Expression of glucoamylase gene usingSUC2 promoter inSaccharomyces cerevisiae

Summary The cloning of glucoamylase geneSTA using theSUC2 promoter intoSaccharomyces cerevisiae was performed. The signal sequence ofSTA gene was used for the secretion of glucoamylase protein. The plasmid constructed in this way was named YEpSUCSTA and its expression was identified. The expression of YEpSUCSTA was repressed in the presence of glucose in growth medium, but derepressed when glucose became depleted. YEpSUCSTA showed the similar efficiency of glucoamylase secretion as YEpSTA-F which has the entireSTA gene. Glucoamylase activity in starch-glucose medium was largely increased because cell mass and plasmid stability were high in biosynthesis phase compared to extracellular glucoamylase activities in media which starch or glucose was the only carbon source.     

Evidence for a role of specific isoacceptor species of tRNA in amino acid transport.

Soybean leghemoglobins ā and b?were compared by microscale peptide mapping after heme removal with acid-acetone. Maps generated by trypsin or the combined action of trypsin and thermolysin indicated a large amount of homology between the proteins with the only variations detected being the N-terminal peptides. The N-terminal tryptic peptide of leghemoglobin b? was found to be both blocked and to lack the first amino acid of the corresponding leghemoglobin ā peptide. Nuclear magnetic resonance and gas chromatography/mass spectroscopy studies showed that the N-terminal of leghemoglobin b? was N-acetyl-alanine. It is possible that leghemoglobin b? arises from leghemoglobin ā by a two-stage modification involving cleavage of the N-terminal valyl residue and subsequent acetylation of the exposed alanyl residue.     

Electron microscopic localization of monoamine oxidase in the rat liver

Summary Two methods have been employed to localize monoamine oxidase activity in the cells of rat liver, using either 2-(2′-benzothiazolyl)-5-stryl-3-(4′-phtalhydrazidyl) tetrazolium chloride (BSPT) or ferricyanide as electron acceptor. With both methods monoamine oxidase activity was found both in the inner and the outer mitochondral membrane, although the outer membrane appeared the most probable location. In addition the BSPT method but not the ferricyanide method, revealed monoamine oxidase activity in the endoplasmatic reticulum. The results obtained by the two methods have been compared and are discussed in view of available biochemical data on monoamine oxidase. Supported by research grants from the National Research Council of Canada (A 3651), The Swedish Medical Research Council (4145) and M. Bergwall's Foundation, Stockholm.     

Molecular Modeling Study on Tunnel Behavior in Different Histone Deacetylase Isoforms

Histone deacetylases (HDACs) have emerged as effective therapeutic targets in the treatment of various diseases including cancers as these enzymes directly involved in the epigenetic regulation of genes. However the development of isoform-selective HDAC inhibitors has been a challenge till date since all HDAC enzymes possess conserved tunnel-like active site. In this study, using molecular dynamics simulation we have analyzed the behavior of tunnels present in HDAC8, 10, and 11 enzymes of class I, II, and IV, respectively. We have identified the equivalent tunnel forming amino acids in these three isoforms and found that they are very much conserved with subtle differences to be utilized in selective inhibitor development. One amino acid, methionine of HDAC8, among six tunnel forming residues is different in isoforms of other classes (glutamic acid (E) in HDAC10 and leucine (L) in HDAC 11) based on which mutations were introduced in HDAC11, the less studied HDAC isoform, to observe the effects of this change. The HDAC8-like (L268M) mutation in the tunnel forming residues has almost maintained the deep and narrow tunnel as present in HDAC8 whereas HDAC10-like (L268E) mutation has changed the tunnel wider and shallow as observed in HDAC10. These results explained the importance of the single change in the tunnel formation in different isoforms. The observations from this study can be utilized in the development of isoform-selective HDAC inhibitors.     

A Novel Electrochemically Active and Fe(III)-reducing Bacterium Phylogenetically Related to Clostridium butyricum Isolated from a Microbial Fuel Cell

An obligatory anaerobic bacterium was isolated from a mediator-less microbial fuel cell using starch processing wastewater as the fuel and designated as EG3. The isolate was Gram-positive, motile and rod (2.8–3.0 μm long, 0.5–0.6 μm wide). The partial 16S rRNA gene sequence and analysis of the cellular fatty acids profile suggested that EG3 clusters with Clostridium sub-phylum and exhibited the highest similarity (98%) with Clostridium butyricum. The temperature and pH optimum for growth were 37°C and 7.0, respectively. The major products of glucose and glucose/Fe(O)OH metabolism were lactate, formate, butyrate, acetate, CO₂and H₂. Growth was faster at the initial phase and the cell yield was higher when the medium was supplemented with Fe(O)OH than without Fe(O)OH. These results suggest that Fe(III) ion is utilised as an electron sink. Cyclic voltammetry showed that Clostridium butyricum EG3 cells were electrochemically active. It is a novel characteristic of strict anaerobic Gram-positive bacteria.     

Model-Based Simulation and Prediction of an Antiviral Strategy against Influenza A Infection

There is a strong need to develop novel strategies in using antiviral agents to efficiently treat influenza infections. Thus, we constructed a rule-based mathematical model that reflects the complicated interactions of the host immunity and viral life cycle and analyzed the key controlling steps of influenza infections. The main characteristics of the pandemic and seasonal influenza strains were estimated using parameter values derived from cells infected with Influenza A/California/04/2009 and Influenza A/NewCaledonia/20/99, respectively. The quantitative dynamics of the infected host cells revealed a more aggressive progression of the pandemic strain than the seasonal strain. The perturbation of each parameter in the model was then tested for its effects on viral production. In both the seasonal and pandemic strains, the inhibition of the viral release (k_C), the reinforcement of viral attachment (k_V), and an increased transition rate of infected cells into activated cells (k_I) exhibited significant suppression effects on the viral production; however, these inhibitory effects were only observed when the numerical perturbations were performed at the early stages of the infection. In contrast, combinatorial perturbations of both the inhibition of viral release and either the reinforcement of the activation of infected cells or the viral attachment exhibited a significant reduction in the viral production even at a later stage of infection. These results suggest that, in addition to blocking the viral release, a combination therapy that also enhances either the viral attachment or the transition of the infected cells might provide an alternative for effectively controlling progressed influenza infection.     

Ablation of Gadd45β ameliorates the inflammation and renal fibrosis caused by unilateral ureteral obstruction

The growth arrest and DNA damage‐inducible beta (Gadd45β) protein have been associated with various cellular functions, but its role in progressive renal disease is currently unknown. Here, we examined the effect of Gadd45β deletion on cell proliferation and apoptosis, inflammation, and renal fibrosis in an early chronic kidney disease (CKD) mouse model following unilateral ureteral obstruction (UUO). Wild‐type (WT) and Gadd45β‐knockout (KO) mice underwent either a sham operation or UUO and the kidneys were sampled eight days later. A histological assay revealed that ablation of Gadd45β ameliorated UUO‐induced renal injury. Cell proliferation was higher in Gadd45β KO mouse kidneys, but apoptosis was similar in both genotypes after UUO. Expression of pro‐inflammatory cytokines after UUO was down‐regulated in the kidneys from Gadd45β KO mice, whereas UUO‐mediated immune cell infiltration remained unchanged. The expression of pro‐inflammatory cytokines in response to LPS stimulation decreased in bone marrow‐derived macrophages from Gadd45β KO mice compared with that in WT mice. Importantly, UUO‐induced renal fibrosis was ameliorated in Gadd45β KO mice unlike in WT mice. Gadd45β was involved in TGF‐β signalling pathway regulation in kidney fibroblasts. Our findings demonstrate that Gadd45β plays a crucial role in renal injury and may be a therapeutic target for the treatment of CKD.     

High‐Voltage‐Driven Surface Structuring and Electrochemical Stabilization of Ni‐Rich Layered Cathode Materials for Li Rechargeable Batteries

Layered lithium–nickel–cobalt–manganese oxide (NCM) materials have emerged as promising alternative cathode materials owing to their high energy density and electrochemical stability. Although high reversible capacity has been achieved for Ni‐rich NCM materials when charged beyond 4.2 V versus Li⁺/Li, full lithium utilization is hindered by the pronounced structural degradation and electrolyte decomposition. Herein, the unexpected realization of sustained working voltage as well as improved electrochemical performance upon electrochemical cycling at a high operating voltage of 4.9 V in the Ni‐rich NCM LiNi_0.895Co_0.085Mn_0.02O₂ is presented. The improved electrochemical performance at a high working voltage at 4.9 V is attributed to the removal of the resistive Ni²⁺O rock‐salt surface layer, which stabilizes the voltage profile and improves retention of the energy density during electrochemical cycling. The manifestation of the layered Ni²⁺O rock‐salt phase along with the structural evolution related to the metal dissolution are probed using in situ X‐ray diffraction, neutron diffraction, transmission electron microscopy, and X‐ray absorption spectroscopy. The findings help unravel the structural complexities associated with high working voltages and offer insight for the design of advanced battery materials, enabling the realization of fully reversible lithium extraction in Ni‐rich NCM materials.