The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the major molecular target of selenodeficiency in rodents, accounting for most of the histopathological and structural abnormalities of testicular tissue and male germ cells. PHGPx exists as a cytosolic form, mitochondrial form, and nuclear form (nPHGPx) predominantly expressed in late spermatids and spermatozoa. Here, we demonstrate that mice with a targeted deletion of the nPHGPx gene were, unlike mice with the full knockout (KO) of PHGPx, not only viable but also, surprisingly, fully fertile. While both morphological analysis of testis and epididymis and sperm parameter measurements did not show any apparent abnormality, toluidine blue and acridine orange stainings of spermatozoa indicated defective chromatin condensation in the KO sperm isolated from the caput epididymis. Furthermore, upon drying and hydrating, KO sperm exhibited a significant proportion of morphologically abnormal heads. Monobromobimane labeling and protein-free thiol titration revealed significantly less extensive oxidation in the cauda epididymis when compared to that in the wild type. We conclude that nPHGPx, by acting as a protein thiol peroxidase in vivo, contributes to the structural stability of sperm chromatin.     

Anopheles (Anopheles) neomaculipalpus: a new malaria vector in the Amazon basin?

Abstract. Anopheles (Anopheles) neomaculipalpus Curry (Diptera: Culicidae) collected by human landing catches and light traps in southern Venezuela were assayed by enzyme-linked immunosorbent assay (ELISA) for detection of Plasmodium circumsporozoite (CS) protein. A total of 356 An. neomaculipalpus were collected, of which three (0.84%) were positive for P. vivax, two for the variant 247 and one for the variant 210. The overall sporozoite rate in An. neomaculipalpus was similar to that for the principal vector An. (Nyssorhynchus) darlingi Root (0.82%) and higher than in An. (Nys.) marajoara Galvão & Damasceno (0.27%). This is the first report of An. neomaculipalpus naturally infected with Plasmodium parasites in Venezuela.     

Neurotoxic dopamine quinone facilitates the assembly of tau into fibrillar polymers

Aberrant aggregation of microtubule associated protein tau is the main characteristic of different disorders known as tauopathies. Different compounds have been described to facilitate tau aberrant aggregation. In this work, we demonstrate that oxidized products of dopamine (neurotoxic dopamine quinone), a neurotransmitter involved in Parkinson's disease, promote tau polymerization. Curiously, neurons expressing dopamine (substantia nigra) show a low content of tau protein and seldom have tau aggregation in tauopathies. In non-dopaminergic neurons, quinone oxidation products may be involved in tau polymerization. These results support a link between oxidative damage and the onset of tauopathies. (Mol Cell Biochem 278: 203–212, 2005)     

Chimeric epitopes delivered by polymeric synthetic linear peptides induce protective immunity to malaria

Polymeric linear peptide chimeras (LPCs) that incorporate Plasmodium vivax promiscuous T cell epitopes and the P. falciparum circumsporozoite protein B cell epitope have been shown to induce a high level of immunogenicity and overcome genetic restriction when tested as vaccine immunogens in BALB/c mice. The present study evaluates the biological relevance of several LPCs using a well characterized rodent malaria model. Polymeric peptide constructs based on P. berghei and P. yoelii sequences, and orthologous to the human malaria sequences included in the original LPCs, were designed and tested for immunogenicity in mice of different H-2 haplotypes. We demonstrate that robust immune responses are induced and that peptides containing the orthologous rodent Plasmodium sequences exhibited similar immunogenic capabilities. Unique to this report, we show that LPCs can also prime MHC class I-restricted cytotoxic T lymphocytes (CTLs) and, most relevantly, that a peptide construct prototype incorporating single B, T and CTL epitopes induced protection against an experimental challenge with P. berghei or P. yoelii sporozoites. Collectively, these results suggest that polymeric polypeptide chimeras can be used as a platform to deliver subunit vaccines.     

Isolation and identification of bacteria from spent nuclear fuel pools

The aim of the present research was to isolate and identify bacteria from spent nuclear fuel pools of a Spanish nuclear power plant. Water samples were collected and inoculated onto different culture media to isolate the highest number of species. 16S rDNA fragments from colonies growing on solid media were amplified and analysed by denaturing gradient gel electrophoresis. Sequencing revealed the presence of 21 different bacteria belonging to several phylogenetic groups (, , and -Proteobacteria, Actinomycetales, Flavobacterium, and the Bacillus/Staphylococcus group). The isolation of these microorganisms in this particular environment (oligotrophic and radioactive) is highly interesting because of the possibility of their being used for the bioremediation of radionuclide-contaminated waters.     

Native crystal structure of a nitric oxide-releasing lectin from the seeds of Canavalia maritima

Here, we report the crystallographic study of a lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal binding sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide.     

The polarized expression of Na+,K+-ATPase in epithelia depends on the association between beta-subunits located in neighboring cells

The polarized distribution of Na+,K+-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca2+, K+, Cl-, and CO3H- across the whole epithelium. We report here that the beta-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na+,K+-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na+,K+-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with beta1-subunit from the dog kidney (CHO-beta). 4) This may be attributed to the adhesive property of the beta1-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-beta cells shows that the expression of dog beta1-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of beta1-subunit forces CHO-beta cells to coexpress endogenous alpha-subunit. Together, our results indicate that MDCK cells express Na+,K+-ATPase at a given border provided the contacting cell expresses the dog beta1-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na+,K+-ATPase in epithelial cells.     

Monitoring proton dissociation and solution conformation of chiral ytterbium complexes with near-IR CD

The ytterbium complex [Yb((S)-THP)](3+) ((S)-THP = (1S,4S,7S,10S-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane) is investigated in solution through NMR, near-IR absorption, and CD spectroscopy. Quantitative analysis of the paramagnetic pseudocontact NMR shift shows Lambda helicity of the ligand cage around the metal. The NIR CD spectrum recorded at acidic pH is found to be very similar to that of [Yb((R)-DOTMA)](-) ((R)-DOTMA = (1R,4R,7R,10R)-alpha,alpha',alpha',alpha'-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), which in solution assumes a twisted square antiprism (TSA) conformation. The similarity of the NIR CD spectra is discussed, and it is the first proof of the Lambda(lambda,lambda,lambda,lambda) conformation of [Yb((S)-THP)](3+). NIR CD spectra recorded in the pH range of 2-9 allow one to easily follow proton dissociation and to calculate the pK of this equilibrium in water (pK(A) = 6.4 +/- 0.1). This value agrees well with that determined for [Lu((S)-THP)](3+) using potentiometric methods. This demonstrates once again that NIR CD spectroscopy is a powerful technique for investigating the solution structure and dynamics of these complexes.     

A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events

Meiosis is a specialized form of cell division by which sexually reproducing diploid organisms generate haploid gametes. During a long prophase, telomeres cluster into the bouquet configuration to aid chromosome pairing, and DNA replication is followed by high levels of recombination between homologous chromosomes (homologs). This recombination is important for the reductional segregation of homologs at the first meiotic division; without further replication, a second meiotic division yields haploid nuclei. In the fission yeast Schizosaccharomyces pombe, we have deleted 175 meiotically upregulated genes and found seven genes not previously reported to be critical for meiotic events. Three mutants (rec24, rec25, and rec27) had strongly reduced meiosis-specific DNA double-strand breakage and recombination. One mutant (tht2) was deficient in karyogamy, and two (bqt1 and bqt2) were deficient in telomere clustering, explaining their defects in recombination and segregation. The moa1 mutant was delayed in premeiotic S phase progression and nuclear divisions. Further analysis of these mutants will help elucidate the complex machinery governing the special behavior of meiotic chromosomes.