Role of methylation in aerotaxis in Bacillus subtilis.

Taxis to oxygen (aerotaxis) in Bacillus subtilis was characterized in a capillary assay and in a temporal assay in which the concentration of oxygen in a flow chamber was changed abruptly. A strong aerophilic response was present, but there was no aerophobic response to high concentrations of oxygen. Adaptation to a step increase in oxygen concentration was impaired when B. subtilis cells were depleted of methionine to prevent methylation of the methyl-accepting chemotaxis proteins. There was a transient increase in methanol release when wild-type B. subtilis, but not a cheR mutant that was deficient in methyltransferase activity, was stimulated by a step increase or a step decrease in oxygen concentration. The methanol released was quantitatively correlated with demethylation of methyl-accepting chemotaxis proteins. This indicated that methylation is involved in aerotaxis in B. subtilis in contrast to aerotaxis in Escherichia coli and Salmonella typhimurium, which is methylation independent.     

Bacterial cellulose production by Acetobacter xylinum in a 50-L internal-loop airlift reactor

Bacterial cellulose (BC) production was realized in a batch cultivation of Acetobacter xylinum subsp. sucrofermentans BPR2001 in a 50-L internal-loop airlift reactor. When the bacterium was cultivated with air supply, 3.8 g/L of BC was produced after 67 hours. When oxygen-enriched gas was supplied, the concentration of BC was doubled and the production rate of BC was 0.116 g/L. h, which was two times higher than that of air-supplied culture and comparable to that in a mechanically agitated stirred-tank fermentor. Bacterial cellulose produced by the airlift reactor formed a unique ellipse pellet (BC pellet), different from the fibrous form which was produced in an agitated stirred-tank fermentor. The BC-pellet suspension was demonstrated to have a higher volumetric oxygen transfer coefficient than the fibrous BC suspension in a 50-L internal-loop airlift reactor. The mixing time of BC-pellet suspension in the airlift reactor was also shorter than that in water.     

Molecular Characterization of Recombinant Mus a 5 Allergen from Banana Fruit

Allergy to banana fruit appears to have become an important cause of fruit allergy in Europe. Among five allergens that have been found, beta-1,3-glucanase denoted as Mus a 5 was identified as a candidate allergen for the component-resolved allergy diagnosis of banana allergy. Because of the variations in protein levels in banana fruit, in this study Mus a 5 was produced as a fusion protein with glutathione-S-transferase in Escherichia coli. The recombinant Mus a 5 was purified under native conditions by a combination of affinity, ion-exchange, and reversed phase chromatography. N-terminal sequence was confirmed by Edman degradation and 55 % of the primary structure was identified by mass fingerprint, while the secondary structure was assessed by circular dichroism spectroscopy. IgG reactivity of recombinant protein was shown in 2-D immunoblot with anti-Mus a 5 antibodies, while IgG and IgE binding to natural Mus a 5 was inhibited with the recombinant Mus a 5 in immunoblot inhibition test. IgE reactivity of recombinant Mus a 5 was shown in ELISA within a group of ten persons sensitized to banana fruit. Recombinant Mus a 5 is a novel reagent suitable for the component-resolved allergy diagnosis of banana allergy.     

Female-induced sexual arousal in male mice and rats: behavioral and testosterone response

Exposure of a male mouse to a female mouse separated from it by a holed partition induced specific behavior and an increase in blood testosterone in the male. The male made more approaches to the partition and spent more time at it. The time spent by the male mouse over the first 10 min at the partition, behind which an estrus female was placed, was increased sixfold compared to the time spent by a male mouse exposed to the vacant neighboring compartment; and 1.5-fold compared to that spent by a male mouse exposed to a nonreceptive female or a male. Increased blood testosterone level was detected at 20 min of exposure to a receptive female in winter and at 40 min in summer. No variation in blood testosterone levels in the male mouse exposed to a nonreceptive female or a male was observed. Similar response to a receptive female placed in the neighboring compartment was shown in a male rat. The time spent by the male rat at the partition was 12 times higher when there was an estrus female behind it than in control. Blood testosterone in the male rat increased in response to a female rat and did not change in response to a male rat indicating female-induced motivation. It was concluded that the partition time might serve as a quantitative measure of sexual motivation in the males and that the model of female-induced sexual arousal used was suitable for studying both motivational and hormonal components of sexual arousal in male mice and rats.     

豆豉纤溶酶基因在大肠杆菌中的可溶性表达及纯化(英文)

豆豉纤溶酶是从豆豉中发现的新型纤溶酶.从枯草杆菌DC-12中提取总DNA,PCR法扩增pro-DFE基因.将pro-DFE基因插入载体pET-32a,构建表达质粒pET-pro-DFE,转化大肠杆菌BL21(DE3),对重组菌株进行变温诱导表达,重组菌株于37℃培养至OD600为0.6时,加入终浓度为0.4mmol/L的IPTG于24℃进行诱导,SDS-PAGE显示可溶性重组融合蛋白约占重组蛋白的60%.且少量融合重组蛋白发生自切割,形成成熟的豆豉纤溶酶,破菌上清中含有成熟的豆豉溶栓酶,纤溶活性为200IU/mL.重组酶经纯化后比酶活为1280IU/mg.     

DEDIFFERENTIATION OF THE DISAGGREGATED SLUG CELL OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM

It was previously shown that differentiation of the prespore cell in the pseudoplasmodium (slug) of the cellular slime molds is characterized by the synthesis of a specific substance which is detectable by a heteroplastic antispore serum (T akeuchi , 1963). When a prespore cell which was already differentiated was disaggregated from a slug of Dictyostelium discoideum and was incubated in salt solution under a sparsely populated condition, it gradually lost its specific substance and dedifferentiated. The dedifferentiation proceeded without accompanying cell growth and was completed within 5 hr of incubation. This process was inhibited at a low temperature and also in the presence of cyclohexamide, actinomycin D, and cyclic AMP. The dedifferentiation was induced and proceeded at a normal rate in the absence of bacteria. When a disaggregated slug cell was incubated in the presence of bacteria, however, every prestalk and prespore cell was able to grow and underwent its first cell division after about 9–10 hr of incubation, and then multiplied with the generation time of 3 hr. The relationship between the dedifferentiation and the growth of a disaggregated slug cell was discussed.     

A Nuclear Stain for Escherichia Coli And Related Organisms

The dye base of new fuchsin was precipitated by adding potassium hydroxide to the dye solution. The precipitate was filtered out and washed with water. It was then suspended in water, brought into solution and adjusted to a pH of about 5.0 with nitric acid. The staining solution was prepared by adding 0.3 ml. of a 14% aqueous solution of pyrogallol and 0.1 ml. of a 1% aqueous solution of boric acid to 3.0 ml. of the dye solution. Smears of cells were made in water on a slide and allowed to dry before covering with the staining solution which was also permitted to air dry. The smear was then washed in water and mordanted for 5-20 seconds in a 0.1% aqueous solution of mercuric nitrate. After rinsing in water, the smear was air dried. When dry, the slide was placed on a 50° C. warm plate for a few seconds before covering with a very thin film of a 5% aqueous solution of nigrosin which had a pH of about 5.0.     

Prefabricated buccal mucosa-lined flap in an animal model that could be used for vaginal reconstruction

Congenital vaginal aplasia, gynecological tumor excision, and male-to-female sex surgery are three clinical conditions in which the plastic surgeon is involved in vaginal reconstruction. Skin-lined or skin-grafted local flaps are currently used, but for many reasons, keratinized skin is not the ideal lining for such a moist cavity because it leads to dryness, desiccation, maceration of the skin, and even hair growth in the cavity. The purpose of this study was to create a subcutaneous cavity lined with mucosa in an area with a predictable blood supply. The abdominal area supplied by the deep circumflex iliac vessels was chosen. Six minipigs were used. Strips of tongue buccal mucosa formed the lining; if additional tissue was required, it was taken from the mucosal aspect of the cheek. The mucosa was expanded by using multiple stab incisions. The mucosa was sutured onto the fascia supplied by the deep circumflex iliac vessels, and the skin incision was closed over a silicone sheet to prevent adhesion to the underlying mucosa. This was left for 1 week to allow the mucosa to take. The prefabricated fascial flap was rolled over a silicone stent and was closed longitudinally to form a cylindrical shape. The flap was placed in a subcutaneous pocket in the right inguinal area. The caudal end was left open and was sutured to the surrounding skin. The silicone stent was used to keep the cavity patent and to prevent adhesions in the early stage of the healing process. Regular digital examination was performed to assess patency and contour; endoscopy allowed assessment of mucosa viability. This method of producing a mucosa-lined flap may provide a solution to the difficult problem of vaginal reconstruction.     

Inter-plot competition in yield trials of potatoes (Solanum tuberosum L.) with single-drill plots

An experiment was carried out to assess the significance of inter-plot competition in a yield trial of potato cultivars. Seventeen cultivars were deliberately chosen and assessed for yield in single-drill and four-drill plots. Inter-plot competition for fresh-weight yield was a significant factor in the single-drill plots. It was modelled using a common competition coefficient with a covariate based on neighbour fresh-weight yields. In contrast, there was no statistically significant inter-plot competition for specific gravity. After adjustment for inter-plot competition, varietal ranking in estimated monoculture yield differed little from that based on unadjusted means. However, there was a reduction in the range of yield estimates, and a closer agreement with the observed pure-stand yields from the inner two drills of the four-drill plots. The adjustment for monoculture performance was most pronounced for the higher and lower yielding varieties, as expected from the assumption that the performance of high yielding varieties was enhanced in a competitive environment at the expense of low yielding ones. A general and flexible method of estimating competition coefficients in variety trials, together with a suitable algorithm, was developed and is explained in an appendix. It was used to check for inter-plot competition in a number of potato trials with single-drill plots and a large number of entries. Competition was found in some trials but not in others. Thus, where potato tubers are grown in single-drill plots for assessment of fresh-weight yield, adjustment should be made for inter-plot competition when evidence of inter-drill competition is found.     

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. Metabolite identification

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 in a reconstituted system and by rat liver microsomes was examined. Eleven metabolites were detected. Two of these, found in spots 2 and 4 of a thin layer plate, were only formed by the rat liver microsomes and may represent reductive metabolites of testosterone. A number of monohydroxy metabolites were conclusively identified by gas chromatography-mass spectrometry. These include the 2-, 6 beta-, 7 alpha-, and 16 alpha-hydroxy isomers. Liver microsomes formed the 2 alpha- and 2 beta-epimers in a 1:2 ratio and both co-chromatographed with a third reduced metabolite in thin layer plate spot 4. In contrast with RLM5 about 90% of the 2-hydroxy isomer was the 2 alpha-epimer. RLM3 did not perform the 2-hydroxylation in detectable amounts. The 6 beta-isomer was a major metabolite of RLM3 and microsomes, but a minor product of metabolism by RLM5. In contrast, the 7 alpha-isomer was a minor metabolite of RLM3, was not formed by RLM5, and was a major microsomal metabolite. Hydroxylation at position 16 alpha was a major activity of RLM5 and the heterogeneous microsomal cytochromes, but with RLM3 it was a minor reaction. One new metabolite was found which appeared to be hydroxylated in the D-ring, had a mass spectrum different from both 16 alpha- and 16 beta-hydroxytestosterone, and was tentatively identified as a 15-hydroxy isomer. In agreement with the literature, androstene-3,17-dione was found to be an oxidative metabolite of testosterone by both microsomes and purified cytochrome P-450. It was a major metabolite of RLM5 but was not produced by RLM3. Studies with 18O2 and H218O conclusively show that oxidation of testosterone at C-17 does not involve transient incorporation of an oxygen atom in this position. A mechanism is suggested whereby cytochrome P-450 acts as a peroxidase in the formation of androstenedione.     

Probing the yeast 5 S RNA-protein complex by fluorescence and controlled proteolytic digestion

The nature of the interaction between the RNA and the protein component in the yeast 5 S rRNA-L1a complex was assessed using fluorescence and controlled proteolytic and RNase digestion. (a) Influence of L1a on the RNA conformation was monitored by ethidium fluorescence and controlled RNase T1 digestion. The complex was digested with alpha-chymotrypsin, Staphylococcus aureus protease V8, subtilisin, or trypsin. Both termini of L1a in the complex were readily accessible to proteases. Proteolytic digestion of the complex resulted in a reduction in fluorescence intensity if ethidium was added after proteolysis. No change was observed when ethidium was allowed to react with the complex prior to proteolysis. Neither the rate of proteolysis nor the resultant peptide pattern was affected by the presence of ethidium. T1 digestion of intact RNP and trypsin-treated RNP produced different oligonucleotide patterns. Both the fluorescence and the T1 digestion data suggest that the conformation of the RNA moiety was influenced by the protein. (b) Influence of the RNA molecule on L1a conformation in the complex was monitored by limited proteolysis. Whereas the protein in the complex was relatively sensitive to proteases, free protein was completely resistant to digestion under identical conditions. The trypsin sensitivity of L1a in complexes containing different truncated 5 S RNA molecules was studied also. Upon removal of residues 31-49 of the 5 S RNA molecule, L1a in the complex became resistant to proteolysis. These results are interpreted in a model in which specific regions of both the RNA and the protein are involved in the interaction.     

Diel profile of behaviour in the smooth newt, Triturus vulgaris (L.): an analysis of environmental cues and endogenous timing

Diel activity in a population of smooth newts during the aquatic period, was compared with rhythmic activity in newts under controlled light-dark and temperature cycles in the laboratory. The light-dark cycle was the primary synchronizer of activity and entrained a bimodal activity rhythm. This rhythm was endogenous and persisted for several cycles under constant conditions. Under natural conditions this rhythm was modified by illumination and temperature to produce a crepuscular activity pattern with a dominant second peak in activity. Diel activity under natural conditions was partitioned into sexual behaviour and feeding. Whereas sexual behaviour was entirely crepuscular, feeding behaviour was nocturnal. Feeding was probably unrelated to the underlying circadian rhythm and was engendered by the diel migration of zooplankton in the ponds. Ascents to the pond surface for air were most frequent during periods of sexual behaviour.     

Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus.

The gene encoding the thermostable direct hemolysin of Vibrio parahaemolyticus was characterized. This gene (designated tdh) was subcloned into pBR322 in Escherichia coli, and the functional tdh gene was localized to a 1.3-kilobase HindIII fragment. This fragment was sequenced, and the structural gene was found to encode a mature protein of 165 amino acid residues. The mature protein sequence was preceded by a putative signal peptide sequence of 24 amino acids. A putative tdh promoter, determined by its similarity to concensus sequences, was not functional in E. coli. However, a promoter that was functional in E. coli was shown to exist further upstream by use of a promoter probe plasmid. A 5.7-kilobase SalI fragment containing the structural gene and both potential promoters was cloned into a broad-host-range plasmid and mobilized into a Kanagawa phenomenon-negative V. parahaemolyticus strain. In contrast to E. coli, where the hemolysin was detected only in cell lysates, introduction of the cloned gene into V. parahaemolyticus resulted in the production of extracellular hemolysin.