大鼠大脑皮层中钙调神经磷酸酶活力的时空变化

以ＰＮＰＰ为底物测定了超离心制备的大鼠出生后早期和成年大脑皮层亚细胞各组分中钙调神经磷酸酶的活力。实验结果表明：（ｌ）钙调神经磷酸酶活力广泛地存在于胞液和突触部分,并且各亚细胞组分有明显差异。成年大鼠大脑皮层中ＣａＮ活力相对最高水平是在突触体,突触质,胞液,重的和轻的突触膜部分。（２）大鼠大脑皮层突触体中ＣａＮ活力在出生后第２周和第３周出现高峰的平台期,这与突触发生的高峰期是一致的。在胞液和重的突触膜中ＣａＮ活力最高水平是在出生后的第７ｄ,而在突触质和轻的突触膜中是在第２０ｄ。总之,这些发现证实,在脑发育期间,ＣａＮ活力是依照区域和时间性控制的,提示ＣａＮ可能参与了突触功能作用。     

Epipolarization Microscopic Immunogold Assay: a Combination of Immunogold Silver Staining, Enzyme-Linked-Immunosorbent Assay and Epipolarization Microscopy

We describe a new immunoassay which combines an immunosorbent assay, Immunogold silver staining and epipolarization microscopy. Our new assay procedure features multiple samples on a single microscope slide, and high sensitivity of epipolarization microscope for detection of silver-enhanced colloidal gold as a final immunoassay product. We call the new immunoassay “on slide immunogold assay” (OSIGA). This new method uses biotinylated antibody and streptavidin-gold reaction with silver enhancement technique. With OSIGA it is possible to investigate 30 samples on a single microscopic slide. Our preliminary studies used 10-20 μ1 samples and detected nanogram quantities of a standardized protein solution. Unlike enzyme linked immunosorbent assay (ELISA), which has a limited time for reading the final color products, the OSIGA specimens can be dried or resin mounted for longer storage and future reference.     

A reporter gene analysis of penicillin biosynthesis gene expression in Penicillium chrysogenum and its regulation by nitrogen and glucose catabolite repression.

Vectors which possess a truncated niaD gene encoding nitrate reductase were developed to allow targeted gene integration during transformation of an niaD mutant Penicillium chrysogenum host. The Penicillium genes pcbC and penAB are immediately adjacent to each other and are divergently transcribed, with an intergenic control region serving as their promoters. Gene fusions were constructed with a reporter gene, uidA, which encodes beta-glucuronidase. The pcbC-penAB intergenic region was fused to the uidA gene in both orientations so that regulated expression of each structural gene could be investigated. These fusion genes were targeted to the chromosomal site of the niaD locus of P. chrysogenum, and their expression was examined under different growth conditions. The expression of each of these penicillin biosynthesis genes was found to be regulated by nitrogen repression, glucose repression, and growth stage control.     

Effects of low root zone temperatures on the early stages of symbiosis establishment between soybean [Glycine max (L.) Merr.] and Bradyrhizobium japonicum

It is known that low root zone temperatures (RZT) have moreeffect on infection and early nodule development than on nitrogenfixation by soybean [Glycine max (L.) Merr.]. However, therehave been no studies regarding how the low RZT inhibit the infectionstages of soybean. Two controlled environment experiments wereconducted to examine the effect of low RZT on bacterial attachmentto, and infection thread penetration of, soybean root hairs.The experimental designs were (1) plants maintained at 25, 17.5or 15C RZT, or transferred from 25 or 17.5 to 15C RZT at either0.5, 1, 2, or 7d after inoculation (DAI), (2) early symbioticestablishment between soybean and Bradyrhizobium japonicum wasexamined microscopically under three RZT (15, 17.5 and 25C).These results indicated that (1) keeping plants at 25C only0.5 DAI prior to transfer to a 15C RZT accelerates the onsetof N₂ fixation at 15C RZT by 6 d, (2) at RZT between 25 and17.5C the infection processes were progressively delayed astemperature declined, (3) RZT less than 17C strongly inhibitedinfection steps, such that when RZT dropped 8.5C from 25 to17.5C infection initiation was delayed 1 d, while when RZTdropped only 2.5C from 17.5 to 15C, infection initiation wasdelayed another 2 d. Key words: Bradyrhizobium japonicum, low temperature, nodulation, soybean     

Expression of mouse metallothionein-I gene confers cadmium resistance in transgenic tobacco plants

Transgenic tobacco plants containing a mouse metallothionein-I (MT-I) gene fused to the cauliflower mosaic virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming tobacco leaf discs with an Agrobacterium tumefaciens strain carrying the chimaeric gene. Transformants were directly selected and rooted on medium containing cadmium and kanamycin. A total of 49 individual transgenic tobacco plants were regenerated. Among them 20% showed a very high expression level and their growth was unaffected by up to 200 M cadmium, whereas the growth of control plants was severely affected leaf chlorosis occurred on medium containing only 10 M cadmium. The concentration of MT-I in leaves of control and transgenic tobacco was determined with Cd/haemoglobin saturation assay, a polarographic method and western blotting. In addition, seeds from self-fertilized transgenic plants were germinated on medium containing toxic levels of cadmium and scored for tolerance/susceptibility to this heavy metal. The ratio of tolerant to susceptible plants was 3:1 indicating that the metallothionein gene is inherited as a single locus.     

New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA ⁺ gene were grown at 30° C and then transferred to 42° C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB ⁺ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42° C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.     

Plant growth and nutrient uptake characteristics of Fe-deficiency chlorosis susceptible and resistant subclovers

The objective of this study was to evaluate the growth and nutrient-uptake characteristics of Fe-deficiency resistant and susceptible subclover (Trifolium subterraneum L., T. yanninicum Katzn. and Morley, T. brachcalycinum Katzn. and Morley) cultivars on a calcareous soil. Ten subclover cultivars showing varying susceptibilities to Fe-deficiency chlorosis (Karridale, Nangeela, Geraldton, Mt. Barker, Woogenellup, Larisa, Trikkala, Rosedale, Koala and Clare) were grown on a low-Fe, calcareous soil (Petrocalcic Paleustoll) under moist (18% water content, 85% of water holding capacity) and water-saturated conditions using a Cone-tainer^® culture system. Chlorosis and its correlation with growth traits and mineral nutrition of the 10 cultivars were examined. The Fe-deficiency susceptibilities of the 10 cultivars decreased in the above order under the moist condition, but in slightly different order under the saturated condition. Shoot and root dry weights, total dry weight, and root-to-shoot ratio were each negatively correlated with chlorosis under both soil-moisture conditions, as was total shoot content of P, Ca, Fe, Mn and Zn. Shoot P and Fe concentrations were each positively correlated with chlorosis under the moist soil condition. Iron and Cu utilization efficiencies (biomass per unit weight of nutrient) in the shoot were each negatively correlated with chlorosis under the moist soil condition. These results suggest that there may be several characteristics of Fe-deficiency chlorosis resistance in subclovers, such as a more effective soil-Fe mobilizing mechanism(s), more balanced nutrition, lower required Fe concentration in the shoot, higher shoot-Fe utilization efficiency, and higher root/shoot ratio under Fe-deficiency stress conditions.