Production of daunorubicin by immobilized growing Streptomyces peucetius cells

Summary Streptomyces peucetius cells were immobilized by entrapment in calcium alginate and a photosensitive synthetic polymer, and used for the production of daunorubicin (daunomycin), which is known to be an antitumour reagent. The use of cultivation media removed insoluble components in a natural medium prevented rapid decrease in daunorubicin titer after maximum production. These entrapped cells could be used at least five times for repeated daunorubicin production; the total cultivation period was 45 days.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

Bovine high molecular weight kininogen. The amino acid sequence, positions of carbohydrate chains and disulfide bridges in the heavy chain portion

The existence of two types of circulating bovine plasma high molecular weight kininogen (HMWK) was predicted from analyses of complementary DNAs coding for this protein (Kitamura, N., Takagaki, Y., Furuto, S., Tanaka, T., Nawa, H., and Nakanishi, S. (1983) Nature 305, 545-549). The present protein-based study provided evidence in support of the proposed amino acid sequence derived from analysis of the cDNA clone, and the results confirm the existence of two types of circulating HMWK. Type I HMWK contains a heavy chain composed of 361 residues, while the heavy chain of type II HMWK contains 359 residues. The amino acid sequences of type I and type II HMWK determined in this study were identical to that inferred from the cDNA sequence with the exception of microheterogeneity observed in the cDNA at position 87 (Glu/Gln) and 168 (Lys/Arg). The heavy chain of type I HMWK contains 4 asparagine-linked carbohydrate chains at Asn-69, -150 (or -151), -179, and -186, while the heavy chain of type II HMWK contains these and an additional carbohydrate chain at Asn-264. In addition, a carbohydrate chain was found to be O-glycosidically linked to Thr-118 in both chains. Among nine disulfide linkages found in HMWK, eight intrachain disulfide pairs were established in the heavy chain. One interchain disulfide bridge occurs between the heavy chain and the light chain. This disulfide pairing, as well as repeating amino acid sequences observed in the heavy chain, provides strong evidence for the existence of three homologous domains in the heavy chain of bovine HMWK.     

The role of glucose in the pH regulation of germ-tube formation in Candida albicans

It has been reported that Candida albicans can form germ-tubes only in the narrow pH range of 6-8, and that by changing only the pH one can regulate germ-tube formation. We found that the pH minimum for germ-tube formation could be dramatically lowered by eliminating the glucose present in many induction solutions. Lee's medium lacking glucose, ethanol, N-acetyl-D-glucosamine, and proline induced germ-tubes at pH values as low as 3 under most conditions. The presence of as little as 1 mM-glucose in these induction solutions was sufficient to cause the cells to grow either as yeasts with multiple buds or as pseudohyphae when the pH was 3.7. However, when C. albicans was grown in any of the above induction solutions (with the exception of ethanol), containing 200 mM-glucose buffered at pH 5.8, not only were germ-tubes formed, but their rate of formation and length were also increased. Preincubation of the cells in a solution buffered at pH 3.7 and containing 200 mM-glucose, before exposure to induction solutions lacking glucose at pH 3.7 or at pH 5.8, did not inhibit germ-tube formation. Likewise, addition of glucose after 45 min exposure to an induction solution was without effect. Theophylline and dibutyryl cAMP did not counteract the action of glucose. Other sugars which suppressed germ-tube formation at low pH were fructose, galactose, mannose, xylose, gluconic acid and the nonmetabolizable sugar 3-O-methylglucose. These results indicate that pH does not directly regulate dimorphism in C. albicans, and that glucose or its metabolites may play an important role.     

Demonstration of up-regulated IL 2 receptor expression on an in vitro cloned BCL1 subline

We have established BCL1 CL-3 cells capable of responding to B15-TRF and interleukin 2 (IL 2). This clone has both high affinity and low affinity receptors for IL 2 (IL 2R), but IL 2 by itself did not stimulate either proliferation or immunoglobulin (Ig) secretion. B15-TRF, which possesses both growth and differentiation activity, causes an increase in size of CL-3 cells and renders CL-3 cells responsive to IL 2, including an increased expression of IL 2R (eight-fold to 10-fold) and the differentiation of CL-3 cells into Ig secretion (60 to 80% of cultured cells). CL-3 cells pretreated with B15-TRF for 12 hr become competent to respond to IL 2 by up-regulation of IL 2R within 12 hr. In contrast CL-3 cells pretreated with IL 2 for 12 hr required 24 hr B15-TRF stimulation to result in IL 2R up-regulation. Thus the ordered action of B15-TRF and IL 2 is the most effective operational pathway for the up-regulation of IL 2R. This IL 2-mediated IL 2R up-regulation and induction of Ig synthesis depends upon the concentration of IL 2 in the culture. Both responses seem to be caused by IL 2 molecules bound to high affinity IL 2R. However, the possibility of involvement of low affinity IL 2R can not be vigorously excluded. In fact the level of IL 2 required for a response is far higher than that needed for activated T cell proliferation. This cloned BCL1 subline promises to be a useful tool for studying the regulation and mechanisms of B cell responses.     

An activated S6 kinase in regenerating rat liver

S6 kinase activity was increased in the regenerating liver 5 h after partial hepatectomy compared with sham-operated liver. The protein kinase activity was eluted from DE-52 column at approximately 250 mM NaCl and was not affected by known regulators of protein kinases. The S6 kinase was further purified by chromatography on peptide R1A13-Sepharose 4B and Sephadex G-150. The molecular weight of the enzyme was estimated to be 4.5 X 10(4) by gel filtration. The enzyme catalyzes the phosphorylation of whole histone, mainly H2B histone, at 75 mM Mg2+. These properties are similar to those of a proteolytically modified Ca2+/phospholipid-independent form of protein kinase C.     

Inhibition of cell proliferation by putative metabolites and non-degradable analogs of methotrexate-gamma-dimyristoylphosphatidylethanolamine

Previous investigations have shown that untargeted liposomes, in which methotrexate is anchored to the lipid bilayers as methotrexate-gamma-dimyristoylphosphatidylethanolamine (methotrexate-gamma-DMPE), can inhibit in vitro cell proliferation. To test the possibility that this inhibition may involve extracellular metabolism of methotrexate-gamma-DMPE, we have degraded it chemically (dilute alkali) or enzymatically (phospholipase A2, phospholipase C, phospholipase C plus phosphatase), and assayed the products using human lymphoblastoid T cells or a subline that has a defective methotrexate transport system. Neither methotrexate-gamma-(1-myristoyl)-glycerophosphorylethanolamine, methotrexate-gamma-glycerophosphorylethanolamine, methotrexate-gamma-phosphorylethanolamine, nor methotrexate-gamma-ethanolamine resemble methotrexate-gamma-DMPE sensitized liposomes or the free derivative in their ability to block tritiated deoxyuridine incorporation into DNA. When added extracellularly, these putative metabolites manifest a higher ID50 concentration and/or, unlike the liposomes or unincorporated methotrexate-gamma-DMPE, utilize the methotrexate transport system to enter cells. Additionally, we have synthesized methotrexate-gamma-dihexadecylphosphatidylethanolamine and methotrexate-gamma-hexadecylphosphorylethanolamine, analogs of methotrexate-gamma-DMPE that cannot be hydrolyzed by phospholipases A2, C and D; liposomes prepared with these derivatives are markedly less potent cytotoxic agents than methotrexate-gamma-DMPE sensitized liposomes. All together, these results are consistent with the conclusion that methotrexate-gamma-DMPE must undergo intracellular metabolism to exert optimal inhibition; they also bear on possible mechanisms by which methotrexate-gamma-DMPE may enter cells.     

Distribution and fine structure of ependymal cells possessing intracellular cysts in the aqueductal wall of the rat brain

Summary The wall of the cerebral aqueduct was examined in 20 male rats at the light- and electron-microscopic levels. Disorder in ciliary orientation was occasionally seen in ordinary ependymal cells. Ependymal cells possessing intracellular cysts of 5 to 30 urn in diameter were observed within and beneath the aqueductal ependyma in all animals examined. Light-microscopic reconstruction from serial, 10-m thick frontal sections revealed an extensive distribution of cystic ependymal cells (CECs), especially along the ependymal ridges in the rostral half of the aqueduct, and along the dorsal region of the aqueductal lining in the caudal half. Both cystic and surface membranes of CECs bore microvilli and cilia. Ectopic ependymal cells (EECs) characterized by densely packed microvilli, well-developed intermediate junctions and cilia, but without cysts, were situated in the subependymal region adjacent to a CEC or another EEC. The ependymal ridges were long, narrow and sporadically stratified ependymal linings extending rostrocaudally and bilaterally along the aqueductal surface. Tanycyte-like cells filled the surface region of the ridge, and CECs and EECs were frequently seen in the core. Intraventricularly injected microperoxidase was detected among densely packed microvilli but not in the cystic lumina of CECs, indicating that EECs and CECs are distinct entities.     

Changes in cerebral level of monoamines by Japanese encephalitis virus infection

The changes in monoamine levels of different brain regions following Japanese encephalitis virus (JEV) intraperitoneal inoculation were examined in experimentally JEV-infected mice. In addition, virus distribution was studied using infectivity assay and immuno-histochemistry of viral antigen. 1) The level of monoamines in brain tissues was not affected by 48 hours after viral inoculation, but marked effects were elicited at 96 hours after the inoculation. The cerebral concentration of 5-hydroxyindole-3-acetic acid (5 HIAA) was increased, while that of dopamine (DA) showed a decrease. Especially these alteration were observed in the cerebral cortex, but not in the cerebellum. 2) The viral growth in the brain was observed at 48 hours after the inoculation. The growth in the cerebellum, however, was found to be lower than those in other cerebral regions. 3) The viral antigen was detected in the cerebral cortex, hippocampus, mesencephalon and diencephalon in addition to the substantia nigra and striatum. From these results, it is presumed that clinical manifestation of JEV infection may involve the changes in the metabolism of neurotransmitter, especially those of DA and serotonin in the brain.     

Two distinct O-methyltransferases in aflatoxin biosynthesis

The substances belonging to the sterigmatocystin group bear a close structural relationship to aflatoxins. When demethylsterigmatocystin (DMST) was fed to Aspergillus parasiticus NIAH-26, which endogenously produces neither aflatoxins nor precursors in YES medium, aflatoxins B1 and G1 were produced. When dihydrodemethylsterigmatocystin (DHDMST) was fed to this mutant, aflatoxins B2 and G2 were produced. Results of the cell-free experiment with S-adenosyl-[methyl-3H]methionine showed that first the C-6-OH groups of DMST and DHDMST are methylated to produce sterigmatocystin and dihydrosterigmatocystin (O-methyltransferase I) and then the C-7-OH groups are methylated to produce O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin (DHOMST) (O-methyltransferase II). However, no methyltransferase activity was observed when either OMST, DHOMST, 5,6-dimethoxysterigmatocystin, 5-methoxysterigmatocystin, or sterigmatin was incubated with the cell extract. Treatment of the cell extract with N-ethylmaleimide inhibited O-methyltransferase I activity but not that of O-methyltransferase II. Furthermore, these O-methyltransferases were different in their protein molecules and were involved in both the reactions from DMST to OMST and DHDMST to DHOMST. The reactions described in this paper were not observed when the same mold had been cultured in YEP medium.