Developmental change in follicular cell-enhanced amino acid uptake into mouse oocytes that depends on intact gap junctions and transport system Gly

Uptake of L-alanine, L-lysine, and choline into both preantral and antral mouse oocytes was enhanced by follicular cells. Follicular cells also enhanced glycine uptake into oocytes at the preantral stage of development, but no effect of these cells was observed at the antral stage. Glycine uptake was predominantly Na+ dependent and inhibited almost completely by 10 mM sarcosine, moderately by proline and its analog pipecolate, and poorly or not at all by other amino acids. By these criteria, glycine transport was mainly via system Gly in follicular cells and the oolemma at both the preantral and antral stages. Moreover, an increase in glycine transport via the oolemma between the preantral and antral stages was more than threefold larger than was the increase in transport of alanine or lysine. This relatively large increase in glycine-specific transport in the oolemma appears to obscure the ability of follicular cells to enhance glycine uptake into antral oocytes. In contrast to other amino acids, leucine uptake into oocytes was not enhanced by follicular cells unless 14 other amino acids were also present at their concentrations in mouse serum. An inhibitor of gap junctional communication, 18-alpha-glycyrrhetinic acid, abolished follicular cell-enhanced uptake of glycine and choline into preantral oocytes. Therefore, the extent to which follicular cells enhance uptake of a particular amino acid into oocytes depends on at least three physiologically important variables. Namely, enhancement may depend on the stage of follicular development, the presence of other amino acids in the environment, and gap junctional communication.     

Responses in Primary Astrocytes and C6-Glioma Cells to Ammonium Chloride and Dibutyryl Cyclic-AMP

Elevated brain ammonia levels are a major factor in the genesis of hepatic encephalopathy (HE). The mechanism of ammonium chloride (NH₄Cl) neurotoxicity involves interruption of oxidative metabolism. This leads to decreased levels of ATP concentration and subsequent glial fibrillary acidic protein (GFAP) degradation of astrocytes and fibrous C6-glioma cells. Our study investigates NH₄Cl toxicity by evaluating changes in ATP concentration and mitochondrial function as well as by evaluating alterations in GFAP expression. NH₄Cl induced decreases in ATP were detected after 15 minutes in C6-glioma cells and 24 hours in both cell types. Mitochondrial function, assessed by MTT (2–4,5-dimethylthiazol A-yl)-2, 5-diphenyltetrazolium bromide) assay, was impaired in both cell types at 24 hours following NH₄Cl treatment. GFAP was also significantly decreased in both cell types. Morphologic and metabolic toxicities were greater in C6-glioma cells. The data clearly indicate that NH₄Cl interrupts oxidative metabolism. The greater toxicity seen in C6-glioma cells may be due to their greater dependence on oxidative metabolism. Lastly, the decrease in GFAP is probably a consequence of diminished ATP.     

Toxicity of Artemisia annua (Asteraceae) essential oil on the tea mealy bug,Pseudococcus viburni Sigornet (Hemiptera: Pseudococcidae)

A combination of bioassay and biochemical approaches were used to determine toxicity of Artemisia annua essential oil (AaEO) Pseudococcus viburni. AaEO via leaf dipping bioassay showed LC₅₀ values of 0.693 and 0.419% after two time exposures. Different concentrations of AaEO caused deterrence index between 28.58 to 86.26% by the calculated ED₅₀ of 0.4%. Although, α-esterase activity using α-naphtyl acetate increased in the treated nymphs by AaEO after 24 hours but it showed the lower activity in the treated nymphs using β-naphtyl acetate. Glutathione S-transferase assayed by CDNB showed the higher activity in the treated nymphs than control after 24 hours while the adverse results gained not only after 48 hours but also after 24 hours by using DCNB. No significant differences were found in the activity of alanine aminotransferase versus control, but aspartate aminotransferase and γ-glutamyl transferase showed the statistically higher activities in the treated nymphs in comparison with control. Activities of aldolase and lactate dehydrogenase were significantly lower than those of control. Only acid phosphatase showed the significantly altered activity in the treated nymphs in comparison with control after 24 hours. Results of our study indicated significant toxicity, deterrence and physiological effects of AaEO on P. viburni.     

Modification of catalase and MAPK in Vicia faba cultivated in soil with high natural radioactivity and treated with a static magnetic field

The effects of a static magnetic field (SMF) and high natural radioactivity (HR) on catalase and MAPK genes in Vicia faba were investigated. Soil samples with high natural radioactivity were collected from Ramsar in north Iran where the annual radiation absorbed dose from background radiation is higher than 20 mSv/year. The specific activity of the radionuclides of ²³²Th, ²³⁶Ra, and ⁴⁰K was measured using gamma spectrometry. The seeds were planted either in the soil with high natural radioactivity or in the control soils and were then exposed to a SMF of 30 mT for 8 days; 8 h/day. Levels of expression of catalase and MAPK genes, catalase activity and H₂O₂ content were evaluated. The results demonstrated significant differences in the expression of catalase and MAPK genes in SMF- and HR-treated plants compared to the controls. An increase in catalase activity was accompanied by increased expression of its gene and accumulation of H₂O₂. Relative expression of the MAPK gene in treated plants, however, was lower than those of the controls. The results suggest that the response of V. faba plants to SMF and HR may be mediated by modification of catalase and MAPK.     

The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5' secondary structure

Eukaryotic initiation factor (elF) 4A functions as a subunit of the initiation factor complex elF4F, which mediates the binding of mRNA to the ribosome. elF4A possesses ATPase and RNA helicase activities and is the prototype for a large family of putative RNA helicases (the DEAD box family). It is thought that the function of elF4A during translation initiation is to unwind the mRNA secondary structure in the 5' UTR to facilitate ribosome binding. However, the evidence to support this hypothesis is rather indirect, and it was reported that elF4A is also required for the translation of mRNAs possessing minimal 5' UTR secondary structure. Were this hypothesis correct, the requirement for elF4A should correlate with the degree of mRNA secondary structure. To test this hypothesis, the effect of a dominant-negative mutant of mammalian elF4A on translation of mRNAs with various degrees of secondary structure was studied in vitro. Here, we show that mRNAs containing stable secondary structure in the 5' untranslated region are more susceptible to inhibition by the elF4A mutant. The mutant protein also strongly inhibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents. UV crosslinking of elF4F subunits and elF4B to the mRNA cap structure is dramatically reduced by the elF4A mutant and RNA secondary structure. Finally, the elF4A mutant forms a more stable complex with elF4G, as compared to the wild-type elF4A, thus explaining the mechanism by which substoichiometric amounts of mutant elF4A inhibit translation.     

Cyclical differentiation of Trypanosoma brucei involves changes in the cellular complement of calmodulin-binding proteins

The present study was undertaken to evaluate changes in the complement of calmodulin-binding proteins which accompany cyclical differentiation in Trypanosoma brucei. An [125I]trypanosome calmodulin overlay procedure was used to detect calmodulin-binding proteins with Mr of 126,000 and 106,000 that were present in homogenates of slender bloodstream froms but were absent in procyclic culture forms. Competition assays with unlabeled bovine brain or trypanosome calmodulins indicated that the developmentally regulated proteins associated with calmodulins from either source. Moreover, [125I]bovine brain calmodulin associated with the same proteins as trypanosome calmodulin. Homogenates of T. evansi exhibited the same pattern of calmodulin-binding activity as T. brucei slender bloodstream forms; however, T. cruzi and Leishmania tarentolae contained distinct patterns of calmodulin-binding activity. Mouse serum contained no detectable binding proteins while mouse brain contained predominantly proteins of Mr 210,000, 60,000, and 49,000 which were associated with the trypanosome calmodulin probe. The developmentally regulated calmodulin-binding proteins from T. brucei were in the 10,000g pellet. We conclude that the cellular complement of calmodulin-binding proteins varies during the trypanosome life cycle.     

Mutations of Arabidopsis TBL32 and TBL33 Affect Xylan Acetylation and Secondary Wall Deposition

Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls.     

Titanium oxide nanoparticles fabrication,hemoglobin interaction,white blood cells cytotoxicity,and antibacterial studies

This study is focused on the fabrication and characterization of titanium oxide (TiO₂) NPs. Afterwards; the interaction of TiO₂ NPs with human hemoglobin (Hb) was investigated by FTIR spectroscopy, fluorescence spectroscopy, and molecular docking studies. Also, the cytotoxic effect of fabricated TiO₂ NPs against human white blood cells (WBCs) was considered by MTT assay. The antibacterial effect of synthesized NPs was examined on Pseudomonas aeruginosa (ATCC 27853); Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). TEM and DLS investigations showed that the synthesized TiO₂ NPs have a narrow nano-sized distribution. XRD pattern of the fabricated NPs exhibited that the TiO₂ NPs contain anatase phase. Similarity in amide I and II signal intensities showed that secondary structure of the adsorbed Hb is preserved. The intrinsic fluorescence study revealed that the fluorescence quenching of Hb was done by complex formation between Hb and TiO₂ NPs trough the hydrogen bond and van der Waals interactions. Synchronous fluorescence spectroscopy determined that interaction of TiO₂ NPs with Hb did not unfold the Hb structure in the vicinity of the Tyr and Trp residues. Molecular docking study depicted that Glu-95, Thr-134 and Tyr-140 are involved in the formation of hydrophilic bonds. MTT data and antibacterial assays indicated that TiO₂ NPs endow distinguished antibacterial activities against Gram-negative and Gram positive strains at safe concentrations. This study may reveal that fabricated TiO₂ NP can be used as a safe and potent antibacterial agent.

Communicated by Ramaswamy H. Sarma     

A prion protein epitope selective for the pathologically misfolded conformation

Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of beta-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry. Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein. Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.     

A new ATP7B gene mutation with severe condition in two unrelated Iranian families with Wilson disease

Wilson disease is associated with a defect in copper metabolism and caused by different mutations in ATP7B gene. The aim of this study was to determine mutation frequency of ATP7B exons 8 and 14 in Wilson disease patients from the south of Iran. The exons 8 and 14 of ATP7B gene were analyzed in 65 unrelated Wilson disease patients by Denaturing High Performance Liquid Chromatography, and samples with abnormal peak profile were selected for direct DNA sequencing. Seven out of 65 (10.8%) patients had mutations at exon 14, including c.3061-1G>A in four and c.3207C>A in three patients. In addition, four different mutations were identified at exon 8 of six patients (9.2%). Three of these mutations have been previously reported, including c.2304delC in two patients, c.2293G>A and 2304dupC each in one patient. Furthermore, a novel mutation, c.2335T>G (p.Trp779Gly), was identified in two unrelated patients. The patients with this novel mutation demonstrated severe neuropsychiatric condition. All together, 13 out of 65 (20%) patients had mutations within exons 8 and 14. We also identified a lower frequency of the most common mutations of exons 8 and 14 in the southern Iranian population.