Isolation of oligosaccharides from a partial-acid hydrolysate of pneumococcal type 3 polysaccharide for use in conjugate vaccines

A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part.     

Importance of the N-terminal region of the phage GA-1 single-stranded DNA-binding protein for its self-interaction ability and functionality

The single-stranded DNA-binding protein (SSB) of phage GA-1 displays higher efficiency than the SSBs of the related phages phi 29 and Nf. In this work, the self-interaction ability of GA-1 SSB has been analyzed by visualization of the purified protein by electron microscopy, glycerol gradient sedimentation, and in vivo cross-linking of bacterial cultures infected with phage GA-1. GA-1 SSB contains an insert at its N-terminal region that is not present in the SSBs of phi 29 and Nf. Three deletion mutant proteins have been characterized, Delta N19, Delta N26, and Delta N33, which lack the 19, 26 or 33 amino acids, respectively, that follow the initial methionine of GA-1 SSB. Mutant protein Delta N19 retains the structural and functional behavior of GA-1 SSB, whereas mutant proteins Delta N26 and Delta N33 no longer stimulate viral DNA replication or display helix-destabilizing activity. Analysis of the mutant proteins by ultracentrifugation in glycerol gradients and electron microscopy indicates that deletion of 26 or 33 but not of 19 amino acids of the N-terminal region of GA-1 SSB results in the loss of the oligomerization ability of this protein. Our data support the importance of the N-terminal region of GA-1 SSB for the differential self-interaction ability and functional behavior of this protein.     

Characterization of Ejl,the cell-wall amidase coded by the pneumococcal bacteriophage Ej-1

The Ejl amidase is coded by Ej-1, a temperate phage isolated from the atypical pneumococcus strain 101/87. Like all the pneumococcal cell-wall lysins, Ejl has a bimodular organization; the catalytic region is located in the N-terminal module, and the C-terminal module attaches the enzyme to the choline residues of the pneumococcal cell wall. The structural features of the Ejl amidase, its interaction with choline, and the structural changes accompanying the ligand binding have been characterized by CD and IR spectroscopies, differential scanning calorimetry, analytical ultracentrifugation, and FPLC. According to prediction and spectroscopic (CD and IR) results, Ejl would be composed of short beta-strands (ca. 36%) connected by long loops (ca. 17%), presenting only two well-predicted alpha-helices (ca. 12%) in the catalytic module. Its polypeptide chain folds into two cooperative domains, corresponding to the N- and C-terminal modules, and exhibits a monomer <--> dimer self-association equilibrium. Choline binding induces small rearrangements in Ejl secondary structure but enhances the amidase self-association by preferential binding to Ejl dimers and tetramers. Comparison of LytA, the major pneumococcal amidase, with Ejl shows that the sequence differences (15% divergence) strongly influence the amidase stability, the organization of the catalytic module in cooperative domains, and the self-association state induced by choline. Moreover, the ligand affinity for the choline-binding locus involved in regulation of the amidase dimerization is reduced by a factor of 10 in Ejl. Present results evidence that sequence differences resulting from the natural variability found in the cell wall amidases coded by pneumococcus and its bacteriophages may significantly alter the protein structure and its attachment to the cell wall.     

Reproductive seasonality in female Iberian red deer (Cervus elaphus hispanicus)

This study characterized the seasonal pattern of luteal cyclicity in Iberian red deer (n=16), by measuring plasma progesterone concentrations in hinds (female red deer) twice per week from calving (May and June) 1996 until May 1997. In eight of these hinds we also examined plasma prolactin profiles to assess seasonal responses to photoperiod. Plasma progesterone concentration in the 16 hinds studied indicated that the reproductive pattern is seasonal, and lasts for 5.73 +/- 0.27 months. After calving, progesterone levels remained basal (no luteal activity) for several months, except in a hind that lost her calf just after calving, and thus did not have to suckle it. This hind showed two consecutive estrus cycles in the month following calving, which suggests that suckling has an inhibiting effect on the resumption of ovarian activity. These results also showed that as long as the hinds do not become pregnant, they show between 5 and 10 estrus cycles per reproductive season (8.06 +/- 0.35), ranging between 105 and 249 days from onset of the first cycle to end of the last one. Uninterrupted cycling lasted for 3.5-6.4 months (mean, 4.6 +/- 0.24). Cyclic luteal activity was found from October to February in all hinds, with a smaller, but notable proportion in September (56.25%) and March (68.8%), whereas it was negligible in the remaining period. Our results show a reproductive season similar to or longer than that recorded by other authors. Prolactin plasma concentrations showed a yearly trend following that of photoperiod, with peak concentrations from April to July, a decrease in August, minimal concentrations from September to February and a sharp increase in March.     

In vitro and in vivo assays of 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives against Trypanosoma cruzi

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, thereafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives S-2d was selected to be assayed in vivo, because of its remarkable trypanocidal properties. To determine toxicity against J774 macrophages, a method based on quantification of cell damage, after 24 h, was used. Cell respiration, an indicator of cell viability, was assessed by the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. Anti-amastigote activity was estimated after 48 h by microscopic counts of May Grünwald-Giemsa-stained monolayers. Nifurtimox and benznidazole were used as reference drugs. For the in vivo experiences, mice were infected with 10(4) blood trypomastigotes and then treated during 15 days with S-2d or nifurtimox by oral route. All of the compounds were highly toxic at 100 micro g/ml for macrophages and a few of them maintained this cytotoxicity even at 10 microg/ml. Of the derivatives assayed against amastigotes 3k and S-2d showed an interesting activity, that was held even at 1microg/ml. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after administration of S-2d to infected mice.     

Mouse novel Ly9: a new member of the expanding CD150 (SLAM) family of leukocyte cell-surface receptors

Human CS1, also known as novel Ly9, 19A24, or CRACC, is a member of the immunoglobulin gene superfamily (IgSF) expressed on natural killer cells and other leukocytes. Here we describe the cloning of the mouse homologue of this gene. The mouse novel Ly9 gene is shown to encode a transmembrane protein composed of two extracellular immunoglobulin-like domains, a transmembrane region and an 88-amino acid cytoplasmic domain. Mouse novel Ly9 is structurally similar to the extracellular domains of CD84 and CD229 (Ly9). Both mouse and human novel Ly9 genes mapped close to the CD229gene in a region where other members of the CD150 family have also been mapped, and analysis of their genomic sequences showed that they have an identical intron/exon organization. Northern blot analysis revealed that the expression of mouse and human novel Ly9 was predominantly restricted to hematopoietic tissues, with the exception of testis. Here we show that SAP (SH2D1A), an adapter protein responsible for the X-linked lymphoproliferative disease, binds to the phosphorylated cytoplasmic tail of human but not mouse novel Ly9. Taken together, these data indicate that mouse novel Ly9 is a new member of the expanding CD150 family of cell surface receptors.     

3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine, a new prodrug of zidovudine. Synthesis, solid state characterization, and anti HIV-1 activity

Synthesis, solid state characterization and anti HIV-1 activity of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (2), a new prodrug of zidovudine (AZT, 1), are described. Two solid forms of 2 prepared by crystallization from ethyl acetate-petroleum ether (form alpha) and from a melt sample of form alpha (amorphous form) were characterized by X-ray diffractometry, infrared spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA) techniques. The novel nucleoside exhibited antiviral activity against standard and resistant strain panels of HIV-1 as well as cytotoxicity similar to that of AZT.     

Structural transitions in polycytidylic acid: proton buffer capacity data

The pH-dependences of proton buffer capacity of poly(C) were computed on the basis of the literature data. In these curves there were observed four peaks: two narrow and two wide ones. The first narrow peak reflects the process of cooperative formation of double helices, which is induced by protonation of the N3 atom of nucleotide bases. The first wide peak is assigned to noncooperative process of poly(C) double helices protonation at the N3 nitrogen atom. It is proposed that the second wide peak corresponds to noncooperative protonation of the neutral cytosine bases at the oxygen atom. This reaction causes cooperative dissociation of the poly(C) double helices. The second narrow peak reflects the dissociation process.     

Cryptococcus neoformans arthritis in a renal transplant recipient

We present a Cryptococcus neoformans knee joint infection in a diabetic renal transplant patient treated with steroids, cyclosporin, and mycofenolate mofetil. We discuss reported cases of cryptococcal arthritis.     

Population structure in the endangered Blanca Cacereña bovine breed demonstrated by RAPD analyses

RAPD analyses have been used to determine the genetic diversity and the population structure of the endangered Blanca Cacere?a bovine breed. Genetic variability was evaluated on the basis of 1048 loci produced by 71 primers. RAPD produced a number of polymorphic loci (30.44%), and it has been proved to be a useful method for evaluating polymorphisms in this breed. The dendrograms based on simililarity indexes and on Nei's genetic distances between 60 animals and the value of genetic differentiation among subpopulations (F(ST)) showed a clear population substructure defined by herds and a scarce genetic flow among herds. Analysis of molecular variance (AMOVA) showed that 32.4% of the total variance was due to differences among herds and confirmed the clustering found. The results of the present study allow us to plan more adequate mating in order to maintain the genetic diversity and to improve the efficiency of conservation for the Blanca Cacere?a bovine breed.