Synthesis and application of a new phosphorylating agent: S-(N-monomethoxytritylaminoethyl)-O-(o-chlorophenyl)phosphorothi oate

A new phosphorylating agent, S-(N-monomethoxytritylaminoethyl)-O-(O-chlorophenyl) phosphorothioate, was prepared and reacted with a 5'-hydroxyl group of an oligonucleotide using 1-mesitylene-sulfonyl-3-nitrotriazole (MSNT) as a condensing agent. After base labile protecting groups were removed, the partially deprotected oligonucleotide was separated on a reversed phase column and converted to the oligonucleotide with an aminoethyl or a phosphoryl group at the 5'-end by treatment with 80% acetic acid or iodine-water, respectively. The syntheses of ppT, pppT, A5'pp5'T and A5'ppp5'T were also performed by treatment of 5'-O-(N-monomethoxytritylaminoethylthiophosphoryl) thymidine with tri-n-octylammonium salt of phosphoric acid, pyrophosphoric acid, pA and ppA, respectively.     

Studies on the structure and stabilizing factor of the CUUCGG hairpin RNA using chemically synthesized oligonucleotides.

A tridecaribonucleotide, r-UGAGCUUCGGCUC, and two analogues r(UGAGC)d(UUCG)r(GCUC) and r-UGAGCUUCIGCUC, which form a hairpin structure with a four-base-paired stem and a UUCG loop, were synthesized by the solid-phase phosphoramidite method. Properties of these three oligomers and d-TGAGCTTCGGCTC, the DNA analogue, were studied by UV, CD and NMR spectroscopy. The melting temperature (Tm) data suggest that the 2'-hydroxy1 groups and the 2-amino group of guanosine in the loop (9G) stabilize the CUUCGG hairpin which is known to have an unusually high Tm. NMR studies show that this 9G takes a syn conformation and the phosphodiester backbone has a turn at 9G-10G which is a junction of the stem and loop.     

Effects of short-term treatment with calcium on the parathyroid gland of the rat,under particular consideration of the alteration of storage granules

Summary Short-term effects of CaCl₂-treatment on parathyroid cells of the rat, especially on their storage granules, were studied at the ultrastructural level. After an injection of 4% CaCl₂, serum calcium levels (SCL) rapidly increased from 9.1 mg/dl (controls) to a maximum of 14.9 mg/dl at 20 min. At 5 min after the injection, the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged, in spite of elevated SCL (12.4 mg/dl). As soon as SCL rose to 13.2 mg/dl at 7.5 min, NSG-I gradually decreased to a minimum at 30 min; in contrast, NSG-II gradually increased to a maximum at 30 min. Vacuolar bodies also increased together with the augmentation of type-II storage granules. The average diameter of the core of the storage granules decreased significantly after the injection. Protein A-gold method for immunocytochemistry showed that the cores of these granules contain parathormone. Acid-phosphatase activity was occasionally found in storage granules of both types, especially in those of type II. It is concluded (i) that type-I storage granules may be transformed into vacuolar bodies via type-II granules as a result of hydrolysis, and (ii) that these processes may be accelerated during hypercalcemia.     

Purification and characterization of exudate gelatinases in the chronic-phase of carrageenin-induced inflammation in rats

Gelatinases have been purified from the exudate in the chronic-phase (day 7) of carrageenin-induced inflammation in rats. The day-7 exudate gelatinases gave two peaks on Sephadex G-150 gel filtration, the initial step of the purification. The molecular weights of the gelatinases corresponding to the two peaks were about 300 kDa (HMW fraction) and about 110 kDa (LMW fraction), respectively. The gelatinase in the HMW fraction has been purified to homogeneity; the purified gelatinase gave a single band corresponding to a molecular weight of 57 kDa on both SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-gelatin PAGE. On the other hand, the gelatinase purified from the LMW fraction was found to consist of three species, with molecular weights of 66, 64, and 57 kDa, as judged on SDS-gelatin PAGE. Granulation tissue-derived fibroblasts in culture mainly produced the 64-kDa species, which was converted to a 57-kDa species on treatment with 4-amino-phenylmercuric acetate, while rat macrophages and polymorphonuclear leukocytes mainly secreted the 96-kDa species. These results suggest that exudate gelatinases are largely produced by fibroblasts in granulation tissue and that they bind to exudate proteins, resulting in the formation of complexes with molecular weights of about 300 kDa and about 110 kDa. The gelatinases purified from the HMW and LMW fractions are metalloproteinases, as judged from the results of inhibitor experiments. Both the gelatinases degraded gelatin, but showed to proteolytic activity toward alpha-casein or type I collagen. Type IV collagen was degraded at 35 degrees C by the gelatinases purified from the LMW fraction but not by that from the HMW fraction.     

Comparison of wild type and genetically engineered nuclear polyhedrosis viruses of Autographa californica for mortality, virus replication and polyhedra production in Trichoplusia ni larvae

Virus replication and polyhedra production of two polyhedron-positive recombinant nuclear polyhedrosis viruses of Autographa californica, AcJHE.KK and AcAaIT which encode juvenile hormone esterase and scorpion toxin, respectively, were compared with those of a plaque purified wild-type nuclear polyhedrosis virus, AcMNPV-C6, in Trichoplusia ni larvae. Though average times required to kill the T. ni larvae increased with the age of the larvae, killing time by either recombinant virus was significantly shorter than that by wild-type virus. Killing time was reduced ca. 30% for AcAaIT-infected larvae and 5 to 8% for AcJHE.KK-infected larvae as compared to that for AcMNPV-C6-infected larvae. The average weight of larvae infected with AcAaIT was significantly lower than that of larvae infected with AcJHE.KK and AcMNPV-C6. The mean numbers of polyhedra produced in each larva inoculated with AcAaIT and AcJHE.KK were ca. 20% and 60%, respectively, of those for AcMNPV-C6. Total virus titers in AcMNPV-C6-infected larvae were significantly higher than those in AcJHE.KK- and AcAaIT-infected larvae until 2 days post infection.     

Parietal control of hand action

Recent advances suggest that neurons of the anterior intraparietal area play a critical role in the visual guidance of hand action. The parietal cortex appears to process in-coming binocular visual signals of the three-dimensional features of objects and matches these signals with the motor signals, which come from the ventral premotor cortex, that will be required for hand manipulation of the object.     

Termination of early pregnancy by ONO-802 suppositories (16,16-dimethyl-trans-Δ-PGE1 methyl ester)

ONO-802 was used in the form of vaginal suppositories for the termination of early pregnancy in 63 healthy volunteers. Fifty-four (86%) of the 63 cases had complete abortions and remaining 9 (14%) had incomplete abortions.One (1.6%) of the 63 cases complained of nausea and vomiting, and 3 (4.8%) complained of headaches. No other side effects were observed.These results suggest that ONO-802 is acceptable in the form of vaginal suppositories for the termination of early pregnancy.     

Role of glycosaminoglycans in the regulation of T cell proliferation induced by thymic stroma-derived T cell growth factor

The present study investigates the regulatory effects of glycosaminoglycans such as heparin and heparan sulfate on T cell proliferation induced by thymic stromal cell monolayer or its derived T cell growth factor (TCGF). A thymic stromal cell clone (MRL104.8a) supported the growth of Ag-specific, IL-2-dependent Th cell clone (9-16) in the absence of Ag and IL-2 by producing a unique TCGF designated as thymic stroma-derived T cell growth factor (TSTGF). The addition of heparin to cultures in which the growth of 9-16 Th cells was otherwise stimulated by the MRL104.8a monolayer or a semipurified sample of the TSTGF resulted in heparin dose-dependent inhibition of 9-16 Th proliferation. The dose of heparin required for inducing 50% reduction of TSTGF-induced proliferation of Th at a given cell number was found to be proportional to the magnitude of the TSTGF added to cultures, suggesting that heparin exerted its inhibitory effect by binding to the TSTGF rather than by acting on Th cells. A similar growth-inhibiting effect of heparin was observed in IL-7-dependent proliferation of pre-B cell line or Th, but not in IL-2-dependent T cell proliferation or IL-3-dependent myeloid cell proliferation. A strong affinity of TSTGF and IL-7 for heparin was confirmed by the fact that both TSTGF and IL-7 adhered to columns of heparin-agarose and were eluted by salt. When various glycosaminoglycans were tested for the heparin-like Th growth-regulatory capacity, heparan sulfate exhibited Th growth-inhibiting ability comparable to that observed for heparin. These results indicate that the activity of thymic and/or bone marrow stroma-derived lymphocyte growth factor (TSTGF/IL-7) but not of Th-producing TCGF (IL-2) is negatively regulated by heparin or heparan sulfate, which would represent major glycosaminoglycans in the extra-cellular matrix of stromal cells.     

Structure and regulated expression of Kunitz chymotrypsin inhibitor genes in winged bean [Psophocarpus tetragonolobus (L.) DC.]

We analyzed the structure and the expression of Kunitz chymotrypsin inhibitor (WCI) genes in winged bean. WCI was encoded by a multigene family which comprised at least seven members. From their primary structures, four genes (WCI-2, WCI-3a, WCI-3b, and WCI-x) were expected to be functional ones and the other three (WCI-P1, WCI-P2, and WCI-P3) to be pseudogenes. The nucleotide sequences of the WCI-3a and WCI-3b genes were nearly identical, and they encoded the WCI-3 protein, the major chymotrypsin inhibitor in seeds. The WCI-2 gene also encoded the chymotrypsin inhibitor found in seeds and the WCI-x gene was expected to encode an unidentified chymotrypsin inhibitor. WCI messenger RNA and protein accumulated mainly in developing seeds and tuberous roots, small amounts of WCI mRNA being present in stems and pericarps. In seeds, transient accumulation of WCI mRNA was observed during the seed maturation period. These results suggest that the expression of WCI genes is regulated organ-specifically and developmentally in winged bean.