Properties of the Relaxation Complexes of Supercoiled Deoxyribonucleic Acid and Protein of the R Plasmids R64, R28K, and R6K

The presence of supercoiled deoxyribonucleic acid in the form of a relaxation complex is described for the antibiotic resistance plasmids R64, R28K, and R6K. The properties of these relaxation complexes indicate that they consist of a covalently closed circular deoxyribonucleic acid molecule associated with an activable, single strand-specific endonuclease.     

Multiple Meiotic Drive Systems in the DROSOPHILA MELANOGASTER Male

The behaviour of two "meiotic drive" systems, Segregation-Distorter (SD) and the sex chromosome sc(4)sc(8) has been examined in the same meiocyte. It has been found that the two systems interact in a specific way. When the distorting effects of SD and sc(4)sc(8) are against each other, there is no detectable interaction. Each system is apparently oblivious to the presence of the other, gametes being produced according to independence expectations. However when the affected chromosomes are at the same meiotic pole an interaction occurs; the survival probability of the gamete containing both distorted chromosomal products is increased, rather than being decreased by the combined action of two systems.     

Developmental Genetics of Loci at the Base of the X Chromosome of Drosophila Melanogaster

We have conducted a genetic and developmental analysis of the 26 contiguous genetic complementation groups within the 19D3-20F2 interval of the base of the X chromosome, a region of 34 polytene bands delimited by the maroon-like and suppressor of forked loci. Within this region there are four loci which cause visible phenotypes but which have little or no effect on zygotic viability (maroon-like, little fly, small optic lobes and sluggish). There are 22 loci which, when mutated, are zygotic lethals and three of these, legless/runt, folded gastrulation and 13E3, have severe effects on embryonic development. In addition, three visible phenotypes have been defined only by overlapping deficiencies (melanized-like, tumorous head, and varied outspread). We have analyzed the lethal phases and maternal requirement of 58 mutations at 22 of the zygotic lethal loci by means of germline clone analysis using the dominant female sterile technique. Additionally, all lethal complementation groups, as well as a specific subset of deficiencies, have been studied histologically for defects in the development of the central and peripheral embryonic nervous systems.     

CYK4 Promotes Antiparallel Microtubule Bundling by Optimizing MKLP1 Neck Conformation

Centralspindlin, a constitutive 2:2 heterotetramer of MKLP1 (a kinesin-6) and the non-motor subunit CYK4, plays important roles in cytokinesis. It is crucial for the formation of central spindle microtubule bundle structure. Its accumulation at the central antiparallel overlap zone is key for recruitment and regulation of downstream cytokinesis factors and for stable anchoring of the plasma membrane at the midbody. Both MKLP1 and CYK4 are required for efficient microtubule bundling. However, the mechanism by which CYK4 contributes to this is unclear. Here we performed structural and functional analyses of centralspindlin using high-speed atomic force microscopy, Fӧrster resonance energy transfer analysis, and in vitro reconstitution. Our data reveal that CYK4 binds to a globular mass in the atypically long MKLP1 neck domain between the catalytic core and the coiled coil and thereby reconfigures the two motor domains in the MKLP1 dimer to be suitable for antiparallel microtubule bundling. Our work provides insights into the microtubule bundling during cytokinesis and into the working mechanisms of the kinesins with non-canonical neck structures.     

NOX5 in human spermatozoa: expression, function, and regulation

Physiological and pathological processes in spermatozoa involve the production of reactive oxygen species (ROS), but the identity of the ROS-producing enzyme system(s) remains a matter of speculation. We provide the first evidence that NOX5 NADPH oxidase is expressed and functions in human spermatozoa. Immunofluorescence microscopy detected NOX5 protein in both the flagella/neck region and the acrosome. Functionally, spermatozoa exposed to calcium ionophore, phorbol ester, or H(2)O(2) exhibited superoxide anion production, which was blocked by addition of superoxide dismutase, a Ca(2+) chelator, or inhibitors of either flavoprotein oxidases (diphenylene iododonium) or NOX enzymes (GKT136901). Consistent with our previous overexpression studies, we found that H(2)O(2)-induced superoxide production by primary sperm cells was mediated by the non-receptor tyrosine kinase c-Abl. Moreover, the H(V)1 proton channel, which was recently implicated in spermatozoa motility, was required for optimal superoxide production by spermatozoa. Immunoprecipitation experiments suggested an interaction among NOX5, c-Abl, and H(V)1. H(2)O(2) treatment increased the proportion of motile sperm in a NOX5-dependent manner. Statistical analyses showed a pH-dependent correlation between superoxide production and enhanced sperm motility. Collectively, our findings show that NOX5 is a major source of ROS in human spermatozoa and indicate a role for NOX5-dependent ROS generation in human spermatozoa motility.     

Automated generation of turn mimetics: proof of concept study for the MC4 receptor

An algorithm has been devised for the automatic design of peptide turn mimetics, particularly applicable to peptide-activated GPCRs. The method is based on flexible alignments using a new design paradigm and scoring system that aims to reduce the molecular weight of the compound and preferentially lead to drug like molecules. The process can be applied either as a de novo design or a virtual screening tool. Its use has been demonstrated by the design of novel double digit nanomolar ligands for the melanocortin 4 receptor (MC4). The method is, in principle, applicable to any type of receptor, including orphan receptors.     

Analysis of the qualitative dermatoglyphics of the digito-palmar complex in patients with primary open angle glaucoma

The primary open-angle glaucomas are a group of diseases that have in common characteristic morphological changes at the optic nerve head and retinal nerve fiber layer, progressive retinal ganglion cells death and characteristic visual field loss. The risk for primary open angle glaucoma rises continuously with the level of the intraocular pressure. The disease advances slowly and there are no symptoms. Primary open angle glaucoma is caused by abnormal aqueous humour outflow in the trabecular meshwork in the open angle. Etiopathogenesis of primary open angle glaucoma is unclear. The increased risk of glaucoma in relatives has long been recognized. Frequency for manifestation of the disease is 10-30% in family members. The discovery of the specific gene loci responsible for the manifestation of glaucoma has helped us to understand its mechanism of origin and definitely confirmed the hereditary nature of this disease. Digito-palmar dermatoglyphs were already used to determine hereditary base of many diseases and it was the reason for investigation of their qualitative patterns in patients with glaucoma (22 males and 23 females), their immediate relatives (19 males and 23 females) in comparison to a group of phenotypically healthy population (52 males and 56 females). The results pointed a connection with the dermatoglyphic traits of the digito-palmar complex between patients with glaucoma and their immediate relatives. There is a possible discrimination of patients and their immediate relatives from phenotypically healthy population, too.     

Revisiting the Role of Cystic Fibrosis Transmembrane Conductance Regulator and Counterion Permeability in the pH Regulation of Endocytic Organelles

Organellar acidification by the electrogenic vacuolar proton-ATPase is coupled to anion uptake and cation efflux to preserve electroneutrality. The defective organellar pH regulation, caused by impaired counterion conductance of the mutant cystic fibrosis transmembrane conductance regulator (CFTR), remains highly controversial in epithelia and macrophages. Restricting the pH-sensitive probe to CFTR-containing vesicles, the counterion and proton permeability, and the luminal pH of endosomes were measured in various cells, including genetically matched CF and non-CF human respiratory epithelia, as well as cftr^+/+ and cftr^−/− mouse alveolar macrophages. Passive proton and relative counterion permeabilities, determinants of endosomal, lysosomal, and phagosomal pH-regulation, were probed with FITC-conjugated transferrin, dextran, and Pseudomonas aeruginosa, respectively. Although CFTR function could be documented in recycling endosomes and immature phagosomes, neither channel activation nor inhibition influenced the pH in any of these organelles. CFTR heterologous overexpression also failed to alter endocytic organellar pH. We propose that the relatively large CFTR-independent counterion and small passive proton permeability ensure efficient shunting of the proton-ATPase–generated membrane potential. These results have implications in the regulation of organelle acidification in general and demonstrate that perturbations of the endolysosomal organelles pH homeostasis cannot be linked to the etiology of the CF lung disease.