Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.     

Salt tolerance and glycerol accumulation of a respiration-deficient mutant isolated from the petite-negative, salt-tolerant yeast Zygosaccharomyces rouxii

A respiration-deficient (RD) mutant was isolated from the petite-negative, salt-tolerant yeast Zygosaccharomyces rouxii. One strain among sixteen glycerol-non-utilizing mutants exhibited vigorous liberation of CO2 but no uptake of O2. Furthermore, this strain lacked cytochrome aa3 and had a reduced level of cytochrome b. The few mitochondria found in cells of this strain contained few or no cristae. Salt tolerance and intracellular accumulation of glycerol by the RD strain were almost equal to that of the wild-type strain in media containing NaCl up to 2.5 M. In media with more than 3 M NaCl, the growth of the RD mutant was retarded and the intracellular accumulation of glycerol was depressed in spite of ample production.     

Bacterial cometabolic degradation of chlorinated paraffins

Summary Cometabolic dechlorination of chlorinated paraffins was demonstrated in the presence of n-hexadecane by bacterial strains (HK-3, HK-6, HK-8, and HK-10) isolated from soil samples.Eleven per cent of chlorine of chlorinated paraffin-150 (CP-150) was released by strain HK-3. The mixed culture of strain HK-3, catalyzing the dechlorination of terminal chlorine of chloroalkane, and strain H15-4, capable of releasing the chlorine from 2-chlorinated fatty acids, dechlorinated CP-150 up to 13%. The mixed culture of the four strains (HK-3, HK-6, HK-8, and HK-10) performed the dechlorination of CP-150 by cometabolism in a jar fermentor pH at 7.0. The amount of chloride released from the chlorinated paraffins tested was in the range of 15–57%.The activated sludge acclimatized to n-hexadecane for 60 days showed a little dechlorination activity to CP-150.     

Mutagenesis of human granulocyte colony stimulating factor

To define the structure-function relationship, we have made a number of mutants of human granulocyte colony-stimulating factor (hG-CSF) by in vitro mutagenesis. The results indicate that most of the mutations located in the internal and C-terminal regions of the molecule abolished the activity, whereas the mutants without N-terminal 4, 5, 7, or 11 amino acids retained the activity. N-terminal amino acids were also altered by cassette mutagenesis using a synthetic oligonucleotide mixture. Among them, KW2228, in which Thr-1, Leu-3, Gly-4, Pro-5 and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg and Ser, showed more potent granulopoietic activity than that of intact hG-CSF both in vitro and in vivo.     

Immunohistochemical localization of proteoglycans in interstitial elements of human pancreas and biliary system

Summary The immunohistochemical localization of large proteoglycan and small proteoglycan was observed, using antibodies 2B1 and 6B6 (Sobueet al., 1988, 1989a), in fetal and adult pancreas and biliary system as well as in tumour tissues, obtained from 11 autopsies and 74 biopsies. The distribution of chondroitin 4- and 6-sulphate side chains, type I and IV collagen and elastin were also studied. In adult pancreas and all the biliary tracts examined, periductal fibrous tissues consisted mainly of dermatan sulphate small proteoglycan with networks of fibrous elements, which were composed of large proteoglycan, elastin, type I collagen and type IV collagen. In the interstitial components of cystadenoma of pancreas and biliary duct carcinoma, similar small proteoglycan-rich components were relatively abundant, although large proteoglycan was present in much larger amounts than that in non-neoplastic adult tissues. In some cholangiomas, the extra-and intracellular hyaline globules formed by the carcinoma cells were found to contain chondroitin sulphate large proteoglycan, laminin and fibronectin.The distribution of proteoglycans was observed to be different in the arterial walls of the interlobular tissues of the adult and the fetal pancreas. The biological significance of large and small proteoglycans in the interstitial connective tissues was discussed.     

Role of Chitinase and Chitin Oligosaccharides in Lignification Response of Cultured Carrot Cells Treated with Mycelial Walls

Chitinase activity was induced in cultured carrot cells by incubationwith mycelial walls of a fungus, Chaetomium globosum. Both intra-and extracellular chitinases were resolved into four componentsby gel filtration chromatography. The extracellular enzymesliberated soluble oligosaccharides of different sizes from insolublechitin, suggesting that these carrot chitinases are endo-hydrolases.The solubilized chitinase digests obtained from insoluble mycelialwalls of C. globosum and chitin were fractionated by gel filtrationchromatography, and the elicitor activity of each fraction forthe accumulation of phenolic acids in cultured carrot cellswas determined. In both solubilized fragments of fungal wallsand of chitin, elicitor-active oligosaccharides were distributedin many fractions, however, potent activity for inducing phenolicacid synthesis was observed in the high molecular weight fractions. (Received October 5, 1987; Accepted February 12, 1988)     

The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM Alpha Modification of Eagle's Minimal essential medium - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate - CS Calf Serum - EGF Epidermal Growth Factor - FGF Fibroblast Growth Factor - HPLC High-Performance Liquid Chromatography - NGF Nerve Growth Factor - NOG 1-O-n-octyl--D-glucopyranoside - NP-40 Nonidet P-40 - PBS Phosphate-Buffered Saline - SB 12 3-(dodecylmethylammonio)-1-propane sulfonate - SDS Sodium Dodecyl Sulfate - TGF- and Transforming Growth Factor type and      

Preventive effect of MCI-186 on 15-HPETE induced vascular endothelial cell injury in vitro

Using cultured bovine aortic endothelial cells, the effects of MCI-186, a radical scavenger, were studied on arachidonic acid metabolism and on the cell injury caused by 15-HPETE. MCI-186 at 3 X 10(-5) M enhanced prostacyclin production in the intact endothelial cells without affecting phospholipase A2. When endothelial cell homogenates were used as an enzyme source, it was found that MCI-186 stimulated the conversion of arachidonic acid to prostacyclin like phenol, perhaps by trapping OH radicals produced in the process of the conversion of PGG2 to PGH2. On the other hand, MCI-186 was found to inhibit lipoxygenase metabolism of arachidonic acid in cell free homogenates of rat basophilic leukemia cells. The lipoxygenase inhibition caused by 3 X 10(-5) M MCI-186 was almost equivalent to that caused by 3 X 10(-6) M BW 755C. MCI-186 remarkably protected against endothelial cell damage caused by 15-HPETE. 3 X 10(-5) M of 15-HPETE caused endothelial cell death in about 60% of the population: however, pretreatment of the cells with 10(-5) M of MCI-186 or concomitant addition of 10(-5) M of MCI-186 with 15-HPETE to the cultures prevented the cell death completely. These results suggest that MCI-186 may become an unique anti-ischemic drug.     

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/10⁵ cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.