Relationship between first polar body morphology before intracytoplasmic sperm injection and fertilization rate, cleavage rate, and embryo quality

Objectives: To evaluate the influence of the morphology of the first polar body (PB) on intracytoplasmic sperm injection (ICSI) outcomes. Methods: The morphology of the first PB was assessed in 3177 metaphase 11 oocytes and classified as: intact and normal size, fragmented, or enlarged size. The rates of fertilization, cleavage, and embryo quality were evaluated on day 2. Results: The rates of fertilization, cleavage, and formation of good quality embryos resulting from the insemination of oocytes with an enlarged first PB (20.7%, 18.7%, and 5.0%, respectively) were significantly lower than those for oocytes with an intact first PB of normal size (70.8%, 62.5%, and 19%, respectively) or a fragmented first PB (69.7%, 60.5%, and 17.1%, respectively). Rates did not differ significantly between oocytes with an intact first PB of normal size and oocytes with a fragmented first PB (P>0.05). Conclusions: The presence of an enlarged PB is related to poorer rates of fertilization, cleavage, and top quality embryos. However, identification of first PB fragmentation does not seem to interfere with ICSI outcomes. (C) 2008 International Federation ofGynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

Precursor scavenging of the resistive grain-boundary phase in 8 mol% yttria-stabilized zirconia: Effect of trace concentrations of SiO2

The influence that trace concentrations Of SiO2 have on improving grain-boundary conduction via precursor scavenging using additional heat treatment at 1200 degreesC for 40 h before sintering was investigated. At a SiO2-impurity level (SIL) less than or equal to 160 ppm by weight, the grain-boundary resistivity (p(gb)) decreased to 20% of its value, while no improvement in grain-boundary conduction was found at a SIL greater than or equal to 310 ppm. The correlation between the resistance per unit grain-boundary area, p(gb), and average grain size indicated that the inhomogeneous distribution of the siliceous phase in the sample with a SIL greater than or equal to 310 ppm. hampered the scavenging reaction.

A longitudinal analysis of cytotoxic T lymphocyte precursor frequencies to the hepatitis B virus in chronically infected patients

Individuals with acute hepatitis B virus (HBV) infection characteristically mount a strong, multispecific cytotoxic T lymphocyte (CTL) response that is effective in eradicating virus. In contrast, this response in chronic carriers is usually weak or undetectable. Since it is generally acknowledged that HBV pathogenesis is immune-mediated, the occurrence of episodes of active liver disease in many carriers suggests that these individuals can mount active CTL responses to HBV. To see whether the detection of circulating CTLs is related to these flare episodes, we have determined the CTL precursor (CTLp) frequencies to HLA-A2-restricted viral peptides in seven patients over a 12-24-month period of their disease. Limiting dilution analyses (LDA) were performed longitudinally to five epitopes comprising the viral capsid (HBc), envelope (HBs) and polymerase (pol) proteins. Assays were performed against a mixture of peptides, or against each individual peptide, to measure overall CTL activity and the multispecificity of the responses, respectively. Since two of the patients were treated with recombinant human interleukin-12 (rHuIL-12) at the time, with one individual achieving complete disease remission a year later after being treated with interferon-alpha, we were also able to examine the effects of these cytokines on HBV cytotoxicity. Our results indicate that weak but detectable CTL responses do occur in chronic carriers which are generally associated with disease flares, although CTLps were also seen occasionally during minimal disease activity. The range of specificities varied between individuals and within each individual during the course of the disease. Finally, we also provide evidence that CTL reactivity is stimulated following treatment with certain cytokines, but is dependent on the time of administration.

The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata

A two-domain portion of the proteinase inhibitor precursor from Nicotiana alata (NaProPI) has been expressed and its structure determined by NMR spectroscopy. NaProPI contains six almost identical 53 amino acid repeats that fold into six highly similar domains; however, the sequence repeats do nut coincide with the structural domains. Five of the structural domains comprise the C-terminal portion of one repeat and the N-terminal portion of the next. The sixth domain contains the C-terminal portion of the sixth repeat and the N-terminal portion of the first repeat. Disulphide bonds link these C and N-terminal fragments to generate the clasped-bracelet fold of NaProPI. The three-dimensional structure of NaProPI is not known, but it is conceivable that adjacent domains in NaProPI interact to generate the circular bracelet with the N and C termini in close enough proximity to facilitate formation of the disulphide bonds that form the clasp The expressed protein, examined in the current study, comprises residues 25-135 of NaProPI and encompasses the first two contiguous structural domains, namely the chymotrypsin inhibitor C1 and the trypsin inhibitor T1, joined by a five-residue linker, and is referred to as C1-T1. The tertiary structure of each domain in C1-T1 is identical to that found in the isolated inhibitors. However, no nuclear Overhauser effect contacts are observed between the two domains and the five-residue linker adopts an extended conformation. The absence of interactions between the domains indicates that adjacent domains do not specifically interact to drive the circularisation of NaProPI. These results are in agreement with recent data which describe similar PI precursors from other members of the Solanaceae having two, three, or four repeats. The lack of strong interdomain association is likely to be important for the function of individual inhibitors by ensuring that there is no masking of reactive sites upon release from the precursor. (C) 2001 Academic Press.

The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

Cleavage of hemoglobin by hookworm cathespin D aspartic proteases and its potential contribution to host specificity

Hookworms routinely reach the gut of nonpermissive hosts but fail to successfully feed, develop, and reproduce. To investigate the effects of host-parasite coevolution on the ability of hookworms to feed in nonpermissive hosts, we cloned and expressed aspartic proteases from canine and human hookworms. We show here that a cathepsin D-like protease from the canine hookworm Ancylosotoma caninum (Ac-APR-1) and the orthologous protease from the human hookworm Necator americanus (Na-APR-1) are expressed in the gut and probably exert their proteolytic activity extracellularly. Both proteases were detected immunologically and enzymatically in somatic extracts of adult worms. The two proteases were expressed in baculovirus, and both cleaved human and dog hemoglobin (Hb) in vitro. Each protease digested Hb from its permissive host between twofold (whole molecule) and sixfold (synthetic peptides) more efficiently than Hb from the nonpermissive host, despite the two proteases' having identical residues lining their active site clefts. Furthermore, both proteases cleaved Hb at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor for host-parasite compatibility and host species range.

Precursor scavenging of resistive grain-boundary phase in 8 mol % ytterbia-stabilized zirconia

The grain-boundary conduction of 8 mol % ytterbia-stabilized zirconia (8YbSZ) was improved markedly by precursor scavenging via the two-stage sintering process. The most significant increase in the grain-boundary conductivity was found when the sample, whose conductivity was higher than that via Al2O3-derived scavenging, was heat-treated at 1250degreesC for greater than or equal to 20 h. The formation of a stable Si-containing phase such as ZrSiO4 during the first-stage heat-treatment was suggested as one probable scavenging route from the optimal heat-treatment temperature (HTT), long duration time (>20 h) at HTT, and the stability of the formed phase up to sintering temperatures (1500degrees C). (C) 2002 The Electrochemical Society.

Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-I, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 mu M. Their activities against HIV-1 protease (K-i 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC50 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC50 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 Angstrom (1) and 1.85 Angstrom (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.

Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

Cobalt complexes bearing scorpionate ligands: Synthesis, characterization, cytotoxicity and DNA cleavage

A number of novel, water-stable redox-active cobalt complexes of the C-functionalized tripodal ligands tris(pyrazolyl)methane XC(pz)(3) (X = HOCH2, CH2OCH2Py or CH2OSO2Me) are reported along with their effects on DNA. The compounds were isolated as air-stable solids and fully characterized by IR and FIR spectroscopies, ESI-MS(+/-), cyclic voltammetry, controlled potential electrolysis, elemental analysis and, in a number of cases, also by single-crystal X-ray diffraction. They showed moderate cytotoxicity in vitro towards HCT116 colorectal carcinoma and HepG2 hepatocellular carcinoma human cancer cell lines. This viability loss is correlated with an increase of tumour cell lines apoptosis. Reactivity studies with biomolecules, such as reducing agents, H2O2, plasmid DNA and UV-visible titrations were also performed to provide tentative insights into the mode of action of the complexes. Incubation of Co(II) complexes with pDNA induced double strand breaks, without requiring the presence of any activator. This pDNA cleavage appears to be mediated by O-centred radical species.

Synthesis of titanate nanostructures using amorphous precursor material and their adsorption/photocatalytic properties

This article reports on a new and swift hydrothermal chemical route to prepare titanate nanostructures (TNS) avoiding the use of crystalline TiO2 as starting material. The synthesis approach uses a commercial solution of TiCl3 as titanium source to prepare an amorphous precursor, circumventing the use of hazardous chemical compounds. The influence of the reaction temperature and dwell autoclave time on the structure and morphology of the synthesised materials was studied. Homogeneous titanate nanotubes with a high length/diameter aspect ratio were synthesised at 160 degrees C and 24 h. A band gap of 3.06 +/- 0.03 eV was determined for the TNS samples prepared in these experimental conditions. This value is red shifted by 0.14 eV compared to the band gap value usually reported for the TiO2 anatase. Moreover, such samples show better adsorption capacity and photocatalytic performance on the dye rhodamine 6G (R6G) photodegradation process than TiO2 nanoparticles. A 98% reduction of the R6G concentration was achieved after 45 min of irradiation of a 10 ppm dye aqueous solution and 1 g L-1 of TNS catalyst.

Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG

Antigenic preparations from Sporothrix schenckii usually involve materials from mixed cultures of yeast and mycelia presenting cross-reactions with other deep mycoses. We have standardized pure yeast phase with high viability of the cells suitable to obtain specific excretion-secretion products without somatic contaminations. These excretion-secretion products were highly immunogenic and did not produce noticeable cross-reactions in either double immunodiffusion or Western blot. The antigenic preparation consists mainly of proteins with molecular weights between 40 and 70 kDa, some of them with proteolytic activity in mild acidic conditions. We also observed cathepsin-like activity at two days of culture and chymotrypsin-like activity at four days of culture consistent with the change in concentration of different secreted proteins. The proteases were able to cleave different subclasses of human IgG suggesting a sequential production of antigens and molecules that could interact and interfere with the immune response of the host.

Is land surface temperature an earthquake precursor?

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies

Trans-Synaptic Signaling by Activity-Dependent Cleavage of Neuroligin-1

Throughout the brain, patterns of activity in postsynaptic neurons influence the properties of synaptic inputs. Such feedback regulation is central to neural network stability that underlies proper information processing and feature representation in the central nervous system. At the cellular level, tight coupling of presynaptic and postsynaptic function is fundamental to neural computation and synaptic plasticity. The cohort of protein complexes at the pre and postsynaptic membrane allows for tight synapse-specific segregation and integration of diverse molecular and electrical signals.(...)