The specificity of alcohol dehydrogenase with cis-retinoids. Activity with 11-cis-retinol and localization in retina.

Studies in knockout mice support the involvement of alcohol dehydrogenases ADH1 and ADH4 in retinoid metabolism, although kinetics with retinoids are not known for the mouse enzymes. Moreover, a role of alcohol dehydrogenase (ADH) in the eye retinoid interconversions cannot be ascertained due to the lack of information on the kinetics with 11-cis-retinoids. We report here the kinetics of human ADH1B1, ADH1B2, ADH4, and mouse ADH1 and ADH4 with all-trans-, 7-cis-, 9-cis-, 11-cis- and 13-cis-isomers of retinol and retinal. These retinoids are substrates for all enzymes tested, except the 13-cis isomers which are not used by ADH1. In general, human and mouse ADH4 exhibit similar activity, higher than that of ADH1, while mouse ADH1 is more efficient than the homologous human enzymes. All tested ADHs use 11-cis-retinoids efficiently. ADH4 shows much higher k(cat)/K(m) values for 11-cis-retinol oxidation than for 11-cis-retinal reduction, a unique property among mammalian ADHs for any alcohol/aldehyde substrate pair. Docking simulations and the kinetic properties of the human ADH4 M141L mutant demonstrated that residue 141, in the middle region of the active site, is essential for such ADH4 specificity. The distinct kinetics of ADH4 with 11-cis-retinol, its wide specificity with retinol isomers and its immunolocalization in several retinal cell layers, including pigment epithelium, support a role of this enzyme in the various retinol oxidations that occur in the retina. Cytosolic ADH4 activity may complement the isomer-specific microsomal enzymes involved in photopigment regeneration and retinoic acid synthesis.     

Bleomycin-induced pulmonary fibrosis is attenuated by a monoclonal antibody targeting HER2.

The importance of HER2/HER3 signaling in decreasing the effects of lung injury was recently demonstrated. Transgenic mice unable to signal through HER2/HER3 had significantly less bleomycin-induced pulmonary fibrosis and showed a survival benefit. Based on these data, we hypothesized that pharmacological blockade of HER2/HER3 in vivo in wild-type mice would have the same beneficial effects. We tested this hypothesis in a bleomycin lung injury model using 2C4, a monoclonal antibody directed against HER2 that blocks HER2/HER3 signaling. The administration of 2C4 before injury decreased the effects of bleomycin at days 15 and 21 after injury. HER2/HER3 blockade resulted in less collagen deposition (362.8 +/- 37.9 compared with 610.5 +/- 27.1 microg/mg; P = 0.03) and less lung morphological changes (injury score of 1.99 +/- 1.55 vs. 3.90 +/- 0.76; P < 0.04). In addition, HER2/HER3 blockade resulted in a significant survival advantage with 50% vs. 25% survival at 30 days (P = 0.04). These results confirm that HER2 signaling can be pharmacologically targeted to reduce lung fibrosis and remodeling after injury.     

The carboxylic acid transporters Jen1 and Jen2 affect the architecture and fluconazole susceptibility of Candida albicans biofilm in the presence of lactate

Candida albicans has the ability to adapt to different host niches, often glucose-limited but rich in alternative carbon sources. In these glucose-poor microenvironments, this pathogen expresses JEN1 and JEN2 genes, encoding carboxylate transporters, which are important in the early stages of infection. This work investigated how host microenvironments, in particular acidic containing lactic acid, affect C. albicans biofilm formation and antifungal drug resistance. Multiple components of the extracellular matrix were also analysed, including their impact on antifungal drug resistance, and the involvement of both Jen1 and Jen2 in this process. The results show that growth on lactate affects biofilm formation, morphology and susceptibility to fluconazole and that both Jen1 and Jen2 might play a role in these processes. These results support the view that the adaptation of Candida cells to the carbon source present in the host niches affects their pathogenicity.     

A New Depigmenting‐Antifungal Methylated Grindelane from Grindelia chiloensis

The new methylated grindelane diterpenoid, 7β ‐hydroxy‐8(17)‐dehydrogrindelic acid ( 1b ), together with the known 7α ‐hydroxy‐8(17)‐dehydrogrindelic acid ( 2a ), 6‐oxogrindelic acid ( 3a ), 4β ‐hydroxy‐6‐oxo‐19‐norgrindelic ( 4a ), 19‐hydroxygrindelic acid ( 5a ), 18‐hydroxygrindelic acid ( 6a ), 4α ‐carboxygrindelic acid ( 7a ), 17‐hydroxygrindelic acid ( 8a ), 6α ‐hydroxygrindelic acid ( 9a ), 8,17‐bisnor‐8‐oxagrindelic acid ( 10a ), 7α ,8α ‐epoxygrindelic acid ( 11a ), and strictanonic acid ( 12a ) as methyl esters were obtained from an Argentine collection of Grindelia chiloensis (Cornel .) Cabrera . Their structures and relative configurations were established on the basis of spectroscopic analysis. CHC l₃ extract from the aerial parts and their pure compounds were evaluated for their antifungal and depigmenting effects. Methyl ester derivative of 10a ( 10b ) exhibited a remarkable mycelial growth inhibition against Botritis cinerea with an IC ₅₀ of 13.5 μg ml^?1. While the new grindelane 1b exerted a clear color reduction of the yellow‐orange pigment developed by Fusarium oxysporum against UV ‐induced damage.     

Loss of nucleoplasmic LAP2alpha-lamin A complexes causes erythroid and epidermal progenitor hyperproliferation

Lamina-associated polypeptide (LAP) 2alpha is a chromatin-associated protein that binds A-type lamins. Mutations in both LAP2alpha and A-type lamins are linked to human diseases called laminopathies, but the molecular mechanisms are poorly understood. The A-type lamin-LAP2alpha complex interacts with and regulates retinoblastoma protein (pRb), but the significance of this interaction in vivo is unknown. Here we address the function of the A-type lamin-LAP2alpha complex with the use of LAP2alpha-deficient mice. We show that LAP2alpha loss causes relocalization of nucleoplasmic A-type lamins to the nuclear envelope and impairs pRb function. This causes inefficient cell-cycle arrest in dense fibroblast cultures and hyperproliferation of epidermal and erythroid progenitor cells in vivo, leading to tissue hyperplasia. Our results support a disease-relevant model in which LAP2alpha defines A-type lamin localization in the nucleoplasm, which in turn affects pRb-mediated regulation of progenitor cell proliferation and differentiation in highly regenerative tissues.     

New retinoid chemotypes: 9-cis-retinoic acid analogs with hydrophobic rings derived from terpenes as selective RAR agonists

A series of 9-cis-retinoic acid analogs modified at the hydrophobic ring with a (bi)cyclohexenyl moiety derived from natural terpenes has been stereoselectively prepared using a Suzuki cross-coupling as key step. Transient transactivation studies indicate that modification of the hydrophobic ring impacts dramatically on RXR-binding and transactivation, with most retinoids being inactive on RXRbeta, while preserving their RAR pan-agonist profile. Furthermore, only the RARgamma subtype was capable of enantiomeric discrimination with some pairs of enantiomeric terpene-retinoids.     

Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains

Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.     

Vibrio parahaemolyticus ScrC modulates cyclic dimeric GMP regulation of gene expression relevant to growth on surfaces

In Vibrio parahaemolyticus, scrC participates in controlling the decision to be a highly mobile swarmer cell or a more adhesive, biofilm-proficient cell type. scrC mutants display decreased swarming motility over surfaces and enhanced capsular polysaccharide production. ScrC is a cytoplasmic membrane protein that contains both GGDEF and EAL conserved protein domains. These domains have been shown in many organisms to respectively control the formation and degradation of the small signaling nucleotide cyclic dimeric GMP (c-di-GMP). The scrC gene is part of the three-gene scrABC operon. Here we report that this operon influences the cellular nucleotide pool and that c-di-GMP levels inversely modulate lateral flagellar and capsular polysaccharide gene expression. High concentrations of this nucleotide prevent swarming and promote adhesiveness. Further, we demonstrate that ScrC has intrinsic diguanylate cyclase and phosphodiesterase activities, and these activities are controlled by ScrAB. Specifically, ScrC acts to form c-di-GMP in the absence of ScrA and ScrB; whereas ScrC acts to degrade c-di-GMP in the presence of ScrA and ScrB. The scrABC operon is specifically induced by growth on a surface, and the analysis of mutant phenotypes supports a model in which the phosphodiesterase activity of ScrC plays a dominant role during surface translocation and in biofilms.     

Molecular Cloning of α-Amylases from Cotton Boll Weevil, Anthonomus grandis and Structural Relations to Plant Inhibitors: An Approach to Insect Resistance

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of α-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two α-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5′ and 3′ RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7 kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several α-amylase inhibitors from plants were assayed against A. grandis α-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and α-AI1 inhibitors on A. grandis α-amylase activity. This work suggests that genetic engineering of cotton to express α-amylase inhibitors may offer a novel route to A. grandis resistance.