Free malonaldehyde determination in tissues by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the quantification of free malonaldehyde (MA) in tissues. HPLC separation was performed using a TSK G1000 PW column (7.5-mm i.d. X 30 cm) with a mobile phase of 0.1 M Na3PO4 buffer, pH 8.0, at a flow rate of 0.6 ml/min. The eluant was monitored at 267 nm. Free MA in the tissue sample was separated and quantified in approximately 50 min. The lowest amount of MA that can be determined by this HPLC technique is approximately 1 ng per injection. This method was successfully applied to rat liver and beef, pork, and chicken muscle and was compared to the thiobarbituric acid (TBA) test. It was found to be more sensitive, accurate, and specific for the determination of free MA than the TBA method.     

Mycoplasma agalactiae MAG_5040 is a Mg2+-Dependent,Sugar-Nonspecific SNase Recognised by the Host Humoral Response during Natural Infection

In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl₂ at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.     

Capillary column gas-liquid chromatographic resolution of oxidized cholesterol derivatives

Fused-silica capillary columns were evaluated for the resolution of oxidized cholesterol derivatives. Thermal instability of diol derivatives, epimeric 7 alpha- and 7 beta-hydroxy, 4 beta-hydroxy, and 25-hydroxycholesterol, was observed during gas chromatography. After derivatization as trimethylsilyl ethers the foregoing diols, alpha-epoxide, cholestane-triol, 7-ketocholesterol, and cholesta-3,5-dien-7-one were completely resolved on a DB-1 column. Each oxidized sterol revealed excellent response linearity as the trimethylsilylated sterol, enabling reliable quantification. The identity of each derivatized sterol was confirmed by mass spectrometry.     

Purification of mitochondrial creatine kinase: Two interconvertible forms of the active enzyme

The purification of creatine kinase from beef heart mitochondria is described. The purified enzyme appears as a single band after electrophoresis on SDS gels. Electrophoresis on cellulose acetate followed by staining for creatine kinase activity shows two forms of the enzyme. The slower migrating (m-1) form upon concentration is converted to the more rapidly migrating form (m-2). The reverse conversion occurs if the m-2 is incubated with β-mercaptoethanol. These results are consistent with a reversible oxidation of protein sulfhydryl group (s).     

CMIP and ATP2C2 Modulate Phonological Short-Term Memory in Language Impairment

Specific language impairment (SLI) is a common developmental disorder characterized by difficulties in language acquisition despite otherwise normal development and in the absence of any obvious explanatory factors. We performed a high-density screen of SLI1, a region of chromosome 16q that shows highly significant and consistent linkage to nonword repetition, a measure of phonological short-term memory that is commonly impaired in SLI. Using two independent language-impaired samples, one family-based (211 families) and another selected from a population cohort on the basis of extreme language measures (490 cases), we detected association to two genes in the SLI1 region: that encoding c-maf-inducing protein (CMIP, minP = 5.5 × 10⁻⁷ at rs6564903) and that encoding calcium-transporting ATPase, type2C, member2 (ATP2C2, minP = 2.0 × 10⁻⁵ at rs11860694). Regression modeling indicated that each of these loci exerts an independent effect upon nonword repetition ability. Despite the consistent findings in language-impaired samples, investigation in a large unselected cohort (n = 3612) did not detect association. We therefore propose that variants in CMIP and ATP2C2 act to modulate phonological short-term memory primarily in the context of language impairment. As such, this investigation supports the hypothesis that some causes of language impairment are distinct from factors that influence normal language variation. This work therefore implicates CMIP and ATP2C2 in the etiology of SLI and provides molecular evidence for the importance of phonological short-term memory in language acquisition.     

Biotechnological Process for Production of β-Dipeptides from Cyanophycin on a Technical Scale and Its Optimization

A triphasic process was developed for the production of β dipeptides from cyanophycin (CGP) on a large scale. Phase I comprises an optimized acid extraction method for technical isolation of CGP from biomass. It yielded highly purified CGP consisting of aspartate, arginine, and a little lysine. Phase II comprises the fermentative production of an extracellular CGPase (CphE_al) from Pseudomonas alcaligenes strain DIP1 on a 500-liter scale in mineral salts medium, with citrate as the sole carbon source and CGP as an inductor. During optimization, it was shown that 2 g liter⁻¹ citrate, pH 6.5, and 37°C are ideal parameters for CphE_al production. Maximum enzyme yields were obtained after induction in the presence of 50 mg liter⁻¹ CGP or CGP dipeptides for 5 or 3 h, respectively. Aspartate at a concentration of 4 g liter⁻¹ induced CphE_al production with only about 30% efficiency in comparison to that with CGP. CphE_al was purified utilizing its affinity for the substrate and its specific binding to CGP. CphE_al turned out to be a serine protease with maximum activity at 50°C and at pH 7 to 8.5. Phase III comprises degradation of CGP to β-aspartate-arginine and β-aspartate-lysine dipeptides with a purity of over 99% (by thin-layer chromatography and high-performance liquid chromatography), employing a crude CphE_al preparation. Optimum degradation parameters were 100 g liter⁻¹ CGP, 10 g liter⁻¹ crude CphE_al powder, and 4 h of incubation at 50°C. The overall efficiency of phase III was 91%, while 78% (wt/wt) of the used CphE_al powder with sustained activity toward CGP was recovered. The optimized process was performed with industrial materials and equipment and is applicable to any desired scale.     

The flagellated parasite Trichomonas vaginalis: new insights into cytopathogenicity mechanisms

Our knowledge concerning cytopathogenicity of Trichomonas vaginalis has been enriched in the past by numerous findings. In this paper, we review the latest advances in the field and discuss the different mechanisms and molecules responsible for the parasite's virulence.     

Novel HPLC analysis of tocopherols, tocotrienols, and cholesterol in tissue

Tocopherols and tocotrienols are being increasingly recognized to have an important role in the prevention of atherosclerosis. It has been reported that they protect low-density lipoprotein (LDL) and tissues from oxidative stress and that tocotrienols can reduce plasma cholesterol levels. Two isocratic high-performance liquid chromatography (HPLC) methods for simultaneous analysis of tocopherols, tocotrienols, and cholesterol in muscle tissue were developed. Method A involves basic saponification of the sample, but causes losses of the gamma- and delta-homologs of vitamin E. Method B does not involve saponification, thereby protecting the more sensitive homologs. Both permit rapid analysis of multiple samples and neither requires specialized equipment. These methods may provide techniques useful in simultaneous assessment of oxidative stress status (OSS) and cholesterol levels.