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Context: Gastrointestinal symptoms are a common complication of influenza virus infection in children, which the gut-lung axis become involved in its biological progress. The protective effect of 999 XiaoErGanMao granules (XEGMG) on multi-organ injury in viral pneumonia remains unclear., Objective: To investigate the therapeutic effect of XEGMG on lungs and intestines injury in A/FM/1/47 (H1N1) influenza virus-infected mice., Materials and Methods: Male BALB/c mice were infected with the 2LD 50 H1N1 influenza virus and then treated with XEGMG (6 or 12 g/kg) intragastrically once a day for 4 days. The lung and colon samples were then collected for pathological observation, and assays for inflammatory cytokines and intestinal barrier. Mouse feces were collected to evaluate the intestinal microbiota., Results: Treating with XEGMG (12 g/kg) can mitigate body weight loss caused by 2LD 50 H1N1 infection. It can also reduce lung index and pathological damage with the decreased inflammatory cytokines such as IL-6 and IL-1β. Furthermore, XEGMG (12 g/kg) can maintain the goblet cell number in the colons to protect the intestinal barrier and regulate the major flora such as Firmicutes, Bacteroidetes, and Muribaculaceae back to normal. Meanwhile, the expression of IL-17A in the colon tissues was significantly lower in the group of XEGMG (6, 12 g/kg) compared to H1N1 group., Discussion and Conclusions: XEGMG can protect against H1N1 invasion involved in gut-lung axis regulation. The results provide new evidence for the protective effect of XEGMG, which is beneficial to vulnerable children.

Arg-Gly-Asp (RGD) motif mediates cell adhesion as a major determinant in interactions of disintegrins with cell surface receptors. In order to obtain a mutant trypsin with both high affinity to integrins and retained proteolytic activity, RGDS and RGD were inserted into a rigid turn region and a flexible loop of trypsin, respectively. Wild type trypsin and substituted mutant trypsins, 37RGDS and 77RGD, were expressed in E. coli and purified. Kinetic properties, autolytic stability as well as platelet aggregation inhibitory activity of both 37RGDS and 77RGD were determined. 37RGDS and 77RGD give retained proteolytic activities of 34% and 87%, respectively, and both become less stable to autolysis. 37RGDS shows an obvious inhibition rate of 29% for platelet aggregation and 77RGD gives a rather weak rate of 14% at the same protein concentration of 3.5 microM, while the wild type trypsin shows no inhibitory activity.

One mutant proinsulin gene was constructed through PCR with the code of A19Tyr changed into that of Phe. The mutant proinsulin, (A19Phe)-lysproinsulin (F19KPI) was expressed in E. coli and purified. After trypsin and carboxypeptidase B cleavage and Resource Q separation, (A19Phe)-human insulin (F19HI) was obtained. With native insulin as standard, activity assay and structural analysis were carried out. It was found that the conformation of F19HI is very similar to that of native insulin according to the data from native PAGE, reverse-phase FPLC and circular dichroism spectrum, which are also in agreement with the result of radioimmune assay. F19HI retains almost full immune activity, but displays only 13.3% of receptor binding activity. These results suggest that the hydroxyl group of insulin A19Tyr is essential for receptor binding.

A mutant proinsulin gene was constructed through PCR mediated mutagenesis. The code of A19Tyr was deleted. The mutant proinsulin, (deltaY)19-lys-proinsulin [(deltaY)19KPI], was expressed in E. coli and purified. After treatment with trypsin and carboxypeptidase B, and Resource Q separation, (deltaY)19-human insulin [(deltaY)19HI] was obtained. It retains 63.6% of receptor binding activity but only 2.2% of immune activity, and shows a longer retaining time on reverse-phase FPLC and a slower mobility by native PAGE analysis. These results suggest that the deletion of A19Tyr causes some conformational changes on insulin, which plays a minor role on the affinity of insulin to its receptor, and a major role on immunogenicity of the hormone.

The autolytic site Arg105 of rat trypsin was replaced with Cys by DNA site-directed mutagenesis method. Comparison of expression and purification of R105C trypsin along with the wild type and some other Arg105 mutants indicates that R105C trypsin could be expressed as well but with a lower expression level. It is unexpected that R105C trypsin has no detectable activity toward trypsin substrate TAME, quite different from the wild type and other Arg105 mutants. Native gel electrophoresis analysis indicates that R105C trypsin has a similar mobility rate to that of wild type trypsin. FPLC also gives similar retaining time. The loss of activity of R105C trypsin may result from the conformational change around active site, but not the dimer formation.

Des-B30 single-chain insulin gene was constructed from chemically synthesized DNA encoding human proinsulin by oligo-nucleotide induced deletion, cloned into the expression plasmid pBV220, and expressed in E. coli with a level of around 5%. The expressed product was in the form of inclusion bodies. After downstream processing, 45 mg of des-B30 single-chain insulin with a purity of up to 90% was obtained from 1 liter of high density fermentation medium. It is suggested that the fusion protein model to increase the stability of small proteins seems inadequate to some extent, the formation of inclusion bodies may be the key factor for the stability of some expressed products, large or small, in the cytoplasm of E. coli cells.

A fusion gene encoding double-C-peptide human proinsulin was constructed by insertion of a DNA fragment encoding human C-peptide into the 5'-terminal C-peptide coding sequence of a synthetic human proinsulin gene with correct reading frame and over-expressed in E. coli. The purified double-C-peptide human proinsulin shows decreased activity in receptor binding and insulin immune assays as compared with human proinsulin. Disulphide bond reconstitution studies demonstrate that there is not much more influence of the protein concentration on the yield of refolded double-C-peptide human proinsulin. The double-C-peptide human proinsulin shows a 1.86-fold human C-peptide immune activity as compared with that of human proinsulin and gives a good yield of the molecule with correct disulphide bonds in reconstitution studies strongly suggesting the existence of very flexible conformation of the C-peptide.

The mutant proinsulin gene was constructed with the codons for A6 and A11 Cys changed to Ser to delete intra-A chain disulfide bond. After expression and purification, the mutations in the protein were further confirmed by amino acid composition. Electrophoretic mobility of the mutant proinsulin is similar to that of human proinsulin, so are the products of tryptic digestions. The mutant proinsulin, which retains its full radioimmuno activity, shows only 5.4% of receptor binding activity of human proinsulin. This suggests that though the intra-A chain disulfide bond disappears, the other two inter-chain disulfide bonds are still correctly paired, and hence the three dimensional structure has not been altered significantly. This intra-chain disulfide bond is essential for insulin displaying its activity.