The ATC (ataxia-telangiectasia complementation group C) locus localizes to 11q22-q23.

The multisystem autosomal recessive disease ataxia-telangiectasia (A-T) is determined by several genes, as evidenced by the existence of four complementation groups in this disorder. Using linkage analysis, the ATA (A-T complementation group A) gene was previously localized to chromosome 11, region q22-q23. Analysis of the segregation of RFLP markers from this region in a Jewish-Moroccan family assigned to group C indicates that the ATC (A-T complementation group C) gene localizes to chromosome 11q22-q23 as well.     

Linkage studies do not confirm the cytogenetic location of incontinentia pigmenti on Xp11

Summary Linkage studies have been performed in 5 incontinentia pigmenti (IP) families totaling 29 potentially informative meioses. Ten probes of the Xp arm were used, six of them were precisely localized on the X chromosome, using hamster x human somatic cell hybrids containing a broken X chromosome derived from an incontinentia pigmenti patient carrying an X;9 translocation [46,XX,t(X;9)(p11.21;q34)]. The following order for probes is proposed: pter-(DXS7, DXS146, DXS255)-IP1-(DXS14, DXS90)-DXS106-qter. The negative lod scores obtained exclude the possibility that in the families studied, the gene for IP is located in Xp11 or in the major part of the Xp arm.     

Putrescine distribution in Escherichia coli studied in vivo by 13C nuclear magnetic resonance

In order to study the intracellular polyamine distribution in Escherichia coli, ¹³C-NMR spectra of [1,4-¹³C]putrescine were obtained after addition of the latter to intact bacteria. The ¹³C-enriched methylene signal underwent line broadening. When the cells were centrifuged after 90 min the cell-bound putrescine peak had a linewidth of 23 Hz, while the supernatant liquid showed an unbound putrescine signal with a linewidth smaller than 1 Hz. By using ¹³C-enriched internal standards it could be shown that the linewidening was not due to the heterogeneity of the medium or to an in vivo paramagnetic effect. Cell-bound putrescine was liberated by addition of trichloroacetic acid and was therefore non-covalently linked to macromolecular cell structures. Cell-bound [¹³C]putrescine could be displaced by addition of an excess of [¹²C]putrescine. When samples of membranes, soluble protein, DNA, tRNA and ribosomes from E. coli were incubated with [1,4-¹³C]putrescine, strong binding was detected only in the ribosomal and membrane fractions. The ribosome-putrescine complex showed properties similar to those determined with the intact cells. By measuring the nuclear Overhauser enhancements η, it was possible to estimate that only about 50% of the polyamine was linked to the macromolecules. Determination of the T₁ values of free and ribosomal-bound putrescine allowed the calculation of a correlation time, τ_c = 4·10^?7 s for the latter. T₁ and τ_c value for the ribosome-putrescine complex were those expected for a motional regime of slowly tumbling molecules.     

Apoptose et ségrégation méiotique dans les spermatozoïdes d'hommes porteurs de translocations

Men with a chromosomal translocation produce a significant percentage of unbalanced spermatozoa. In order to determine a correlation between chromosomal anomalies and apoptosis in human sperm, we analysed DNA fragmentation and meiotic segregation in sperm from men with a (13;14) Robertsonian translocation. We studied sperm from 12 (13;14) translocation carriers and 9 proven fertile men with a normal karyotype. Meiotic segregation of chromosomes 13 and 14 was analysed using dual-colour fluorescencein situ hybridization with locus-specific probes for chromosomes 13 and 14. Apoptosis in spermatozoa was measured byin situ TUNEL assay. The meiotic segregation study showed a significantly increased frequency of unbalanced spermatozoa for chromosomes 13 and 14 in (13;14) carriers (15.9%) compared to the control population (1.3%) (p=0.00016). The study of apoptosis showed an increase of DNA fragmentation in (13;14) carriers (34.9%) compared to the control population (13.8%) (p=0.0036). This increased apoptosis was observed in spermatozoa presenting an increase of unbalanced chromosomal anomalies concerning chromosomes 13 and 14, but with a predominance of balanced spermatozoa compared to the theoretical risk of meiotic segregation. These results suggest that apoptosis could be involved as a regulatory mechanism to eliminate unbalanced chromosomal spermatozoa in men with a (13;14) Robertsonian translocation.     

Disappearance of porphobilinogen deaminase activity in leaves before the onset of senescence

The activity of porphobilinogen deaminase was measured in young and senescent or mature leaves of pepper (Capsicum annuum), and poinsettia (Euphorbia pulcherrima). Whereas high activity was found in the crude extracts of the young leaves, almost no activity was found in the extracts of senescent or mature leaves. The decrease in deaminase activity was not due to the presence of an isolatable inhibitor. By purifying the crude enzyme extracts from leaves of different ages on DEAE-cellulose columns it was shown that the decrease in deaminase activity was due to a real decrease in the amount of enzyme. Fruiting also decreased porphobilinogen deaminase activity. Several kinetic constants of the C. annuum deaminase were determined.     

Hsp90 Inhibitors Exhibit Resistance-Free Antiviral Activity against Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in young children, leading to significant morbidity and mortality worldwide. Despite its medical importance, no vaccine or effective therapeutic interventions are currently available. Therefore, there is a pressing need to identify novel antiviral drugs to combat RSV infections. Hsp90, a cellular protein-folding factor, has been shown to play an important role in the replication of numerous viruses. We here demonstrate that RSV requires Hsp90 for replication. Mechanistic studies reveal that inhibition of Hsp90 during RSV infection leads to the degradation of a viral protein similar in size to the RSV L protein, the viral RNA-dependent RNA polymerase, implicating it as an Hsp90 client protein. Accordingly, Hsp90 inhibitors exhibit antiviral activity against laboratory and clinical isolates of RSV in both immortalized as well as primary differentiated airway epithelial cells. Interestingly, we find a high barrier to the emergence of drug resistance to Hsp90 inhibitors, as extensive growth of RSV under conditions of Hsp90 inhibition did not yield mutants with reduced sensitivity to these drugs. Our results suggest that Hsp90 inhibitors may present attractive antiviral therapeutics for treatment of RSV infections and highlight the potential of chaperone inhibitors as antivirals exhibiting high barriers to development of drug resistance.     

Heme oxygenase induction by CoCl2, Co-protoporphyrin IX, phenylhydrazine, and diamide: evidence for oxidative stress involvement.

The induction of heme oxygenase in rat liver by cobaltous chloride (CoCl2) and Co-protoporphyrin IX is entirely prevented by the administration of alpha-tocopherol and allopurinol. CoCl2 was converted in the liver into Co-protoporphyrin IX before it induced heme oxygenase activity. Actinomycin and cycloheximide affected to a similar degree the induction of heme oxygenase by both CoCl2 and Co-protoporphyrin IX. Administration of either CoCl2 or Co-protoporphyrin strongly decreased the intrahepatic GSH pool, a decrease which was completely prevented by the administration of either alpha-tocopherol or allopurinol. The latter compounds prevented heme oxygenase induction as well as the decrease in hepatic GSH when administered 2 h before, together with, or 2 h after CoCl2. However, when given 5 h after administration of CoCl2, alpha-tocopherol and allopurinol showed no preventive effect. Similar results were obtained when Co-protoporphyrin IX was used, with the difference that when alpha-tocopherol and allopurinol were given 2 h after administration of the inducer, they showed no protective effect. Phenylhydrazine and diamide also induced heme oxygenase activity in rat liver. This inductive effect was preceded by a decrease in the intrahepatic GSH pool, which took place several hours before induction of the oxygenase. Administration of alpha-tocopherol and allopurinol prevented induction of the oxygenase but had no effect on the decrease in GSH levels. These results suggest that the induction of heme oxygenase by phenylhydrazine and the diamide is preceded by an oxidative stress which very likely originates in the depletion of GSH. The induction of heme oxygenase by hemin was not prevented by administration of alpha-tocopherol or allopurinol. Coprotoporphyrin IX did not affect the pattern of the molecular forms of hepatic biliverdin reductase, at variance with CoCl2, which is known to convert molecular form 1 of the enzyme into molecular form 3.     

Human Eosinophils Express the High Affinity IgE Receptor,FcεRI,in Bullous Pemphigoid

Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE≥400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils.     

The interplay between basicity, conformation, and enzymatic reduction in biliverdins.

Biliverdins with extended conformations are reduced by biliverdin reductase (BvR) at higher rates than biliverdins with helical conformations. To find out the molecular basis for this important feature of BvR mechanism, helical and extended biliverdins were titrated for their acid-base equilibria in a protic solvent (methanol). It was found that the basicity of biliverdins increases with the stretching of the conformation. Biliverdin IX gamma (all-syn) has a pKa = 3.6; 5,10,15-syn,syn,anti-biliverdin has a pKa = 3.7; 5,10,15-syn,anti,syn-biliverdin has a pKa = 6.1; 5,10,15-syn,anti,anti-biliverdin has a pKa = 6.4; and 5,10,15-all-anti-biliverdin has a pKa = 7.9. The increase in basicity with progressive stretching of conformations closely parallels the increase in the reduction rates by BvR. A biliverdin constrained by a four carbon chain to a helical conformation and which is a very weak base (pKa = 0.4) is not reduced by BvR. Nucleophilic additions of 2-mercaptoethanol at the C10 in biliverdins closely parallel their basicities, as can be expected if the formation of a positive mesomeric species at C10 is linked to the basicity (i.e., the ease of protonation) of the N23 on the pyrrolenine ring.     

Review: cellular substrates of the eukaryotic chaperonin TRiC/CCT

The TCP-1 ring complex (TRiC; also called CCT, for chaperonin containing TCP-1) is a large (approximately 900 kDa) multisubunit complex that mediates protein folding in the eukaryotic cytosol. The physiological substrate spectrum of TRiC is still poorly defined. Genetic and biochemical data show that it is required for the folding of the cytoskeletal proteins actin and tubulin. Recent years have witnessed a steady stream of reports that describe other proteins that require TRiC for proper folding. Furthermore, analysis of the transit of newly synthesized proteins through TRiC in intact cells suggests that the chaperonin contributes to the folding of a distinct subset of cellular proteins. Here we review the current understanding of a role for TRiC in the folding of newly synthesized polypeptides, with a focus on some of the individual proteins that require TRiC.