Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli

Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8-1%, in which approximately 20-50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.     

Cloning and characterization of a SnRK1-encoding gene from <Emphasis Type="Italic">Malus hupehensis</Emphasis> Rehd. and heterologous expression in tomato

Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) plays an important role in metabolic regulation in plant. To understand the molecular mechanism of amino acids and carbohydrate metabolism in Malus hupehensis Rehd. var. pinyiensis Jiang (Pingyi Tiancha, PYTC), a full-length cDNA clone encoding homologue of SnRK1 was isolated from PYTC by Rapid Amplification of cDNA Ends (RACE). The clone, designated as MhSnRK1, contains 2063 nucleotides with an open reading frame of 1548 nucleotides. The deduced 515 amino acids showed high identities with other plant SnRK1 genes. Quantitative real-time PCR analysis revealed this gene was expressed in roots, stems and leaves. Exposing seedlings to nitrate caused and initial decrease in expression of the MhSnRK1 gene in roots, leaves and stems in short term. Ectopic expression of MhSnRK1 in tomato mainly resulted in higher starch content in leaf and red-ripening fruit than wild-type plants. This result supports the hypothesis that overexpression of SnRK1 causes the accumulation of starch in plant cells. All the results suggest that MhSnRK1 may play important roles in carbohydrate and amino acid metabolisms.     

Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma

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Molecular determinants of voltage-dependent slow inactivation of the Ca2+ channel.

Ba(2+) current through the L-type Ca(2+) channel inactivates essentially by voltage-dependent mechanisms with fast and slow kinetics. Here we found that slow inactivation is mediated by an annular determinant composed of hydrophobic amino acids located near the cytoplasmic ends of transmembrane segments S6 of each repeat of the alpha(1C) subunit. We have determined the molecular requirements that completely obstruct slow inactivation. Critical interventions include simultaneous substitution of A752T in IIS6, V1165T in IIIS6, and I1475T in IVS6, each preventing in additive manner a considerable fraction of Ba(2+) current from inactivation. In addition, it requires the S405I mutation in segment IS6. The fractional inhibition of slow inactivation in tested mutants caused an acceleration of fast inactivation, suggesting that fast and slow inactivation mechanisms are linked. The channel lacking slow inactivation showed approximately 45% of the sustained Ba(2+) or Ca(2+) current with no indication of decay. The remaining fraction of the current was inactivated with a single-exponential decay (pi(f) approximately 10 ms), completely recovered from inactivation within 100 ms and did not exhibit Ca(2+)-dependent inactivation properties. No voltage-dependent characteristics were significantly changed, consistent with the C-type inactivation model suggesting constriction of the pore as the main mechanism possibly targeted by Ca(2+) sensors of inactivation.     

Isoliquiritigenin prevents hyperglycemia-induced renal injuries by inhibiting inflammation and oxidative stress via SIRT1-dependent mechanism

Diabetic nephropathy (DN) as a global health concern is closely related to inflammation and oxidation. Isoliquiritigenin (ISL), a natural flavonoid compound, has been demonstrated to inhibit inflammation in macrophages. Herein, we investigated the effect of ISL in protecting against the injury in STZ-induced type 1 DN and in high glucose-induced NRK-52E cells. In this study, it was revealed that the administration of ISL not only ameliorated renal fibrosis and apoptosis, but also induced the deterioration of renal function in diabetic mice. Mediated by MAPKs and Nrf-2 signaling pathways, respectively, upstream inflammatory response and oxidative stress were neutralized by ISL in vitro and in vivo. Moreover, as further revealed by the results of molecular docking, sirtuin 1 (SIRT1) binds to ISL directly, and the involvement of SIRT1 in ISL-mediated renoprotective effects was confirmed by studies using in vitro models of SIRT1 overexpression and knockdown. In summary, by reducing inflammation and oxidative stress, ISL has a significant pharmacological effect on the deterioration of DN. The benefits of ISL are associated with the direct binding to SIRT1, the inhibition of MAPK activation, and the induction of Nrf-2 signaling, suggesting the potential of ISL for DN treatment.Subject terms: Pharmacology, Molecular biology     

Immunological characterization of cytochrome H-450 in rat tissues

Rabbit antiserum produced against rat liver cytochrome H-450 was specific for cytochrome H-450. The antiserum did not react with hemolysate, microsomal and mitochondrial fractions of liver, and tissue extracts from heart, lung skeletal muscle, and testis of rat. With the monospecific antiserum, a rocket immunoelectrophoretic assay method was developed for the quantitation of the antigen with a sensitivity of 25 ng. By using rocket immunoelectrophoresis, the total amounts of the antigen found in liver, kidney, and brain of 20 rats were 33.6, 3.6, and 1.3 mg, respectively. It appears that the antigens in liver, kidney, and brain are immunologically identical. From immunological studies with subcellular fractions of rat liver, the antigen was found only in the postmicrosomal fraction. This indicates that the antigen is not a precursor or a proteolytic product of known cytochromes in mitochondria or microsomes. Therefore, cytochrome H-450 is a unique cytosolic protein found in brain, kidney, and liver.