Establishment of a human anaplastic thyroid cancer cell line secreting granulocyte colony-stimulating factor in response to cytokines

Summary A human anaplastic thyroid cancer cell line K-119, derived from a 77-yr-old woman who had developed marked neutrophilia and underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike and polygonal cells in shape are stably proliferating since the beginning of its culture 2 yr ago. The cells grow rapidly and the population doubling time is 26 h. The chromosomes show many abnormalities and many marker chromosomes have been observed. Heterotransplantation of the cells into nude mice has resulted in the formation of tumors that are histologically interpreted as anaplastic cancer. The most noteworthy characteristics of the cell line are the many Ki-67-positive cells (86.3%) and that the cell line spontaneously secretes granulocyte colony-stimulating factor (G-CSF) and releases increased amounts of G-CSF in response to the stimulation of tumor necrosis factor, interleukin 1α, and interleukin 1β. The conditioned medium obtained from K-119 cells contains an autocrine factor stimulating the proliferation of themselves.     

Tissue distribution of antihypertensive dipeptide, Val-Tyr, after its single oral administration to spontaneously hypertensive rats.

The distribution of an antihypertensive dipeptide, Val-Tyr (VY), in the tissues of spontaneously hypertensive rats (SHR) was investigated in this study. A single oral administration of VY (10 mg/kg) to 18-week-old SHR resulted in a prolonged reduction of systolic blood pressure (SBP) up to 9 h (SBP0h 198.0+/-3.6 mmHg; SBP9h 154.6+/-3.5 mmHg). As a result of VY determination, a roughly 10-fold higher increment of plasma VY level was observed at 1 h than that at 0 h, whereas thereafter the level declined rapidly. In tissues, VY was widely accumulated in the kidney, lung, heart, mesenteric artery and abdominal aorta with the area under the curve over 9 h of more than 40 pmol h/g tissue; of these a higher VY level was observed in the kidney and lung. In addition, a mean resident time (MRT) for each tissue (>5 h except for liver) revealed that VY preferably accumulated in the tissues rather than in the plasma (MRT 3.8 h). Significant reductions of tissue angiotensin I-converting enzyme activity and angiotensin II level were found in the abdominal aorta as well as in the kidney, suggesting that these organs could be a target site associated with the antihypertensive action of VY.     

Genetic diagnosis of cytoplasmic male sterile cybrid plants of rice

Twelve Japanese rice cultivars were converted to CMS by asymmetric protoplast fusion with MTC-5A, the cytoplasm of which was derived from an indica rice, Chinsurah Boro II. With the exception of the cybrids that had a nucleus from Hoshiyutaka, most of these cybrid plants were sterile. The unique sequence downstream from the mitochondrial atp6 of MTC-5A was specifically amplified in the sterile cybrid plants by PCR. All progenies of the cybrid plants carrying this unique sequence were sterile. On the other hand, in some of the sterile cybrid plants in which the unique sequence was not amplified by PCR, fertility was recovered in their progenies. Somaclonal mutation may have caused sterility in these cybrids. Only the cybrid plants that had the unique sequence detected by PCR were CMS. Thus, the CMS plants can be selected rapidly and easily by PCR, at an early stage of plant regeneration. Soon after transplanting the regenerated plants to a green house, fertile cybrids and sterile cybrids produced by somaclonal mutation can be removed. These findings also show that the unique region downstream from atp6 is tightly linked with the CMS phenotype.     

ET-1-induced bronchoconstriction is mediated via ETB receptor in mice

Nagase, Takahide, Tomoko Aoki, Teruaki Oka, YoshinosukeFukuchi, and Yasuyoshi Ouchi. ET-1-induced bronchoconstriction ismediated via ET_B receptor in mice.J. Appl. Physiol. 83(1): 46-51, 1997.Endothelin (ET)-1 is one of the most potent agonists of airwaysmooth muscle and can act via two different ET receptor subtypes, i.e.,ET_A andET_B. To determine the effects ofET-1 on in vivo pulmonary function and which ET receptors are involved in murine lungs, we investigated 1)the effects of ET and sarafotoxin S6c (S6c), a selectiveET_B agonist, on pulmonary functionand 2) the effects of BQ-123 andBQ-788, specific ET_A- andET_B-receptor antagonists, onET-1-induced bronchoconstriction. ICR mice were anesthetized and mechanically ventilated (frequency = 2.5 Hz, tidalvolume = 8 ml/kg, positive end-expiratory pressure = 3 cmH₂O). Intravenous ET-1, ET-2,and ET-3 increased lung resistance similarly and equipotently, whereasS6c elicited a greater degree of bronchoconstriction. Mice were thenpretreated with saline (Sal), BQ-123 (0.2, 1, and 5 mg/kg), or BQ-788(0.2, 1, and 5 mg/kg) before administration of ET-1(10⁷ mol/kg iv). No dose ofBQ-123 blocked ET-1-induced constriction, whereas pretreatment witheach dose of BQ-788 significantly inhibited ET-1-induced responses.There were significant differences in morphometrically assessed airwayconstriction between Sal and BQ-788 and between BQ-123 and BQ-788,whereas no significant difference was observed between Sal and BQ-123.There were no significant morphometric differences in the airway wallarea among the three groups. These observations suggest that theET_B- but notET_A-receptor subtype may mediatethe changes in murine pulmonary function in response to ET-1. Inaddition, the ET_B-receptorantagonist reduces ET-1-induced airway narrowing by affecting airwaysmooth muscle contraction in mice.

     

Histological Observations on Initiation and Morphogenesis in Immature and Mature Embryo Derived Callus of Barley (Hordeum vulgare L.)

Callus was induced from immature and mature embryos of barley(cv. Haruna Nijo) on Murashige and Skoog medium containing 2mg l^-1 2,4-D and 5 mg l^-1 picloram, respectively. Paraffin sections(10 µm thick) were prepared for histology during callusinitiation and plant regeneration. Meristems were regeneratedfrom nodular compact callus (NC) derived from scutellar epidermisin immature embryos, whereas they were regenerated from NC derivedfrom epidermal cells of leaf or coleoptile bases in mature embryos.Regardless of the explant source, regeneration was predominantlythrough organogenesis, although regeneration through somaticembryogenesis infrequently occurred. Thus, the callus inducedfrom immature and mature embryos of barley was regarded as 'nodularcompact' rather than 'embryogenic'.Copyright 1995, 1999 AcademicPress Barley, callus, Hordeum vulgare, histology, immature embryo, mature embryo, regeneration     

Enzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

Abstract The serodiagnosis of melioidosis is commonly performed with tests using protein or polysaccharide as antigen. However, due to the low sensitivity, specificity and difficulty in the preparation of the antigens, more simple, precise and reproducible diagnostic tests were required. A purified glycolipid antigen (GL) which is a specific lipid component of Burkholderia pseudomallei has been used in an ELISA. With this antigen, specific immunoglobulin G (IgG) was detected in 49 out of 50 melioidosis sera. IgG was also detected in 2 out of 185 (Japanese) and 16 out of 181 (Vietnamese) control sera. Thus, the sensitivity was 98.0%, and specificity was 98.9% and 91.1% in the Japanese and Vietnamese sera, respectively. When the ELISA and indirect haemagglutination (IHA) tests were combined, a sensitivity of 100% and specificity of 97.8% were achieved. The advantages of the glycolipid antigen are ease of preparation, stability, high sensitivity and specificity.     

The study of differentiative potential of the lactating mouse mammary gland in organ culture

Summary The organ culture of the mammary gland of lactating mice was used to examine the response of the differentiated gland to lactogenic stimuli, insulin, cortisol, and prolactin. Time course studies showed that casein synthesis in cultured tissue decreased rapidly during the first 2 d despite the presence of the three hormones, but on the 3rd d tissue cultured with either insulin and prolactin or all three hormones regained the ability to synthesize milk proteins, casein, and α-lactalbumin: a greater increase occurred in the three hormone system. The delayed addition of prolactin on Day 2 to the culture system containing insulin and cortisol also stimulated casein synthesis. The addition of cytarabine, which inhibited insulin-dependent cell proliferation in cultured explants, did not block the rebound of milk protein synthesis. The results indicate that in the presence of insulin, cortisol, and prolactin mammary epithelial cells in culture first lose and then regain the ability of synthesizing milk protein without requiring the formation of new daughter cells.     

A unique electrophoretic slow-moving glucose 6-phosphate dehydrogenase variant (G6PD Asahikawa) with a markedly acidic pH optimum

Summary A new glucose 6-phosphate dehydrogenase (G6PD) variant associated with chronic nonspherocytic hemolytic anemia was discovered in Japan. The patient showed hemolytic crises after upper respiratory infections. The enzyme activity was about 3.8% of the normal. The partially purified enzyme revealed slow anodal electrophoretic mobility, high Km NADP, marked thermal-instability, and increased affinity for a substrate analogue (deamino-NADP). A particular characteristic of this enzyme was a biphasic pH curve with a greatly increased activity at low pH values. From these results, this variant was clearly different from hitherto observed G6PD variants, and was designated G6PD Asahikawa.     

Evidence that endogenous 6-(ARG or LYS)-opioid peptides can interact with kappa-receptors as agonists

Evidence is provided for the abilities of endogenous 6-(Arg or Lys)-opioid peptides to interact with kappa-receptors as agonists. Dynorphin-(1-17) and -(1-8), alpha- and beta-neo-endorphin, [Met5]-enkephalin-Arg6-Phe7 and des acetyl salmon endorphin I significantly inhibited the electrically-evoked contractions of rabbit vas deferens which had been shown to contain kappa-receptors exclusively, indicating that endogenous 6-(Arg or Lys)-opioid peptides could act on kappa-receptors as agonists. Additionally, the inhibition of contractions of rabbit vas deferens by 6-(Arg or Lys)-opioid peptides was antagonized more effectively by Mr 2266 which had a high affinity to both mu- and kappa-receptors, than naloxone which had a high affinity only to mu-receptors. This also suggested that 6-(Arg or Lys)-opioid peptides acted as kappa-receptor agonists. The rank order of the inhibitory potency of 6-(Arg or Lys)-opioid peptides against contractions of rabbit vas deferens was as follows: dynorphin-(1-17) greater than alpha-neo-endorphin greater than beta-neo-endorphin .=. dynorphin-(1-8) greater than des acetyl salmon endorphin I greater than [Met5]-enkephalin-Arg6-Phe7. Since other endogenous opioid peptides such as [Met5]- and [Leu5]-enkephalin and beta-endorphin have been shown not to act on kappa-receptors as agonist, data in the present study suggest that endogenous opioid peptides can be classified into two groups in terms of an ability to interact with kappa-receptors as an agonist.     

Achromobacter protease I-catalyzed conversion of porcine insulin into human insulin

We have established a procedure for converting porcine insulin into human insulin using a serine protease from $\text{Achromobacter}\text{lyticus}$ M497-1 which shows unique specificity against lysine residues on the carboxyl side of the splitting point. Desalanine-(B30)-insulin (DAI) was prepared by digestion of porcine insulin with $\text{Achromobacter}$ protease. The coupling between DAI and Thr-OBu^t was performed by the same enzyme at pH 6.5 with a large excess of the amine component (Thr-OBu^t) in the presence of high concentrations of organic co-solvents. The highest yield was 85% by 20 h reaction at 37°C. The synthesized [Thr-OBu^t-B30]-insulin was isolated, then deprotected with trifluoroacetic acid in the presence of anisole to obtain semisynthetic human insulin.