The mitochondrial and nuclear genetic structure of Myotis capaccinii (Chiroptera: Vespertilionidae) in the Eurasian transition, and its taxonomic implications

Allopatric isolation in glacial refugia has caused differentiation and speciation in many taxa globally. In this study, we investigated the nuclear and mitochondrial genetic differentiation of the long fingered bat, Myotis capaccinii during the ice ages in south-eastern Europe and Anatolia. The mitochondrial DNA (mtDNA) analyses indicated a suture zone similar to those recorded in other animal species, including bats, suggesting the association of more than one refugium with the region. Contrary to most of the other species where a suture zone was seen in Anatolia, for M. capaccinii the geographical location of the genetic break was in south-eastern Europe. This mitochondrial differentiation was not reflected in the nuclear microsatellites, however, suggesting that the lack of contact during the ice ages did not result in reproductive isolation. Hence taxonomically, the two mitochondrial clades cannot be treated as separate species.     

Evidence for the deficiency of β-glucosidase-activating factor in fibroblasts of patients with I-cell disease

Summary Reduced activity of -glucosidase was shown in the cultured skin fibroblasts of four patients with I-cell disease when the enzyme was tested without the use of detergents. In the presence of taurocholate and triton X100 -glucosidase activity was normal. This suggested a deficiency of a -glucosidase-activating factor in I-cell fibroblasts rather than of the enzyme itself. The deficiency of -glucosidase activity was corrected to some extent by mixing cell lysates, and more effectively by cocultivation and fusion of I-cell disease and Gaucher fibroblasts. These results present evidence for the presence of a -glucosidase-activating factor in normal and Gaucher fibroblasts. In fibroblasts of patients with I-cell disease this activator is probably deficient, as is the case for most lysosomal enzymes.     

Agonist Leukadherin-1 Increases CD11b/CD18-Dependent Adhesion Via Membrane Tethers

Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn²⁺ additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1^−/− neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn²⁺-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn²⁺ induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.     

Role of Cysteine Residues in Human Plasma Phospholipid Transfer Protein

Phospholipid transfer protein (PLTP) belongs to a family of human plasma lipid transfer proteins that bind to small amphophilic molecules. PLTP contains cysteines at residues 5, 129, 168, and 318. Bactericidal/permeability-increasing protein, which is a member of the same gene family, contains an essential disulfide bond between Cys₁₃₅ and Cys₁₇₅; these residues, which correspond to Cys₁₂₉ and Cys₁₆₈ in PLTP, are conserved among all known members of the gene family. To identify the importance of these and the remaining cysteine residues to PLTP secretion and activity, each was replaced by a glycine by site-directed mutagenesis. The mutant as well as wild-type PLTP cDNAs were cloned into the mammalian expression vector pSV·SPORT1, and the PLTP cDNAs were transfected to COS-6 cells for expression. PLTP Cys₁₂₉ Gly and PLTP Cys₁₆₈ Gly were secretion incompetent. Neither PLTP mass nor activity was detectable in cell lysates and culture medium. Relative to wild-type PLTP, PLTP Cys₅ Gly and PLTP Cys₃₁₈ Gly exhibited similar specific activities but partially impaired PLTP synthesis and secretion. Intracellular PLTP appeared as two bands of 75 and 51 kDa corresponding to reported molecular masses for the glycosylated and nonglycosylated forms. The specific activities of PLTP Cys₅ Gly and PLTP Cys₃₁₈ Gly were similar in the cell lysates and medium, suggesting that glycosylation does not affect transfer activity.     

Application of isotopic ratio mass spectrometry for the in vitro determination of demethylation activity in human liver microsomes using N-methyl-13C-labeled substrates

The reaction of demethylation mediated by cytochrome P450 (CYP) leads to the equimolar production of demethylated metabolite and formaldehyde. From a 13C-substrate labeled on a carbon of the methyl moiety, [13C]formaldehyde (H13CHO) is liberated. A highly sensitive and specific assay involving the oxidation of H13CHO to 13CO(2) by a double-enzymatic-step reaction is reported. The 13CO(2) was quantified by the method of reverse isotopic dilution based on gas chromatography-isotope ratio mass spectrometry analysis. The method first involves the limiting step of the CYP-dependent reaction, which is stopped with a mixture of zinc sulfate 5 mM and trichloroacetic acid 100 mM. Then, the transformation of H13CHO to 13CO(2) is performed with the formaldehyde (0.2 unit) and the formate (0.2 unit) dehydrogenase NAD-dependent enzymes. The recovery of 13CO(2) from the incubation mixture was equal to 91.4 +/- 3.0%. The accuracy and the precision of the present method were within 12 and 10%, respectively. The limit of quantification was set to 25 pmol. The performance of the assay was validated on human liver microsomes with five probes: [13C]erythromycin, [1-13C]caffeine, [3-13C]caffeine, [7-13C]caffeine, and [13C(2)]aminopyrine. This method is useful for the rapid determination of N-demethylase activity of human liver microsomes from methyl-13C-substrates.     

Protectome Analysis: A New Selective Bioinformatics Tool for Bacterial Vaccine Candidate Discovery

New generation vaccines are in demand to include only the key antigens sufficient to confer protective immunity among the plethora of pathogen molecules. In the last decade, large-scale genomics-based technologies have emerged. Among them, the Reverse Vaccinology approach was successfully applied to the development of an innovative vaccine against Neisseria meningitidis serogroup B, now available on the market with the commercial name BEXSERO® (Novartis Vaccines). The limiting step of such approaches is the number of antigens to be tested in in vivo models. Several laboratories have been trying to refine the original approach in order to get to the identification of the relevant antigens straight from the genome. Here we report a new bioinformatics tool that moves a first step in this direction. The tool has been developed by identifying structural/functional features recurring in known bacterial protective antigens, the so called “Protectome space,” and using such “protective signatures” for protective antigen discovery. In particular, we applied this new approach to Staphylococcus aureus and Group B Streptococcus and we show that not only already known protective antigens were re-discovered, but also two new protective antigens were identified.Although vaccines based on attenuated pathogens as pioneered by Luis Pasteur have been shown to be extremely effective, safety and technical reasons recommend that new generation vaccines include few selected pathogen components which, in combination with immunostimulatory molecules, can induce long lasting protective responses. Such approach implies that the key antigens sufficient to confer protective immunity are singled out among the plethora of pathogen molecules. As it turns out, the search for such protective antigens can be extremely complicated.Genomic technologies have opened the way to new strategies in vaccine antigen discovery (1, 2, 3). Among them, Reverse Vaccinology (RV)¹ has proved to be highly effective, as demonstrated by the fact that a new Serogroup B Neisseria meningitidis (MenB) vaccine, incorporating antigens selected by RV, is now available to defeat meningococcal meningitis (4, 5). In essence, RV is based on the simple assumption that cloning all annotated proteins/genes and screening them against a robust and reliable surrogate-of-protection assay must lead to the identification of all protective antigens. Because most of the assays available for protective antigen selection involve animal immunization and challenge, the number of antigens to be tested represents a severe bottleneck of the entire process. For this reason, despite the fact that RV is a brute force, inclusive approach (“test-all-to-lose-nothing” type of approach) in their pioneered work of MenB vaccine discovery, Pizza and co-workers did not test the entire collection of MenB proteins but rather restricted their analysis to the ones predicted to be surface-localized. This was based on the evidence that for an anti-MenB vaccine to be protective bactericidal antibodies must be induced, a property that only surface-exposed antigens have. For the selection of surface antigens Pizza and co-workers mainly used PSORT and other available tools like MOTIFS and FINDPATTERNS to find proteins carrying localization-associated features such as transmembrane domains, leader peptides, and lipobox and outer membrane anchoring motifs. At the end, 570 proteins were selected and entered the still very labor intensive screening phase. Over the last few years, our laboratories have been trying to move to more selective strategies. Our ultimate goal, we like to refer to as the “Holy Grail of Vaccinology,” is to identify protective antigens by “simply” scanning the genome sequence of any given pathogen, thus avoiding time consuming “wet science” and “move straight from genome to the clinic” (6).With this objective in mind, we have developed a series of proteomics-based protocols that, in combination with bioinformatics tools, have substantially reduced the number of antigens to be tested in the surrogate-of-protection assays (7, 8). In particular, we have recently described a three-technology strategy that allows to narrow the number of antigens to be tested in the animal models down to less than ten (9). However, this strategy still requires high throughput experimental activities. Therefore, the availability of in silico tools that selectively and accurately single out relevant categories of antigens among the complexity of pathogen components would greatly facilitate the vaccine discovery process.In the present work, we describe a new bioinformatics approach that brings an additional contribution to our “from genome to clinic” goal. The approach has been developed on the basis of the assumption that protective antigens are protective in that they have specific structural/functional features (“protective signatures”) that distinguish them from immunologically irrelevant pathogen components. These features have been identified by using existing databases and prediction tools, such as PFam and SMART. Our approach focuses on protein biological role rather than its localization: it is completely protein localization unbiased, and lead to the identification of both surface-exposed and secreted antigens (which are the majority in extracellular bacteria) as well as cytoplasmic protective antigens (for instance, antigens that elicit interferon γ producing CD4+ T cells, thus potentiating the killing activity of phagocytic cells toward intracellular pathogens). Should these assumptions be valid, PS could be identified if: (1) all known protective antigens are compiled to create what we refer to as “the Protectome space,” and (2) Protectome is subjected to computer-assisted scrutiny using selected tools. Once signatures are identified, novel protective antigens of a pathogen of interest should be identifiable by scanning its genome sequence in search for proteins that carry one or more protective signatures. A similar attempt has been reported (10), where the discrimination of protective antigens versus nonprotective antigens was tried using statistical methods based on amino acid compositional analysis and auto cross-covariance. This model was implemented in a server for the prediction of vaccine candidates, that is, Vaxijen (www.darrenflower.info/Vaxijen); however, the selection criteria applied are still too general leading to a list of candidates that include ca. 30% of the total genome ORFs very similarly to the number of antigens predicted by classical RV based on the presence of localization signals.Here we show that Protectome analysis unravels specific signatures embedded in protective antigens, most of them related to the biological role/function of the proteins. These signatures narrow down the candidate list to ca. 3% of the total ORFs content and can be exploited for protective antigen discovery. Indeed, the strategy was validated by demonstrating that well characterized vaccine components could be identified by scanning the genome sequence of the corresponding pathogens for the presence of the PS. Furthermore, when the approach was applied to Staphylococcus aureus and Streptococcus agalactiae (Group B Streptococcus, GBS) not only already known protective antigens were rediscovered, but also two new protective antigens were identified.     

Effects of Environmental Lead Pollution, Smoking, and Smokeless Tobacco (Maras Powder) Use on Blood Lead Level

One hundred sixty-four adult male volunteers (29 controls [Group 1] and 135 combi drivers) enrolled in the study. The combi drivers were divided into three groups as nonusers of either Maras powder or cigarette (Group 2), smokers (Group 3), and users of Maras powder (Group 4). Blood lead levels (BLLs) were analyzed by atomic absorption spectrophotometer. BLL was detected as 2.8 ± 2.3 μg/dL in Group 1 (n = 29); however, it was 3.5 ± 1.6 μg/dL in Group 2 (n = 33), 3.8 ± 2.4 μg/dL in Group 3 (n = 62), and 3.9 ± 2.4 μg/dL in Group 4 (n = 40). BLL in Group 1 was found significantly lower than other groups (p < 0.05). The use of cigarette or Maras powder by the drivers did not give rise to a marked difference on the BLLs (p > 0.05). BLL of (combi) drivers was detected to be significantly higher than nondrivers; however, it was still under the hazardous level of 10 μg/dL announced by WHO. Although there are publications reporting that usage of tobacco increases the level of lead in blood, both smoking and use of Maras powder did not affect BLL markedly in our study. Poster presented (the abstract section published in Congress Book) at the 7th Congress of Turkish Family Physicians, 23–26 May 2006, Cesme-IZMIR, Turkey.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation from young and mature explants of thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> and <Emphasis Type="Italic">T. longicaulis</Emphasis>) resulting in genetically stable shoots

An efficient in vitro propagation protocol, applicable both to young and mature explants of two Thymus spp., results in genetically stable plantlets. In vitro-grown shoot tips of Thymus vulgaris L. were exposed to cytokinins (6-benzyladenine, kinetin, and thidiazuron) alone or in combination with auxins, gibberellic acid (GA₃) and/or silver nitrate in order to optimize in vitro shoot proliferation. Optimum shoot proliferation (97% regeneration rate, with 8.6 shoots produced per explant) was obtained when semi-solid Murashige and Skoog (MS) medium was supplemented with 1 mg L⁻¹ kinetin and 0.3 mg L⁻¹ GA₃. Rooting of the shoots was easily obtained on semi-solid MS medium that was either hormone-free or supplemented with auxins. However, the best root apparatus (92.5% rooting rate, with 19 adventitious roots per shoot) developed on MS medium supplemented with 0.05 mg L⁻¹ 2,4-dichlorophenoxyacetic acid. Genetic stability was confirmed in the in vitro-germinated mother plant as well as the shoots that underwent two, four, six, eight, or ten cycles of in vitro subculturing by random amplified polymorphic DNA (RAPD) analysis. When applied to the micropropagation of mature shoot tips of T. longicaulis C. Presl subsp. longicaulis var. subisophyllus (Borbás) Jalas, the optimized in vitro propagation protocol resulted in a 97.5% shoot regeneration rate, with five shoots formed per explant, and 100% rooting. Rooted plantlets of both species were transferred to 250-mL plastic pots and successfully acclimatized by gradually reducing the relative humidity.