Use of sodium lauroyl sarcosinate in a high-sensitivity protein assay by resonance light scattering technique

A simple and high-sensitivity method has been developed for the determination of proteins in aqueous solutions by resonance light scattering (RLS) technique. At pH 3.4 and ionic strength 1.2 x 10(-3), the weak RLS intensity of sodium lauroyl sarcosinate was greatly enhanced by the addition of proteins with the maximum peak located at 391 nm. Under the optimum conditions, the enhanced RLS intensities were in proportion to the concentrations of proteins in the range of 0.04 to 2.1 microg/mL for lysozyme, 0.0025 to 1.2 microg/mL for bovine serum albumin, 0.0075 to 0.9 microg/mL for human serum albumin, 0.02 to 1.4 microg/mL for gamma-globulin, 0.02 to 0.8 microg/mL for egg albumin, and 0.01 to 0.6 microg/mL for hemoglobin. Low detection limits ranging from 0.8 ng/mL to 4.3 ng/mL depending on the kind of proteins that have been achieved. The protein concentrations in synthetic samples and real biochemical samples were determined with satisfactory results. This method presented here is not only sensitive and simple but also reliable and suitable for practical bioassay applications.     

Plant community complexity in the arid valley of Minjiang River, southwestern China

Biocomplexity theory is becoming increasingly important in understanding natural vegetation dynamics and interrelation among all components of the ecosystem. In this study, based on the field investigation of plant species and environmental factors (altitude, microtopography, soil water content, and soil nutrients) in an arid valley of the upper reaches of Minjiang River, Sichuan Province, southwestern China, plant community complexity and its relationship with environmental factors, community diversity, species evenness and richness were studied. Both total and structural complexities of the communities showed a “high- low-high” tendency with the increase in altitude of the area, which meant that the complexity of communities was the highest at the sites of low and high altitude, whereas it was the lowest at the sites of intermediate altitude. It was found that the total community complexity had significant quadratic correlations with soil organic matter (SOM) content, total nitrogen (N), hydrolyzable N, soil water content, and available potassium (K), whereas it had no significant correlations with soil total K, total phosphorus (P), available P, and pH value. The total community complexity positively correlated with community diversity, species evenness and species richness, whereas the structural complexity negatively correlated with the community evenness. Of the two components of the total community complexity, namely, the structural complexity and the structural diversity, the structural complexity was more sensitive than the structural diversity to the changes of species in the community, which was not only related to the community evenness but also to the community richness. The relative contribution of both the structural complexity and the structural diversity to the total complexity would be different for different study areas or ecosystems.     

High level expression and characterization of the cyclophilin B gene from the anaerobic fungus Orpinomyces sp. strain PC-2

Cyclophilins are an evolutionarily conserved family of peptidyl-prolyl cis-trans isomerases (PPIases). A cyclophilin B (cypB) gene from the anaerobic fungus Orpinomyces sp. strain PC-2 was cloned and overexpressed in Escherichia coli. It was expressed as an amino-terminal 6 x His-tagged recombinant protein to facilitate purification. Highly purified protein (26.5 kDa) was isolated by two chromatographic steps involving affinity and gel filtration for biochemical studies of the enzyme. The recombinant CypB displayed PPIase activity with a k(cat)/K(m) of 8.9 x 10(6) M(-1) s(-1) at 10 degrees C and pH 7.8. It was inhibited by cyclosporin A (CsA) with an IC(50) of 23.5 nM, similar to those of the native protein and other cyclophilin B enzymes from animals. Genomic DNA analysis of cypB revealed that it was present as a single copy in Orpinomyces PC-2 and contained two introns, indicating it has a eukaryotic origin. It is one of the most heavily interrupted genes with intron sequences found in anaerobic fungi. The three-dimensional model of Orpinomyces PC-2 CypB was predicted with a homology modeling approach using the Swiss-Model Protein Modeling Server and three dimensional structure of human CypB as a template. The overall architecture of the CypB molecule is very similar to that of human CypB.     

Cutting edge: induction of B7-H4 on APCs through IL-10: novel suppressive mode for regulatory T cells

Multiple modes of suppressive mechanisms including IL-10 are thought to be implicated in CD4+CD25+ regulatory T (Treg) cell-mediated suppression. However, the cellular source, role, and molecular mechanism of IL-10 in Treg cell biology remain controversial. We now studied the interaction between Treg cells and APCs. We demonstrate that Treg cells, but not conventional T cells, trigger high levels of IL-10 production by APCs, stimulate APC B7-H4 expression, and render APCs immunosuppressive. Initial blockade of B7-H4 reduces the suppressive activity mediated by Treg cell-conditioned APCs. Further, APC-derived, rather than Treg cell-derived, IL-10 is responsible for APC B7-H4 induction. Therefore, Treg cells convey suppressive activity to APCs by stimulating B7-H4 expression through IL-10. Altogether, our data provide a novel cellular and molecular mechanism for Treg cell-mediated immunosuppression at the level of APCs, and suggest a plausible mechanism for the suppressive effect of IL-10 in Treg cell-mediated suppression.     

Biodegradable amphiphilic triblock copolymer bearing pendant glucose residues: preparation and specific interaction with Concanavalin A molecules

A novel biodegradable amphiphilic block copolymer PLGG-PEG-PLGG bearing pendant glucose residues is successfully prepared by the coupling reaction of 3-(2-aminoethylthio)propyl-alpha-D-glucopyranoside with the pendant carboxyl groups of PLGG-PEG-PLGG in the presence of N,N'-carbonyldiimidazole. The polymer PLGG-PEG-PLGG, i.e., poly{(lactic acid)-co-[(glycolic acid)-alt-(L-glutamic acid)]}-block-poly(ethylene glycol)-block- poly{(lactic acid)-co-[(glycolic acid)-alt-(L-glutamic acid)]}, is prepared by ring-opening copolymerization of L-lactide (LLA) with (3s)-benzoxylcarbonylethylmorpholine-2,5-dione (BEMD) in the presence of dihydroxyl PEG with molecular weight of 2000 as macroinitiator and Sn(Oct)2 as catalyst, and then by catalytic hydrogenation. The glucose-grafted copolymer shows a lower degree of cytotoxicity to ECV-304 cells and improved specific recognition and binding with Concanavalin A (Con A). Therefore, this kind of glucose-grafted copolymer may find biomedical applications.     

The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process

Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation.     

Using pseudo-amino acid composition and support vector machine to predict protein structural class

As a result of genome and other sequencing projects, the gap between the number of known protein sequences and the number of known protein structural classes is widening rapidly. In order to narrow this gap, it is vitally important to develop a computational prediction method for fast and accurately determining the protein structural class. In this paper, a novel predictor is developed for predicting protein structural class. It is featured by employing a support vector machine learning system and using a different pseudo-amino acid composition (PseAA), which was introduced to, to some extent, take into account the sequence-order effects to represent protein samples. As a demonstration, the jackknife cross-validation test was performed on a working dataset that contains 204 non-homologous proteins. The predicted results are very encouraging, indicating that the current predictor featured with the PseAA may play an important complementary role to the elegant covariant discriminant predictor and other existing algorithms.     

Inhibition of In Vivo HIV Infection in Humanized Mice by Gene Therapy of Human Hematopoietic Stem Cells with a Lentiviral Vector Encoding a Broadly Neutralizing Anti-HIV Antibody

Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/γ_c^null mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/γ_c^null mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1_JR-CSF, mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/γ_c^null mice inoculated with equivalent high-titer HIV-1_JR-CSF. These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.While broadly neutralizing human immunodeficiency virus (HIV)-specific antibodies have the capacity to prevent or suppress HIV infection, they are rarely produced by infected individuals, thereby markedly compromising the ability of the humoral response to control HIV infection (reviewed in reference 28). The high degree of sequence variability in the gp120 structure limits the number of highly conserved epitopes available for targeting by neutralizing antibodies (40). In addition, HIV utilizes several mechanisms to shield the limited number of conserved neutralizing epitopes from the potentially potent antiviral effects of HIV envelope-specific antibodies (14). First, the envelope protein is heavily glycosylated, and the linkage of the most immunoreactive envelope peptide structures to poorly immunogenic glycans shields them from antibody binding (37). Second, exposure of neutralizing epitopes not protected from antibody binding by glycosylation is greatly reduced by trimerization of the gp120-gp41 structure (5). Third, susceptibility of other neutralizing epitopes to antibodies is greatly reduced by limiting their accessibility to antibody binding to the brief transient phase of conformational changes that occur only during binding of the envelope protein to its cellular receptors, CD4 and CCR5 or CXCR4 (41). These intrinsic structural features of gp120 greatly reduce the capacity of natural HIV infection or vaccination to generate broadly neutralizing antibodies able to prevent or control infection. Despite these constraints, rare human antibodies with broad anti-HIV neutralizing activity, i.e., 2G12, b12, 2F5, and 4E10, have been isolated (2).The capacity of passive immunization with neutralizing antibodies to prevent infection was suggested by challenge studies demonstrating that transferred neutralizing antibodies protected monkeys from infection by simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) (15). These studies were extended to humans, including several studies that examined the effect of passive immunotherapy using 2G12, 2F5, and 4E10 on inhibition of HIV replication in infected individuals (20). Passive immunotherapy with a triple combination of 2G12, 2F5, and 4E10 delayed viral rebound after the cessation of highly active antiretroviral therapy (HAART), and activity of 2G12 was critical for inhibitory activity by this antibody combination (18). The key role of 2G12 in suppressing HIV replication was supported by the development of viral rebound in parallel with the emergence of HIV isolates resistant to neutralization by 2G12 (19).While HIV infection may be controlled by the lifelong treatment of HIV-infected individuals with periodic infusions of neutralizing-antibody cocktails every few weeks, this is not a practical or cost-effective therapeutic approach. Eliciting these antibodies by vaccination has not been successful. Therefore, we investigated whether we could circumvent the mechanisms that limit the endogenous production of broadly neutralizing HIV-specific antibodies using a molecular genetic approach to generate B cells that secrete these protective antibodies. In a proof-of-concept study, we examined the capacity of a single lentiviral vector to express the heavy and light chains of the 2G12 antibody, a well-studied anti-HIV human antibody that has broad neutralizing activity both against T cell line-adapted and primary HIV isolates (31). The 2G12 antibody was generated by applying murine/human xenohybridoma technology to establish human hybridoma cell lines from B cells isolated from HIV-infected individuals (16), and it targets the high-mannose and/or hybrid glycans of residues 295, 332, and 392 and peripheral glycans from residues 386 and 448 on gp120. In the current study we demonstrated that a lentiviral vector encoding the heavy and light chains of the 2G12 antibody reprogrammed B cells in vitro to secrete 2G12 with functional neutralizing activity. Furthermore, we demonstrated that the 2G12 lentiviral vector genetically modified human hematopoietic stem cells (hu-HSC), enabling them to differentiate in vivo into progeny cells that secreted 2G12 antibody that inhibited the development of in vivo HIV infection in humanized mice.