Elimination of apoptotic spermatozoa from rabbit insemination dose using annexin V associated with the MACS technique. A preliminary study

The aim of this study was to verify whether the separation and elimination of the apoptotic fraction in rabbit semen using a MACS technique may improve sperm fertility potential and consequently rabbit kindling rate. Semen samples from 25 New Zealand White (NZW) rabbit males were collected using an artificial vagina and evaluated using the CASA system for concentration and motility. For artificial insemination the best 11 bucks were chosen based on motility parameters. Their ejaculates were mixed to make a heterospermic pool and routinely diluted in a commercial insemination diluent (MiniTüb, Tiefenbach, Germany) at a ratio of 1:6. Diluted heterospermic spermatozoa were filtered through a Sartorius filter to wash out seminal plasma, re-diluted in binding buffer (Annexin V Microbead Kit, Miltenyi Biotec, Germany) at a ratio of 1:3.66 and divided into two groups: an experimental group intended for MACS separation and control group without MACS separation. Then hormonally treated females of NZW rabbits were inseminated with fresh doses of filtered heterospermic semen (n = 27; 0.5 ml I.D. per female) and MACS separated semen (n=28; 0.5 ml I.D. per female). Separation and subsequent elimination of apoptotic spermatozoa (positive selection) from the insemination dose (after negative MACS selection) was verified under in vivo conditions on the basis of increased kindling rate in the experimental group in comparison with kindling rate in the control group (81.3% vs. 73.8%). In conclusion, elimination of apoptotic spermatozoa by the use of the MACS technique results in a slight improvement in kindling rate of rabbit does.     

TRAIL dependent fratricidal killing of gp120 primed hepatocytes by HCV core expressing hepatocytes

The mechanism by which HIV and HCV cooperatively accelerate hepatocyte damage is not clearly understood; however, each virus affects the TRAIL: TRAIL-receptor system. We, therefore, questioned whether the independent effects of HCV and HIV combine to synergistically result in TRAIL dependent hepatocyte killing. We describe that Huh7 hepatocytes treated with HIV gp120 results in both increase TRAIL-R2 expression and an acquired sensitivity to TRAIL mediated killing. Moreover HCV infection and HCV core expression alone in Huh7 cells upregulates TRAIL. Co-incubation of HIV gp120 primed hepatocytes with HCV core expressing hepatocytes results in the selective death of the HIV gp120 primed hepatocytes that is selectively blocked by TRAIL-R2-Fc fusion protein. Liver biopsies from HIV mono-infected patients have increased TRAIL-R2; biopsies from HCV infected patients have increased TRAIL, while co-infected liver biopsies have increased PARP cleavage within hepatocytes indicating enhanced apoptosis. These findings suggest a pathogenic model to understand why HIV/HCV co-infection accelerates liver injury.     

Comparison of the effect of phenoxyherbicides on human erythrocyte membrane (in vitro)

The molecular mechanisms of phenoxyherbicides action in animals have been insufficiently studied. Now, we have investigated the interaction of sodium salts of phenoxyherbicides, e.g., 2,4-dichlorophenoxyacetic acid (2,4-D-Na), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T-Na) and 4-chloro-2-methylphenoxyacetic acid (MCPA-Na) with human erythrocytes. In this study, we evaluated the effect of these compounds on erythrocyte membrane fluidity as well as changes in membrane proteins content. It was observed that all of the compounds studied altered membrane fluidity, changed the size and shape of the erythrocytes and provoked echinocytes formation. It was also revealed that 2,4-D-Na and 2,4,5-T-Na changed the content of erythrocyte membrane proteins mainly by a decrease in the level of spectrin and low molecular weight proteins. The comparison of the action of phenoxyherbicides examined showed that 2,4,5-T-Na caused the greatest changes in the erythrocytes membrane, whereas MCPA-Na induced the lowest alterations in the incubated cells. It must be noted that changes of the investigated parameters were observed only at presence of significant concentrations of these compounds that may penetrate human organism only as a result of acute poisoning.     

Cloning, characterization and sulfonamide inhibition studies of an α-carbonic anhydrase from the living fossil sponge Astrosclera willeyana

The α-carbonic anhydrase (CA, EC 4.2.1.1) Astrosclerin-3 previously isolated from the living fossil sponge Astrosclera willeyana (Jackson et al., Science 2007, 316, 1893), was cloned, kinetically characterized and investigated for its inhibition properties with sulfonamides and sulfamates. Astrosclerin-3 has a high catalytic activity for the CO(2) hydration reaction to bicarbonate and protons (k(cat) of 9.0×10(5) s(-1) and k(cat)/K(m) of 1.1×10(8) M(-1) × s(-1)), and is inhibited by various aromatic/heterocyclic sulfonamides and sulfamates with inhibition constants in the range of 2.9 nM-8.85 μM. Astrosclerin, and the human isoform CA II, display similar kinetic properties and affinities for sulfonamide inhibitors, despite more than 550 million years of independent evolution. Because Astrosclerin-3 is involved in biocalcification, the inhibitors characterized here may be used to gain insights into such processes in other metazoans.     

Drastic decrease in dopamine receptor levels in the striatum of acetylcholinesterase knock-out mouse

BackgroundThe acetylcholinesterase knock-out mouse lives to adulthood despite 60-fold elevated acetylcholine concentrations in the brain that are lethal to wild-type animals. Part of its mechanism of survival is a 50% decrease in muscarinic and nicotinic receptors and a 50% decrease in adrenoceptor levels.HypothesisThe hypothesis was tested that the dopaminergic neuronal system had also adapted.MethodsRadioligand binding assays measured dopamine receptor level and binding affinity in the striatum. Immunohistochemistry of brain sections with specific antibodies visualized dopamine transporter. Effects on the intracellular compartment were measured as cAMP content, PI-phospholipase C activity.ResultsDopamine receptor levels were decreased 28-fold for the D₁-like, and more than 37-fold for the D₂-like receptors, though binding affinity was normal. Despite these huge changes in receptor levels, dopamine transporter levels were not affected. The intracellular compartment had normal levels of cAMP and PI-phospholipase C activity.ConclusionSurvival of the acetylcholinesterase knock-out mouse could be linked to adaptation of many neuronal systems during development including the cholinergic, adrenergic and dopaminergic. These adaptations balance the overstimulation of cholinergic receptors caused by high acetylcholine concentrations and thus maintain homeostasis inside the cell, allowing the animal to live.     

Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozyme IX with aromatic and heterocyclic sulfonamides

The inhibition of the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isozyme has been investigated with a series of aromatic and heterocyclic sulfonamides, including the six clinically used derivatives acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide and brinzolamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) were also provided for comparison. A very interesting and unusual inhibition profile against CA IX with these sulfonamides has been observed. Several nanomolar (K(I)-s in the range of 14-50 nM) CA IX inhibitors have been detected, both among the aromatic (such as orthanilamide, homosulfonilamide, 4-carboxy-benzenesulfonamide, 1-naphthalenesulfonamide and 1,3-benzenedisulfonamide derivatives) as well as the heterocylic (such as 1,3,4-thiadizole-2-sulfonamide, etc.) sulfonamides examined. Because CA IX is a highly active isozyme predominantly expressed in tumor tissues with poor prognosis of disease progression, this finding is very promising for the potential design of CA IX-specific inhibitors with applications as anti-tumor agents.     

Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards

From Mycobacterium avium species Mycobacterium avium subsp. paratuberculosis (n=961), Mycobacterium a. avium (n=677), Mycobacterium a. silvaticum (n=5), and Mycobacterium a. hominissuis (n=1566) were examined, and from Mycobacterium tuberculosis complex M. tuberculosis (n=2), Mycobacterium bovis (n=13), M. bovis BCG (n=4), and Mycobacterium caprae (n=10) were examined. From other mycobacterial species Mycobacterium intracellulare (n=60) and atypical mycobacteria (n=256) including Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium scrofulaceum, Mycobacterium gastri and other species of conditionally pathogenic mycobacteria were analysed. The internal standard molecules corresponding to insertion sequences IS900, IS901, IS1245, and flanking region (FR300) of IS901 were produced by PCR of alfalfa genome segment and inserted into plasmid vector. The resulting recombinant plasmid molecules were used as internal standards in coamplification with a total of 4729 mycobacterial collection strains and field isolates between 1996 and 2003. The size differences between amplicons obtained from IS900 (258 bp), IS901 (1108 bp), IS1245 (427 bp), and FR300 (300 bp) and from corresponding internal standard molecules ISIS900 (591 bp), ISIS901 (1 336 bp), ISIS1245 (583 bp), and IS901 flanking region of 300 bp ISFR300 (488 bp), respectively, allowed easy discrimination. The internal amplicons were visible by naked aye on agarose gel when 10(1), 10(3), 10(2), and 10(2) molecules for ISIS900, ISIS901, ISIS1245, and ISFR300 were used in the PCR, respectively, when no bacterial DNA was added to the reaction. The system was tested to define the amount of internal standards that could be used in the PCR without affecting the amplification of the specific segment. Non-specific amplifications were observed in M. fortuitum with IS1245 PCR and mixed infections with M. a. avium and M. a. hominissuis from pigs and cattle were found. PCR results of typing were compared with serotyping and Accu-Probes analyses in selected field isolates.     

Long-term biomanipulation of Rimov Reservoir (Czech Republic)

The history of fish, zooplankton and phytoplankton in RimovReservoir during its 14 years existence is documented. Afterthe filling of the reservoir in autumn 1978, the biomass ofplanktivorous fish increased from near zero to 650 kg ha-1 bythe fourth year of the reservoirs existence, after which thefish stock continually decreased under various forms ofbiomanipulative pressure, down to a value of about 100 kg ha-1. Thisdevelopment of fish stock was accompanied by changes in zooplanktonsize structure, but a cascading effect down to reduction ofphytoplankton biomass was not pronounced. The changes ofzooplankton body size structure were: increasing or decreasingaverage size of Daphnia galeata (the main cladoceranherbivore species) and changes in relative proportion ofcladoceran species, but none of the main zooplankton species was replacedor disappeared. The variation in phytoplankton biomass wasexplained by variation in phosphorus concentration and not related tozooplankton structure. It was concluded that the criticalbiomass of planktivorous fish below which the effect of loweringphytoplankton biomass could be observed lies below 100 kg ofplanktivorous fish per hectare. An attempt is made to quantifythe relationship between fish stock density and the size structureof zooplankton potentially able to influence top-down effects onphytoplankton.     

Polymorphic DNA haplotypes at the phenylalanine hydroxylase (PAH) locus in European families with phenylketonuria (PKU)

DNA haplotype data from the phenylalanine hydroxylase (PAH) locus are available from a number of European populations as a result of RFLP testing for genetic counseling in families with phenylketonuria (PKU). We have analyzed data from Hungary and Czechoslovakia together with published data from five additional countries--Denmark, Switzerland, Scotland, Germany, and France--representing a broad geographic and ethnographic range. The data include 686 complete chromosomal haplotypes for eight RFLP sites assayed in 202 unrelated Caucasian families with PKU. Forty-six distinct RFLP haplotypes have been observed to date, 10 unique to PKU-bearing chromosomes, 12 unique to non-PKU chromosomes, and the remainder found in association with both types. Despite the large number of haplotypes observed (still much less than the theoretical maximum of 384), five haplotypes alone account for more than 76% of normal European chromosomes and four haplotypes alone account for more than 80% of PKU-bearing chromosomes. We evaluated the distribution of haplotypes and alleles within these populations and calculated pairwise disequilibrium values between RFLP sites and between these sites and a hypothetical PKU "locus." These are statistically significant differences between European populations in the frequencies of non-PKU chromosomal haplotypes (P = .025) and PKU chromosomal haplotypes (P much less than .001). Haplotype frequencies of the PKU and non-PKU chromosomes also differ significantly (P much less than .001. Disequilibrium values are consistent with the PAH physical map and support the molecular evidence for multiple, independent PKU mutations in Caucasians. However, the data do not support a single geographic origin for these mutations.(ABSTRACT TRUNCATED AT 250 WORDS)