Partial purification of α-amylase from culture supernatant of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in aqueous two-phase systems

A study was made of the partition and purification of -amylase from a culture supernatant of Bacillus subtilis in the polyethylene glycol (PEG)—citrate aqueous two-phase system (ATPS). Factors that influenced the partition of the protein in this system, including the molecular weight of the PEG, the tie line length of ATPS, the pH value and the sodium chloride concentration, were investigated. Purification of -amylase was attained with a purification factor (PF) of 1.8 and 90% yield at pH 6.0 in a PEG1000-citrate ATPS with short tie line length. By utilizing the salt-out effect of neutral salt, the purification of -amylase was further improved to 2.0 of PF and 80% yield in a PEG3350-citrate ATPS with 4% sodium chloride.     

Regulators of G-protein signaling (RGS) 4, insertion into model membranes and inhibition of activity by phosphatidic acid

Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a G alpha(i1)-[gamma-(32)P]GTP single turnover assay with an IC(50) approximately 4 microm and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into m1 acetylcholine receptor-G alpha(q) vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC(50) of RGS4 was increased from 1.5 to 7 nm. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys(20) to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.     

Identification and characterization of a nuclear interacting partner of anaplastic lymphoma kinase (NIPA)

Anaplastic large-cell lymphoma is a subtype of non-Hodgkin lymphomas characterized by the expression of CD30. More than half of these lymphomas carry a chromosomal translocation t(2;5) leading to expression of the oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). NPM-ALK is capable of transforming fibroblasts and lymphocytes in vitro and of causing lymphomas in mice. Previously, we and others demonstrated phospholipase C-gamma and phosphatidylinositol 3-kinase as crucial downstream signaling mediators of NPM-ALK-induced oncogenicity. In this study, we used an ALK fusion protein as bait in a yeast two-hybrid screen identifying NIPA (nuclear interacting partner of ALK) as a novel downstream target of NPM-ALK. NIPA encodes a 60-kDa protein that is expressed in a broad range of human tissues and contains a classical nuclear translocation signal in its C terminus, which directs its nuclear localization. NIPA interacts with NPM-ALK and other ALK fusions in a tyrosine kinase-dependent manner and is phosphorylated in NPM-ALK-expressing cells on tyrosine and serine residues with serine 354 as a major phosphorylation site. Overexpression of NIPA in Ba/F3 cells was able to protect from apoptosis induced by IL-3 withdrawal. Mutations of the nuclear translocation signal or the Ser-354 phosphorylation site impaired the antiapoptotic function of NIPA. In NPM-ALK-transformed Ba/F3 cells, apoptosis triggered by wortmannin treatment was enhanced by overexpression of putative dominant-negative NIPA mutants. These results implicate an antiapoptotic role for NIPA in NPM-ALK-mediated signaling events.     

Optimization of conditions for bacteriocin extraction in PEG/salt aqueous two-phase systems using statistical experimental designs

The bacteriocin nisin was extracted in PEG/salt aqueous two-phase systems (ATPS) using the property that the systems can extract hydrophobic proteins. The concentrations of the phase-forming components, PEG 4000 and Na(2)SO(4), were optimized for nisin recovery by means of statistical experimental designs, and it was found that they strongly influenced nisin recovery. The optimal composition of ATPS was found to be 15.99% (w/w) PEG 4000 and 15.85% (w/w) Na(2)SO(4) (pH 2), and the optimal ATPS allowed an 11.60% increase of nisin recovery compared to the standard method of nisin assay.     

Tyrosylprotein sulfotransferase inhibitors generated by combinatorial target-guided ligand assembly

Tyrosylprotein sulfotransferases (TPSTs) catalyze the sulfation of tyrosine residues within secreted and membrane-bound proteins. The modification modulates protein-protein interactions in the extracellular environment. Here we use combinatorial target-guided ligand assembly to discover the first known inhibitors of human TPST-2.     

Dopamine transport function is elevated in cocaine users

Dopaminergic transmission has been suggested to be a primary mechanism mediating reinforcement, withdrawal and craving associated with psychostimulant addiction. Pyscho-stimulants attenuate dopamine transporter (DAT) clearance efficiency, resulting in a net increase in synaptic dopamine levels. Re-uptake rate is determined by the number of functional DAT molecules at the membrane surface. Previous in vivo imaging studies in humans and in vitro studies in post-mortem human brain have demonstrated that chronic cocaine abuse results in a neuroadaptive increase in DAT-binding site density in the limbic striatum. Whether this increase in DAT availability represents an increase in the functional activity of the transporter is unknown. Here, we present evidence that DAT function is elevated by chronic cocaine abuse. The effect of increasing post-mortem interval on the functional viability of synaptosomes was modeled in the baboon brain. Baboon brains sampled under conditions similar to human brain autopsies yielded synaptosomal preparations that were viable up to 24 h post-mortem. Dopamine (DA) uptake was elevated twofold in the ventral striatum from cocaine users as compared to age-matched drug-free control subjects. The levels of [3H]DA uptake were not elevated in victims of excited cocaine delirium, who experienced paranoia and marked agitation prior to death. In keeping with the increase in DAT function, [3H]WIN 35,428 binding was increased in the cocaine users, but not in the victims of excited delirium. These results demonstrate that DA uptake function assayed in cryopreserved human brain synaptosomes is a suitable approach for testing hypotheses of the mechanisms underlying human brain disorders and for studying the actions of addictive drugs in man.     

Functional rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon

The centromere of eukaryotic chromosomes is essential for the faithful segregation and inheritance of genetic information. In the majority of eukaryotic species, centromeres are associated with highly repetitive DNA, and as a consequence, the boundary for a functional centromere is difficult to define. In this study, we demonstrate that the centers of rice centromeres are occupied by a 155-bp satellite repeat, CentO, and a centromere-specific retrotransposon, CRR. The CentO satellite is located within the chromosomal regions to which the spindle fibers attach. CentO is quantitatively variable among the 12 rice centromeres, ranging from 65 kb to 2 Mb, and is interrupted irregularly by CRR elements. The break points of 14 rice centromere misdivision events were mapped to the middle of the CentO arrays, suggesting that the CentO satellite is located within the functional domain of rice centromeres. Our results demonstrate that the CentO satellite may be a key DNA element for rice centromere function.     

The prevalence of connexin 26 ( GJB2) mutations in the Chinese population

Mutations in GJB2, encoding gap junction beta 2 protein (connexin 26), are responsible for the commonest form of non-syndromic recessive deafness in many populations. It has been reported recently that the most common 35delG mutation in GJB2 is exceptionally low in Japanese and Korean populations, but another deletion, 235delC, is relatively frequent. Since the Chinese constitute approximately one fifth of the global population, the frequency of GJB2 mutations in the population has important implications for understanding worldwide causes of genetic deafness. To determine whether GJB2 mutations are an important cause of deafness in Chinese, we conducted mutation screening for GJB2 in 118 deaf Chinese probands, including 60 from simplex and 58 from multiplex families with non-syndromic deafness, and 150 normal hearing Chinese controls. Four mutations, including 235delC, 299-300delAT, V37I, and 35delG, were found in the patients. Thirty-nine percent of the probands had a GJB2mutation. Of the 118 probands, 19 carried two definitely pathogenic mutations: three among the 58 multiplex cases (5.2%) and 16 among the 60 simplex cases (26.7%). Twenty-seven probands (22.9%) were found to carry only single GJB2 mutations. None of them had mutations in exon 1 of GJB2 and or the 342-kb deletion of GJB6. The 235delC mutation was the most prevalent mutation (20.3% of alleles), accounting for 81% of the pathologic alleles in multiplex cases and 67% in simplex cases. Analysis of the affected haplotypes in the patients with the homozygous 235delC mutation yielded evidence for a single origin of the mutation. The carrier frequency of the 235delC mutation in control subjects with normal hearing was 1.3%. The 35delG mutation was only noted as a heterozygous change in two simplex cases (1.2% of alleles). These results indicated that mutations in GJB2 are a major cause of inherited and sporadic congenital deafness in the Chinese population. The 235delC mutation, rather than 35delG, is the most common mutation found in the Chinese deaf population. Our data support the view that specific combinations of GJB2 mutation exist in different populations.     

Biooxidation of ferrous iron by immobilized Acidithiobacillus ferrooxidans in poly(vinyl alcohol) cryogel carriers

PVA-cryogels entrapping about 10⁹ cells of Acidithiobacillus ferrooxidans per ml of gel were prepared by freezing-thawing procedure, and the biooxidation of Fe²⁺ by immobilized cells was investigated in a 0.365 l packed-bed bioreactor. Fe²⁺ oxidation fits a plug-flow reaction model well. A maximum oxidation rate of 3.1 g Fe²⁺ l^–1 h^–1 was achieved at the dilution rate of 0.4 h^–1 or higher, while no obvious precipitate was determined at this time. In addition, cell-immobilized PVA-cryogels packed in bioreactor maintained their oxidative ability for more than two months under non-sterile conditions. Nomenclature: C _A0 – Concentration of Fe²⁺ in feed stream (g l^–1) C _A – Concentration of Fe^{2 +} in outlet stream (g l^{– 1}) D – Dilution rate of the packed-bed bioreactor (h^–1) F – Volumetric flow rate of iron solution (l h^–1) F _A0 – Mass flow rate of Fe²⁺ in the feed stream (g h^–1) K – Kinetic constant (l l^–1 h^–1) r _A – Oxidation rate of Fe²⁺ (g l^–1 h^–1) V – Volume of packed-bed bioreactor (l) X _A – Conversion ratio of Fe²⁺ (%)