Genetic structure of three marine fishes from the Gulf of Pagasitikos (Greece) based on allozymes, RAPD, and mtDNA RFLP markers

In the present work we used three molecular techniques (allozymes, RAPDs and mtDNA RFLPs) in order to study the genetic structure of three commercial marine species (Mullus surmuletus, Mullus barbatus, and Pagellus erythrinus). Each species was sampled from three locations within the Gulf of Pagasitikos, Greece and from two neighbouring locations outside the Gulf (Trikeri and Alonissos). Values of genetic heterozygosity and nucleotide diversity for all populations studied were similar or above the mean values observed in marine fishes. None of the three types of molecular markers used revealed diagnostic patterns, which could allow the allocation of individuals to one of the populations. The analyses revealed that the three populations within Pagasitikos were homogenous representing thus a panmictic stock. However, there were evidences of genetic population subdivision between localities from inside and outside of the Pagasitikos Gulf. The results provide essential information for the design of a sustainable management plan of the Gulf of Pagasitikos and its demersal fish resources.     

Abolished circadian rhythm of salivary cortisol in elite artistic gymnasts

Objective

The aim of this study was to evaluate the effects of intensive physical exercise and acute psychological stress during high level athletic competition as reflected on the levels of salivary cortisol in elite artistic gymnasts (AGs).

Design

The study included 239 AGs (142 females-97 males) who participated in the European Championship of Gymnastics in 2006 and 81 adolescents (40 females-41 males), matched for age, as controls. All athletes participated voluntarily in all or parts of the study, providing samples or data for each of the variables measured. Height, weight, body fat, lean body mass (LBM), bone age and Tanner stage of puberty were assessed and data concerning the time of thelarche, adrenarche and menarche as well as, the onset and the intensity (hours per week) of training were obtained.

Methods

Saliva samples were collected, the morning before training and in the afternoon shortly after the competition. From controls, the saliva samples were collected in the morning. Cortisol concentrations were measured using a chemiluminescence method. Acute stress was assessed using a questionnaire designed for the study.

Results

No difference was found between morning and afternoon salivary cortisol levels in both male and female AGs (females: AM: 15.45 ± 7.45 nmol/l vs PM: 15.73 ± 9.38 nmol/l; males: AM: 10.21 ± 5.52 nmol/l vs PM: 9.93 ± 13.8 nmol/l, p > 0.05).Female AGs presented higher levels of morning salivary cortisol than female controls (p < 0.05). Both male and female AGs had higher degree of psychological stress in comparison with controls (p < 0.001, p < 0.013, respectively). Female AGs had higher morning and afternoon salivary cortisol levels (p < 0.01, p < 0.01, respectively) and higher degree of stress (p < 0.003) than males.

Conclusions

In elite AGs the diurnal rhythm of salivary cortisol has been abolished, probably due to the strenuous training and competition conditions. Female AGs presented higher levels of morning salivary cortisol and psychological stress compared to both male AGs and female controls. The long term consequences of these modifications of the HPA axis remain to be elucidated.     

Airway allergen exposure stimulates bone marrow eosinophilia partly via IL-9

Background

Interleukin (IL)-9 is a Th2-derived cytokine with pleiotropic biological effects, which recently has been proposed as a candidate gene for asthma and allergy. We aimed to evaluate the therapeutic effect of a neutralizing anti-IL-9 antibody in a mouse model of airway eosinophilic inflammation and compared any such effect with anti-IL-5 treatment.

Methods

OVA-sensitized Balb/c mice were intraperitoneally pretreated with a single dose (100 μg) of an anti-mouse IL-9 monoclonal antibody (clone D9302C12) or its vehicle. A third group was given 50 μg of a monoclonal anti-mouse IL-5 antibody (TRFK-5) or its vehicle. Animals were subsequently exposed to OVA on five days via airways. Newly produced eosinophils were labelled using 5-bromo-2''-deoxyuridine (BrdU). BrdU⁺ eosinophils and CD34⁺ cell numbers were examined by immunocytochemistry. After culture and stimulation with OVA or PMA+IC, intracellular staining of IL-9 in bone marrow cells from OVA-exposed animals was measured by Flow Cytometry. The Mann-Whitney U-test was used to determine significant differences between groups.

Results

Anti-IL-9 significantly reduced bone marrow eosinophilia, primarily by decrease of newly produced (BrdU⁺) and mature eosinophils. Anti-IL-9 treatment also reduced blood neutrophil counts, but did not affect BAL neutrophils. Anti-IL-5 was able to reduce eosinophil numbers in all tissue compartments, as well as BrdU⁺ eosinophils and CD34⁺ progenitor cells, and in all instances to a greater extent than anti-IL-9. Also, FACS analysis showed that IL-9 is over-expressed in bone marrow CD4⁺ cells after allergen exposure.

Conclusions

Our data shows that a single dose of a neutralizing IL-9 antibody is not sufficient to reduce allergen-induced influx of newly produced cells from bone marrow to airways. However, in response to allergen, bone marrow cells over-express IL-9. This data suggest that IL-9 may participate in the regulation of granulocytopoiesis in allergic inflammation.     

Directional operations in the motor cortex modeled by a neural network of spiking neurons

A neural network with realistically modeled, spiking neurons is proposed to model ensemble operations of directionally tuned neurons in the motor cortex. The model reproduces well directional operations previously identified experimentally, including the prediction of the direction of an upcoming movement in reaching tasks and the rotation of the neuronal population vector in a directional transformation task.     

The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.Poxviruses are large DNA viruses that infect a wide range of hosts. The smallpox virus devastated human populations until its eradication 3 decades ago. Other poxviruses are emerging, such as monkeypox virus, which also infects humans and causes disease (61). The smallpox vaccine is a model of vaccine efficacy, but how the vaccine induces protection is not well understood. Knowledge of how the vaccine produces protection will also likely be important for efforts to produce vaccines that are effective against other pathogens. Though highly successful in the population overall, the smallpox vaccine uses an infectious strain of vaccinia virus and has a significant level of serious side effects (6). One focus of current research is to develop vaccines using recombinant poxvirus proteins that are as protective as the live virus vaccine but produce fewer complications. A more detailed structural characterization of the protein antigens that are important for conferring protection will improve our knowledge of how the smallpox vaccine works and lead to a better understanding of poxvirus biology.There are two morphologically distinct forms of poxviruses: the mature virion (MV) and the enveloped virion (EV) (53). The MV, also known as the intracellular mature virion (IMV), is found inside the infected cell (62, 65). Enveloped virions are formed from MVs that have been wrapped by modified Golgi or endosomal membranes (53). MVs are thought to be responsible for host-to-host proliferation of the virus, while the EVs mediate virus spread within a host (40, 49). EVs that are attached to the cell surface, also termed cell-associated enveloped virions (CEV), are thought to propagate viral infection to neighboring cells (65). EVs that are released from the cell surface, also termed extracellular enveloped virus (EEV), mediate longer-range dissemination in the host (65). The outer membranes of the MV and EV forms each have a distinct assemblage of proteins. Candidate subunit vaccines have been shown to require proteins from both virus forms to be most effective (17, 27, 28).A33 is a type II integral membrane glycoprotein found on the surface of the EV form of the virus and is also expressed on the host cell membrane (62). A33 is a disulfide-bonded homodimer with both N- and O-linked glycosylation (57, 62). Deletion of the A33R gene in vaccinia virus results in a small-plaque phenotype, defects in actin tail formation, and inefficient cell-to-cell spread in cell culture (63). Evidence implicates A33 in the spreading of virus from cell to cell by a mechanism that is antibody resistant (40). Antibodies against A33 in cell culture prevent the formation of comet-shaped viral plaques, which are assayed in an overlay of plaques with liquid and are indicative of cell-to-cell spreading of the EV (2, 17). A33 has been shown to interact through its cytoplasmic and transmembrane regions with the EV proteins A36 (18, 64, 72, 76) and B5 (58, 64). Vaccination with A33 is protective in a number of animal models of poxvirus infection as a component of protein subunit vaccination (16, 17, 19, 77), DNA vaccination (19, 27-29), or a combination of the two methods (24). Despite the inclusion of A33 in vaccination studies, the functions of A33 in the virus are unclear.Members of the C-type lectin-like domain (CTLD) superfamily of proteins are found in organisms ranging from bacteria to humans (13, 78). The first crystal structures of carbohydrate-binding domains with this fold gave rise to the name “lectin” for the family, but many family members do not bind carbohydrates. The classification of a domain as being C type lectin-like is currently based on similarities in protein sequence and fold (74). CTLDs have been shown to bind noncarbohydrate small molecules, lipids, proteins, and other structures, such as ice (13, 78). One group of type II transmembrane proteins that contain CTLDs is comprised of the natural killer (NK)-cell receptors of the innate immune system (78). The NK-cell receptors are composed of dimers of CTLDs, which are the only dimers out of the several hundred C-type lectin-like structures that are known. Protein binding by NK-cell receptors occurs on a surface formed by the “long loop” and nearby residues that are on the opposite side of the dimer from the N and C termini (60). A second group of proteins contains a monomeric CTLD, termed a “Link module” CTLD, that binds glycosaminoglycans but lacks the “long loop” region that is present in almost all other CTLDs (78).To gain a better understanding of the structure and possible functions of A33 and to further its development in vaccines, we determined the X-ray crystal structure of the A33 ectodomain from vaccinia virus. Based on the structure and sequence of A33, the carbohydrate-binding site of the canonical CTLD is not present. The structure revealed A33 to have dimers of CTLDs. Comparison of A33 with other CTLDs, including dimers from NK-cell receptors and monomers from Link modules, indicates that A33 contains an unusual CTLD that likely binds ligands of host or virus origins.     

Cariporide Counteracts Atherosclerosis‐related Functions in Monocytes from Obese and Normal Individuals

Objective: This study aimed to show the effect of high glucose concentrations in combination with a pharmaceutical analog of the Na⁺/H+ antiport inhibitor, cariporide, on scavenger receptor CD36 expression, cell adhesion, and cell migration of human monocytes derived from obese and normal individuals. Research Methods and Procedures: Monocytes were isolated from six healthy obese individuals and six healthy age‐ and sex‐matched controls by use of whole blood Percoll sedimentation and plastic surface monocyte binding. The density of CD36 scavenger receptors on the surface of monocytes was assessed by the use of a fluorescent fluorescein isothiocyanate (FITC)‐linked monoclonal antibody. Transmigration of monocytes through laminin‐1–coated filters was performed on 5‐μm pore Transwell culture inserts. Monocyte attachment to laminin was estimated by a solid phase assay. Results: High glucose concentrations caused an increase in monocytes from normal and obese individuals in the expression of CD36 receptors and positively influenced monocyte migration and adhesion to laminin. Cariporide together with glucose counteracted these effects. The effects of migration and adhesion of monocytes to laminin were specific to glucose, because the effect was significantly higher when monocytes were incubated in the presence of 20 mM of glucose than in the presence of 20 mM of fructose. Monocytes from obese subjects showed greater response than in normal to all of the studied effects, with the highest response in laminin attachment. Discussion: The data of this study suggest that cariporide counteracts atherosclerosis‐related functions through Na⁺/H+ antiport inhibition in monocytes from both normal and obese individuals.     

A comparative approach towards thylakoid membrane proteome analysis of unicellular green alga Scenedesmus obliquus

The chlorophyll (Chl)-containing membrane protein complexes from the green alga Scenedesmus obliquus have been isolated from the thylakoid membranes by solubilization with dodecyl-β-maltoside and fractionation using a sucrose density gradient. The Chl-containing protein fractions were characterized by absorption spectroscopy, tricine SDS PAGE, BN-PAGE, and dynamic light scattering (DLS). BN-PAGE showed the presence of seven protein complexes with molecular weights in the range of 68, 118, 157, 320, 494, 828 and 955 kDa, respectively. Furthermore, light scattering reveals the simultaneous presence of particles of different sizes in the 3-4 nm and 6.0-7.5 nm range, respectively. The smaller size is related to the hydrodynamic radius of the trimer Light Harvesting Complex (LHCII), whereas the larger size is associated with the presence of photosystem I and photosystem II reaction centers. Additionally, functional information regarding protein-protein interactions was deconvoluted using coupling 2-D BN-PAGE, MALDI-TOF MS and a detailed mapping of S. obliquus photosynthetic proteome of the solubilized thylakoid membranes is therefore presented.