Expression of alternative oxidase inhibits programmed cell death-like phenomenon in bloodstream form of Trypanosoma brucei rhodesiense

Trypanosoma brucei rhodesiense is one of the causative agents of African Trypanosomiasis. Programmed cell death (PCD) is fundamental in the development, homeostasis and immune mechanisms of multicellular organisms. It has been shown that, other than occurring in multicellular organisms, the PCD phenomenon also takes place in unicellular organisms. In the present study, we have found that under high-density axenic culture conditions, bloodstream form of T. b. rhodesiense depicts a PCD-like phenomenon. We investigated the association of the PCD-like phenomenon with expression of trypanosome alternative oxidase (TAO) under low-temperature stress conditions. We observed that bloodstream form of T. b. rhodesiense did not show any PCD but had up-regulated expression of TAO. Inhibition of TAO by the addition of ascofranone caused the development of PCD in bloodstream T. b. rhodesiense under low-temperature stress, implying that expression of TAO may contribute to the inhibition of PCD.     

Role of L-ficolin/mannose-binding lectin-associated serine protease complexes in the opsonophagocytosis of type III group B streptococci

Serotype III group B streptococci (GBS) are a common cause of neonatal sepsis and meningitis. Although deficiency in maternal capsular polysaccharide (CPS)-specific IgG correlates with susceptibility of neonates to the GBS infection, serum deficient in CPS-specific IgG mediates significant opsonophagocytosis. This IgG-independent opsonophagocytosis requires activation of the complement pathway, a process requiring the presence of both Ca(2+) and Mg(2+), and is significantly reduced by chelating Ca(2+) with EGTA. In these studies, we defined a role of L-ficolin/mannose-binding lectin-associated serine protease (MASP) complexes in Ca(2+)-dependent, Ab-independent opsonophagocytosis of serotype III GBS. Incubation of GBS with affinity-purified L-ficolin/MASP complexes and C1q-depleted serum deficient in CPS-specific Ab supported opsonophagocytic killing, and this killing was inhibited by fluid-phase N-acetylglucosamine, the ligand for L-ficolin. Binding of L-ficolin was proportional to the CPS content of individual strains, and opsonophagocytic killing and C4 activation were inhibited by fluid-phase CPS, suggesting that L-ficolin binds to CPS. Sialic acid is known to inhibit alternative complement pathway activation, and, as expected, the bactericidal index (percentage of bacteria killed) for individual strains was inversely proportional to the sialic acid content of the CPS, and L-ficolin-initiated opsonophagocytic killing was significantly increased by addition of CPS-specific IgG2, which increased activation of the alternative pathway. We conclude that binding of L-ficolin/MASP complexes to the CPS generates C3 convertase C4b2a, which deposits C3b on GBS. C3b deposited by this lectin pathway forms alternative pathway C3 convertase C3bBb whose activity is enhanced by CPS-specific IgG2, leading to increased opsonophagocytic killing by further deposition of C3b on the GBS.     

Anticonflict effects of rose oil and identification of its active constituents

The present study investigates the pharmacologically active constituents of rose oil, which possesses anti-conflict effects. Analysis using GC/MS revealed that rose oil contains 9 substances that were identified as myrcene, benzyl alcohol, 2-phenethyl alcohol, citronellol, geraniol, citronellyl acetate, eugenol, geranyl acetate and methyl eugenol. We examined the effects of each of these substances using the Geller and Vogel conflict tests in ICR mice. Myrcene, benzyl alcohol and citronellyl acetate did not produce any effects in either tests. Geranyl acetate and methyl eugenol produced no effect in the Geller conflict test. Geraniol and eugenol decreased the response rate during the safe period of the Geller conflict test, but did not affect the response rate during the alarm period. In contrast, 2-phenethyl alcohol and citronellol, like rose oil, produced an increasing effect on the response rate during the alarm period in the Geller conflict test. In addition, both chemicals increased the number of electric shocks mice received in the Vogel conflict test in a manner similar to that of rose oil. Given that 2-phenethyl alcohol and citronellol produced the same anti-conflict effects in both tests as rose oil, we concluded that they are the pharmacologically active constituents of anti-anxiety-like effect of rose oil.     

cDNA cloning and characterization of porcine histamine H4 receptor

Fast muscle myosin responds in similar way to F-actin and to phalloidin F-actin. It is activated 7.5 fold at infinite F-actin concentration and 6.8 fold at infinite phalloidin F-actin. The actomyosin dissociation constants are 0.89 +/- 0.34 microM with F-actin and 0.90 +/- 0.71 microM with phalloidin F-actin. Slow muscle myosin responds differently to F-actin and to phalloidin F-actin. It is activated 3.76 fold at infinite F-actin concentration and only 2.27 fold at infinite phalloidin F-actin concentration. The actomyosin dissociation constants are 1.95 +/- 1.27 microM with F-actin and 0.27 +/- 0.16 microM with phalloidin F-actin. At first glance this means that substitution of F-actin with phalloidin F-actin magnifies the difference between fast muscle and slow muscle myosins. Furthermore the change of the dissociation constants may affect the contractile force of the attached crossbridge.     

Functional characterization of cysteinyl leukotriene CysLT(2) receptor on human coronary artery smooth muscle cells

Cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) are a class of biologically active lipids that exert potent effects on the heart. To assess their roles, we investigated the distribution of their receptors, CysLT(1) and CysLT(2), in the cardiovascular system. CysLT(2) mRNA was detected at high levels in the human atrium and ventricle and at intermediate levels in the coronary artery, whereas CysLT(1) mRNA was barely detected. Further analysis by in situ hybridization revealed that CysLT(2) mRNA was expressed in myocytes, fibroblasts, and vascular smooth muscle cells, but not in endothelial cells. When human coronary smooth muscle cells were stimulated with LTC(4), the intracellular calcium concentration increased in a dose-dependent manner, and this action was partially inhibited by nicardipine. Additionally, these cells showed chemotactic responses to LTC(4). This is the first report on the physiological role of CysLT(2), and the findings suggest that CysLT(2) has biological significance in the cardiovascular system.     

Phosphorylation of vimentin head domain inhibits interaction with the carboxyl-terminal end of alpha-helical rod domain studied by surface plasmon resonance measurements

The amino-terminal head domain of vimentin is the target site for several protein kinases and phosphorylation induces disassembly of the vimentin intermediate filaments in vivo and in vitro. To better understand molecular mechanisms involved in phosphorylation-dependent disassembly, we examined domain interactions involving the head domain and the effect of phosphorylation on the interaction, using surface plasmon resonance. We observed that the head domain binds to the carboxyl-terminal helix 2B in the rod domain, under physiological ionic strength. This interaction was interfered with by A-kinase phosphorylation of the head domain. Deletion of the carboxyl-terminal 20 amino acids of helix 2B resulted in loss of the interaction. Furthermore, peptide representing the carboxyl-terminal 20 residues of helix 2B had a substantial affinity with the head domain but not with the phosphorylated one. These findings support the idea that the interaction between the head domain and the last 20 residues of helix 2B is essential for association of vimentin tetramers into the intermediate filaments and that the phosphorylation-dependent disassembly is the result of loss of the interaction.     

A new beta-1,2-N-acetylglucosaminyltransferase that may play a role in the biosynthesis of mammalian O-mannosyl glycans

Recent studies have shown that O-mannosyl glycans are present in several mammalian glycoproteins. Although knowledge on the functional roles of these glycans is accumulating, their biosynthetic pathways are poorly understood. Here we report the identification and initial characterization of a novel enzyme capable of forming GlcNAc beta 1-2Man linkage, namely UDP-N-acetylglucosamine: O-linked mannose beta-1,2-N-acetylglucosaminyltransferase in the microsome fraction of newborn rat brains. The enzyme transfers GlcNAc to beta-linked mannose residues, and the formed linkage was confirmed to be beta 1-2 on the basis of diplococcal beta-N-acetylhexosaminidase susceptibility and by high-pH anion-exchange chromatography. Its activity is linearly dependent on time, protein concentration, and substrate concentration and is enhanced in the presence of manganese ion. Its activity is not due to UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT-I) or UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,2-D-acetylglucosaminyltransferase II (GnT-II), which acts on the early steps of N-glycan biosynthesis, because GnT-I or GnT-II expressed in yeast cells did not show any GlcNAc transfer activity against a synthetic mannosyl peptide. Taken together, the results suggest that the GlcNAc transferase activity described here is relevant to the O-mannosyl glycan pathway in mammals.     

Production of Maltohexaose by α-Amylase from Bacillus circulans G-6

An α-amylase which produces maltohexaose as the main product from strach was found in the culture filtrate of Bacillus circulans G-6 which was isolated from soil and identified by the author.

The enzyme was purified by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex G-200 column chromatography. The purified enzyme was homogeneous on disc electrophoresis. The optimum pH and temperature of the enzyme were around pH 8.0 and around 60°C, respectively. The enzyme was stable in the range of pH 5–10. Metal ions such as Hg²⁺, Cu²⁺, Zn²⁺, Fe²⁺ and Co²⁺ inhibited the enzyme activity. The molecular weight was about 76,000. The yield of maltohexaose from soluble starch of DE (dextrose equivalent*) 1.8-12.6 was about 30%, and the combined action of the enzyme and pullulanase or isoamylase increased the yield of maltohexaose.     

What Triggers a Diagnosis of HIV Infection in the Tokyo Metropolitan Area? Implications for Preventing the Spread of HIV Infection in Japan

Background

Japan has not succeeded in reducing the annual number of new HIV-infected patients, although the prevalence of HIV infection is low (0.02%).

Methods

A single-center observational study was conducted at the largest HIV clinic in Tokyo, which treats 15% of the total patients in Japan, to determine the reasons for having diagnostic tests in newly infected individuals. HIV-infected patients who visited our clinic for the first time between 2011 and 2014 were analyzed.

Results

The 598 study patients comprised one-third of the total reported number of new patients in Tokyo during the study period. 76% were Japanese MSM. The reasons for being tested which led to the diagnosis was voluntary testing in 32%, existing diseases in 53% (AIDS-defining diseases in 22%, sexually transmitted infections (STI) in 8%, diseases other than AIDS or STIs in 23%) and routine pre-surgery or on admission screening in 15%. 52% and 74% of the study patients and patients presented with AIDS, respectively, had never been tested. The median CD4 count in patients with history of previous testing (315/μL) was significantly higher than that of patients who had never been tested (203/μL, p<0.001).

Conclusions

Only 32% of the newly HIV diagnosed patients were diagnosed because of voluntary testing, and 53% were diagnosed due to presence of other diseases. These results remain unchanged from our previous report 10 years earlier (2000–2004) on newly diagnosed patients at the same clinic. HIV testing has not been widely used by newly diagnosed patients in the Tokyo metropolitan area.