Genotoxicity assessment in patients with thalassemia minor

Thalassemia is an inherited blood disorder that affects both genders and results in reduced synthesis of hemoglobin, and thus causing anemia. Previous studies have shown that the severe form of this disease, thalassemia major, is associated with genotoxicity. This includes increases in the level of sister chromatid exchange (SCEs), chromosomal aberrations (CAs) and micronuclei. In this study, we assessed genotoxicity in the lymphocytes of thalassemia minor subjects using sister chromatid exchange (SCE) and chromosomal aberration (CA) assays. In addition, we investigated the level of oxidative DNA damage by measuring 8-hydroxy-2'-deoxyguanosine (8OHdG) biomarker in urine samples. Eighteen thalassemia minor subjects and eighteen matched normal healthy controls were volunteered in the study. In addition, seven thalassemia major patients were recruited as positive controls. The results showed increases in the frequency of SCEs (P<0.05) in thalassemia minor compared to healthy controls. However, no difference in CAs frequency was detected between thalassemia minor and controls (P>0.05). Both SECs and CAs in thalassemia major patients were significantly higher compared to other groups (P<0.05). Regarding urine 8OHdG levels, the result showed a slight increase in thalassemia minor compared to healthy controls but the difference was not significant (P>0.05). In conclusion, our results showed that thalassemia minor is associated with genotoxicity to blood lymphocytes as indicated by SCEs assay.     

Identification and characterization of the vitellogenin receptor in Macrobrachium rosenbergii and its expression during vitellogenesis

In oviparous organisms, oocyte maturation depends on massive production of the egg yolk-precursor protein, vitellogenin (Vg). Vg is taken up by the developing oocytes through receptor-mediated endocytosis (RME), a process essential to successful reproduction. The aims of this study were to identify and characterize the yet-unknown vitellogenin receptor (VgR) from the pleocyamate crustacean Macrobrachium rosenbergii, and to investigate its expression levels during vitellogenesis and its interaction with Vg. The VgR gene was cloned, and its translated protein was specifically located at the oocyte membrane. Moreover, for the first time, a VgR protein was identified and sequenced by mass spectrometry. The putative MrVgR displayed high sequence similarity to VgRs from crustaceans, insects, and vertebrates, and its structure includes typical elements, such as an extracellular, lipoprotein-binding domain (LBD), EGF-like, and O-glycosylation domains, a transmembrane domain, and a short, C-terminal, cytosolic tail. In this article, we identify the first crustacean VgR protein, and present data demonstrating its high affinity for a Vg column followed by elution with suramin and EDTA. Additionally we demonstrate that VgR expression in the oocyte is elevated during vitellogenesis. Our results contribute to the fundamental understanding of oocyte maturation in crustaceans, and particularly elucidate Vg uptake through RME via the VgR.     

Biodegradation Kinetics of Four Substituted Chlorobenzoic Acids by Enterobacter aerogenes

Enterobacter aerogenes is generally found in soil, sewage plants, and human gastrointestinal tract. Thus, this study was conducted to evaluate the ability of Enterobacter aerogenes to degrade four chlorobenzoic acid compounds (2-chlorobenzoic acid (2-CBA), 3-chlorobenzoic acid (3-CBA), 4-chlorobenzoic acid (4-CBA), and 3,4-dichlorobenzoic acid (3,4-dCBA)) in minimal salt medium. Enterobacter aerogenes was partially able to degrade and dechlorinate these CBAs at concentration of 3.5 mM within 72 h of incubation. According to Haldane single-substrate model, the values of maximum predicted growth rate (μ_max), half saturation constant (K _s), and inhibition constant (K _i) fell in the range of 0.2–0.8 h^?1, 8–41 mM, and 5–53 mM, respectively. Based on the estimated values of both α, a growth-associated constant, and β, a non–growth-associated constant, the production of chloride was predominantly growth associated, since negligible values of the β were determined. Haldane model gave a good prediction of the CBA substrate utilization and degradation, and was in a very good agreement with the experimental data. Because of the capability of Enterobacter aerogenes to utilize these aromatic compounds as carbon and energy sources, this microorganism can be a valuable and promising candidate for use in the biotreatment of wastewater and soil samples contaminated with mixtures of chlorobenzoates.     

The Crustacean Androgen: A Hormone in an Isopod and Androgenic Activity in Decapods

The androgenic gland has been described in a variety of crustaceanspecies—isopods, amphipods and decapods. It has been shownto play a role in the regulation of male differentiation andin the inhibition of female differentiation. Upon its applicationfor endocrine manipulation, it inhibits female characteristics.Recently, the androgenic hormone from the isopod Armadillidiumvulgare was purified and characterized on the basis of a morphologicalbioassay. The hormone is a glycosylated protein composed oftwo peptide chains connected each to the other by two disulfidebridges. The pro-hormone consists of the same two chains connectedby a third peptide in a complex that resembles the insulin superfamily hormones. The study of the androgenic gland in decapodslags behind that in the isopods, and a decapod androgenic hormonehas yet to be identified. In this review, five decapod speciesare described as models, in which the androgenic gland exertsmorphological, anatomical, physiological and behavioral effects.These models could serve as the basis of possible bioassaysfor the study of the structure and mode of action of the androgenichormone in decapod crustaceans.     

Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification

Acrylamide concentration, urea content, and the trailing ion used for sodium dodecyl sulfate (SDS)-gels modify electrophoretic protein mobilities in a protein-dependent way. Varying these parameters we coupled two SDS-gels to a two-dimensional (2-D) electrophoresis system. Protein spots in 2-D gels are dispersed around a diagonal. Hydrophobic proteins are well separated from water-soluble proteins which is the essential advantage of the novel technique. Mass spectrometric identification of previously unaccessible hydrophobic proteins is now possible.     

Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation

Inhibiting the activity of the beta-amyloid converting enzyme 1 (BACE1) or reducing levels of BACE1 in vivo decreases the production of amyloid-beta. The reticulon family of proteins has four members, RTN1, RTN2, RTN3 and RTN4 (also known as Nogo), the last of which is well known for its role in inhibiting neuritic outgrowth after injury. Here we show that reticulon family members are binding partners of BACE1. In brain, BACE1 mainly colocalizes with RTN3 in neurons, whereas RTN4 is more enriched in oligodendrocytes. An increase in the expression of any reticulon protein substantially reduces the production of Abeta. Conversely, lowering the expression of RTN3 by RNA interference increases the secretion of Abeta, suggesting that reticulon proteins are negative modulators of BACE1 in cells. Our data support a mechanism by which reticulon proteins block access of BACE1 to amyloid precursor protein and reduce the cleavage of this protein. Thus, changes in the expression of reticulon proteins in the human brain are likely to affect cellular amyloid-beta and the formation of amyloid plaques.     

Determination of glimepiride in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS-MS) method has been developed at our center for the determination of glimepiride in human plasma. After the addition of the internal standard, plasma samples were extracted by liquid-liquid extraction technique using diethyl ether. The compounds were separated on a prepacked C18 column using a mixture of acetonitrile, methanol and ammonium acetate buffer as mobile phase. A Finnigan LCQDUO ion trap mass spectrometer connected to an Alliance Waters HPLC was used to develop and validate the method. The analytical method was validated according to the FDA bioanalytical method validation guidance. The results were within the accepted criteria as stated in the aforementioned guidance. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 5.0-500.0 ng/ml with a coefficient of determination (r2) of 0.9998. Accuracy for glimepiride ranged from 100.58 to 104.48% at low, mid and high levels. The intra-day precision was better than 12.24%. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 5.0 ng/ml with a precision of 7.96%. The proposed method enables the unambiguous identification and quantitation of glimepiride for pharmacokinetic, bioavailability or bioequivalence studies.