Mycobacteria Exploit Host Hyaluronan for Efficient Extracellular Replication

In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.     

Chemical and Genetic Studies on Hybrid of Ligularia subspicata and Ligularia cyathiceps Collected in Yunnan Province of China

Two morphologically ambiguous Ligularia samples (samples A and B), and samples with morphology of Ligularia subspicata (sample C), Ligularia lamarum (sample D), or Ligularia cyathiceps (sample E), were collected at Tianchi Pond, Shangrila County, Yunnan Province, China. Analysis of the nucleotide sequence of the internal transcribed spacers (ITSs) in the nuclear ribosomal RNA gene cluster indicated that not only sample B but also sample D was a hybrid of L. cyathiceps and L. lamarum/L. subspicata. Although the morphology of sample A suggested that it was also a hybrid, the ITS sequence of sample A was that of L. cyathiceps. Twenty compounds were isolated from the five samples, and the structures of two new compounds 7 and 14 were determined. Furanoeremophilanes typical of L. lamarum/L. subspicata were detected not only in samples C and D, but also in samples A and B. These results indicate that the ability to produce root chemicals can spread through introgression.     

Identification of genes and pathways involved in the synthesis of Mead acid (20:3n − 9), an indicator of essential fatty acid deficiency

In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n − 9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1–6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n − 9, 20:1n − 9 and 20:2n − 9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n − 9 → (Fads2) → 18:2n − 9 → (Elovl5) → 20:2n − 9 → (Fads1) → 20:3n − 9 and pathway 2) 18:1n − 9 → (Elovl5) → 20:1n − 9 → (Fads2) → 20:2n − 9 → (Fads1) → 20:3n − 9.     

Hypomethylated Pollen Bypasses the Interploidy Hybridization Barrier in Arabidopsis

Plants of different ploidy levels are separated by a strong postzygotic hybridization barrier that is established in the endosperm. Deregulated parent-of-origin specific genes cause the response to interploidy hybridizations, revealing an epigenetic basis of this phenomenon. In this study, we present evidence that paternal hypomethylation can bypass the interploidy hybridization barrier by alleviating the requirement for the Polycomb Repressive Complex 2 (PRC2) in the endosperm. PRC2 epigenetically regulates gene expression by applying methylation marks on histone H3. Bypass of the barrier is mediated by suppressed expression of imprinted genes. We show that the hypomethylated pollen genome causes de novo CHG methylation directed to FIS-PRC2 target genes, suggesting that different epigenetic modifications can functionally substitute for each other. Our work presents a method for the generation of viable triploids, providing an impressive example of the potential of epigenome manipulations for plant breeding.     

Developmental Changes in the NGF Content in the Brain of Young,Growing, Low-Birth-Weight Rats

The NGF content in each region of the brain of four-week-old rats was ranked in the decreasing order of cerebral cortex, hippocampus, cerebellum, midbrain/diencephalon, and pons/medulla ob-longata, and the NGF concentration, in the decreasing order of hippocampus, cerebral cortex, cerebellum, midbrain/diencephalon, and pons/medulla oblongata in both AFD and SFD groups. The NGF content and concentration in the cerebral cortex were about the same value at each age between those in the AFD and SFD groups. Those in the hippocampus were a little higher in the SFD group than in the AFD group at the ages of three and four weeks, unlike those in the other regions, where the values for the cerebellum, midbrain/diencephalon and pons/medulla oblongata tended to be somewhat higher in the AFD group than in the SFD group. The NGF concentrations in the hippocampus and cerebral cortex increased with growth: the concentration in the hippocampus at four weeks of age was about 4-fold of that at one week in the AFD group and about 5.7-fold of that at one week in the SFD group; and likewise the concentration in the cerebral cortex at four weeks of age was about 5.3-fold in the AFD group and about 7-fold in the SFD group. The NGF concentrations in the cerebellum decreased, and those in midbrain/diencephalon and pons/medulla oblongata hardly changed with growth in either AFD or SFD group. From these results NGF may have stronger implications for the neuronal growth in the hippocampus compared with those in the lower brain regions of the SFD rats.     

Activation of peroxisome proliferator-activated receptor-alpha stimulates both differentiation and fatty acid oxidation in adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a dietary lipid sensor, whose activation results in hypolipidemic effects. In this study, we investigated whether PPARα activation affects energy metabolism in white adipose tissue (WAT). Activation of PPARα by its agonist (bezafibrate) markedly reduced adiposity in KK mice fed a high-fat diet. In 3T3-L1 adipocytes, addition of GW7647, a highly specific PPARα agonist, during adipocyte differentiation enhanced glycerol-3-phosphate dehydrogenase activity, insulin-stimulated glucose uptake, and adipogenic gene expression. However, triglyceride accumulation was not increased by PPARα activation. PPARα activation induced expression of target genes involved in FA oxidation and stimulated FA oxidation. In WAT of KK mice treated with bezafibrate, both adipogenic and FA oxidation-related genes were significantly upregulated. These changes in mRNA expression were not observed in PPARα-deficient mice. Bezafibrate treatment enhanced FA oxidation in isolated adipocytes, suppressing adipocyte hypertrophy. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα was recruited to promoter regions of both adipogenic and FA oxidation-related genes in the presence of GW7647 in 3T3-L1 adipocytes. These findings indicate that the activation of PPARα affects energy metabolism in adipocytes, and PPARα activation in WAT may contribute to the clinical effects of fibrate drugs.     

Inhibitory effect of naringenin chalcone on inflammatory changes in the interaction between adipocytes and macrophages

Obese adipose tissue is characterized by an enhanced infiltration of macrophages. It is considered that the paracrine loop involving monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-alpha between adipocytes and macrophages establishes a vicious cycle that augments the inflammatory changes and insulin resistance in obese adipose tissue. Polyphenols, which are widely distributed in fruit and vegetables, can act as antioxidants and some of them are also reported to have anti-inflammatory properties. Tomato is one of the most popular and extensively consumed vegetable crops worldwide, which also contains many flavonoids, mainly naringenin chalcone. We investigated the effect of flavonoids, including naringenin chalcone, on the production of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated macrophages and in the interaction between adipocytes and macrophages. Naringenin chalcone inhibited the production of TNF-alpha, MCP-1, and nitric oxide (NO) by LPS-stimulated RAW 264 macrophages in a dose-dependent manner. Coculture of 3T3-L1 adipocytes and RAW 264 macrophages markedly enhanced the production of TNF-alpha, MCP-1, and NO compared with the control cultures; however, treatment with naringenin chalcone dose-dependently inhibited the production of these proinflammatory mediators. These results indicate that naringenin chalcone exhibits anti-inflammatory properties by inhibiting the production of proinflammatory cytokines in the interaction between adipocytes and macrophages. Naringenin chalcone may be useful for ameliorating the inflammatory changes in obese adipose tissue.     

Changes in the levels of neural cell specific proteins in the developing rat brain

The levels of S-100 protein (S-100) and neuron-specific enolase (NSE) in the developing rat brain were determined by a sensitive enzyme immunoassay and the results were compared with those obtained by other methods. Changes with development in the levels of S-100, NSE, and 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase), biochemical markers for astroglia, neurons and oligodendroglia respectively, were determined in various brain regions including the cerebral hemisphere (CH), brain stem (BS) and cerebellum (Ce). The peak increments of S-100, NSE, and CNPase activity were reached later than that of the brain weight in all of the regions. The ratios of S-100/NSE and CNPase/NSE rose during the 21 days after birth in the CH and BS; the S-100/NSE ratio in the CH began to decrease from the 21st day, whereas the CNPase/NSE ratio continued to rise even after the 30th day, suggesting different maturation periods of the different glial cells. In the Ce, the change of these ratios showed a pattern different from those in the other regions. In the CH of rats with experimental microencephaly induced by methylazoxymethanol (MAM), the ratios were almost normal, in spite of the reduction of the brain weight to about 50% of the control.Dedicated to Professor Yasuzo Tsukada.     

Characterization of an aryl-alcohol oxidase from the plant saprophytic basidiomycete <Emphasis Type="Italic">Coprinopsis cinerea</Emphasis> with broad substrate specificity against aromatic alcohols

Objectives

The aim of the study was to obtain information about the enzymatic properties of aryl-alcohol oxidase from the plant saprophytic basidiomycete Coprinopsis cinerea (rCcAAO), which is classified into the auxiliary activities family 3 subfamily 2 (AA3_2).

Results

The gene encoding AAO from the plant saprophytic basidiomycete Coprinopsis cinerea (CcAAO) was cloned, and the recombinant CcAAO (rCcAAO) was heterologously expressed in the methylotrophic yeast Pichia pastoris. The purified rCcAAO showed significant activity not only against trans,trans-2,4-hexadien-1-ol but also against a broad range of aromatic alcohols including aromatic compounds that were reported to be poor substrates for known AAOs. Moreover, site-directed mutagenesis analysis demonstrated that mutants with substitutions from leucine to phenylalanine and tryptophan at position 416 exhibited decreases of activity for aromatic alcohols but still maintained the activity for trans,trans-2,4-hexadien-1-ol.

Conclusions

Leucine 416 in CcAAO contributes to the broad substrate specificity against various aromatic alcohols, which is useful for the production of hydrogen peroxide using this enzyme.

     

Detection and normalization of biases present in spotted cDNA microarray data: a composite method addressing dye, intensity-dependent, spatially-dependent, and print-order biases.

Microarrays are often used to identify target genes that trigger specific diseases, to elucidate the mechanisms of drug effects, and to check SNPs. However, data from microarray experiments are well known to contain biases resulting from the experimental protocols. Therefore, in order to elucidate biological knowledge from the data, systematic biases arising from their protocols must be removed prior to any data analysis. To remove these biases, many normalization methods are used by researchers. However, not all biases are eliminated from the microarray data because not all types of errors from experimental protocols are known. In this paper, we report an effective way of removing various types of biases by treating each microarray dataset independently to detect biases present in the dataset. After the biases contained in each dataset were identified, a combination of normalization methods specifically made for each dataset was applied to remove biases one at a time.