The influence of blood glucose on neutrophil function in individuals without diabetes

We assessed the association of neutrophil function with glycated hemoglobin (HbA1c) levels in a Japanese general population. Participants were 809 males and females who were over 20 years old living in the Iwaki region in Aomori Prefecture located in northern Japan. Lifestyle parameters (smoking, alcohol consumption, and exercise habits), HbA1c and neutrophil function such as reactive oxygen species (ROS) production capability and phagocytic activity (PA) were measured. ROS production capability was measured before and after phagocytic stimulus to obtain basal ROS production and stimulated ROS production. Level of HbA1c had a positive correlation with basal ROS production (p=0.053), a negative correlation with stimulated ROS production (p=0.072) and PA (p=0.059) only in post‐menopausal groups, and not in pre‐menopausal groups. However, there were no correlations between levels of HbA1c and neutrophil functions in male. In conclusion, in the present study, despite the presence of diabetes, chronic hyperglycemia was found to cause an increase in daily basal ROS production of neutrophils, and increased susceptibility to infection caused by reduced neutrophilic reaction in females in their menopause. Therefore, from the oxidative point of view, strict glycemic control is necessary to prevent post‐menopausal females from developing diabetic complications in spite of the presence of diabetes.     

Investigations on the Influence of a Low Casein Diet on the Free Fatty Acids in Liver Homogenate by a Method with Some Devices in Purification Procedure Using KOH

Previously, some changes were noticed in energy metabolism of rats fed a low casein diet. In connection with these phenomena, influence of a low casein diet on the composition and amounts of free fatty acids in liver homogenate after autolyzing for a few hours was investigated. For the measurement of free fatty acids, they were purified by a method with some devices in purification procedure using KOH. It was found that amounts of free fatty acids in liver homogenate after autolyzing for a few hours were lower in rats fed a low casein diet.     

A Novel Assay System for Anti-Human Immunodeficiency Virus Type 1 (HIV-1) Activity Using a Subclone of a Monocytic Cell Line,U937

A subclonal cl.1–14 cell was established from a monocytic cell line U937 by a limiting dilution method. The anti-HIV-1 activity of some antiviral compounds was evaluated in HIV-1-infected cl.1–14 cells. The results demonstrated that although AZT was a potent inhibitor of HIV-1 replication in cl.1–14 cells, its 50% effective concentration (EC₅₀) values was 80 times higher than that in HIV-1 infected MT-4 cells; the EC₅₀ of AZT was 0.16 μM and 0.002 μM in cl.1–14 and MT-4 cells, respectively. In contrast, the anti-HIV-1 activity of ddA, ddI and ddC in cl.1–14 cells was comparable to that in MT-4 cells. The antiviral activity of nevirapine, dextran sulfate, curdlan sulfate and T22 did not differ significantly between the cl. 1–14 and MT-4 cells. The antiviral activity of several compounds in the HIV-1-infected cl.1–14 cells was similar to that in the HIV-1jr -fl -infected human peripheral macrophages. Our results suggest that cl.1–14 cell cultures are very useful for estimating antiviral activity and more advantageous than the use of peripheral blood macrophages.     

Involvement of Protein Kinase C Activation in L-Leucine-Induced Stimulation of Protein Synthesis in L6 Myotubes

Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [³H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, α-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A₂ and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C.     

Identification of 4-Trimethylaminobutyraldehyde Dehydrogenase (TMABA-DH) as a Candidate Serum Autoantibody Target for Kawasaki Disease

Kawasaki disease (KD), an acute vasculitis that preferentially affects coronary arteries, is still the leading cause of acquired heart disease in children. Although the involvement of immune system malfunction in the onset of KD is suggested, its etiology still remains to be clarified. We investigated autoantibodies in KD patients, which are frequently found in sera from patients with autoimmune diseases, vasculitides and arteritides. We performed two-dimensional western blotting and LC-MS/MS to analyze the antigens of autoantibodies, detected two protein spots with 4 out of 24 sera from KD patients but not with 6 control sera, and identified the antigens as 4-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH). A slot blot analysis with TMABA-DH as an antigen also revealed higher reactivities of patients'' sera than control sera (positive rates: 18/43 vs 3/41). Using an enzyme-linked immunosorbent assay (ELISA), we found that the reactivity of anti-TMABA-DH antibodies in sera from KD patients was significantly higher than that in sera from age-matched controls. The optimal cut-off value of 0.043 had a sensitivity of 83.7% and a specificity of 80.0% in detecting KD patients (positive rates: 37/43 for KD patients, 9/41 for controls). Immunohistochemistry performed on thin sections of rat heart revealed that TMABA-DH colocalized with myosin light chains in cardiac myocytes. Patient sera with high reactivity gave similar immunostaining pattern. These results suggest that the detection of anti-TMABA-DH autoantibody could be a potential strategy for a diagnosis of KD.     

Identification of cis-localization elements of the maize 10-kDa δ-zein and their use in targeting RNAs to specific cortical endoplasmic reticulum subdomains

The RNAs for the storage proteins of rice ( Oryza sativa ), prolamines and glutelins, which are stored as inclusions in the lumen of the endoplasmic reticulum (ER) and storage vacuoles, respectively, are targeted by specific cis -localization elements to distinct subdomains of the cortical ER. Glutelin RNA has one or more cis -localization elements (zip codes) at the 3' end of the RNA, whereas prolamine has two cis -elements; one located in the 5' end of the coding sequence and a second residing in the 3'-untranslated region (UTR). We had earlier demonstrated that the RNAs for the maize zeins ('prolamine' class) are localized to the spherical protein body ER (PB-ER) in developing maize endosperm. As the PB-ER localization of the 10-kDa δ-zein RNA is maintained in developing rice seeds, we determined the number and proximate location of their cis -localization elements by expressing GFP fusions containing various zein RNA sequences in transgenic rice and analyzing their spatial distribution on the cortical ER by in situ RT-PCR and confocal microscopy. Four putative cis -localization elements were identified; three in the coding sequences and one in the 3'-UTR. Two of these zip codes are required for restricted localization to the PB-ER. Using RNA targeting determinants we show, by mis-targeting the storage protein RNAs from their normal destination on the cortical ER, that the coded proteins are redirected from their normal site of deposition. Targeting of RNA to distinct cortical ER subdomains may be the underlying basis for the variable use of the ER lumen or storage vacuole as the final storage deposition site of storage proteins among flowering plant species.     

Stable nuclear transformation of the Closterium peracerosum-strigosum-littorale complex

Although charophycean algae form a relevant monophyly with embryophytes and hence occupy a fundamental place in the development of Streptophyta, no tools for genetic transformation in these organisms have been developed. Here we present the first stable nuclear transformation system for the unicellular Zygnematales, the Closterium peracerosum-strigosum-littorale complex (C. psl complex), which is one of the most useful organisms for experimental research on charophycean algae. When a vector, pSA106, containing the dominant selectable marker ble (phleomycin-resistant) gene and a reporter cgfp (Chlamydomonas-adapted green fluorescent protein) gene was introduced into cells via particle bombardment, a total of 19 phleomycin-resistant cells were obtained in the presence of a low concentration of phleomycin. Six isogenic strains isolated using conditioned medium showed consecutive cgfp expression and long-term stability for phleomycin resistance. DNA analyses verified single or tandem/redundant integration of ~10 copies of pSA106 into the C. psl complex genome. We also constructed an overexpression vector, pSA1102, and then integrated a CpPI gene encoding minus-specific sex pheromone into pSA1102. Ectopic overexpression of CpPI and the pheromonal function were confirmed when the vector pSA1102_CpPI was introduced into mt(+) cells. The present efficient transformation system for the C. psl complex should provide not only a basis for molecular investigation of Closterium but also an insight into important processes in early development and evolution of Streptophyta.