Effect of formulation factors on in vitro permeation of moxifloxacin from aqueous drops through excised goat, sheep, and buffalo corneas

The purpose of this investigation was to evaluate the effect of formulation factors on in vitro permeation of moxifloxacin from aqueous drop through freshly excised goat, sheep, and buffalo corneas. Aqueous isotonic ophthalmic solutions of moxifloxacin hydrochloride of different concentrations (pH 7.2) or 0.5% (wt/vol) solutions of different pH or 0.5% solutions (pH 7.2) containing different preservatives were made. Permeation characteristics of drug were evaluated by putting 1 mL formulation on freshly excised cornea (0.50 cm²) fixed between donor and receptor compartments of an all-glass modified Franz diffusion cell and measuring the drug permeated in the receptor (containing 10 mL bicarbonate ringer at 37°C under stirring) by spectrophotometry at 291 nm, after 120 minutes. Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Dunnett’s test. Increase in drug concentration in the formulation resulted in an increase in the quantity permeated but a decrease in percentage permeation. Increase in pH of the solution from 5.5 to 7.2 increased drug permeation, indicating pH-dependent transport. Compared with control formulation, moxifloxacin 0.5% (wt/vol) solution (pH 7.2) containing disodium edetate (EDTA) (0.01% wt/vol) produced significantly (P<.05) higher permeation with all the corneas. Formulation with benzyl alcohol significantly (P<.05) increased permeation with buffalo cornea compared with its control. Presence of benzalkonium chloride (BAK) (0.01% wt/vol) and EDTA (0.01% wt/vol) in the formulation increased permeation to the maximum with all the corneas. The results suggest that moxifloxacin 0.5% ophthalmic solution (pH 7.2) containing BAK (0.01%) and EDTA (0.01%) provides increased in vitro ocular availability through goat, sheep, and buffalo corneas. Published: February 10, 2006 Formerly College of Pharmacy, University of Delhi, Pushp Vihar, Sector III, New Delhi-110017, India     

V-ATPase V1 sector is required for corpse clearance and neurotransmission in Caenorhabditis elegans

The vacuolar-type ATPase (V-ATPase) is a proton pump composed of two sectors, the cytoplasmic V(1) sector that catalyzes ATP hydrolysis and the transmembrane V(o) sector responsible for proton translocation. The transmembrane V(o) complex directs the complex to different membranes, but also has been proposed to have roles independent of the V(1) sector. However, the roles of the V(1) sector have not been well characterized. In the nematode Caenorhabditis elegans there are two V(1) B-subunit genes; one of them, vha-12, is on the X chromosome, whereas spe-5 is on an autosome. vha-12 is broadly expressed in adults, and homozygotes for a weak allele in vha-12 are viable but are uncoordinated due to decreased neurotransmission. Analysis of a null mutation demonstrates that vha-12 is not required for oogenesis or spermatogenesis in the adult germ line, but it is required maternally for early embryonic development. Zygotic expression begins during embryonic morphogenesis, and homozygous null mutants arrest at the twofold stage. These mutant embryos exhibit a defect in the clearance of apoptotic cell corpses in vha-12 null mutants. These observations indicate that the V(1) sector, in addition to the V(o) sector, is required in exocytic and endocytic pathways.     

Diversity array technology markers: genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.)

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.     

Comparative Analysis of FLC Homologues in Brassicaceae Provides Insight into Their Role in the Evolution of Oilseed Rape

We identified nine FLOWERING LOCUS C homologues (BnFLC) in Brassica napus and found that the coding sequences of all BnFLCs were relatively conserved but the intronic and promoter regions were more divergent. The BnFLC homologues were mapped to six of 19 chromosomes. All of the BnFLC homologues were located in the collinear region of FLC in the Arabidopsis genome except BnFLC.A3b and BnFLC.C3b, which were mapped to noncollinear regions of chromosome A3 and C3, respectively. Four of the homologues were associated significantly with quantitative trait loci for flowering time in two mapping populations. The BnFLC homologues showed distinct expression patterns in vegetative and reproductive organs, and at different developmental stages. BnFLC.A3b was differentially expressed between the winter-type and semi-winter-type cultivars. Microsynteny analysis indicated that BnFLC.A3b might have been translocated to the present segment in a cluster with other flowering-time regulators, such as a homologue of FRIGIDA in Arabidopsis. This cluster of flowering-time genes might have conferred a selective advantage to Brassica species in terms of increased adaptability to diverse environments during their evolution and domestication process.     

Wired to the roots: Impact of root-beneficial microbe interactions on aboveground plant physiology and protection

Often, plant-pathogenic microbe interactions are discussed in a host-microbe two-component system, however very little is known about how the diversity of rhizospheric microbes that associate with plants affect host performance against pathogens. There are various studies, which specially direct the importance of induced systemic defense (ISR) response in plants interacting with beneficial rhizobacteria, yet we don’t know how rhizobacterial associations modulate plant physiology. In here, we highlight the many dimensions within which plant roots associate with beneficial microbes by regulating aboveground physiology. We review approaches to study the causes and consequences of plant root association with beneficial microbes on aboveground plant-pathogen interactions. The review provides the foundations for future investigations into the impact of the root beneficial microbial associations on plant performance and innate defense responses.     

The role of alpha 6 integrin in prostate cancer migration and bone pain in a novel xenograft model

Of the estimated 565,650 people in the U.S. who will die of cancer in 2008, almost all will have metastasis. Breast, prostate, kidney, thyroid and lung cancers metastasize to the bone. Tumor cells reside within the bone using integrin type cell adhesion receptors and elicit incapacitating bone pain and fractures. In particular, metastatic human prostate tumors express and cleave the integrin A6, a receptor for extracellular matrix components of the bone, i.e., laminin 332 and laminin 511. More than 50% of all prostate cancer patients develop severe bone pain during their remaining lifetime. One major goal is to prevent or delay cancer induced bone pain. We used a novel xenograft mouse model to directly determine if bone pain could be prevented by blocking the known cleavage of the A6 integrin adhesion receptor. Human tumor cells expressing either the wildtype or mutated A6 integrin were placed within the living bone matrix and 21 days later, integrin expression was confirmed by RT-PCR, radiographs were collected and behavioral measurements of spontaneous and evoked pain performed. All animals independent of integrin status had indistinguishable tumor burden and developed bone loss 21 days after surgery. A comparison of animals containing the wild type or mutated integrin revealed that tumor cells expressing the mutated integrin resulted in a dramatic decrease in bone loss, unicortical or bicortical fractures and a decrease in the ability of tumor cells to reach the epiphyseal plate of the bone. Further, tumor cells within the bone expressing the integrin mutation prevented cancer induced spontaneous flinching, tactile allodynia, and movement evoked pain. Preventing A6 integrin cleavage on the prostate tumor cell surface decreased the migration of tumor cells within the bone and the onset and degree of bone pain and fractures. These results suggest that strategies for blocking the cleavage of the adhesion receptors on the tumor cell surface can significantly prevent cancer induced bone pain and slow disease progression within the bone. Since integrin cleavage is mediated by Urokinase-type Plasminogen Activator (uPA), further work is warranted to test the efficacy of uPA inhibitors for prevention or delay of cancer induced bone pain.     

Picornavirus genome replication. Identification of the surface of the poliovirus (PV) 3C dimer that interacts with PV 3Dpol during VPg uridylylation and construction of a structural model for the PV 3C2-3Dpol complex

Picornaviruses have a peptide termed VPg covalently linked to the 5'-end of the genome. Attachment of VPg to the genome occurs in at least two steps. First, Tyr-3 of VPg, or some precursor thereof, is used as a primer by the viral RNA-dependent RNA polymerase, 3Dpol, to produce VPg-pUpU. Second, VPg-pUpU is used as a primer to produce full-length genomic RNA. Production of VPg-pUpU is templated by a single adenylate residue located in the loop of an RNA stem-loop structure termed oriI by using a slide-back mechanism. Recruitment of 3Dpol to and its stability on oriI have been suggested to require an interaction between the back of the thumb subdomain of 3Dpol and an undefined region of the 3C domain of viral protein 3CD. We have performed surface acidic-to-alanine-scanning mutagenesis of 3C to identify the surface of 3C with which 3Dpol interacts. This analysis identified numerous viable poliovirus mutants with reduced growth kinetics that correlated to reduced kinetics of RNA synthesis that was attributable to a change in VPg-pUpU production. Importantly, these 3C derivatives were all capable of binding to oriI as well as wild-type 3C. Synthetic lethality was observed for these mutants when placed in the context of a poliovirus mutant containing 3Dpol-R455A, a residue on the back of the thumb required for VPg uridylylation. These data were used to guide molecular docking of the structures for a poliovirus 3C dimer and 3Dpol, leading to a structural model for the 3C(2)-3Dpol complex that extrapolates well to all picornaviruses.     

Picornavirus genome replication: roles of precursor proteins and rate-limiting steps in oriI-dependent VPg uridylylation

The 5' ends of all picornaviral RNAs are linked covalently to the genome-encoded peptide, VPg (or 3B). VPg linkage is thought to occur in two steps. First, VPg serves as a primer for production of diuridylylated VPg (VPg-pUpU) in a reaction catalyzed by the viral polymerase that is templated by an RNA element (oriI). It is currently thought that the viral 3AB protein is the source of VPg in vivo. Second, VPg-pUpU is transferred to the 3' end of plus- and/or minus-strand RNA and serves as primer for production of full-length RNA. Nothing is known about the mechanism of transfer. We present biochemical and biological evidence refuting the use of 3AB as the donor for VPg uridylylation. Our data are consistent with precursors 3BC and/or 3BCD being employed for uridylylation. This conclusion is supported by in vitro uridylylation of these proteins, the ability of a mutant replicon incapable of producing processed VPg to replicate in HeLa cells and cell-free extracts and corresponding precursor processing profiles, and the demonstration of 3BC-linked RNA in mutant replicon-transfected cells. These data permit elaboration of our model for VPg uridylylation to include the use of precursor proteins and invoke a possible mechanism for location of the diuridylylated, VPg-containing precursor at the 3' end of plus- or minus-strand RNA for production of full-length RNA. Finally, determinants of VPg uridylylation efficiency suggest formation and/or collapse or release of the uridylylated product as the rate-limiting step in vitro depending upon the VPg donor employed.     

In vitro propagation of Spilanthes mauritiana DC., an endangered medicinal herb, through axillary bud cultures

Summary Spilanthes mauritiana DC., (Compositae), a East African medicinal herb containing pharmaceutically promising secondary metabolites, has successfully been raised in vitro. We have developed a clonal propagation protocol that uses juvenile plants as starting material. The addition of benzylaminopurine (BA) (1.0 μM) and naphthaleneacetic acid (NAA) (0.1 μM) to the culture medium resulted in maximum shooting response with minimal callusing. Shoots rooted best in vitro in MS medium supplemented with indole-3-acetic acid (IAA; 0.2 μM), and plants that had already developed roots showed better growth, with maximum survival rate, in the greenhouse after an initial hardening.