Purification and properties of cathepsin D from the mammary glands of lactating rabbits

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.     

Retention of the skill of adaptive biocontrol of cortical bioelectric activity synchronization in argon-nitrogen-oxygen atmosphere with different oxygen concentrations

A decreased oxygen concentration in the air (hypoxia) is an efficient preventive measure against fire in spacecrafts and other habitable pressurized modules. Addition of argon to the air may increase the human tolerance of this hypoxia. In order to estimate the human mental performance in argon-nitrogenoxygen atmosphere (ANOA) with different oxygen concentrations, the following characteristics were determined: (i) the retention of the skill of adaptive biocontrol (ABC) of bioelectric activity synchronization in the frontoparietal and parietooccipital cortical areas with the use of EEG; (ii) emotional and mental states estimated using Luscher’s test; and (iii) performance parameters estimated using the computer games Tetris and Minesweeper as models. Despite fluctuations of the anxiety level, ANOA did not decrease the parameters of the performance of the model activity or long-term retention of the ABC skill (for 24 h), specific characteristics of the ABC skill retention being related to the type of activity.     

The structural and dynamic model of the serotonin 5-HT3 receptor. Comparative analysis of the structure of the channel domain of pentameric ligand-gated ion channels

The three-dimensional structure of the 5-HT3 receptor is currently unknown. An available structure of the nicotinic acetylcholine receptor closely related by homology to the 5-HT3 receptor was used as a template for the computer-based homology modeling of the 5-HT3 receptor. The study of the ion migration through the channel by the steered molecular dynamics method has shown that the steric factor in the region of residue Thr279 and the region of Glu272, Asp293 influences the ion transmission. The characteristic of the close interaction between the ion and the amino acid substitutions of the 5-HT3 channel was studied by computing the energy profile using constraint force molecular dynamic simulations. The amino acid sequence responsible for selective ion transmission has been investigated. The structure of the channel domain of the serotonin 5-HT3 receptor as a universal functional unit of the ligand-gated ion channels was discussed.     

The effect of glutoxim on Na+ transport in frog skin: the role of cytoskeleton

Using voltage-clamp technique, the possible role of the cytoskeleton in the effect of pharmacological analogue of oxidized glutathione (GSSG), drug glutoxim, on Na+ transport in the frog Rana temporaria skin was investigated. It was shown for the first time that preincibation of the skin with the microtubular disrupter, nocodazole, actin filament disrupter, cytochalasin D or protein phosphatase PP1/PP2A inhibitor, calyculin A, significantly decrease the stimulatory effect of glutoxim on Na+ transport. The data suggest the involvement of microtubules and microfilaments in the regulatory effect of glutoxim on Na+ transport in frog skin and that reorganization of actin filaments or microtubules leads to inhibition of stimulatory effect of glutoxim on Na+ transport in frog skin epithelia.     

Inhibition of GABAergic Transmission as a Model of Hyperactivation of Purkinje Cells in the Rat Cerebellum

Biophysics - Abstract—Pathological changes in the cerebellum are often associated with dysfunction of Purkinje cells, which is manifested in excessive spike activity. In our experiments, the...     

Mobilization of a cryptic plasmid from the melioidosis pathogen in heterologous species of microorganisms

Cryptic plasmids with different molecular weights have been detected in B. pseudomallei strains. Mobilization of B. pseudomallei plasmid DNA in heterologous B. mallei species was performed by the conjugate plasmid RP1::Tn10. Possibility of detecting phenotypical characteristics of plasmids and behavior of B. pseudomallei non-chromosomal replicons in B. mallei have been determined.     

Sex allocation in relation to host races in the brood-parasitic common cuckoo (Cuculus canorus)

Sex allocation theory and empirical evidence both suggest that natural selection should favour maternal control of offspring sex ratio in relation to their ability to invest in the offspring. Generalist parasites constitute a particularly interesting group to test this theory as different females commonly utilize different host species showing large variation in provisioning ability. The common cuckoo (Cuculus canorus) is a generalist brood parasite that lays its eggs in the nest of many different passerine birds, but each female tends to specialize on one particular host species giving rise to highly specialized host races. The different host species show large variation in their ability to invest in the parasitic offspring, presenting an opportunity for female cuckoos to bias offspring sex ratio in relation to host species quality. Here, we investigate host-race specific sex allocation controlling for maternal identity in the common cuckoo. We found no evidence of any significant relationship between host race and sex ratio in one sympatric population harbouring three different host races, or in a total of five geographically separated populations. There was also no significant association between host quality, as determined by species-specific female host body mass, and cuckoo sex ratio. Finally, we found no significant relationship between individual cuckoo maternal quality, as determined by her egg volume, and sex ratio within each host race. We conclude that the generalist brood-parasitic common cuckoo show no significant sex-ratio bias in relation to host race and discuss this finding in light of gene flow and host adaptations.     

Molecular phylogenetic analysis of the ant genus <Emphasis Type="Italic">Formica</Emphasis> L. (Hymenoptera: Formicidae) from Palearctic region

Sixty-five sequences of the mitochondrial DNA cytochrome b gene fragment (759 bp) and 23 sequences of the NADH dehydrogenase subunit 6 gene fragment (224 bp) were compared in ants of the genus Formica L. from different regions of the Palearctic and in Polyergus rufescens Latr. as outgroup. In total, 28 species of the genus Formica were examined. As a result, dated trees with a molecular clock were constructed showing the phylogenetic relationships of Formica ants. The topology of the obtained tree based on the Cyt-b sequences was found to be not consistent with the generally accepted opinion on the Formica rufa and F. rufibarbis groups. New data on the formation history of the present-day fauna of Formica ants of the Palearctic were obtained. It was demonstrated that a considerable fraction of the examined species (about a third) were formed in the Quaternary Period.     

General patterns of sexual dimorphism in graylings (Thymallus), with a comparison to other salmonid species

Among fishes, salmonids (family Salmonidae) have attracted a great deal of research attention focused on sexual dimorphism and associated selective forces. Most of this research has been directed toward anadromous and mostly semelparous salmon and trout (Oncorhynchus, Salmo), and comparatively little is known about intersexual variability in strictly iteroparous freshwater salmonids. We examined a comprehensive data set of 28 linear morphometric characters in 11 of 15 currently recognised species of grayling (Thymallinae, Thymallus), a genus consisting of iteroparous species only, to identify general patterns of intersexual morphological variability. Overall, we found that all grayling species show common sex-specific traits particularly relating to size dimensions of the dorsal, anal, pelvic and pectoral fins. Although the magnitude of sexual dimorphism differed among species, there was no significant phylogenetic signal associated with these differences across the genus. These results are discussed in terms of the assumed selection pressures driving sexual dimorphism in graylings and are compared to existing knowledge in Salmonidae as a whole where similarities and differences with both Salmoninae and Coregoninae exist. The present study provides the first detailed genus-wide comparison of sexually dimorphic phenotypic characters in graylings, and highlights the need for more large-scale comparative studies in multiple salmonid species to better understand general macroevolutionary trends among this important group of freshwater fishes.

     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.