Photochemical reaction of 9-cis-retro-γ-rhodopsin at low temperatures

9-cis-Retro-γ;rhodopsin (λ_max = 420 nm) was prepared from 9-cis-retro-γ-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-γ-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-γ-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of bathorhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.     

Microtubules inside the plasma membrane of squid giant axons and their possible physiological function

Summary The effects of application of the microtubule-disassembling reagents to squid giant axons upon resting potential, the height of the propagated action potential, and the threshold to evoke action potential were studied using colchicine, podophyllotoxin, vinblastine, griseofulvin, sulfhydryl reagents including NEM, diamide, DTNB and PCMB, and Ca²⁺ ions. At the same time, the effects of concentrations of K halides and K glutamate on the above physiological properties were studied in comparison within vitro characteristics of microtubule assembly from purified axoplasmic tubulin.It was found that there was good correlation between conditions supporting maintenance of membrane excitability and microtubule assembly. The experiments suggest that associated with the internal surface of the plasma membrane there are microtubules which regulate in part both resting and action potentials.     

Relationships between nucleic acid,nitrogen, and growth rate of tobacco cells in suspension culture

Summary Changes in the amount of nucleic acid and nitrogen, and the relationships between these amounts and the growth rate of tobacco cells (Nicotiana tabacum L. cv. Bright Yellow-2) at different initial nitrogen concentrations in the medium, were examined in batch cultures. During culture in basal medium, the amount of intracellular nucleic acid expressed per unit of dry biomass was 36.3 mg RNA g^–1 cell and 8.1 mg DNA g^–1 cell at the beginning of batch culture. These values increased 2.5 fold for RNA and 1.5 fold for DNA during the exponential growth phase and then gradually decreased with the decline in the growth rate. Similar changes were also observed in the medium containing less nitrogen. The specific growth rate, (day^–1), of the culture corresponded to the magnitude of the intracellular RNA content (mg RNA g^–1 cell), and the linear relationship, RNA=38+23 was obtained. In addition, there were remarkable positive correlations between the total and protein nitrogen, and during the cultures. The mononucleotide composition of total RNA (AMP+UMP)/(GMP+CMP) which was suggested to be a convenient index of metabolic activity was nearly constant (0.78 to 0.80) during tobacco cell culture in the basal medium.     

Fungal contamination and mycotoxin-producing potential of dried beans

A total of 604 samples of about 7 different types of beans was examined to determine their mycological profiles, and suitability for use as solid substrates for mycotoxin production.All of the samples were collected from bean jam makers in Tokyo by the official food examiners.Genera Penicillium and Aspergillus were predominant, and genus Wallemia was also found commonly in all types of beans.Mycotoxin-producing Aspergillus strains were isolated from 52 samples of beans, approximately 9% of the total. The highest incidence of toxigenic Aspergillus (14.1%) was found in kidney beans. Red beans and peas inoculated with Aspergillus ochraceus were found to produce about 7 to 8 times more toxin than was obtained in a liquid medium, and red beans inoculated with A. versicolor produced more toxin than was obtained in yeast extract sucrose broth. Green peas inoculated with Fusarium graminearum produced about 8 times more T-2 toxin than was obtained in 1% peptone containing Czapek solution under comparable culture conditions.     

Dye-coupled electrode system for the rapid determination of cell populations in polluted water.

We determined cell populations in polluted waters by using a fuel cell-type electrode. The electrode was constructed from a platinum anode, a silver peroxide cathode, and a membrane filter for retaining microorganisms. The principle of cell number determination is based on sensing a redox dye reduced by the microorganisms with the electrode. Sample solutions containing microorganisms were membrane filtered, and the resulting filter containing microbial cells was attached to the surface of a platinum anode. The electrode was immersed in phosphate buffer solution (0.05 M, pH 7) containing a redox dye (2,4-dichlorophenol-indophenol), and the current generated was measured. The response time of the electrode system was 10 to 20 min, and the current generated was proportional to cell populations above 10(4) cells/ml.     

Animalizing Effect of A23187 on Sea Urchin Embryos

Pulse treatment of sea urchin embryos with 3 μM A23187 for 2 hr starting at a stage in initial 10 hr period of development at 20°C, followed by a culture in normal sea water up to the pluteus corresponding stage (45 hr after fertilization), yielded many large exogastrulae with thin embryo walls. The pulse treatment starting at a time between 10 and 13 hr after fertilization yielded considerable number of large prisms and gastrulae having thin embryo walls. Probably, the pulse treatment exerts stimulating effects on ectodermal cell determination in whole span of pre-hatching period to produce animalized embryos. On the other hand, pulse treatment with A23187 in pre-hatching period exerts stage-specific effects on gut formation. Embryos, thus treated for 2 hr starting at stages between 3 and 5 hr after fertilization, produced quite small exoguts but those treated at stages between 7 and 8 hr formed well developed and long exoguts. In embryos treated at the other stages than above, guts or exoguts were almost the same in their size to those in normal ones. These effects of A23187 on morphogenesis were canceled by procaine, tetracaine and ruthenium red. Probably, artificial Ca²⁺signal induced by A23187 alters the determination of cell fates, programmed in pre-hatching period.     

Subcortical Rotation and Specification of the Dorsoventral Axis in Newt Eggs

The specification of the dorsoventral axis in naturally polyspermic eggs of the Japanese newt, Cynops pyrrhogaster , was first examined by studies on the spatial relationship between the dorsal midline of the future body plan and the sperm entrance points (SEPs ¹ ). On local insemination, the dorsal blastopore lip was usually found to be formed opposite the SEPs, as in anuran monospermic eggs. Next the movements of the subcortical layer and the cortex were analyzed. "Subcortical rotation" was observed, similar to that of Xenopus laevis eggs with respect to its timing and extent, and its direction was shown to predict the embryonic axis of the eggs. Thus, the dorsoventral axis was concluded to be determined by essentially the same mechanism in the newt as in Xenopus .
Owing to their large size and long first cell cycle, newt eggs appear to be suitable material for study of subcortical rotation, but their behavior is unique in that subcortical rotation occurs in only the vegetal hemisphere so that the subcortical layer stretches in the future dorsal side. Studies on the movement of Nile blue spots suggested that the cytoplasm under the cortex in newt eggs consists of two layers.     

Conversion of growth hormone-secreting cells into prolactin-secreting cells and its promotion by insulin and insulin-like growth factor-1 in vitro

The newly established rat pituitary cell line, MtT/S, has pituitary somatotroph (growth hormone-producing cell)-like characteristics, i.e., the cells produce growth hormone (GH), possess GH-immunopositive secretory granules, and respond to GH-releasing hormone. When MtT/S cells were cultured in regular medium no prolactin (PRL) cells were observed and PRL was not detected, by radioimmunoassay or Western blot analysis, in the medium or the cells. However, GH production and the GH cell population decreased markedly when the cells were incubated with insulin or insulin-like growth factor-1 (IGF-1). After stimulation with insulin or IGF-1 there was a 2-day lag period, then some PRL was detected in the medium; after 5 days a number of PRL cells appeared. Double immunocytochemistry indicated clearly that no cell contained both PRL and GH. These results show that insulin and IGF-1 stimulate conversion of MtT/S cell line GH cells to PRL cells. This suggests that the MtT/S cell line is an excellent model system which shows the GH-cell/PRL-cell lineage.     

Directed mutagenesis in an asporogenous methylotrophic yeast: cloning, sequencing, and one-step gene disruption of the 3-isopropylmalate dehydrogenase gene (LEU2) of Candida boidinii to derive doubly auxotrophic marker strains.

A model system for one-step gene disruption for an asporogenous methylotrophic yeast, Candida boidinii, is described. In this system, the 3-isopropylmalate dehydrogenase gene (C. boidinii LEU2) was selected as the target gene for disruption to derive new host strains for transformation. First, the C. boidinii LEU2 gene was cloned, and its complete nucleotide sequence was determined. Next, the LEU2 disruption vectors, which had the C. boidinii URA3 gene as the selectable marker, were constructed. Of the Ura+ transformants obtained with these plasmids, more than half showed a Leu- phenotype. Finally, the double-marker strains of C. boidinii were derived. When vectors with repeated flanking sequences of the C. boidinii URA3 gene were used for gene disruption, Leu- Ura+ transformants changed spontaneously to a Leu- Ura- phenotype ca. 100 times more frequently than they did when plasmids without the repeated sequences were used. Southern analysis showed that these events included a one-step gene disruption and a subsequent popping out of the C. boidinii URA3 sequence from the transformant chromosome.     

Alpha-elastin coacervate as a protein liquid membrane: effect of pH on transmembrane potential responses.

A protein liquid membrane composed of coacervated alpha-elastin, a chemical fragmentation product of the biological elastic fiber protein, functioned as an amphoteric liquid ion-exchange membrane. Ionic permselectivities of the alpha-elastin coacervate membrane to a series of metal chlorides were investigated for the concentration-cell systems by the ordinary electrochemical measurements. Effects of pH on the transmembrane potential responses for NaCl, CaCl2, and MgCl2 systems were examined. Only in the Ca(2+)-containing system did potential responses stay at constant levels against the pH changes, whereas in the other systems, increasing pH caused potential changes, indicating an improvement of cationic permselectivity across the alpha-elastin coacervate membrane. It was suggested that the characteristic Ca2+ transport mechanisms across the alpha-elastin coacervate membrane are related in some way to the polypeptide backbone interactions specific and selective to Ca2+ ions.