Reconstituted basement membrane potentiates in vivo adipogenesis of 3T3-F442A cells

Adipocytes forming fat pad in vivo are surrounded by well developed basement membranes. Synthesis of basement membrane is enhanced during in vitro differentiation of preadipocyte line. In order to know the role of basement membrane in adipogenesis in vivo, we injected 3T3-F442A preadipocytes subcutaneously into nude mice together with or without the reconstituted basement membrane, Matrigel. Histological sections of the fat pads newly formed by injecting the cell alone showed dense population of immature adipocytes and microvessels within 2 weeks and they matured rapidly. In contrast, injection of the cells together with Matrigel showed sparse adipocytes after 2 weeks and they matured slowly over the period of 6 weeks. Quantification of the process by measuring the weight, DNA content, triglyceride content and glycerophosphate dehydrogenase (GPDH) activity of the fat pads showed that injection of the cell alone resulted in early maturation of adipose tissue with fewer adipocytes while the presence of Matrigel decelerated but potentiated the maturation of adipose tissue with 2 fold contents of DNA, triglyceride and GPDH activity. We thus showed that reconstituted basement membrane (Matrigel) supported the survival and maturation of adipocytes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ascorbic acid stimulates barrier function of cultured endothelial cell monolayer

The macromolecular permeability of cultured bovine aortic, bovine venous, and human umbilical vein endothelial cell monolayers was decreased significantly in culture medium containing L-ascorbic acid (Asc Acid; 0.01–0.1 mM) and L-ascorbic acid 2-phosphate (Asc 2-P). Dithiothreitol, which shows reducing activity equivalent to that of Asc Acid, did not affect endothelial permeability. Asc Acid induced a sixfold increase in collagen synthesis by the endothelial cells. The coexistence of L-azetidine 2-carboxylic acid, an inhibitor of collagen synthesis, attenuated the effect of Asc 2-P in a dose-dependent manner. Another collagen synthesis inhibitor, ethyl-3,4-dihydroxybenzoate, also inhibited collagen synthesis and increased endothelial permeability. The decrease in permeability of the endothelial monolayer was dependent on a reduction of the permeability coefficient of the endothelial monolayer. These findings indicate that endothelial barrier function is stimulated by Asc Acid via an increase in collagen synthesis. © 1995 Wiley-Liss, Inc.     

The effect of linker length on binding affinity of a photoswitchable molecular glue for DNA

Molecular glue for DNA is a small synthetic ligand that adheres two single-stranded DNAs to produce a double-stranded DNA. We previously devised a photoswitchable molecular glue (PMG) that uses external light stimuli to reversibly control DNA hybridization. To optimize the structure of PMG, we synthesized a series of PMGs and evaluated the effect of changing the methylene linker length on the binding affinity and photoresponse. From the comprehensive T_m and CSI-TOF-MS measurements, a PMG possessing a three-methylene linker with carbamate linkage produced maximum binding affinity and photoswitching ability. These results indicate that a small difference in the linker can significantly affect PMG function. These findings are useful for designing new photoswitchable DNA-binding ligands.     

An attelabid weevil (Euops splendida) cultivates fungi

An attelabid weevil,Euops splendida, the female of which makes leaf-roll cradles, is symbiotic with a fungus. She possesses a set of fungus-cultivating structures: spore reservoir, spore incubator, and spore bed, and also comb plate which is used for sowing spores in the nibbled sites on the cradle leaf. Some parts of this paper were presented in a symposium at the 45th Annual Meeting of the Entomological Society of Japan (Sakurai 1985).     

Differentiation of Human Skeletal Muscle Stem Cells into Odontoblasts Is Dependent on Induction of α1 Integrin Expression

Skeletal muscle stem cells represent an abundant source of autologous cells with potential for regenerative medicine that can be directed to differentiate into multiple lineages including osteoblasts and adipocytes. In the current study, we found that α7 integrin-positive human skeletal muscle stem cells (α7⁺hSMSCs) could differentiate into the odontoblast lineage under specific inductive conditions in response to bone morphogenetic protein-4 (BMP-4). Cell aggregates of FACS-harvested α7⁺hSMSCs were treated in suspension with retinoic acid followed by culture on a gelatin scaffold in the presence of BMP-4. Following this protocol, α7⁺hSMSCs were induced to down-regulate myogenic genes (MYOD and α7 integrin) and up-regulate odontogenic markers including dentin sialophosphoprotein, matrix metalloproteinase-20 (enamelysin), dentin sialoprotein, and alkaline phosphatase but not osteoblastic genes (osteopontin and osteocalcin). Following retinoic acid and gelatin scaffold/BMP-4 treatment, there was a coordinated switch in the integrin expression profile that paralleled odontoblastic differentiation where α1β1 integrin was strongly up-regulated with the attenuation of muscle-specific α7β1 integrin expression. Interestingly, using siRNA knockdown strategies revealed that the differentiation-related expression of the α1 integrin receptor positively regulates the expression of the odontoblastic markers dentin sialophosphoprotein and matrix metalloproteinase-20. These results strongly suggest that the differentiation of α7⁺hSMSCs along the odontogenic lineage is dependent on the concurrent expression of α1 integrin.     

Seasonal variations in bacterial communities and antibiotic-resistant strains associated with green bottle flies (Diptera: Calliphoridae)

Green bottle flies occur frequently around human environments in Japan. Many species of green bottle flies have been studied with regard to their importance in forensic examinations or clinical therapies, but the bacterial communities associated with this group of flies have not been comprehensively investigated. In this research, 454 pyrosequencing was used to reveal the bacterial communities in green bottle flies collected in different seasons. Meanwhile, the bacteria were screened with selective media and tested for antibiotic susceptibility. Samples collected in three different seasons harbored distinctive bacterial communities. The predominant genera associated with green bottles flies were Staphylococcus in spring, Ignatzschineria in summer, and Vagococcus, Dysgonomonas, and an unclassified Acetobacteraceae in autumn. An upward trend in bacterial community diversity was observed from spring to autumn. Changes in climatic conditions could be the cause of these seasonal variations in fly-associated bacterial communities. The species of isolated antibiotic-resistant bacteria also differed across seasons, but it was difficult to correlate seasonal changes in antibiotic-resistant bacteria with changes in whole communities. A number of multiple-antibiotic-resistant bacteria were isolated, and some of these strains were closely affiliated with pathogens such as Enterococcus faecalis and Enterococcus faecium, which could cause serious threats to public health. Overall, this research provided us with information about the composition and seasonality of bacterial communities in green bottle flies, and highlighted the risks of fly-mediated dissemination of antibiotic-resistant pathogens.     

Study of the role of anaerobic metabolism in succinate production by Enterobacter aerogenes

Succinate is a core biochemical building block; optimizing succinate production from biomass by microbial fermentation is a focus of basic and applied biotechnology research. Lowering pH in anaerobic succinate fermentation culture is a cost-effective and environmentally friendly approach to reducing the use of sub-raw materials such as alkali, which are needed for neutralization. To evaluate the potential of bacteria-based succinate fermentation under weak acidic (pH <6.2) and anaerobic conditions, we characterized the anaerobic metabolism of Enterobacter aerogenes AJ110637, which rapidly assimilates glucose at pH 5.0. Based on the profile of anaerobic products, we constructed single-gene knockout mutants to eliminate the main anaerobic metabolic pathways involved in NADH re-oxidation. These single-gene knockout studies showed that the ethanol synthesis pathway serves as the dominant NADH re-oxidation pathway in this organism. To generate a metabolically engineered strain for succinate production, we eliminated ethanol formation and introduced a heterogeneous carboxylation enzyme, yielding E. aerogenes strain ΔadhE/PCK. The strain produced succinate from glucose with a 60.5 % yield (grams of succinate produced per gram of glucose consumed) at pH <6.2 and anaerobic conditions. Thus, we showed the potential of bacteria-based succinate fermentation under weak acidic conditions.     

Ozakia,a new genus of winged fruit shared between the Miocene of Japan and western North America

A new genus is recognized based on winged fruits with a single species shared between the Miocene of southwestern Honshu, Japan, and the Miocene of Oregon and Idaho, USA. Calyces of Ozakia emryi gen. et sp. n. were formerly attributed to Heptacodium (Caprifoliaceae) and Amelanchier (Rosaceae); however, newly recovered specimens reveal additional characters that contradict these assignments. The pedicellate fruits are obovate, tapering basally and truncate apically, with about 10 longitudinal ribs, a prominent epigynous synsepalous calyx of five lobes, each with a midvein and a pair of weaker, ascending intramarginal primary veins. The single style has a capitate stigma. Ozakia is considered to represent an extinct eudicot genus, the familial affinities of which remain uncertain. The eastern Asian–western North American disjunction of Ozakia occurrences suggests that this plant traversed the Beringia land bridge during or prior to the Middle Miocene. Relatively few extinct angiosperm genera are known as late as the Miocene.