Immunochemical studies of human urinary trypsin inhibitor

Antisera against purified urinary trypsin inhibitor (UTI-I, molecular weight 67,000) and UTI-III (molecular weight 23,000) were first produced in rabbits. Both anti-UTI-I and anti-UTI-III sera formed a single immunoprecipitin line with human plasma inter-alpha-trypsin inhibitor (I alpha TI), whereas two immunoprecipitin lines were formed with crude urine. It was speculated that both UTI-I and UTI-II might be present in normal human urine. In the present study, the inhibitory effects of anti-UTI sera on UTI activity were examined by three different assay methods. The results indicated that the inhibitory effect was almost immediate. Although the inhibitory effect of anti-UTI-III serum on UTI-III was almost of the same degree of completeness for the three assay methods. UTI-I was partially inhibited by the anti-UTI-I serum when residual trypsin activity was measured by the caseinolytic or fibrinolytic assay method. This discrepancy was considered to be due to the difference in conformational change between UTI-I and UTI-III by antigen-antibody reaction.     

Isolation and properties of fecal proteins and fecal alkaline phosphatase from germfree and conventional rats.

Fecal proteins from germfree and conventional rats were isolated. The proteins from the two kinds of feces differed in molecular weight, judging from Sephadex gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The conventional feces contained a greater amount of high-molecular-weight and a lesser amount of low-molecular-weight proteins than did the germfree feces. The fecal proteins of both kinds contained carbohydrates. Both feces contained considerable enzyme activity. The germfree feces contained extremely high activity in alkaline phosphatase and leucine aminopeptidase. Both feces showed the same level of trehalase activity. The conventional feces contained higher levels of activity of protease and acid phosphatase than did the germfree feces. Lactase activity was observed only in the conventional feces. The fecal alkaline phosphatase resembled the intestinal enzyme in response to L-phenylalanine inhibition and urea denaturation. From these results it was inferred that the germfree feces contained some of the intestinal proteins and that the conventional feces contained bacterial proteins in addition to intestinal proteins.     

Structural evolution and function of stress associated proteins in regulating biotic and abiotic stress responses in plants

Adverse environmental conditions greatly influence crop production every year and threaten food security. Plants have a range of signaling networks to combat these stresses, in which several stress-responsive genes and regulatory proteins function together. One such important family of proteins, the Stress Associated Protein (SAP) family, has been identified as a novel regulator of multiple stresses. The SAPs possess a characteristic N-terminal A20 zinc-finger domain combined with either AN1 or C₂H₂ at the C-terminus. SAPs provide tolerance against various abiotic stresses, including cold, salt, drought, heavy metal, and wounding. The majority of SAPs are stress-inducible and have a function in conferring stress tolerance in transgenics. The role of SAPs in regulating biotic stress responses is a newly emerging field among researchers. SAPs interact with many other proteins to execute their functions; however, the detailed mechanism of these interactions needs to be elucidated. In this context, the present review provides a detailed view of the evolution and functions of SAPs in plants. The involvement in crosstalk between abiotic and biotic stress signaling pathways makes SAPs ideal targets to develop crops with tolerance against multiple stresses without any yield penalty. Altogether, we provide current knowledge on SAPs for investigating their role in stress response, which can further be exploited to develop climate-resilient crops through transgene-based, breeding-mediated, or genome-editing approaches.

     

Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.     

Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution

Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit–subunit interactions through mutations.     

The Neuroprotective Effects of Coccomyxa Gloeobotrydiformis on the Ischemic Stroke in a Rat Model

Stroke is a major cause of mortality and the leading cause of permanent disability. In this study, we adopted the classic middle cerebral artery occlusion(MCAO) stroke model to observe the therapeutic effects of coccomyxa gloeobotrydiformis(CGD) on ischemic stroke, and discuss the underlying mechanisms. Low dose (50 mg/kg.day) and high dose (100 mg/kg.day) concentrations of the drug CGD were intragastrically administrated separately for 8 weeks. Infarct volumes, neurologic deficits and degree of stroke-induced brain edema were measured 24 hours after reperfusion. Furthermore, oxidative stress related factors (SOD and MDA), mitochondrial membrane potential, and apoptosis regulatory factors (mitochondrial Cyt-C, Bcl-2, Bax, and caspase-3) were all investigated in this research. We found that CGD attenuated cerebral infarction, brain edema and neurologic deficits; CGD maintained the mitochondrial membrane potential and decreased mitochondrial swelling. It also prevented oxidative damage by reducing MDA and increasing SOD. In addition, CGD could effectively attenuate apoptosis by restoring the level of mitochondrial Cyt C and regulating the expression of Bcl-2, Bax and caspase 3. These results revealed that CGD has a therapeutic effect on ischemic stroke, possibly by inducing mitochondrial protection and anti-apoptotic mechanisms.     

Quercetin glucosides promote ischemia-induced angiogenesis,but do not promote tumor growth

Aims

Dietary flavonoid intake shows a significant inverse association with mortality from coronary heart disease, incidence of myocardial infarction and stroke. Quercetin is one of the most common flavonoids in our diet and has several favorable biological activities. Quercetin glucosides, which are enzymatically trans-glycosylated isoquercitrin, have high water-solubility and bioavailability compared with quercetin. Here, we investigated the effects of quercetin glucosides on collateral development in a murine hindlimb ischemia model.

Main methods

We induced hindlimb ischemia in 24- to 32-week-old male C3H/HeJ mice by resecting the right femoral artery. Then, 0.5% carboxymethyl cellulose (control) or quercetin glucosides (100 mg/kg/day) were administered daily by gavage. Blood flow was monitored weekly by laser Doppler imaging.

Key findings

Recovery of blood flow to the ischemic leg was significantly enhanced by quercetin glucosides (blood flow ratio at 4 weeks: control, 0.57 ± 0.11; quercetin glucosides, 0.95 ± 0.10, p < 0.05). Furthermore, anti-CD31 immunostaining revealed that quercetin glucosides increased capillary density in the ischemic muscle (control, 200 ± 24/mm²; quercetin glucosides, 364 ± 41/mm², p < 0.01). Quercetin glucosides did not promote tumor growth. The beneficial effect of quercetin glucosides was abrogated in eNOS-deficient mice.

Significance

These results suggest that quercetin glucosides may have therapeutic potential to promote angiogenesis in ischemic tissue.     

Reduced Serum Selenium Concentration in Miscarriage Incidence of Indonesian Subjects

Selenium is an essential nutrient for human health, and maternal selenium concentration has been reported to be associated with pregnancy outcome. To further investigate the possible role of selenium (Se) in miscarriage, we conducted a case–control study to evaluate the correlations among selenium status, glutathione peroxidase activity, and spontaneous abortion. A total of 46 subjects with normal pregnancies and 25 subjects with spontaneous abortion were recruited, and their serum selenium concentrations and serum glutathione peroxidase activities were analyzed. The total serum selenium concentrations in subjects with normal pregnancies were significantly higher than those of subjects with spontaneous abortion; however, the glutathione peroxidase activities were similar in both groups. We further separated the subjects into smoking and nonsmoking groups, and the logistic regression analysis suggested that total serum selenium concentration, but not serum glutathione peroxidase activity or smoking, was significantly correlated with the incidence of miscarriage. The present study thus reaffirms that low serum selenium levels are associated with miscarriage and that selenium plays an important role in pregnancy maintenance.