A synthetic peptide reproducing the mitochondrial targeting motif of AKAP121: a conformational study

The conformational features of a peptide derived by the 10-30 sequence of the mitochondrial domain of AKAP121 [Ac-1XKKPLALPGMLALLGWWWFFSRKKX25-NH2 (X=beta-Ala)] in water and in a water/trifluoroethanol (TFE) mixture at 298 K have been determined by NMR and CD spectroscopy. Backbone clustering analysis of NMR-derived structures led to the identification of a single representative structure in water/TFE. The structure of the peptide consists mainly of an alpha-helix, whose core is the region 7-23, with a less ordered N-terminal part. These data are confirmed by CD analysis. It is noteworthy that the high hydrophobic Trp16-Phe20 segment, that might also mediate interaction with tubulin, is organized in an alpha-helical wheel. Our conformational data can be the starting point for the development of highly selective peptides that interfere with the biological function of the Protein Kinase A scaffold protein AKAP121.     

Tonic and phasic guanidinium toxin-block of skeletal muscle Na channels expressed in Mammalian cells

The blockage of skeletal muscle sodium channels by tetrodotoxin (TTX) and saxitoxin (STX) have been studied in CHO cells permanently expressing rat Nav1.4 channels. Tonic and use-dependent blockage were analyzed in the framework of the ion-trapped model. The tonic affinity (26.6 nM) and the maximum affinity (7.7 nM) of TTX, as well as the "on" and "off" rate constants measured in this preparation, are in remarkably good agreement with those measured for Nav1.2 expressed in frog oocytes, indicating that the structure of the toxin receptor of Nav1.4 and Nav1.2 channels are very similar and that the expression method does not have any influence on the pore properties of the sodium channel. The higher affinity of STX for the sodium channels (tonic and maximum affinity of 1.8 nM and 0.74 nM respectively) is explained as an increase on the "on" rate constant (approximately 0.03 s(-1) nM(-1)), compared to that of TTX (approximately 0.003 s(-1) nM(-1)), while the "off" rate constant is the same for both toxins (approximately 0.02 s(-1)). Estimations of the free-energy differences of the toxin-channel interaction indicate that STX is bound in a more external position than TTX. Similarly, the comparison of the toxins free energy of binding to a ion-free, Na(+)- and Ca(2+)-occupied channel, is consistent with a binding site in the selectivity filter for Ca(2+) more external than for Na(+). This data may be useful in further attempts at sodium-channel pore modeling.     

Modulating action of the new polymorphism L436F detected in the<Emphasis Type="Italic"> GLB1</Emphasis> gene of a type-II GM1 gangliosidosis patient

Mutations in the GJB2 gene encoding connexin 26 (Cx26) are a major cause of autosomal recessive and sporadic cases of congenital deafness in most populations. The 235delC mutation of GJB2 is the most frequent known mutation in some east Asian populations, with a carrier frequency of approximately 1%. In order to study the origin of 235delC among east Asians, we analyzed single-nucleotide polymorphisms (SNPs) within the coding region of GJB2 and flanking the 235delC mutation. We observed significant linkage disequilibrium between 235delC and five linked polymorphic markers, suggesting that 235delC arose from a common founder. The detection of 235delC only in east Asians, but not in Caucasians, and the small chromosomal interval of the shared haplotype suggest that 235delC is an ancient mutation that arose after the divergence of Mongoloids and Caucasians. Similarly, the finding that this mutation appears on a single haplotype provides no support for the possibility that recurrent mutation is the explanation for the high frequency of the allele.D. Yan, H.-J. Park and X.M. Ouyang contributed equally to this work     

Characterization of Cr(VI)-Resistant Bacteria Isolated from Chromium-Contaminated Soil by Tannery Activity

Bacterial strains, previously isolated from a chromium-polluted soil, were identified on the basis of Gram reaction and biochemical characteristics (Biolog system). Moreover, chromate MICs, chromate reduction capability, multiple heavy metal tolerance, and antibiotic susceptibility were tested for each isolate. All strains but one were Gram-positive and resistant to high concentrations of chromate. The most Cr(VI)-resistant isolate (22mM) was identified as Corynebacterium hoagii. All Cr(VI)-resistant strains except the isolate ChrC20 were capable of catalyzing the reduction of Cr(VI) to Cr(III), a less toxic and less water-soluble form of chromium. The only isolate Cr(VI)-sensitive, attributed to the Pseudomonas genus, also exhibited Cr(VI)-reduction. Isolates were also screened for the presence of plasmid DNA. The strains ChrC20 and ChrB20 harbored one and two plasmids of high molecular mass, respectively. This approach permitted selection of some bacterial strains, which could be used for bioremediation of Cr(VI)-polluted environments. Received: 21 February 2002 / Accepted: 27 March 2002     

Proteomic identification of nitrated proteins in Alzheimer's disease brain

Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with O2- leads to formation of peroxynitrite ONOO-, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration.     

Different heparan sulfate proteoglycans serve as cellular receptors for human papillomaviruses

Papillomaviruses replicate in stratified epithelia of skin and mucosa. Infection with certain human papillomavirus (HPV) types is the main cause of anogenital neoplasia, in particular cervical cancer. Early events of papillomavirus infectivity are poorly understood. While heparan sulfate proteoglycans (HSPGs) mediate initial binding to the cell surface, the class of proteins carrying heparan sulfates has not been defined. Here we examined two processes of papillomavirus infection, attachment of virus-like particles (VLP) to cells and infection with authentic HPV type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major epithelial form and is strongly upregulated in wound edge keratinocytes. We employed K562 cells, which lack HSPGs except minor amounts of endogenous betaglycan, and stable clones that express cDNAs of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated with levels of heparan sulfate on the cell surface. Parental K562 bound HPV16 VLP weakly, whereas all three K562 transfectants demonstrated enhanced binding, with the highest binding capacity observed for syndecan-1-transfected cells, which also expressed the most HSPG. For HPV11 infectivity assays, a high virion inoculum was required to infect K562 cells, whereas ectopic expression of syndecan-1 increased permissiveness eightfold and expression of syndecan-4 or glypican-1 fourfold. Infection of keratinocytes was eliminated by treatment with heparitinase, but not phospholipase C, further implicating the syndecan family of integral membrane proteins as receptor proteins. Human keratinocytes with a homozygous deletion of alpha6 integrin are permissive for HPV11 infection. These results indicate that several HSPGs can serve as HPV receptors and support a putative role for syndecan-1, rather than alpha6 integrin, as a primary receptor protein in natural HPV infection of keratinocytes.