Lipid rafts determine clustering of STIM1 in endoplasmic reticulum-plasma membrane junctions and regulation of store-operated Ca2+ entry (SOCE)

Store depletion induces STIM1 to aggregate and relocate into clusters at ER-plasma membrane junctions where it functionally interacts with and activates plasma membrane channels that mediate store-operated Ca(2+) entry (SOCE). Thus, the site of peripheral STIM1 clusters is critical for the regulation of SOCE. However, what determines the location of the STIM1 clusters in the ER-PM junctional regions, and whether these represent specific sites in the cell is not yet known. Here we report that clustering of STIM1 in the subplasma membrane region of the cell and activation of TRPC1-dependent SOCE are determined by lipid raft domains (LRD). We show that store depletion increased partitioning of TRPC1 and STIM1 into plasma membrane LRD. TRPC1 and STIM1 associated with each other within the LRD, and this association was dynamically regulated by the status of the ER Ca(2+) store. Peripheral STIM1 clustering was independent of TRPC1. However, sequestration of membrane cholesterol attenuated thapsigargin-induced clustering of STIM1 as well as SOCE in HSG and HEK293 cells. Recruitment and association of STIM1 and TRPC1 in LRD was also decreased. Additionally STIM1(D76A), which is peripherally localized and constitutively activates SOCE in unstimulated cells, displayed a relatively higher partitioning into LRD and interaction with TRPC1, as compared with STIM1. Disruption of membrane rafts decreased peripheral STIM1(D76A) puncta, its association with TRPC1 and the constitutive SOCE. Together, these data demonstrate that intact LRD determine targeting of STIM1 clusters to ER-plasma membrane junctions following store depletion. This facilitates the functional interaction of STIM1 with TRPC1 and activation of SOCE.     

Evaluation of pentachlorophenol-degrading potentiality of Pseudomonas sp. in a soil microcosm

Pseudomonas sp. strain IST103 obtained from a stable consortium was capable of degrading pentachlorophenol (PCP) as sole carbon and energy source. The PCP-degrading potentiality of the strain was determined by growth of bacteria in culture medium, utilization of PCP by high performance liquid chromatography (HPLC), chloride release and ring cleavage. The strain was applied in two set of soil microcosms containing 20 and 40% moisture, each having different concentrations, 0, 10, 100, 500, and 1000 mg/l, of PCP. The result showed significant utilization of PCP (77% in 45 days) and higher growth of bacterial strain when PCP was applied in 100 mg/l concentration at 40% moisture. Inhibitory effects on the growth of bacterial strain were seen in 500 and 1000 mg/l concentration.     

Do Antenatal Parasite Infections Devalue Childhood Vaccination?

On a global basis, both potent vaccine efficacy and high vaccine coverage are necessary to control and eliminate vaccine-preventable diseases. Emerging evidence from animal and human studies suggest that neglected tropical diseases (NTDs) significantly impair response to standard childhood immunizations. A review of efficacy and effectiveness studies of vaccination among individuals with chronic parasitic infections was conducted, using PUBMED database searches and analysis of data from the authors'' published and unpublished studies. Both animal models and human studies suggest that chronic trematode, nematode, and protozoan infections can result in decreased vaccine efficacy. Among pregnant women, who in developing countries are often infected with multiple parasites, soluble parasite antigens have been shown to cross the placenta and prime or tolerize fetal immune responses. As a result, antenatal infections can have a significant impact on later vaccine responses. Acquired childhood parasitic infections, most commonly malaria, can also affect subsequent immune response to vaccination. Additional data suggest that antiparasite therapy can improve the effectiveness of several human vaccines. Emerging evidence demonstrates that both antenatal and childhood parasitic infections alter levels of protective immune response to routine vaccinations. Successful antiparasite treatment may prevent immunomodulation caused by parasitic antigens during pregnancy and early childhood and may improve vaccine efficacy. Future research should highlight the varied effects that different parasites (alone and in combination) can have on human vaccine-related immunity. To optimize vaccine effectiveness in developing countries, better control of chronic NTDs may prove imperative.     

Can Prenatal Malaria Exposure Produce an Immune Tolerant Phenotype?: A Prospective Birth Cohort Study in Kenya

Background

Malaria in pregnancy can expose the fetus to malaria-infected erythrocytes or their soluble products, thereby stimulating T and B cell immune responses to malaria blood stage antigens. We hypothesized that fetal immune priming, or malaria exposure in the absence of priming (putative tolerance), affects the child''s susceptibility to subsequent malaria infections.

Methods and Findings

We conducted a prospective birth cohort study of 586 newborns residing in a malaria-holoendemic area of Kenya who were examined biannually to age 3 years for malaria infection, and whose malaria-specific cellular and humoral immune responses were assessed. Newborns were classified as (i) sensitized (and thus exposed), as demonstrated by IFNγ, IL-2, IL-13, and/or IL-5 production by cord blood mononuclear cells (CBMCs) to malaria blood stage antigens, indicative of in utero priming (n = 246), (ii) exposed not sensitized (mother Plasmodium falciparum [Pf]+ and no CBMC production of IFNγ, IL-2, IL-13, and/or IL-5, n = 120), or (iii) not exposed (mother Pf−, no CBMC reactivity, n = 220). Exposed not sensitized children had evidence for prenatal immune experience demonstrated by increased IL-10 production and partial reversal of malaria antigen-specific hyporesponsiveness with IL-2+IL-15, indicative of immune tolerance. Relative risk data showed that the putatively tolerant children had a 1.61 (95% confidence interval [CI] 1.10–2.43; p = 0.024) and 1.34 (95% CI 0.95–1.87; p = 0.097) greater risk for malaria infection based on light microscopy (LM) or PCR diagnosis, respectively, compared to the not-exposed group, and a 1.41 (95%CI 0.97–2.07, p = 0.074) and 1.39 (95%CI 0.99–2.07, p = 0.053) greater risk of infection based on LM or PCR diagnosis, respectively, compared to the sensitized group. Putatively tolerant children had an average of 0.5 g/dl lower hemoglobin levels (p = 0.01) compared to the other two groups. Exposed not sensitized children also had 2- to 3-fold lower frequency of malaria antigen-driven IFNγ and/or IL-2 production (p<0.001) and higher IL-10 release (p<0.001) at 6-month follow-ups, when compared to sensitized and not-exposed children. Malaria blood stage–specific IgG antibody levels were similar among the three groups.

Conclusions

These results show that a subset of children exposed to malaria in utero acquire a tolerant phenotype to blood-stage antigens that persists into childhood and is associated with an increased susceptibility to malaria infection and anemia. This finding could have important implications for malaria vaccination of children residing in endemic areas. Please see later in the article for Editors'' Summary     

Angiotensin II type 1 receptor blockade corrects cutaneous nitric oxide deficit in postural tachycardia syndrome

Low-flow postural tachycardia syndrome (POTS) is associated with increased plasma angiotensin II (ANG II) and reduced neuronal nitric oxide (NO), which decreases NO-dependent vasodilation. We tested whether the ANG II type 1 receptor (AT(1)R) antagonist losartan would improve NO-dependent vasodilation in POTS patients. Furthermore, if the action of ANG II is dependent on NO, then the NO synthase inhibitor nitro-L-arginine (NLA) would reverse this improvement. We used local heating of the skin of the left calf to 42 degrees C and laser-Doppler flowmetry to assess NO-dependent conductance [percent maximum cutaneous vascular conductance (%CVC(max))] in 12 low-flow POTS patients aged 22.5 +/- 0.8 yr and in 15 control subjects aged 22.0 +/- 1.3 yr. After measuring the baseline local heating response at three separate sites, we perfused individual intradermal microdialysis catheters at those sites with 2 microg/l losartan, 10 mM NLA, or losartan + NLA. The predrug heat response was reduced in POTS, particularly the plateau phase reflecting NO-dependent vasodilation (50 +/- 5 vs. 91 +/- 7 %CVC(max); P < 0.001 vs. control). Losartan increased baseline flow in both POTS and control subjects (from 6 +/- 1 to 21 +/- 3 vs. from 10 +/- 1 to 21 +/- 2 %CVC(max); P < 0.05 compared with predrug). The baseline increase was blunted by NLA. Losartan increased the POTS heat response to equal the control subject response (79 +/- 7 vs. 88 +/- 6 %CVC(max); P = 0.48). NLA decreased both POTS and control subject heat responses to similar conductances (38 +/- 4 vs. 38 +/- 3 %CVC(max); P < 0.05 compared with predrug). The addition of NLA to losartan reduced POTS and control subject conductances compared with losartan alone (48 +/- 3 vs. 53 +/- 2 %CVC(max)). The data suggest that the reduction in cutaneous NO-dependent vasodilation in low-flow POTS is corrected by AT(1)R blockade.     

Allelic variation in the cg2 gene does not correlate with chloroquine resistance among Indian Plasmodium falciparum isolates.

The cg2 gene of Plasmodium falciparum has been proposed to be associated with chloroquine resistance. Here we describe PCR amplification and sequencing of all the four repeat regions (kappa (κ), gamma (γ), psi (φ) and omega (ω)) of this gene, from Indian isolates. There were variant forms for each of these repeat regions (two for κ and γ, and three for φ and ω) among the 123 Indian isolates of P. falciparum. Among these isolates certain forms of φ and ω repeats were uniquely present while some of the reported forms of the κ and ω repeats were absent. The pattern of combination of all four repeat regions of cg2 gene (genotype) was analysed from 52 isolates. A total of 11 different genotypes were observed among these cases, of which 10 were unique to Indian isolates. Certain genotypes were more common than others. The nucleotide sequencing of all the four repeat regions revealed that Indian isolates have some unique repeating units within the γ and ω domains. Altogether, the PCR and sequencing results showed that there was an unrelatedness between cg2 repeats and chloroquine resistance.     

Interaction of Melanin with Proteins – The Importance of an Acidic Intramelanosomal pH

Melanin is a highly irregular heteropolymer consisting of monomeric units derived from the enzymatic oxidation of the amino acid tyrosine. The process of melanin formation takes place in specialized acidic organelles (melanosomes) in melanocytes. The process of melanin polymerization requires an alkaline pH in vitro, and therefore, the purpose of an acidic environment in vivo remains a mystery. It is known that melanin is always bound to protein in vivo. It is also seen that polymerization in vitro at an acidic pH necessarily requires the presence of proteins. The effect of various model proteins on melanin synthesis and their interaction with melanin was studied. It was seen that many proteins could increase melanin synthesis at an acidic pH, and that different proteins resulted in the formation of different states of melanin, i.e., a precipitate or a soluble, protein‐bound form. We also present evidence to show that soluble protein‐bound melanin is present in vivo (in B16 cells as well as in B16 melanoma tissue). An acidic pH appeared to be necessary to ensure the formation of a uniform, very high molecular weight melano–protein complex. The interaction between melanin and proteins appears to be largely charge‐dependent as evidenced by zeta potential measurements, and this interaction is also increased in an acidic pH. Thus, it appears that an acidic intramelanosomal pH is essential to ensure maximum interaction between protein and melanin, and also to ensure that all the melanin formed is protein‐bound.     

Membrane-induced structure of the mammalian tachykinin neuropeptide gamma

Neuropeptide gamma (NPgamma) is a neurokinin-2 (NK-2) receptor selective agonist, which plays an important role in mediation of asthma and elicits a wide range of biological responses like bronchoconstriction, vasodepression and regulation of endocrine functions. The structure determination of this peptide agonist is important in understanding the molecular basis of peptide ligand recognition by the receptor and for rational drug design. In the present study we report the solution structure of NPgamma characterized by circular dichroism (CD) spectropolarimetry and 2D (1)H NMR spectroscopy in both aqueous and membrane mimetic solvents. Effect of calcium ions on the conformation of NPgamma was also studied using CD spectropolarimetry. Sequence-specific resonance assignments of protons have been made with the aid of correlation spectroscopy experiments and nuclear Overhauser effect spectroscopy experiments. The distance constraints obtained from the NMR data have been utilized to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that in water NPgamma prefers to be in an extended chain conformation whereas a helical conformation is induced in the central core and the C-terminal region of the peptide (K13-M21) in the presence of perdeuterated dodecylphosphocholine micelles, a membrane model system. A type II' beta turn from H9 to R11 precedes the helical core in the C-terminus of NPgamma. N-terminus of NPgamma also displays some degree of order and a possible turn structure. Conformation adopted by NPgamma in presence of lipid micelles represents a structural motif typical of NK-2 selective agonists and is similar to that observed for Neurokinin A in hydrophobic environment. The observed conformational features have been correlated to the binding ability and biological activity of NPgamma.