[Effect of triptolide on the expression of RANTES in the renal tissue of diabetic nephropathy rats].

OBJECTIVE: To investigate the effect of triptolide (TPL) on the renal tissue of diabetic rats and its possible mechanisms. METHODS: SD rats were randomly divided into the normal control group (as the normal group), the diabetic model group (the model group), the low dose TPL treatment group (the low dose TPL group, TPL 0.2 mg/kg by gastrogavage), the high dose TPL treatment group (the high dose TPL group, TPL 0.4 mg/kg by gastrogavage). Equal volume of normal saline was given to rats in the normal group and the model group. Five rats were randomly selected from each group at week 4, 8, and 12 of the experiment to detect body weight, kidney weight, 24 h urinary albumin (24 h UAL), plasma glucose (FBG), total cholesterol (TC), total triglyeride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), white blood cell (WBC), and hemoglobin A1c (HbA1c). The mRNA and protein expression of regulated upon activation normal T-cell expressed and secreted (RANTES) in the renal tissue was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). The renal tissue was pathologically stained by HE, PAS, and Masson staining. The glomerular and renal tubular interstitial lesions were observed at each time point. The glomerular sclerosis index (GSI) was observed by PAS staining, and the renal interstitial filrosis index (RIFI) was calcutated. RESULTS: Compared with the same group at week 4, the expression of 24 h UAL, RANTES, GSI, and RIFI at week 12 significantly decreased in two TPL groups (P <0.01). Compared with the same group at week 8, the expression of 24 h UAL, RANTES, GSI, and RIFI at week 12 also significantly decreased in the two TPL groups (P <0. 05, P <0.01). Compared with the normal group, body weight and the kidney weight obviously decreased at week 4, 8, and 12 in the model group (P <0. 01); 24 h UAL, FBG, TG, TC, HbA1c, RANTES, GSI, and RIFI were obviously elevated (P <0.01). Compared with the model group, 24 h UAL, RANTES, GSI, and RIFI also decreased in the two TPL treatment groups (P <0.01). Compared with the low dose TPL group, they were attenuated in the high dose TPL group (P <0. 05, P <0. 01). CONCLUSION: TPL could not only inhibit the over-expression of RANTES, but also improve the glomerular sclerosis and renal interstitial fibrosis in the renal tissue of diabetic rats.

A new marine bacterial laccase with chloride-enhancing, alkaline-dependent activity and dye decolorization ability.

A bacterial laccase gene designated as lac21 was screened from a marine microbial metagenomic library of the South China Sea based on sequence screening strategy. The protein encoded by lac21 shared less than 40% sequence identities with all of the laccases found. Lac21, which was recombinantly expressed in Escherichia coli, showed high activity toward syringaldazine at an optimum pH of 7.5 and temperature of 45°C. Lac21 was stable at pH values ranging from 5.5 to 9.0 and temperatures lower than 40°C. Interestingly, chloride enhanced the laccase activity, with concomitant increase in substrate affinity. Furthermore, Lac21 has high decolorization capability toward azo dyes in the absence of redox mediators, with 80% of Reactive Deep Blue M-2GE (50mg/L) being decolorized by 15U/L enzyme after 24h incubation at 20°C. These unusual properties demonstrate that the new bacterial laccase Lac21 has potentials in specific industrial or environmental applications.

Effect of Tang No.1 granule (1) in treating patients with impaired glucose tolerance.

OBJECTIVE: To observe the therapeutic effect of Tang No.1 granule (1, T1G) in treating patients with impaired glucose tolerance (IGT). METHODS: One hundred and forty patients with IGT and with Pi-Wei dampness-heat syndrome type were assigned randomly according to their visiting sequence into two equal groups. The control group received only general knowledge about IGT, but to the treated group, based on current knowledge available, T1G was given additionally for 6 months. Changes in related laboratory indexes, including fasting plasma glucose and insulin (FPG and FINS), plasma glucose 2 h after meal (2hPG), glycosylated hemoglobin (HbA1c), serum triglyceride (TG), low density lipoprotein cholesterol (LDL) and insulin resistance index (HOMA-IR), were observed. RESULTS: The levels of FPG, 2hPG, HbA1c, FINS, TG and HOMA-IR were significantly decreased after treatment in the treated group, showing a significant difference compared to the control group (P<0.01). Among them, HbA1c decreased from 7.08+or-1.41% to 6.56+or-1.29% in the treated group, while in the control group, it decreased from 7.02+or-1.37% to 6.93+or-1.31%. The level of LDL was also reduced in the treated group after treatment (P<0.05). In the treated group, 13 out of 68 patients (19.12%) had their glucose tolerance reversed to normal, while in the control group, only 2/64 (3.1%) got it reverse; a comparison between the two groups in terms of reversion rate showed a significant difference (P<0.01). No severe adverse reaction was found in the therapeutic course. CONCLUSIONS: T1G has good clinical effect as a treatment intervention for IGT, as it could improve glycometabolism, significantly depress the levels of post-prandial blood sugar and blood lipids, alleviate clinical symptoms of patients, and effectively cut-off and reverse the yielding and development of diabetes mellitus.

[Cloning and heterologous expression of the gene of laccase C from Trametes sp. 420 and potential of recombinant laccase in dye decolorization].

A new laccase gene (lacC) was cloned from the genomic DNA isolated from Trametes sp. 420, a new laccase-producing fungus, using the degenerate primers based on the conserved copper-binding regions in fungal laccases. Long distance-inverse PCR (LD-IPCR) was used to amplify the flanking sequences of the gene. The lacC DNA sequence obtained was 3640 base pairs (bp), including the entire open reading frame (2263bp) and the 5'-and 3'-noncoding regions. The lacC cDNA sequence is 1560bp, encoding a 519 amino acid protein. The deduced peptide sequence of LacC contains ten putative N-glycosylation sites and four conserved copper-binding regions. The lacC cDNA without its signal sequence was cloned into the expression vector pPIC9K through the pPIC9 plasmid and transformed into the Pichia pastoris strain GS115.The positive transformant was cultured at 20 degrees C in BMM medium containing 0.3mmol/L CuSO4 and 0.8% alanine, with the yield of the recombinant laccase rLacC being 1.62 x 10(4) U/L after a 9-day cell growth. Furthermore, the crude enzyme was used to decolorize several synthetic dyes at a final concentration of 50mg/L. The results showed that rLacC (6U/L) possessed the valuable ability to decolorize dyes of triarylmethane and azo types tested. The presence of low molecular weight redox mediators of ABTS and HBT increased the efficiency and velocity of dye decolorization significantly.

Cloning of a laccase gene from a novel basidiomycete Trametes sp. 420 and its heterologous expression in Pichia pastoris.

The laccase gene lacD, cloned from a novel laccase-producing basidiomycete Trametes sp. 420, contained 2,052 base pairs (bp) interrupted by 8 introns. lacD displayed a relatively high homology with laccase genes from other white rot fungi, whereas the homology between lacD and laccase genes from plants, insects, or bacteria was less than 25%. A 498-amino acid peptide encoded by the lacD cDNA was heterologously expressed in the Pichia pastoris strain GS115, resulting in the highest yield of laccase (8.3 x 10(4) U/l) as determined with ABTS (2,2'-azinobis [3-ethylbenzothia-zoline-6-sulfonic acid]) as the substrate. Additionally, the enzyme activity of recombinant laccase on decolorization of some industrial dyes was assessed.

[High output of a Trametes laccase in Pichia pastoris and characterization of recombinant enzymes].

A laccase gene (lacD) from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21 x 10(5) u/L and 7.38 x 10(4) u/L, respectively, as determined with 2,2'-azinobis(3-ethylbenzothia-zoline- 6-sulfonic acid) (ABTS) as substrate. The yield of rLacDx was further increased to 2.39 x 10(5) u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50 degrees C and at a pH range from 3 to 10. However, the specific activity of rLacDx (1761 u/mg) for ABTS was higher than that of rLacDe (1122 u/mg), and the apparent Km value of rLacDx (427 microM) was less than that of rLacDe (604 microM).

[Inducing synthesis of LacA from Trametes sp. AH28-2 and cloning & analysis of 5'-terminal sequence of transcription control of the gene].

Copper ion was necessary for the transcription of all laccase isozyme genes from Trametes sp. AH28-2, with higher concentrations of Cu2+ (1-2 mmol/L) being more favorable to the synthesis of laccase. In the glucose media containing 0.5 mmol/L Cu2+, the laccase activity of the supernate was rather low (44.3 u/L) and had an increase of 60.3% (71.0 u/L) when 4.0 mmol/L o-toluidine was added. Moreover, the activity reached up to 2584 u/L as the glucose was replaced by cellobiose. And Native-PAGE showed that LacA was the main laccase component if fungus was induced by o-toluidine or copper ions. It had been demonstrated by quantitative RT-PCR that the regulation of lacA expression, induced by o-toluidine, occurred at the transcriptional level, with the accumulation of mRNA transcripts being accompanied by the increase of laccase activity of the culture fluid. In addition, the structural gene of lacA interrupted by 10 introns was 2110 bp in length and the corresponding cDNA sequence was 1560 bp encoding a 520 aa protein, which had high similarities with other laccases from basidiomycetes. Furthermore, a length of 1881 bp of 5'-terminal sequence of transcription control of lacA, amplified by the improved inverse PCR, contained a TATA box, seven CAAT boxes as well as a number of putative cis-acting elements important for its expression, including five MREs, nine CreA-binding sites, four XREs, two STREs and seven nitrogen factor binding sites. The existence of these elements was well in agreement with the data obtained from Trametes sp. AH28-2 shaken-flask cultures.