Structural mechanism of immune evasion of HIV-1 gp120 by genomic, computational, and experimental science

The third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope gp120 subunit participates in determination of viral infection co-receptor tropism and host humoral immune responses. Positive charge of the V3 plays a key role in determining viral co-receptor tropism. In our previous papers, we showed a key role of the V3's net positive charge in the immunological escape and co-receptor tropism evolution in vivo. On the other hand, the several papers suggested that trimeric gp120s are protected from immune system by occlusion on the oligomer, by mutational variation, by carbohydrate masking and by conformational masking. If we can reveal the mechanism of neutralization escape, we expect that we will regulate the neutralization of HIV-1. In this review, we will overview the structural mechanism of neutralization escape of HIV-1 gp120 examined by computational science. The computational sciences for virology can provide more valuable information in combination with genomic and experimental science.     

Generation of 'Unnatural Natural Product' library and identification of a small molecule inhibitor of XIAP

Natural products have been utilized for drug discovery. To increase the source diversity, we generated a new chemical library consisting of chemically modified microbial metabolites termed 'Unnatural Natural Products' by chemical conversion of microbial metabolites in crude broth extracts followed by purification of reaction products with the LC-photo diode array-MS system. Using this library, we discovered an XIAP inhibitor, C38OX6, which restored XIAP-suppressed enzymatic activity of caspase-3 in vitro. Furthermore, C38OX6 sensitized cancer cells to anticancer drugs, whereas the unconverted natural product did not. These findings suggest that our library could be a useful source for drug seeds.     

Cadaverine turnover in soybean seedlings using 15N-labelled lysine and cadaverine

The synthesis and translocation of the diamine cadaverine during soybean (Glycine max L. Meer cv. Sakai) germination were studied using ¹⁵N-labelled lysine (the cadaverine precursor) and ¹⁵N-labelled cadaverine, both under light/dark (12 h/12 h) and total dark germinating conditions. ¹⁵N-cadaverine and non-labelled polyamines were simultaneously detected using ionspray ionization–mass spectrometry. Both ¹⁵N-cadaverine and ¹⁵N-lysine were taken up by soybean. ¹⁵N-lysine was transported to the shoot and root and converted into ¹⁵N-cadaverine, whereas relatively little ¹⁵N-cadaverine was formed from ¹⁵N-lysine in the cotyledon. The acropetal translocation of ¹⁵N-cadaverine from the cotyledon to the shoot seemed to predominate over basipetal transport to the root. Although no other ¹⁵N-derivatised polyamines were found, supplying exogenous ¹⁵N-lysine seemed to indirectly affect the metabolism of ¹⁴N putrescine, spermidine and spermine, while no significant effect was detected after supplying ¹⁵N-cadaverine.     

Effects of reconstituted HDL on charge-based LDL subfractions as characterized by capillary isotachophoresis

Modified LDL in human plasma including small, dense LDL (sdLDL) and oxidized LDL carries a more negative charge than unmodified LDL and is atherogenic. We examined the effects of apolipoprotein A-I (apoA-I)/POPC discs on charge-based LDL subfractions as determined by capillary isotachophoresis (cITP). Three normal healthy subjects and seven patients with metabolic disorders were included in the study. LDL in human plasma was separated into two major subfractions, fast- and slow-migrating LDL (fLDL and sLDL), by cITP. Normal LDL was characterized by low fLDL, and mildly oxidized LDL in vitro and mildly modified LDL in human plasma were characterized by increased fLDL. Moderately oxidized LDL in vitro and moderately modified LDL in a patient with hypertriglyceridemia and HDL deficiency were characterized by both increased fLDL and a new LDL subfraction with a faster mobility than fLDL [very-fast-migrating LDL as determined by cITP (vfLDL)]. cITP LDL subfractions with faster electrophoretic mobility (fLDL vs. sLDL, vfLDL vs. fLDL) were associated with an increased content of sdLDL. Incubation of a plasma fraction with d>1.019 g/ml (depleted of triglyceride-rich lipoproteins) in the presence of apoA-I/POPC discs at 37 degrees C greatly decreased vfLDL and fLDL but increased sLDL. Incubation of whole plasma from patients with an altered distribution of cITP LDL subfractions in the presence of apoA-I/POPC discs also greatly decreased fLDL but increased sLDL. ApoA-I/POPC discs decreased the cITP fLDL level, the free cholesterol concentration, and platelet-activating factor acetylhydrolase activity in the sdLDL subclasses (d=1.040-1.063 g/ml) and increased the size of LDL. ApoA-I/POPC discs reduced charge-modified LDL in human plasma by remodeling cITP fLDL into sLDL subfractions.     

Delayed treatment with cannabidiol has a cerebroprotective action via a cannabinoid receptor-independent myeloperoxidase-inhibiting mechanism

We examined the neuroprotective mechanism of cannabidiol, non-psychoactive component of marijuana, on the infarction in a 4 h mouse middle cerebral artery (MCA) occlusion model in comparison with Delta(9)-tetrahydrocannabinol (Delta(9)-THC). Release of glutamate in the cortex was measured at 2 h after MCA occlusion. Myeloperoxidase (MPO) and cerebral blood flow were measured at 1 h after reperfusion. In addition, infarct size and MPO were determined at 24 and 72 h after MCA occlusion. The neuroprotective effect of cannabidiol was not inhibited by either SR141716 or AM630. Both pre- and post-ischemic treatment with cannabidiol resulted in potent and long-lasting neuroprotection, whereas only pre-ischemic treatment with Delta(9)-THC reduced the infarction. Unlike Delta(9)-THC, cannabidiol did not affect the excess release of glutamate in the cortex after occlusion. Cannabidiol suppressed the decrease in cerebral blood flow by the failure of cerebral microcirculation after reperfusion and inhibited MPO activity in neutrophils. Furthermore, the number of MPO-immunopositive cells was reduced in the ipsilateral hemisphere in cannabidiol-treated group. Cannabidiol provides potent and long-lasting neuroprotection through an anti-inflammatory CB(1) receptor-independent mechanism, suggesting that cannabidiol will have a palliative action and open new therapeutic possibilities for treating cerebrovascular disorders.     

Highly conserved configuration of catalytic amino acid residues among calicivirus-encoded proteases

A common feature of caliciviruses is the proteolytic processing of the viral polyprotein catalyzed by the viral 3C-like protease encoded in open reading frame 1 (ORF1). Here we report the identification and structural characterization of the protease domains and amino acid residues in sapovirus (SaV) and feline calicivirus (FCV). The in vitro expression and processing of a panel of truncated ORF1 polyproteins and corresponding mutant forms showed that the functional protease domain is 146 amino acids (aa) in SaV and 154 aa in FCV. Site-directed mutagenesis of the protease domains identified four amino acid residues essential to protease activities: H(31), E(52), C(116), and H(131) in SaV and H(39), E(60), C(122), and H(137) in FCV. A computer-assisted structural analysis showed that despite high levels of diversity in the primary structures of the protease domains in the family Caliciviridae, the configurations of the H, E, C, and H residues are highly conserved, with these residues positioned closely along the inner surface of the potential binding cleft for the substrate. These results strongly suggest that the H, E, C, and H residues are involved in the formation of a conserved catalytic surface of the SaV and FCV 3C-like proteases.     

Sphingosine 1-phosphate induces cyclooxygenase-2 via Ca2+-dependent, but MAPK-independent mechanism in rat vascular smooth muscle cells

The effects of sphingosine 1-phosphate (S1P) on prostaglandin I(2) (PGI(2)) production and cyclooxygenase (COX) expression in cultured rat vascular smooth muscle cells (VSMCs) were investigated. S1P stimulated PGI(2) production in a concentration-dependent manner, which was completely suppressed by NS-398, a selective COX-2 inhibitor, as determined by radioimmunoassay. S1P stimulated COX-2 protein and mRNA expressions in a concentration- and time-dependent manner, while it had no effect on COX-1 expression. S1P(2) and S1P(3) receptors mRNA were abundantly expressed in rat VSMCs. Suramin, an antagonist of S1P(3) receptor, almost completely inhibited S1P-induced COX-2 expression. Pretreatment of VSMCs with pertussis toxin (PTX) partially, but significantly inhibited S1P-induced PGI(2) production and COX-2 expression. S1P also activated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). However, neither PD 98059, a selective inhibitor of ERK activation, nor SB 203580, a selective inhibitor of p38 MAPK, had a significant inhibitory effect on S1P-induced COX-2 expression, suggesting that the MAPK activation does not play main roles in S1P-induced COX-2 induction. S1P-induced COX-2 expression was inhibited by PP2, an inhibitor of Src-family tyrosine kinase, Ca(2+) depletion, and GF 109203X, an inhibitor of protein kinase C (PKC). These results suggest that S1P stimulates COX-2 induction in rat VSMCs through mechanisms involving Ca(2+)-dependent PKC and Src-family tyrosine kinase activation via S1P(3) receptor coupled to PTX-sensitive and -insensitive G proteins.