Quantum-mechanical kinetic study of the primary reaction of the photochemical cycle of Halobacterium halobium

A two-frequency oscillator model for the primary photochemical reaction bacteriorhodopsin batho-bacteriorhodopsin (K₆₁₀) is proposed. According to this model two conformational changes in the reaction are considered to take place: the first one is a distortion of the retinal in the bacteriorhodopsin active site and the second one is a conformational transition of the bacterioopsin, affecting the native structure hydrogen bonds. On the basis of this model the temperature dependences of the rate constants for normal and deuterated reactants are calculated in good agreement with the available experimental data. The relations of the reaction considered to the primary photochemical reaction of vision are discussed.     

The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins

BACKGROUND:Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. MATERIALS AND METHODS: Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay. RESULTS: Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II). CONCLUSIONS:On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition.     

Identification of diagnostic biomarkers for infection in premature neonates

Infection is a leading cause of neonatal morbidity and mortality worldwide. Premature neonates are particularly susceptible to infection because of physiologic immaturity, comorbidity, and extraneous medical interventions. Additionally premature infants are at higher risk of progression to sepsis or severe sepsis, adverse outcomes, and antimicrobial toxicity. Currently initial diagnosis is based upon clinical suspicion accompanied by nonspecific clinical signs and is confirmed upon positive microbiologic culture results several days after institution of empiric therapy. There exists a significant need for rapid, objective, in vitro tests for diagnosis of infection in neonates who are experiencing clinical instability. We used immunoassays multiplexed on microarrays to identify differentially expressed serum proteins in clinically infected and non-infected neonates. Immunoassay arrays were effective for measurement of more than 100 cytokines in small volumes of serum available from neonates. Our analyses revealed significant alterations in levels of eight serum proteins in infected neonates that are associated with inflammation, coagulation, and fibrinolysis. Specifically P- and E-selectins, interleukin 2 soluble receptor alpha, interleukin 18, neutrophil elastase, urokinase plasminogen activator and its cognate receptor, and C-reactive protein were observed at statistically significant increased levels. Multivariate classifiers based on combinations of serum analytes exhibited better diagnostic specificity and sensitivity than single analytes. Multiplexed immunoassays of serum cytokines may have clinical utility as an adjunct for rapid diagnosis of infection and differentiation of etiologic agent in neonates with clinical decompensation.     

Optimisation of synthetic medium composition for levorin biosynthesis by Streptomyces levoris 99/23 and investigation of its accumulation dynamics using mathematical modelling methods

The composition of a synthetic culture medium for levorin biosynthesis by Streptomyces levoris 99/23 was optimised using mathematical modelling methods. The optimal concentrations of the medium components were established by means of an optimum composition design at three factor variation levels. An adequate regression model was obtained. Levorin biosynthesis by Streptomyces levoris 99/23 in the optimised synthetic medium was over 38% higher than in the initial medium. The antibiotic biosynthesis dynamics in the optimised culture medium was studied by means of a non-linear differential equation system. The resultant model was valid.     

Human ribonuclease A superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation

A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-alpha, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.     

X-ray and Fe Mössbauer study on a Fe(III) complex of 2-acetyl-1,3-indandione

Single-crystal X-ray structure of an iron(III) complex of 2-acetyl-1,3-indandione is resolved which reveals that a racemic mixture, composed of Δ-fac and Λ-fac stereoisomers, is formed. Both species have octahedral geometry, but slightly distinguishable. Detailed Mössbauer data indicate a high-spin electronic structure of the Fe(III) centres Fe1 and Fe2 with spin-spin magnetic relaxation process. Two different approaches for computer processing of the experimental Mössbauer spectra are considered.     

Epizootic Hemorrhagic Disease Virus Induces and Benefits from Cell Stress,Autophagy, and Apoptosis

The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.     

Multiplexed protein profiling on microarrays by rolling-circle amplification

Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-alpha treatment. Because microarrays can accommodate approximately 1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.     

Isolation, purification and characterization of levorin produced by Streptomyces levoris 99/23

Levorin produced by Streptomyces levoris 99/23 was isolated, purified and characterized. It was established that 80% of the levorin was localized in the mycelium and only 20% was in the cell-free supernatant. Amorphous yellow levorin with activity of 24 000 IU/mg and 96% purity was obtained. The preparation exhibited three absorption maxima: at λ 362, 382 and 404 nm. The antibiotic contained seven components: A₀, A₁, A₂, A₃, A₄, and two unidentified ones. According to its composition, the preparation corresponded to the levorin used for medicinal purposes. However, the levorin produced by S. levoris 99/23 contained half as much levorin A₂ and a more than 100 times larger quantity of the more active and less toxic component levorin A₃.