Mint3 is dispensable for pancreatic and kidney functions in mice

Munc-18 interacting protein 3 (Mint3) is an activator of hypoxia-inducible factor-1 in cancer cells, macrophages, and cancer-associated fibroblasts under pathological conditions. However, exactly which cells highly express Mint3 in vivo and whether Mint3 depletion affects their physiological functions remain unclear. Here, we surveyed mouse tissues for specific expression of Mint3 by comparing Mint3 expression in wild-type and Mint3-knockout mice. Interestingly, immunohistochemical analyses revealed that Mint3 was highly expressed in islet cells of the pancreas, distal tubular epithelia of the kidney, choroid plexus ependymal cells of the cerebrum, medullary cells of the adrenal gland, and epithelial cells of the seminal gland. We also studied whether Mint3 depletion affects the physiological functions of the islets and kidneys. Mint3-knockout mice did not show any abnormalities in glucose-tolerance and urine-biochemical tests, indicating that Mint3 depletion was compensated for in these organs. Thus, loss of Mint3 might be compensated in the islets and kidneys under physiological conditions in mice.     

Formation of new polyhydroxyalkanoate containing 3-hydroxy-4-methylvalerate monomer in Burkholderia sp

Burkholderia sp. synthase has been shown to polymerize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate, and 3-hydroxy-4-pentenoic acid monomers. This study was carried out to evaluate the ability of Burkholderia sp. USM (JCM 15050) and its transformant harboring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae to incorporate the newly reported 3-hydroxy-4-methylvalerate (3H4MV) monomer. Various culture parameters such as concentrations of nutrient rich medium, fructose and 4-methylvaleric acid as well as harvesting time were manipulated to produce P(3HB-co-3H4MV) with different 3H4MV compositions. The structural properties of PHA containing 3H4MV monomer were investigated by using nuclear magnetic resonance and Fourier transform infrared spectroscopy (FTIR). The relative intensities of the bands at 1,183 and 1,228 cm⁻¹ in the FTIR spectra enabled the rapid detection and differentiation of P(3HB-co-3H4MV) from other types of PHA. In addition, the presence of 3H4MV units in the copolymer was found to considerably lower the melting temperature and enthalpy of fusion values compared with poly(3-hydroxybutyrate) (P(3HB)). The copolymer exhibited higher thermo-degradation temperature but similar molecular weight and polydispersity compared with P(3HB).     

Inhibitory effects of excess sympathetic activity on parasympathetic vasodilation in the rat masseter muscle

The present study was designed to examine the effect of sympathetic tonic activity on parasympathetic vasodilation evoked by the trigeminal-mediated reflex in the masseter muscle in urethane-anesthetized rats. Sectioning of the superior cervical sympathetic trunk (CST) ipsilaterally increased the basal level of blood flow in the masseter muscle (MBF). Electrical stimulation of the peripheral cut end of the CST for 2 min using 2-ms pulses ipsilaterally decreased in a dependent manner the intensity (0.5-10 V) and frequency (0.1-5 Hz) of the MBF. The CST stimulation for 2 min at <0.5 Hz with 5 V using 2-ms pulses seems to be comparable with the spontaneous activity in the CST fibers innervating the masseter vasculature, because this stimulation restored the basal level of the MBF to the presectioned values. Parasympathetic vasodilation evoked by electrical stimulation of the central cut end of the lingual nerve in the masseter muscle was markedly reduced by CST stimulation for 2 min with 5 V using 2-ms pulses in a frequency-dependent manner (0.5-5 Hz). Intravenous administration of phentolamine significantly reduced the vasoconstriction induced by CST stimulation in a dose-dependent manner (0.1-1 mg/kg), but pretreatment with either phentolamine or propranolol failed to affect the sympathetic inhibition of the parasympathetic vasodilation. Our results suggest that 1) excess sympathetic activity inhibits parasympathetic vasodilation in the masseter muscle, and 2) alpha- and beta-adrenoceptors do not contribute to sympathetic inhibition of parasympathetic vasodilation, and thus some other types of receptors must be involved in this response.     

Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick Haemaphysalis longicornis

We characterize here a cDNA from the ixodid tick Haemaphysalis longicornis, which encodes an asparaginyl endopeptidase, legumain (HlLgm), that was present as a functional molecule in the midgut of this tick. Endogenous HlLgm was detected as a 38-kDa antigen in H. longicornis extracts and was seen throughout all developmental stages. Endogenous HlLgm was mainly localized in the midgut epithelium by immunohistochemistry, and was shown to be up-regulated by the host blood-feeding process. Recombinant HlLgm (rHlLgm) produced in Escherichia coli was shown to hydrolyze the synthetic substrate Z-Ala-Ala-Asn-MCA at the rate of 6.42x10(-4)mumol/min/mg protein. Its activity was inhibited by the thiol blocking reagents iodoacetamide and N-ethylmaleimide. The enzyme was shown to possess a unique feature of having an autocatalyzed cleavage at asparagines(364-365) at the C-terminus of both endogenous HlLgm and rHlLgm. rHlLgm degraded bovine hemoglobin and bovine serum albumin (BSA) showing its strict specificity for hydrolysis of the peptide on the carboxyl side of the asparagines, as demonstrated by internal amino acid sequence analysis of proteolytic product of BSA cleavage. These results suggest that HlLgm plays an important role in host blood-meal digestion and may be critical for the final process of digestion of blood components.     

Structural analysis of four and half LIM protein-2 in dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a cardiac disease characterized by dilated ventricle and systolic dysfunction. Most of the DCM patients are sporadic cases, but a certain population of DCM patients can be familial cases caused by mutations in genes for sarcomere/Z-disc components including titin/connectin. However, disease-causing mutations could be identified only in a part of the familial DCM patients, suggesting that there should be other disease causing genes for DCM. To explore a novel disease gene for DCM, we searched for mutations in FHL2, encoding for four and half LIM protein 2 (FHL2) in DCM patients, because FHL2 is known to associate with titin/connectin. A missense mutation, Gly48Ser, was identified in a patient with familial DCM. Functional analysis demonstrated that the FHL2 mutation affected the binding to titin/connectin. Because FHL2 protein is known to tether metabolic enzymes to titin/connectin, these observations suggest that the Gly48Ser mutation may be involved in the pathogenesis of DCM via impaired recruitment of metabolic enzymes to the sarcomere.     

Characterization of N-deoxyribosyltransferase from Lactococcus lactis subsp. lactis

A nucleoside N-deoxyribosyltransferase-homologous gene was detected by homological search in the genomic DNA of Lactococcus lactis subsp. lactis. The gene yejD is composed of 477 nucleotides encoding 159 amino acids with only 25% identity, which is low in comparison to the amino acid sequences of the N-deoxyribosyltransferases from other lactic acid bacteria, i.e. Lactobacillus leichmannii and Lactobacillus helveticus. The residues responsible for catalytic and substrate-binding sites in known enzymes are conserved at Gln49, Asp73, Asp93 (or Asp95), and Glu101, respectively. The recombinant YejD expressed in Escherichia coli shows a 2-deoxyribosyl transfer activity to and from both bases of purine and pyrimidine, showing that YejD should be categorized as a class II N-deoxyribosyltransferase. Interestingly, the base-exchange activity as well as the heat stability of YejD was enhanced by the presence of monovalent cations such as K(+), NH(4)(+), and Rb(+), indicating that the Lactococcus enzyme is a K(+)-activated Type II enzyme. However, divalent cations including Mg(2+) and Ca(2+) significantly inhibit the activity. Whether or not the yejD gene product actually participates in the nucleoside salvage pathway of Lc. lactis remains unclear, but the lactic acid bacterium possesses the gene coding for the nucleoside N-deoxyribosyltransferase activated by K(+) on its genome.     

Significance of PGR5-dependent cyclic electron flow for optimizing the rate of ATP synthesis and consumption in Arabidopsis chloroplasts

Photosynthesis Research - The proton motive force (PMF) across the chloroplast thylakoid membrane that is generated by electron transport during photosynthesis is the driving force for ATP...     

Microbial Secretion Platform for 3‐Hydroxybutyrate Oligomer and Its End‐Capped Forms Using Chain Transfer Reaction‐Mediated Polyhydroxyalkanoate Synthases

The biodegradable polyester 3‐hydroxybutyrate (3HB) polymer [P(3HB)] is intracellularly synthesized and accumulated in recombinant Escherichia coli. In this study, native polyhydroxyalkanoate (PHA) synthases are used to attempt to microbially secrete 3HB homo‐oligomers (3HBOs), which are widely distributed in nature as physiologically active substances. High secretory production is observed, especially for the two PHA synthases from Aeromonas caviae and Bacillus cereus YB4. Surprisingly, an ethyl ester at the carboxy terminus (ethyl ester form) of 3HBOs is identified for most of the PHA synthases tested. Next, 3HBOs with a functional carboxyl group (carboxyl form of 3HBO) are obtained by using the alcohol dehydrogenase gene (adhE)‐deficient mutant strain, suggesting that the endogenous ethanol produced in E. coli acts as a chain transfer (CT) agent in the generation of 3HBOs. Furthermore, an in vitro polymerization assay reveals that CT agents such as ethanol and free 3HB are involved in the generation of ethyl ester and carboxyl form of 3HBO, respectively. The microbial platform established herein allows the secretion of 3HBOs with desirable end structures by supplementation with various CT agents. The obtained 3HBOs and their end‐capped forms may be used as physiologically active substances and building blocks for polymeric materials.     

Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaC_Ac). In in vitro assays, among the three PhaCs tested, PhaC_Ac was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaC_Ac), whereas the prepolymerization PhaC_Re (derived from Ralstonia eutropha) and PhaC_Da (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaC_Ac showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C₆). PhaP_Ac also activated PhaC_Ac when it was added during polymerization (polymer-elongating PhaC_Ac), while this effect was not observed for PhaC_Re. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaP_Ac expression on PHA synthesis by PhaC_Ac or PhaC_Re was examined. As PhaP_Ac expression increased, PHA production was increased by up to 2.3-fold in the PhaC_Ac-expressing strain, whereas it was slightly increased in the PhaC_Re-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaC_Ac both in vitro and in vivo but do not activate PhaC_Re. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaC_Ac.     

Alcoholytic Cleavage of Polyhydroxyalkanoate Chains by Class IV Synthases Induced by Endogenous and Exogenous Ethanol

Polyhydroxyalkanoate (PHA)-producing Bacillus strains express class IV PHA synthase, which is composed of the subunits PhaR and PhaC. Recombinant Escherichia coli expressing PHA synthase from Bacillus cereus strain YB-4 (PhaRC_YB-4) showed an unusual reduction of the molecular weight of PHA produced during the stationary phase of growth. Nuclear magnetic resonance analysis of the low-molecular-weight PHA revealed that its carboxy end structure was capped by ethanol, suggesting that the molecular weight reduction was the result of alcoholytic cleavage of PHA chains by PhaRC_YB-4 induced by endogenous ethanol. This scission reaction was also induced by exogenous ethanol in both in vivo and in vitro assays. In addition, PhaRC_YB-4 was observed to have alcoholysis activity for PHA chains synthesized by other synthases. The PHA synthase from Bacillus megaterium (PhaRC_Bm) from another subgroup of class IV synthases was also assayed and was shown to have weak alcoholysis activity for PHA chains. These results suggest that class IV synthases may commonly share alcoholysis activity as an inherent feature.