Structural and functional characterization of a prothrombin activator from the venom of Bothrops neuwiedi

A prothrombin activator from the venom of Bothrops neuwiedi was purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on a Zn2+-chelate column. The overall purification was about 200-fold, which indicates that the prothrombin activator comprises about 0.5% of the crude venom. The venom activator is a single-chain protein with an apparent molecular weight of 60 kDa. It readily activated bovine prothrombin with a Km of 38 microM and a Vmax of 120 mumol prothrombin activated per min per mg of venom activator. Venom-catalyzed prothrombin activation was not accelerated by the so-called accessory components of the prothrombinase complex, phospholipids plus Ca2+ and Factor Va. Gel-electrophoretic analysis of prothrombin activation indicated that the venom activator only cleaved the Arg-323-Ile-324 bond of bovine prothrombin, since meizothrombin was the only product of prothrombin activation. The activator did not hydrolyze commercially available p-nitroanilide substrates and its prothrombin-converting activity was not inhibited by benzamidine, phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethyl ketone and soy-bean trypsin inhibitor. However, chelating agents such as EDTA, EGTA and o-phenanthroline rapidly destroyed the enzymatic activity of the venom activator. The activity of chelator-treated venom activator could be partially restored by the addition of an excess CaCl2. These results indicate that the venom activator remarkably differs from Factor Xa and that the enzyme is not a serine proteinase, but likely belongs to the metalloproteinases. The structural and functional properties of the venom prothrombin activator from B. neuwiedi are similar to those reported for the venom activator from Echis carinatus.     

Visceral larva migrans: examinations by means of enzyme-linked immunosorbent assay of human sera for antibodies to excretory-secretory antigens of the second-stage larvae of Toxocara canis

An enzyme-linked immunosorbent assay is described for the detection of serum antibodies to visceral larva migrans (Toxocariasis). Excretory-secretory antigens of the second-stage larvae of Toxocara canis were used as antigen to coat the polystyrene plates. With sera from patients high antibody titers were observed in both ocular and visceral disorders. Cross-reactions due to other parasitic infections could be excluded, including other migrating larval infections such as ascariasis, trichinellosis, strongyloidiasis, filariasis, and anisakiasis. In a small seroepidemiologic survey of healthy primary schoolchildren, a remarkably high percentage (7.1) reacted positively to this method. These children showed eosinophilia as compared to the seronegative group. The data were compared with those observed in other countries and the results prompt reconsideration of the significance of T. canis for public health.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

Dietary vitamin A intake below the recommended daily intake during pregnancy and the risk of congenital diaphragmatic hernia in the offspring

BACKGROUND

Vitamin A has been related to the etiology of congenital diaphragmatic hernia (CDH). We performed a case‐control study to investigate whether maternal dietary vitamin A intake is related to CDH in the offspring.

METHODS

Thirty‐one pregnancies diagnosed with CDH and 46 control pregnancies were included during the study. After CDH diagnosis and inclusion of controls by risk set sampling, maternal vitamin A intake was investigated with a food frequency questionnaire. Serum retinol and retinol‐binding protein were determined. Univariable and multivariable logistic regression models were used to calculate risk estimates with adjustment for potential confounders.

RESULTS

We found no significant differences in the overall nutrient and vitamin A intake between case and control mothers. After stratification in body mass index (BMI) categories, case mothers with normal weight showed a lower energy adjusted vitamin A intake (685 vs. 843 μg retinol activity equivalents [RAEs] / day; p = 0.04) and a slightly lower serum retinol (1.58 vs. 1.67 μmol/L; p = 0.08) than control mothers. Vitamin A intake <800 μg retinol activity equivalents (recommended daily intake) in normal weight mothers was associated with a significantly increased CDH risk (odds ratio [OR], 7.2; 95% confidence interval [CI], 1.5–34.4; p = 0.01). Associations were not significantly different in underweight and overweight mothers.

CONCLUSIONS

In normal‐weight mothers, dietary vitamin A intake during pregnancy below the recommended daily intake is significantly associated with an increased risk of a child with CDH. This finding supports the retinoid hypothesis in human CDH, but warrants further investigation in larger study populations. Birth Defects Research (Part A), 2013. © 2013 Wiley Periodicals, Inc.     

Assessing the impact of thermal acclimation on physiological condition in the zebrafish model

The zebrafish has become a valuable vertebrate model organism in a wide range of scientific disciplines, but current information concerning the physiological temperature response of adult zebrafish is rather scarce. In this study, zebrafish were experimentally acclimated for 28 days to 18, 26 or 34 °C and a suite of non-invasive and invasive methods was applied to determine the thermal dependence of zebrafish physiological condition. With decreasing temperature, the metabolic rate of zebrafish decreased, as shown by the decreasing oxygen uptake and ammonia excretion rates, limiting the critical swimming speed, probably due to a decreased muscle fibre power output. In response to exercise, fuel stores were mobilized to the liver as shown by the increased hepatosomatic index, liver total absolute energetic value and liver carbohydrate concentration but due to the low metabolic rate they could not be adequately addressed to power swimming activity at 18 °C. Conversely, the increased metabolic performance at high temperature came with an increased metabolic cost resulting in decreased energy status reflected particularly well by the non-invasive condition factor and invasive measures of carcass protein concentration, carcass total absolute energetic value and liver carbohydrate concentration. We showed that the combined measurement of the relative condition factor and critical swimming speed is a powerful non-invasive tool for long-term follow-up studies. Invasive methods were redundant for measuring general energy status but they provided detailed information concerning metabolic reorganization. With this study we proved that the usefulness of the zebrafish as a model organism can easily be expanded to include physiological studies and we provided a reference dataset for the selection of measures of physiological responses for future studies using the zebrafish.     

GABA Shapes the Dynamics of Bistable Perception

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Multifactorial diversity sustains microbial community stability

Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty.     

Immunohistochemical localization of irisin in skin,eye, and thyroid and pineal glands of the crested porcupine (Hystrix cristata)

Irisin was first identified in muscle cells. We detected irisin immunoreactivity in various organs of the crested porcupine (Hystrix cristata). In the epidermis, irisin immunoreactivity was localized mainly in stratum basale, stratum spinosum and stratum granulosum layers; immunoreactivity was not observed in the stratum corneum. In the dermis, irisin was found in the external and internal root sheath, cortex and medulla of hair follicles, and in sebaceous glands. Irisin immunoreactivity was found in the neural retina and skeletal muscle fibers associated with the eye. The pineal and thyroid glands also exhibited irisin immunoreactivity.