Tat-vaccinated macaques do not control simian immunodeficiency virus SIVmac239 replication

The regulatory proteins of human immunodeficiency virus may represent important vaccine targets. Here we assessed the role of Tat-specific cytotoxic T lymphocytes (CTL) in controlling pathogenic simian immunodeficiency virus SIVmac239 replication after using a DNA-prime, vaccinia virus Ankara-boost vaccine regimen. Despite the induction of Tat-specific CTL, there was no significant reduction in either peak or viral set point compared to that of controls.     

Sinorhizobium fredii HH103 has a truncated nolO gene due to a -1 frameshift mutation that is conserved among other geographically distant S. fredii strains

Strain SVQ121 is a mutant derivative of Sinorhizobium fredii HH103 carrying a transposon Tn5-lacZ insertion into the nolO-coding region. Sequence analysis of the wild-type gene revealed that it is homologous to that of Rhizobium sp. NGR234, which is involved in the 3 (or 4)-O-carbamoylation of the nonreducing terminus of Nod factors. Downstream of nolO, as in Rhizobium sp. NGR234, the noeI gene responsible for methylation of the fucose moiety of Nod factors was found. SVQ121 Nod factors showed lower levels of methylation into the fucosyl residue than those of HH103-suggesting a polar effect of the transposon insertion into nolO over the noel gene. A noeI HH103 mutant was constructed. This mutant, SVQ503, produced Nod factors devoid of methyl groups, confirming that the S. fredii noeI gene is functional. Neither the nolO nor the noeI mutation affected the ability of HH103 to nodulate several host plants, but both mutations reduced competitiveness to nodulate soybean. The Nod factors produced by strain HH103, like those of other S. fredii isolates, lack carbamoyl residues. By using specific polymerase chain reaction primers, we sequenced the nolO gene of S. fredii strains USDA192, USDA193, USDA257, and 042B(s). All the analyzed strains showed the same -1 frameshift mutation that is present in the HH103 nolO-coding region. From these results, it is concluded that, regardless of their geographical origin, S. fredii strains carry the nolO-coding region but that it is truncated by the same base-pair deletion.     

Performance comparison of experimental constructed wetlands with different filter media and macrophytes treating industrial wastewater contaminated with lead and copper

The aim of this study was to investigate the treatment efficiency of passive vertical-flow wetland filters containing different macrophytes (Phragmites and/or Typha) and granular media with different adsorption capacities. Gravel, sand, granular activated carbon, charcoal and Filtralite (light expanded clay) were used as filter media. Different concentrations of lead and copper sulfate were added to polluted urban stream inflow water to simulate pretreated mine wastewater. The relationships between growth media, microbial and plant communities as well as the reduction of predominantly lead, copper and five-day biochemical oxygen demand (BOD5) were investigated. An analysis of variance showed that concentration reductions (mg l(-1)) of lead, copper and BOD5 were significantly similar for the six experimental wetlands. Microbial diversity was low due to metal pollution and similar for all filters. There appears to be no additional benefit in using adsorption media and macrophytes to enhance biomass performance during the first 10 months of operation.     

The interaction between ADAM22 and 14-3-3β

ADAM family consists of a number of transmembrane proteins that contain a disintegrin and metalloprotease domain. ADAMs are involved in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis and inflammatory response. The ADAM proteins have both cell adhesion and protease activities.Adam22 is highly expressed in human brain. Theadam22-/- mice presented severe ataxia and died before weaning, but the function of ADAM22 is still unknown. 14-3-3 β interacting with ADAM22 was detected by using yeast two-hybrid assay. The specificity of interaction between ADAM22 and 14-3-3β was proved byin vitro binding assay and immunoprecipitation. The major 14-3-3β binding site was located in the last 28 amino acid residues of ADAM22 cytoplasmic tail. Protein 14-3-3β is abundant and plays an important role in mediating cell diffusion, migration and cell cycle control. The interaction of ADAM22 and 14-3-3β suggests that the ADAM22 may play a crucial role in neural function and development.     

Adeno-associated virus-mediated gene transfer to the neonatal brain

For many metabolic diseases, early treatment is necessary to prevent irreversible developmental damage. This is particularly true for childhood diseases that affect the central nervous system (CNS). The development of effective techniques for gene transfer to the neonatal brain would provide a new set of therapeutic options for many of these disorders. Vectors based on adeno-associated virus (AAV) have shown promise as agents for neonatal CNS transduction. In preclinical animal models, a single treatment with AAV vectors at birth has been shown to produce persistent CNS expression of transduced genes into adulthood. Transduction of the neonatal brain has been accomplished by a variety of methods, including direct intraparenchymal injection, intraventricular infusion, and intravenous administration. Of these methods, intraparenchymal injection provides the highest levels of localized activity, while intraventricular infusion results in a more widespread distribution of activity when performed in the neonate. Here we describe a method for direct, intraparenchymal injection of AAV into the neonatal brain. This technique provides a method for investigators to evaluate the effects of in vivo expression of exogenous genes on the process of early brain development.     

Soluble expression in Escherichia coli,purification and characterization of a human TF-1 cell apoptosis-related protein TFAR19

A novel human TF-1 cell apoptosis-related protein, TFAR19, cloned from a human leukemia cell line, TF-1, was first overexpressed in Escherichia coli with the sequence Met-Gly-His(6)-Gly-Thr-Asn-Gly, a hexahistidine sequence followed by a hydroxylamine cleavage site attached to its amino terminus. The resulting protein was soluble and single-step purified to homogeneity by metal chelating affinity chromatography. After cleavage of the purified His(6)-tagged TFAR19 sample with hydroxylamine, highly purified untagged TFAR19 protein was then obtained through an FPLC Resource Q column. The structural characteristics and function of the His(6)-tagged and untagged TFAR19 proteins were studied using circular dichroism, intrinsic fluorescence, and ANS-binding fluorescence spectra and apoptosis activity assay. The results show that alpha-helix is the main secondary structure of the proteins and the two forms of TFAR19 protein fold properly, which correspond well to their apoptosis activity expression. The results also indicate that the extra sequence including the His(6)-tag fused to the N-terminus of TFAR19 protein has a minimal effect on its structure and function, suggesting that the His(6)-tagged TFAR19 protein could be further used as an immobilized target for finding potential proteins which interact with TFAR19 from a cDNA library using in vitro ribosome display technique.     

Endoenzymes associated with haemocyte types in the scallop (Chlamys farreri)

Haemocyte types of the scallop (Chlamys farreri) were identified by Giemsa stain and flow cytometry (FCM). Additionally, the activities of peroxidase (POD), phenoloxidase (PO) and alkaline phosphatase (ALP) in haemocytes were analysed by immunocytochemical and biochemical methods. The results indicate that there were two types of haemocytes in the scallop, hyalinocytes and granulocytes, and that POD, PO and ALP were more abundant and more active in granulocytes than in hyalinocytes.     

The Drosophila nuclear lamina protein YA binds to DNA and histone H2B with four domains

Dramatic changes occur in nuclear organization and function during the critical developmental transition from meiosis to mitosis. The Drosophila nuclear lamina protein YA binds to chromatin and is uniquely required for this transition. In this study, we dissected YA's binding to chromatin. We found that YA can bind to chromatin directly and specifically. It binds to DNA but not RNA, with a preference for double-stranded DNA (linear or supercoiled) over single-stranded DNA. It also binds to histone H2B. YA's binding to DNA and histone H2B is mediated by four domains distributed along the length of the YA molecule. A model for YA function at the end of Drosophila female meiosis is proposed.     

A unique approach for high level expression and production of a recombinant cobra neurotoxin in Escherichia coli

In this report, we describe a simple approach to produce a large quantity of a recombinant cobra neurotoxin containing four pairs of disulfide bonds. A cDNA encoding the toxin was fused, in frame, to the carboxyl termini of thioredoxin via a linker sequence encoding two amino acids, Asp and Pro. Due to the presence of thioredoxin, a soluble form of the fusion protein was expressed in a compartment, sensitive to osmotic pressure, in Escherichia coli. The fusion protein was released into the solution with low ionic strength under an osmotic shock treatment, and purified in a single step using an ion exchange chromatography column. The purified protein was treated in diluted hydrochloric acid to induce hydrolysis of the protein at the Asp-Pro linker site. Then, the recombinant neurotoxin was purified by gel filtration of the acid-treated sample. When the biological activity of the purified toxin was assayed, it was as potent as the natural toxin. Using this protocol, approximately 12 mg of pure recombinant neurotoxin can be produced from one liter of bacterial culture. More importantly, this protocol can be easily used for the production of the toxin at a larger scale with low cost. The approach outlined in this report will be suitable for the production of other recombinant proteins especially those of the 'three-finger' family.