Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world’s leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1–893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/c mice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocular mucosa was well tolerated without signs of inflammation. N-PmpC-specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFNγ immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage.     

The dicationic derivatives of DBTAA: Interactions with DNA/RNA and antiproliferative effects on human cell lines

Three dibenzotetraaza[14]annulenes non-covalently interacted with double-stranded DNA and RNA by mixed minor groove and/or intercalative binding mode. Observed interactions were strongly dependent on the steric exposure of positive charges and the length of the linkers of studied compounds as well as on the secondary structure and basepair composition of DNA/RNA. Compound 2 showed pronounced selectivity toward dA-dT-rich sequences and binding mode switch from dominant minor groove binding to ds-DNA to dominant intercalation into ds-RNA. Antiproliferative effect of studied compounds on human tumor and normal cell lines was in good agreement with the strength of observed interactions with DNA/RNA.     

Phosphorylation-dependent Changes in Nucleotide Binding,Conformation, and Dynamics of the First Nucleotide Binding Domain (NBD1) of the Sulfonylurea Receptor 2B (SUR2B)

The sulfonylurea receptor 2B (SUR2B) forms the regulatory subunit of ATP-sensitive potassium (K_ATP) channels in vascular smooth muscle. Phosphorylation of the SUR2B nucleotide binding domains (NBD1 and NBD2) by protein kinase A results in increased channel open probability. Here, we investigate the effects of phosphorylation on the structure and nucleotide binding properties of NBD1. Phosphorylation sites in SUR2B NBD1 are located in an N-terminal tail that is disordered. Nuclear magnetic resonance (NMR) data indicate that phosphorylation of the N-terminal tail affects multiple residues in NBD1, including residues in the NBD2-binding site, and results in altered conformation and dynamics of NBD1. NMR spectra of NBD1 lacking the N-terminal tail, NBD1-ΔN, suggest that phosphorylation disrupts interactions of the N-terminal tail with the core of NBD1, a model supported by dynamic light scattering. Increased nucleotide binding of phosphorylated NBD1 and NBD1-ΔN, compared with non-phosphorylated NBD1, suggests that by disrupting the interaction of the NBD core with the N-terminal tail, phosphorylation also exposes the MgATP-binding site on NBD1. These data provide insights into the molecular basis by which phosphorylation of SUR2B NBD1 activates K_ATP channels.     

Phylogeography of Y-chromosome haplogroup I reveals distinct domains of prehistoric gene flow in europe

To investigate which aspects of contemporary human Y-chromosome variation in Europe are characteristic of primary colonization, late-glacial expansions from refuge areas, Neolithic dispersals, or more recent events of gene flow, we have analyzed, in detail, haplogroup I (Hg I), the only major clade of the Y phylogeny that is widespread over Europe but virtually absent elsewhere. The analysis of 1,104 Hg I Y chromosomes, which were identified in the survey of 7,574 males from 60 population samples, revealed several subclades with distinct geographic distributions. Subclade I1a accounts for most of Hg I in Scandinavia, with a rapidly decreasing frequency toward both the East European Plain and the Atlantic fringe, but microsatellite diversity reveals that France could be the source region of the early spread of both I1a and the less common I1c. Also, I1b*, which extends from the eastern Adriatic to eastern Europe and declines noticeably toward the southern Balkans and abruptly toward the periphery of northern Italy, probably diffused after the Last Glacial Maximum from a homeland in eastern Europe or the Balkans. In contrast, I1b2 most likely arose in southern France/Iberia. Similarly to the other subclades, it underwent a postglacial expansion and marked the human colonization of Sardinia ~9,000 years ago.     

Status of the DPC4 tumor suppressor gene in sporadic colon adenocarcinoma of Croatian patients: identification of a novel somatic mutation

Loss of heterozygosity (LOH) of loci on chromosome 18q occurs in a majority of colorectal cancers. The DPC4 (Smad4) tumor suppressor gene, located at 18q21.1, may be a predisposing gene for Juvenile Polyposis Syndrome. To investigate alterations of the DPC4 gene in sporadic colon adenocarcinoma, a panel of 60 tumor specimens from Croatian patients was surveyed for evidence of LOH and also for mutations within the entire DPC4 coding region (exons 1-11). Using three pairs of specific primers for the three DPC4 microsatellite repetitive sequences, we investigated the frequency of LOH. The presence of single nucleotide change at restriction sites of specific codons in exons 2, 8, 10, and 11 (which belong to the conserved region of the gene) was examined by RFLP analysis. The investigation was extended to search for any other mutation within the entire coding region of the DPC4 gene by single strand conformation polymorphism (SSCP) analysis. Our results show a high frequency of heterozygosity in 58 of 60 (97%) colon adenocarcinoma samples. LOH at any one of the three flanking markers was observed in 26 (45%) of the 58 informative cases. The loss of one allele of the DPC4 gene was negatively correlated with tumor size; more frequent in smaller tumors (<5 cm) than in larger ones. A mutation was found in exon 11 in only one tumor sample (T18), and the mutation was verified by sequencing. Sequencing demonstrated a novel mutation-a deletion in exon 11 (134-153 del TAGACGAAGTACTTCATACC) of the DPC4 gene in the MH2 domain. These data suggest that inactivation of the DPC4 gene contributes to the genesis of colorectal carcinoma through allelic loss whereas mutation in the coding region of the DPC4 gene is infrequently detected in Croatian patients with A, B or C stages of colorectal cancers.     

Morphological, motor and situation-motor characteristics of elite female handball players according to playing performance and position

The aim of the study was to define biomotor characteristics that determine playing performance and position in female handball. A battery of 13 variables consisting of somatotype components (3 variables), basic motor abilities (5 variables) and specific motor abilities (5 variables) were applied in a sample of 52 elite female handball players. Differences in biomotor characteristics according to playing performance and position of female handball players were determined by use of the analysis of variance (ANOVA) and discriminative analysis. Study results showed the high-quality female handball players to predominantly differ from the less successful ones in the specific factor of throw strength and basic dash factor, followed by the specific abilities of movement without and with ball, basic coordination/agility and specific ability of ball manipulation, and a more pronounced mesomorphic component. Results also revealed the wing players to be superior in the speed of movement frequency (psychomotor speed), run (explosive strength) and speed of movement with ball as compared with players at other playing positions. Also, endomorphic component was less pronounced in players at the wing and back player positions as compared with goalkeeper and pivot positions, where endomorphic component was considerably more pronounced.     

Insecticidal potential of natural zeolite and diatomaceous earth formulations against rice weevil (Coleoptera: Curculionidae) and red flour beetle (Coleoptera: Tenebrionidae)

Insecticidal potential of natural zeolites and diatomaceous earths originating from Serbia against Sitophilus oryzae (L.) and Tribolium castaneum (Herbst) was evaluated. Two natural zeolite formulations (NZ and NZ Modified) were applied to wheat at rates of 0.50, 0.75, and 1.0 g/kg, while two diatomaceous earth (DE) formulations (DE S-1 and DE S-2) were applied at rates of 0.25, 0.50, 0.75, and 1.0 g/kg. A bioassay was conducted under laboratory conditions: temperature of 24 +/- 1 degrees C, relative humidity in the range 50-55%, in tests with natural zeolites, and 60-65%, in tests with DEs, and in all combinations for progeny production. Mortality was assessed after 7, 14, and 21 d of insect contact with treated wheat, and the total mortality after an additional 7-d recovery on untreated broken wheat. Progeny production was also assessed after 8 wk for S. oryzae and 12 wk for T. castaneum. The highest mortality for S. oryzae and T. castaneum was found after the longest exposure period and 7 d of recovery, on wheat treated with NZ at the highest rate and DEs at rates of 0.50 -1.0 g/kg. Progeny reduction higher than 90% was achieved after 14 and 21 d of contact of both beetle pests with wheat treated with DE S-1 at 0.50-1.0 g/kg and DE S-2 at 0.75-1.0 g/kg, while the same level of reduction was achieved only for T. castaneum after its contact with the highest rate of NZ formulation. NZ Modified, applied even at the highest rate, revealed much lower insecticidal potential.     

The thermal stability of the external invertase isoforms from Saccharomyces cerevisiae correlates with the surface charge density

Understanding the effect of surface charge on the stability of proteins is one prerequisite for "tailoring" proteins with increased thermal stability. Here, we investigated the origin of the altered thermal stability observed between the four recently isolated isoforms (EINV1-EINV4) of external invertase. External invertase from yeast Saccharomyces cerevisiae, a homodimeric glycoprotein, represents a widely used model for studying the influence of the glyco component on protein stability. The stability of the four isoforms of invertase decreases from EINV1 to EINV4, which is accompanied by an increase in negative surface charge density. Mass spectrometry analysis revealed that the isoforms share identical protein parts indicating that the differences in stability are the result of post-translational modifications. (31)P NMR analysis revealed that the isoforms contain negatively charged phosphate groups in diester and monoester forms attached to the glycan part. The total amount of phosphate bound to the polymannan component varies between the different isoforms. These results, together with the analysis of the amount of polymannan components, show that negative surface charge density does not entirely depend on the amount of phosphate but rather on its distribution. This suggests that charged groups bound to the glyco-component of a protein can influence the stability of glycoproteins.     

In vitro conditions affect photosynthetic performance and crassulacean acid metabolism in Mammillaria gracilis Pfeiff. tissues

Mammillaria gracilis Pfeiff. plants cultivated in the pot (pot plants, PP), as well as in vitro-grown normal shoots (NS), habituated callus (HC), hyperhydric shoots (HS), and tumour tissue (TT) were investigated in order to reveal the influence of in vitro culture on functionality of the photosynthetic apparatus and CAM photosynthesis in cactus M. gracilis Pfeiff. Photosynthetic pigments content as well as maximum (F _v/F _m) and effective (??_PSII) quantum yield of photosystem II (PSII) decreased in all in vitro-grown tissues in comparison to PP. The decrease observed in hyperhydric HC, HS and TT correlated with a low expression of Rubisco large subunit (RbcL) and ?? subunit of ATP synthase (?? ATP synt) and almost undetectable levels of protein D1, light-harvesting chlorophyll a/b-binding protein (LHCII) and cytochrome f protein of thylakoid Cyt b ₆ /f-complex (Cyt f) found in these tissues. As for crassulacean acid metabolism (CAM) pattern, PP and NS expressed diurnal acid fluctuation, while HC, HS and TT failed to show it. Nevertheless, all M. gracilis tissues exhibited diurnal changes of phosphoenolpyruvate carboxylase (PEPC) activity indicating the typical CAM physiology. In conclusion, the photosynthesis was down-regulated in all in vitro-grown tissues. NS maintained typical CAM photosynthesis, while HC, HS and TT withheld PEPC activity, but not acid accumulation specific for CAM. Minor changes observed in NS in comparison to PP could be attributed to the sugar supplementation while the more prominent deviations found in HC, HS and TT could be correlated with hyperhydricity and the loss of characteristic tissue organisation pattern.