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The determination of the maritime boundary is related to the selection of the tidal datum used in relation to the zero-depth reference. This research was conducted to determine the impact of the zero-depth reference that has been used, the Mean Low Water Spring (MLWS), against the zero-depth recommended by the International Hydrographic Organization (IHO), the Lowest Astronomical Tide (LAT). Tidal data is obtained from BIG's tidal station processing, while the MLWS value data is obtained from a combination of analysis from the Hydrographic Office of Indonesia (Pushidros). Based on these data, it is found that 25 out of 37 tidal stations indicate that the LAT value is below the MLWS in its vertical position. This shows that sea boundary claims can potentially be shifted by changing the zero-depth reference from the original by switching from MLWS to LAT. Lastly, this study presents the benefits of implementing the vertical system reference recommended by the IHO, the LAT, as an international chart datum. [ABSTRACT FROM AUTHOR]

• HSV-1 latently infected mice exhibit enhanced neuroinflammation in brainstem. • Principal sensory nucleus of spinal trigeminal tract (Pr5) of brainstem was examined. • Locus coeruleus (LC) has indirect synaptic connections to Pr5 and was examined. • Neuroinflammation is enhanced in females latently infected with LAT null mutant. • Aged mice generally contained reduced neuroinflammation compared to young mice. Human alphaherpesvirus 1 (HSV-1) establishes life-long latency in sensory neurons in trigeminal ganglia (TG), brainstem neurons, and other CNS neurons. Two important segments of the brainstem were examined in this study: principal sensory nucleus of the spinal trigeminal tract (Pr5) because it receives direct afferent inputs from TG, and locus coeruleus (LC) because it is indirectly connected to Pr5 and LC sends axonal projections to cortical structures, which may facilitate viral spread from brainstem to the brain. The only viral gene abundantly expressed during latency is the latency associated transcript (LAT). Previous studies revealed 8-week old female C57Bl/6 mice infected with a LAT null mutant (dLAT2903) versus wild-type (wt) HSV-1 exhibit higher levels of senescence markers and inflammation in LC of females. New studies revealed 1-year old mice latently infected with wt HSV-1 or dLAT2903 contained differences in neuroinflammation and senescence in Pr5 and LC versus young mice. In summary, these studies confirm HSV-1 promotes neuro-inflammation in the brainstem, which may accelerate neurodegenerative disease. [Display omitted] [ABSTRACT FROM AUTHOR]

Living-apart-together (LAT) partnerships are gaining prominence in many high-income societies, prompting ongoing discussions about their significance and their role in the family formation process. This study provides a contemporary update on LAT relationships in Spain, with a specific emphasis on variations across different life stages. The study focuses on several key aspects: (1) recent trends in the prevalence of LAT relationships, (2) socio-demographic factors associated with being in a LAT relationship, (3) joint influence of both partners' characteristics, and (4) short-term intentions to co-reside. Using data from the 2018 Spanish Fertility Survey, we employ logistic regression models to analyze the factors influencing individuals' likelihood of being in a LAT relationship as opposed to a co-residential partnership. Our findings reveal a noticeable rise in LAT partnerships in Spain over the past two decades, except among the youngest age group. Personal motivations and socially attributed meanings of LAT relationships, however, differ depending on an individual's life stage. Among young adults, LAT partnerships largely serve as a transitional phase in the family formation process, preceding co-residence with a partner. In this early adulthood stage, unemployment and temporary work contracts – affecting any of the partners – often hinder household formation, but intentions to co-reside in the near future remain strong. In contrast, LAT partnerships in the mid-life stage often stem from a desire to maintain personal independence and are frequently linked to prior partnership and reproductive biographies. ● The prevalence of LAT relationships has risen in Spain over the last two decades. ● Among young adults, LAT serves as a transitional phase preceding co-residence. ● Young couples in which any of the partners lacks stable work are more likely to LAT. ● Middle-aged adults with children from prior partnerships are more likely to LAT. ● The desire for autonomy is a relatively common reason to LAT in the mid-life stage. [ABSTRACT FROM AUTHOR]

Clustering is a prominent feature of receptors at the plasma membrane (PM). It plays an important role in signaling. Liquid–liquid phase separation (LLPS) of proteins is emerging as a novel mechanism underlying the observed clustering. Receptors/transmembrane signaling proteins can be core components essential for LLPS (such as LAT or nephrin) or clients enriched at the phase-separated condensates (for example, at the postsynaptic density or at tight junctions). Condensate formation has been shown to regulate signaling in multiple ways, including by increasing protein binding avidity and by modulating the local biochemical environment. In moving forward, it is important to study protein LLPS at the PM of living cells, its interplay with other factors underlying receptor clustering, and its signaling and functional consequences. [Display omitted] • Protein phase separation is a mechanism for plasma membrane receptor clustering. • Membrane proteins can be core phase separating components or enriched clients. • Increased binding avidity at condensates promotes signaling molecule activation. • Phase separation regulates actin nucleation, enzyme activity and RNA processing. • Signaling complexes and pathways may consist of multiple phase separating modules. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is protozoan parasite with ability of causing disease in wide-spectrum of animals; many species of animals in captivity died of clinical toxoplasmosis. The monitoring of T. gondii antibodies in zoo animals can be an important indicator of T. gondii circulation in zoo. The aim of this study was to examine sera of animals from eight Czech zoos by latex agglutination test with statistical evaluation and detect T. gondii DNA in stray cats and rodents captured in the zoos. T. gondii antibodies were detected in 33% of 1043 zoo animals without statistical difference between birds (27%, n = 74) and mammals (33%, n = 969). In birds, the chance to be infected with T. gondii was higher in Accipitriformes (71%) compared to Pelecaniformes (6%) (p < 0.0001). In mammals, the chance to be infected with T. gondii was higher in Carnivora (63%) compared to Cetarodactyla (30%), Perissodactyla (26%), Primates (28%) and Rodentia (13%) (p < 0.0001) and higher in Felidae (70%) compared to Bovidae (28%) and Equidae (28%) (p < 0.0001). Mammals with carnivore/scavenger way of feeding were in a higher risk of T. gondii infection compared to herbivores and omnivores (p < 0.0001). T. gondii DNA was detected in tissue of one stray cat while in none of 77 rodents caught in zoo. This study is the first report on toxoplasmosis in zoos from the Czech Republic including seroepidemiology and molecular detection. [ABSTRACT FROM AUTHOR]

Vav1 is a hematopoietic-specific Rho GDP/GTP exchange factor and signaling adaptor. Although these activities are known to be stimulated by direct Vav1 phosphorylation, little information still exists regarding the regulatory layers that influence the overall Vav1 activation cycle. Using a collection of cell models and activation-mimetic Vav1 mutants, we show here that the dephosphorylated state of Vav1 in nonstimulated T cells requires the presence of a noncatalytic, phospholipase Cγ1–Slp76-mediated inhibitory pathway. Upon T cell stimulation, Vav1 becomes rapidly phosphorylated via the engagement of Lck and, to a much lesser extent, other Src family kinases and Zap70. In this process, Lck, Zap70 and the adaptor protein Lat contribute differently to the dynamics and amplitude of the Vav1 phosphorylated pool. Consistent with a multiphosphosite activation mechanism, the optimal stimulation of Vav1 can only be recapitulated by the combination of several activation-mimetic phosphosite mutants. The analysis of these mutants has also unveiled the presence of different Vav1 signaling competent states that are influenced by phosphosites present in the N- and C-terminal domains of the protein. [ABSTRACT FROM AUTHOR]

Mast cells play a central role in the pathogenesis of allergic reaction. Activation of mast cells by antigens is strictly dependent on the influx of extracellular calcium that involves a complex interaction between signalling molecules located within the cells. We have previously reported that tHGA, an active compound originally isolated from a local shrub known as Melicope ptelefolia , prevented IgE-mediated mast cell activation and passive systemic anaphylaxis by suppressing the release of interleukin-4 (IL-4) and tumour necrosis factor (TNF)-α from activated rat basophilic leukaemia (RBL)-2H3 cells. However, the mechanism of action (MOA) as well as the molecular target underlying the mast cell stabilising effect of tHGA has not been previously investigated. In this study, DNP-IgE-sensitised RBL-2H3 cells were pre-treated with tHGA before challenged with DNP-BSA. To dissect the MOA of tHGA in IgE-mediated mast cell activation, the effect of tHGA on the transcription of IL-4 and TNF-α mRNA was determined using Real Time-Polymerase Chain Reaction (qPCR) followed by Calcium Influx Assay to confirm the involvement of calcium in the activation of mast cells. The protein lysates were analysed by using Western Blot to determine the effect of tHGA on various important signalling molecules in the LAT-PLCγ-MAPK and PI3K-NFκB pathways. In order to identify the molecular target of tHGA in IgE-mediated mast cell activation, the LAT and LAT2 genes in RBL-2H3 cells were knocked-down by using RNA interference to establish a LAT/LAT2 competition model. The results showed that tHGA inhibited the transcription of IL-4 and TNF-α as a result of the suppression of calcium influx in activated RBL-2H3 cells. The results from Western Blot revealed that tHGA primarily inhibited the LAT-PLCγ-MAPK pathway with partial inhibition on the PI3K-p65 pathway without affecting Syk. The results from RNAi further demonstrated that tHGA failed to inhibit the release of mediators associated with mast cell degranulation under the LAT/LAT2 competition model in the absence of LAT. Collectively, this study concluded that the molecular target of tHGA could be LAT and may provide a basis for the development of a mast cell stabiliser which targets LAT. [ABSTRACT FROM AUTHOR]

Summary CD4 + T cells differentiate into T helper cell subsets in feedforward manners with synergistic signals from the T cell receptor (TCR), cytokines, and lineage-specific transcription factors. Naive CD4 + T cells avoid spontaneous engagement of feedforward mechanisms but retain a prepared state. T cells lacking the adaptor molecule LAT demonstrate impaired TCR-induced signals yet cause a spontaneous lymphoproliferative T helper 2 (T H 2) cell syndrome in mice. Thus, LAT constitutes an unexplained maintenance cue. Here, we demonstrate that tonic signals through LAT constitutively export the repressor HDAC7 from the nucleus of CD4 + T cells. Without such tonic signals, HDAC7 target genes Nur77 and Irf4 are repressed. We reveal that Nur77 suppresses CD4 + T cell proliferation and uncover a suppressive role for Irf4 in T H 2 polarization; halving Irf4 gene-dosage leads to increases in GATA3 + and IL-4 + cells. Our studies reveal that naive CD4 + T cells are dynamically tuned by tonic LAT-HDAC7 signals. [ABSTRACT FROM AUTHOR]

Introduction Low expression of linker for activation of T cells (LAT) is observed in asthma. LAT and its downstream regulator, phospholipase C-gamma 1 (PLC-γ1) play important roles in the T cell antigen receptor signaling pathway, and their interaction is associated with CD4 + cell polarization. Here, we investigated whether LAT can alleviate the imbalance among CD4 + cell subgroups and the possible mechanism. Methods An ovalbumin-induced allergic asthma mouse model was established and LAT plasmid was delivered. The pathological changes in lung were evaluated by hematoxylin and eosin and periodic acid-Schiff staining. The typical cytokines released by T helper 2 (Th2) and regulatory T (Treg) cells were measured using enzyme-linked immunosorbent assay and the number of Th1, Th2, and Treg cells were determined using flow cytometry. Lung CD4 + T cells were isolated by magnetic isolation. The mRNA expression of LAT and PLC-γ1 was determined by real-time PCR. Co-Immunoprecipitation was performed to confirm the interaction between LAT and PLC-γ1. The protein expression of LAT, PLC-γ1 and corresponding downstream signaling factors were determined by western blotting. Results The delivery of LAT DNA to the lung could suppress an overactive Th2 response by decreasing allergic response and Th2 cytokine secretion, and by increasing Treg cytokine secretion. The Th2/Treg imbalance in lung and decreased phosphorylated PLC-γ1 expression in lung CD4 + T cells were rectified by LAT DNA delivery. Excessive activation of the Raf-MEK-ERK and PI3K-AKT-CREB pathways after asthma is attenuated by LAT. Conclusion The site-specific delivery of LAT DNA to the lung could suppress an overactive Th2 response and rectify the Th2/Treg imbalance in asthmatic mouse model. LAT-PLC-γ1 interaction may contribute to LAT activity in vivo and LAT protects against asthma partly via Raf-MEK-ERK and PI3K-AKT-CREB pathways. The delivery of LAT DNA could offer a novel and safe strategy for asthma prevention. [ABSTRACT FROM AUTHOR]

A novel Com1 synthetic peptide-based latex agglutination test (LAT) was developed and evaluated against commercial ELISA kit for sero-screening of coxiellosis in cattle. The developed test is economical, has field applicability and can serve as an important rapid tool for sero-screening of coxiellosis in cattle. [ABSTRACT FROM AUTHOR]

Brominated flame-retardants (BFRs) are used in industrial products to reduce the risk of fire. However, their continuous release into the environment is a concern as they are often persistent, bioaccumulating and toxic. Information on the impact these compounds have on human health and wildlife is limited and only a few of them have been identified to disrupt hormone receptor functions. In the present study we used in silico modeling to determine the interactions of selected BFRs with the human androgen receptor (AR). Three compounds were found to dock into the ligand-binding domain of the human AR and these were further tested using in vitro analysis. Allyl 2,4,6-tribromophenyl ether (ATE), 2-bromoallyl 2,4,6-tribromophenyl ether (BATE) and 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE) were observed to act as AR antagonists. These BFRs have recently been detected in the environment, in house dust and in aquatic animals. The compounds have been detected at high concentrations in both blubber and brain of seals and we therefore also assessed their impact on the expression of L -type amino acid transporter system (LAT) genes, that are needed for amino acid uptake across the blood–brain barrier, as disruption of LAT gene function has been implicated in several brain disorders. The three BFRs down-regulated the expression of AR target genes that encode for prostate specific antigen (PSA), 5 α- reductases and β-microseminoprotein. The potency of PSA inhibition was of the same magnitude as the common prostate cancer drugs, demonstrating that these compounds are strong AR antagonists. Western blot analysis of AR protein showed that ATE, BATE and DPTE decreased the 5 α- dihydrotestosterone-induced AR protein levels, further confirming that these BFRs act as AR antagonists. The transcription of the LAT genes was altered by the three BFRs, indicating an effect on amino-acid uptake across cellular membranes and blood–brain barrier. This study demonstrated that ATE, BATE and DPTE are potent AR antagonists and the alterations in LAT gene transcription suggest that these compounds can affect neuronal functions and should be considered as potential neurotoxic and endocrine disrupting compounds. [ABSTRACT FROM AUTHOR]

Abstract: Transmembrane adaptor proteins (TRAPs) are structurally related proteins that have no enzymatic function, but enable inducible recruitment of effector molecules to the plasma membrane, usually in a phosphorylation dependent manner. Numerous surface receptors employ TRAPs for either propagation or negative regulation of the signal transduction. Several TRAPs (LAT, NTAL, PAG, LIME, PRR7, SCIMP, LST1/A, and putatively GAPT) are known to be palmitoylated that could facilitate their localization in lipid rafts or tetraspanin enriched microdomains. This review summarizes expression patterns, binding partners, signaling pathways, and biological functions of particular palmitoylated TRAPs with an emphasis on the three most recently discovered members, PRR7, SCIMP, and LST1/A. Moreover, we discuss in silico methodology used for discovery of new family members, nature of their binding partners, and microdomain localization. [Copyright &y& Elsevier]

Abstract: Phospholipase C-γ1 (PLC-γ1) is a key regulator of T cell receptor (TCR)-induced signaling. Activation of the TCR enhances PLC-γ1 enzymatic function, resulting in calcium influx and the activation of PKC family members and RasGRP. The current model is that phosphorylation of LAT tyrosine 132 facilitates the recruitment of PLC-γ1, leading to its activation and function at the LAT complex. In this study, we examined the phosphorylation kinetics of LAT and PLC-γ1 and the cellular localization of activated PLC-γ1. We observed that commencement of the phosphorylation of LAT tyrosine 132 and PLC-γ1 tyrosine 783 occurred simultaneously, supporting the current model. However, once begun, PLC-γ1 activation occurred more rapidly than LAT tyrosine 132. The association of LAT and PLC-γ1 was more transient than the interaction of LAT and Grb2 and a pool of activated PLC-γ1 translocated away from LAT to cellular structures containing the TCR. These studies demonstrate that LAT and PLC-γ1 form transient interactions that catalyze the activation of PLC-γ1, but that activated PLC-γ1 resides in both LAT and TCR clusters. Together, this work highlights that our current model is incomplete and the activation and function of PLC-γ1 in T cells is highly complex. [Copyright &y& Elsevier]

Abstract: The dog tapeworm Echinococcus granulosus is the causative agent of cystic hydatid disease in domestic/wild herbivores animals and man. Accurate immunodiagnosis of the infection requires highly specific and sensitive antigens. The aim of this study was to develop and evaluate various immunoassays with principles of precipitation, agglutination and enzyme immunoassays for the identification of sheep infected with hydatid cyst which would allow the monitoring of animals from endemic areas and identifying infected animals prior to slaughter. The immunoassays were developed and validated using hydatid specific, non-cross reactive low molecular weight 8kDa hydatid cyst fluid protein. Sera used for the assay validations were obtained from 150 sheep infected naturally with hydatid cyst and 150 non-infected sheep. The highest diagnostic sensitivity was obtained in enzyme linked immune electro transfer blot (EITB) at 99.33 per cent followed by latex agglutination test (98.67 per cent) and counter immunoelectrophoresis (94.67 per cent). The study demonstrated that EITB was most sensitive immunological test for the detection of cystic echinococcosis in sheep. It should be useful for the conformation of hydatid cyst infected individual sheep. However, CIEP and LAT methods can be applied to practical use for screening studies. [Copyright &y& Elsevier]

Abstract: The specific TLR2/1 complex activator Pam3CSK4 has been shown to provoke prominent activation and aggregation of human non-nucleated platelets. As Pam3CSK4-evoked platelet activation does not employ the major signalling pathway established in nucleated immune cells, we investigated if the TLR2/1 complex on platelets may initiate signalling pathways known to be induced by physiological agonists such as collagen via GPVI or thrombin via PARs. We found that triggering TLR2/1 complex-signalling with Pam3CSK4, in common with that induced via GPVI, and in contrast to that provoked by PARs, involves tyrosine phosphorylation of the adaptor protein LAT as well as of PLCγ2 in a src- and Syk-dependent manner. In this respect, we provide evidence that Pam3CSK4 does not cross-activate GPVI. Further, by the use of platelets from a Glanzmann's thrombasthenia patient lacking β3, in contrast to findings in nucleated immune cells, we show that the initiation of platelet activation by Pam3CSK4 does not involve integrin β3 signalling; whereas the latter, subsequent to intermediate TXA2 synthesis and signalling, was found to be indispensable for proper dense granule secretion and full platelet aggregation. Together, our findings reveal that triggering the TLR2/1 complex with Pam3CSK4 initiates human platelet activation by engaging tyrosine kinases of the src family and Syk, the adaptor protein LAT, as well as the key mediator PLCγ2. [Copyright &y& Elsevier]

Abstract: In the search for an efficient, fluorine-18 labeled amino acid based radiotracer for tumor imaging with positron emission tomography (PET), two new tryptophan analogs were synthesized and characterized in vitro and in vivo. Both are tryptophan alkyl-derivatives, namely 2-(3-[18F]fluoropropyl)-dl-tryptophan ([18F]2-FPTRP) and 5-(3-[18F]fluoro-propyl)-dl-tryptophan ([18F]5-FPTRP). Standard reference compounds and precursors were prepared by multi step approaches. Radiosynthesis was achieved by no-carrier-added nucleophilic [18F]fluorination in 29–34% decay corrected yields with radiochemical purity over 99%. In vitro cell uptake assays showed that both compounds are substrates for amino acid transport and enter small cell lung cancer cells (NCI-H69) most probably almost exclusively via large neutral amino acids transporter(s) (LAT). Small animal PET imaging with xenograft bearing mice revealed high tumor/background ratios for [18F]2-FPTRP comparable to the well established tyrosine analog O-(2-[18F]fluroethyl)-l-tyrosine ([18F]FET). Radiometabolite studies showed no evidence of involvement of a biotransformation step in tumor accumulation. [Copyright &y& Elsevier]

Abstract: The present study was aimed to investigate and compare the efficacy of duloxetine (DLX) loaded nanostructured lipid carriers (NLC) with DLX solution pharmacodynamically following intranasal administration. The study was further conducted to estimate DLX concentration in brain and blood. DLX was administered to albino Wistar rats either intranasally or orally in solution form (DLX solution) or encapsulated in NLC (DLX–NLC). These were evaluated in-vivo for pharmacodynamic studies for depression by forced swimming test and locomotor activity test. Intranasal DLX–NLC treatment exhibited improved behavioural analysis results (swimming, climbing, and immobility) than the DLX solution after 24 h of study. Furthermore, DLX–NLC significantly increased the total swimming and climbing time when compared with control and significantly reduced the immobility period. The intranasal DLX–NLC demonstrated improved locomotor activity when compared with DLX solution. Amount of DLX was quantified in blood and brain after the forced swimming test. The intranasal DLX–NLC demonstrated higher concentration in brain compared with DLX solution. Thus, intranasal DLX–NLC was found to be a promising formulation for the treatment of depression. [Copyright &y& Elsevier]

Abstract: Purpose: We evaluated new 111In-labeled amino acid derivatives, in which the amino acids are conjugated with1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A) or 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A). Methods: DOTA-aminoalanine (DOTA-A), DOTA-aminohomoalanine (DOTA-H), DOTA-lysine (DOTA-L), DO2A-alanine (DO2A-A), DO3A-alanine (DO3A-A) and DO3A-homoalanine (DO3A-H) were labeled with 111In. In vitro cell uptake assays were performed usingHep3B (a human hepatoma cell line), CT26 (a mouse colon cancer cell line) and U87MG (a human glioma cell line). In vitro cell uptake inhibition assays were performed using U87MG and 111In-DO3A-H. U87MG bearing xenografted mice were subject to biodistribution, SPECT imaging, autoradiography, and immunohistochemistry studies. Results: Of the amino acid derivatives and cell lines examined, U87MG and 111In-DO3A-H showed highest uptake in vitro. This uptake was blocked by 2-aminobicyclo-[2,2,1] heptane-2-carboxylic acid (BCH) and by tryptophan. 111In-DO3A-HSPECT imaging of U87MG bearing xenografted mice visualized tumors (mean tumor-to-muscle ratio 3.16±0.74). Autoradiography and immunohistochemistry revealed that 111In-DO3A-H uptake matched L-type amino acid transporter 1 expression. Conclusion: Tumor uptake was successfully imaged using 111In-DO3A-H in U87MG bearing xenografted mice. 111In-DO3A-H appears to be useful for imaging tumors expressing L-type amino acid transporter. [Copyright &y& Elsevier]

Abstract: T cell response initiated by engagement of T cell receptor (TCR) is dependent on signal transduction events composed of protein kinases and adaptor proteins. However, the molecular mechanism for gene expression of these proteins is not entirely understood. Here we identified Heat Shock Protein 90 (HSP90) as an essential regulator for gene expression of Linker for activation of T cells (LAT) in primarily activated human T cells. Primarily activated T cells continuously synthesized LAT protein and treatment of cells with 17-AAG, a pharmacological inhibitor of HSP90, decreased LAT protein level following reduction of LAT mRNA. Furthermore, promoter activity of LAT gene was dramatically inhibited by 17-AAG. These results reveal a novel role of HSP90 as a positive regulator for expression of LAT gene in activated T cells. [Copyright &y& Elsevier]

Abstract: We investigated IgE-mediated allergic responses in a metabolic syndrome model rat strain, SHRSP.Z, which develops obesity and hypertension to cast light on the relationship between metabolic disturbances and allergic responses. IgE-mediated cutaneous anaphylactic responses were severely attenuated in this strain regardless of the presence of fa/fa mutation, compared with the parental WKY/Izm strain. Furthermore, in the peritoneal mast cells of both the SHRSP.Z and SHRSP/Izm strains, IgE-mediated activation, such as degranulation and protein tyrosine phosphorylation, was severely impaired whereas no significant differences were found in morphology and number of peritoneal mast cells. Immunoblot analyses revealed that phosphorylation levels of Syk upon IgE-mediated antigen stimulation were significantly decreased and basal expression of linker for activation of T cells (LAT) was down-regulated in peritoneal mast cells of the SHRSP strains. These results suggest that attenuated cutaneous allergic responses in the SHRSP.Z strain might be attributed to impaired FcɛRI-mediated signal transduction in mast cells. [Copyright &y& Elsevier]

Abstract: The Large Area Telescope (LAT) on-board the Fermi Gamma-ray Space Telescope began its on-orbit operations on June 23, 2008. Calibrations, defined in a generic sense, correspond to synchronization of trigger signals, optimization of delays for latching data, determination of detector thresholds, gains and responses, evaluation of the perimeter of the South Atlantic Anomaly (SAA), measurements of live time, of absolute time, and internal and spacecraft boresight alignments. Here we describe on-orbit calibration results obtained using known astrophysical sources, galactic cosmic rays, and charge injection into the front-end electronics of each detector. Instrument response functions will be described in a separate publication. This paper demonstrates the stability of calibrations and describes minor changes observed since launch. These results have been used to calibrate the LAT datasets to be publicly released in August 2009. [Copyright &y& Elsevier]

Abstract: Transmembrane adaptor proteins are key mediators of antigen receptor signaling in lymphocytes. By influencing proliferation and differentiation, these molecules might play a role in ethiopathogenesis of acute lymphoblastic leukemia (ALL). The aim of this study was to characterize expression of PAG, LAT, NTAL and LIME adaptors at the mRNA and protein levels in normal B- and T-precursors. Moreover, diagnostic samples of childhood ALL cases were analyzed. During normal lymphocyte development, some adaptors show significant dynamics (gradual decrease of NTAL and increase of LAT and LIME during the T-cell maturation, decrease of PAG in B-precursors, high levels of LIME in peripheral B-lymphocytes). Analysis of childhood ALL samples revealed that in B-cell precursor ALL, the TEL/AML1 subgroup have unique adaptor profile compared to other leukemias. Moreover, NTAL expression separates T lineage leukemias into two subgroups with good and poor response to initial prednisone therapy showing prognostic impact of this molecule in T-ALL. [Copyright &y& Elsevier]

Abstract: The entire gene encoding microneme protein 3 (MIC3) from Toxoplasma gondii was cloned into the plasmid pGEX-KG and subsequently expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The recombinant MIC3 (rMIC3) was purified and evaluated in a latex agglutination test (LAT) as the diagnostic antigen for the detection of antibodies to T. gondii in pig sera. The specificity, stability, and reproducibility of the test were examined. No agglutination was found when the sensitized latex beads were mixed with phosphate-buffered saline (PBS), borate-buffered saline (BBS), normal saline, and negative serum samples. There was no cross-reactivity with the standard positive sera of other pathogens. But intense agglutination occurred with T. gondii antibody positive serum samples. In our study, the coincidence rate of tested positive-sera of the LAT with rMIC3-sensitized latex particles and the ELISA with rSAG1 was up to 92.8%, T. gondii specific antibodies were detected by the LAT in all piglets that were experimentally infected with T. gondii tachyzoites from 8 to 42 days after infection. Our results indicated that the rMIC3 based latex agglutination test appears to be suitable for the detection of T. gondii antibodies at the early stage of infection. [Copyright &y& Elsevier]

Abstract: syn- and anti-1-amino-3-[2-iodoethenyl]-cyclobutane-1-carboxylic acid (syn-, anti-IVACBC 16, 17) and their analogue 1-amino-3-iodomethylene-cyclobutane-1-carboxylic acid (gem-IVACBC 18) were synthesized and radioiodoinated with [123I] in 34–43% delay-corrected yield. All these amino acids entered 9L gliosarcoma cells primarily via L-type transport in vitro with high uptake of 8–10% ID/1×106 cells. Biodistribution studies of [123I]16, 17 and 18 in rats with 9L gliosarcoma brain tumors demonstrated high tumor to brain ratios (4.7–7.3:1 at 60min post-injection). In this model, syn-, anti-, and gem-[123I]IVACBC are promising radiotracers for SPECT brain tumor imaging. [Copyright &y& Elsevier]

Abstract: LAT is a linker protein essential for activation of T lymphocytes. Its rapid tyrosine-phosphorylation upon T cell receptor (TCR) stimulation recruits downstream signaling molecules for membrane targeting and activation. LAT is physically concentrated in cholesterol-enriched membrane microdomains and is known a substrate for Syk/Zap70 kinase. In this study, we demonstrate that LAT serves as a dual substrate for both Lck and Syk kinases. LAT phosphorylation is absent in Lck-deficient J.CaM1.6 cells and Lck is co-precipitated with LAT in pervanadate-activated Jurkat cells. Further, the in vitro kinase assay using purified Lck and LAT shows that Lck directly phosphorylates LAT. Both Lck and Syk, phosphorylate the ITAM-like motifs on LAT at Y171Y191, which is essential for induction of the interaction of LAT with downstream signaling molecules such as Grb2, PLC-γ1 and c-Cbl, and for activation of MAPK-ERK. Collectively, our data indicate that LAT is an immediate substrate for Lck in one of the earliest events of T cell activation. [Copyright &y& Elsevier]

Abstract: A 1.7kb fragment of lat was obtained from Streptomyces clavuligerus NRRL 3585, and recombinant plasmid pKC1139-lat, which was used to disrupt the lat gene was constructed. pKC1139-lat was introduced into S. clavuligerus by bi-parental conjugation from Escherichia coli ET12567 to S. clavuligerus. The apramcin-resistant transformants were obtained and through homogeneous single-crossover between recombinant plasmid pKC1139-lat and the S. clavuligerus chromosome lat disrupted mutant strains were obtained. The genome of S. clavuligerus NRRL 3585 and the lat disrupted mutants were analyzed by PCR technique, the bioactivity of cephamycin C in the two kinds of strains were also tested. Both results proved that lat was disrupted by the insertion of pKC1139 in the lat disrupted mutants. And the production of clavulanic acid of these two kinds of strains were analyzed by HPLC with different incubation time interval (96 and 120h), and the yield in the lat mutants was approximately 2.6 fold higher at their highest production point. [Copyright &y& Elsevier]

Abstract: The melanogenic marine bacterium M. mediterranea synthesizes marinocine, a protein with antibacterial activity. We cloned the gene coding for this protein and named it lodA [P. Lucas–Elío, P. Hernández, A. Sanchez-Amat, F. Solano, Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea. Biochim. Biophys. Acta 1721 (2005) 193–203; P. Lucas-Elío, D. Gómez, F. Solano, A. Sanchez-Amat, The antimicrobial activity of marinocine, synthesized by M. mediterranea, is due to the hydrogen peroxide generated by its lysine oxidase activity. J. Bacteriol. 188 (2006) 2493–2501]. Now, we show that this protein is a new type of lysine oxidase which catalyzes the oxidative deamination of free l-lysine into 6-semialdehyde 2-aminoadipic acid, ammonia and hydrogen peroxide. This new enzyme is compared to other enzymes related to lysine transformation. Two different groups have been used for comparison. Enzymes in the first group lead to 2-aminoadipic acid as a final product. The second one would be enzymes catalyzing the oxidative deamination of lysine releasing H2O2, namely lysine-α-oxidase (LαO) and lysyl oxidase (Lox). Kinetic properties, substrate specificity and inhibition pattern show clear differences with all above mentioned lysine-related enzymes. Thus, we propose to rename this enzyme lysine-ε-oxidase (lod for the gene) instead of marinocine. Lod shows high stereospecificity for free l-lysine, it is inhibited by substrate analogues, such as cadaverine and 6-aminocaproic acid, and also by β-aminopropionitrile, suggesting the existence of a tyrosine-derived quinone cofactor at its active site. [Copyright &y& Elsevier]

Abstract: Recent genetic evidence demonstrated that Shc is a critical molecule for T cell activation and differentiation. However, how Shc is coupled to the T cell antigen receptor (TCR) has not been clearly characterized. Here we report that the tyrosine kinase Lck functions as a connecting molecule for TCR and Shc. Lck plays a critical role in TCR signal transduction by phosphorylating the immuno-receptor tyrosine based activation motif (ITAM). Our data shows that the PTB domain of Shc binds the SH2/3 domains of Lck in a phosphotyrosine-independent manner. Inhibition of the Lck/Shc interaction led to the loss of IL-2 promoter activation, confirming that the role of Shc in IL-2 production requires its interaction with Lck. Together, the data show that Shc is connected to the activated TCR via direct interaction with Lck. [Copyright &y& Elsevier]

Accumulation of [3H]l-serine in crude synaptosomal fractions freshly prepared from rat brain has been found to be temperature-sensitive and to consist of both Na+-dependent and Na+-independent components. The accumulation of [3H]l-serine measured at submicromolar concentrations had a distinct substrate selectivity, different from the uptake of [3H]l-proline, [3H]l-glutamate and [3H]GABA. It was fully inhibited by l-glutamine, l-asparagine, l-cysteine, l-alanine, l-leucine, l-isoleucine, l-tyrosine, l-phenylalanine, l-threonine and by the synthetic marker for the large neutral amino acid transport systems 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, but not influenced by β-alanine, taurine, glycine nor was it inhibited by the marker for the A system, l-2-methylamino isobutyric acid. d-Serine at 1 mM concentration produced no significant inhibition of the accumulation of 10 nM [3H]l-serine. We conclude that l-serine uptake observed in the present study is mediated by at least two distinct transport systems: a Na+-dependent one of lower affinity (Km in mM range) and a Na+-independent system of higher affinity (Km&ap;20–100 μM). Characteristics of [3H]l-serine accumulation displayed at low substrate concentrations suggest that it was mediated neither by the typical ‘A’, nor by the ‘large neutral’, amino acid transport systems but predominantly by transporters belonging to the recently identified LAT (l-amino acid transporter) family. [Copyright &y& Elsevier]

To monitor the non-specific agglutination (NSA) in latex immunoagglutination assay, antigen-coated structured latex particles, which have carboxyl and sulphonate groups as hydrophilic domains, were tested for an antibody assay. Sulphonated particles showed NSA in high antibody concentrations, where no surface antigen left to match with. This was further justified with the more stable highly sulphonated particles, which showed higher degree of NSA. It can therefore be confirmed that sulphonate groups cause (or at least promote) NSA, while carboxyl groups do not. Surface coverage over 17% was not fully utilized for antigen–antibody reaction, due to the prozone effect. The difference in sensitivity of particles was explained in terms of our new explanations on the governing interactions of protein adsorption. [Copyright &y& Elsevier]

: ObjectivesWe sought to define the prevalence of cardiac involvement in female patients with Anderson–Fabry disease (AFD).: BackgroundAnderson–Fabry disease is a rare inborn X-linked lysosomal storage disorder. Globotriaosylceramide (Gb3), the major substrate of the deficient α-galactosidase A enzyme, accumulates progressively in vulnerable cells, including the cardiovascular system. It has been believed that heterozygous females have less cardiac involvement than hemizygous males with AFD.: MethodsWe performed two-dimensional echocardiographic examinations of female patients heterozygous for AFD.: ResultsSince 1997, a total of 55 female patients (mean age, 39.6 years; range, 6.1 to 70.8 years) with proven AFD have been investigated prospectively at our hospital. Of these, 13 (23.6%) had normal left ventricular (LV) geometry and LV mass (LVM). Seven patients (12.7%) had concentric remodeling, 29 patients (52.7%) concentric LV hypertrophy (LVH), and 6 patients (10.9%) eccentric LVH (2 with subaortic pressure gradients). There was a strong correlation between age and the severity of LVH (r2 = 0.905; p < 0.0001), and all patients older than 45 years had LVH. With increasing LVM, there was a significant age-independent decrease in systolic and diastolic LV function. Mild thickening of the aortic valve leaflets was present in 25.5% of patients, with the same percentage demonstrating mild thickening of the mitral valve leaflets. Mild mitral valve prolapse was documented in 10.9% of patients.: ConclusionsCardiac involvement, with LVH and structural valve abnormalities, is very common and worsens with age in females who are heterozygous for AFD, and they should therefore be considered candidates for enzyme replacement therapy. [Copyright &y& Elsevier]

Crosslinking the high affinity IgE receptor, Fc&epsiv;RI, on basophils and mast cells initiates cascades of biochemical events leading to degranulation, membrane ruffling and other physiological responses. Downstream of Fc&epsiv;RI and its coupled tyrosine kinases, Lyn and Syk, scores of different proteins and lipids are implicated in these signaling cascades and new players are being identified continuously. Here, we use immunogold probes to label receptors and signaling proteins on the cytoplasmic face of membrane sheets prepared from RBL-2H3 mast cells and transmission electron microscopy to examine their distributions in relationship to each other and to features of the membrane. New topographical data are integrated with existing knowledge of the biochemistry of Fc&epsiv;RI signaling and of cell shape during signaling to implicate at least two distinct membrane domains in Fc&epsiv;RI signaling. “Primary signaling domains”, also called osmiophilic patches, are recognized by their dark staining with osmium, adjacency to coated pits (previously mapped to planar membrane between lamellae) and by the characteristic presence of receptor, Syk and PLCγ2, but not Lyn. “Secondary signaling domains” are characterized by the presence of large elliptical linker for activation of T cells (LAT) rafts and of PLCγ1 (previously mapped to lamellae) but not receptor. The signaling proteins, Vav, Grb2, Cbl and Gab2, and the endocytic proteins, AP2 and clathrin, all map to the primary domains, while the p85 regulatory subunit of phosphatidylinositol 3 (PI 3)-kinase maps to both domains. Recognition that Fc&epsiv;RI signaling is controlled not only by which chemical species are available for interaction, but also by where the interactions occur, may provide new opportunities for the modeling of signaling cascades and new targets for the development of drugs to treat allergies and asthma. [Copyright &y& Elsevier]

The generation of signals following engagement of cell surface receptors is an ordered process that requires tight regulation as spurious signals could result in unwanted, and possibly deleterious, cellular responses. Like other cell surface receptors, stimulation of a mast cell via the high affinity IgE receptor (Fc&epsiv;RI) causes multiple biochemical events that ultimately result in cell activation and effector responses. Recently, our knowledge of how these events are ordered has increased. We now have identified some of the molecules involved, how they are organized into macromolecular complexes by Fc&epsiv;RI stimulation, and the role of some of the constituents of these macromolecular signaling complexes in mast cell effector responses. In brief, we review the knowledge on macromolecular signaling complexes used by Fc&epsiv;RI in mast cell activation and provide our view on the regulation of signal generation and its effect on mast cell activation. [Copyright &y& Elsevier]

: ObjectivesWe sought to develop a prognostic model to predict the disappearance of left atrial thrombi (LAT) among candidates for percutaneous transvenous mitral commissurotomy (PTMC).: BackgroundComplete LAT resolution can be achieved with oral anticoagulation, allowing a number of patients to safely undergo PTMC.: MethodsWe randomly allocated 108 PTMC candidates with LAT into two subsets—one to derive the model and the other to validate it. The existence of LAT and its size were measured by transesophageal echocardiography. Patients were given oral anticoagulation and followed up for 6 to 34 months. There was a 62% disappearance rate of LAT.: ResultsWe developed the following model: P = 1/(1 + exponential [−8.1 + 1.8 NYHA + 0.7 area]), where NYHA = New York Heart Association functional class (from I to IV), and area = LAT area (in cm2). The model was well calibrated (goodness-of-fit test, p = 0.82) and well discriminated (area under the receiver-operating characteristics [ROC] curve = 0.92). Performance in the validating sample was equally good (area under the ROC curve = 0.94; goodness-of-fit test, p = 0.16). When a cut-off point of p > 0.7 was used to designate the LAT disappearance in the validating set, the model had a sensitivity, specificity and positive and negative predictive values of 93.3%, 79.2%, 84.9% and 90.5%, respectively.: ConclusionsCombined clinical (NYHA functional class) and echocardiographic (LAT area) variables are predictive of the 34-month outcome of oral anticoagulation for LAT resolution among PTMC candidates. This simple and highly predictive model might be potentially useful for clinical assessment and proper management. [Copyright &y& Elsevier]

Toxoplasmosis is a globally spread disease, affecting humans and many animal species, including birds. Antibodies to Toxoplasma gondii were detected in ostriches from South and North America, Africa and Asia. Except for one study from Spain, there is a lack of information about T. gondii seroprevalence in ostriches from Europe. For this reason, the aim of the study was to detect antibodies to T. gondii in farm-reared ostriches from the Czech Republic. Serum samples of 409 ostriches (Struthio camelus), collected at 9 farms were tested by Latex agglutination test. Antibodies to T. gondii were detected in 149 (36%) birds with a statistical difference for individual farms (8%–71%, p = 0.0121), and regions (8%–65%, p = 0.002). Seropositivity did not statistically differ (p > 0.05) in size of farms (50% and 35% on small and large farms, respectively), sex of birds (38% and 35% in males and females, respectively), season and year of collection. Tissue samples (brain, heart, and pectoral muscle) of 105 birds were also tested by PCR to detect T. gondii DNA. The parasite T. gondii was detected in the brain and heart of one seronegative ostrich (1%) from a small farm. Based on our results, we can assume that ostriches may present high risk of toxoplasmosis for humans through consumption of raw or undercooked ostrich meat and even seronegative individuals could harbor T. gondii in their tissues. To our knowledge, this is the first serological detection of T. gondii in ostriches in the Czech Republic, and the first PCR detection in Europe. • There were 36% ostriches tested positive for antibodies against Toxoplasma gondii. • High seropositivity may indicate risk of infection from ostrich meat to humans. • Brain and heart tissue are suitable sample for PCR detection of T. gondii DNA. • Ostriches may presumably act as serological non-responders to T. gondii infection. [ABSTRACT FROM AUTHOR]

The primary activating receptor for T cells is the T cell receptor (TCR), which is stimulated upon binding to an antigen/MHC complex. TCR activation results in the induction of regulated signaling pathways vital for T cell differentiation, cellular adhesion and cytokine release. A critical TCR-induced signaling protein is the adaptor protein LAT. Upon TCR stimulation, LAT is phosphorylated on conserved tyrosines, which facilitates the formation of multiprotein complexes needed for propagation of signaling pathways. Although the role of the conserved tyrosines in LAT-mediated signaling has been investigated, few studies have examined the role of larger regions of LAT in TCR-induced pathways. In this study, a sequence alignment of 97 mammalian LAT proteins was used to identify several "functional" domains on LAT. Using LAT mutants expressed in Jurkat E6.1 cells, we observed that the membrane proximal, proline-rich region of LAT and the correct order of domains containing conserved tyrosines are necessary for optimal TCR-mediated early signaling, cytokine production, and cellular adhesion. Together, these data show that LAT contains distinct regions whose presence and correct order are required for the propagation of TCR-mediated signaling pathways. • Alignment of 97 species of LAT identifies several "functional" domains in LAT. • LAT proline-rich region is required for optimal TCR-mediated signaling and function. • The order of tyrosines on LAT is vital for TCR-induced signaling and function. • LAT is unstructured and has functional domains critical for it's signaling capacity. [ABSTRACT FROM AUTHOR]

T cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that ≥90% of T cell receptor (TCR) complex molecules TCRαβ and TCRζ, as well as the co-receptor CD4 (cluster of differentiation 4) and the co-stimulatory molecule CD2, reside on microvilli of resting human T cells. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, including the protein tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase) and the key adaptor LAT (linker for activation of T cells), are also enriched on microvilli. Notably, phosphorylated proteins of the ERM (ezrin, radixin, and moesin) family colocalize with TCRαβ as well as with actin filaments, implying a role for one or more ERMs in linking the TCR complex to the actin cytoskeleton within microvilli. Our results establish microvilli as key signaling hubs, in which the TCR complex and its proximal signaling molecules and adaptors are preassembled prior to activation in an ERM-dependent manner, facilitating initial antigen sensing. • Single-molecule microscopy maps signaling molecules on the resting T cell membrane • TCRαβ, TCRζ, CD4, CD2, Lck, and LAT are all highly enriched on microvilli • TCR localization is mediated by ERM proteins • Microvilli are hubs for TCR signaling, facilitating fast initial immune response T-cell surfaces are covered with microvilli, actin-rich and flexible protrusions. Ghosh et al. show that T cell receptor complex molecules, as well as several proximal signaling molecules, are preassembled on microvilli in an ERM-dependent manner. These results establish microvilli as key signaling hubs that facilitate initial antigen sensing by T cells. [ABSTRACT FROM AUTHOR]