Venom of the Peruvian snake Bothriopsis oligolepis: Detection of antibacterial activity and involvement of proteolytic enzymes and C-type lectins in growth inhibition of Staphylococcus aureus.

There is a rising interest in snake venoms proteins (SVPs) because these macromolecules are related to pharmacological properties that manifest themselves during poisoning and can lead to secondary microbial infections. Interestingly, researchers have somehow neglected the antimicrobial activity of SVPs. The aims of this study were: (i) to verify whether the venom of the Peruvian snake Bothriopsis oligolepis displays such activity; (ii) to isolate and identify some of its antimicrobial constituents. Liquid growth inhibition assays revealed that the crude venom inhibited the growth of Gram-positive and Gram-negative bacteria, but not of Candida species. Fractionation of the venom by anion-exchange chromatography provided fractions P2, P4 and P8 active against S. aureus. Fractionation of P2 or P8 by gel-filtration chromatography and of P4 by RP-HPLC furnished the sub-fractions P2-I, P8-II and P4-II, respectively, being those fractions active against S. aureus. Analyses of these sub-fractions by SDS-PAGE under denaturing/reducing conditions evidenced SVPs with 59-73, 27 and 14-28 kDa, respectively. Their in-gel tryptic digestion gave peptide fragments, whose sequencing by MALDI-TOF/MS followed by protein BLAST analysis allowed identifying PIII metalloprotease(s) [SVMP(s)] in P2-I, serine protease(s) [SVSP(s)] in P4-II and lectin(s) in P8-II. Detection of gelatinolytic activity in P2-I and P4-II reinforced the existence of PIII-SVMP(s) and SVSP(s), respectively. Activation of the coagulation cascade intrinsic pathway by P8-II (probably by interaction with factors IX and/or X as some snake C-type lectins do) supported the presence of C-type lectin(s). Altogether, these new findings reveal that the venom of the Peruvian snake Bothriopsis oligolepis displays antibacterial activity and that the isolated SVMP(s), SVSP(s) and C-type lectin(s) are associated to its ability to inhibit the growth of S. aureus.

Venom of the peruvian snake bothriopsis oligolepis: detection of antibacterial activity and involvement of proteolytic enzymes and C-type lectins in growth inhibition of Staphylococcus aureus

There is a rising interest in snake venoms proteins (SVPs) because these macromolecules are related to pharmacological properties that manifest themselves during poisoning and can lead to secondary microbial infections. Interestingly, researchers have somehow neglected the antimicrobial activity of SVPs. The aims of this study were: (i) to verify whether the venom of the Peruvian snake Bothriopsis oligolepis displays such activity; (ii) to isolate and identify some of its antimicrobial constituents. Liquid growth inhibition assays revealed that the crude venom inhibited the growth of Gram-positive and Gram-negative bacteria, but not of Candida species. Fractionation of the venom by anion-exchange chromatography provided fractions P2, P4 and P8 active against S. aureus. Fractionation of P2 or P8 by gel filtration chromatography and of P4 by RP-HPLC furnished the sub-fractions P2-I, P8-II and P4-II, respectively, being those fractions active against S. aureus. Analyses of these sub-fractions by SDS-PAGE under denaturing/reducing conditions evidenced SVPs with 59-73, 27 and 14-28 kDa, respectively. Their in-gel tryptic digestion gave peptide fragments, whose sequencing by MALDI-TOF/MS followed by protein BLAST analysis allowed identifying Pill metalloprotease(s) [SVMP(s)] in P2-I, serine protease(s) [SVSP(s)] in P4-II and lectin(s) in P8-II. Detection of gelatinolytic activity in P2-I and P4-II reinforced the existence of PIII-SVMP(s) and SVSP(s), respectively. Activation of the coagulation cascade intrinsic pathway by P8-II (probably by interaction with factors IX and/or X as some snake C-type lectins do) supported the presence of C-type lectin(s). Altogether, these new findings reveal that the venom of the Peruvian snake Bothriopsis oligolepis displays antibacterial activity and that the isolated SVMP(s), SVSP(s) and C-type lectin(s) are associated to its ability to inhibit the growth of S. aureus.

Astronomy. ASASSN-15lh: A highly super-luminous supernova.

We report the discovery of ASASSN-15lh (SN 2015L), which we interpret as the most luminous supernova yet found. At redshift z = 0.2326, ASASSN-15lh reached an absolute magnitude of Mu ,AB = -23.5 ± 0.1 and bolometric luminosity Lbol = (2.2 ± 0.2) × 10(45) ergs s(-1), which is more than twice as luminous as any previously known supernova. It has several major features characteristic of the hydrogen-poor super-luminous supernovae (SLSNe-I), whose energy sources and progenitors are currently poorly understood. In contrast to most previously known SLSNe-I that reside in star-forming dwarf galaxies, ASASSN-15lh appears to be hosted by a luminous galaxy (MK ≈ -25.5) with little star formation. In the 4 months since first detection, ASASSN-15lh radiated (1.1 ± 0.2) × 10(52) ergs, challenging the magnetar model for its engine.

ACO1, a gene for aminocyclopropane-1-carboxylate oxidase: effects on internode elongation at the heading stage in rice.

Although reports on a gene for 1-amino-cyclopropane-1-carboxylate (ACC) oxidase (ACO1) in rice (Oryza sativa L.) suggest that high levels of its transcript are associated with internode elongation of deep-water rice during submergence, the role of ACO1 in rice development is largely unknown. The tissue-specificity of ACO1 expression indicated that its transcript significantly accumulated in lower parts of elongating internodes at the heading stage. Histochemical analysis and in situ hybridization showed that the ACO1 expression was localized in the basal parts of leaf sheaths immediately above nodes or the lower parts of elongating internodes. To further examine the role of ACO1, ACO1-deficient (aco1) and overexpressing (ACO1-OX) mutants were characterized. The total length of the elongated internodes of aco1 mutants was slightly shorter than that of wild-type plants and that of ACO1-OX mutants was longer. Interestingly, expression of the ACC synthase gene ACS1 and ethylene signalling gene OsEIN2 was up-regulated in the aco1 mutants. This study suggests that the ACO1 has a little effect on internode elongation at the heading stage, and that up-regulation of the ACS1 and OsEIN2 expression may attenuate inhibition of internode elongation.

Two Kunitz-type inhibitors with activity against trypsin and papain from Pithecellobium dumosum seeds: purification, characterization, and activity towards pest insect digestive enzyme.

Two trypsin inhibitors (called PdKI-3.1 and PdKI-3.2) were purified from the seeds of the Pithecellobium dumosum tree. Inhibitors were obtained by TCA precipitation, affinity chromatography on Trypsin-Sepharose and reversed-phase-HPLC. SDS-PAGE analysis with or without reducing agent showed that they are a single polypeptide chain, and MALDI-TOF analysis determined molecular masses of 19696.96 and 19696.36 Da, respectively. The N-terminal sequence of both inhibitors showed strong identity to the Kunitz family trypsin inhibitors. They were stable over a wide pH (2-9) and temperature (37 to 100 degrees C) range. These inhibitors reduced over 84% of trypsin activity with inhibition constant (Ki) of 4.20 x 10(-8) and 2.88 x 10(-8) M, and also moderately inhibited papain activity, a cysteine proteinase. PdKI-3.1 and PdKI-3.2 mainly inhibited digestive enzymes from Plodia interpunctella, Zabrotes subfasciatus and Ceratitis capitata guts. Results show that both inhibitors are members of the Kunitz-inhibitor family and that they affect the digestive enzyme larvae of diverse orders, indicating a potential insect antifeedant.

Aspectos étnicos, biológicos e químicos de Senna occidentalis (Fabaceae) / Ethnic, biological and chemical aspects of Senna occidentalis (Fabaceae)

Senna occidentalis (sin. Cassia occidentalis) é um arbusto perene nativo da América do Sul e distribuída em regiões tropicais ao redor do mundo, frequentemente contaminando pastos e culturas de cereais. Inúmeros estudos demonstraram que esta planta é tóxica para animais. Na medicina popular, tribos americanas, africanas e indianas usam preparações da S. occidentalis como tônico, estomáquico, febrífugo, laxante e antimicrobiano. Diversas propriedades biológicas da espécie já foram comprovadas, tais como a antibacteriana, antifúngica, antimalárica, antitumoral e hepatoprotetora. As análises fitoquímicas evidenciaram que as antraquinonas, os flavonóides e outros derivados fenólicos são os seus principais constituintes. Esta revisão apresenta dados etnofarmacológicos, químicos e biológicos publicados na literatura sobre S. occidentalis.

Senna occidentalis (syn. Cassia occidentalis) is a perennial shrub, native to South America and indigenous to tropical regions throughout the world, often contaminating pastures and cereal crops. There have been many reports showing that S. occidentalis is toxic to animals. In traditional medicine, some American, African and Indian ethnic groups use S. occidentalis preparations in stomach treatments and as a tonic, febrifuge, laxative and topical antimicrobial agent. Several biological properties of this species have been proved, such as antibacterial, antifungal, antimalarial, antitumor and hepatoprotective activity. Phytochemical analysis has shown that anthraquinones, flavonols and other phenolics are its major constituents. In this paper we present an overview of the ethnopharmacological, chemical and biological data published in the literature on S. occidentalis.

Identification of a Kunitz-type proteinase inhibitor from Pithecellobium dumosum seeds with insecticidal properties and double activity.

A trypsin inhibitor, PdKI, was purified from Pithecellobium dumosum seeds by TCA precipitation, trypsin-sepharose chromatography, and reversed-phase-HPLC. PdKI was purified 217.6-fold and recovered 4.7%. SDS-PAGE showed that PdKI is a single polypeptide chain of 18.9 kDa and 19.7 kDa by MALDI-TOF. The inhibition on trypsin was stable in the pH range 2-10 and at a temperature of 50 degrees C. The Ki values were 3.56 x 10(-8)and 7.61 x 10(-7) M with competitive and noncompetitive inhibition mechanisms for trypsin and papain, respectively. The N-terminal sequence identified with members of Kunitz-type inhibitors from the Mimosoideae and Caesalpinoideae subfamilies. PdKI was effective against digestive proteinase from Zabrotes subfasciatus, Ceratitis capitata, Plodia interpunctella, Alabama argillaceae, and Callosobruchus maculatus, with 69, 66, 44, 38, and 29% inhibition, respectively. Results support that PdKI is a member of the Kunitz inhibitor family and its insecticidal properties indicate a potent insect antifeedant.

Structure and function of fibroblast growth factor.

We describe some structural requirements of the fibroblast growth factor (FGF) signaling system for the stimulation of the mitogenic response in terms of the design, synthesis and mitogenic activity of linear peptides related to the human FGF-1 sequence and the structural requirements of heparin for the potentiation of the mitogenic activity of FGF-1. The best mitogenic peptide we have synthesized so far is Ac-WFVGLKKNGSSKRGPRT-NH2, that has been shown: 1) to bind to heparin-Sepharose columns with moderate affinity, requiring about 0.5 M NaCl to be eluted from the resin; 2) to be mitogenic upon BALB/c 3T3 fibroblasts in culture (ED50 = 10-20 microM) and 3) to compete with human FGF-1 for cellular binding (ID50 = 30-50 microM). The potentiating activity of heparin upon FGF-1 has shown to be dependent on the oligosaccharide size, degree of sulfation and carboxylation. Apparently, these same requirements hold for the heparan sulfate molecules. Based on the reported studies, we propose some important requirements of an oligosaccharide to potentiate FGF-1 mitogenic activity: 1) to have a minimum of twelve units, organized as disaccharides where one of the units is a uronic acid and the second is glycosamine; 2) to have at least one iduronic acid sulfated at position 2 and 3) to have N-sulfated glycosamines, preferentially 6-O-sulfated. To have groups of negative charges is not enough: they need to be localized in a correct conformation.

Structure and function of fibroblast growth factor

We describe some structural requirements od the fibroblast growth factor (FGF) signaling system for the stimulation of the mitogenic response in terms of the design, synthesis and mitogenic activity of linear peptides related to the human FGF-1 sequence and the structural requirements of heparin for the potentiation of the mitogenic activity of FGF-1. The best mitogenic peptide we have synthesized so far is Ac-WFVGLKKNGSSKRGPRT-NH2, that has been shown: 1)to bind to heparin-Sepharose columns with moderate affinity, requiring about 0.5 M NaCl to be eluted from the resin; 2) to be mitogenic upon BALB/c 3T3 fibroblasts in culture (ED50=10-20 muM) and 3)to compete with human FGF-1 for cellular binding (ID50=30-50 muM). The potentiating activity of heparin upon FGF-1 has shown to be dependent on the oligosaccharide size, degree of sulfation and carboxylation. Apparently, these same requirements hold for the heparan sulfate molecules. Based on the reported studies, ee propose some important requirements of an oligosaccharide to potentiate FGF-1 mitogenic activity: 1) to have a minimum of twelve units, organized as disaccharides where one of the units is a uronic acid and the second is glycosamine; 2) to have at least one iduronic acid sulfated at position 2 and 3) to have N-sulfated glycosamines, preferentially 6-O-sulfated. To have groups of negative charges is not enough: they need to be localized in a correct conformation.

[Epidemiologic survey of pleural plaques among inhabitants of Matsubase exposed to asbestos].

A high prevalence of pleural plaques (41.5%, 148/357) was found during a mass screening for lung cancer in Matsubase town in 1988. The inhabitants of this town were carefully studied each year from 1988 to 1993. The vast majority (81.2%) of inhabitants over the age of 20 years underwent chest roentgenography at least once during this period. Pleural plaques were detected by CT in 938 subjects, which is 17.3% of those studied and 4.1% of the total population. A total of 89 had an occupational history of asbestos exposure, 64 (71.9%) of whom had pleural plaques. However, these subjects with occupational exposure accounted for only 6.8% of the 938 subjects, and therefore most of the pleural plaques seemed to have been caused by general environmental exposure. The incidence of plaques was greater in older subjects: among those in the seventh decade of life it was more than eight times higher than among those in the fourth decade of life. Anthophyllite was detected in the main asbestos mill. The concentrations of asbestos fibers in the air and water near the old asbestos mills and factories were not high. The death rates and the adjusted mortality rates due to lung cancer in Matsubase were lower than in surrounding towns and lower than in Kumamoto prefecture as a whole. These results indicate that there is now no environmental contamination by asbestos fibers in Matsubase town. No cases of malignant mesothelioma have been confirmed in this town during the past 17 years.

Lack of Control in Inorganic Phosphate Uptake by Catharanthus roseus (L.) G. Don Cells (Cytoplasmic Inorganic Phosphate Homeostasis Depends on the Tonoplast Inorganic Phosphate Transport System?).

Inorganic phosphate (Pi) uptake by Catharanthus roseus (L.) G. Don cells was studied in relation to its apparent uncontrolled uptake using 31P-nuclear magnetic resonance spectroscopy. Kinetics of Pi uptake by the cells indicated that apparent Km and Vm were about 7 [mu]M and 20 [mu]mol g-1 fresh weight h-1, respectively. Pi uptake in Murashige-Skoog medium under different Pi concentrations and different initial cell densities followed basically the same kinetics. When supplied with abundant Pi, cells absorbed Pi at a constant rate (Vm) for the first hours and accumulated it in the vacuole. As the endogenous pool expanded, the rate of Pi uptake gradually decreased to nil. Maximum Pi accumulation was 100 to 120 [mu]mol g-1 fresh weight if cell swelling during Pi uptake (about 2-fold in cell volume) was not considered. Results indicated that (a) the rate of Pi uptake by Catharanthus cells was independent of initial cell density and was constant over a wide range of Pi concentrations (2 mM to about 10 [mu]M) unless the cells were preloaded with excess Pi, and (b) there was no apparent feedback control over the Pi uptake process in the plasma membrane to avoid Pi toxicity. The importance of the tonoplast Pi transport system in cytoplasmic Pi homeostasis is discussed.

[Analysis of pleural plaque found at lung cancer screening examination].

In the screening test of lung cancer, we found that there was a high prevalence of cases with pleural plaque recognized by chest X-ray film in inhabitants living in A town in Kumamoto Prefecture. We detected abnormal pleural plaque in 148 (41.5%) of 357 cases received lung cancer screening. These pleural plaques resulted in pleural thickening and calcification. Two or three mines of serpentine and an asbestos factory existed in this region from 1883 until 1970. Although twelve cases had a history of factory work, none had fibrous changes in the lung fields on chest X-ray films. It was considered that the pleural plaque probably resulted from exposure to low doses of asbestos in the atmosphere or contact with asbestos workers in their homes. The incidence of lung cancer in this region was not higher than that in other regions in Kumamoto Prefecture. There were no cases of malignant mesothelioma in our hospital during the past eleven years.

[Crystalline glass ceramics model teeth].

The feeling of hardness, stickiness and the rate of cutting during the preparation of a tooth were compared using an extracted tooth, melamine resin and crystalline glass ceramics model tooth (Bioram M) which are used in dental education and practice. Diamond burs were used with an air-turbine handpiece in this experiment. The results indicated that Bioram M had the desirable characteristics of a model tooth and there was a strong resemblance feeling of cutting between Bioram M and a natural vital tooth.

Multiple regression analysis of sixteen risk factors including serum apolipoproteins in angiographically documented coronary artery disease.

Multiple regression analysis of 16 risk factors, including serum apolipoproteins in angiographically measured coronary stenosis, was carried out in 239 consecutive patients (159 males and 80 females, ranging in age from 30-80 years and mean 56.4 years) who underwent coronary angiography for suspected coronary artery disease during the past five years (1981-1985). The risk factors (independent variables) were age, total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), HDL-C/TC, apolipoprotein (Apo) A-I, A-II, B, C-II, C-III, E, YRSMOK (average number of packs per day X years of smoking), weight index (WI), glucose tolerance (GT), and blood pressure (BP). Severity of coronary atherosclerosis was scored as the extent of disease seen at arteriography (coronary score: CS). The order of importance of risk factors to CS in the five groups of subjects studied were as follows. (1) All patients: YRSMOK greater than Apo A-I greater than TC greater than GT, (2) Male group: Apo A-I greater than TC greater than Age greater than GT, (3) Female group: TC, (4) Young group (age below 54 years): BP greater than YRSMOK, and (5) Old group (age over 55 years): YRSMOK greater than TG greater than TC greater than GT. The results clearly indicated the importance of Apo A-I but not other apolipoproteins including Apo B in males, and that of blood pressure in the young group of the patients studied.

Role of alpha 2-adrenergic receptor in platelet activation during insulin-induced hypoglycemia in normal subjects.

The effects of alpha 2-adrenergic-receptor blocker mianserin on the responses of blood glucose, plasma beta-thromboglobulin (beta-TG), and various counterregulatory hormones to insulin-induced hypoglycemia were studied in nine healthy male subjects. The alpha 2-adrenoceptor-blocking action of mianserin was confirmed by its inhibitory effect on platelet activation in vitro. Mianserin was given orally 90 min before insulin injection; the same study without mianserin was performed on another day as the control study. The time courses of blood glucose and serum C-peptide (0, 20, 45, and 180 min after the insulin injection) were identical in both studies, indicating that mianserin has no effect on these parameters. However, a significant increase of beta-TG at 45 min after insulin injection was completely suppressed by the administration of mianserin (mean +/- SE, 68.5 +/- 6.0 vs. 28.8 +/- 7.6 ng/ml, n = 6, P less than .05). No significant differences were obtained between the two studies in the responses of plasma or serum catecholamines, cortisol, glucagon, growth hormone, thromboxane B2, and 6-ketoprostaglandin F1 alpha. These results suggest that epinephrine is responsible for some, if not all, of the beta-TG release from the platelets during insulin-induced hypoglycemia.