Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology.

Small ubiquitin-related modifier (SUMO) technology has been widely used in Escherichia coli expression systems to produce antimicrobial peptides. However, E. coli is a pathogenic bacterium that produces endotoxins and can secrete proteins into the periplasm, forming inclusion bodies. In our work, cathelicidin-BF (CBF), an antimicrobial peptide purified from Bungarus fasciatus venom, was produced in a Bacillus subtilis expression system using SUMO technology. The chimeric genes his-SUMO-CBF and his-SUMO protease 1 were ligated into vector pHT43 and expressed in B. subtilis WB800N. Approximately 22 mg of recombinant fusion protein SUMO-CBF and 1 mg of SUMO protease 1 were purified per liter of culture supernatant. Purified SUMO protease 1 was highly active and cleaved his-SUMO-CBF with an enzyme-to-substrate ratio of 1:40. Following cleavage, recombinant CBF was further purified by affinity and cation exchange chromatography. Peptide yields of ~3 mg/l endotoxin-free CBF were achieved, and the peptide demonstrated antimicrobial activity. This is the first report of the production of an endotoxin-free antimicrobial peptide, CBF, by recombinant DNA technology, as well as the first time purified SUMO protease 1 with high activity has been produced from B. subtilis. This work has expanded the application of SUMO fusion technology and may represent a safe and efficient way to generate peptides and proteins in B. subtilis.

Limits to sustained energy intake. XVIII. Energy intake and reproductive output during lactation in Swiss mice raising small litters.

Limits to sustained energy intake (SusEI) during lactation in Swiss mice have been suggested to reflect the secretory capacity of the mammary glands. However, an alternative explanation is that milk production and food intake are regulated to match the limited growth capacity of the offspring. In the present study, female Swiss mice were experimentally manipulated in two ways - litter sizes were adjusted to be between 1 and 9 pups and mice were exposed to either warm (21°C) or cold (5°C) conditions from day 10 of lactation. Energy intake, number of pups and litter mass, milk energy output (MEO), thermogenesis, mass of the mammary glands and brown adipose tissue cytochrome c oxidase activity of the mothers were measured. At 21 and 5°C, pup mass at weaning was almost independent of litter size. Positive correlations were observed between the number of pups, litter mass, asymptotic food intake and MEO. These data were consistent with the suggestion that in small litters, pup requirements may be the major factor limiting milk production. Pups raised at 5°C had significantly lower body masses than those raised at 21°C. This was despite the fact that milk production and energy intake at the same litter sizes were both substantially higher in females raising pups at 5°C. This suggests that pup growth capacity is lower in the cold, perhaps due to pups allocating ingested energy to fuel thermogenesis. Differences in observed levels of milk production under different conditions may then reflect a complex interplay between factors limiting maternal performance (peripheral limitation and heat dissipation: generally better when it is cooler) and factors influencing maximum pup growth (litter size and temperature: generally better when it is hotter), and may together result in an optimal temperature favouring reproduction.

(E)-N'-(2,5-Dimethoxy-benzyl-idene)-2,4-dihydroxy-benzohydrazide.

In the title compound, C(16)H(16)N(2)O(5), the dihedral angle between the two benzene rings is 4.2 (2)° and an intra-molecular O-H⋯O hydrogen bond generates an S(6) ring. In the crystal, mol-ecules are linked into layers lying parallel to the bc plane by O-H⋯O and N-H⋯O hydrogen bonds.

(E)-N'-(3,4-Dimethoxy-benzyl-idene)-2,4-dihydroxy-benzohydrazide methanol solvate.

The title compound, C(16)H(16)N(2)O(5)·CH(3)OH, was obtained from a condensation reaction of 3,4-dimethoxy-benzaldehyde and 2,4-dihydroxy-benzohydrazide. The non-H atoms of the Schiff base mol-ecule are approximately coplanar (r.m.s. deviation = 0.043 Å) and the dihedral angle between the two benzene rings is 1.6 (1)°. The mol-ecule adopts an E configuration with respect to the C=N double bond. An intra-molecular O-H⋯O hydrogen bond is observed. The Schiff base and methanol mol-ecules are linked into a two-dimensional network parallel to (10) by inter-molecular N-H⋯O, O-H⋯N and O-H⋯O hydrogen bonds.

(Z)-N'-(4-Hydr-oxy-4-methyl-pentan-2-yl-idene)-2-(8-quinol-yloxy)acetohydrazide.

The title compound, C(17)H(21)N(3)O(3), has a Z configuration about the N=N double bond. The molecular conformation is stabilized by intramolecular N-H⋯O and O-H⋯N hydrogen bonds.

Acute electrical stimulation of nucleus ambiguus enhances immune function in rats.

BACKGROUND: Up to now, many "immunoactive" brain areas have been identified, such as hypothalamic nuclei, brain reward system; but the nucleus ambiguous (Amb), a nucleus nervi vagis of medulla oblongata, was less well studied in neuroimmunomodulation. METHODS: In order to obtain more profound comprehension and more knowledge on Amb, we studied the effect of acute electrical stimulation of Amb on thymus and spleen activity in rat. A stimulator was applied to stimulate the Amb of the anaesthetic rats using the parameter at 100 microgA x 5 ms x 100 Hz every 1 s for 1 min. The levels of TGF-13 and thymosin-beta4 mRNA in thymus, the release of IL-2 and IL-6 at splenocyte in vitro and splenic lymphocyte proliferation were measured at hour 0.5, 1, 2, 3 following the electrical stimulation. RESULTS: The results showed that concanavalin A (Con A)-induced splenic lymphocyte proliferation and the release of IL-2 and IL-6 were all significantly enhanced at 0.5, 1, and 2 h following effective Amb stimulation as compared to in the control group. However, as compared to in the control group, the levels of TGF-beta and thymosin-beta4 mRNA in the thymus were both remarkably reduced at 0.5, 1, and 2 h following effective Amb stimulation. CONCLUSIONS: These findings reveal that the Amb participates in the modulation of animal immune functions.

[Isolation and identification of coxsackievirus in Sichuan golden monkey].

A coxsackievirus B strain was successfully isolated by cells culture from cardiac muscle tissues of a dead Sichuan golden monkey with myocarditis from a zoo of Changchun in China. The isolate was consistent with CVB by morphology, physicochemistry test, animal regression test and RT-PCR. Analysis of VP1 partial gene sequence and detection of mice specific serum IgG showed that the strain isolated was a coxsackievirus B3. It was the first CVB case report in Sichuan golden monkey and the strain isolated was named CVB/SGM-05.

[Analysing genetic distance of national wistar rat with DNA fingerprinting].

DNA fingerprinting of Wistar rat were studied with JL-02 Mulilocus probe and Southern hybridization, and comparing with different individuals of nine groups Wistar rat from six national classical area and different groups. It was indicated that DNA fingerprinting could reflect genetic material of outbred strain rat,and were more polymorphic. The genetic distances of Wistar rat were distributed for 0.2-0.6 among different individuals within same groups, and the genetic distances of Wistar rat were distributed for 0.2-0.7 among different groups.