A Randomized, Controlled Phase I/II Study to Evaluate the Safety and Efficacy of MGV354 for Ocular Hypertension or Glaucoma.

Purpose: To assess the clinical safety, tolerability, and efficacy of topically administered MGV354, a soluble guanylate cyclase (sGC) activator, in patients with ocular hypertension (OH) or glaucoma.

Design: Double-masked, randomized, and vehicle-controlled study.

Methods: Parts 1 and 2 evaluated safety and tolerability to identify the maximum tolerated dose (MTD) of once-daily MGV354 in 32 healthy volunteers (Part 1) and 16 patients with OH or glaucoma (Part 2) at a single clinical site.

A Novel Selective Soluble Guanylate Cyclase Activator, MGV354, Lowers Intraocular Pressure in Preclinical Models, Following Topical Ocular Dosing.

Purpose: The nitric oxide/soluble guanylate cyclase/protein kinase G (NO/sGC/PKG) is known to be involved in the regulation of intraocular pressure (IOP) and may be dysregulated in glaucoma. The purpose is to demonstrate that the sGC activator MGV354 lowers IOP in a monkey model of glaucoma and could be considered as a possible new clinical drug candidate.

Methods: Changes to cGMP were assessed in primary human trabecular meshwork (hNTM) cells and binding studies were conducted using human sGC full-length protein.

Discovery of Highly Potent and Selective Small-Molecule Reversible Factor D Inhibitors Demonstrating Alternative Complement Pathway Inhibition in Vivo.

The highly specific S1 serine protease factor D (FD) plays a central role in the amplification of the complement alternative pathway (AP) of the innate immune system. Genetic associations in humans have implicated AP activation in age-related macular degeneration (AMD), and AP dysfunction predisposes individuals to disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The combination of structure-based hit identification and subsequent optimization of the center (S)-proline-based lead 7 has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties.

Inflammation and immunogenicity limit the utility of the rabbit as a nonclinical species for ocular biologic therapeutics.

The nonclinical safety evaluation of therapeutic drug candidates is commonly conducted in two species (rodent and non-rodent) in keeping with international health authority guidance. Biologic drugs typically have restricted species cross-reactivity, necessitating the evaluation of safety in non-human primates and thus limiting the utility of lower order species. Safety studies of cross-reactive ocular biologic drug candidates have been conducted in rabbits as a second toxicology species, despite the fact that rabbits are not a rodent species.

Okadaic acid and trifluoperazine enhance Agrobacterium-mediated transformation in eastern white pine.

Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (beta-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated.

Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine.

An efficient transgenic eastern white pine (Pinus strobus L.) plant regeneration system has been established using Agrobacterium tumefaciens strain GV3850-mediated transformation and the green fluorescent protein (gfp) gene as a reporter in this investigation. Stable integration of transgenes in the plant genome of pine was confirmed by polymerase chain reaction (PCR), Southern blot, and northern blot analyses.

Efficient delivery of small interfering RNA to plant cells by a nanosecond pulsed laser-induced stress wave for posttranscriptional gene silencing.

Small interfering RNA (siRNA) induced posttranscriptional gene silencing (PTGS) has been an efficient method for genetic and molecular analysis of certain developmental and physiological processes and represented a potential strategy for both controlling virus replication and developing therapeutic products. However, there are limitations for the methods currently used to deliver siRNA into cells. We report here, to our knowledge, the first efficient delivery of siRNA to plant cells by a nanosecond pulsed laser-induced stress wave (LISW) for posttranscriptional gene silencing.

Expression of a transcription factor from Capsicum annuum in pine calli counteracts the inhibitory effects of salt stress on adventitious shoot formation.

Transcription factors regulating the stress-responsive gene expression play an important role in plant stress adaptation. In this study, we examined the salt stress tolerance of transgenic Virginia pine (Pinus virginiana Mill.) overexpressing a Capsicum annuum ERF/AP2-type transcription factor (CaPF1), which may enhance the ability of transgenic plants to tolerate various kinds of stresses during vegetative growth.

Non-destructive analysis of the nuclei of transgenic living cells using laser tweezers and near-infrared raman spectroscopic technique.

Transgenic cell lines of loblolly pine (Pinus taeda L.) were analyzed by a compact laser-tweezers-Raman-spectroscopy (LTRS) system in this investigation. A low power diode laser at 785 nm was used for both laser optical trapping of single transgenic cells and excitation for near-infrared Raman spectroscopy of the nuclei of synchronized cells, which were treated as single organic particles, at the S-phase of the cell cycle.

Plant regeneration through multiple adventitious shoot differentiation from callus cultures of slash pine (Pinus elliottii).

A plant regeneration system through multiple adventitious shoot differentiation from callus cultures has been established in slash pine (Pinus elliottii). Influences of seven different basal media on callus induction, adventitious shoot formation, and rooting were investigated. Among the different basal media, B5, SH, and TE proved to be suitable for callus induction and plantlet regeneration.

Mevalonate kinase activity during different stages of plant regeneration from nodular callus cultures in white pine (Pinus strobus).

Mevalonate kinase (MK) catalyzes a step in the isoprenoid biosynthetic pathway, which leads to a huge number of compounds that play important roles in plant growth and development. Here, we report on changes in MK activity in white pine (Pinus strobus L.) during plant regeneration by adventitious shoot organogenesis from cotyledons of mature embryos, including nodular callus induction, shoot formation and rooting.

Differential gene silencing induced by short interfering RNA in cultured pine cells associates with the cell cycle phase.

The double-stranded short interfering RNA (siRNA) molecules can silence targeted genes through sequence-specific cleavage of the cognate RNA transcript. The rapid adoption of technologies based on this siRNA interference mechanism has been a widely used method to analyze gene function in plants, invertebrates, and mammalian systems. In order to understand the dynamics of siRNA-mediated gene inactivation during cell division, we have investigated the relationship between the cell cycle phase and the post-transcriptional gene silencing mediated by siRNA in gfp transgenic Virginia pine (Pinus virginiana Mill.

Overexpression of the pepper transcription factor CaPF1 in transgenic Virginia pine (Pinus Virginiana Mill.) confers multiple stress tolerance and enhances organ growth.

Transcription factors play an important role in regulating gene expression in response to stress and pathogen tolerance. We describe here that overexpression of an ERF/AP2 pepper transcription factor (CaPF1) in transgenic Virginia pine (Pinus virginiana Mill.) confers tolerance to heavy metals Cadmium, Copper, and Zinc, to heat, and to pathogens Bacillus thuringiensis and Staphylococcus epidermidis, as by the survival rate of transgenic plants and the number of decreasing pathogen cells in transgenic tissues.

Polyamines promote root elongation and growth by increasing root cell division in regenerated Virginia pine (Pinus virginiana Mill.) plantlets.

Polyamines have been demonstrated to play an important role in adventitious root formation and development in plants. Here, we present a detailed analysis of influence of exogenously added polyamines on adventitious root development and its relationship to cold tolerance in Virginia pine (Pinus virginia Mill.).

High efficiency inducible gene expression system based on activation of a chimeric transcription factor in transgenic pine.

Inducible gene expression systems are needed in functional genomics of tree species. A glucocorticoid-inducible gene expression system was established in a gymnosperm species Virginia pine (Pinus virginiana Mill.) through Agrobacterium tumefaciens-mediated genetic transformation.

Peroxidase and catalase activities are involved in direct adventitious shoot formation induced by thidiazuron in eastern white pine (Pinus strobus L.) zygotic embryos.

We reported establishment of an efficient plant regeneration procedure through direct adventitious shoot (DAS) formation from cotyledons and hypocotyls of eastern white pine (Pinus strobus L.) mature embryos in this investigation. Multiple DASs were initiated from cotyledons of embryos on PS medium containing N6-benzyladenine (BA), thidiazuron (TDZ), or kinetin (KIN).

Relationships between genomic, cell cycle, and mutagenic responses of TK6 cells exposed to DNA damaging chemicals.

Genotoxic stress causes a variety of cellular and molecular responses in mammalian cells, including cell cycle arrest, DNA repair, and apoptosis. These responses result from the interplay between the genotoxic events themselves, and the biological context in which they occur. To better understand this interplay, we investigated cytotoxicty, mutagenesis, cell cycle profile, and global gene expression in the human TK6 lymphoblastoid cell line exposed to six genotoxicants.

Inducible antisense-mediated post-transcriptional gene silencing in transgenic pine cells using green fluorescent protein as a visual marker.

An inducible post-transcriptional gene silencing (PTGS) system was established in Virginia pine (Pinus virginiana Mill.) cells. This system is based on the activation of an antisense gfp gene construct by a chimeric transcriptional activator GVG (Gal4-binding domain-VP16 activation domain-glucocorticoid receptor fusion) upon application of the inducer to gfp transgenic cell lines.

Administration of a PPARalpha agonist increases serum apolipoprotein A-V levels and the apolipoprotein A-V/apolipoprotein C-III ratio.

Apolipoprotein A-V (apoA-V) first gained attention as a regulator of triglycerides through transgenic mouse studies. Furthermore, peroxisome proliferator-activated receptor alpha (PPARalpha) agonists such as fenofibrate increase apoA-V mRNA expression. Our group recently developed the first assay to quantitate serum apoA-V levels.

Enhanced tolerance to salt stress in transgenic loblolly pine simultaneously expressing two genes encoding mannitol-1-phosphate dehydrogenase and glucitol-6-phosphate dehydrogenase.

A reproducible approach to improve salt tolerance of conifers has been established by using the technology of plant genetic transformation and using loblolly pine (Pinus taeda L.) as a model plant. Mature zygotic embryos of three genotypes of loblolly pine were infected with Agrobacterium tumefaciens strain LBA 4404 harboring the plasmid pBIGM which carrying two bacterial genes encoding the mannitol-1-phosphate dehydrogenase (Mt1D, EC 1.

Plant regeneration from callus cultures derived from mature zygotic embryos in white pine (Pinus strobus L.).

Plant regeneration via adventitious shoot organogenesis from callus cultures initiated from mature embryos in white pine (Pinus strobus L.) was achieved in this study. Callus cultures were induced from mature embryos cultured on PS medium supplemented with 2,4-dichlorophenoxyacetic acid, alpha-naphthaleneacetic acid, or indole-3-acetic acid.

Post-transcriptional gene silencing induced by short interfering RNAs in cultured transgenic plant cells.

Short interfering RNA (siRNA) is widely used for studying post-transcriptional gene silencing and holds great promise as a tool for both identifying function of novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which were designed against different specific areas of coding region of the same target green fluorescent protein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice (Oryza sativa L.; OS), cotton (Gossypium hirsutum L.

Comparison of gene expression changes induced in mouse and human cells treated with direct-acting mutagens.

Exposure to DNA-damaging agents can elicit a variety of stress-related responses that may alter the gene expression of numerous biological pathways. We used Affymetrix microarrays to detect gene expression changes in mouse lymphoma (L5178Y) and human lymphoblastoid (TK6) cells in response to methyl methanesulfonate (MMS; a prototypical alkylating agent) and bleomycin (a prototypical oxidative mutagen). Cells were treated for 4 hr, and RNA was isolated either at the end of the treatment or after a 20-hr recovery period.

Regulated gene expression by glucocorticoids in cultured Virginia pine (Pinus virginiana Mill.) cells.

The effects of six glucocorticoids (dexamethasone, hydrocortisone, 6-methylprednisolone, prednisolone, prednisone, and triamcinolone) on inducible gene expression, based on the chimaeric transcriptional activator GVG and carried by the binary expression vector pINDEX3-m-gfp5-ER, were evaluated in transgenic Virginia pine cell cultures. The concentration that activated GVG transcription factor activity, the level of inducible m-gfp5-ER expression, and the kinetics of inducible m-gfp5-ER expression were determined for each glucocorticoid. Transgenic cells produced green fluorescence upon blue light excitation after treatment with prednisolone, prednisone, 6-methylprednisolone, dexamethasone, triamcinolone, and hydrocortisone.