This review describes the relationship between hetero-oligosaccharides and microorganisms. It is possible to prepare aminosugar nucleotides as donors for hetero-oligosaccharide synthesis with a combination of yeast fermentation and bacterial enzymes, and to use the product to test for a rare human blood group. We have isolated various glycosidases produced by microorganisms, mainly from soil, to elucidate the structure and function of hetero-oligosaccharides. Among them, a mold endoglycosidase was found to have specific transglycosylation activity in addition to hydrolysis activity, and we have used it to synthesize chemo-enzymatically various bioactive glycopeptides by the attachment of a hetero-oligosaccharide to a peptide. We found that lactic acid bacteria bound to a hetero-oligosaccharide on the intestinal tract cell surface in animals. We also analyzed the bifidobacterial hetero-oligosaccharide-hydrolyzing enzymes involved in the degradation of mucin glycoprotein in the host intestinal tract and human milk oligosaccharides, and identified a specific saccharide that acted as a bifidobacteria growth factor.

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The highly reactive α-dicarbonyl compounds, glyoxal, methylglyoxal (MGO), and 3-deoxyglucosone, react with the amino groups of proteins to form advanced glycation end-products (AGEs) which have been implicated in diabetic complications, aging, and Alzheimer's disease. We found that a test sample of terpinen-4-ol (T4) containing hydroperoxides showed cleaving activity toward an α-dicarbonyl compound, but that the freshly isolated pure sample did not. Prepared terpinen-4-ol hydroperoxide (T4-H) also efficiently cleaved the C-C bond of the α-dicarbonyl compounds via Baeyer-Villiger-like rearrangement and subsequent hydrolysis of an acid anhydride moiety in the rearranged product to give carboxylic acids. Other terpene hydroperoxides, as well as T4-H, showed significant cleaving activities, and all these hydroperoxides protected RNase A from the lowering of enzyme activity induced by MGO. The cleaving mechanism via Baeyer-Villiger-like rearrangement was confirmed by time-interval NMR measurements of the reaction mixture of the symmetrical α-dicarbonyl compound, diacetyl with T4-H.

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The enantiomers of citronellal were converted to all the stereoisomers of 6-methyl-2-octadecanone, 14-methyl-2-octadecanone, and 6,14-dimethyl-2-octadecanone, the female-produced sex pheromone components of the Lyclene dharma dharma moth. Three well-established procedures, the Wittig reaction, alkylation of alkynes, and acetoacetic ester synthesis, were employed for the carbon-carbon bond formation to connect the building blocks.

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We identified 4-oxo-(E)-2-hexenal (4-OHE) as a common component of the secretion from both Dolycoris baccarum nymphs (66.5 ± 34.7 µg/bug) and adults (87.4 ± 48.0 µg/bug) by GC/MS. We also found that this compound inhibited the growth of bacteria starting at 10 µg. The stronger antibacterial activity of 4-OHE than that of (E)-2-hexenal and (E)-2-octenal might be explained by the reactivity of α,β-unsaturated aldehydes with nucleophilic molecules.

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Dactylellina cionopaga is a potential biocontrol agent of phytoparasitic nematodes. Here the functions of snodprot of D. cionopaga were analysed. The gene was transcribed with a higher level under inducing conditions with nematodes. The recombinant protein expressed in Pichia pastoris had a molecular weight of 14 kDa and might form polymers in its native state. In a concentration-dependent manner, snodprot changed the chemotaxis and increased the body-bend frequency of Caenorhabditis elegans, but did not induce immunity in the indicated plants significantly. The results of an immunofluorescence assay proved that snodprot was expressed during the development of traps and conidia. According to the parasitism mechanisms of nematophagous fungi and the chemotaxis and locomotion mechanisms of C. elegans, the possible active sites of snodprot were speculated to be ASE or ASI. The gene identification indicated that snodprot is a novel parasitism-related protein of nematophagous fungi, and possesses novel activity, different from other members of the cerato-platanin family.

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Human platelet factor 4 (hPF4) was evaluated as a clinical alternative to protamine for heparin neutralization, a protector against radiation injury and an anti-neoplastic. To achieve high-level expression of hPF4, expression vectors pET-28a(+)-nf PF4 (n=4, 5, 6) containing n tandem repeats of PF4 were constructed and transformed into the Escherichia coli BL21(DE3) strain. A higher expression level, about 45% of the total proteins (TP), was obtained for E. coli BL21(DE3)/pET28a(+)-nf PF4 (n=4, 5, 6). The purified His-PF4 protein was further identified by cleavage with enterokinase and MS, and its heparin-neutralizing activity was determined by colony formation assay. This study represents a novel approach to large-scale production of PF4 in E. coli, one that might be applied to large-scale production of PF4 protein for possible clinical application. It also provides theoretical points for the expression and purification of other small-molecule peptides.

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To investigate the effects of the JAK2/STAT3 pathway on skeletal muscle development and energy metabolism, AG490 and IL-11 were used as agonist and inhibitor in treating mice and the mouse skeletal muscle cells. Average body weight (ABW) was reduced significantly in the mice treated with AG490 (p<0.05), while IL-11 had the opposite effect (p<0.05), as compared with the controls, average body temperature (ABT) remained at normal levels in both groups. Western blotting was used to determine the protein levels of JAK2, STAT3, p-JAK2, and p-STAT3. AG490 caused significant decreases in p-JAK2 and p-STAT3 (p<0.05), while IL-11 did the opposite (p<0.05, p<0.01). Quantitative RT-PCR also showed significantly decreased expression levels of Myf5, MyoD, LXRa, and UCP3 in the AG490 group (p<0.01), but in the IL-11 group, the expression of Myf5, MyoD, and UCP3 was increased (p<0.05), except that LXRa whose expression did not change. In cultured skeletal muscle cells, the expression of MyoD, Myf5, LXRa, and UCP3 (p<0.05) exhibited the same trend as that in the skeletal muscles of both treated groups (p<0.05). These results implicate the JAK2/STAT3 in skeletal muscle development and energy metabolism.

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Glechoma hederacea L. (Labiatae) has been used in folk medicine to treat various ailments for centuries. We investigated the effects of G. hederacea extract on melanogenesis in B16 melanoma cells. It significantly reduced both the cellular melanin content and tyrosinase activity in a concentration-dependent manner. An MTT assay did not reveal any obvious cytotoxicity. Furthermore, we found that G. hederacea extract decreased tyrosinase and microphthalmia-associated transcription factor protein expression, but did not inhibit tyrosinase-related protein-1 and tyrosinase-related protein-2 expression. RT-PCR analysis indicated that the antimelanogenic effect of G. hederacea extract might be due to inhibition of tyrosinase gene transcription. Moreover, this effect is regulated via suppression of microphthalmia-associated transcription factor protein expression. Our data indicate that G. hederacea extract inhibits melanin synthesis in B16 melanoma cells but is not cytotoxic. Hence it might prove a useful therapeutic agent for treating hyperpigmentation and an effective component of whitening cosmetics.

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Vanillin is one of the most valuable compounds in the flavoring and fragrance industries, and many attempts to produce natural vanillin have been made in recent years. Isoeugenol monooxygenase (Iem) converts the phenylpropanoid compound isoeugenol to vanillin. In Pseudomonas nitroreducens Jin1, the positive regulatory protein IemR is divergently expressed from Iem, and the promoter region is located between the genes. In this study, we investigated the transcriptional regulation of iem in Escherichia coli. We focused on inducers and regulatory protein IemR. Transcription of iem was found to be dependent on the amounts of isoeugenol and IemR. Isoeugenol was found to be the best inducer of iem, followed by trans-anethole, which induced iem to 58% of the transcription level observed for isoeugenol. Overproduction of IemR in E. coli significantly increased the transcription of iem, up to 96-fold, even in the absence of isoeugenol, as compared to basally expressed IemR. Results of this study indicate that the transcription of iem iss dependent on the type of inducers and on IemR. They should contribute to the development of bioengineering strategies for increased production of vanillin through high-level expression of the isoeugenol monooxygenase gene in microorganisms.

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An optimized method based on tetracycline-inducible gene expression system T-REx was developed to screen and evaluate Tet repressor (TetR)-expressing cell lines using enhanced green fluorescence protein (EGFP) as reporter gene. To verify the effectiveness of the method, two TetR-expressing Chinese hamster ovary (CHO) cell lines, CHO-TR B2 (stringent) and B5 (less stringent), in which the EGFP genes were variantly controlled by tetracycline, were used to construct cell lines expressing the anti-apoptosis gene survivin upon induction with tetracycline. The resulting stable clones were analyzed for survivin expression. The analysis showed that all four B5-derived clones exhibited leaky survivin expression in the absence of tetracycline, while the B2-derived clones did not. DNA laddering and annexin V/PI staining assays further indicated that although tetracycline-inducible expression of survivin conferred resistance to NH₄Cl- and staurosporine-induced apoptosis in both the B2- and the B5-derived stable cell lines, the B2-derived cell lines showed more stringent regulation in the absence of tetracycline. This represents successful utilization of the present screening method.

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Iron is an essential element for virtually all kingdoms of life, and especially for primary producers in ocean ecosystems. To date, the molecular mechanism of iron utilization by macroalgae remains largely unknown. To elucidate the strategy of iron acquisition and storage in macroalgae, we focused on the function of the iron storage protein ferritin in the sea lettuce, Ulva pertusa, which has abundant iron content. Judging from the primary structure, U. pertusa ferritin (UpFer) can be classified as a land-plant-type ferritin, which is usually found in plastids. The gene of UpFer was expressed in the peripheral, central and rhizoid parts. Western blot analysis showed that UpFER was present and functioned in processed 26- and 22-kDa forms. Furthermore, recombinant UpFER had iron incorporation activity comparable to other ferritins. These results suggest that ferritin also functions as an iron storage protein as in unicellular algae and land plants.

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The hydrolytic specificities of chitosanases were determined using N¹,N⁴-diacetylchitohexaose [(GlcN)₂-GlcNAc-(GlcN)₂-GlcNAc]. The results for the hydrolytic specificities of chitosanases belonging to subclasses I, II, and III toward chitohexaose and N¹,N⁴-diacetylchitohexaose agreed with previous results obtained by analysis of the hydrolysis products of partially N-acetylated chitosan. N¹,N⁴-Diacetylchitohexaose is a useful substrate to determine the hydrolytic specificity of chitosanase. On the other hand, chitosanases from Amycolatopsis sp. CsO-2 and Pseudomonas sp. A-01 showed broad cleavage specificity. They cleaved both the GlcNAc-GlcN and the GlcN-GlcNAc bonds in addition to the GlcN-GlcN bond in the substrate. Thus, both enzymes were new chitosanases. The chitosanases were divided into four subclasses according to their specificity for hydrolysis of the β-glycosidic linkages in partially N-acetylated chitosan.

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Ecl1, a product of the YGR146C gene in Saccharomyces cerevisiae, was identified as a factor involved in chronological lifespan. In this study we found evidence that the function of Ecl1 in the extension of chronological lifespan is dependent on mitochondrial function. The respiratory activity of cells increased when Ecl1 was overexpressed or cells were grown under calorie restriction, but there was no additive effect of calorie restriction and Ecl1 overexpression on increases in respiratory activity or on the extension of chronological lifespan. Based on these results, we propose that overexpression of Ecl1 has same effect as caloric restriction and that its function also depends on mitochondria, just like caloric restriction.

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The mechanism of regulation of the expression of copA and copB, encoding putative copper-translocating P_1B-type ATPases in Corynebacterium glutamicum, was investigated. The levels of copA and copB mRNAs were upregulated in response to excess copper as well as excess zinc. Disruption of csoR, encoding a transcriptional regulator, resulted in constitutive expression of copA and copB. The CsoR protein bound to the promoter regions of the copA-csoR and the cgR_0124-copB-cgR_0126 operon. In vitro DNA binding activity was strongly inhibited by copper, but much less inhibited by zinc. A csoR-deficient mutant showed slightly increased resistance to copper, but slightly decreased resistance to zinc. These findings indicate that CsoR acts as a transcriptional repressor not only of the cognate copA-csoR operon but also of the cgR_0124-copB-cgR_0126 operon, which is not physically linked to csoR on the chromosome, and that CsoR plays a major role in copper homeostasis.

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Prokaryotic ubiquitin-like protein (Pup) is a functional analog of ubiquitin. Post-translationally modified pupylated proteins are selectively degraded by a proteasome-dependent proteolytic system. Deaminase of Pup (Dop) activates Pup by deaminating the C-terminal from glutamine to glutamate, and subsequently activated Pup is conjugated to target proteins by proteasome accessory factor A. Dop is also involved in the removal of Pup from pupylated proteins. Deconjugated free Pup is capable of religating to target proteins. Although the pupylation system is well studied in Mycobacterium, little is known about it in other actinomycetes. Both Rhodococcus and Mycobacterium Dop remove Pup from pupylated proteins, but in these two bacteria, no accumulation of deconjugated free Pup from Rhodococcus is observed. Analysis of a model pupylated protein revealed that Rhodococcus Pup is degraded at multiple sites by Dop. The endopeptidase activity of Dop can be detected using a fluorogenic substrate in conjunction with aminopeptidase. Moreover, the enzymatic activity of the model enzyme increases when Pup is deconjugated. These results suggest that depupylated Rhodococcus Pup is not recycled for religation with target proteins, and that Pup not only functions as a degradation signal, but also regulates the enzymatic activity of target proteins by conjugation and deconjugation to them.

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Honeybees, Apis mellifera, possess three α-glucosidase isozymes, HBG-I, HBG-II, and HBG-III, which belong to glycoside hydrolase family 13. They show high sequence similarity, but clearly different enzymatic properties. HBG-III preferred sucrose to maltose as substrate and formed only α-1,4-glucosidic linkages by transglucosylation, while HBG-II preferred maltose and formed the α-1,6-linkage. Mutation analysis of five amino acids in conserved region II revealed that Pro226-Tyr227 of HBG-III and the corresponding Asn226-His227 of HBG-II were crucial to the discriminating properties. By replacing these two amino acids, the substrate specificities and regioselectivity in transglucosylation were changed drastically toward the other. The HBG-III mutant, Y227H, and the HBG-II mutant, N226P, which harbor HBG-I-type Pro-His at the crucial positions, resembled HBG-I in enzymatic properties with marked increases in reaction velocities on maltose and transglucosylation ratios. These findings indicate that the two residues are determinants of the enzymatic properties of glycoside hydrolase family 13 (GH-13) α-glucosidases and related enzymes.

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Pyridylamino derivatization is suitable for the microanalysis of glycans but there is a problem in that by-products of the labeling reaction and fluorescent substances from samples occasionally interfere with the detection of pyridylamino glycans. We have reported a three-step purification method (S. Natsuka et al., FEBS J., 278, 452–460 (2011)). That method gives high purity and high yield for various glycans, but it is rather complicated. In this study I checked the efficiency of a one-step method with a spin column for the purification of pyridylamino glycans and found that it was excellent in respect of throughput. High-throughput analysis of N-glycans is desirable in glycomics.

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A cytochrome bc-type complex of Roseobacter denitrificans OCh114 was thought to be a novel cytochrome c oxidase. To determine its function, we deleted the genes encoding the complex. The mutant grew normally by aerobic respiration, but failed to grow by denitrification and lacked nitric oxide reductase activity, indicating that the physiological function of the gene product is nitric oxide reduction.

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Skunk cabbage (Symplocarpus renifolius) spadices contain abundant transcripts for cysteine protease (CP). From thermogenic spadices, we isolated SrCPA, a highly expressed CP gene that encoded a papain-type CP. SrCPA is structurally similar to other plant CPs, including the senescence-associated CPs found in aroids. The expression of SrCPA increased during floral development, and was observed in all floral tissues except for the stamens.

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We analyzed structural features of N-glycans linked to glycoproteins expressed in various seaweeds to identify new sources of biologically-important N-glycans or N-glycopeptides. Structural analysis of the N-glycans of glycopeptides prepared from pepsin digests of 15 species of seaweed revealed that only high-mannose type N-glycans occur in seaweed glycoproteins, and the Man₉GlcNAc₂ structure predominates in Sargassum fulvellum and Zostera marina, while no typical plant complex type N-glycans bearing β1-2 xylosyl and α1-3 fucosyl residues present in either algae or seagrass. These results indicate that seaweeds lack the activities of several of the glycosyltransferases required for the biosynthesis of the complex type N-glycans found in terrestrial plants, and that the context of N-glycan processing in seaweeds is different from that in terrestrial plant cells.

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The carbohydrate-binding activity of lectins was examined using polyamidoamine dendrimer conjugated with carbohydrates (sugar-PD). When a C-type lectin, CEL-IV, was mixed with melibiose-PD, large complexes with a diameter of about 1 μm were formed. Changes in the amount of CEL-IV/melibiose-PD complex as an indication of lectin activity were measured sensitively by Rayleigh scattering. The carbohydrate specificity of the lectin was determined on the basis of inhibition of complex-formation by individual carbohydrates. It is suggested that various lectins can also be measured using sugar-PDs to which different carbohydrates are attached.

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Hydrogenated resistant maltodextrin (H-RMD) is a dietary fiber whose energy value has not previously been reported. We evaluated the energy value of H-RMD. We conducted an in vitro digestion test, in vivo blood glucose measurement after ingestion, in vitro fermentability test, excretion test by rats and indirect calorimetry combined with breath hydrogen measurement for humans. H-RMD was hydrolyzed in vitro in a very small amount by human salivary amylase and by the rat small intestinal mucosal enzyme. Ingestion of H-RMD did not increase the blood glucose level of human subjects. An examination of in vitro fermentability suggested that H-RMD was fermented by several enterobacteria. Oral administration of H-RMD showed a saccharide excretion ratio of 42% by rats. A combination of indirect calorimetry and breath hydrogen measurement evaluated the metabolizable energy of H-RMD as 1.1 kcal/g in humans. We concluded from these results that H-RMD was not digested or absorbed in the upper gastrointestinal tract and was fermented in the colon to produce short-chain fatty acids which provided a lower amount of energy than that of resistant maltodextrin.

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Abnormal activation of astrocytes (e.g., the overproduction of cytokines and nitric oxide) is relevant to neurodegenerative disease. It is important, therefore, to search for inhibitors of the abnormal activation of astrocytes that can be derived from natural substances. This study focused on the effects of extracts from young fruits of Citrus unshiu on lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in rat primary astrocytes. The methanol extract of young citrus inhibited NO production in a concentration-dependent manner. After reverse-phase extraction of the extract, we found that polymethoxyflavone, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeletin inhibited NO production by primary astrocytes. These polymethoxyflavones also inhibited LPS-induced iNOS protein and mRNA expression by suppressing nuclear factor-κB (NF-κB) activation and p38-mitogen-activated protein kinase (MAPK) phosphorylation. To evaluate possible applications of these neuroprotective agents in vivo, we examined the effects of young citrus fruit on delayed neurodegeneration in hippocampal CA1 neurons of the Mongolian gerbil after global ischemia. Oral administration of young citrus fruit significantly suppressed delayed neuronal death in hippocampal CA1 neurons. This suggests a possible application of young citrus fruit as a neuroprotective agent.

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Vitamin B₆ (B₆) deficiency affects homocysteine metabolism, and this leads to hyperhomocysteinemia. In this study, we examined i) the effects of B₆-deficiency and graduated levels of dietary methionine on homocysteine metabolism, and ii) the effects of fortified folate on homocysteine metabolism. In experiment 1, Wistar male rats were fed a control or a B₆-deficient diet supplemented with L-methionine at a level of 3, 6, or 9 g/kg of diet for 5 weeks. The resulting plasma homocysteine levels in the B₆-deficient groups increased in relation to the increase in dietary methionine level. Next, in experiment 2, rats were fed a control, B₆-deficient, or folate enriched (10 mg pteroylmonoglutamic acid/kg) B₆-deficient diet containing L-methionine at 9 g/kg for 5 weeks. Although the B₆-deficient diet induced hyperhomocysteinemia, folate fortification ameliorated the plasma homocysteine concentration. Overall, our results indicate that folate fortification ameliorates the hyperhomocysteinemia induced by B₆ deficiency and supplemental methionine intake.

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The aim of this study was to purify an acidic α-glucan–protein complex from the fruiting bodies of Pleurotus sajor-caju by using the cell wall-degrading enzymes, xylanase and cellulase. The acidic glucan–protein complex was separated from a polysaccharide extract by using DEAE Toyopearl 650M anion-exchange and Sepharose CL-6B chromatography. Its homogeneity was ensured by high-performance size-exclusion chromatography and agarose gel electrophoresis. The acidic glucan–protein complex had a molecular weight of approximately 182 kDa. Fourier transform infrared spectroscopy of the acidic glucan–protein complex revealed an α-glycosidic bond and the typical characteristics of polysaccharides and proteins. The amino acid composition of the protein moiety was dominated by proline, glycine, glutamic acid and aspartic acid, indicating that the protein was highly flexible and had a negative charge. Atomic force microscopy proved that the acidic α-glucan–protein complex existed in a spherical conformation. The acidic α-glucan–protein complex stimulated the activation of macrophages, including the production of nitric oxide and tumor necrosis factor-α.

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The anti-inflammatory effect of lactic acid bacteria (LAB) has been reported in several models for autoimmune diseases. It was considered in those studies that IL-10 induced by LAB might have been involved in such anti-inflammatory activity. We therefore examined the IL-10-inducing activity of LAB in detail by using an in vitro culture system of DO11.10 splenocytes. Most strains of LAB tested in this study increased IL-10 production. A further study using one of the tested strains with potent immune-regulatory activity, Lactobacillus plantarum NRIC1832, showed that the enhanced IL-10 was mainly produced by T cells. However, this enhancement required several types of cells other than T cells. NRIC1832 enhanced IL-10 production after short-term exposure to T cells, but this effect diminished after long-term exposure, indicating that the enhancement of IL-10 production by NRIC1832 was temporary, in contrast to the enhancement of IFN-γ production which was still apparent after long-term exposure.

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The epitopes for OVA323-339-specific CD4⁺T cells from OVA23-3 food allergy model and DO11.10 tolerant induction model mice were analyzed. We found that OVA23-3 CD4⁺T cells recognized the N-terminal region, showing strong proliferation and the Th2-phenotype, and that DO11.10 CD4⁺T cells recognized the C-terminal region, showing milder proliferation and a Th1-skewed response. These differences may regulate the responses of those mice to OVA-feeding, inflammation and tolerance.

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A silk fibroin hydrolysate (SFH) exhibited a pronounced in vivo blood pressure-lowering effect on spontaneously hypertensive rats (SHRs) accompanied with decreases in the plasma angiotensin II, endothelin (ET), TNF-α and NO concentrations. We also observed a markedly decreased LPO level, a parameter of oxidative tissue damage, in the medium- and high-dose SFH-treated groups which was accompanied by an increased SOD level in erythrocytes. Our data suggest ACE inhibition together with an improved antioxidative status as the underlying antihypertensive mechanism for the silk fibroin hydrolysate. Since SFH could markedly lower the blood pressure and improve several physiological parameters involved in the occurrence of hypertension, it could be used as a possible supplement against cardiovascular diseases and as a functional food ingredient.

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The avian infectious bronchitis virus (IBV) multi-epitope based peptide EpiC was found to be effective in inducing strong humoral and cellular responses against IBV. In this study, the gene EpiC was introduced into Lactococcus lactis NZ3900, and three recombinant strains expressing EpiC in intracellular and extracellular forms were constructed. SDS–PAGE and Western blot results indicated that EpiC was successfully expressed and had good immunoreactivity with chicken anti-IBV serum. Fusion of the signal pepitide gene SPusp45 and the nine-peptide LEISSTCDA encoding oligonucleotide to EpiC increased the secretion of EpiC, but reduced the total yields of EpiC. Oral immunization to specific-pathogen-free (SPF) chickens with recombinant strains induced significantly higher levels of humoral immune responses, and provided protection against lethal dose challenge by the IBV SAIBk strain. These results indicate that it is feasible to use L. lactis as an antigen delivery vehicle in developing oral vaccines against IBV infection.

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To explore potential probiotics in the traditional foods of Indonesia, fermented mare milk produced in Sumbawa Island was investigated in this study. Gram stain, catalase activity, gas production, cell morphology, carbohydrate utilization pattern, and 16S rDNA sequencing were performed to identify isolated lactic acid bacteria. To assess their probiotic ability, tolerance of low pH, bile salts, artificial gastrointestinal fluids, and adhesion properties to extracellular matrices, were examined. In total 27 strains, 25 Lactobacillus rhamnosus and two Lactobacillus fermentum, were obtained. Among the isolated lactobacilli, three Lb. rhamnosus strains, FSMM15, FSMM22, and FSMM26, were selected as candidates for probiotics, using Lb. rhamnosus GG as index. In vitro binding assay of the three strains against several extracellular matrix proteins revealed that FSMM15 and FSMM26 gave greater binding ratios of mucin/bovine serum albumin (BSA) and significantly higher adhesive abilities to fibronectin than Lb. rhamnosus GG. FSMM22 showed significantly higher adhesion to laminin than Lb. rhamnosus GG.

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Serratia marcescens 2170 produces three chitinases and the chitin-binding protein CBP21, and efficiently degrades insoluble and crystalline chitin. The three chitinases and CBP21 are induced by N,N'-diacetylchitobiose [(GlcNAc)₂], the major product of chitin hydrolysis by S. marcescens chitinases. We have found that uptake of both (GlcNAc)₂ and N-acetylglucosamine (GlcNAc) is important for the efficient utilization of (GlcNAc)₂ because (GlcNAc)₂ is less efficiently fermented by S. marcescens 2170 in the absence of chitobiase. In order to determine the mechanism of utilization of the degradation products of chitin by S. marcescens, chitobiase deficient transposon mutants were screened. A transposon present in chitobiase-deficient mutants was inserted into the ybfMN-ctb cluster. The mutants produced chitinases, except for TT327, in which a transposon was inserted into the 5'-untranslated region (5'-UTR) of ybfM. Ectopic expression of this region in TT327 restored chitinase production. These results indicate that the 5'-UTR of ybfM is important for chitinase induction in S. marcescens.

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Vba5p is closest to Vba3p in the vacuolar transporter for basic amino acids (VBA) family of Saccharomyces cerevisiae. We found that green fluorescence protein (GFP)-tagged Vba5p localized exclusively to the plasma membrane. The uptake of lysine and arginine by whole cells was little affected by deletion of the VBA5 gene, but was stimulated by overexpression of the VBA5 gene. The inhibitory effect of 4-nitroquinoline N-oxide on cell growth was accelerated by expression of the VBA5 gene, and was lessened by the addition of arginine. These results suggest that Vba5p is a plasma membrane protein involved in amino acid uptake and drug sensitivity.

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An active antifouling diterpene was isolated from marine actinomycete strain PK209 and productivity was induced in a co-culture experiment. The active constituent was identified as the diterpene lobocompactol by interpretion of nuclear magnetic resonance and mass spectroscopy data. A PK209 co-culture was designed and a lobocompactol-resistant bacterium, KNS-16, was selected as co-culture competitor to induce lobocompactol production. Adding a small volume of 16-h-old KNS-16 culture to the 96-h-old PK209 culture caused rapid induction of lobocompactol production. The final yield was 2.7 mg/L, 10.4-fold higher than that collected from a single PK209 culture. The two bacteria, strains PK209 and KNS-16, were identified as Streptomyces cinnabarinus and Alteromonas sp. based on 16S rDNA sequencing. Lobocompactol showed significant antifouling activity, of 0.18 and 0.43 µg/mL, for EC₅₀ against the macroalga Ulva pertusa and the diatom Navicula annexa respectively. It showed activity with MIC of 61–112 µg/mL against fouling bacteria.

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