Construction and characterization of partiallyntrC-deleted mutants inAlcaligenes faecalis

To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Km^r fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC ^- ) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifH-lacZ gene was introduced into the ntrC^- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC^-mutant was nif ⁺ , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC^- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.     

Characterization of the flagellar biosynthesis regulatory geneflbD inAzospirillum brasilense

A flagellar gene cluster fragment includingflbD ofAzospirillum brasilense was cloned and sequenced. TheflbD mutant strain was found to be nonmotile—losing both polar and lateral flagella (Fla⁻Laf⁻). Motility and flagella were regained by complementation with plasmid-borne multicopyflbD, but altered with larger swarming circle and fewer lateral flagella on the semisolid plate. This result indicated that FlbD plays an important role in the regulation of both polar and lateral flagellar biosynthesis inA. brasilense.     

An explanation to the high efficiency ofm-THPC (temporfin) used in photodynamic therapy

Meso-tetrahydroxylphenyl chlorin (m-THPC) is one of the most efficient prospective sensitizers used in photodynamic therapy (PDT). ESR spectroscopy, fluorescence quenching experiments and cyclic voltammogram measurement were used to study its redox properties. The results showed that the ability of m-THPC generating superoxide radical anions was very strong, and the rate constant of m-THPC fluorescence quenching by oxygen k_q (O₂)=1.46×10¹⁰ mol^-1·s^-1. The values of fluorescence quen- ching rate constant of m-THPC by some other electron acceptors, such as methyl viologen (MV²⁺) and anthraquinone (An), were also measured. And they were k_q (MV²⁺)=5.51×10⁹ mol^-1·s^-1, k_q (An)=7.81×10⁹ mol^-1·s^-1. The oxidation potential of m-THPC was examined to be +0.62 V (vs. NHE) in acetonitrile. All these suggested that m-THPC should be a much stronger electron donor than photofrin, the currently used in clinical photodrug, and may react easily through electron transfer with biological matter to yield various radicals. So it seemed reasonable that the type Ⅰ reaction may play an important role in the high activity of m-THPC-PDT.     

A tumor suppressor genefhit prokaryotic expression and identification

The recombinant expression vector pGEMD-fhit which contains full encoding region offhit gene was constructed. The recombinant was introduced into the BL21 (DE3) strain ofE. coli and induced by 1 mmol/L IPTG to express a 29×10³ polypeptide offhit fusion protein. And the 29×10³ protein was sensitive and specific in reaction with anti-fhit antibody in Western blot. Foundation item: Supported by the National Natural Science Foundation of China (39770373) Biography: SUN Yan (1975−), female, Master of science, Research direction: gene engineering     

Microcalorimetric study on the antibiotic activity of clarithromycin and erythromycin

By using LKB-2277 Bioactivity Monitoring System, the heat effect changes in the process of inhibitory action of clarithromycin and erythromycin onEscherichia coli at 37°C were determined. Quantitative analysis showed that relationship between antibiotic concentrationc and rate contantk ofEscherichia coli growth, and half inhibitory ratio concentration IC₅₀: clarithromycin:k=0. 030 03–1. 1736×10⁻³ c, 8. 45 mg ·L⁻¹; erythromycin:k=0.031 08–8.4657×10⁻⁴ c, 14. 45 mg·L⁻¹. As a result of the microcalorimetry experiments, it not only indicated that antibacterial activity of clarithromycin was stronger than that of erythromycin, but also reported the changeable features of thermodynamics of the bacterial cell in biological, biochemical and metabolic process under different drug action. Foundation item: Supported by Natinal Natural Science Fundation of China (29973030), Natural Science Fundation of Hubei Province (98J052) and Post-doctoral Science Fundation of China Biography, SHEN Xue-song (1956-), Associate professor Research direction: biothermochemistry.     

Cloning and expression analysis ofMBLL cDNA

Thembl (muscleblind) gene ofDrosophila encodes a nuclear protein which contains two Cys₃His motifs. The mutation ofmbl gene will disturb the differentiation of all theDrosophila’s photoreceptors. Primers have been designed according to human EST086139, which is highly homologous tombl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designatedMBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys₃His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology toDrosophila’s mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show thatMBLL is a widely expressed gene, but the expression amounts differ in these tissues.     

Root and xylem ABA changes in response to soil drying in two woody plants

Two woody plants, Platycladus orientalise (tolerant to drought) and Acacia auriculi-formis (sensitive to drought), have been subjected to rapid and slow soil drying. ABA levels in their roots and xylem sap have been determined using radioimmunoassay (RIA, sensitivity is 0.4 pmol per assay vial) with a monoclonal antibody against ( + )-ABA. ABA contents of P. orientalise and A. auriculiformis growing in well watered soil are 0.3 and 2.5 nmol·gDW-1 in roots and 1.6 and 0.4 μmol in xylem saps, respectively. A rapid soil drying has been applied to these two plants with soil water content (SWC) being reduced to 0.02 and 0.06 g·gDW-1 respectively. Under such treatment, ABA was increased by 22 times and 2 times in roots and by 7 times and 34 times in xylem saps respectively for P. orientalise and A. auriculiformis. After rewatering for 6 d, ABA in roots and xylem sap of both species returned to control levels. When a slow soil drying was applied, SWC was reduced to 0.1 and 0.13 g·gDW-1 respectively for P. orientalise and A. auriculiformis. ABA was increased by 5 times and 1.6 times in roots and by 6 times and 19 times in xylem saps respectively for these two plants. ABA in roots and xylem saps decreased to near control levels 8 d after watering. Plant leaf water potentials of both plants hardly changed at times when root and xylem ABA showed substantial increase in response to soil drying. It is concluded that ABA levels in the roots and xylem saps of P. orientalise and A. auriculiformis are more sensitively regulated than leaf water potential in response to soil drying and can act as a chemical signal in root-shoot communications of the drought stress.     

Improving DNA damage repair ability ofE. coli to UV-radiosensitivity by DNA-mediated gene transfer of low energy ion beam

Using calcium chloride method of transfer gene as control, a new technique of transferring gene by low energy ion beam has been applied to the study of improving DNA damage repair ability ofE. coli to UV-radiosensitivity. The genome DNA pieces ofDeinococcus radiodurans, as “foreign” genetic materials, were introduced into the UV-radiosensitive strains ofE. coli by implantation of 20 keV Ar⁺ at doses ranging from 1 × 10¹⁵ to 2 × 10¹⁵ ions/cm². Results show that the transfected strains present higher UV-radioresistance than that of un-transfected ones and start ones. The survival rate of transfected strains and their unscheduled DNA synthesis (UDS) ability is increased, indicating that the transfer gene is a success.     

K-uniformly rotund spaces andk-uni formly smooth spaces

The conception ofk-uniform smoothness (KUS) is introduced. It is the extension of the conception of uniform smoothness. It is proved that thek-uniform smoothness and Sullivan’sK-uniform rotundity (KUR) are the daul notions. X* is a KUR space if and only if X is a KUS space, X* is a KUS space if and only if X is a KUR space. If X is a KUS space, then X is a (K + 1) US space. It is also proved that the KUS space includes the Nan’sk-strongly smooth space.     

Efficient transfer and expression of human clotting factor IX cDNA in neonatal hemophilia B mice mediated by VSV-G pseudotyped retrovirus

The feasibility of in vivo gene therapy for hemophilia B by VSV-G pseudotyped retroviral vector was introduced. The novel packaging cell line 293GPG was used to produce VSV-G/G1NaBAIX pseudotyped virus with the highest titers up to 8.5×10⁸ cfu·mL^-1. In contrast to the conventional retrovirus, VSV-G pseudotyped virus was more resistant to inactivation by serum complements (P＜0.001). Our results also demonstrated that VSV-G pseudotyped virus was more stable in neonatal mice serum than in adult mice serum (P＜0.01). After intraperitoneal injection of different doses of virus, hFIX antigen was detected and lasted for more than 120 d, the highest level reached (72.5±6.1) ng·mL^-1. Moreover, the functional activity was improved to some extent in all hFIX-treated mice, the most remarkable improvement was observed in the mice treated with higher dose of virus whose clotting activity increased to (3.4±1.5)% and APTT (activated partial thromboplastin time) reduced to (43.2±7.2) s. The anti-hFIX antibody was not detected by the method of Bethesda, no germ line transmission and any side effects associated with gene transfer were found. Our results indicated that neonatal gene therapy for hemophilia B mice by VSV-G pseudotyped retrovirus is promising.     

Synoptic-scale variation of δ18O in summer monsoon rainfall at Lijiang, China

Based on the daily δ~(18)O data in June―September 2003 at Lijiang and the daily mean NCEP/ NCAR reanalysis data, synoptic-scale variation of δ~(18)O in summer monsoon rainfall was investigated. The 'precipitation amount effect' is obvious for the daily δ~(18)O variation, whereas the 'temperature effect' is insignificant. Alternate occurrences of active phase and break phase of the southwest monsoon probably influence the synoptic-scale δ~(18)O variation prominently. Moreover, the isotopic composition in precipitation during the late monsoon months is presumably influenced significantly by recycling of monsoon precipitation. Both the above factors disturb the 'amount effect' of isotopic variation in the monsoon region. This study also indicates that the synoptic-scale rainfall δ~(18)O variation at Lijiang in summer is domi-nated by the Indian monsoon depression (low pressure) system at large scale. These results are important for further studying the 'amount effect' and reconstructing paleoclimate in the monsoon region.     

Molecular characterization of a K+ channel blocker in the scorpionButhus martensii Karsch

K⁺ channel blockers of scorpion venoms are of important value in studying pharmacology and physiology of specific K⁺ channel of cells. Based on the amino acid sequences of BmP01 previously characterized as a small-conductance Ca²⁺-activated K⁺ channel blocker, two “back to back” degenarate primers have been designed and synthesized for inverse PCR strategy, its full-length cDNA has been cloned from the venom gland of the Chinese scorpionButhus martensii. The cDNA is composed of 3 parts: 5′ UTR, ORF and 3′ UTR. The flanking sequence of translation initiation codon ATG is AAAATGA, which is highly conserved in scorpion Na⁺ channel toxin and protozoan genes, suggesting that these genes may have followed a common mechanism for translation initiation. The 3′ UTR contains poly(A) signal AATAAA. The open reading frame encodes a precursor of 57 residues with a signal peptide of 28 residues and a mature peptide of 29 residues. The signal peptide is rich in hydrophobic amino acid residues and its length is significantly different from that of the determined scorpion Na⁺ channel toxin. The deduced amino acid sequence of mature peptide is completely consistent with BmP01 previously determined by primary structure analysis.     

Multi-parameter infinite-dimensional (r,δ)-OU process

The definition of n-parameter infinite-dimensional (r,δ) -Ornstein-Uhlenbeck process {xt (·) } ((r, δ)-OUPn∞ for short) is given. The absolute continuity of distribution μt of Xt(·) and the limit of Xt(·) when |t| →∞ is discussed.     

Oligocarpia kepingensis sp. nov. from the Lower Permian of the northern Tarim Basin, Xinjiang and itsin situ spores

A Palaeozoic gleicheniaceous fernOligocarpia kepingensis sp. nov. is described from the Lower Permian of the northern Tarim Basin, Xinjiang, China. The material comprises fertile organs including sori, sporangia, spores and associated sterile leaf of theSphenopteris type. The sori are circular and 0.6–0.8 mm in diameter, and each sorus consists of 4–6 oval sporangia without an indusium. A transverse annulus completely encircles the sporangium. Each sporangium produces probably 256 trilete spores resembling the dispersed genusLeiotriletes. Comparisons are made betweenO. kepingensis and other species ofOligocarpia in the soral organization and spores. It is reasonable to includeOligocarpia in Gleicheniaceae based on its similarities of fertile character to the extant gleicheniaceous members.     

Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.