Backbone dynamics and hydrogen exchange of Pseudomonas aeruginosa ferricytochrome c 551

A model-free analysis of Pseudomonas aeruginosa ferricytochrome c(551) dynamics based on (15)N R(1), (15)N R(2), and [(1)H]-(15)N heteronuclear nuclear Overhauser effect data is reported. The protein backbone is highly rigid (< S(2)>=0.924+/-0.005) and displays little variation in picosecond-nanosecond time scale dynamics over the structure. The loop structure containing the axial methionine ligand (loop 3) displays anomalous rigidity, which is attributed to its high proline content. Also reported are protection factors calculated from hydrogen-exchange rates. These data reveal that loop 3 residues, including the axial methionine, are protected from exchange as a result of long-range hydrogen-bonding interactions. These results are contrasted with data reported for Saccharomyces cerevisiae iso-1-ferricytochrome c, which displays higher overall flexibility (< S(2)>=0.80+/-0.07), greater variation of dynamics as a function of structure, and low protection factors for loop 3. This analysis reveals that heme proteins with similar functions and topologies may display diverse dynamical properties.     

Sequence, distribution and quantification of the motilin precursor in the cat

Due to motilin's relation to the migrating motor complex (MMC), the physiology of motilin has been mostly studied in man and dog. The cat does not have an MMC pattern, and little is known about cat motilin. Therefore we identified the cat motilin precursor (GenBank accession no. AF127917) and developed a quantitative polymerase chain reaction (PCR) to explore its distribution in the gastrointestinal tract and in the central nervous system (CNS). The precursor is closely related to the dog precursor and consists of an open reading frame of 348bp encoding the signal peptide (25 amino acids), the motilin sequence (22 amino acids) and the motilin associated peptide (69 amino acids). One amino acid of the signal peptide was subject to gene polymorphism. Quantification of motilin messenger RNA (mRNA) was for the first time achieved. It is most abundant in the gastrointestinal tract, with the highest concentration in the duodenum, the lowest in the colon and is not detectable in the corpus. However an important expression was also observed in several regions of the CNS, except the striatum and cerebral cortex. The highest level was in the hypothalamus (although 23-fold lower than in the duodenum), the lowest level in the pons. Moderate levels were found in the thyroid. These data suggest that the physiological role of motilin may extend beyond its effect on gastrointestinal motility.     

Study of the factors affecting the extraction of soybean protein by reverse micelles

In this work, the forward and back extraction of soybean protein by reverse micelles was studied. The reverse micellar systems were formed by anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT), isooctane and KCl solution. The effects of AOT concentration, aqueous pH, KCl concentration and phase volume ratio on the extraction efficiency of soybean protein were tested. Suitability of reverse micelles of AOT and Triton-X-100/AOT mixture in organic solvent toluene for soybean protein extraction was also investigated. The experimental results lead to complete forward extraction at the AOT concentration 120 mmol l⁻¹, aqueous pH 5.5 and KCl concentration 0.8 mol l⁻¹. The backward extraction with aqueous phase (pH 5.5) resulted in 100% extraction of soybean protein from the organic phase.     

Differentiation of Bacillus anthracis, B. cereus, and B. thuringiensis by Using Pulsed-Field Gel Electrophoresis

A pulsed-field gel electrophoresis (PFGE) method was developed for discriminating Bacillus anthracis from B. cereus and B. thuringiensis. A worldwide collection of 25 B. anthracis isolates showed high-profile homology, and these isolates were unambiguously distinguished from B. cereus and B. thuringiensis isolates by cluster analysis of the whole-genome macrorestriction enzyme digestion patterns generated by NotI.     

转双价抗虫基因毛白杨无性系85号抗虫性研究

以优化的毛白杨无性系85号最适遗传转化体系为基础,通过农杆菌介导法将Bt(CryⅠAc)和API双价抗虫基因导入毛白杨无性系85号基因组.在高浓度卡那霉素的培养基上诱导不定芽和根,初步选择到200株转化植株,PCR检测显示,66株呈阳性反应.Southern杂交和ELISA检测进一步证明,Bt(CryⅠAc)和API双价抗虫基因已整合到毛白杨无性系85号基因组中,并得到了表达.对转化植株用舞毒蛾幼虫进行饲虫试验,结果显示昆虫幼虫的死亡率高达60.0%~77.8%,且存活幼虫的生长发育也受到了明显抑制.研究结果为杨树抗虫育种提供了新的种质资源.     

Isolation and characterization of a novel Ganoderma lucidum gene that differentially expressed between shaking culture and liquid static culture

Suppression subtractive hybridization (SSH) was preformed to investigate the differences of gene expression between the shaking culture mode and the liquid static culture mode which favors ganoderic acids production in Ganoderma lucidum. One novel gene preferentially expressed in liquid static culture was identified and analyzed. Its full length cDNA sequence and the 5′-flanking region were then obtained by rapid amplification of cDNA ends (RACE) and self-formed adaptor PCR (SEFA-PCR), respectively. Nucleotide sequence of the gene is not homologous to any of the known Ganoderma genes. The sequence analysis revealed that the open reading frame of this gene encodes a protein of 371 amino acids that has high homology with the mitogen-activated protein kinase (MAPK) of other five species-Postia (97%), Coprinopsis (91%), Neurospora (86%), Aspergillus (83%), and Saccharomyces (80%)-so that it can be defined as a G. lucidum MAPK gene (GenBank accession number: JF781125). Computer assisted analysis revealed that this new G. lucidum MAPK gene contains thirteen exons and twelve introns. The quantitative real-time RT-PCR (qRT-PCR) analysis showed that this new gene had a much higher expression level in liquid static culture than in traditional shaking culture. Results of this research established a good foundation for further study on the functions of the G. lucidum MAPK at the molecular level.     

Ganoderic acid Mf and S induce mitochondria mediated apoptosis in human cervical carcinoma HeLa cells

In this work, the effects of a pair of positional isomer of ganoderic acids (GAs), namely ganoderic acid Mf (GA-Mf) and ganoderic acid S (GA-S) purified from the fermented mycelia of Ganoderma lucidum, on induction of cell apoptosis and the apoptotic pathway in HeLa cells were investigated. The results demonstrate that both isomers decreased cell population growth on various human carcinoma cell lines by MTT assay, while GA-Mf had better selectivity between normal and cancer cells. The flow cytometry analysis indicated that treatment of HeLa cells with GA-S caused cell cycle arrest in the S phase, while GA-Mf caused cell cycle arrest in the G1 phase. Compared with GA-S, GA-Mf had more potent increase in the number of early and late apoptotic cells. Treatment of HeLa cells with each isomer decreased the mitochondria membrane potential and caused the release of cytochrome c from mitochondria into the cytosol. In addition, stimulation of caspase-3 and caspase-9 activity was observed. The Bax/Bcl-2 ratio was also increased in GA-treated HeLa cells. The results demonstrated that both isomers GA-Mf and GA-S induced apoptosis of human HeLa cells through a mitochondria mediated pathway, but they had the different cell cycle arrest specificity. The findings will be helpful to the development of useful cancer chemopreventive compounds from G. lucidum.