Electrochemical screening of recombinant protein solubility in Escherichia coli using scanning electrochemical microscopy (SECM)

A microbial array chip with collagen gel spots entrapping living Escherichia coli (E. coli) DH5alpha was applied for the screening of recombinant protein solubilities. The alpha-fragment of beta-galactosidase (betaGal) was fused to the target protein, namely, maltose-binding protein (MBP), to monitor the solubility of MBP. Scanning electrochemical microscopy (SECM) was used to detect the release of p-aminophenol from E. coli cells catalyzed by intracellular betaGal. Comparison of the SECM-based method with the Western blotting-based method indicated that the current response obtained using SECM increased with an increase in the betaGal activity and therefore, with the soluble fraction of MBP in the host cells.     

Legumain/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.     

AMPK mediates autophagy during myocardial ischemia in vivo

We have recently shown that autophagy is induced by ischemia and reperfusion in the mouse heart in vivo. Ischemia stimulates autophagy through an AMP activated protein kinase (AMPK)-dependent mechanism, whereas reperfusion after ischemia stimulates autophagy through a Beclin 1-dependent, but AMPK-independent, mechanism. Autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion. We will discuss the role of AMPK in mediating autophagy during myocardial ischemia in vivo.     

Evaluation of the Duty Ratio of the Bacterial Flagellar Motor by Dynamic Load Control

The bacterial flagellar motor is one of the most complex and sophisticated nanomachineries in nature. A duty ratio D is a fraction of time that the stator and the rotor interact and is a fundamental property to characterize the motor but remains to be determined. It is known that the stator units of the motor bind to and dissociate from the motor dynamically to control the motor torque depending on the load on the motor. At low load, at which the kinetics such as proton translocation speed limits the rotation rate, the dependency of the rotation rate on the number of stator units N implies D: the dependency becomes larger for smaller D. Contradicting observations supporting both the small and large D have been reported. A dilemma is that it is difficult to explore a broad range of N at low load because the stator units easily dissociate, and N is limited to one or two at vanishing load. Here, we develop an electrorotation method to dynamically control the load on the flagellar motor of Salmonella with a calibrated magnitude of the torque. By instantly reducing the load for keeping N high, we observed that the speed at low load depends on N, implying a small duty ratio. We recovered the torque-speed curves of individual motors and evaluated the duty ratio to be 0.14 ± 0.04 from the correlation between the torque at high load and the rotation rate at low load.     

Screening and development of enzymes for determination and transformation of amino acids

ABSTRACT

The high stereo- and substrate specificities of enzymes have been utilized for micro-determination of amino acids. Here, I review the discovery of l-Phe dehydrogenase and its practical use in the diagnosis of phenylketonuria in more than 5,400,000 neonates over two decades in Japan. Screening and uses of other selective enzymes for micro-determination of amino acids have also been discussed. In addition, novel enzymatic assays with the systematic use of known enzymes, including assays based on a pyrophosphate detection system using pyrophosphate dikinase for a variety of l-amino acids with amino-acyl-tRNA synthetase have been reviewed. Finally, I review the substrate specificities of a few amino acid-metabolizing enzymes that have been altered, using protein engineering techniques, mainly for production of useful chemicals, thus enabling the wider use of natural enzymes.     

Thin-layer chromatography and high-performance liquid chromatography for the assay of fatty acid compositions of individual phospholipids in platelets from non-insulin-dependent diabetes mellitus patients: effect of eicosapentaenoic acid ethyl ester administration

Eight major phospholipids were separated by a TLC method with a one-dimensional developing system without any pretreatment of the plate and the fatty acids incorporated into each phospholipid class were analysed by an improved HPLC method with a simple elution system, which has advantages with respect to resolution and analysis time. The fatty acid compositions of individual phospholipids in platelets were investigated following administration of ethyl cis-5,8,11,14,17-eicosapentaenoate for more than 13 weeks to patients with non-insulin-dependent diabetes mellitus. The cis-5,8,11,14,17-eicosapentaenoic acid compositions of all phospholipid classes were significantly increased with decreasing platelet aggregation rates after the administration. These results suggested that the present method provides the complete separation of individual phospholipids in sufficient amounts to allow fatty acid analysis on the isolated phospholipid moieties.     

Inhibition of human T-lymphocyte activation by macrolide antibiotic, roxithromycin.

Effects of macrolide antibiotic, roxithromycin (RXM) on human lymphocytes in culture were studied. The drug showed a dose-dependent inhibition of 3H-thymidine and 35S-methionine uptake responding to T cell mitogens and purified protein derivative of tuberculin (PPD). Activation by PPD, as assessed by 3H-thymidine uptake, was more sensitive to inhibition than the response to T cell mitogens. The drug produced a loss of blasts when added soon after transformation commenced. Immunosuppressive effects of RXM were further characterized by using four different types of metabolized RXM, RU 28111, RU 39001, RU 44981 and RU 45179. The most potent inhibitor of lymphocyte transformation was RU 45179, followed by RU 44981, RU 39001 and RU 28111 have little activity.     

Three types of polymorphisms in exon 14 in porcine Mx1 gene

Much is known about the antiviral activity of Mx proteins in species such as mouse and human. In the mouse, loss of resistibility to influenza virus has been shown to be due to specific polymorphisms in the Mx gene. This gene is therefore an interesting candidate gene for disease resistance in farm animals. The porcine Mx1 gene has already been identified and characterized based on its homology with mouse Mx1; however, until now no evidence of polymorphisms in the porcine gene has been reported. In this study, we have found two new polymorphisms in exon 14 of porcine Mx1 by DNA sequencing and confirmed their presence in different breeds, using polymerase chain reaction (PCR)–restriction fragment length polymorphisms (RFLP) with NarI and NaeI restriction enzymes. On the basis of the deduced amino acid sequence, one allele contains a deletion that may result in a frameshift to yield several amino acid substitutions and extension of the carboxyl terminal region of Mx1 protein. The deletion allele, Mx1 ^c, was found to be segregating in Landrace, Berkshire, Duroc, Hampshire, and Yucatan miniature pig. A second point mutation, Mx1 ^b, was detected in Meishan and two Vietnamese native pig breeds. All other breeds tested were fixed for the Mx1 ^a allele that is identical to the sequence reported previously. It will be interesting to determine if the Mx1 ^c deletion is associated with variation in resistance to the myxovirus family in the pig.     

3-phosphoinositide-dependent protein kinase 1, an Akt1 kinase, is involved in dephosphorylation of Thr-308 of Akt1 in Chinese hamster ovary cells

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using anti-phosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulin-stimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulin-stimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.