High tetramethylpyrazine production by the endophytic bacterial Bacillus subtilis isolated from the traditional medicinal plant Ligusticum chuanxiong Hort.

Tetramethylpyrazine (TMP) with significant protective effects on cardiovascular is the active ingredient of traditional Chinese medicine Rhizoma Chuanxiong (RC). However, many studies have reported the low content of TMP in RC. The endophytes of medicinal plants have the biosynthetic potential to produce the same or similar active metabolites as the host, while few reports were conducted to explore the endophytic bacteria of Ligusticum chuanxiong Hort. and its productive capacity for the important ingredient TMP. The present paper focuses on the isolation and identification of TMP producing endophytic bacteria from RC. In this study, the endophytic bacteria were isolated from the rhizome of Ligusticum chuanxiong Hort. (Umbelliferae). Yeast extract peptone glucose medium (YP) was used for fermentation medium (37 °C, 220 rpm agitation, 144 h). GC and GC/MS were performed to determine and verify the product, the fermentation characteristics were investigated. Morphological observation, physiological and biochemical indexes combining with 16S rRNA sequence analysis were carried out to identify the endophytic bacteria. As a result, five strains of endophytic Bacillus subtilis were firstly isolated and identified from RC, named as LB3, LB3-2-1, LB6-2, LB4, LB5 respectively. All five strains of endophytic B. subtilis produced TMP, while LB5 had the highest production of 10.69 g/L at the 144 h fermentation. This work demonstrates the fact that the endophytic B. subtilis of RC can produce a high level of TMP, indicating the endophytic B. subtilis might play a role in the accumulation of TMP during the growth period of RC.

Treatment of rotaviral gastroenteritis with Qiwei Baizhu powder.

AIM: To observe the effects of Qiwei Baizhu Powder (QWBZP) on rotaviral gastroenteritis in children and in animal models. METHODS: Enrolled patients were divided into two groups, and one group was treated with oral rehydration solution (ORS) and the other treated with oral liquid of QWBZP. Neonate mice were orally infected with 50 microL rotavirus suspension (4 X 10(8) PFU/mL) and treated with ORS or oral liquid of QWBZP, respectively. RESULTS: Eighty-three cases of rotaviral gastroenteritis treated with QWBZP revealed a better efficacy than that treated with ORS (X(2)=10.87, P < 0.05). The contents of sodium and glucose as well as number of patients with positive human rotavirus antigen in stool in QWBZP group were all less than that in ORS group. In animal models, QWBZP was found effective in treating rotavirus gastroenteritis in neonate NIH mice, as compared with control groups. In QWBZP group, the mortality of infected mice was decreased by 73.3%, the body weight of infected mice was increased, the contents of sodium and glucose as well as number of mice with positive rotavirus antigen in feces were significantly reduced, and the pathological changes such as damage of small intestinal mucosa and villi were also obviously alleviated. CONCLUSION: QWBZP has effects on improving the absorptive function of small intestine, shortening the duration of diarrhea and rotavirus shedding from stool and alleviating the pathological changes of small intestine induced by rotavirus.

Radiation inactivation analysis of H(+)-pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings.

Radiation inactivation analysis was employed to determine the functional masses of enzymatic activity and proton translocation of H(+)-pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings. The activities of H(+)-pyrophosphatase decayed as a simple exponential function with respect to radiation dosage. D(37) values of 6.9+/-0.3 and 7.5+/-0.5 Mrad were obtained for pyrophosphate hydrolysis and its associated proton translocation, yielding molecular masses of 170+/-7 and 156+/-11 kDa, respectively. In the presence of valinomycin and 50 mM KCl, the functional size of H(+)-pyrophosphatase of tonoplast was decreased, while that of submitochondrial particles remained the same, indicating that they are two distinct types of proton pump using PP(i) as their energy source.

Localization of a carboxylic residue possibly involved in the inhibition of vacuolar H+-pyrophosphatase by N, N'-dicyclohexylcarbodi-imide.

A vacuolar H(+)-pyrophosphatase (EC 3.6.1.1) that catalyses PP(i) hydrolysis and the electrogenic translocation of protons from the cytosol to the vacuole lumen, was purified from etiolated hypocotyls of mung bean seedlings (Vigna radiata L.). Group-specific modification was used to identify a carboxylic residue involved in the inhibition of vacuolar H(+)-pyrophosphatase. Carbodi-imides, such as N,N'-dicyclohexylcarbodi-imide (DCCD) and 1-ethyl-3-(3-dimethylamino-propyl)carbodi-imide, and Woodward's reagent K caused a progressive decline in the enzymic activity of vacuolar H(+)-pyrophosphatase in a time- and concentration-dependent manner. The stoichiometry of labelling of the vacuolar H(+)-pyrophosphatase by [(14)C]DCCD determined that DCCD modifies one carboxylic residue per subunit of the enzyme. Protection studies suggest that the DCCD-reactive carboxylic residue resides at or near the substrate-binding site. Furthermore, peptide mapping analysis reveals that Asp(283), located in the putative loop V of a tentative topological model of vacuolar H(+)-pyrophosphatase on the cytosolic side, was labelled by radioactive [(14)C]DCCD. Cytosolic loop V contains both DCCD-sensitive Asp(283) and a conserved motif sequence, rendering it a candidate for the catalytic site of vacuolar H(+)-pyrophosphatase. A topological picture of the active domain of vacuolar H(+)-pyrophosphatase is tentatively proposed.

Subunit interaction of vacuolar H+-pyrophosphatase as determined by high hydrostatic pressure.

Vacuolar H+-pyrophosphatase (H+-PPase) from etiolated hypocotyls of mung bean (Vigna radiata L.) is a homodimer with a molecular mass of 145 kDa. The vacuolar H+-PPase was subjected to high hydrostatic pressure to investigate its structure and function. The inhibition of H+-PPase activity by high hydrostatic pressure has a pressure-, time- and protein-concentration-dependent manner. The Vmax value of vacuolar H+-PPase was dramatically decreased by pressurization from 293.9 to 70.2 micromol of PPi (pyrophosphate) consumed/h per mg of protein, while the Km value decreased from 0.35 to 0.08 mM, implying that the pressure treatment increased the affinity of PPi to vacuolar H+-PPase but decreased its hydrolysis. The physiological substrate and its analogues enhance high pressure inhibition of vacuolar H+-PPase. The HPLC profile reveals high pressure treatment of H+-PPase provokes the subunit dissociation from an active into inactive form. High hydrostatic pressure also induces the conformational change of vacuolar H+-PPase as determined by spectroscopic techniques. Our results indicate the importance of protein-protein interaction for this novel proton-translocating enzyme. Working models are proposed to interpret the pressure inactivation of vacuolar H+-PPase. We also suggest that association of identical subunits of vacuolar H+-PPase is not random but proceeds in a specific manner.

High-pressure effects on vacuolar H+-ATPase from etiolated mung bean seedlings.

A high-hydrostatic-pressure technique was employed to study the structure-function relationship of plant vacuolar H+-ATPase from etiolated mung bean seedlings (Vigna radiata L.). When isolated vacuolar H+-ATPase was subjected to hydrostatic pressure, the activity of ATP hydrolysis was markedly inhibited in a time-, protein concentration- and pressure-dependent manner. The pressure treatment decreased both Vmax and Km of solubilized vacuolar H+-ATPase, implying an increase in ATP binding affinity, but a decrease in the ATP hydrolysis activity. Physiological substrate, Mg2+-ATP, augmented the loss of enzymatic activity upon pressure treatment. However, ADP, AMP, and Pi exerted substantial protective effects against pressurization. Steady-state ATP hydrolysis was more sensitive to pressurization than single-site ATPase activity. The inactivation of solubilized vacuolar H+-ATPase by pressure may result from changes in protein-protein interaction. The conformational change of solubilized vacuolar H+-ATPase induced by hydrostatic pressure was further determined by spectroscopic techniques. The inhibition of vacuolar H+-ATPase under pressurization involved at least two steps. Taken together, our work indicates that subunit-subunit interaction is crucial for the integrity and the function of plant vacuolar H+-ATPase. It is also suggested that the assembly of the vacuolar H+-ATPase complex is probably not random, but follows a sequestered pathway.

Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation.

Vacuolar proton-pyrophosphatase (H(+)-PPase) of mung bean seedlings contains a single kind of polypeptide with a molecular mass of approx. 73 kDa. However, in this study, a molecular mass of approx. 140 kDa was obtained for the purified vacuolar H(+)-PPase by size-exclusion gel-filtration chromatography, suggesting that the solubilized form of this enzyme is a dimer. Radiation inactivation analysis of tonoplast vesicles yielded functional masses of 141.5 +/- 10.8 and 158.4 +/- 19.5 kDa for PP1 hydrolysis activity and its supported proton translocation respectively. These results confirmed the in situ dimeric structure of the membrane-bound H(+)-PPase of plant vacuoles. Further target-size analysis showed that the functional unit of purified vacuolar H(+)-PPase was 71.1 +/- 6.7 kDa, indicating that only one subunit of the purified dimeric complex would sufficiently display its enzymic reaction. Moreover, in the presence of valinomycin and KCl, the functional size of membrane-bound H(+)-PPase was decreased to approx. 63.4 +/- 6.3 kDa. A working model was proposed to elucidate the structure of native H(+)-PPase on vacuolar membrane as a functional dimer. Factors that would disturb the membrane, e.g. membrane solubilization and the addition of valinomycin and KCl, may induce an alteration in its enzyme structure, subsequently resulting in a different functional size.

Involvement of tyrosine residue in the inhibition of plant vacuolar H(+)-pyrophosphatase by tetranitromethane.

Plant vacuolar vesicles contain a novel H(+)-translocating pyrophosphatase (H(+)-PPase, EC 3.6.1.1). Modification of tonoplast vesicles and purified vacuolar H(+)-PPase from etiolated mung bean seedlings with tetranitromethane (TNM) resulted in a progressive decline in H(+)-translocating pyrophosphatase activity. The half-maximal inhibition was brought about by 0.6, 1.0, and 0.8 mM TNM for purified and membrane-bound H(+)-PPases, and its associated proton translocation, respectively. The maximal inhibition of vacuolar H(+)-PPase by TNM occurred at a pH value above 8. Loss of activity of purified H(+)-pyrophosphatase followed pseudo-first order rate kinetics, yielding a first-order rate constant (k2) of 0.039 s(-1) and a steady-state dissociation constant of inactivation (Ki) of 0.02 mM. Covalent modification of vacuolar H(+)-PPase by TNM increased Km value of the enzyme for its substrate without a significant effect on Vmax. Double logarithmic plots of the pseudo-first order rate constant (kobs) versus TNM concentration exhibited a slope of 0.88, suggesting that at least one tyrosine residue was involved in the inactivation of H(+)-PPase enzymatic activity. Further spectrophotometric measurements of the nitrated H(+)-pyrophosphatase indicated that TNM could modify approximately two tyrosine residues/subunit of the enzyme. However, Tsou's analysis revealed that only one of those modified tyrosine residues directly participated in the inhibition of enzymatic activity of vacuolar H(+)-PPase. The physiological substrate, i.e., dimagnesium pyrophosphate, provided substantial protection against inactivation by TNM. Moreover, NEM pretreatment of the enzyme decreased the number of subsequent nitration of vacuolar H(+)-PPase. Taken together, we suggest that vacuolar H(+)-pyrophosphatase contains a substrate-protectable tyrosine residue conferring to the inhibition of its activity and this tyrosine residue may be located in a domain sensitive to the modification of Cys-634 by NEM.

[Changes in the deformability of erythrocytes and 2,3-DPG in coronary artery stenosis].

The changes of deformability of RBC and 2,3-DPG in RBC in patients with coronary artery stenosis and the influence of some factors on them were studied. The results showed that: (1) the deformability index was inversely proportional to the degree of lesions of coronary vessels (P less than 0.01); (2) the 2,3-DPG was inversely proportional to the degree of lesions of coronary vessels (P less than 0.01); (3) there was a significant decrease in deformability index after using contrast medium; (4) Saliva may increase deformability of RBC and 2,3-DPG; (5) Nifedipine may increase deformability of RBC. These results showed: (1) the possible existence of microcirculatory dysfunction in coronary artery disease and in parallel of impaired deformability of RBC; (2) the possibility of normalization of the decreased deformability of 2,3-DPG in RBC by Salvia; (3) the possibility of increase of velocity of blood flow in microcirculation in coronary artery disease by Salvia.