Analysis of high dysmorphogenic activity of Ro 13-6307, a tetramethylated tetralin analog of retinoic acid.

Certain synthetic retinoids differ widely from retinoic acid (RA) in teratogenic potency, being much more or much less effective than RA. It is assumed that the potency of a retinoid may depend on the nature of its interaction with cellular binding components (nuclear retinoic acid receptors or cytoplasmic binding proteins) and, as in the case of retinoids that are mammalian teratogens, on factors that determine its accessibility to the embryo. To investigate some of the factors that contribute to potency, we used a new synthetic retinoid Ro 13-6307 that differs in structure from RA in having an aromatic ring inserted in its side chain along with gem dimethyl modification of the natural cyclohexenyl ring. Pregnant ICR mice were given a single oral dose (0, 1, or 10 mg/kg) on day 11 of gestation, and the resultant teratogenic outcome was monitored on day 17. Direct effects on cell differentiation were obtained by exposing high density cultures of limb bud mesenchymal cells to a range of concentrations (0.3 ng/ml-3 micrograms/ml) of Ro 13-6307 and scoring for chondrogenic suppression. Concentrations reaching the embryo after maternal administration of Ro 13-6307 were measured by HPLC to quantify the analog for a period of 4 h after administration of the oral dose. We found that this retinoid was 40-fold as active as RA in both inducing teratogenesis and suppressing chondrogenesis, yet its concentration in the affected embryo was only a fraction of that achieved after an equivalent dose of RA was employed in a similar protocol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic and molecular analysis of light-regulated plant development

Light regulates many physiological and developmental events in plants through the action of multiple sensory pigment systems. Although our understanding of the regulatory photoreceptors, including phytochromes (that principally absorb red and far-red energy) and blue light receptors, has advanced considerably in the recent past, the mechanisms of light signal transduction in higher plants are poorly understood. To unravel the molecular events associated with light-regulated plant development, a large number of photomorphogenic mutants have been isolated in several different plant species, including Arabidopsis, cucumber, tomato, pea, Brassica and Sorghum, which are either impaired in normal perception of light signal (photoreceptor mutants) or are affected in some specific or a sub-set of phenotypic traits (signal transduction mutants). Their physiological and molecular analysis is proving to be valuable in (1) assigning specific function to discrete phytochrome species, (2) elucidation of elements that constitute the transduction pathway downstream of signal perception, and (3) determining how different photosensory systems regulate many diverse responses. The progress made in the analysis of photomorphogenic mutants, as reviewed in this article, clearly indicates that multiple photoreceptors, either of the same or different class, interact through an intricate network of signal transduction pathways to finally determine the light-dependent phenotype of both monocots and dicots.     

Transport genes and chemotaxis in Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is a Gram-negative, sea gull-shaped rod associated with community-acquired gastroenteritis. The bacterium has been found in diverse freshwater environments including fish, frogs and drinking water reservoirs. Using the complete genome sequence data of L. hongkongensis, we performed a comprehensive analysis of putative transport-related genes and genes related to chemotaxis, motility and quorum sensing, which may help the bacterium adapt to the changing environments and combat harmful substances.

Results

A genome-wide analysis using Transport Classification Database TCDB, similarity and keyword searches revealed the presence of a large diversity of transporters (n = 457) and genes related to chemotaxis (n = 52) and flagellar biosynthesis (n = 40) in the L. hongkongensis genome. The transporters included those from all seven major transporter categories, which may allow the uptake of essential nutrients or ions, and extrusion of metabolic end products and hazardous substances. L. hongkongensis is unique among closely related members of Neisseriaceae family in possessing higher number of proteins related to transport of ammonium, urea and dicarboxylate, which may reflect the importance of nitrogen and dicarboxylate metabolism in this assacharolytic bacterium. Structural modeling of two C⁴-dicarboxylate transporters showed that they possessed similar structures to the determined structures of other DctP-TRAP transporters, with one having an unusual disulfide bond. Diverse mechanisms for iron transport, including hemin transporters for iron acquisition from host proteins, were also identified. In addition to the chemotaxis and flagella-related genes, the L. hongkongensis genome also contained two copies of qseB/qseC homologues of the AI-3 quorum sensing system.

Conclusions

The large number of diverse transporters and genes involved in chemotaxis, motility and quorum sensing suggested that the bacterium may utilize a complex system to adapt to different environments. Structural modeling will provide useful insights on the transporters in L. hongkongensis.     

Safety and Immunogenicity of DNA and MVA HIV-1 Subtype C Vaccine Prime-Boost Regimens: A Phase I Randomised Trial in HIV-Uninfected Indian Volunteers

Study Design

A randomized, double-blind, placebo controlled phase I trial.

Methods

The trial was conducted in 32 HIV-uninfected healthy volunteers to assess the safety and immunogenicity of prime-boost vaccination regimens with either 2 doses of ADVAX, a DNA vaccine containing Chinese HIV-1 subtype C env gp160, gag, pol and nef/tat genes, as a prime and 2 doses of TBC-M4, a recombinant MVA encoding Indian HIV-1 subtype C env gp160, gag, RT, rev, tat, and nef genes, as a boost in Group A or 3 doses of TBC-M4 alone in Group B participants. Out of 16 participants in each group, 12 received vaccine candidates and 4 received placebos.

Results

Both vaccine regimens were found to be generally safe and well tolerated. The breadth of anti-HIV binding antibodies and the titres of anti-HIV neutralizing antibodies were significantly higher (p<0.05) in Group B volunteers at 14 days post last vaccination. Neutralizing antibodies were detected mainly against Tier-1 subtype B and C viruses. HIV-specific IFN-γ ELISPOT responses were directed mostly to Env and Gag proteins. Although the IFN-γ ELISPOT responses were infrequent after ADVAX vaccinations, the response rate was significantly higher in group A after 1^st and 2^nd MVA doses as compared to the responses in group B volunteers. However, the priming effect was short lasting leading to no difference in the frequency, breadth and magnitude of IFN-γELISPOT responses between the groups at 3, 6 and 9 months post-last vaccination.

Conclusions

Although DNA priming resulted in enhancement of immune responses after 1^st MVA boosting, the overall DNA prime MVA boost was not found to be immunologically superior to homologous MVA boosting.

Trial Registration

Clinical Trial Registry CTRI/2009/091/000051     

Roles of his205, his296, his303 and Asp259 in catalysis by NAD+-specific D-lactate dehydrogenase.

The role of three histidine residues (His205, His296 and His303) and Asp259, important for the catalysis of NAD+-specific D-lactate dehydrogenase, was investigated using site-directed mutagenesis. None of these residues is presumed to be involved in coenzyme binding because Km for NADH remained essentially unchanged for all the mutant enzymes. Replacement of His205 with lysine resulted in a 125-fold reduction in kcat and a slight lowering of the Km value for pyruvate. D259N mutant showed a 56-fold reduction in kcat and a fivefold lowering of Km. The enzymatic activity profile shifted towards acidic pH by approximately 2 units. The H303K mutation produced no significant change in kcat values, although Km for pyruvate increased fourfold. Substitution of His296 with lysine produced no significant change in kcat values or in Km for substrate. The results obtained suggest that His205 and Asp259 play an important role in catalysis, whereas His303 does not. This corroborates structural information available for some members of the D-specific dehydrogenases family. The catalytic His296, proposed from structural studies to be the active site acid/base catalyst, is not invariant. Its function can be accomplished by lysine and this has significant implications for the enzymatic mechanism.     

Growth and differentiation of callus cultures of Pinus

Segments of hypocotyl and cotyledons of aseptically-grown seedlings of Pinus strobus L. (white pine) and P. echinata Mill (shortleaf pine) were used as explants for establishing tissue cultures. Growth and differentiation of callus were studied on a modified Murashige and Skoog's medium containing nutrients and plant growth regulators. Meristems below the surface of callus tissue of P. strobus could be induced on media supplemented with -naphthaleneacetic acid alone or in combination with certain other plant growth regulators. Occasionally, differentiation of shoot buds also occurred on callus cultures. These shoot buds could be grown in vitro but roots did not develop.Abbreviations ABA abscisic acid - BA 6-Benzyl-aminopurine - 2-ip N⁶-(²-isopentanyl)-adenine - GD Gresshoff and Doy's medium - GE Gamborg and Eveleigh's medium - MS Modified Murashige and Skoog's medium - NAA -naphthaleneacetic acid - SC Sommer and Caldas' medium - TIBA 2,3,5-Triiodobenzoic acid