Steroidal saponins from the fruits of Asparagus racemosus

Three steroidal saponins, racemosides A (1), B (2) and C (3), were isolated from the methanolic extract of the fruits of Asparagus racemosus, and characterized as (25S)-5beta-spirostan-3beta-ol-3-O-{beta-D- glucopyranosyl (1-->6)-[alpha-L-rhamnopyranosyl (1-->6)-beta-D-glucopyranosyl (1-->4)]-beta-D-glucopyranoside}, (25S)-5beta-spirostan-3beta-ol-3-O-alpha-L-rhamnopyranosyl (1-->6)-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranoside and (25S)-5beta-spirostan-3beta-ol-3-O-{alpha-L-rhamnopyranosyl-(1-->6)-[alpha-L-rhamnopyranosyl (1-->4)]-beta-D-glucopyranoside}, respectively, by spectrometric analysis and some chemical strategies.     

Enhanced clavulanic acid production inStreptomyces clavuligerus NRRL3585 by overexpression of regulatory genes

We constructed four recombinant plasmids to enhance the production of clavulanic acid (CA) inStreptomyces clavuligerus NRRL3585: (1) plBRHL1, which includesccaR, a pathway-specific regulatory gene involved in cephamycin C and CA biosynthesis; (2) plBRHL2, containingclaR, again a regulatory gene, which controls the late steps of CA biosynthesis; (3) pGIBR containingafsR-p, a global regulatory gene fromStreptomyces peucetius, and (4) pKS, which harbors all of the genes (ccaR/claR/afsR-p). The plasmids were expressed inS. clavuligerus NRRL3585 along with theermE ^* promoter. All of them enhanced the production of CA; 2.5-fold overproduction for plBRHL1, 1.5-fold for plBRHL2, 1.6-fold for pGIBR, and 1.5-fold for pKS compared to the wild type.     

Anticancer effects of the novel 1alpha, 25-dihydroxyvitamin D3 hybrid analog QW1624F2-2 in human neuroblastoma

Vitamin D3 analogs are potential anti-cancer agents with theoretically wide therapeutic index, but there have been limited studies directed towards human neuroblastoma. The antiproliferative ability of the novel vitamin D3 hybrid analog QW-1624F2-2 (QW, 1-hydroxymethyl-16-ene-24, 24-F2-26, 27-bishomo-25-hydroxyvitamin D3) was examined in two human neuroblastoma-derived cell-lines. Analog QW inhibited cell-cycle progression of IMR5 cells with accumulation in G1 phase. QW induced the differentiation of CHP134 as evidenced by increased neurite length. These effects were accompanied by decreased expression of MYCN in both the cell-lines treated with QW. Furthermore, QW inhibited the migration of CHP134 cells in matrigel invasion assays, indicating its anti-invasive ability. In athymic nude mice, we found that QW was less calcemic than EB1089 (1alpha, 25-dihydroxy-22, 24-diene-24, 26,27-trishomovitamin D3). Systemic administration of QW in a mouse xenotransplantation model revealed that it is more effective than EB1089 in suppressing the growth of CHP134 flank tumors. In summary, the low-calcemic hybrid analog QW showed significant anti-tumor activity in vivo and thus exhibits potential as a novel cancer therapeutic.     

Genomic characterization of the Yersinia genus

Background

New DNA sequencing technologies have enabled detailed comparative genomic analyses of entire genera of bacterial pathogens. Prior to this study, three species of the enterobacterial genus Yersinia that cause invasive human diseases (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) had been sequenced. However, there were no genomic data on the Yersinia species with more limited virulence potential, frequently found in soil and water environments.

Results

We used high-throughput sequencing-by-synthesis instruments to obtain 25- to 42-fold average redundancy, whole-genome shotgun data from the type strains of eight species: Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. mollaretii, Y. rohdei, and Y. ruckeri. The deepest branching species in the genus, Y. ruckeri, causative agent of red mouth disease in fish, has the smallest genome (3.7 Mb), although it shares the same core set of approximately 2,500 genes as the other members of the species, whose genomes range in size from 4.3 to 4.8 Mb. Yersinia genomes had a similar global partition of protein functions, as measured by the distribution of Cluster of Orthologous Groups families. Genome to genome variation in islands with genes encoding functions such as ureases, hydrogeneases and B-12 cofactor metabolite reactions may reflect adaptations to colonizing specific host habitats.

Conclusions

Rapid high-quality draft sequencing was used successfully to compare pathogenic and non-pathogenic members of the Yersinia genus. This work underscores the importance of the acquisition of horizontally transferred genes in the evolution of Y. pestis and points to virulence determinants that have been gained and lost on multiple occasions in the history of the genus.     

Binary polypeptide system for permanent and oriented protein immobilization

Background  

Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling.     

Neuropeptide Y Has a Protective Role during Murine Retrovirus-Induced Neurological Disease

Viral infections in the central nervous system (CNS) can lead to neurological disease either directly by infection of neurons or indirectly through activation of glial cells and production of neurotoxic molecules. Understanding the effects of virus-mediated insults on neuronal responses and neurotrophic support is important in elucidating the underlying mechanisms of viral diseases of the CNS. In the current study, we examined the expression of neurotrophin- and neurotransmitter-related genes during infection of mice with neurovirulent polytropic retrovirus. In this model, virus-induced neuropathogenesis is indirect, as the virus predominantly infects macrophages and microglia and does not productively infect neurons or astrocytes. Virus infection is associated with glial cell activation and the production of proinflammatory cytokines in the CNS. In the current study, we identified increased expression of neuropeptide Y (NPY), a pleiotropic growth factor which can regulate both immune cells and neuronal cells, as a correlate with neurovirulent virus infection. Increased levels of Npy mRNA were consistently associated with neurological disease in multiple strains of mice and were induced only by neurovirulent, not avirulent, virus infection. NPY protein expression was primarily detected in neurons near areas of virus-infected cells. Interestingly, mice deficient in NPY developed neurological disease at a faster rate than wild-type mice, indicating a protective role for NPY. Analysis of NPY-deficient mice indicated that NPY may have multiple mechanisms by which it influences virus-induced neurological disease, including regulating the entry of virus-infected cells into the CNS.The early innate immune response to virus infection in the central nervous system (CNS) plays an important regulatory role in controlling both viral infection and pathogenesis. The neuroinflammatory response can limit virus replication through production of type I interferons and recruitment of virus-specific T cells to the CNS (5, 9, 12, 15, 19). However, the neuroinflammatory response can also lead to chronic gliosis, the production of cytokines that are toxic to neurons, and the recruitment of virus-infected cells to the CNS (6, 8, 18, 35). Understanding the relationship between the innate immune response and viral disease is essential in order to manipulate this response to control virus infection in the CNS.To better understand the role of the innate immune responses in viral pathogenesis in the CNS, we have utilized a mouse model of polytropic retrovirus infection. In this model, neuropathogenesis is indirect, since the polytropic retroviruses do not productively infect neurons. Instead, the viruses predominantly infect macrophages and microglia in the CNS (32). Despite severe neurological disease development following polytropic retrovirus infection, the only histologic changes observed in the brain are the activations of microglia and astrocytes (31). In addition, we have found high levels of proinflammatory cytokines and chemokines in brain tissue from infected mice, including tumor necrosis factor (TNF); interleukin 1 alpha (IL-1α), IL-1β, and IL-6; and the chemokines chemokine ligand 2 (CCL2/MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), and chemokine (C-X-C motif) ligand 10 (CXCL10/IP-10) (28). Studies with different chemokine receptors and cytokine-deficient mice demonstrated that at least two of these proinflammatory cytokines, CCL2 and TNF, can contribute to retrovirus-induced neurological disease (26, 27). However, neither of these molecules was necessary for neurological disease for all of the neurovirulent polytropic retroviruses studied, suggesting that other host factors contribute to retroviral pathogenesis.Analysis of the envelope protein of the neurovirulent polytropic retrovirus identified key residues in the envelope protein that influence the ability of the virus to induce neurological disease (28). In this study, we utilized neurovirulent and nonneurovirulent chimeric viruses that differ by only a few amino acid residues in these envelope regions to identify host response factors whose expression correlated with neurovirulence. We also utilized two different mouse strains, Inbred Rocky Mountain White (IRW) and 129S6, to confirm that expression of these host response factors is consistently induced or suppressed during neurovirulent virus infection. We determined that, although a number of host response genes are induced by polytropic retrovirus infection of the CNS, the expression of several of these factors correlated only with neuroinvasion and was not strongly correlative of neurovirulence. However, we have identified a neurotrophin, neuropeptide Y (NPY), whose expression strongly correlates with neurovirulence. We found that NPY had a protective influence on retroviral neuropathogenesis and examined the mechanisms by which NPY influences retrovirus infection of the CNS.     

Determination of free and liposomal Amphotericin B in human plasma by liquid chromatography–mass spectroscopy with solid phase extraction and protein precipitation techniques

Amphotericin B is available in various drug delivery systems such as cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The separation and measurement of free drug (drug which is not bound with liposomal lipids) and liposomal drug (drug which is entrapped in liposomes) in the human plasma after injection of liposomal Amphotericin B is of prime importance due to toxicity concerns. A robust, specific and sensitive method has been developed to effectively separate and then quantify the free drug and liposomal drug, present in human plasma. This method utilizes solid phase extraction Oasis HLB cartridges, which retains the free drug and the liposomal Amphotericin B was eluted from the cartridge in first step. The eluted liposomal Amphotericin B was then extracted from lipids by protein precipitation method using 2% dimethylsulfoxide (DMSO) in acetonitrile. After separation and extraction, the quantification of free and liposomal fractions of Amphotericin B was performed by HPLC–MS–MS technique. The chromatographic separation was performed using Chromolith Performance RP 18e column. The mobile phase composed of 5 mM ammonium acetate, methanol and acetonitrile and a gradient elution program was used. The calibration curves were found to be linear for free Amphotericin B (0.25–15.0 μg/ml) and liposomal Amphotericin B (1.0–100.0 μg/ml). The recovery was about 96% for free Amphotericin B and about 92% for liposomal Amphotericin B. Recoveries were consistent over the linearity ranges defined. The intra-batch and inter-batch accuracy and precision fulfilled the international requirements. The stability of free and liposomal Amphotericin B was assessed under different storage conditions.     

Metabolism of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene by Alcaligenes denitrificans

Metabolism of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), a persistent metabolite of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), by an Alcaligenes denitrificans was optimal under `non-shaking' conditions, was accelerated by adding 1 g glucose l^–1, and inhibited by 1 g sodium acetate l^–1 or 1 g sodium succinate l^–1. Addition of biphenyl, in the vapor form, to the reaction mixture did not enhance DDE metabolism. During the reaction, accumulation of conventional metabolites, 1-chloro-2,2-bis(4-chlorophenyl)ethylene (DDMU) and 4-chlorobenzoate, was not observed.     

Particle bombardment and the genetic enhancement of crops: myths and realities

DNA transfer by particle bombardment makes use of physical processes to achieve the transformation of crop plants. There is no dependence on bacteria, so the limitations inherent in organisms such as Agrobacterium tumefaciens do not apply. The absence of biological constraints, at least until DNA has entered the plant cell, means that particle bombardment is a versatile and effective transformation method, not limited by cell type, species or genotype. There are no intrinsic vector requirements so transgenes of any size and arrangement can be introduced, and multiple gene cotransformation is straightforward. The perceived disadvantages of particle bombardment compared to Agrobacterium-mediated transformation, i.e. the tendency to generate large transgene arrays containing rearranged and broken transgene copies, are not borne out by the recent detailed structural analysis of transgene loci produced by each of the methods. There is also little evidence for major differences in the levels of transgene instability and silencing when these transformation methods are compared in agriculturally important cereals and legumes, and other non-model systems. Indeed, a major advantage of particle bombardment is that the delivered DNA can be manipulated to influence the quality and structure of the resultant transgene loci. This has been demonstrated in recently reported strategies that favor the recovery of transgenic plants containing intact, single-copy integration events, and demonstrating high-level transgene expression. At the current time, particle bombardment is the most efficient way to achieve plastid transformation in plants and is the only method so far used to achieve mitochondrial transformation. In this review, we discuss recent data highlighting the positive impact of particle bombardment on the genetic transformation of plants, focusing on the fate of exogenous DNA, its organization and its expression in the plant cell. We also discuss some of the most important applications of this technology including the deployment of transgenic plants under field conditions.     

Enzymatic production and biological activities of chitosan oligosaccharides (COS): A review

Many researchers have focused chitosan as a source of potential bioactive material during past few decades. However, chitosan has several drawbacks to be utilized in biological applications, including poor solubility under physiological conditions. Therefore, a new interest has recently been emerged on partially hydrolyzed chitosan, chitosan oligosaccharides (COS). During the resent past, several technological approaches have been taken to prepare COS and, enzymatic preparation methods captured a great interest due to safe and non-toxic concerns. With time, new improvements were introduced to enzymatic production and presently it has been developed to a continuous production process. Many of the biological activities reported for COS, such as antimicrobial, anticancer, antioxidant, and immunostimulant effects are depend on their physico-chemical properties. In this review, we have summarized different enzymatic preparation methods of COS and some of their reported biological activities.