Dynamic, ligand-dependent conformational change triggers reaction of ribose-1,5-bisphosphate isomerase from Thermococcus kodakarensis KOD1

Ribose-1,5-bisphosphate isomerase (R15Pi) is a novel enzyme recently identified as a member of an AMP metabolic pathway in archaea. The enzyme converts d-ribose 1,5-bisphosphate into ribulose 1,5-bisphosphate, providing the substrate for archaeal ribulose-1,5-bisphosphate carboxylase/oxygenases. We here report the crystal structures of R15Pi from Thermococcus kodakarensis KOD1 (Tk-R15Pi) with and without its substrate or product. Tk-R15Pi is a hexameric enzyme formed by the trimerization of dimer units. Biochemical analyses show that Tk-R15Pi only accepts the α-anomer of d-ribose 1,5-bisphosphate and that Cys(133) and Asp(202) residues are essential for ribulose 1,5-bisphosphate production. Comparison of the determined structures reveals that the unliganded and product-binding structures are in an open form, whereas the substrate-binding structure adopts a closed form, indicating domain movement upon substrate binding. The conformational change to the closed form optimizes active site configuration and also isolates the active site from the solvent, which may allow deprotonation of Cys(133) and protonation of Asp(202) to occur. The structural features of the substrate-binding form and biochemical evidence lead us to propose that the isomerase reaction proceeds via a cis-phosphoenolate intermediate.     

Protein-tyrosine phosphatase 1B associates with insulin receptor and negatively regulates insulin signaling without receptor internalization

Phosphorylated platelet-derived growth factor (PDGF) receptor becomes internalized and then is dephosphorylated by protein-tyrosine phosphatase (PTP) 1B at the endoplasmic reticulum (ER). However, it remains unclear where PTP1B dephosphorylates insulin receptor and inhibits its activity. To clarify how and where PTP1B could interact with insulin receptor, we overexpressed a phosphatase-inactive mutant, PTP1BC/S, in 3T3-L1 adipocytes. Although PDGF receptor was maximally associated with PTP1BC/S at 30 min after PDGF stimulation, the maximal association of insulin receptor with PTP1BC/S was attained at 5 min after insulin stimulation. Furthermore, dansylcadaverine, a blocker of receptor internalization, inhibited this PDGF-induced association of PTP1BC/S with its receptor. However, dansylcadaverine did not affect the insulin-stimulated association of PTP1BC/S with insulin receptor, as well as dephosphorylation of insulin receptor by PTP1B. These results indicate that PTP1B might interact with insulin receptor and deactivate it without internalization. Finally, we overexpressed the wild-type and cytosolic-form of PTP1B to determine the role of ER-anchoring of PTP1B, and found that both inhibited insulin signaling equally. Thus, our data indicate that localization of PTP1B at the ER is not needed for insulin receptor dephosphorylation by PTP1B.     

Adsorption and infectivity of human immunodeficiency virus type 1 are modified by the fluidity of the plasma membrane for multiple-site binding

Based on the assumption that fluidity of the plasma membrane and viral envelope is necessary for recruiting additional receptors and ligands to the initial attachment site for "multiple-site binding," we determined the effect of increased temperature on viral infectivity. Infection of human immunodeficiency virus type 1 (HIV-1) and a pseudotyped luciferase-expressing chimeric virus using MAGI and GHOST/CXCR4 cells showed that in 1 hr of viral adsorption the extent of virus infection and the amount of tightly adsorbed viruses depended on temperature; and that membrane fluidity increased according to increased temperature. Augmented infection was observed as post-attachment enhancement (PAE) when cells were washed and incubated at 40 C for 1 hr after viral adsorption. PAE was completely inhibited by 1 micro M of anti-CXCR4 peptide T140, and addition of T140 at 20 min resulted in a gradual loss of inhibition of PAE, indicating the need for a 30 to 40 min timelag to ensure tight multiple-site binding. These data suggest that the accumulation of gp120 and receptor complex (multiple-site binding) was needed to complete the infection. Treatments of cells with 0.05% Tween 20 or 2 micro g/ml of anti-HLA-II antibody resulted in increases or decreases, respectively, of attached viruses and the infectivity. As well, Tween 20 and anti-HLAII antibody enhanced and suppressed the fluidity of the plasma membrane, respectively. Amounts of adsorbed viruses and degrees of viral infectivity correlated with the intensity of fluidity of the plasma membrane, probably due to the formation of multiple-site binding.     

Gender-related differences in expression and function of hepatic P-glycoprotein and multidrug resistance-associated protein (Mrp2) in rats

To clarify whether gender-related differences exist in the expression and function of hepatic P-glycoprotein- and/or multidrug resistance-associated protein (Mrp2), we measured the hepatobiliary excretion of doxorubicin and their protein levels in male and female Sprague-Dawley rats. When rats received a single intravenous injection of doxorubicin (5 mg/kg), a delay in the disappearance of doxorubicin from plasma was observed in male rats. When rats received a constant-rate infusion of doxorubicin, no significant gender-related differences in the apparent biliary clearance of doxorubicin based on the steady state plasma concentrations were observed between male and female rats. However, the net biliary clearance of doxorubicin based on the liver concentration, which represents the actual function of P-glycoprotein and/or Mrp2, was higher in female rats than in male rats. These results suggest that the actual function of the hepatobiliary transport of doxorubicin is greater in female than in male rats. Western blot analysis revealed that the expression of P-glycoprotein and Mrp2 in the liver of female rats was significantly higher than in male rats, similar to results of hepatobiliary excretion experiments. The expression of hepatic cytochrome P450 (CYP) 2B1, which is involved in the metabolism of doxorubicin, was significantly higher in male than in female rats. By pretreatment with testosterone (10 mg/day for 7 days), the actual biliary clearance of doxorubicin in female rats was nearly that of male rats. The protein levels of P-glycoprotein and Mrp2 in female rats were also lowered by treatment with testosterone so as to be nearer those in male rats. These results suggest that gender-related differences exist in P-glycoprotein- and Mrp2-mediated hepatobiliary transport and that these two transporters may be regulated by sex hormones.     

Aspirin prevents adhesion of T lymphoblasts to vascular smooth muscle cells

In the development of atherosclerosis, inflammatory cells adhere to and migrate into the vascular walls by interacting with vascular smooth muscle cells. To investigate the mechanism of aspirin’s anti-atherogenic activity, we examined whether aspirin inhibits the adhesion of lymphocytes to human aortic smooth muscle cells (AoSMC). Aspirin inhibited T-cell adhesion to AoSMC activated by interleukin 1β (IL-1β) in a dose-dependent manner. Antibodies to the adhesion molecules ICAM-1 or VCAM-1, but not to E-selectin, prevented T-cell adhesion. ICAM-1 and VCAM-1 expression stimulated by IL-1β was reduced by the treatment with aspirin, whereas the expression of E-selectin was unaffected. Nuclear factor κB (NF-κB) activity was enhanced by IL-1β and reduced by aspirin, indicating that decreased ICAM-1 and VCAM-1 expression was due to reduced NF-κB activity.Thus, aspirin inhibits the adhesion of Jurkat T cells to IL-1β-activated AoSMC by reducing NF-κB activity and decreasing expression of ICAM-1 and VCAM-1, and may prevent the development of atherosclerosis.     

<Emphasis Type="Italic">Phytophthora colocasiae</Emphasis> from Vietnam,China, Hawaii and Nepal: intra- and inter-genomic variations in ploidy and a long-lived,diploid Hawaiian lineage.

Phytophthora colocasiae is an important pathogen of taro and is widely distributed. Our goal was to develop whole genome sequence and single nucleotide polymorphism (SNP) markers to characterize historical and current populations from Hawaii (2010 and 2016, HA), historical isolates from Vietnam and China (2010, VN and CH) and current isolates from Nepal (2016, NEP). Seven isolates (VN = 2, CH = 1, HA = 1, NEP = 3) were sequenced (NCBI BioProject PRJNA378784) and compared using the reference genome of the closely related vegetable pathogen P. capsici. Genome-wide SNP analysis using 27,537 markers revealed genomes of diploid, triploid, tetraploid and higher ploidy. Ploidy varied within and between populations, with HA being primarily diploid, CH primarily triploid, VN containing diploid and triploid isolates, and NEP having predominantly triploid, tetraploid and higher ploidy. A total of 37 SNP markers were genotyped in 89 samples (grown in culture or directly from infected tissue) using targeted-sequencing. Analyses indicate a single clonal lineage dominated populations in HA from 2010 to 2016 and targeted-sequencing was useful to estimate ploidy. The implications for adaptation and evolution of P. colocasiae are discussed, as well as consequences for selection and breeding of resistant taro cultivars.     

Allozyme divergence between two groups of the Japanese spinous loach, <Emphasis Type="Italic">Cobitis takatsuensis</Emphasis>

Phylogenetic relationships of the Chugoku-Kyushu and Shikoku groups of the Japanese spinous loach, Cobitis takatsuensis, among the diploid congeners in Japan, C. biwae and Cobitis sp. complex with Niwaella delicata as an outgroup, were investigated by analyzing 20 protein-coding loci. The two groups of C. takatsuensis are clearly diverged genetically, with the genetic distance corresponding to the species level (average D=0.27). The two groups form a monophyletic cluster (bootstrap probability, P=94.9%) that is a sister cluster of C. biwae (P=63.1%). The monophyletic cluster of the Cobitis sp. complex (P=93.2%) is the most distantly related in the genus. The present results differ mostly from the mitochondrial phylogeny previously known in which the Chugoku-Kyushu group of C. takatsuensis is connected to the eastern group of C. biwae and the Shikoku group is connected to the Cobitis sp. complex with the western group of C. biwae. The contradiction between the allozymic and mitochondrial phylogenies suggests that the Shikoku group of C. takatsuensis and the western group of C. biwae received mitochondrial introgression from the Cobitis sp. complex.     

Lactate utilization as an energy substrate in ischemic preconditioned rat brain slices

We examined the utilization of lactate as an energy substrate in ischemic preconditioned slices obtained from the rat brain left hemisphere, of which the contralateral middle cerebral artery was occluded 48 h before the slice preparation. The levels of high-energy phosphates in the brain slices were measured using 31P NMR with a time resolution of 4 min at 25 degrees C. When iodoacetic acid-pretreated brain slices were further treated with fluorocitrate, a glial toxin, for 2 h (neuron-rich slices), the recovery of the phosphocreatine (PCr) level in artificial cerebrospinal fluid (ACSF) containing lactate after high-K+ stimulation was completely abolished in intact slices, whereas the PCr level in ischemic preconditioned slices well recovered in otherwise similar conditions. These results indicated that neurons, when preconditioned with ischemia, acquire the ability to utilize lactate as an energy substrate. In parallel experiments, we recorded population excitatory postsynaptic potentials and spikes from granule cells in hippocampal slices. Population spikes of intact slices in ACSF containing lactate were completely abolished in 30 min, but those of the ischemic preconditioned slices were maintained well over 50%. These results show that ischemic preconditioning may induce certain systematic changes in neurons, such as the expression of lactate transporters and/or the activation of lactate dehydrogenase.     

Analysis of the mechanism of the tight-junctional permeability increase by capsaicin treatment on the intestinal Caco-2 cells

In a previous experiment (Isoda et al., 2001), we showed that the tight-junctional (TJ) permeability increase in Caco-2 cells during capsaicin exposure was through binding of the capsaicin molecule to a capsaicin receptor-like protein. In the present study, we examined how actin, which modulates TJ permeability, is influenced by capsaicin. We showed that after treatment of the Caco-2 cells with capsaicin, the volume of F-actin decreased. Moreover, we also examined protein kinase C (PKC) and heat shock protein 47 (HSP47), which act as probable second messengers in causing TJ permeability increase. We showed that after capsaicin treatment, HSP47 was activated. However, PKC activity was the same in both control and treatment setups. These results suggest that, while PKC is not involved, it is highly possible that HSP47plays a role in TJ permeability increase in intestinal Caco-2 cells exposed to capsaicin. This revised version was published online in August 2006 with corrections to the Cover Date.