Thyroid hormones and the reactivities of genetic variants of human erythrocytic glucose-6-phosphate dehydrogenase

Thyroid hormones, throxine (T4) and triiodothyronine (T3) which are known to activate glucose-6-phosphate dehydrogenase (G6PD) activity in vivo act as substrate inhibitors of G6PD in vitro. T4 competitively inhibits NADP in human erythrocyte G6PD variants G6PDA, G6PDB and G6PDA- with inhibition constants of 2.40 +/- 0.90 X 10(-6), 3.44 +/- 0.63 X 10(-6) and 6.53 +/- 0.60 X 10(-6) mol/l, respectively. The inhibition is, however, noncompetitive with respect to G6P in the three variants. T3 also has similar inhibition pattern to T4 with inhibition constants for NADP of 1.9 +/- 0.08 X 10(-5) and 1.28 +/- 0.17 X 10(-5) mol/l for G6PDB and G6PDA-, respectively. cAMP on the other hand inhibits G6P competitively with inhibition constants 1.50 +/- 0.22 X 10(-4), 1.06 +/- 0.24 X 10(-4) and 1.76 +/- 0.14 X 10(-4) mol/l for G6PDB, G6PDA and G6PDA-, respectively. There are significant differences in the inhibition effects of T4 and cAMP with respect to NADP as substrates for the normal enzyme G6PDA or G6PDB and the deficient enzyme G6PDA- when NADP is the substrate, the latter being much more inhibited. The activation effect of thyroid hormones in vivo may therefore not be a direct result of thyroid hormone binding to the G6PD enzyme nor mediated through the action of cAMP but plausibly be through complexation of inhibitory trace metal ions by the thyroid hormones T4 and T3.     

Association of ABCA1 with syntaxin 13 and flotillin-1 and enhanced phagocytosis in tangier cells

The ATP-binding cassette transporter A1 (ABCA1) facilitates the cellular release of cholesterol and choline-phospholipids to apolipoprotein A-I (apoA-I) and several studies indicate that vesicular transport is associated with ABCA1 function. Syntaxins play a major role in vesicular fusion and have also been demonstrated to interact with members of the ABC-transporter family. Therefore, we focused on the identification of syntaxins that directly interact with ABCA1. The expression of syntaxins and ABCA1 in cultured human monocytes during M-CSF differentiation and cholesterol loading was investigated and syntaxins 3, 6, and 13 were found induced in foam cells together with ABCA1. Immunoprecipitation experiments revealed a direct association of syntaxin 13 and full-length ABCA1, whereas syntaxin 3 and 6 failed to interact with ABCA1. The colocalization of ABCA1 and syntaxin 13 was also shown by immunofluorescence microscopy. Silencing of syntaxin 13 by small interfering RNA (siRNA) led to reduced ABCA1 protein levels and hence to a significant decrease in apoA-I-dependent choline-phospholipid efflux. ABCA1 is localized in Lubrol WX-insoluble raft microdomains in macrophages and syntaxin 13 and flotillin-1 were also detected in these detergent resistant microdomains along with ABCA1. Syntaxin 13, flotillin-1, and ABCA1 were identified as phagosomal proteins, indicating the involvement of the phagosomal compartment in ABCA1-mediated lipid efflux. In addition, the uptake of latex phagobeads by fibroblasts with mutated ABCA1 was enhanced when compared with control cells and the recombinant expression of functional ABCA1 normalized the phagocytosis rate in Tangier fibroblasts. It is concluded that ABCA1 forms a complex with syntaxin 13 and flotillin-1, residing at the plasma membrane and in phagosomes that are partially located in raft microdomains.     

mTORC2 regulates PGE2-mediated endothelial cell survival and migration

Prostaglandin E₂ (PGE₂) promotes angiogenesis by in part inducing endothelial cell survival and migration. The present study examined the role of mTOR and its two complexes, mTORC1 and mTORC2, in PGE₂-mediated endothelial cell responses. We used small interfering RNA (siRNA) to raptor or rictor to block mTORC1 or mTORC2, respectively. We observed that down-regulation of mTORC2 but not mTORC1 reduced baseline and PGE₂-induced endothelial cell survival and migration. At the molecular level, we found that knockdown of mTORC2 inhibited PGE₂-mediated Rac and Akt activation two important signaling intermediaries in endothelial cell migration and survival, respectively. In addition, inhibition of mTORC2 by prolonged exposure of endothelial cells to rapamycin also prevented PGE₂-mediated endothelial cell survival and migration confirming the results obtained with the siRNA approach. Taken together these results show that mTORC2 but not mTORC1 is an important signaling intermediary in PGE₂-mediated endothelial cell responses.     

ApoA-I induces a preferential efflux of monounsaturated phosphatidylcholine and medium chain sphingomyelin species from a cellular pool distinct from HDL(3) mediated phospholipid efflux

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used for a detailed analysis of cellular phospholipid and cholesterol efflux in free cholesterol (FC) loaded human primary fibroblasts and human monocyte-derived macrophages (HMDM) loaded with enzymatically modified LDL (E-LDL). Although both cell models differed significantly in their cellular lipid composition, a higher apoA-I specific efflux was found for monounsaturated phosphatidylcholine (PC) species together with a decreased contribution of polyunsaturated PC species in both cell types. Moreover, medium chain sphingomyelin (SPM) species SPM 14:0 and SPM 16:1 were translocated preferentially to apoA-I in both cell types. In contrast to fibroblasts, HMDM displayed a considerable proportion of cholesteryl esters (CE) in basal and apoA-I specific efflux media, most likely due to secretion of CE associated to apoE. Analysis of HDL(3) mediated lipid efflux from HMDM using D(9)-choline and (13)C(3)-FC stable isotope labeling revealed significantly different D(9)-PC and D(9)-SPM species pattern for apoA-I and HDL(3) specific efflux media, which indicates a contribution of distinct cellular phospholipid pools to apoA-I and HDL(3) mediated efflux. Together with a partial loading of fibroblasts and HMDM with HDL(3)-derived CE species, these data add further evidence for retroendocytosis of HDL. In summary, analysis of apoA-I/ABCA1 and HDL(3) mediated lipid efflux by ESI-MS/MS demonstrated a preferential efflux of monounsaturated PC and medium chain SPM to apoA-I. Moreover, this is the first study, which provides evidence for distinct cellular phospholipid pools used for lipid transfer to apoA-I and HDL(3) from the analysis of phospholipid species pattern in HMDM.     

Pseudomonas putida Strain FStm2 Isolated from Shark Skin: A Potential Source of Bacteriocin

Bacteriocin-producing Pseudomonas putida strain FStm2 isolated from shark showed broad range of antibacterial activity against all pathogens tested except Bacillus subtilis ATCC11774, MRSA N32064, Proteus mirabilis ATCC12453, Enterococcus faecalis ATCC14506, Salmonella typhimurium ATCC51312, Salmonella mutan ATCC25175, and Aeromonas hydrophila Wbf314. Of the three growth media tested in this study, TSB was observed to support the bacteriocin activity the most. While the highest bacteriocin activity was observed for media supplemented with 1 % NaCl, there was an observed reduction in bacteriocin activity with increasing salt concentration. Although the least bacteriocin activity was observed for marine broth, addition of increasing amounts of tryptone, glucose, or yeast extract increased bacteriocin activity. This was, however, contrary to the effect observed when MgSO₄ and MnSO₄ were added as supplements. In the presence of α-amylase, lipase, DNase, and RNase, a positive effect on bacteriocin production was observed. Proteinase K strongly inhibited bacteriocin production. Furthermore, the bacteriocins produced were heat stable within the temperature range of 30–70 °C. Bacteriocin activity also was not affected within a wide pH range of 3–9. Exposure to detergents did not inhibit the activity of the bacteriocin at the concentrations tested. Instead, a positive effect on the relative activity of produced bacteriocin was observed as sodium dodecyl sulfate (SDS), EDTA, and Tween 20 at 1 % concentration all improved bacteriocin activity when the cell-free supernatant was tested against Serratia marcescens ATCC 13880. The bacteriocin was purified by ammonium sulfate precipitation and gel filtration on a Superdex-200 column. SDS-PAGE analysis of the partially purified bacteriocin revealed an apparent molecular weight of ~32 kDa.     

Basal gonadotropin hormone release rates during the period of selective follicle-stimulating hormone release in the juvenile female rat

There are situations in which adult female rats release increased amounts of follicle-stimulating hormone (FSH) independent of increased luteinizing hormone (LH) release. This results from, at least in part, a selective increase in the basal FSH release rate. We investigated whether an increase in the basal FSH release rate is contributory to the rise in serum FSH levels which occurs independent of a rise in serum LH levels in the immature female rat. Rats had high serum FSH concentrations on days 7 and 15 after birth, low serum FSH levels on day 23, and low serum LH levels on all three days. In contrast, anterior pituitary gland (APG) FSH and LH concentrations and contents increased from day 7 to day 15 and the contents increased further from day 15 to day 23. Similarly, basal FSH and LH release rates per mg APG or per APG, as assessed by measurement of FSH and LH released into culture medium containing APG(s) from different aged rats, increased from day 7 to day 15 but did not increase further between days 15 and 23. The results indicate that unlike situations observed to date in adult female rats, a mechanism(s) other than an increase in the basal FSH release rate is involved in selective FSH release in the immature female rat.     

Dopamine-induced exocytosis of Na,K-ATPase is dependent on activation of protein kinase C-epsilon and -delta

The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 μM DA or a dopaminergic D₁ agonist, fenoldopam, but not with the dopaminergic D₂ agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase α1 and β1 protein abundance in the basolateral membrane (BLM) of AT2 cells. This increase in protein abundance was mediated by the exocytosis of Na,K-pumps from late endosomal compartments into the BLM. Down-regulation of diacylglycerol-sensitive types of protein kinase C (PKC) by pretreatment with phorbol 12-myristate 13-acetate or inhibition with bisindolylmaleimide prevented the DA-mediated increase in Na,K-ATPase activity and exocytosis of Na,K-pumps to the BLM. Preincubation of AT2 cells with either 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983), a selective inhibitor of PKC-δ, or isozyme-specific inhibitor peptides for PKC-δ or PKC-ε inhibited the DA-mediated increase in Na,K-ATPase. PKC-δ and PKC-ε, but not PKC-α or -β, translocated from the cytosol to the membrane fraction after exposure to DA. PKC-δ– and PKC-ε–specific peptide agonists increased Na,K-ATPase protein abundance in the BLM. Accordingly, dopamine increased Na,K-ATPase activity in alveolar epithelial cells through the exocytosis of Na,K-pumps from late endosomes into the basolateral membrane in a mechanism-dependent activation of the novel protein kinase C isozymes PKC-δ and PKC-ε.     

High density lipoprotein modulates platelet function.

BACKGROUND: Platelet activation by atherogenic lipoproteins can be antagonized by high density lipoprotein (HDL), probably via interaction with the ATP-binding cassette transporter A1 (ABCA1). METHODS: ABCA1 expression and its association with cholesterol rich membrane domains was analyzed by mRNA and Western blot analysis. HDL effects on platelet receptor clustering were analyzed by flow cytometric analysis of fluorescence resonance energy transfer between fluorochrome-labeled antibodies. RESULTS: ABCA1 expression increased upon megakaryocytic differentiation of human stem cells and ABCA1 protein partially associated to LubroIWX-resistant membrane domains. Plasma HDL-cholesterol in healthy donors negatively correlated to the platelet membrane cholesterol content. Receptor cluster analysis revealed a decrease in the association of Gplb and FcgammaRII upon incubation of platelets with HDL3. CONCLUSION: Our results suggest that HDL modulates platelet reactivity by altering lipid raft associated receptor clustering.     

DAZL 260A > G and MTHFR 677C > T variants in sperm DNA of infertile Indian men

DAZL (deleted in azoospermia-like) 260A > G and MTHFR (methylene tetrahydrofolate reductase) 677C > T are two important autosomal variants associated with impaired spermatogenesis. In this study, we investigated DAZL 260A > G and MTHFR 677C > T variants in sperm DNA and their frequency in oligozoospermic infertile men of Indian origin. The study on sperm DNA was performed, since it is more prone to oxidative stress-induced damage and mutation. One hundred oligozoopsermic infertile men having normal chromosomal complement with intact Y chromosome and 100 age- and ethnically-matched fertile controls were investigated for these variants in their sperm genome. Spermatozoa were separated by gradient centrifugation and DNA was isolated and analyzed for the single nucleotide polymorphisms (SNPs) by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The results showed no significant differences in the frequency of DAZL AG (P = 0.58) and MTHFR CT (P = 0.44) between oligozoospermic infertile men and controls. However, 8% (8/100) oligozoospermic infertile men harbored both the variants and showed significantly (P < 0.0001) lower sperm count (3.28 +/- 1.1 vs 12.50 +/- 4.09) compared to infertile men with either of the single variant. None of the fertile controls showed the presence of the both variants. In conclusion, the combined effect of both DAZL 260A > G and MTHFR 677C > T variants may have role in compromised sperm count. However, further studies are required to find the pathological role of these combined variants in male infertility.     

Iron metabolism: microbes,mouse, and man

Recent advances in research on iron metabolism have revealed the identity of a number of genes, signal transduction pathways, and proteins involved in iron regulation in mammals. The emerging paradigm is a coordination of homeostasis within a network of classical iron metabolic pathways and other cellular processes such as cell differentiation, growth, inflammation, immunity, and a host of physiologic and pathologic conditions. Iron, immunity, and infection are intricately linked and their regulation is fundamental to the survival of mammals. The mutual dependence on iron by the host and invading pathogenic organisms elicits competition for the element during infection. While the host maintains mechanisms to utilize iron for its own metabolism exclusively, pathogenic organisms are armed with a myriad of strategies to circumvent these measures. This review explores iron metabolism in mammalian host, defense mechanisms against pathogenic microbes and the competitive devices of microbes for access to iron.