Microarray phenotyping in Dictyostelium reveals a regulon of chemotaxis genes

MOTIVATION: Coordinate regulation of gene expression can provide information on gene function. To begin a large-scale analysis of Dictyostelium gene function, we clustered genes based on their expression in wild-type and mutant strains and analyzed their functions. RESULTS: We found 17 modes of wild-type gene expression and refined them into 57 submodes considering mutant data. Annotation analyses revealed correlations between co-expression and function and an unexpected correlation between expression and function of genes involved in various aspects of chemotaxis. Co-regulation of chemotaxis genes was also found in published data from neutrophils. To test the predictive power of the analysis, we examined the phenotypes of mutations in seven co-regulated genes that had no published role in chemotaxis. Six mutants exhibited chemotaxis defects, supporting the idea that function can be inferred from co-expression. The clustering and annotation analyses provide a public resource for Dictyostelium functional genomics.     

Spectrofluorimetric assay of ciprofloxacin hydrochloride in tablets

OBJECTIVE: To describe the spectrofluorimetric method for the determination of ciprofloxacin hydrochloride in tablets under optimum conditions. STUDY DESIGN: Examination by spectrofluorimetry. RESULTS: The wavelengths of excitation and emission were 290 and 450 nm, respectively. The fluorescence intensity was linearly related to the drug concentration over the range 2-8 microg/mL, with a limit of detection of 1.4 microg/mL and a limit of quantitation of 2.0 microg/mL. The method was highly accurate and precise, having a relative SD of < 1.0 %. The validity of the method was tested by recovery studies of the standard addition to pharmaceuticals, and the results were satisfactory. The results obtained by the application of this method and the official one were in good agreement, and statistical comparison by Student's t test and the variance ratio F test showed no significant difference between the 2 methods. CONCLUSION: The proposed method is simple and sufficiently precise for quality control purposes.     

An alternative parameter for monitoring the therapeutic benefits of allopurinol simultaneously in renal ischaemia-reperfusion injury: MDA/ATP Ratio

The therapeutic benefits of allopurinol pretreatment in renal ischaemia-reperfusion injury were investigated by monitoring renal malondialdehyde (MDA) and ATP levels together with calculated MDA/ATP ratio in ischaemic (45 min) and reperfused (15 min) rat kidneys. MDA levels remained unchanged during ischaemia, but increased after the subsequent reperfusion. ATP content of the ischaemic kidney was decreased significantly and the recovery of ATP was incomplete after the reperfusion, whereas the MDA/ATP ratio increased at both periods. Allopurinol pretreatment (40 mg kg(-1) iv) maintained higher ATP levels during the ischaemia and inhibited the MDA formation during the reperfusion and decreased the MDA/ATP ratio at both periods. Our findings demonstrate that allopurinol exerts a biphasic protective action by preserving tissue ATP and by inhibiting lipid peroxidation during ischaemia and the reperfusion period, respectively. These findings suggest the selective involvement of two protective mechanisms in the different periods of renal ischaemia-reperfusion injury. The MDA/ATP ratio could be a useful parameter for monitoring these protective actions of allopurinol simultaneously.     

Nogo-Receptor 1 Deficiency Has No Influence on Immune Cell Repertoire or Function during Experimental Autoimmune Encephalomyelitis

The potential role of Nogo-66 Receptor 1 (NgR1) on immune cell phenotypes and their activation during neuroinflammatory diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), is unclear. To further understand the function of this receptor on haematopoietically-derived cells, phenotypic and functional analyses were performed using NgR1-deficient (ngr1-/-) animals. Flow cytometry-based phenotypic analyses performed on blood, spleen, thymus, lymph nodes, bone marrow and central nervous-system (CNS)-infiltrating blood cells revealed no immunological defects in naïve ngr1-/- animals versus wild-type littermate (WTLM) controls. EAE was induced by either recombinant myelin oligodendrocyte glycoprotein (rMOG), a model in which B cells are considered to contribute pathogenically, or by MOG_35–55 peptide, a B cell-independent model. We have demonstrated that in ngr1-/- mice injected with MOG_35–55, a significant reduction in the severity of EAE correlated with reduced axonal damage present in the spinal cord when compared to their WTLM controls. However, despite a reduction in axonal damage observed in the CNS of ngr1-/- mice at the chronic stage of disease, no clinical differences could be attributed to a specific genotype when rMOG was used as the encephalitogen. Following MOG_35–55-induction of EAE, we could not derive any major changes to the immune cell populations analyzed between ngr1-/- and WTLM mice. Collectively, these data demonstrate that NgR1 has little if any effects on the repertoire of immune cells, their activation and trafficking to the CNS.     

Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1

The anti-apoptotic proteins Bcl-2 and Bcl-X(L) bind and inhibit Beclin-1, an essential mediator of autophagy. Here, we demonstrate that this interaction involves a BH3 domain within Beclin-1 (residues 114-123). The physical interaction between Beclin-1 and Bcl-X(L) is lost when the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-X(L) is mutated. Mutation of the BH3 domain of Beclin-1 or of the BH3 receptor domain of Bcl-X(L) abolishes the Bcl-X(L)-mediated inhibition of autophagy triggered by Beclin-1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin-1 and Bcl-2/Bcl-X(L), antagonizes autophagy inhibition by Bcl-2/Bcl-X(L) and hence stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain-of-function mutation of the sole BH3-only protein from Caenorhabditis elegans, EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy. These results reveal a novel autophagy-stimulatory function of BH3-only proteins beyond their established role as apoptosis inducers. BH3-only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin-1 and Bcl-2 or Bcl-X(L).     

Self-association of the APC tumor suppressor is required for the assembly,stability, and activity of the Wnt signaling destruction complex

The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling through its activity in the destruction complex with Axin, GSK3β, and CK1 that targets β-catenin/Armadillo (β-cat/Arm) for proteosomal degradation. The destruction complex forms macromolecular particles we termed the destructosome. Whereas APC functions in the complex through its ability to bind both β-cat and Axin, we hypothesize that APC proteins play an additional role in destructosome assembly through self-association. Here we show that a novel N-terminal coil, the APC self-association domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates β-cat degradation in Drosophila and human cells. Consistent with this, removal of the ASAD from the Drosophila embryo results in β-cat/Arm accumulation and aberrant Wnt pathway activation. These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine.     

Investigation of a green process for the polymerization of catechin

Flavonoids are polyphenolic secondary plant metabolites which possess antioxidant and anti-inflammatory properties. Besides, they have been shown to exhibit increased antioxidant properties in their polymerized form. Catechins are one of the attractive class of flavonoids which belong to the group of flavan-3-ols. Polymerization of catechins have been investigated in numerous studies indicating the requirement of certain amount of organic solvent to provide the solubility of the monomer. However, many research projects have been conducted recently to replace toxic organic contaminants of the processes with environmentally friendly solvents. In this aspect, deep eutectic solvents (DESs) that are regarded as “green solvents” have been studied extensively in various enzyme catalyzed reactions. In the present study, we focused on establishing a green pathway for laccase catalyzed polycatechin synthesis by replacing organic solvent content with DESs as green solvents. For this aim, various parameters were investigated, such as DES types and concentrations laccase amount and reaction time. Consequently, the highest molecular weight polycatechin was obtained using 5% (v/v) B–M, 125?U laccase in 1?hr of reaction time, at 30°C, as 4,354?±?678?g?mol^?1. Corresponding X/XO inhibitory activity and superoxide radical scavenging activities were achieved as, 59 and 50%, respectively.     

A new polyaniline–catalase–glutaraldehyde-modified biosensor for hydrogen peroxide detection

A new biosensor based on catalase enzyme immobilized on electrochemically constructed polyaniline (PANI) film modified with glutaraldehyde has been developed for the determination of hydrogen peroxide (H₂O₂) in milk samples. Assembly processes of polyaniline and immobilization of the enzyme were monitored with the help of electrochemical impedance spectroscopy. Amperometric measurements have been performed at cathodic peak (?0.3?V vs. Ag/AgCI) which was attributed to reduction of PANI. Hydrogen peroxide was determined by using amperometric method at ?0.3?V. The biosensor responses were correlated linearly with the hydrogen peroxide concentrations between 5.0?×?10^?6 and 1.0?×?10^?4?M by amperometric method. Detection limit of the biosensor is 2.18?×?10^?6?M for H₂O₂. In the optimization studies of the biosensor, some parameters such as optimum pH, temperature, concentration of aniline, amount of enzyme, and number of scans during electropolymerization were investigated.