Continuous culture of reuber hepatoma cells in serum-free,arginine-, glutamine-and tyrosine-deprived chemically defined medium

Summary The rat hepatoma cell line, H4-II-E, was grown serially over a I-year period and about 30 passages in arginine-, glutamine-, and tyrosine-deprived and ornithine-supplemented Eagle's mininum essential medium with no supplements other than biotin. The adapted cel line, R-Y121B, proliferates in the above mentioned medium with a doubling time of about 4 days and maintains hepatic “marker” enzymes such as tyrosine aminotransferase, phenylalanine hydroxylase, and all the enzymes of the urea cycle. This work was supported in part by Grant-in-Aid for Cancer Research 301050 and Science Research Grant 337013 from the Ministry of Education, Science and Culture, Japan.     

Identification of some abnormal metabolites in psoriatic nail using gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric analysis was used to separate and identify abnormal compounds in the nail of psoriatic patients. The nail was extracted with heated ethanol, and the extract was analyzed with and without trimethylsilylation. Tetradecanoic acid octadecyl ester, hexadecanoic acid octadecyl ester and octadecanoic acid octadecyl ester were first identified in the psoriatic nail, but were not detected in normal nail.     

Steering responses of adult and nymphal crickets to light,with special reference to the head rolling movement

Most tethered adult crickets (Gryllus bimaculatus) assumed flight postures with or without flapping their wings in a windstream. Nymphal crickets (sixth and seventh, i.e. final, instars) also displayed the flight posture in spite of the incompleteness of wing development. These adult nymphal crickets rolled their heads towards the light source in response to unequal illumination of the compound eyes only while maintaining the flight posture. The amphtude of the head rolling movements was proportional to the change of light position up to 120°C, and independent of the light intensity if the duration was longer than 1 sec. The unequal illumination could also induce a transient increase in discharge frequency of the wing muscles on both sides, a decrease in wing beat amplitude of the ipsilateral wing on the illuminated side, and bending movements of the legs and abdomen towards the light. Cutting either of the nerve connectives at any level between the subosophageal and metathoracic ganglia did not affect the response of either the head or the abdomen to illumination. These results are discussed in relation to the steering mechanism associated with the dorsal light reaction.     

One step purification procedure of elastase from pancreatic powder by affinity chromatography

Pancreatic elastase was isolated from acetone powder of porcine pancreas by a one step purification procedure on a trialanyl CH-Sepharose 4B affinity column. This column exhibited affinity not only for active elastase but also for trypsin and chymotrypsin which were present in the same pancreatic powder. However, as the extent of affinity toward elastase is considerably higher, the proper conditions were determined with which the adsorbed elastase was isolated in a highly purified form. The yield of elastolytic activity ranged from 60–85% and the purified elastase was shown to be one component by polyacrylamide disc electrophoresis.     

Breakup and re-formation of colony in the first-instar larvae of the winter cherry bug,Acanthocoris sordidus thunberg (Hemiptera: Coreidae), in relation to the defence against their enemies

The winter cherry bug, Acanthocoris sordidusThunberg , lives in aggregation especially in their early larval instars. Using the 1st-instar larvae of this species, the author tried to clarify both the processes and the mechanisms of the breakup and later re-formation of colony in relation to the defence against their enemies. The results obtained were summarized as follows.

1. In the field population, there is a high possibility of dispersal of the 1st-instar larvae from a colony possibly through the disturbance by some predators but they can re-form a colony with each other or join, with colonies of different instar larvae.
2. The individuals in a colony immediately disperse through the attack of predatory coccinellid beetle, Harmonica axyridis but tend to re-form a colony in a short time.
3. The breakup of colony is caused by the secretion from the attacked individual.
4. The formation of colony is attributed to the habit closely related with the senses of smell and/or contact.

From these results, it was concluded that the dispersal of 1st-instar larvae from a colony, followed by the re-formation of a colony, is an an adaptive behaviour to escape from the attack by their predators.     

The RNA-binding protein Puf5 and the HMGB protein Ixr1 contribute to cell cycle progression through the regulation of cell cycle-specific expression of CLB1 in Saccharomyces cerevisiae

Puf5, a Puf-family RNA-binding protein, binds to 3´ untranslated region of target mRNAs and negatively regulates their expression in Saccharomyces cerevisiae. The puf5Δ mutant shows pleiotropic phenotypes including a weakened cell wall, a temperature-sensitive growth, and a shorter lifespan. To further analyze a role of Puf5 in cell growth, we searched for a multicopy suppressor of the temperature-sensitive growth of the puf5Δ mutant in this study. We found that overexpression of CLB2 encoding B-type cyclin suppressed the temperature-sensitive growth of the puf5Δ mutant. The puf5Δ clb2Δ double mutant displayed a severe growth defect, suggesting that Puf5 positively regulates the expression of a redundant factor with Clb2 in cell cycle progression. We found that expression of CLB1 encoding a redundant B-type cyclin was decreased in the puf5Δ mutant, and that this decrease of the CLB1 expression contributed to the growth defect of the puf5Δ clb2Δ double mutant. Since Puf5 is a negative regulator of the gene expression, we hypothesized that Puf5 negatively regulates the expression of a factor that represses CLB1 expression. We found such a repressor, Ixr1, which is an HMGB (High Mobility Group box B) protein. Deletion of IXR1 restored the decreased expression of CLB1 caused by the puf5Δ mutation and suppressed the growth defect of the puf5Δ clb2Δ double mutant. The expression of IXR1 was negatively regulated by Puf5 in an IXR1 3´ UTR-dependent manner. Our results suggest that IXR1 mRNA is a physiologically important target of Puf5, and that Puf5 and Ixr1 contribute to the cell cycle progression through the regulation of the cell cycle-specific expression of CLB1.     

Production and Accumulation of Xylooligosaccharides with Long Chains by Growing Culture and Xylanase of a Mutant Strain of Bacillus pumilus X-6-19

Bacillus pumilus X-6-9 isolated from soil and subsequently identified, produced xylooligosacchatides with long chainsfrom xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increasein the production of the xylooligosaccharides was established, when compared to the original culture conditions of B. pumilus X-6-19.The addition of D-glucose to the culture of the mutant swain U-3 of B. pumilus X-6-9 repressed the synthesis of β-xylosidase, but notxylanase. Thus, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharideswith long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized.The hydrolyzates generated by the purified xylanase contained xylobiose, xylotrinse, xylotewaose, and xylopentaose, but not xylose.     

Phytosulfokine stimulates somatic embryogenesis in Cryptomeria japonica

Phytosulfokine (PSK), which has been identified as a plant growth factor, had a dramatic stimulatory effect on the formation of somatic embryos of sugi (Cryptomeria japonica) in the presence of polyethylene glycol. The resultant somatic embryos germinated with synchronous sprouting of cotyledons, hypocotyls and roots, and most of the seedlings grew normally. A cDNA clone for the precursor to the PSK peptide of C. japonica was identified in an expressed sequence tags database. Our results support the existence of a PSK signaling pathway in C. japonica.     

Prevention of PERV infections in pig to human xenotransplantation by the RNA interference silences gene

The possibility of preventing the transmission of porcine endogenous retrovirus (PERV) to human cells using short interfering RNAs (siRNA) was investigated. The siRNA for the p30 of PERV gag region was cloned into pSUPER, the polymerase-III H1-RNA gene promoter. A green fluorescence protein (GFP) was also cloned into pSUPER to establish pSXGH. Pig endothelial cells (PEC) were transduced with the LacZ gene by pseudotype infection, and infected with PERV subtype B, resulting in the formation of PEC(LacZ)/PB. The PEC(LacZ)/PB was next transfected with pSXGH-siRNA. The expression of siRNA was provisionally checked by determining the level of expression of GFP. Culture supernatants of infected cells were then inoculated into HEK293 cells. The siRNA clearly destroyed the PERV infectivity of PEC(LacZ)/PB in both transient cell lines and stable clones. Moreover, the decreased levels of mRNA and gag protein were evidenced in the stable clones by real-time PCR and Western blotting, respectively. The final goal of our study was to establish a transgenic pig expressing the siRNA for PERV. The results suggest that siRNA represents a novel approach for controlling PERV infections in clinical xenotransplantation.