Effects of aurothiomalate and gold(III) complexes on spontaneous motility of isolated human oviduct

Organic gold complexes have different biological activity, depending on their potential for interactions with key functional molecules.The aim of this study was to investigate potential of several newly synthesized organic gold complexes to influence spontaneous motility of the Fallopian tubes.The effects of [Au(bipy)Cl(2)](+) (dichloride(2,2'-bipyridyl)aurate(III)-ion), aurothiomalate, [Au(DMSO)(2)Cl(2)]Cl and DMSO on spontaneous motility of Fallopian tubes were tested on the isolated tube segments in vitro. Aurothiomalate (from 2.9?×?10(-9) to 4.9?×?10(-4)?M/l), [Au(bipy)Cl(2)]Cl (from 3.3?×?10(-9) to 4.2?×?10(-5)?M/l) and DMSO (from 1.9?×?10(-8) to 1.0?×?10(-5)?M/l) did not affect spontaneous contractions of the isolated Fallopian tube ampulla, while [Au(DMSO)(2)Cl(2)]Cl (from 2.9?×?10(-9) to 4.2?×?10(-5)?M/l) showed concentration-dependent increase (stimulation) of spontaneous contractions of the isolated Fallopian tube isthmus, and remained without effect on the isolated ampulla.The drugs designed as organic gold complexes with weaker bonds between the gold itself and organic part of a molecule could adversely affect motility of the Fallopian tubes, and theoretically fertility of women taking such drugs in their reproductive age.     

Self-organization of domain structures by DNA-loop-extruding enzymes

The long chromosomal DNAs of cells are organized into loop domains much larger in size than individual DNA-binding enzymes, presenting the question of how formation of such structures is controlled. We present a model for generation of defined chromosomal loops, based on molecular machines consisting of two coupled and oppositely directed motile elements which extrude loops from the double helix along which they translocate, while excluding one another sterically. If these machines do not dissociate from DNA (infinite processivity), a disordered, exponential steady-state distribution of small loops is obtained. However, if dissociation and rebinding of the machines occurs at a finite rate (finite processivity), the steady state qualitatively changes to a highly ordered ‘stacked’ configuration with suppressed fluctuations, organizing a single large, stable loop domain anchored by several machines. The size of the resulting domain can be simply regulated by boundary elements, which halt the progress of the extrusion machines. Possible realizations of these types of molecular machines are discussed, with a major focus on structural maintenance of chromosome complexes and also with discussion of type I restriction enzymes. This mechanism could explain the geometrically uniform folding of eukaryote mitotic chromosomes, through extrusion of pre-programmed loops and concomitant chromosome compaction.     

Increase in specific density of levobupivacaine and fentanyl solution ensures lower incidence of inadequate block

The clinical presentation of a subarachnoid block (SAB) is dependent upon the intrathecal spread of local anesthetic (LA). Intrathecal distribution depends on the chemical and physical characteristics of LA, puncture site, technique used, patient anatomical characteristics and hydrodynamic properties of cerebrospinal fluid. We tried to determine whether a combined glucose/LA solution can render a clinically significant difference in sensory block distribution and motor block intensity.This was a controlled, randomized and double blinded study. The surgical procedures were stripping of the great or small saphenous vein and extirpation of remaining varicose veins. The study included 110 patients distributed into two groups: Hyperbaric (7.5 mg levobupivacaine (1.5 ml 0.5% Chirocaine) + 50 microg Fentanyl (0.5 ml Fentanil) and 1 ml 10% glucose (Pliva)) vs. Hypobaric (7.5 mg levobupivacaine (1.5 ml 0.5% Chirocaine) + 50 microg Fentanyl (0.5 ml Fentanil) and 1 ml 0.9% NaCl (Pliva, Zagreb)) adding to a total volume of 3.5 ml per solution. Spinal puncture was at L3-L4 level. Spinal block distribution was assessed in five minute intervals and intensity of motor block was assessed according to the modified Bromage scale. Pain was assessed with the Visual Analogue Scale. A statistically significant difference in sensory block distribution, motor block intensity and recovery time was established between hyperbaric and hypobaric solutions. By increasing the specific density of anesthetic solution, a higher sensory block, with lesser variability, a diminished influence of Body Mass Index, decreased motor block intensity and faster recovery time may be achieved.     

Vaginal delivery and continuous epidural analgesia: should we change our clinical approach?

The aim of the study was to investigate the effects of continuous epidural analgesia (EA) on the course of vaginal delivery with an emphasis on duration of labor and instrumental interventions. In a prospective 2-year trial, the study group included singleton vaginal births between 35 and 41 gestational weeks with a vertex fetus, in which continuous EA with bupivacaine or chirocaine in concentration of 0.125% combined with 2-4 microg of fentanyl or 0.5 microg of sufenta was used. The control group was created randomly from laboring patients with singleton pregnancies but without EA. The groups were adjusted for epidemiological characteristics and compared regarding the obstetric data and perinatal outcome. Student t-test and Mann-Whitney U-test were performed for normally and non-normally distributed results, respectively. Out of 1284 patients, 551 pregnant women were included in the study group and 733 in the control group. The statistically significant differences between the groups related to duration of the first and second stage of labor, frequency of premature rupture of membranes, intrapartal complications, and incidence of operative deliveries were found. Both stages of labor were significantly protracted and the incidence of operative deliveries was higher in the study group of patients compared with controls. There is a need for an active obstetric approach and management of vaginal deliveries of women who receive continuous EA, particularly if it is medically indicated.     

Chromosome elasticity and mitotic polar ejection force measured in living Drosophila embryos by four-dimensional microscopy-based motion analysis

BACKGROUND: Mitosis involves the interaction of many different components, including chromatin, microtubules, and motor proteins. Dissecting the mechanics of mitosis requires methods of studying not just each component in isolation, but also the entire ensemble of components in its full complexity in genetically tractable model organisms. RESULTS: We have developed a mathematical framework for analyzing motion in four-dimensional microscopy data sets that allows us to measure elasticity, viscosity, and forces by tracking the conformational movements of mitotic chromosomes. We have used this approach to measure, for the first time, the basic biophysical parameters of mitosis in wild-type Drosophila melanogaster embryos. We found that Drosophila embryo chromosomes are significantly less rigid than the much larger chromosomes of vertebrates. Anaphase kinetochore force and nucleoplasmic viscosity were comparable with previous estimates in other species. Motion analysis also allowed us to measure the magnitude of the polar ejection force exerted on chromosome arms during metaphase by individual microtubules. We find the magnitude of this force to be approximately 1 pN, a number consistent with force generation either by collision of growing microtubules with chromosomes or by single kinesin motors. CONCLUSIONS: Motion analysis allows noninvasive mechanical measurements to be made in complex systems. This approach should allow the functional effects of Drosophila mitotic mutants on chromosome condensation, kinetochore forces, and the polar ejection force to be determined.     

Characterization of the human type XVIII collagen gene and proteolytic processing and tissue location of the variant containing a frizzled motif.

Human type XVIII collagen was found to be expressed as three variants, termed NC1-303, NC1-493 and NC1-728, differing in their N-terminal non-collagenous domains (NC1). The corresponding gene was found to be approximately 105 kb in size and contain 43 exons. The short variant is derived from utilization of an upstream promoter associated with the first two exons of the gene. The two other variants are derived from a downstream promoter and alternative splicing of exon 3, resulting in 192 residues of shared sequences characterized by a putative approximately 30 residue conserved coiled-coil motif and 235 residues of sequences specific to NC1-728. The NC1-728 variant has a conserved cysteine-rich domain homologous with the ligand-binding part of the frizzled proteins. A polyclonal antibody specific to the NC1-728 variant was generated, and immunostaining of fetal tissues revealed staining in lung and skeletal muscle. Human serum contained 173- and 144-kDa alpha1(XVIII) chains corresponding to the NC1-728 and NC1-493 variants, respectively. A 200-kDa polypeptide was detected in cells transfected with a cDNA construct corresponding to the full-length NC1-728 variant, and EBNA-293 cells endogenously synthesizing low amounts of type XVIII collagen had a 45-kDa fragment in their culture medium that corresponded to most of the NC1 domain of the NC1-728 variant, suggesting processing of the N-terminal frizzled-containing domain.     

Inhibition of aurora B kinase blocks chromosome segregation,overrides the spindle checkpoint,and perturbs microtubule dynamics in mitosis

How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.     

Limits of Ligand Selectivity from Docking to Models: In Silico Screening for A1 Adenosine Receptor Antagonists

G protein-coupled receptors (GPCRs) are attractive targets for pharmaceutical research. With the recent determination of several GPCR X-ray structures, the applicability of structure-based computational methods for ligand identification, such as docking, has increased. Yet, as only about 1% of GPCRs have a known structure, receptor homology modeling remains necessary. In order to investigate the usability of homology models and the inherent selectivity of a particular model in relation to close homologs, we constructed multiple homology models for the A₁ adenosine receptor (A₁AR) and docked ∼2.2 M lead-like compounds. High-ranking molecules were tested on the A₁AR as well as the close homologs A_2AAR and A₃AR. While the screen yielded numerous potent and novel ligands (hit rate 21% and highest affinity of 400 nM), it delivered few selective compounds. Moreover, most compounds appeared in the top ranks of only one model. These findings have implications for future screens.     

Resurrected Ceriodaphnia quadrangula highlight differences between pheno‐ and genotypic expressions

The hatching of cladoceran ephippia from a 15‐cm long sediment core was investigated, and Ceriodaphnia quadrangula clones were isolated from different sediment layers. Bosmina microfossil data were also analyzed, and compared with the corresponding data from a Pb210 dated core, which allowed us to infer the age of the sediment layers. Using changes in Bosmina microfossil morphologies, we were, furthermore, able to infer the presence of different regimes of fish predation. C. quadrangula was found to hatch in layers with an inferred age of approximately a century. Newly hatched individuals had smaller eye‐size in sediment layers corresponding to high predation by young‐of‐the‐year perch. Newly hatched individuals also generally had a marked neck‐spine. In contrast, morphological characters of C. quadrangula clones reared in the laboratory over several generations showed no variation in relation to predation regime, indicating the absence of fixed genotype level changes. Furthermore, the laboratory grown clones only rarely produced a neck‐spine. The results suggest phenotypic variation in response to the regime under which ephippia were produced.     

A simple and efficient protocol for the production of recombinant cathepsin V and other cysteine cathepsins in soluble form in Escherichia coli

Cysteine cathepsins are major players in numerous physiologic and pathologic processes and important drug targets. Several different expression systems have been developed for the production of these enzymes. Here we describe a novel, simple and efficient protocol for the production of recombinant cathepsin V and other cysteine cathepsins. Recombinant procathepsin V was expressed in soluble form in the cytoplasm of Escherichia coli and purified in one step by immobilized nickel ion-affinity chromatography, yielding approximately 0.7 mg procathepsin V per liter bacterial culture. The recombinant proenzyme was then autocatalytically activated in vitro by incubation at pH 4.0 and 30 °C. The yield of proenzyme conversion was over 95% and the mature enzyme exhibited potent activity towards several commonly used synthetic substrates. The same protocol also proved successful in the production of several other cysteine procathepsins, such as cathepsin B, demonstrating that this procedure is widely applicable for the production of recombinant papain-like cysteine peptidases.