Complementation of the tomato HWS gene with its Arabidopsis counterpart demonstrates conservation of the gene function between both species

The HAWAIIAN SKIRT (HWS) gene was originally described in Arabidopsis for the characteristic fusion of sepals in the mutant. A tomato line mutated in the putative ortholog gene was isolated in a previous study. The tomato hws-1 mutant showed facultative parthenocarpy and produced fruits with elevated Brix, revealing the gene as a hopeful resource for crop improvement. To confirm the orthology relationship between the Arabidopsis and tomato HWS genes, the hws-1 mutant was complemented with either the tomato wild-type genomic fragment or the Arabidopsis sequence of the gene. In both complementation experiments, defective phenotypes of hws-1 are rescued, albeit to different extents. Recovery of these phenotypes, which include parthenocarpic fruit production, increased Brix, loss of leaflet serration, alteration of bud and petal shape, firmly establishes SlHWS as an ortholog of the originally described HWS in Arabidopsis. This work indicates that the function of HWS is likely to be conserved in a wide range of plant species.     

The kinesin KIF4 mediates HBV/HDV entry through the regulation of surface NTCP localization and can be targeted by RXR agonists in vitro

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection. KIF4 knockdown suppressed both HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 mutant regained the surface localization of NTCP and significantly restored HBV permissiveness in these cells. IF revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study revealed that KIF4 interacts with NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent HBV infection in both HepG2-hNTCP and primary human hepatocyte (PXB) (Bexarotene, IC₅₀ 1.89 ± 0.98 μM) cultures. Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates targeting HBV and HDV entry.     

Aerobic degradation of lindane (γ-hexachlorocyclohexane) in bacteria and its biochemical and molecular basis

γ-Hexachlorocyclohexane (γ-HCH, also called γ-BHC and lindane) is a halogenated organic insecticide that causes serious environmental problems. The aerobic degradation pathway of γ-HCH was extensively revealed in bacterial strain Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26. γ-HCH is transformed to 2,5-dichlorohydroquinone through sequential reactions catalyzed by LinA, LinB, and LinC, and then 2,5-dichlorohydroquinone is further metabolized by LinD, LinE, LinF, LinGH, and LinJ to succinyl-CoA and acetyl-CoA, which are metabolized in the citrate/tricarboxylic acid cycle. In addition to these catalytic enzymes, a putative ABC-type transporter system encoded by linKLMN is also essential for the γ-HCH utilization in UT26. Preliminary examination of the complete genome sequence of UT26 clearly demonstrated that lin genes for the γ-HCH utilization are dispersed on three large circular replicons with sizes of 3.5 Mb, 682 kb, and 191 kb. Nearly identical lin genes were also found in other HCH-degrading bacterial strains, and it has been suggested that the distribution of lin genes is mainly mediated by insertion sequence IS6100 and plasmids. Recently, it was revealed that two dehalogenases, LinA and LinB, have variants with small number of amino acid differences, and they showed dramatic functional differences for the degradation of HCH isomers, indicating these enzymes are still evolving at high speed.     

The transformation-sensitive heat shock protein (hsp47) binds specifically to Fetuin

The transformation-sensitive heat shock protein of Mr = 47,000 (hsp47) has been shown to bind to collagen and gelatin. We examined the binding specificity of hsp47. The binding of hsp47 to gelatin-Sepharose 4B was competitively inhibited by fetuin as effectively as by gelatin or collagen, whereas a variety of other proteins tested had no effect. Fetuin-coupled Sepharose 4B was found to bind hsp47 even at high ionic strength, but the complex was dissociated at pH less than or equal to 5.5.)     

Heterogeneous dissolved organic nitrogen supply over a coral reef: first evidence from nitrogen stable isotope ratios

Dissolved organic nitrogen (DON) potentially plays a major role in sustaining the high productivity and biological diversity of coral reefs. However, data are scarce regarding sources and sinks of DON. This study, for the first time, determined the ¹⁵N isotopic composition of total dissolved nitrogen (δ¹⁵N_TDN), reflecting the isotopic signature of DON, in the water column over a coral reef. The uniformity in δ¹⁵N_TDN during high tide (3.2 ± 0.3 ‰) indicated that the DON was mainly derived from offshore waters. In contrast, higher spatial heterogeneity of δ¹⁵N_TDN (3.1 ± 0.9 ‰) and DON concentrations during low tide indicated the existence of local DON sources patchily distributed over the reef. Low δ¹⁵N_TDN values located mid-reef were indicative of DON release from organisms that obtained their N via N₂ fixation, whereas high δ¹⁵N_TDN appeared to reflect localized release of DON by organisms exposed to dissolved inorganic nitrogen with elevated ¹⁵N, such as from terrestrial and offshore inputs. Collectively, the results highlight the importance of spatial patterns in DON release from reef communities in the N cycling of coral reefs.     

The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen.

The cell surface Fas antigen is a membrane-associated polypeptide which can mediate apoptosis. cDNA clones encoding the Fas antigen were isolated from a cDNA library constructed with mRNA from the mouse macrophage cell line BAM3. The nucleotide sequence and the deduced amino acid sequence of the mouse Fas antigen were 58.5 and 49.3% identical, respectively, to the corresponding sequences of human Fas antigen cDNA. The mouse Fas antigen consists of 306 amino acids with a calculated Mr of 34,971 and contains a single transmembrane domain which divides the molecule into extracellular and cytoplasmic domains. A 2.1-kb mRNA coding for the Fas antigen was detected in the mouse thymus, heart, liver, and ovary but not in brain and spleen. The expression of the Fas antigen gene in mouse fibroblast L929 and macrophage BAM3 cell lines was significantly induced by treatment with IFN-gamma but not by IFN-alpha/beta. Interspecific backcross analysis indicated that the gene coding for the Fas antigen is in the distal region of mouse chromosome 19.     

Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly)₁₀, suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2′ positions, which may be attributed to the larger space available for substrate binding at the S2 and S2′ sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase.     

On the preparation of indoxyl red from indican and some new characteristics

An indole compound with a strong purple–red color was produced by boiling a solution of indican under acidic conditions and purified by chromatographies on DEAE-650S Toyopearl TSK-gel and silica-gel columns. The purple-red compound purified was identified as indoxyl red, on the basis of FAB Mass, ¹³C NMR, ¹H NMR, UV–visible spectra, and IR spectra. Although indoxyl red was first synthesized by Seidel⁹ 70 years ago, very little information has been available on its characteristics. We repot here that the compound was purple-red colored at acidic pH and green at pH 13, and showed antiproliferative and cytotoxic activities to the mouse B cell lymphoma cell line NSF202.     

Changes of colon epithelium proliferation due to individual aging with cyclin proliferating cell nuclear antigen (PCNA/cyclin) immunostaining compared to [3H]-thymidine radioautography

We report a change in the proliferative activity of mouse colonic epithelium due to development and aging. In order to measure the proliferative activity, colonic epithelium was immunostained for cyclin proliferating cell nuclear antigen (PCNA/cyclin), which appears from the Gl to the S phase of the cell cycle, and compared with labeling obtained by [³H]-thymidine radioautography. Litter mice of six age groups from the fetal period (embryonic day 19), newborn period (postnatal day 1), suckling period (postnatal day 5), weaning period (postnatal dy 21), adult period (2 month old) to the senescent period (11 month old) were examined by immunohistochemistry. The descending colons were fixed in methacarn (method-Carnoy) and embedded in paraffin. Sections were stained for PCNA/cyclin activity using 19A2 monoclonal antibody and the avidin-biotin peroxidase complex (ABC) technique. For radioautography, litter mice of nine age groups using in vivo intraperitoneal administration of [³H]-thymidine. The labeling indices of colonic epithelial cells in the proliferative zone were then analyzed and compared between the two investigative methods. Our results show that the prliferative activity of mice colon was high in the fetal and newborn periods and almost constant from the suckling period to senescence, as demonstrated by both PCNA/cyclin immunohistochemistry and [³H]-thymidine radioautography. The labeling index seen by PCNA/cyclin immunohistochemistry was, however, higher than that seen by [³H]-thymidine radioautography.