Formation of N-(2-hydroxyethyl)valine in human hemoglobin-effect of lifestyle factors

The formation of N-(2-hydroxyethyl)valine (HEV) in hemoglobin has been considered as a biomarker to assess exogenous and endogenous exposures to ethylene oxide (EO) and/or ethylene (ET). Factors associated with daily exposures to such compounds might significantly affect the formation of HEV. Tobacco smoke containing EO elicited a significant increase in the levels of HEV amongst smokers, although other factors related to lifestyles may warrant further studies. The objective of this study was to specifically analyze HEV using a modified Edman degradation technique in order to study the association between lifestyle related factors (smoking, second-hand smoke exposure, tea and alcohol consumption) and HEV formation in vivo. Total of 148 Taiwanese volunteers with no history of occupational exposure to either EO or ET were recruited in this study. The HEV levels for smokers (204 +/- 151 pmol HEV/g globin, n = 70 ) were greater than those for non-smokers (57 +/- 46 pmol HEV/g globin, n = 78), HEV level increasing with the number of cigarettes smoked by subjects per day with a rate of 8.8 pmol HEV/g globin per cigarettes per day. Further analysis revealed that the rate of HEV formation in our study subjects was significantly associated with the number of daily cigarettes smoked (P < 0.001), but was not associated with tea or alcohol consumption, second-hand smoke exposure, subject age, or subject gender. These results suggest that the significantly higher levels of HEV for smokers than for non-smokers were mainly due to subject exposure to EO contained in cigarette smoke.     

Expression and purification of human placenta lactogen in Escherichia coli

There are many growth factors secreted by placenta including growth hormone, placenta lactogen (PL), prolactin, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and chorionic gonadotropin. For a systematic study of how these growth factors work together to result in the various biological functions and future clinical applications, it is needed to produce enough quantities of each protein. In this paper, we report the cloning of human PL (hPL) and expression by Escherichia coli (E. coli). Four kinds of expression vectors containing the hPL gene were transformed into several kinds of suitable host strains and grown at 37 and/or 30 degrees C. Determination of the yield of recombinant hPL by SDS-PAGE reveals that among the various conditions, pQE30-PL in E. coli strain M15[pREP4] expressed the largest amount of recombinant hPL at 37 degrees C. However, the expressed recombinant hPL was accumulated in inclusion body forms. The inclusion bodies were solubilized in 8M urea and purified by a His6 tagged affinity column under denaturing condition and the final yield of hPL was determined to be 48 mg/L. Intra-chain disulfide bonds could be formed either by oxidation in the refolding buffer or by air oxidation in the presence of urea. The biological activity was examined by the fact that hPL could stimulate erythroid maturation by the formation of hemoglobin in K-562 cells in the presence of erythropoietin. Initial optimization studies resulted in the production of 282.4 mg/L of hPL.     

Determination of indomethacin in plasma by micellar electrokinetic chromatography with UV detection for premature infants with patent ducts arteriosus

A simple and selective micellar electrokinetic chromatography (MEKC) is described for determination of indomethacin in plasma. Plasma proteins are precipitated by acetonitrile. An aliquot of supernatant was evaporated and reconstituted with Tris buffer for MEKC analysis. The separation of indomethacin was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (30 mM; pH 8.0) with 100 mM sodium octanesulfonate (SOS) as an anionic surfactant. Under this condition, a good separation with high efficiency and short analysis time is achieved. Several parameters affecting the separation of indomethacin were studied, including pH and concentrations of the Tris buffer and SOS. The linear range of the method for the determination of indomethacin was over 0.3-10.0 microg/mL; the detection limit (signal-to-noise ratio=3; injection 0.5 psi 5s) was 0.1 microg/mL. The proposed method for determination of indomethacin in premature infants with patent ducts arteriosus has been demonstrated.     

Mice lacking alpha/beta and gamma interferon receptors are susceptible to junin virus infection

Junin virus (JUNV) causes a highly lethal human disease, Argentine hemorrhagic fever. Previous work has demonstrated the requirement for human transferrin receptor 1 for virus entry, and the absence of the receptor was proposed to be a major cause for the resistance of laboratory mice to JUNV infection. In this study, we present for the first time in vivo evidence that the disruption of interferon signaling is sufficient to generate a disease-susceptible mouse model for JUNV infection. After peripheral inoculation with virulent JUNV, adult mice lacking alpha/beta and gamma interferon receptors developed disseminated infection and severe disease.     

Asparagine of z8 Insert Is Critical for the Affinity,Conformation, and Acetylcholine Receptor-clustering Activity of Neural Agrin

Agrin isoforms with different bioactivities are synthesized by the nerve and the muscle. Neural agrin containing an 8-amino acid insert (z8) introduced by alternative splicing is the active form that induces synaptic differentiation at the neuromuscular junction. In addition to alternative splicing, extracellular calcium is also required for the activity of neural agrin. To understand better how the activity of agrin is regulated by alternative splicing, we have applied alanine substitution mutagenesis to the z8 insert and the calcium binding site in the minimally functional AgG3z8 fragment. Single alanine substitutions in the 4th through the 7th amino acid of the z8 splice insert significantly reduced the function of agrin, in terms of acetylcholine receptor clustering activity and the affinity for binding to the muscle surface. Mutation of the asparagine at the 4th position drastically reduces bioactivity such that it is equivalent to that of muscle form AgG3z0. These reduced activity mutants also show reduced magnitudes of the calcium-induced CD spectrum change from that observed in AgG3z8 fragments, indicating that cross-talk between calcium and the z8 insert is critical for the normal activity of agrin. However, removal of Ca²⁺ binding via mutation of both aspartic acids in the calcium binding site did not totally eliminate the activity of AgG3z8. These results suggest a model wherein the z8 insert is a Ca²⁺-responsive allosteric element that is essential in forming an active conformation in neuronal agrin.