Interferon effect on ribosomal ribonucleic acid related to chromosome 21 ploidy.

Antiviral and cell-growth-inhibitory activities of human interferon were shown to be related to the activity of a gene or genes present on chromosome 21. The 18s rRNA is vital to cell growth; it is capable of a viral-mRNA-recognition function and it is coded for by genes a portion of which are present on chromosome-21. A previously reported ability of human interferon to affect rRNA metabolism is characterized by a decrease in the sucrose-gradient-peak ratio of radiolabelled 28S to 18S rRNA in extracts from the cytoplasm of interferon-treated human fibroblasts. In the present report, interferon dose-response curves are presented demonstrating a direct relationship between a decrease in this ratio and interferon concentrations in the media. By using this virus-independent cytoplasmic rRNA assay, eight human fibroblast lines, differing in chromosome 21 ploidy, were tested for sensitivity to human interferon. Two monosomy-21, two euploid-21 and four trisomy-21 cell lines were tested. The monosomy-21 cell populations were significantly less sensitive to interferon than the other six cell types tested. Of the cell lines tested, the most sensitive, by a wide margin, was a trisomy-21 line. Trisomy-21 cell monolayer sensitivity, however, varied widely within the range from normal to supersensitive. These observations suggest that interferon's ability to affect rRNA metabolism is related to the activity of a gene or genes present on chromosome 21.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Self-association and domains of interactions of an amphipathic helix peptide inhibitor of HIV-1 integrase assessed by analytical ultracentrifugation and NMR experiments in trifluoroethanol/H(2)O mixtures

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147-175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H(2)O mixtures. In pure water the helix content is weak but increases regularly up to 50-60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The N(H) temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four alpha-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147-175 segment in integrase.     

Efficacité et innocuité du prélèvement percutané de sperme testiculaire dans la prise en charge de l’infertilité masculine

Objective

To assess the efficacy and safety of percutaneous testicular biopsy to provide sperm cells for ICSI in male patients with azoospermia not amenable to surgical treatment.

Materials and methods

From October 1995 to December 2001, 175 biopsies were performed in men with azoospermia to provide material for intracytoplasmic sperm injection. Azoospermia was obstructive (OA) in 41 cases and non-obstructive (NOA) in 134 cases. Open biopsy was performed in the first 15 patients in the series and percutaneous biopsy was performed on an outpatient basis, under local anesthesia, with a Biopty Gun® (14G needle), in the subsequent patients as the first step in management. Open surgical biopsies were performed in another 15 patients following a sperm cell-negative percutaneous biopsy.

Results

All biopsies performed for OA were positive, but only 51/134 biopsies (38%) were positive in the NOA group. The material provided by percutaneous biopsy, when positive for sperm cells, was always sufficient to perform ICSI. When percutaneous biopsy was negative, open surgical biopsy failed to give better results. Five men developed minor complications (acute hematocele) following percutaneous biopsies requiring reoperation for hemostasis (3.12%). No major complications were observed. Results were comparable in terms of fertilization and pregnancy rates whether fresh or frozen-thawed sperm was used.

Conclusion

Percutaneous testicular sperm extraction is a safe, well-tolerated and cost-effective procedure in the management of male-factor infertility related to azoospermia.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Hydrogen bonding versus ion pairing in polyelectrolyte multilayers with homopolynucleotides

Homopolynucleotides--poly(adenylic acid), poly(A), and poly(uridylic acid), poly(U)--were assembled, layer-by-layer, into thin films with poly(ethylenimine), PEI. Various combinations and sequences of polynucleotide and PEI were used to highlight contributions of electrostatic versus hydrogen bonding as driving forces for multilayer build-up. Assembly of alternating poly(A) and poly(U) failed to yield growing films, due to excessively strong interactions between these complimentary strands. The surface morphology of multilayers depended on the deposition order and whether films had been annealed by salt. Films assembled from preformed A/U duplexes (having high persistence lengths) were very smooth. Individual adsorption steps, followed by optical waveguide light-mode spectroscopy, showed that only complementary polynucleotides adsorb by H-bonding to the surface of a growing multilayer. In contrast to behavior usually observed for polyelectrolyte multilayer build-up, the films decreased in thickness with increasing salt concentration.     

Circular dichroism and fluorescence of a tyrosine side-chain residue monitors the concentration-dependent equilibrium between U-shaped and coiled-coil conformations of a peptide derived from the catalytic core of HIV-1 integrase

The peptide denoted K159 (30 residues) derives from the catalytic core (CC) sequence of HIV-1 integrase (IN, residues 147-175). In the crystal structure of CC, the corresponding segment belongs to the alpha4 helix (residues 148-168, including residues Glu 152, Lys 156 and Lys 159, crucial for enzyme activity and DNA recognition), a loop (residues 169-171) and a part of the alpha5 helix (171-175), involved in enzyme dimerization. We used the fluorescence and the circular dichroism (CD) properties in the near-UV of the aromatic side chain of a tyrosine residue added at the C-terminal end of K159 in order to analyze the behavior of the concentrated and diluted peptide in aqueous trifluoroethanol (TFE), in an attempt to connect the information obtainable at high (NMR), medium (CD) and low (fluorescence) concentrations of the peptide. Altogether, the C-terminal tyrosine residue provided indirect information on the global conformation of K159 and on the local orientation and environment of the residue. The propensity of TFE to stabilize alpha-helical conformations in peptides was confirmed in CD and fluorescence experiments at relatively high (20-160 microM) and low (2-16 microM) concentrations, respectively. At relatively high concentration, stabilization of the peptide into alpha-helical conformation favored its auto-association likely in parallel coiled-coil dimers, as pointed out in our previous work [Eur. J. Biochem. 253 (1998) 236]. This was further confirmed by ANS (1-anilinonaphtalene-8-sulfonic acid) analysis and fluorescence temperature coefficient measurement. With diluted K159, a Stern-Volmer analysis with positively and negatively charged quenchers indicated that, when the intermolecular interactions were absent, the tyrosine was in a positively charged environment, as if the peptide folded into a U-shaped conformation similar to that present in the crystal structure of the enzyme.     

Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.