of human milk, and they may be responsible for the long term effects of breast feeding.     

Hydrogen Sulfide Regulates Cardiovascular Function by Influencing the Excitability of Subfornical Organ Neurons

Hydrogen sulfide (H₂S), a gasotransmitter endogenously found in the central nervous system, has recently been suggested to act as a signalling molecule in the brain having beneficial effects on cardiovascular function. This study was thus undertaken to investigate the effect of NaHS (an H₂S donor) in the subfornical organ (SFO), a central nervous system site important to blood pressure regulation. We used male Sprague-Dawley rats for both in vivo and in vitro experiments. We first used RT-PCR to confirm our previous microarray analyses showing that mRNAs for the enzymes required to produce H₂S are expressed in the SFO. We then used microinjection techniques to investigate the physiological effects of NaHS in SFO, and found that NaHS microinjection (5 nmol) significantly increased blood pressure (mean AUC = 853.5±105.7 mmHg*s, n = 5). Further, we used patch-clamp electrophysiology and found that 97.8% (88 of 90) of neurons depolarized in response to NaHS. This response was found to be concentration dependent with an EC₅₀ of 35.6 µM. Coupled with the depolarized membrane potential, we observed an overall increase in neuronal excitability using an analysis of rheobase and action potential firing patterns. This study has provided the first evidence of NaHS and thus H₂S actions and their cellular correlates in SFO, implicating this brain area as a site where H₂S may act to control blood pressure.     

Hypertension is associated with marked alterations in sphingolipid biology: a potential role for ceramide

Background

Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function.

Methods and Findings

In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p<0.05). Imaging mass spectrometry and immunohistochemistry indicated that these contractions were most likely mediated by ceramide and dependent on iPLA₂, cyclooxygenase-1 and thromboxane synthase. Expression levels of these enzymes were higher in SHR vessels. In concurrence, infusion of dimethylsphingosine caused a marked rise in blood pressure in anesthetized SHR (42±4%; n = 7), but not in WKY (−12±10%; n = 6). Lipidomics analysis by mass spectrometry, revealed elevated levels of ceramide in arterial tissue of SHR compared to WKY (691±42 vs. 419±27 pmol, n = 3–5 respectively, p<0.05). These pronounced alterations in SHR sphingolipid biology are also reflected in increased plasma ceramide levels (513±19 pmol WKY vs. 645±25 pmol SHR, n = 6–12, p<0.05). Interestingly, we observed similar increases in ceramide levels (correlating with hypertension grade) in plasma from humans with essential hypertension (185±8 pmol vs. 252±23 pmol; n = 18 normotensive vs. n = 19 hypertensive patients, p<0.05).

Conclusions

Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone.     

Probing the Binding Sites of Antibiotic Drugs Doxorubicin and N-(trifluoroacetyl) Doxorubicin with Human and Bovine Serum Albumins

We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with bovine serum albumin (BSA) and human serum albumins (HSA) at physiological conditions, using constant protein concentration and various drug contents. FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding sites, the binding constant and the effect of drug complexation on BSA and HSA stability and conformations. Structural analysis showed that doxorubicin and N-(trifluoroacetyl) doxorubicin bind strongly to BSA and HSA via hydrophilic and hydrophobic contacts with overall binding constants of K _DOX-BSA = 7.8 (±0.7)×10³ M⁻¹, K _FDOX-BSA = 4.8 (±0.5)×10³ M⁻¹ and K _DOX-HSA = 1.1 (±0.3)×10⁴ M⁻¹, K _FDOX-HSA = 8.3 (±0.6)×10³ M⁻¹. The number of bound drug molecules per protein is 1.5 (DOX-BSA), 1.3 (FDOX-BSA) 1.5 (DOX-HSA), 0.9 (FDOX-HSA) in these drug-protein complexes. Docking studies showed the participation of several amino acids in drug-protein complexation, which stabilized by H-bonding systems. The order of drug-protein binding is DOX-HSA > FDOX-HSA > DOX-BSA > FDOX>BSA. Drug complexation alters protein conformation by a major reduction of α-helix from 63% (free BSA) to 47–44% (drug-complex) and 57% (free HSA) to 51–40% (drug-complex) inducing a partial protein destabilization. Doxorubicin and its derivative can be transported by BSA and HSA in vitro.     

Protective action of resveratrol in human skin: possible involvement of specific receptor binding sites

Background

Resveratrol is a plant-derived polyphenol with purported protecting action on various disorders associated with aging. It has been suggested that resveratrol could exert its protective action by acting on specific plasma membrane polyphenol binding sites (Han Y.S., et al. (2006) J Pharmacol Exp Ther 318:238–245). The purpose of this study was to investigate, in human skin, the possible existence of specific binding sites that mediate the protective action of resveratrol.

Methods and Findings

Using human skin tissue, we report here the presence of specific [³H]-resveratrol binding sites (K_D  = 180 nM) that are mainly located in the epidermis. Exposure of HaCaT cells to the nitric oxide free radical donor sodium nitroprusside (SNP; 0.3–3 mM) resulted in cell death which was reduced by resveratrol (EC₅₀  = 14.7 µM), and to a much lesser extent by the resveratrol analogue piceatannol (EC₅₀  = 95 µM) and epigallocatechin gallate (EC₅₀  = 200 µM), a green-tea derived polyphenol. The protective action of resveratrol likely relates to its anti-apoptotic effect since at the same range of concentration it was able to reduce both the number of apoptotic cells as well as mitochondrial apoptotic events triggered by SNP.

Conclusion

Taken together, these findings suggest that resveratrol, by acting on specific polyphenol binding sites in epidermis, may be useful to prevent skin disorders associated with aging.     

Curcumin-loaded apotransferrin nanoparticles provide efficient cellular uptake and effectively inhibit HIV-1 replication in vitro

Background

Curcumin (diferuloylmethane) shows significant activity across a wide spectrum of conditions, but its usefulness is rather limited because of its low bioavailability. Use of nanoparticle formulations to enhance curcumin bioavailability is an emerging area of research.

Methodology/Principal Findings

In the present study, curcumin-loaded apotransferrin nanoparticles (nano-curcumin) prepared by sol-oil chemistry and were characterized by electron and atomic force microscopy. Confocal studies and fluorimetric analysis revealed that these particles enter T cells through transferrin-mediated endocytosis. Nano-curcumin releases significant quantities of drug gradually over a fairly long period, ∼50% of curcumin still remaining at 6 h of time. In contrast, intracellular soluble curcumin (sol-curcumin) reaches a maximum at 2 h followed by its complete elimination by 4 h. While sol-curcumin (GI₅₀ = 15.6 µM) is twice more toxic than nano-curcumin (GI₅₀ = 32.5 µM), nano-curcumin (IC₅₀<1.75 µM) shows a higher anti-HIV activity compared to sol-curcumin (IC₅₀ = 5.1 µM). Studies in vitro showed that nano-curcumin prominently inhibited the HIV-1 induced expression of Topo II α, IL-1β and COX-2, an effect not seen with sol-curcumin. Nano-curcumin did not affect the expression of Topoisomerase II β and TNF α. This point out that nano-curcumin affects the HIV-1 induced inflammatory responses through pathways downstream or independent of TNF α. Furthermore, nano-curcumin completely blocks the synthesis of viral cDNA in the gag region suggesting that the nano-curcumin mediated inhibition of HIV-1 replication is targeted to viral cDNA synthesis.

Conclusion

Curcumin-loaded apotransferrin nanoparticles are highly efficacious inhibitors of HIV-1 replication in vitro and promise a high potential for clinical usefulness.     

Methanocella conradii sp. nov., a thermophilic, obligate hydrogenotrophic methanogen, isolated from Chinese rice field soil

Background

Methanocellales contributes significantly to anthropogenic methane emissions that cause global warming, but few pure cultures for Methanocellales are available to permit subsequent laboratory studies (physiology, biochemistry, etc.).

Methodology/Principal Findings

By combining anaerobic culture and molecular techniques, a novel thermophilic methanogen, strain HZ254^T was isolated from a Chinese rice field soil located in Hangzhou, China. The phylogenetic analyses of both the 16S rRNA gene and mcrA gene (encoding the α subunit of methyl-coenzyme M reductase) confirmed its affiliation with Methanocellales, and Methanocella paludicola SANAE^T was the most closely related species. Cells were non-motile rods, albeit with a flagellum, 1.4–2.8 µm long and by 0.2–0.3 µm in width. They grew at 37–60°C (optimally at 55°C) and salinity of 0–5 g NaCl l⁻¹ (optimally at 0–1 g NaCl l⁻¹). The pH range for growth was 6.4–7.2 (optimum 6.8). Under the optimum growth condition, the doubling time was 6.5–7.8 h, which is the shortest ever observed in Methanocellales. Strain HZ254^T utilized H₂/CO₂ but not formate for growth and methane production. The DNA G+C content of this organism was 52.7 mol%. The sequence identities of 16S rRNA gene and mcrA gene between strain HZ254^T and SANAE^T were 95.0 and 87.5% respectively, and the genome based Average Nucleotide Identity value between them was 74.8%. These two strains differed in phenotypic features with regard to substrate utilization, possession of a flagellum, doubling time (under optimal conditions), NaCl and temperature ranges. Taking account of the phenotypic and phylogenetic characteristics, we propose strain HZ254^T as a representative of a novel species, Methanocella conradii sp. nov. The type strain is HZ254^T ( = CGMCC 1.5162^T = JCM 17849^T = DSM 24694^T).

Conclusions/Significance

Strain HZ254^T could potentially serve as an excellent laboratory model for studying Methanocellales due to its fast growth and consistent cultivability.     

Biochemical comparison of Anopheles gambiae and human NADPH P450 reductases reveals different 2'-5'-ADP and FMN binding traits

NADPH-cytochrome P450 oxidoreductase (CPR) plays a central role in chemical detoxification and insecticide resistance in Anopheles gambiae, the major vector for malaria. Anopheles gambiae CPR (AgCPR) was initially expressed in Eschericia coli but failed to bind 2′, 5′-ADP Sepharose. To investigate this unusual trait, we expressed and purified a truncated histidine-tagged version for side-by-side comparisons with human CPR. Close functional similarities were found with respect to the steady state kinetics of cytochrome c reduction, with rates (k _cat) of 105 s⁻¹ and 88 s⁻¹, respectively, for mosquito and human CPR. However, the inhibitory effects of 2′,5′-ADP on activity were different; the IC₅₀ value of AgCPR for 2′, 5′ –ADP was significantly higher (6–10 fold) than human CPR (hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in affinity for 2′, 5′- ADP. This was confirmed by isothermal titration calorimetry where binding of 2′,5′-ADP to AgCPR (K _d = 410±18 nM) was ∼10 fold weaker than human CPR (K _d = 38 nM). Characterisation of the individual AgFMN binding domain revealed much weaker binding of FMN (K_d = 83±2.0 nM) than the equivalent human domain (K_d = 23±0.9 nM). Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC₅₀ = 28 µM±2 and 361±31 µM respectively). Taken together, these results reveal unusual biochemical differences between mosquito CPR and the human form in the binding of small molecules that may aid the development of ‘smart’ insecticides and synergists that selectively target mosquito CPR.     

α1Proteinase inhibitor regulates CD4+ lymphocyte levels and is rate limiting in HIV-1 disease

Background

The regulation of adult stem cell migration through human hematopoietic tissue involves the chemokine CXCL12 (SDF-1) and its receptor CXCR4 (CD184). In addition, human leukocyte elastase (HLE) plays a key role. When HLE is located on the cell surface (HLE_CS), it acts not as a proteinase, but as a receptor for α₁proteinase inhibitor (α₁PI, α₁antitrypsin, SerpinA1). Binding of α₁PI to HLE_CS forms a motogenic complex. We previously demonstrated that α₁PI deficiency attends HIV-1 disease and that α₁PI augmentation produces increased numbers of immunocompetent circulating CD4⁺ lymphocytes. Herein we investigated the mechanism underlying the α₁PI deficiency that attends HIV-1 infection.

Methods and Findings

Active α₁PI in HIV-1 subjects (median 17 µM, n = 35) was significantly below normal (median 36 µM, p<0.001, n = 30). In HIV-1 uninfected subjects, CD4⁺ lymphocytes were correlated with the combined factors α₁PI, HLE_CS ⁺ lymphocytes, and CXCR4⁺ lymphocytes (r² = 0.91, p<0.001, n = 30), but not CXCL12. In contrast, in HIV-1 subjects with >220 CD4 cells/µl, CD4⁺ lymphocytes were correlated solely with active α₁PI (r² = 0.93, p<0.0001, n = 26). The monoclonal anti-HIV-1 gp120 antibody 3F5 present in HIV-1 patient blood is shown to bind and inactivate human α₁PI. Chimpanzee α₁PI differs from human α₁PI by a single amino acid within the 3F5-binding epitope. Unlike human α₁PI, chimpanzee α₁PI did not bind 3F5 or become depleted following HIV-1 challenge, consistent with the normal CD4⁺ lymphocyte levels and benign syndrome of HIV-1 infected chimpanzees. The presence of IgG-α₁PI immune complexes correlated with decreased CD4⁺ lymphocytes in HIV-1 subjects.

Conclusions

This report identifies an autoimmune component of HIV-1 disease that can be overcome therapeutically. Importantly, results identify an achievable vaccine modification with the novel objective to protect against AIDS as opposed to the current objective to protect against HIV-1 infection.     

Crystallization and preliminary analysis of crystals of the 24-meric hemocyanin of the emperor scorpion (Pandinus imperator)

Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (M_W = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.     

Maximum diastolic potential of human induced pluripotent stem cell-derived cardiomyocytes depends critically on I(Kr)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl₂ (n = 22). Patch-clamp techniques were used to record I_Kr and I_K1 from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl₂, at concentrations that selectively block I_K1 (50–100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I_K1 in 53% (20/38) of the cells studied, but presence of I_Kr in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I_K1 in the majority of cells, but robust expression of I_Kr. In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I_Kr due to the absence of I_K1. Thus, efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications.     

Beneficial effects of resistance exercise on glycemic control are not further improved by protein ingestion

Purpose

To investigate the mechanisms underpinning modifications in glucose homeostasis and insulin sensitivity 24 h after a bout of resistance exercise (RE) with or without protein ingestion.

Methods

Twenty-four healthy males were assigned to a control (CON; n = 8), exercise (EX; n = 8) or exercise plus protein condition (EX+PRO; n = 8). Muscle biopsy and blood samples were obtained at rest for all groups and immediately post-RE (75% 1RM, 8×10 repetitions of leg-press and extension exercise) for EX and EX+PRO only. At 24 h post-RE (or post-resting biopsy for CON), a further muscle biopsy was obtained. Participants then consumed an oral glucose load (OGTT) containing 2 g of [U-¹³C] glucose during an infusion of 6, 6-[²H₂] glucose. Blood samples were obtained every 10 min for 2 h to determine glucose kinetics. EX+PRO ingested an additional 25 g of intact whey protein with the OGTT. A final biopsy sample was obtained at the end of the OGTT.

Results

Fasted plasma glucose and insulin were similar for all groups and were not different immediately post- and 24 h post-RE. Following RE, muscle glycogen was 26±8 and 19±6% lower in EX and EX+PRO, respectively. During OGTT, plasma glucose AUC was lower for EX and EX+PRO (75.1±2.7 and 75.3±2.8 mmol·L⁻¹∶120 min, respectively) compared with CON (90.6±4.1 mmol·L⁻¹∶120 min). Plasma insulin response was 13±2 and 21±4% lower for EX and CON, respectively, compared with EX+PRO. Glucose disappearance from the circulation was ∼12% greater in EX and EX+PRO compared with CON. Basal 24 h post-RE and insulin-stimulated PAS-AS160/TBC1D4 phosphorylation was greater for EX and EX+PRO.

Conclusions

Prior RE improves glycemic control and insulin sensitivity through an increase in the rate at which glucose is disposed from the circulation. However, co-ingesting protein during a high-glucose load does not augment this response at 24 h post-exercise in healthy, insulin-sensitive individuals.