Antioxidant and antidiabetic activities of mycelial and fruit-body extracts from <Emphasis Type="Italic">Mycoleptodonoides aitchisonii</Emphasis>

Mycoleptodonoides aitchisonii (Berk.) Maas Geest. is a culinary mushroom that is recognized as both a nutritious food and an excellent source of bioactive compounds. The purpose of this study was to investigate the antioxidant and antidiabetic properties of M. aitchisonii (MA) both in vitro and in vivo. Total oxyradical scavenging capacity (TOSC) assays revealed that fruit-body extracts had higher antioxidant capacity than mycelial extracts, 0.9-fold higher as measured by peroxynitrite (PN) scavenging assay, 3.7-fold higher as measured by peroxyl radical (PR) scavenging assay, and 1.6-fold as measured by hydroxyl radical (HR) scavenging assay, respectively. The assay of Akt phosphorylation, which is inhibited by Interleukin 6 (IL-6) in the signal transduction pathway for diabetes, was employed to evaluate the antidiabetic activity. Fruit-body extracts significantly increased Akt phosphorylation according to the fruit-body extract concentration, with a maximum increment of 77% at a concentration of 100 μg/mL compared to 51.4% decrement caused by IL-6, but there was no effect of mycelial extracts. Treatment with 5% MA fruit-body powder and streptozotocin (STZ) decreased the blood sugar level to 233.8 mg/dL in diabetic mice compared to 333.8 mg/dL after treatment with STZ alone. Additionally, MA treatment lowered total cholesterol (TC), triglyceride (TG), and LDL-cholesterol levels, while it increased the HDL-cholesterol level. All these findings indicate that fruit-body of M. aitchisonii has potential utility in preventing various diseases such as disorders of sugar and lipid metabolism.     

<Emphasis Type="Italic">Lactobacillus curvatus</Emphasis> WiKim38 isolated from kimchi induces IL-10 production in dendritic cells and alleviates DSS-induced colitis in mice

Probiotics such as lactobacilli and bifidobacteria have healthpromoting effects by immune modulation. In the present study, we examined the immunomodulatory properties of Lactobacillus curvatus WiKim38, which was newly isolated from baechu (Chinese cabbage) kimchi. The ability of L. curvatus WiKim38 to induce cytokine production in bone marrow-derived dendritic cells (BMDCs) was determined by enzyme-linked immunosorbent assay. To evaluate the molecular mechanisms underlying L. curvatus Wikim38-mediated IL-10 production, Western blot analyses and inhibitor assays were performed. Moreover, the in vivo anti-inflammatory effects of L. curvatus WiKim38 were examined in a dextran sodium sulfate (DSS)-induced colitis mouse model. L. curvatus WiKim38 induced significantly higher levels of IL-10 in BMDCs compared with that induced by LPS. NF-κB and ERK were activated by L. curvatus WiKim38, and an inhibitor assay revealed that these pathways were required for L. curvatus WiKim38-induced production of IL-10 in BMDCs. An in vivo experiment showed that oral administration of L. curvatus WiKim38 increased the survival rate of mice with DSS-induced colitis and improved clinical signs and histopathological severity in colon tissues. Taken together, these results indicate that L. curvatus Wikim38 may have health-promoting effects via immune modulation, and may thus be applicable for therapy of various inflammatory diseases.     

The action of philanthotoxin-343 and photolabile analogues on locust (Schistocerca gregaria) muscle

The effects of philanthotoxin-343 (PhTX-343; tyrosyl-butanoyl-spermine) and photolabile analogues of this synthetic toxin on locust (Schistocerca gregaria) skeletal muscle have been investigated using whole muscle preparations (twitch contractions), single muscle fibres (excitatory postsynaptic currents (EPSCs)) and muscle membrane patches containing single quisqualate-sensitive glutamate receptors (qGluR). Analogues containing an azido group attached to either the butanoyl side-chain of PhTX-343 or as a substitute for the hydroxyl moiety of the tyrosyl residue were about 6 fold more potent antagonists than PhTX-343; those with an azido group located at the distal end of the toxin molecule were generally 2–3 fold less potent than PhTX-343. When these compounds were tested in subdued light, they were reversible antagonists of the muscle twitch, EPSC and qGluR. When a muscle was irradiated with U.V. during application of photolabile toxin combined with either neural stimulation of the muscle orl-glutamate application, antagonism of the twitch, EPSC and qGluR was complete and irreversible.     

Purification and characterization of laccase from the white rot fungus Trametes versicolor

Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50 degrees C. The Km value of the enzyme for substrate ABTS is 12.8 micrometer and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.     

Spectrophotometric determination of peptide transport with chromogenic peptide mimetics

A spectrophotometric assay to determine peptide transport has been developed. Using two chromogenic peptide mimetics, L-phenylalanyl-L-2-sulfanilylglycine (PSG) and L-phenylalanyl-L-3-thiaphenylalanine (PSP), the peptide transport patterns in individual cell species can be evaluated effectively. After the addition of PSG to a HeLa cell suspension, sulfanilic acid accumulated progressively inside, but not outside, the cells, demonstrating that PSG was transported wholly intact. The addition of PSP to the same cell suspension was followed immediately by extracellular thiophenol production. Measurement of the rate of thiophenol release thereby provided direct determination of PSP transport. The thiophenol release was consistent with Michaelis-Menten kinetics, with a K(m) of 0.016 mM and a V(max) of 5.07 nmol/min (1 x 10(6) cells/ml, pH 7.4, 37 degrees C). The resulting kinetic constants estimated were in agreement with values determined by single-substrate enzyme kinetics. Using PSP, transport kinetics of various dipeptides was examined by competitive spectrophotometry. As a result, dipeptides tested could be ranked in order of kinetic power for their transport.     

Structural hybridization of pyrrolidine-based T-type calcium channel inhibitors and exploration of their analgesic effects in a neuropathic pain model

Highly effective and safe drugs for the treatment of neuropathic pain are urgently required and it was shown that blocking T-type calcium channels can be a promising strategy for drug development for neuropathic pain. We have developed pyrrolidine-based T-type calcium channel inhibitors by structural hybridization and subsequent assessment of in vitro activities against Ca_v3.1 and Ca_v3.2 channels. Profiling of in vitro ADME properties of compounds was also carried out. The representative compound 17h showed comparable in vivo efficacy to gabapentin in the SNL model, which indicates T-type calcium channel inhibitors can be developed as effective therapeutics for neuropathic pain.     

Targeting CreERT2 expression to keratin 8-expressing murine simple epithelia using bacterial artificial chromosome transgenesis

Keratin 8 (K8) is a type II keratin that is associated with the type I keratins K18 or K19 in single layered epithelia. We generated a bacterial artificial chromosome (BAC) transgenic mouse line that expresses the tamoxifen inducible CreER(T2) inserted into the endogenous murine K8 gene. The transgenic mouse line contains two copies of the BAC transgene. To determine the expression specificity and inducibility of CreER(T2), the K8-CreER(T2) mice were bred with a Gt(ROSA 26)( ACTB-tdTomato-EGFP ) fluorescent protein-based reporter transgenic mouse line. We demonstrated that CreER(T2) and the endogenous K8 gene share the same patterns of expression and that the enzymatic activity of CreER(T2) can be efficiently induced by tamoxifen in all K8-expressing tissues. This mouse line will be useful for studying gene function in development and homeostasis of simple epithelia, and investigating both tissue lineage hierarchy and the identity of the cells of origin for epithelial cancers.     

Deciphering the proteome of the in vivo diagnostic reagent "purified protein derivative" from Mycobacterium tuberculosis

Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.     

In vivo Tracking of Dendritic Cell using MRI Reporter Gene,Ferritin

The noninvasive imaging of dendritic cells (DCs) migrated into lymph nodes (LNs) can provide helpful information on designing DCs-based immunotherapeutic strategies. This study is to investigate the influence of transduction of human ferritin heavy chain (FTH) and green fluorescence protein (GFP) genes on inherent properties of DCs, and the feasibility of FTH as a magnetic resonance imaging (MRI) reporter gene to track DCs migration into LNs. FTH-DCs were established by the introduction of FTH and GFP genes into the DC cell line (DC2.4) using lentivirus. The changes in the rate of MRI signal decay (R₂*) resulting from FTH transduction were analyzed in cell phantoms as well as popliteal LN of mice after subcutaneous injection of those cells into hind limb foot pad by using a multiple gradient echo sequence on a 9.4 T MR scanner. The transduction of FTH and GFP did not influence the proliferation and migration abilities of DCs. The expression of co-stimulatory molecules (CD40, CD80 and CD86) in FTH-DCs was similar to that of DCs. FTH-DCs exhibited increased iron storage capacity, and displayed a significantly higher transverse relaxation rate (R₂*) as compared to DCs in phantom. LNs with FTH-DCs exhibited negative contrast, leading to a high R₂* in both in vivo and ex vivo T₂*-weighted images compared to DCs. On histological analysis FTH-DCs migrated to the subcapsular sinus and the T cell zone of LN, where they highly expressed CD25 to bind and stimulate T cells. Our study addresses the feasibility of FTH as an MRI reporter gene to track DCs migration into LNs without alteration of their inherent properties. This study suggests that FTH-based MRI could be a useful technique to longitudinally monitor DCs and evaluate the therapeutic efficacy of DC-based vaccines.     

The phosphotransferase system of Corynebacterium glutamicum: features of sugar transport and carbon regulation

In this review, we describe the phosphotransferase system (PTS) of Corynebacterium glutamicum and discuss genes for putative global carbon regulation associated with the PTS. C. glutamicum ATCC 13032 has PTS genes encoding the general phosphotransferases enzyme I, HPr and four enzyme II permeases, specific for glucose, fructose, sucrose and one yet unknown substrate. C. gluamicum has a peculiar sugar transport system involving fructose efflux after hydrolyzing sucrose transported via sucrose EII. Also, in addition to their primary PTS, fructose and glucose are each transported by a second transporter, glucose EII and a non-PTS permease, respectively. Interestingly, C. glutamicum does not show any preference for glucose, and thus co-metabolizes glucose with other sugars or organic acids. Studies on PTS-mediated sugar uptake and its related regulation in C. glutamicum are important because the production yield of lysine and cell growth are dependent on the PTS sugars used as substrates for fermentation. In many bacteria, the PTS is also involved in several regulatory processes. However, the detailed molecular mechanism of global carbon regulation associated with the PTS in this organism has not yet been revealed.