Studies on extracellular ribonucleases of Ustilago sphaerogena. Purification and properties

1. Four ribonucleases were isolated from culture media of Ustilago sphaerogena. They were designated ribonucleases U(1), U(2), U(3) and U(4). 2. They were purified about 1600-, 3700-, 1100- and 16-fold respectively. 3. It was shown by gel filtration that ribonucleases U(1), U(2) and U(3) have molecular weights about 10000 like ribonuclease T(1), and that ribonuclease U(4) is much larger. 4. Ribonucleases U(1), U(2) and U(3) are thermostable, but ribonuclease U(4) is not. 5. The pH optimum of ribonucleases U(1) and U(4) is pH8.0-8.5, and that of ribonucleases U(2) and U(3) is pH4.5.     

Establishment and characterization of a human malignant schwannoma cell line (HKMS)

The malignant schwannoma cell line (HKMS) was established from the subcutaneous tumor of Axilla region of a 48-year-old Japanese woman. The HKMS line has the following biological properties. 1. The HKMS cells were spindle in shape and showed neoplastic and pleomorphic features. The monolayer sheet of HKMS cells showed the resemble cell-arrangement with that of the original tumor tissue. 2. The cells showed a stable growth and the serial passages were successively carried out 150 times within 3 years. Their population doubling time is about 40 hours. 3. The chromosome number varied widely, and the modal number was stable at the 78-80. The marker chromosomes were present. 4. The cells were transplanted into the subcutis of nude mice and produced the malignant schwannoma.     

Establishment and characterization of human neuroblastoma cell line (HSNB)

We cultured an aspiration fluid of the sternal bone marrow of the patient having adrenal neuroblastoma and established a neuroblastoma cell line (HSNB). The HSNB line has the following biological properties. 1. They are small round in shape and proliferate in flotation while forming cell aggregate, and often they attach the bottom of plastic dish and process the nerve-like fibers. A rough-endoplasmic reticulum are poorly developed, however, a lot of free ribosomes are scattered in the cytoplasm. In the peripheral area of the cells, small spherical secretory granules (60-140 nm in diameter) are existed. One characteristic of this cell is existence of microtubules in the cell-projections. 2. They show a stable growth and the doubling time is about 50 hours. 3. Their chromosome number varied widely and the mode is 46. The double minute chromosomes were present in 50% of cells. 4. When they are transplanted in the cheek pouch of hamster, they produced the neuroblastoma. 5. They produce neuron specific enolase. 6. N-myc gene was amplified ca 250 folds.     

Covalent structure of a low-molecular-mass protein, meleagrin, present in a turkey (Meleagris gallopavo) ovomucoid preparation

A low-molecular-mass protein, tentatively named meleagrin, was isolated from a commercial preparation of turkey (Meleagris gallopavo) ovomucoid. This 40-amino acid protein contains 3 disulfide bonds and high concentrations of aromatic residues (2 tryptophans and 3 tyrosines). It lacks threonine, methionine, phenylalanine, and arginine residues. The complete amino acid sequence was determined to be the following: less than Glu-Val-Leu-Lys-Tyr-Cys-Pro-Lys-Ile-Gly-Tyr-Cys-Ser-Ser-Lys-Cys-Ser-Lys- Ala- Glu-Val-Trp-Ala-Tyr-Ser-Pro-Asp-Cys-Lys-Val-His-Cys-Cys-Val-Pro-Ala-Asn- Gln-Lys - Trp. One of the three disulfide bonds exists between Cys12 and Cys28, and the two others links Cys32-Cys33 with Cys6 and Cys16. The amino acid sequence of meleagrin shows a strong homology to a similar basic protein, cygnin (Simpson, G.R. & Morgan, F.J. [1983] Int. J. Pep. Protein Res. 22, 476-481), of a rather distantly related aves, black swan (Cygnus atratus), suggesting some vital role of this protein in avian eggs. Similarity to a part (exon 9) of transferrins was also recognized.     

A study on the effectiveness of a thromboxane synthetase inhibitor (OKY-046) in increasing the survival length of skin flaps

In an experimental study to test the thromboxane (TX) synthetase inhibitor OKY-046, two random-pattern skin flaps, each measuring 15.5 x 2 cm, and caudally based, were elevated on the backs of rabbits, and the effect of the test drug on their survival length was evaluated. The results indicated that the survival length of the skin flaps was 4.5 +/- 0.2 cm in the control group and 6.8 +/- 0.3 cm in the OKY-046-treated group, hence exceeding the control value by more than 50 percent, which was statistically significant. A laser speckle flow-meter showed that the OKY-046-treated flaps had significantly greater blood flow as compared with the control group both at 1 and 48 hours after operation. Whereas the blood flow values were significantly lower at 48 hours than at 1 hour after operation in the control group, no such reduction was noted in the OKY-046-treated group. On the other hand, while plasma TXB2 was found elevated at 1 hour postoperatively in the control group, such a response to the surgical intervention was blocked and the plasma TXB2/6-keto prostaglandin (PG) F1a ratio was decreased in the OKY-046-treated group. These results clearly indicated that OKY-046 suppressed a plasma thromboxane elevation induced by surgery, it augmented the flap blood flow, and it thereby increased flap survival length, suggesting that the drug might be helpful clinically and that further investigation must be carried out concerning its application.     

Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes

Sera (fetal calf serum: FCS; and oestrous cow serum: ECS), hormones (2.5 FSH micrograms/ml + 5 micrograms LH/ml + 1 microgram oestradiol/ml) and granulosa cells (5 x 10(6)/ml) were added to culture medium to determine the frequencies of in-vitro maturation, fertilization, cleavage (2- to 8-cell) and development into blastocysts of bovine follicular oocytes. The maturation rates after 24 h in culture were not significantly different among the three factors tested (56-72%). The fertilization rates were significantly affected by serum type and the addition of granulosa cells. FCS gave significantly higher rates of fertilization (57-71%) than did ECS (34-52%), but the proportions of polyspermic fertilization were significantly higher in the former (8-19%) than in the latter (2-3%). The addition of hormones did not affect fertilization, cleavage and development. Neither type of serum affected cleavage and development. The highest rates of blastocyst formation were obtained when granulosa cells alone were added (FCS, 17%; ECS, 16%). The cell numbers of the blastocysts obtained were 100-150, similar to those of blastocysts developed in vivo. Transfer of 6 blastocysts to 3 cows resulted in 1 pregnancy. The present results indicate that the co-culture with granulosa cells is the most important factor for in-vitro fertilization to development into blastocysts of bovine oocytes matured in vitro.