Role of chemical structure in molecular recognition by transferrin

Studies of molecular recognition of chiral compounds by proteins are of importance from many points of view. The biological role of proteins in their interaction with small molecules is of fundamental interest and can be used in many different fields, for instance for in vitro analysis of optically active compounds. Studies in these areas need a detailed study of the interaction sites on the protein surface and the relationship between chemical structure and the complex formation ability of small molecules, such as drugs. The electrophoretic migration of charged compounds through a protein zone may provide information about the surface properties of the macromolecule in the interaction site. The interaction of human serum transferrin with tryptophan-methyl- (TME), ethyl- (TEE) and butyl-esters (TBE) has been investigated by capillary electrophoresis (CE) and model calculations. Differences in the separation of tryptophan derivatives were obtained by varying experimental parameters such as, pH, ionic strength of background electrolyte and the length of transferrin zone. Limited separation of the enantiomer pairs were observed at pH 5 and 7 with a maximum resolution at pH 6. The size of the ligands coupled to the chiral centre has importance in stereoselective recognition; however, a direct comparison of resolution different in same runs may lead to false conclusion if the experimental conditions are not comparable. With a careful evaluation of the data we obtained significant differences between the resolution of the smallest enantiomer pair compared to those of tryptophan derivatives with longer alkyl chains.     

Electromuscular incapacitation results from stimulation of spinal reflexes

Electronic stun devices (ESD) often used in law enforcement, military action or self defense can induce total body uncoordinated muscular activity, also known as electromuscular incapacitation (EMI). During EMI the subject is unable to perform purposeful or coordinated movements. The mechanism of EMI induction has not been reported, but has been generally thought to be direct muscle and nerve excitation from the fields generated by ESDs. To determine the neuromuscular mechanisms linking ESD to induction of EMI, we investigated EMI responses using an anesthetized pig model. We found that EMI responses to ESD application can best be simulated by simultaneous stimulation of motor and sensory peripheral nerves. We also found that application of local anesthetics limited the response of ESD to local muscle stimulation and abolished the total body EMI response. Stimulation of the pure sensory peripheral nerves or nerves that are primarily motor nerves induced muscle responses that are consistent with well defined spinal reflexes. These findings suggest that the mechanism of ESD‐induced EMI is mediated by excitation of multiple simultaneous spinal reflexes. Although direct motor‐neuron stimulation in the region of ESD contact may significantly add to motor reactions from ESD stimulation, multiple spinal reflexes appear to be a major, and probably the dominant mechanism in observed motor response. Bioelectromagnetics 30:411–421, 2009. © 2009 Wiley‐Liss, Inc.     

Mechanism of Activation and Functional Role of Protein Kinase C�� in Human Platelets

The novel class of protein kinase C (nPKC) isoform η is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y₁ receptor antagonist, or YM-254890, a G_q blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y₁ and G_q knock-out mice. However, pretreatment of platelets with P2Y₁₂ receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin α_IIbβ₃ mediated outside-in signaling. Moreover, in the presence of SC-57101, a α_IIbβ₃ receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, α_IIbβ₃ failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y₁ receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by α_IIbβ₃ integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis (1). Vascular injury exposes subendothelial collagen that activates platelets to change shape, secrete contents of granules, generate thromboxane, and finally aggregate via activated α_IIbβ₃ integrin, to prevent further bleeding (2, 3). ADP is a physiological agonist of platelets secreted from dense granules and is involved in feedback activation of platelets and hemostatic plug stabilization (4). It activates two distinct G-protein-coupled receptors (GPCRs) on platelets, P2Y₁ and P2Y₁₂, which couple to G_q and G_i, respectively (5–8). G_q activates phospholipase Cβ (PLCβ), which leads to diacyl glycerol (DAG)² generation and calcium mobilization (9, 10). On the other hand, G_i is involved in inhibition of cAMP levels and PI 3-kinase activation (4, 6). Synergistic activation of G_q and G_i proteins leads to the activation of the fibrinogen receptor integrin α_IIbβ₃. Fibrinogen bound to activated integrin α_IIbβ₃ further initiates feed back signaling (outside-in signaling) in platelets that contributes to the formation of a stable platelet plug (11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate various platelet functional responses such as dense granule secretion and integrin α_IIbβ₃ activation (12, 13). Based on their structure and cofactor requirements, PKCs are divided in to three classes: classical (cofactors: DAG, Ca²⁺), novel (cofactors: DAG) and atypical (cofactors: PIP₃) PKC isoforms (14). All the members of the novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ, η, and ε, are expressed in platelets (15), and they require DAG for activation. Among all the nPKCs, PKCδ (15, 16) and PKCθ (17–19) are fairly studied in platelets. Whereas nPKCδ is reported to regulate protease-activated receptor (PAR)-mediated dense granule secretion (15, 20), nPKCθ is activated by outside-in signaling and contributes to platelet spreading on fibrinogen (18). On the other hand, the mechanism of activation and functional role of nPKCη is not addressed as yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via three mechanisms (14, 21): 1) cofactor binding: upon cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as DAG, Ca²⁺, or PIP3, release autoinhibition, and attain an active conformation exposing catalytic domain of the enzyme. 2) phosphorylations: 3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates conserved threonine residues on activation loop of catalytic domain; this is followed by autophosphorylations of serine/threonine residues on turn motif and hydrophobic region. These series of phosphorylations maintain an active conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind receptors for activated C kinases (RACKs) and are lead to various subcellular locations to access the substrates (22, 23). Although various leading laboratories have elucidated the activation of PKCs, the mechanism of down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein phosphorylations by kinases and dephosphorylations by phosphatases has gained immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the serine/threonine phosphatases reported to date. Among them PP1 and PP2 phosphatases are known to regulate various platelet functional responses (24, 25). Furthermore, PP1c, is the catalytic unit of PP1 known to constitutively associate with α_IIb and is activated upon integrin engagement with fibrinogen and subsequent outside-in signaling (26). Among various PP1 isoforms, recently PP1γ is shown to positively regulate platelet functional responses (27). Thus, in this study we investigated if the above-mentioned phosphatases are involved in down-regulation of nPKCη. Furthermore, reports from other cell systems suggest that nPKCη regulates ERK/JNK pathways (28). In platelets ERK is known to regulate agonist induced thromboxane generation (29, 30). Thus, we also investigated if nPKCη regulates ERK phosphorylation and thereby agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP receptors and its inactivation by an integrin-associated phosphatase PP1γ. We also studied if nPKCη regulates functional responses in platelets and found that this isoform regulates ADP-induced thromboxane generation, but not fibrinogen receptor activation in platelets.     

Impaired Replication Capacity of Acute/Early Viruses in Persons Who Become HIV Controllers

Human immunodeficiency virus type 1 (HIV-1) controllers maintain viremia at <2,000 RNA copies/ml without antiretroviral therapy. Viruses from controllers with chronic infection were shown to exhibit impaired replication capacities, in part associated with escape mutations from cytotoxic-T-lymphocyte (CTL) responses. In contrast, little is known about viruses during acute/early infection in individuals who subsequently become HIV controllers. Here, we examine the viral replication capacities, HLA types, and virus sequences from 18 HIV-1 controllers identified during primary infection. gag-protease chimeric viruses constructed using the earliest postinfection samples displayed significantly lower replication capacities than isolates from persons who failed to control viremia (P = 0.0003). Protective HLA class I alleles were not enriched in these early HIV controllers, but viral sequencing revealed a significantly higher prevalence of drug resistance mutations associated with impaired viral fitness in controllers than in noncontrollers (6/15 [40.0%] versus 10/80 [12.5%], P = 0.018). Moreover, of two HLA-B57-positive (B57⁺) controllers identified, both harbored, at the earliest time point tested, signature escape mutations within Gag that likewise impair viral replication capacity. Only five controllers did not express “protective” alleles or harbor viruses with drug resistance mutations; intriguingly, two of them displayed typical B57 signature mutations (T242N), suggesting the acquisition of attenuated viruses from B57⁺ donors. These data indicate that acute/early stage viruses from persons who become controllers have evidence of reduced replication capacity during the initial stages of infection which is likely associated with transmitted or acquired CTL escape mutations or transmitted drug resistance mutations. These data suggest that viral dynamics during acute infection have a major impact on HIV disease outcome.Human immunodeficiency virus type I (HIV-1)-infected individuals who control viremia spontaneously without antiviral therapy have been termed HIV controllers (3, 18, 21, 48, 52). Unraveling the mechanisms associated with this phenotype should provide important insights regarding HIV pathogenesis and could contribute to vaccine development.Host and viral genetics, as well as host innate and adaptive immune responses, influence the rate of disease progression in HIV-1 infection (reviewed in reference 18). Several studies have reported the correlation between in vitro HIV replication capacity and level of plasma virus loads or disease progression in individuals with chronic infection (6, 13, 35, 45, 50, 55). Studies of HIV-1 elite controllers (EC), who control viremia to below the limit of detection in commercial assays, have revealed the presence of replication-competent viruses in these individuals (7), although these viruses appear to be less fit based on studies of envelope (35) and Gag-protease (45). This fitness defect in the chronic phase of infection is due at least in part to fitness-impairing mutations induced by cytotoxic-T-lymphocyte (CTL) responses restricted by “protective” HLA class I alleles (46).In contrast, little is known about viruses obtained from the acute/early phase of infection in persons who subsequently become HIV-1 controllers, largely due to the difficulty in enrolling such people during the acute/early phase of infection. The characterization of acute/early-phase viruses in individuals who subsequently achieve low set-point virus loads is of paramount importance to our understanding of the mechanisms of HIV-1 control.In the present study, we analyzed acute/early-phase plasma HIV RNA sequences from 18 untreated individuals who were diagnosed during the acute/early phase and subsequently became controllers (<2,000 RNA copies/ml). We compared these to sequences from a group of HIV-1 noncontrollers enrolled similarly during acute/early infection. We also generated chimeric viruses carrying patient-derived gag-protease sequences from acute/early-phase infection and compared the viral replication capacities of the chimeric viruses from controllers and from noncontrollers.We observed that the chimeric viruses derived from controllers have significantly reduced replicative capacities compared to those from noncontrollers. Moreover, we observed that at least 80% of these individuals who go on to become controllers featured transmission of attenuated drug-resistant viruses, transmission of HLA-B57-restricted CTL escape variants to HLA-mismatched recipients, selection of attenuated CTL escape variants in HLA-B57-positive (B57⁺) recipients, or combinations of these factors. Taken together, these results indicate that the initial viral dynamics have a major influence on the subsequent course of disease.     

Complex haplotypes of IRS2 gene are associated with severe obesity and reveal heterogeneity in the effect of Gly1057Asp mutation

In order to understand the role of the insulin receptor substrate-2 (IRS2) gene (chromosome region: 13q34) in obesity, a complex disorder associated with insulin resistance and glucose intolerance, we determined single nucleotide polymorphims (SNPs) and complex haplotypes in women with morbid obesity and a body mass index (BMI) of 41+/-0.8 kg/m2 ( n=99) compared with controls having a BMI of 23.8+/-0.1 kg/m2 ( n=92). Sequencing of unphased DNA or digestion of polymerase chain reaction fragments revealed seven SNPs, including a new C/T(-769) replacement at the 5' untranslated region. Considering four or seven SNPs, we reconstructed with the PHASE program nine or 24 haplotypes, respectively, that were well correlated into the cladogram. Logistic regression analysis with nine haplotypes in the whole sample revealed that obesity was associated with haplotype H3, with P<0.025, an odds ratio (OR) of 1.9 and a 95% confidence interval (CI) of 1.1-3.4, or pairs 3/3 ( P<0.005, OR=8.7, CI=1.9-40.1) and 3/4 ( P<0.023, OR=2.5, CI=1.1-5.6), all containing the the Gly1057Asp allelic variant of IRS2, whereas controls were associated with H5 and H6 ( P<0.02, OR=0.2, CI=0.01-0.81). Although obese H5 carriers (also containing Gly1057Asp mutation) were the most insulin resistant, haplotypes of IRS2 were poorly correlated (analysis of variance) with insulin resistance. By contrast, haplotypes H3, H4 and pairs 3/3 were consistently associated with increased 2-h glucose levels during an oral glucose tolerance test in obese individuals ( P<0.0005, 0.025 and 0.027, respectively). These data indicate that IRS2 is an influential gene in severe obesity and glucose intolerance in this population, whereas gene-based haplotypes of IRS2 have revealed heterogeneity in the behaviour of the Gly1057Asp mutation in relation to insulin resistance.     

Role of stationary phase and eluent composition on the determination of log P values of N-hydroxyethylamide of aryloxyalkylen and pyridine carboxylic acids by reversed-phase high-performance liquid chromatography

The partition coefficients, P, between n-octanol and water of a number of growth stimulating substances, N-hydroxyethylamide of aryloxyalkylen- and pyridine carboxylic acids were obtained from Pomona College (C log P), and Rekker's (log P_Rekker) revised fragmental constant system was used to calculate log P data sets. Both of these data sets were correlated with two different substance lipophilicity parameters, log k_w and ₀. Log k_w was obtained by extrapolation of log retention factor (k) to 0% organic modifier measured in reversed-phase liquid chromatography (RPLC) systems. ₀ values were obtained from the slopes and intercepts of these relationships. The RPLC experiments were performed on four commercially available reversed-phase columns. Binary mixtures of methanol–water, methanol–phosphate buffer (pH 7.0), methanol–tricine buffer (pH 7.0) and acetonitrile–water were used as mobile phases for the determination of log k_w values. For the methanolic eluents linear regression provided satisfactory correlations (r>0.99) for the relationships log k vs. organic modifier content in the eluent, while for the acetonitrile-containing eluents a second-degree polynominal regression was necessary. For all four RPLC columns, by linear regression satisfactory correlations (r>0.99) were obtained between log k_w and log P data using methanolic eluents. In such eluents ₀ values were shown to be the second-best lipophilicity parameters. For acetonitrile-containing eluents the use of second-degree polynominal regression was necessary and, in contrast to methanol, significant influence of the applied column on regression results was observed. For acetonitrile-containing eluents the ₀-index does not provide satisfactory results for our substances. No difference in regression results between the use of buffered and non-buffered eluents was observed.