The nucleotide sequence of tobacco rattle virus RNA-2 (CAM strain).

The nucleotide sequence of the smaller genomic strand (RNA-2) of the bipartite tobacco rattle virus (CAM strain) has been determined. RNA-2 is capped at the 5' terminus and contains 1799 nucleotide residues. There is a single 223 codon long open reading frame extending from nucleotide 574 to 1242 which designates a protein of Mr 23,654. The derived amino acid composition, in percent, matches that previously determined for the virus capsid protein. The long open reading frame is flanked by 5' and 3' untranslated regions of 573 and 554 nucleotides, respectively. The 5' leader sequence contains two different sets of direct repeats, one of 119 nucleotides and the other of 76. It also contains 13 apparently unused AUG codons, four of which lie in the same frame as the capsid protein cistron. The 3' terminal sequence of RNA-2 is identical to that of the larger genomic strand (RNA-1) for 459 nucleotides.     

Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis: the cryV-encoded protein is expressed early in stationary phase.

A CryV-type protein (CGCryV) has been isolated from supernatant fluids of Bacillus thuringiensis AB88 cultures. Previous reports have suggested the cryptic nature of the cryV-type genes on the basis of the absence of CryV-type proteins in parasporal crystals. The CryV-type protein reported here is expressed early in stationary phase, and evidence indicates that it is an exported protein. Analysis of the deduced protein sequence from this gene reveals the presence of an N-terminal domain that likely acts as a signal peptide. The CGCryV protein is the first reported case of a delta-endotoxin being a secreted protein, which may influence the biological relevance of these proteins.     

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco

Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season. We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene. Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development. Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering. Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines. ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue. All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 g ICP per gram fresh weight in leaves from the mid-section of the plant at flowering. The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.     

Protease-armed bacteria in the skin

The skin constitutes a formidable barrier against commensal and pathogenic bacteria, which permanently and transiently colonise the skin, respectively. Commensal and pathogenic species inhabiting skin both express proteases. Whereas proteases secreted by commensals contribute to homeostatic bacterial coexistence on skin, proteases from pathogenic bacteria are used as virulence factors, helping them colonise skin with breached integrity of the epithelial layer. From these initial sites of colonisation, pathogens can disseminate into deeper layers of skin, possibly leading to the spread of infection. Secreted bacterial proteases probably play an important role in this process and in the deterrence of innate defence mechanisms. For example, Staphylococcus aureus proteases are essential for changing the bacterial phenotype from adhesive to invasive by degrading adhesins on the bacterial cell surface. Secreted staphylococcal proteases mediate pathogen penetration by degrading collagen and elastin, essential components of connective tissue in the dermis. The activation of the contact system and kinin generation by Streptococcus pyogenes and S. aureus proteases contributes to an inflammatory reaction manifested by oedema, redness and pain. Kinin-enhanced vascular leakage might help bacteria escape into the circulation thereby causing possible systemic dissemination of the infection. The inflammatory reaction can also be fueled by the activation of protease-activated receptors on keratinocytes. Concomitantly, bacterial proteases are involved in degrading antimicrobial peptides, disarming the complement system and neutrophils and preventing the infiltration of the infected sites with immune cells by inactivation of chemoattractants. Together, this provides protection for colonising and/or invading pathogens from attack by antibacterial forces of the skin.     

The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes

Rhodopsin, the prototypical G-protein-coupled receptor, which is densely packed in the disc membranes of rod outer segments, was proposed to function as a monomer. However, a growing body of evidence indicates dimerization and oligomerization of numerous G-protein-coupled receptors, and atomic force microscopy images revealed rows of rhodopsin dimers in murine disc membranes. In this work we demonstrate by electron microscopy of negatively stained samples, blue native- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, chemical crosslinking, and by proteolysis that native bovine rhodopsin exists mainly as dimers and higher oligomers. These results corroborate the recent findings from atomic force microscopy and molecular modeling on the supramolecular structure and packing arrangement of murine rhodopsin dimers.     

Sexual dimorphism in growth in the relative length of the forearm and relative knee height during adolescence

There are numerous studies concerning sexual dimorphism in body proportions, but only a few have investigated growth in the relative length of particular segments of the upper and lower limbs during adolescence. The aim of the study is an assessment of sex differences of longitudinal growth in the relative length of the forearm and knee height among adolescents. Sample involved 121 boys and 111 girls, participants of the Wroclaw Growth Study, examined annually between 8 and 18 years of age. Sexual dimorphism in six ratios: forearm length and knee height relatively to: trunk, height, and limb length were analyzed using a two‐way analysis of variance with repeated measurements. The sex and age relative to an estimate of maturity timing (3 years before, and after age class at peak height velocity [PHV]) were independent variables. All of the ratios showed significant sex differences in interaction with age relative to age at PHV. The relative length of the forearm, in boys, did not change significantly with the years relative to age at PHV, whereas in girls, was the lowest in the two first age classes and afterward significantly increased just 1 year before and during the adolescent growth spurt, remaining unchanged in further age classes. For relative knee height no clear pattern for sex differences was noticed. It is proposed that relatively longer forearms, particularly in relation to the trunk in girls, could have evolved as an adaptation to more efficient infant carrying and protection during breastfeeding.     

Locally enhanced sampling molecular dynamics study of the dioxygen transport in human cytoglobin

Cytoglobin (Cyg)--a new member of the vertebrate heme globin family--is expressed in many tissues of the human body but its physiological role is still unclear. It may deliver oxygen under hypoxia, serve as a scavenger of reactive species or be involved in collagen synthesis. This protein is usually six-coordinated and binds oxygen by a displacement of the distal HisE7 imidazole. In this paper, the results of 60 ns molecular dynamics (MD) simulations of dioxygen diffusion inside Cyg matrix are discussed. In addition to a classical MD trajectory, an approximate Locally Enhanced Sampling (LES) method has been employed. Classical diffusion paths were carefully analyzed, five cavities in dynamical structures were determined and at least four distinct ligand exit paths were identified. The most probable exit/entry path is connected with a large tunnel present in Cyg. Several residues that are perhaps critical for kinetics of small gaseous diffusion were discovered. A comparison of gaseous ligand transport in Cyg and in the most studied heme protein myoglobin is presented. Implications of efficient oxygen transport found in Cyg to its possible physiological role are discussed.