Amino-terminal charge affects the periplasmic accumulation of recombinant heregulin/EGF hybrids exported using the Escherichia coli alkaline phosphatase signal sequence

An Escherichia coli expression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA-hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated by in vitro replacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein in E. coli periplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed in E. coli using the PhoA signal sequence for protein export.     

Polyethylene nanocomposites by gas‐phase polymerization of ethylene in the presence of a nanosilica‐supported zirconocene catalyst system

BACKGROUND: Much of the current research related to the development of in situ nanocomposites of olefins by polymerizing them with metallocenes in the presence of surface‐treated fillers is carried out in the slurry phase. In slurry‐phase methods a large amount of solvent is required and there is always a need of purification of the final product due to the possibility of traces of solvents present in the product. To overcome these drawbacks, to perform solvent‐free metallocene‐catalysed polymerizations with in situ incorporation of inorganic nanoparticles, we have used a gas‐phase polymerization technique as this does not require solvents and also utilizes monomer feed stocks efficiently. RESULTS: The catalyst used for the synthesis of in situ polyethylene nanocomposites by gas‐phase polymerization was nanosilica‐supported zirconocene. The fillers used were Cloisite‐20A, kaolin and nanosilica. Three different in situ polyethylene nanocomposites, i.e. Cloisite‐20A‐filled polyethylene (CFPE), kaolin‐filled polyethylene (KFPE) and nanosilica‐filled polyethylene (SFPE), were prepared by gas‐phase polymerization. The nanocomposites were obtained in the form of fine powder. The polyethylene content in the developed nanocomposites is in the orthorhombic crystalline phase. Using our approach, it is observed that the nanofillers are completely encapsulated by a thin layer of polyethylene. Significantly higher molecular weight polyethylene was formed in the case of KFPE in comparison to CFPE and SFPE. The thermal decomposition temperature, melting temperature and enthalpy are also observed to be higher for KFPE. CONCLUSIONS: The gas‐phase polymerization technique has been successfully carried out for the synthesis of in situ polyethylene nanocomposites. Copyright © 2007 Society of Chemical Industry     

Arsenic in drinking water and incidence of urinary cancers

To investigate the effects of a new nonNMDA antagonist on the trisynaptic pathways in the hippocampus, the author examined kainate(KA)-induced generalized seizures in rats. A novel nonNMDA antagonist, YM90K, showed the blockade of the Schaffer collaterals in 2-deoxyglucose study (2-DG) and that the CA1-2 pyramidal cells of the hippocampus were preserved seven days after the KA injections. On the other hand, the control and MK-801 (NMDA-antagonist) treated rats did not depress the Schaffer collaterals and showed persistent hypermetabolism of glucose in the CA1 pyramidal cell layer, where neurons were not preserved seven days later. 2-DG was useful to reveal the effects of nonNMDA antagonist on the KA-induced generalized seizures. This suggests that YM90K is a potent nonNMDA antagonist and that it has a neuroprotective effect in rats.     

Synthesis and Characterization of in situ Polyethylene and Polypropylene Nanocomposites: Gas Phase Polymerization by Nanosilica Supported <Emphasis Type="Italic">bis</Emphasis> (Cyclopentadienyl) Zirconium (IV) Dichloride Catalyst System

In the present work, in situ polyethylene and polypropylene nanocomposites have been successfully synthesized by gas phase polymerization of ethylene and propylene in presence of surface treated nanofillers with nanosilica-supported-bis (cyclopentadienyl) zirconium (IV) dichloride as a coordination catalyst. The nanofillers used are Cloisite-20A and nanosilica, respectively. These were pre-treated with MAO (Methylaluminoxane) to inhibit their deactivating action on the catalyst because of the presence of either acidic or basic groups on their surfaces; in this way MAO acts as a co-catalyst for the polymerization reactions being discussed. Two in situ Polyethylene (PE) i.e., CFPE and SFPE (Cloisite-20A filled polyethylene and nanosilica filled polyethylene) and two in situ polypropylene (PP) i.e., CFPP and SFPP (Cloisite-20A filled polypropylene and nanosilica filled polypropylene) nanocomposites have been prepared by gas-phase polymerization. The in situ polyethylene and polypropylene nanocomposites were characterized using Fourier transform infrared Spectroscope (FTIR), Thermogravimetric analyzer (TGA), Differential Scanning Calorimeter (DSC) and Scanning electron microscope (SEM). By our approach, it has been observed that the nanofillers are completely encapsulated by a thin layer of polymer chains.     

Purification of recombinant human rhinovirus 14 3C protease expressed in Escherichia coli

A physiologically relevant thrombopoietin (TPO) must be a humoral regulator with lineage specificity for megakaryocytes and their precursors. It should be capable of stimulating platelet production in normal animals, and elevated levels of TPO should be detectable in the plasma following acute, severe thrombocytopenia. Acute thrombocytopenia provides a model system that is likely to predict the effects of TPO, since many of the effects on megakaryocytes and platelets observed after induction of acute thrombocytopenia would be mediated by TPO. Important questions remain to be answered. Do the currently available data for the c-Mpl ligand explain previously published data that describe elevated levels of Meg-CSF in the circulation following production of bone marrow aplasia? Does the c-Mpl ligand account for all of the megakaryocyte stimulatory factors that have been described? Is there another factor that accounts for at least some of the acute alterations in megakaryocytopoiesis that occur immediately following a decrease in platelet levels?     

Assessing the impact of palmitic,myristic and lauric acids on hydrogen production from glucose fermentation by mixed anaerobic granular cultures

The effects of lauric (LUA), myristic (MA), palmitic (PA), and a mixture of myristic:palmitic (MA:PA) acids on hydrogen (H₂) production from glucose degradation using anaerobic mixed cultures were assessed at 37 °C with an initial pH set at 5.0 and 7.131 mM of each acid. The maximum H₂ yield (2.53 ± 0.18 mol mol⁻¹ glucose) was observed in cultures treated with PA. A principal component analysis (PCA) of the by-products and the microbial population data sets detected similarities between the controls and PA treated cultures; however, differences were observed between the controls and PA treated cultures in comparison to the MA and LUA treated cultures. The flux balance analysis (FBA) showed that PA decreased the quantity of H₂ consumed via homoacetogenesis compared to the other LCFAs. The control culture was dominated by Thermoanaerovibrio acidaminovorans (60%), Geobacillus sp. and Eubacterium sp. (28%), while Clostridium sp. was less than 1%. Treatment with PA, MA, MA:PA, or LUA increased the H₂ producers (Clostridium sp. and Bacillus sp.) population by approximately 48, 67, 86, and 86%, respectively.     

Impact of culture source and linoleic acid (C18:2) on biohydrogen production from glucose under mesophilic conditions

Genomic and statistical methods were used to demonstrate the effects of linoleic acid (LA) on hydrogen (H₂) production in mixed anaerobic cultures from two sources (designated as A and B). The microbial composition of the control cultures CA and CB were statistically different. Bacteroidaceae (26%) and Clostridiaceae (10%) dominated CA whereas Clostridiaceae (33%) and Bacteroidaceae (10%) dominated CB. Homoacetogens directed 42% of the electron equivalents to acetate production and decreased the H₂ yield by 50% in CA compared to CB. The maximum H₂ yields (3.11 ± 0.02 and 3.11 ± 0.07 mol H₂ mol⁻¹ glucose in LA-treated cultures ALA and BLA, respectively) were statistically the same. Cultures ALA and BLA followed the acetate-butyrate pathway while CA and CB followed propionate and homoacetogenic pathways. LA-treated and control cultures were statistically different based on the type and quantity of metabolites; the differences were also confirmed by principal component analysis (PCA).     

Predominance of slow acetylators of N-acetyltransferase in a Hmong population residing in the United States

Pharmacogenetics can be an important determinant of pharmacologic response. To learn more about interpopulation differences in drug metabolism between ethnically diverse populations of subjects cared for by an International Clinic, a study was conducted to describe the prevalence of fast or slow acetylators of N-acetyltransferase (NAT2) in a population of Hmong residing in Minnesota. Ninety-eight healthy Hmong refugees from Laos volunteered to take caffeine as an oral probe drug to establish acetylator phenotype. Participants were classified as either rapid or slow acetylators based on the urinary molar ratio of select metabolites of caffeine. Assignment of phenotype was based on results from analysis of urine collected subsequent to ingestion of caffeine. The ratio of 5-acetylamino-6-formylamino-3-methyluracil (AFMU) to the combined products of the 7-demethylation pathway of paraxanthine (AFMU, 1-methylxanthine (1X), and 1-methylurate (1U)] formed the basis for this determination. A probit plot of the data collected in our subjects qualified a metabolic ratio of 0.34 as an acceptable cut point for phenotype assignment. Participants with an AFMU/(AFMU + 1X + 1U) ratio of < 0.34 were classified as slow acetylators and all others as rapid acetylators. Analysis of the data suggested a bimodal distribution with an excess (74.5%) of slow acetylators in the population. The predominance of slow acetylators found in the Hmong contrast with the prevalence of slow acetylators seen in other ethnic groups. These findings may have important clinical implications given the large number of Hmong treated each year in our International Clinic and the increasing use of medications metabolized by NAT2 in this population.