4-Vinylphenol production from glucose using recombinant <Emphasis Type="Italic">Streptomyces mobaraense</Emphasis> expressing a tyrosine ammonia lyase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Objectives

To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species.

Results

The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme’s k_cat/K_m value was 0.54 mM ^?1 s^?1. Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l^?1 from 10 g glucose l^?1.

Conclusion

A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.

     

Mtu1-Mediated Thiouridine Formation of Mitochondrial tRNAs Is Required for Mitochondrial Translation and Is Involved in Reversible Infantile Liver Injury

Reversible infantile liver failure (RILF) is a unique heritable liver disease characterized by acute liver failure followed by spontaneous recovery at an early stage of life. Genetic mutations in MTU1 have been identified in RILF patients. MTU1 is a mitochondrial enzyme that catalyzes the 2-thiolation of 5-taurinomethyl-2-thiouridine (τm⁵s²U) found in the anticodon of a subset of mitochondrial tRNAs (mt-tRNAs). Although the genetic basis of RILF is clear, the molecular mechanism that drives the pathogenesis remains elusive. We here generated liver-specific knockout of Mtu1 (Mtu1^LKO) mice, which exhibited symptoms of liver injury characterized by hepatic inflammation and elevated levels of plasma lactate and AST. Mechanistically, Mtu1 deficiency resulted in a loss of 2-thiolation in mt-tRNAs, which led to a marked impairment of mitochondrial translation. Consequently, Mtu1^LKO mice exhibited severe disruption of mitochondrial membrane integrity and a broad decrease in respiratory complex activities in the hepatocytes. Interestingly, mitochondrial dysfunction induced signaling pathways related to mitochondrial proliferation and the suppression of oxidative stress. The present study demonstrates that Mtu1-dependent 2-thiolation of mt-tRNA is indispensable for mitochondrial translation and that Mtu1 deficiency is a primary cause of RILF. In addition, Mtu1 deficiency is associated with multiple cytoprotective pathways that might prevent catastrophic liver failure and assist in the recovery from liver injury.     

Combined brain‐derived neurotrophic factor with extinction training alleviate impaired fear extinction in an animal model of post‐traumatic stress disorder

Impaired fear memory extinction (Ext) is one of the hallmark symptoms of post‐traumatic stress disorder (PTSD). However, since the precise mechanism of impaired Ext remains unknown, effective interventions have not yet been established. Recently, hippocampal‐prefrontal brain‐derived neurotrophic factor (BDNF) activity was shown to be crucial for Ext in naïve rats. We therefore examined whether decreased hippocampal‐prefrontal BDNF activity is also involved in the Ext of rats subjected to a single prolonged stress (SPS) as a model of PTSD. BDNF levels were measured by enzyme‐linked immunosorbent assay (ELISA), and phosphorylation of TrkB was measured by immunohistochemistry in the hippocampus and medial prefrontal cortex (mPFC) of SPS rats. We also examined whether BDNF infusion into the ventral mPFC or hippocampus alleviated the impaired Ext of SPS rats in the contextual fear conditioning paradigm. SPS significantly decreased the levels of BDNF in both the hippocampus and mPFC and TrkB phosphorylation in the ventral mPFC. Infusion of BDNF 24 hours after conditioning in the infralimbic cortex (ILC), but not the prelimbic cortex (PLC) nor hippocampus, alleviated the impairment of Ext. Since amelioration of impaired Ext by BDNF infusion did not occur without extinction training, it seems the two interventions must occur consecutively to alleviate impaired Ext. Additionally, BDNF infusion markedly increased TrkB phosphorylation in the ILC of SPS rats. These findings suggest that decreased BDNF signal transduction might be involved in the impaired Ext of SPS rats, and that activation of the BDNF‐TrkB signal might be a novel therapeutic strategy for the impaired Ext by stress.     

N’-acyl-N-methylphenylenediamine as a novel proximity labeling agent for signal amplification in immunohistochemistry

We synthesized novel phenylenediamine derivatives and evaluated them as labeling agents to label proteins in close proximity to a single electron transfer catalyst. We found that N’-acyl-N-methylphenylenediamine labels tyrosine effectively in a model experiment using tris(bipyridine)ruthenium (Ru(bpy)₃²⁺) as the single electron transfer catalyst. By changing the substituents on the nitrogen atom of the phenylenediamine derivatives, the electrochemical properties of the labeling agent can be drastically changed. On the other hand, horseradish peroxidase (HRP) also catalyzes the reaction with almost the same oxidation potential as Ru(bpy)₃²⁺ (～+1.1?V). HRP proximity labeling is applicable to signal amplification in immunohistochemistry. We evaluated the phenylenediamine derivatives as labeling agents for HRP proximity labeling and signal amplification, and found that N’-acyl-N-methylphenylenediamine is a novel and efficient agent for signal amplification using HRP in immunohistochemistry.     

Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2)

Streptomyces and other bacterial actinomycete species produce many important natural products, including the majority of known antibiotics, and cytochrome P450 (P450) enzymes catalyze important biosynthetic steps. Relatively few electron transport pathways to P450s have been characterized in bacteria, particularly streptomycete species. One of the 18 P450s in Streptomyces coelicolor A3(2), P450 105D5, was found to bind fatty acids tightly and form hydroxylated products when electrons were delivered from heterologous systems. The six ferredoxin (Fdx) and four flavoprotein Fdx reductase (FDR) proteins coded by genes in S. coelicolor were expressed in Escherichia coli, purified, and used to characterize the electron transfer pathway. Of the many possibilities, the primary pathway was NADH --> FDR1 --> Fdx4 --> P450 105D5. The genes coding for FDR1, Fdx4, and P450 105D5 are located close together in the S. coelicolor genome. Several fatty acids examined were substrates, including those found in S. coelicolor extracts, and all yielded several products. Mass spectra of the products of lauric acid imply the 8-, 9-, 10-, and 11-hydroxy derivatives. Hydroxylated fatty acids were also detected in vivo in S. coelicolor. Rates of electron transfer between the proteins were measured; all steps were faster than overall hydroxylation and consistent with rates of NADH oxidation. Substrate binding, product release, and oxygen binding were relatively fast in the catalytic cycle; high kinetic deuterium isotope effects for all four lauric acid hydroxylations indicated that the rate of C-H bond breaking is rate-limiting in every case. Thus, an electron transfer pathway to a functional Streptomyces P450 has been established.     

The content of protein, fibre and minerals of leaves of selected Acacia species indigenous to north-western Tanzania

Browse tree leaves of six species of Acacia (A. angustissima L., A. drepanolobium L., A. nilotica L., A. polyacantha L., A. senegal L., A. tortilis L.) were screened for chemical composition, including minerals and trace elements. Crude protein (CP) varied among the species from 145 (A. senegal) to 229 g/kg DM (A. angustissima). The species had moderate to high levels of minerals. The concentrations of Ca, P, Mg and S varied among the species from 14.6-31.5, 3.5-4.9, 1.4-3.0 and 1.7-2.8 g/kg DM, respectively. The forages showed relatively low concentrations of trace elements. Content of trace elements varied among the species from 4.5-23.8, 99.4-173.6, 146.2-432, 41.0-90.1, 10.9-22.2 and 0.05-0.65 mg/kg DM for Cu, Mo, Fe, Mn, Zn and Co, respectively. All leaves of browse species would meet the normal requirements for Ca, P, Mg and S in ruminants, although some species had higher levels of Ca than tabulated mineral requirements in livestock. Assayed Cu, Mn, Zn and Co would satisfy the lower range of recommended requirements of trace elements depending on their bioavailability. Therefore, browse leaves from Acacias could form good sources of CP and mineral supplements to ruminants.     

Identification of human plasma C1 inhibitor as a target protein for staphylococcal superantigen-like protein 5 (SSL5)

The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection.     

Identification of triterpene biosynthetic genes from Momordica charantia using RNA-seq analysis

Cucurbitaceae plants contain characteristic triterpenoids. Momordica charantia, known as a bitter melon, contains cucurbitacins and multiflorane type triterpenes, which confer bitter tasting and exhibit pharmacological activities. Their carbon skeletons are biosynthesized from 2,3-oxidosqualene by responsible oxidosqualene cyclase (OSC). In order to identify OSCs in M. charantia, RNA-seq analysis was carried out from ten different tissues. The functional analysis of the resulting four OSC genes revealed that they were cucurbitadienol synthase (McCBS), isomultiflorenol synthase (McIMS), β-amyrin synthase (McBAS) and cycloartenol synthase (McCAS), respectively. Their distinct expression patterns based on RPKM values and quantitative RT-PCR suggested how the characteristic triterpenoids were biosynthesized in each tissue. Although cucurbitacins were finally accumulated in fruits, McCBS showed highest expression in leaves indicating that the early step of cucurbitacins biosynthesis takes place in leaves, but not in fruits.

Abbreviations: OSC: oxidosqualene cyclase; RPKM: reads perkilobase of exon per million mapped reads     

Suppression of plasma aldosterone by chronic ACTH administration is offset by converting enzyme inhibitor

In order to elucidate the mechanism of suppression of plasma aldosterone by chronic ACTH administration, especially the role of the renin-angiotensin system and dopamine, we administered ACTH with or without MK422, a converting enzyme inhibitor, to reduce the endogenous angiotensin II in rats, and measured the plasma renin activity, plasma corticoid concentrations and urinary dopamine excretion. The plasma aldosterone concentration (PAC) was decreased after chronic ACTH administration. However, in the ACTH + MK422 administered group, aldosterone suppression was not observed. It appeared therefore that the aldosterone suppressing mechanism was independent of the weakened renin-angiotensin system following chronic ACTH administration, since PAC was not decreased in the ACTH + MK422 administered group when angiotensin II might be completely eliminated. The urinary excretion of dopamine was significantly increased in the chronic ACTH + MK422 administered group as well as in the chronic ACTH administered group. This suggested that the inhibitory effect of dopamine on aldosterone did not contribute significantly to the suppression of plasma aldosterone. The present results suggest therefore that the mechanism of suppression of plasma aldosterone following chronic ACTH administration was not dependent on the renin-angiotensin system and dopamine.