Efficient bacterial production of functional antibody fragments using a phagemid vector

The so-called ‘in vitro evolutionary method’ using a phage display system has been applied for protein engineering of the antigen-binding fragment of antibodies (Fab) by conducting random mutagenesis at the antigen-binding site in combination with antigen-based biopanning. However, isolated phage clones displaying Fab cannot necessarily be used for efficient bacterial production of engineered Fab proteins, often due to deleterious defects in their proper folding abilities derived in compensation for the gain of high affinity for a particular antigen. We here report a new method of an efficient and direct bacterial expression system for the phagemid-coded Fab proteins without use of the helper phage. To overcome a low folding efficiency derived from somatic hypermutations, if any, we have established optimum conditions for bacterial cultivation and protein expression, utilizing unusually long cultivation time (>50 h) and very low temperature (25 °C) and thereby leading to the production and extracellular secretion of Fab proteins in a very high yield (3–15 mg/L of culture). The purified Fab folded correctly and could efficiently bind an antigen, as judged by circular dichroism and isothermal titration calorimetry, respectively.     

Muscular outputs during dynamic bench press under stable versus unstable conditions

Previous studies have suggested that resistance training exercise under unstable conditions decreases the isometric force output, yet little is known about its influence on muscular outputs during dynamic movement. The objective of this study was to investigate the effect of an unstable condition on power, force, and velocity outputs during the bench press. Twenty male collegiate athletes (mean age, 21.3 +/- 1.5 years; mean height, 167.7 +/- 7.7 cm; mean weight, 75.9 +/- 17.5 kg) participated in this study. Each subject attempted 3 sets of single bench presses with 50% of 1 repetition maximum (1RM) under a stable condition with a flat bench and an unstable condition with a Swiss ball. Acceleration data were obtained with an accelerometer attached to the center of a barbell shaft, and peak outputs of power, force, and velocity were computed. Although significant loss of the peak outputs was found under the unstable condition (p < 0.017), their reduction rates remained relatively low, approximately 6% for force and 10% for power and velocity outputs, compared with previous findings. Such small reduction rates of muscular outputs may not compromise the training effect. Prospective studies are necessary to confirm whether the resistance training under an unstable condition permits the improvement of dynamic performance and trunk stability.     

Review of factors essential for blastocyst implantation for their modulating effects on the maternal immune system

Pituitary and ovarian hormones prepare the endometrium for successful blastocyst implantation and support its process directly or indirectly through the action of growth factors, cytokines and other molecules. Many of the blastocyst implantation essential factors (BIEFs) are modulators of the maternal immune system. Since little is known as to the action of these molecules on the uterine lymphocytes, its clarification is imperative to the understanding of the process of blastocyst implantation.     

Nutrient control of release of pancreatic enzymes in yellowtail (Seriola quinqueradiata): involvement of CCK and PY in the regulatory loop

Cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptides are key regulators of pancreatic enzyme secretion in vertebrates. CCK stimulates enzyme secretion whereas peptide Y (PY), a NPY-related peptide, plays an antagonistic role to that of CCK. In fish, very little is known about how different nutrients affect the synthesis of CCK and PY in the digestive tract, and the mechanism by which CCK and PY actually regulate digestive enzyme secretion is not well understood. In order to determine how different nutrients stimulate the synthesis of CCK and PY in yellowtail (Seriola quinqueradiata), CCK and PY mRNA levels in the digestive tract were measured after oral administration of a single bolus of either phosphate-buffered saline (PBS: control), starch (carbohydrate), casein (protein), oleic acid (fatty acid) or tri-olein (triglyceride). In addition, in order to confirm the synthesis and secretion of digestive enzymes, the mRNA levels and enzymatic activities of three digestive enzymes (lipase, trypsin and amylase) were also analyzed. Casein, oleic acid and tri-olein increased the synthesis of lipase, trypsin and amylase, while starch and PBS did not affect the activity of any of these enzymes. CCK mRNA levels rose, while PY mRNA levels were reduced in fish administered casein, oleic acid and tri-olein. These results suggest that in yellowtail, CCK and PY maintain antagonistic control of pancreatic enzyme secretion after intake of protein and/or fat.     

Production of recombinant leptin and its effects on food intake in rainbow trout (Oncorhynchus mykiss)

Leptin is a key factor for the regulation of food intake and energy homeostasis in mammals, but information regarding its role in teleosts is still limited. There are large differences between mammalian and teleost leptin at both gene and protein levels, and in order to characterize the function of leptin in fish, preparation of species-specific leptin is therefore a key step. In this study, full-length cDNA coding for rainbow trout leptin was identified. In spite of low amino acid sequence similarity with other animals, leptin is highly conserved between trout and salmon (98.7%). Based on the cDNA, we produced pure recombinant trout leptin (rt-leptin) in E. coli, with a final yield of 20 mg/L culture medium. We then examined the effects of intraperitoneal (IP) injection of rt-leptin on feeding behavior and gene expression of hypothalamic NPY and POMCs (POMC A1, A2 and B) in a short-term (8 h) experiment. The rt-leptin suppressed food intake and led to transient reduction of NPY mRNA levels, while the expression of POMCs A1 and A2, was elevated compared with vehicle-injected controls. These results for rainbow trout are the first that describe a physiological role of leptin using a species-specific orthologue in teleosts, and they suggest that leptin suppresses food intake mediated by hypothalamic regulation. This anorexic effect is similar to that observed in mammals and frogs and supports that the neuroendocrine pathways that control feeding by leptin are ancient and have been conserved through evolution.     

Molecular cloning of urea transporters from the kidneys of baleen and toothed whales

Urea transport in the kidney is important for the production of concentrated urine. This process is mediated by urea transporters (UTs) encoded by two genes, UT-A (Slc14a2) and UT-B (Slc14a1). Our previous study demonstrated that cetaceans produce highly concentrated urine than terrestrial mammals, and that baleen whales showed higher concentrations of urinary urea than sperm whales. Therefore, we hypothesized that cetaceans have unique actions of UTs to maintain fluid homeostasis in marine habitat. Kidney samples of common minke (Balaenoptera acutorostrata), sei (B. borealis), Bryde's (B. brydei) and sperm whales (Physeter macrocephalus) were obtained to determine the nucleotide sequences of mRNAs encoding UT. The sequences of 2.5-kb cDNAs encode 397-amino acid proteins, which are 90-94% identical to the mammalian UT-A2s. Two putative glycosylation sites are conserved between the whales and the terrestrial mammals, whereas consensus sites for protein kinases are not completely conserved; only a single protein kinase A consensus site was identified in the whale UT-A2s. Two protein kinase C consensus sites are present in the baleen whale UT-A2s, however, a single protein kinase C consensus site was identified in the sperm whale UT-A2. These different phosphorylation sites of whale UT-A2s may result in the high concentrations of urinary urea in whales, by reflecting their urea permeability.     

In vivo study of toxoplasmic parasitemia using interferon-gamma-deficient mice: absolute cell number of leukocytes, parasite load and cell susceptibility

Toxoplasma gondii infection was induced in wild type (WT) C57BL/6 and BALB/c mice and same-background interferon-γ knockout (GKO) mice by peroral inoculation of Fukaya strain cysts. We studied parasitemia, absolute cell number of leukocytes, and cell susceptibility in peripheral blood leukocyte (PBL) subsets in vivo. Parasitemia was observed in both WT and GKO mice, although the pattern of the parasite load was totally different among them. After infection, the absolute number of neutrophils in peripheral blood increased in both GKO C57BL/6 and GKO BALB/c mice with statistical significance, while it rose up slightly in susceptible WT C57BL/6 mice, but declined moderately in resistant WT BALB/c mice. The absolute number of lymphocytes in both WT and GKO mice decreased significantly after infection. Although leukocyte number per μl decreased significantly in both strains of WT mice, it increased in GKO BALB/c mice. There were no differences in susceptibility of PBL subsets to T. gondii infection as assessed by quantitative competitive polymerase chain reaction in both WT and GKO mice. These results indicate that each of the PBL subsets was infected in vivo. The increase of neutrophils only among immunocompromised or susceptible strains suggests that the neutrophil may be involved in the lethal process of T. gondii infection. The lack of any difference in cell susceptibility per μg gDNA among the PBL subsets examined implies that the neutrophil may contribute to the whole body dissemination process of the parasite in vivo more than other PBL subsets through the increase in number.     

Tetratricopeptide repeat proteins Tom70 and Tom71 mediate yeast mitochondrial morphogenesis

The maintenance of correct mitochondrial shape requires numerous proteins that act on the surface or inside of the organelle. Although the soluble F-box protein Mfb1 was recently found to associate peripherally with mitochondria and to regulate organelle connectivity in budding yeast, how it localizes to mitochondria is unknown. Here, we show that two tetratricopeptide repeat proteins-the general preprotein import receptor Tom70 (a component of translocase of the outer membrane) and its paralogue Tom71-are required for Mfb1 mitochondrial localization. Mitochondria in cells lacking Tom70 and Tom71 form short tubules and aggregates, aberrant morphologies similar to those observed in the mfb1-null mutant. In addition, Mfb1 interacts with Tom71 in vivo, and binds to mitochondria through Tom70 in vitro. Our data indicate an unexpected role for Tom70 in recruitment of soluble proteins to the mitochondrial surface, and indicate that Tom71 has a specialized role in Mfb1-mediated mitochondrial morphogenesis.     

Plasma brain natriuretic peptide as a surrogate marker for cardioembolic stroke

Background

Cardioembolic stroke generally results in more severe disability, since it typically has a larger ischemic area than the other types of ischemic stroke. However, it is difficult to differentiate cardioembolic stroke from non-cardioembolic stroke (atherothrombotic stroke and lacunar stroke). In this study, we evaluated the levels of plasma brain natriuretic peptide in acute ischemic stroke patients with cardioembolic stroke or non-cardioembolic stroke, and assessed the prediction factors of plasma brain natriuretic peptide and whether we could differentiate between stroke subtypes on the basis of plasma brain natriuretic peptide concentrations in addition to patient's clinical variables.

Methods

Our patient cohort consisted of 131 consecutive patients with acute cerebral infarction who were admitted to Kagawa University School of Medicine Hospital from January 1, 2005 to December 31, 2007. The mean age of patients (43 females, 88 males) was 69.6 ± 10.1 years. Sixty-two patients had cardioembolic stroke; the remaining 69 patients had non-cardioembolic stroke (including atherothrombotic stroke, lacunar stroke, or the other). Clinical variables and the plasma brain natriuretic peptide were evaluated in all patients.

Results

Plasma brain natriuretic peptide was linearly associated with atrial fibrillation, heart failure, chronic renal failure, and left atrial diameter, independently (F_4,126 = 27.6, p < 0.0001; adjusted R² = 0.45). Furthermore, atrial fibrillation, mitral regurgitation, plasma brain natriuretic peptide (> 77 pg/ml), and left atrial diameter (> 36 mm) were statistically significant independent predictors of cardioembolic stroke in the multivariable setting (Χ² = 127.5, p < 0.001).

Conclusion

It was suggested that cardioembolic stroke was strongly predicted with atrial fibrillation and plasma brain natriuretic peptide. Plasma brain natriuretic peptide can be a surrogate marker for cardioembolic stroke.     

Chlorpromazine oligomer is a potentially active substance that inhibits human D-amino acid oxidase, product of a susceptibility gene for schizophrenia

D-amino acid oxidase (DAO), a potential risk factor for schizophrenia, has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia. Here we show the inhibitory effect of an antipsychotic drug, chlorpromazine, on human DAO, which is consistent with previous reports using porcine DAO, although human DAO was inhibited to a lesser degree (K(i) = 0.7 mM) than porcine DAO. Since chlorpromazine is known to induce phototoxic or photoallergic reactions and also to be transformed into various metabolites, we examined the effects of white light-irradiated chlorpromazine on the enzymatic activity. Analytical methods including high-resolution mass spectrometry revealed that irradiation triggered the oligomerization of chlorpromazine molecules. The oligomerized chlorpromazine showed a mixed type inhibition with inhibition constants of low micromolar range, indicative of enhanced inhibition. Taken together, these results suggest that oligomerized chlorpromazine could act as an active substance that might contribute to the therapeutic effects of this drug.