Relationship between DNA chain growth termination and replicon sizes in γ-irradiated mouse cells

Unirradiated mouse leukemic L5178Y cells were pulse-labeled with [³H]thymidine continuously for up to 150 minutes. The resulting DNA fiber autoradiograms showed a continuously increasing fiber length up to 70 μm. However, in cells irradiated with 500 to 2000 rad of γ-rays, the resulting DNA fiber lengths leveled off at approximately 35 μm. Independent experiments to estimate replicon size by measuring the center-to-center distance in tandem tracks yielded a value of 48 μm. These results are consistent with a model proposing that the average replicon size is equal to √2 times the plateau value of the average length of labeled DNA measured in irradiated cells.     

Purification and properties of reductases for aromatic aldehydes and ketones from guinea pig liver.

NADPH-dependent enzymatic reduction of aromatic aldehydes and ketones observed in the cytosol of guinea pig liver was mediated by at least three distinct reductases (AR 1, AR 2, and AR 3), which were separated by DEAE-cellulose chromatography. By several procedures AR 2 and AR 3 were purified to homogeneity, but AR 1 could be purified only 30-fold because of the small amount. These enzymes were found to have similar molecular weights of 34,000 to 36,000 and similar Stokes radii of about 2.5 nm. AR 3 was identical to aldehyde reductase [EC 1.1.1.2] in substrate specificity for aromatic aldehydes and D-glucuronate and specific inhibition by barbiturates. AR 1 and AR 2 acted on aromatic ketones and cyclohexanone as well as aromatic aldehydes at optimal pHs of 5.4 and 6.0, respectively, and were immunochemically distinguished from AR 3. AR 1 was the most sensitive to sulfhydryl reagents, and AR 2 was more stable at 50 degrees C than the other enzymes. Similar heterogeneity was observed in the kidney enzymes, but other tissues had little aldehyde reductase activity and contained only AR 3. In addition, lung contained a high molecular weight aromatic ketone reductase different from the above reductases.     

Reproductive lifespan and reproductive performance in SPF C3H mice: the onset of reproductive life and production efficiency (author's transl)]

To improve the production system, the onset and the termination of reproductive life of C3Hf/HeMsNrs mice mated immediately after weaning and reared for 400 days of life, were studied. From weaning females mated with a full grown male (group A), the first litter was obtained at a mean age of 47 days, suggesting the first copulation at 26 days of age. The age of males at the first copulation was estimated to be at 44 days of age from the age giving the first litters in weanling males mated with weanling (group B) and full grown (group C) females. The sex ratio of litters delivered by young dams tended to be excess in males. The reproductive performance of dams in later life was not affected by the parturition in earlier age. The production efficiency with weaned youngs per pair during the first 200 days after mating was the highest in group A. It was found from these results that the C3H females attained their sexual maturity at 5 to 6 days after weaning, being available for breeding without any deletion in reproductive performance.     

Tremorgenic Mycotoxin from Penicillium paraherquei

A tremorgenic mycotoxin was isolated from Penicillium paraherquei Abe ex G. Smith and identified as verruculogen. It was produced at the rate of approximately 1 mg/g of the dried fungal mycelium cultured on peptone-enriched Czapek-Dox medium at 28°C.     

Ionic mechanism of 5-hydroxytryptamine induced hyperpolarization and inhibitory junctional potential in body wall muscle cells of Hirudo medicinalis

Five-hydroxy tryptamine (5-HT) causes a hyperpolarization and increased conductance of the leech body wall muscle cell membrane. If 5-HT is applied in the absence of the Cl^?ion, the response appears as a depolarization, whereas if 5-HT is applied in the absence of the K⁺ion, the response is a hyperpolarization. In both cases, the conductance of the muscle cell membrane is increased. Stimulation of the peripheral nerve to the body wall muscle produces a complex junctional potential in muscle cells. Exposing the muscle to d-tubocurarine (d-TC) eliminates the excitatory component (EJP) of the complex potential. The inhibitory potential (IJP) that remains has an equilibrium potential at approximately 65 m V. Furthermore, this IJP appears as a depolarization when the nerve is stimulated in the presence of d-TC and low CL^?, whereas this is not the case if the nerve is stimulated in the presence of d-TC and low K⁺. The drugs BOL-148 and cyproheptadine block the IJP's in the body wall muscle. These data are interpreted as indicating that 5'HT acts on leech body wall muscle cells by increasing the conductance to the Cl^?ion and that the IJP's caused by nerve stimulation are probably the result of 5-HT release at nerve terminals. As a final point, it has been shown that the inhibition by 5-HT of the spontaneous EJP's that occur on the leech body wall muscle results from an inhibition of central neurons and not from any direct effect on the muscle cell or on peripheral synapses.     

The action of sphingomyelinase from Bacillus cereus on ATP-depleted bovine erythrocyte membranes and different lipid composition of liposomes

The presence of cholesterol or phosphatidylethanolamine in sphingomyelin liposomes enhanced 2- to 10-fold the breakdown of sphingomyelin by sphingomyelinase from Bacillus cereus. On the other hand, the presence of phosphatidylcholine was either without effect or slightly stimulative at a higher molar ratio of phosphatidylcholine to sphingomyelin (3/1). In the bovine erythrocytes and their ghosts, the increase by 40-50% or the decrease by 10-23% in membranous cholesterol brought about acceleration or deceleration of enzymatic degradation of sphingomyelin by 50 or 40-50%, respectively. The depletion of ATP (less than 0.9 mg ATP/100 ml packed erythrocytes) enhanced K+ leakage from, and hot hemolysis (lysis without cold shock) of, bovine erythrocytes but decelerated the breakdown of sphingomyelin and hot-cold hemolysis (lysis induced by ice-cold shock to sphingomyelinase-treated erythrocytes), either in the presence of 1 mM MgCl2 alone or in the presence of 1 mM MgCl2 and 1 mM CaCl2. Also, ATP depletion enhanced the adsorption of sphingomyelinase onto bovine erythrocyte membranes in the presence of 1 mM CaCl2 up to 81% of total activity, without appreciable K+ leakage and hot or hot-cold hemolysis. These results suggest that the presence of cholesterol or phosphatidylethanolamine in biomembranes makes the membranes more susceptible to the attack of sphingomyelinase from B. cereus and that the segregation of lipids and proteins in the erythrocyte membranes by ATP depletion causes the deceleration of sphingomyelin hydrolysis despite the enhanced enzyme adsorption onto the erythrocyte membranes.     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.     

禽多杀性巴氏杆菌C48-3株ompH基因敲除突变株的构建及其生物学功能

[目的]探讨ompH基因在禽多杀性巴氏杆菌致病过程中的作用.[方法]利用同源重组原理构建中间为四环素抗性基因,两侧为ompH基因上下游同源序列同源的敲除载体pWSK29△ompH,将敲除载体电击转入C48-3株感受态细胞中,通过四环素抗性和菌落PCR筛选ompH基因的敲除突变株,并通过组合PCR、逆转录PCR和DNA测序对突变株进行验证.用生物学功能实验比较野生株、互补株和突变株在生长速率、荚膜结构、粘着能力和致病性等方面的差异.[结果]组合PCR、逆转录PCR和DNA测序结果证实ompH基因的敲除突变株C48-3△ompH构建成功,电镜观察结果证实ompH基因的缺陷影响细菌的荚膜合成能力,粘附实验结果显示与野生株C48-3和互补株C48-3C相比突变株C48-3△ompH对CEF细胞的粘附能力显著降低(P＜0.01),而小鼠毒力实验结果表明突变株C48-3△ompH的致病性相对减弱.[结论]本实验构建的突变株C48-3△ompH,为进一步研究多杀性巴氏杆菌的致病机理奠定基础.     

Occurrence of ommochrome-containing pigment granules in the central nervous system of the silkworm, Bombyx mori

Dark-red pigment granules were found in the brain and ganglion of the normal strain of the silkworm, Bombyx mori, by light microscopy. No other pigmentation was seen in the brain or ganglia. Electron microscopy showed that the granules were electron-dense. The granules were similar to the ommochrome-containing pigment granules that are present in the epidermal cells of the quail mutant, as previously reported. The pigment in the larval central nervous system (CNS) of the normal silkworm was identical to the ommin standard with respect to the absorption spectrum, the infrared spectrum, and the Rf value in thin-layer chromatography (TLC). After acid hydrolysis of the pigment, 3-hydroxykynurenine was detected by TLC. The pigment granules in the CNS contained mainly ommin. An ommochrome-binding protein was also detected in the CNS by in vitro binding studies and Western blotting. The ommochrome granules may have an important function in the CNS of the silkworm.     

A non-destructive screenable marker,OsFAST, for identifying transgenic rice seeds

The production of transgenic plants has contributed greatly to plant research. Previously, an improved method for screening transgenic Arabidopsis thaliana seeds using the FAST (Fluorescence-Accumulating-Seed Technology) method and FAST marker was reported. Arabidopsis seeds containing the FAST marker may be visually screened using a fluorescence stereomicroscope or blue LED handy-type instrument. Although the FAST method was originally designed for Arabidopsis screens, this study endeavors to adapt this method for the screening of other plants. Here, an optimized technology, designated the OsFAST method, is presented as a useful tool for screening transgenic rice seeds. The OsFAST method is based on the expression of the OsFAST-G marker under the control of a seed-embryo-specific promoter, similar to the Arabidopsis FAST-G marker. The OsFAST method provides a simple and non-destructive method for identifying transgenic rice seeds. It is proposed that the FAST method is adaptable to various plant species and will enable a deeper analysis of the floral-dip method.Key words: Oryza sativa, oleosin, seed, green fluorescent protein, transformation, screenable markerThe production of transgenic plants has significantly enhanced many areas of plant science research. Antibiotic/herbicide-resistance genes are traditionally used as screenable markers for the selection of transgenic plants. However, this approach does have disadvantages. First, antibiotics or herbicides occasionally inhibit the growth of transgenic plants, regardless of the incorporation of antibiotic- or herbicide-resistance genes¹ into the transgenic plants. Second, the identification of resistant transgenic plants requires that the seed population be sown onto plates containing antibiotics or herbicides. Third, the selection process is slow and labor intensive, often involving the screening of vast numbers of potentially transgenic seeds on selective plates.To overcome these disadvantages, an improved approach for selecting transgenic Arabidopsis thaliana, designated the FAST (Fluorescence-Accumulating-Seed Technology) method, was developed. This method employs the use of a fluorescent protein that is expressed in seeds and used as a visual screenable marker for the identification of transgenic seeds. The seed-specific protein oleosin, a family of oil-body-membrane proteins,² has an important role as a size regulator of oil bodies.³ AtOLE1, the most abundant oleosin, functions in the freezing tolerance of Arabidopsis seeds.⁴ A plasmid containing an AtOLE1-GFP fusion gene controlled by the AtOLE1 promoter was constructed and designated the FAST-G (Fluorescence-Accumulating-Seed Technology with OLE1-GFP) marker. Interestingly, Arabidopsis seeds containing the FAST-G marker emitted clear fluorescence under a fluorescence stereomicroscope or blue LED handy-type instrument. The transgenic seeds were visually identified by the seed fluorescence without the use of antibiotics or herbicides, thus indicating that the FAST method offers a nondestructive approach. The FAST marker permits the identification of homozygous seeds among the T2 population with a false discovery rate of less than 1% as a co-dominant screenable marker. In contrast to conventional methods using antibiotics or herbicides, the FAST method reduces the amount of time required to acquire homozygous transgenic plants from 7.5 months to 4 months. The fluorescence of the FAST-G marker was limited to a specific organ (i.e., in seeds) and a specific time (i.e., during dormancy), desirable characteristics of selectable and/or screenable markers. Furthermore, the FAST marker does not require sterile seeding and the handling of large numbers of plants.