Studies on the terminal stages of antibody-complement-mediated killing of a tumor cell. II. Inhibition of transformation of T to dead cells by 3'5' cAMP.

Transformation of T to dead cells was prevented by 3'5' cAMP. The effect of 3'5' cAMP was dose, time, and temperature dependent. T washed free of 3'5' cAMP after short-term incubation proceeded to die to the same extent as control cells. After 3 hr of incubation of T with 3'5' cAMP the level of killing was significantly reduced. The 3'5' cyclic nucleotides of uridine, guanine, cytosine, and thymidine and the 2'3' cyclic adenosine nucleotide were not effective. It was concluded that prolonged treatment of T with 3'5' cAMP either irreversibly blocked the damage-producing process or facilitated the reapir of damaged sites.     

Chemical structures of mono-, di-, tri-, and tetraglycosyl glycerides in rice bran.

1. Monoglycosyl monoglyceride, mono-, di-, tri- and tetraglycosyl diglycerides were isolated from rice bran and characterized for their chemical structures. 2. Monoglycosyl monoglycerides were characterized as Gal(beta 1' leads to 3)-1- or 2-monoacyl-sn-glycerol and Glc(beta 1' leads to 3)-1- or 2-monoacyl-sn-glycerol. 3. The structures of monoglycosyl diglyceride were Gal(beta 1' leads to 3)-1,2-diacyl-sn-glycerol and Glc(beta 1' leads to 3)-1,2diacyl-sn-glycerol. Epimeric separation of the galactosyl and glucosyl glycerides was for the first time achieved by thin-layer chromatography. 4. The main diglycosyl diglyceride was shown to be Gal(alpha 1' leads to 6')-Gal(beta 1' leads to 3)-1,2-diacyl-sn-glycerol. 5. The major structure of triglycosyl diglyceride was characterized as Gal(alpha 1' leads to 6')-Gal(alpha 1' leads to 6')-Gal(beta 1' leads to 3)-1,2-diacyl-sn-glycerol. 6. The representative structure of tetraglycosyl diglyceride was for the first time established as Gal(alpha 1' leads to 6')-Gal(alpha 1' leads to 6')-Gal(a-pha 1' leads to 6')-Gal(beta1' leads to 3)-1,2-diacyl-sn-glycerol.     

Homogeneous DNA hybridization assay by using europium luminescence energy transfer

Homogeneous DNA hybridization assay based on luminescence resonance energy transfer (LRET) from a tetradentate beta-diketonate europium chelate, 4,4'-bis(1' ',1' ',1' ',2' ',2' ',3' ',3' '-heptafluoro-4' ',6' '-hexanedion-6' '-yl)-chlorosulfo-o-terphenyl (BHHCT)-Eu(3+) (lambda(ex) = 340 nm and lambda(em) = 615 nm), to an organic dye, Cy5 (lambda(ex) = 643 nm and lambda(em) = 669 nm) has been developed, in which two DNA probes whose sequences comprises the whole complementary strand to the target DNA, are used; one probe having a biotin label on the 3'-terminus and the other a Cy5 label on the 5'-terminus. After hybridization, streptavidin labeled with BHHCT-Eu(3+) was added to the hybridization solution, and in the presence of the target DNA, the sensitized emission of Cy5 was observed when the hybridized complex was irradiated at 340 nm. In the absence of the target DNA, no emission was observed from Cy5.     

2' Derivatives of guanosine and inosine cyclic 3',5'-phosphates. Synthesis, enzymic activity, and the effect of 8-substituents.

A series of representative derivatives of guanosine cyclic 3',5'-phosphate (cGMP) and inosine cyclic 3',5'-phosphate (cIMP) which contained modifications in either the 2' position or the 8 and 2' positions were synthesized. Three types of derivatives were investigated: (1) derivatives in which the 2' position has been altered to produce a 2'-deoxynucleoside cyclic 3',5'-phosphate or a 9-beta-D-arabinofuranosylpurine cyclic 3',5'-phosphate; (2) 2'-omicron-acyl derivatives; and (3) doubly modified derivatives containing a 2' modification [as in (1) and (2)] and an 8-substitution. 2'-Deoxyinosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosylhypoxanthine cyclic 3',5'-phosphate were obtained by HNO2 deamination of 2'-deoxyadenosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosyladenine cyclic 3',5'-phosphate (ara-cAMP), respectively. Treatment of 8-bromo-2'-omicron-(p-toluenesulfonyl) adenosine cyclic 3',5'-phosphate with NaSH yielded the intermediate 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoadenine cyclic 3',5-phosphate, which was converted directly to 2'-deoxyadenosine cyclic 3',5'-phosphate (dcAMP) by treatment with Raney nickel. 8-Bromo-2'-omicron-(p-toluenesulfonyl) guanosine cyclic 3',5'-phosphate was converted to 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate, and the latter was desulfurized with Raney nickel to give 2-deoxyguanosine cyclic 3',5'-phosphate. Ara-cAMP, 9-beta-D-arabinofuranosylguanine cyclic 3',5'-phosphate, and 9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate have been previously reported (Mian et al. (1974), J. Med. Chem. 17, 259). 8-Bromo-2'-omicron-acetylinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]-2'-omicron-acetylinosine cyclic 3',5'-phosphate were produced by acylation of 8-bromoinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]inosine cyclic 3',5'-phosphate, respectively; while 8-bromo-2'-omicron-butyrylguanosine cyclic 3',5'-phosphate was synthesized by bromination of 2'-omicron-butyrylguanosine cyclic 3',5'-phosphate.     

Guanosine tetraphyosphate and its analogues. Chemical synthesis of guanosine 3',5'-dipyrophosphate, deoxyguanosine 3',5'-dipyrophosphate, guanosine 2',5'-bis(methylenediphosphonate), and guanosine 3',5'-bis(methylenediphosphonate).

A new procedure for the synthesis of the pyrophosphate bond has been employed in the preparation of nucleoside dipyrophosphates from nucleoside 3',5'-diphosphates. The method makes use of a powerful phosphorylating agent generated in a mixture of cyanoethyl phosphate, dicyclohexylcarbodiimide, and mesitylenesulfonyl chloride in order to avoid possible intramolecular reactions between the two phosphate groups on the sugar ring. That such reactions can readily occur was shown by the facile cyclization of deoxyguanosine 3',5'-diphosphate to P1,P2-deoxyguanosine 3',5'-cyclic pyrophosphate in the presence of dicyclohexylcarbodiimide alone. The phosphorylation reagent was initially tested in the conversion of deoxyguanosine 3',5'-diphosphate to the corresponding 3',5'-dipyrophosphate and was then used to phosphorylate 2'-O-(alpha-methoxyethyl)guanosine 3',5'-diphosphate, which had been prepared from 2'-O-(alpha-methoxyethyl)guanosine. In the latter case, the addition of the two beta phosphate groups was accomplished in 40% yield. Removal of the methoxyethyl group from the phosphorylated product gave guanosine 3',5'-dipyrophosphate, which was shown to be identical with guanosine tetraphosphate prepared enzymatically from a mixture of GDP and ATP. A modification of published procedures was also necessary to effect the synthesis of guanosine bis(methylenediphosphonate). Guanosine was treated with methylenediphosphonic acid and dicyclohexylcarbodiimide in the absence of added base. The product consisted of a mixture of guanosine 2',5' - and 3',5'-bis(methylenediphosphonate), which was resolved by anion-exchange chromatography. The 2',5' and 3',5' isomers are interconvertible at low pH, with the ultimate formation of an equilibrium mixture having a composition ratio of 2:3. The predominant constituent of this mixture has been unequivocally identified as the 3',5' isomer by synthesis from 2'-O-tetrahydropyranylguanosine.     

Synthesis and separation of diastereomers of uridine 2',3'-cyclic boranophosphate

The first boron-containing 2',3'-cyclic phosphate-modified analogue, uridine 2',3'-cyclic boranophosphate (2',3'-cyclic-UMPB), was synthesized. 5'-O-Protected uridine was cyclophosphorylated by diphenyl H-phosphonate to yield uridine 2',3'-cyclic H-phosphonate, which upon silylation followed by boronation and subsequent acid treatment gave 2',3'-cyclic-UMPB in high yield. The two diastereomers of 2',3'-cyclic-UMPB were separated by HPLC. An alternative method for synthesis of uridine 2',3'-cyclic phosphorothioate (2',3'-cyclic-UMPS) via H-phosphonate was also described.     

Specificities of rabbit anti-(2',5')oligoadenylate antibodies towards phosphorothioate and seco analogs of oligoadenylates.

Rabbit antibodies to 2',5'-linked triadenylate were prepared by immunization with (2',5')A3 conjugated via the 2'3'-levulinic group, (2'5')A3-Lev, to BSA. New radioimmunoassay for (2',5')oligoadenylates was developed using 125I thyrosine labeled derivative of (2',5')A3-Lev. Reactivity of antibodies with phosphorothioate and seco analogs of oligoadenylates was studied. It was found that (i) stereospecific substitution of the diastereotopic oxygens with sulfur in the internucleotide phosphodiester linkages changes the immunoreactivity of such analogs; (ii) the seco analogs of oligoadenylates display in some cases a rather high reactivity.     

A new fertility restorer locus linked closely to the Rfo locus for cytoplasmic male sterility in radish

In this study, we have investigated a new fertility restorer (Rf) locus for cytoplasmic male sterility (CMS) in radish. We have obtained a CMS-Rf system consisting of sterile line '9802A1', maintainer line '9802B1' and restorer line '9802H'. F(1) plants from cross between sterile line '9802A1' and restorer line '9802H' were all male fertile, self pollination of F(1) plants produced an F(2) segregating population consisting of 600 individuals. The segregating population was found to fit a segregation ratio 3:1 for male fertile and sterile types, indicating that male fertility is restored by a single dominant gene (termed Rfo2) in the CMS-Rf system. Based on the DNA sequence of Rfo/Rfk1 (AJ535623), just one full length gene in the sterile line '9802A1', in the restorer line '9802H' and in the male fertile line '2006H', was cloned, respectively. The three sequences correspond to the same gene with two alleles: Rfob in '9802H' and rfob in '9802A1' and '2006H'. These two alleles differ from Rfo/Rfk1 and rfk1 (AJ535624) alleles by two synonymous base substitutions, respectively. Based on the differences between the Rfob and rfob genes, one PCR-based marker was developed, and designated Marker 1, which is identical to the corresponding region of Rfob by sequence analysis. In the F(2) segregating population described above, the Marker 1 was present in 5 sterile plants and in 453 fertile plants, absent in 4 fertile plants and in 138 sterile plants, and was found to fit a segregation ratio 3:1 indicating that Rfob was single copy in '9802H'. Linkage analysis showed that the Rfo2 locus for our CMS-Rf system was distant from the Rfo locus by about 1.6 cM. The sterile line '9802A1' was pollinated by the male fertile line '2006H' and the resulting F(1) plants were all male fertile. These results indicated that the male fertility of radish CMS can be restored by a new Rf locus, which linked tightly to the Rfo locus.     

Inhibition of protein synthesis by the cordycepin analog of (2'-5')ppp(Ap)nA, (2'-5')ppp(3'dAp)n3'dA, in intact mammalian cells

The introduction of the cordycepin analog of (2'-5')An, (2'-5')ppp(3'dAp)n3'dA [referred to as (2'-5')p33'dAn], into mouse L929 cells and cultured human fibroblasts resulted in a dose-dependent inhibition of protein synthesis which was comparable to the inhibition observed by (2'-5')ppp(Ap)nA [referred to as (2'-5')p3An]. The inhibition of protein synthesis by (2'-5')p33'dAn was much more persistent than that of the naturally occurring (2'-5')p3An following prolonged incubation of cells. Furthermore, the (2'-5')p3An was cytotoxic to mammalian cells in culture, whereas the (2'-5')p33'dAn was not.     

Evidence that the 5'' end of lac mRNA starts to decay as soon as it is synthesized.

By monitoring the decay of the first 16% of the beta-galactosidase message, we showed that the 5' end started to decay before the 3' end was completed and at a rate equivalent to that of the whole molecule. Thus, decay was neither from 3' to 5' nor from random internal fragmentation but rather proceeded in a net 5' to 3' direction.     

Mechanism of activation of the mouse c-mos oncogene by the LTR of an intracisternal A-particle gene.

In the mouse myeloma XRPC-24 the DNA of an intracisternal A-particle (IAP) is inserted within the coding region of c-mos. This insertion splits the c-mos into a 3' rc-mos and a 5' rc-mos separated by approximately 4.7 kb of IAP DNA. The insertion is in a head-to-head orientation and brings the 5' LTR of the IAP in juxtaposition to the 3' rc-mos such that the IAP and the 3' rc-mos are transcribed in opposite directions. The intact c-mos gene is usually dormant, whereas the 3' rc-mos is actively transcribed and is capable of transforming NIH3T3 cells. In an effort to understand the nature of this activation we mapped the 5' ends of the 3' rc-mos mRNA present in XPRC-24. We found two main mRNA start sites, one mapping to the junction of the 3' rc-mos and the 5' LTR, and the other located 10 nucleotides upstream to this junction, within the 5' LTR. This result indicates that the 3' rc-mos in XRPC-24 was activated by insertion of a promoter provided by the LTR of an IAP genome. Furthermore, the 5' LTR appears to possess promoter activities in two directions. This conclusion was confirmed by the fact that this 5' LTR, in both orientations, was able to activate the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in the modular vector pSVOCAT.     

Biochemical effects of pure isomers of hexachlorobiphenyl: fatty livers and cell structure.

The effects of a single oral dose (50 mg/kg body wt.) of 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), 2,4,5,2',4',5'-HCB or 2,3,5,2',3',5'-HCB for a period of 72 h have been studied in the male rat. Only 3,4,5,3',4',5'-HCB caused necrosis of thymocytes. 3,4,5,3',4',5'-HCB caused marked pathological changes in the liver with less marked effects being caused by 2,4,5,2',4',5'-HCB and 2,3,5,2',3',5'-HCB. Total lipid content was increased by all the isomers studied, but 3,4,5,3',4',5'-HCB had more pronounced effect on total lipid content. Lipid accumulation was pericentral in the livers obtained from rats treated with 3,4,5,3',4',5'-HCB, but midzonal in the liver obtained from the rats treated with the other two isomers. Analysis of various lipid fractions showed that triacylglycerols were increased seven-fold only by 3,4,5,3',4',5'-HCB, while phospholipids were increased slightly by 2,4,5,2',4',5'-HCB or 2,3,5,2',3',5'-HCB. Only 3,4,5,3',4',5'-HCB increased the level of total and esterified cholesterol. These results show that the fatty livers caused by 3,4,5,3',4',5'-HCB were qualitatively and quantitatively different from those caused by the other two isomers at the same dose. For the first time a hexachlorobiphenyl unchlorinated in the para position, 2,3,5,2',3',5'-HCB has been shown to be a specific inducer of cytochrome P-450. The effects of 2,3,5,2',3',5'-HCB on cell structure and phospholipid content were quantitatively similar to those caused by 2,4,5,2',4',5'-HCB. Thus, chlorination at para (4,4') positions in chlorobiphenyls is not necessarily required for biological activity. It is hypothesized that net stereoelectronic properties of the isomers or resistance to metabolism may be the underlying factor in determining structure-activity relationships.     

Mismatch repair in human nuclear extracts. Quantitative analyses of excision of nicked circular mismatched DNA substrates,constructed by a new technique employing synthetic oligonucleotides

Mammalian mismatch repair (MMR) systems respond to broad ranges of DNA mismatches and lesions. Kinetic analyses of MMR processing in vitro have focused on base mismatches in a few sequence contexts, because of a lack of general and quantitative MMR assays and because of the difficulty of constructing a multiplicity of MMR substrates, particularly those with DNA lesions. We describe here simple and efficient construction of 11 different MMR substrates, by ligating synthetic oligomers into gapped plasmids generated using sequence-specific N.BstNBI nicking endonuclease, then using sequence-specific nicking endonuclease N.AlwI to introduce single nicks for initiation of 3' to 5' or 5' to 3' excision. To quantitatively assay MMR excision gaps in base-mispaired substrates, generated in human nuclear extracts lacking exogenous dNTPs, we used position- and strand-specific oligomer probes. Mispair-provoked excision along the shorter path from the pre-existing nick toward the mismatch, either 3' to 5' or 5' to 3', predominated over longer path excision by roughly 10:1 to 20:1. MMR excision was complete within 7 min, was highly specific (90:1) for the nicked strand, and was strongly mispair-dependent (at least 40:1). Nonspecific (mismatch-independent) 5' to 3' excision was considerably greater than nonspecific 3' to 5' excision, especially at pre-existing gaps, but was not processive. These techniques can be used to construct and analyze MMR substrates with DNA mismatches or lesions in any sequence context.     

The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far.     

New dinucleoside analogues via cross-coupling metathesis

Synthesis of 3'-3', 5-5', and 3'-5' dimeric thymidine, linked by an olefinic chain between glycosidic moieties is described. Cross metathesis reaction of 3' or 5' O-allyl analogues of thymidine led to the expected 3'-3' and 5'-5' dimeric compounds, respectively. In order to obtain the 3'-5' dimer, 5'-O-allyl and 3'-O-allyl monomers were first linked by their free 3' OH and 5' OH groups through a glutaryl spacer; ring closing metathesis was then operated upon this temporary dimer, followed by glutaryl removal.     

Stimulation of cholinergic receptors and changes in cyclic nucleotide levels in the cerebral cortex of the guinea pig

The effect of Atropine and Physostigmine on 3'5' AMP and 3'5' GMP content was investigated in slices of guinea-pig cerebral cortex maintained at rest or electrically-stimulated. Atropine and Physostigmine did not modify either the basal content or the electrically-evoked increase of 3'5' AMP and 3'5' GMP. On the contrary, Betanechol 25 micro M significantly increased 3'5' GMP and 3'5' AMP content in slices kept at rest. The effect was abolished by Atropine 1,5 x 10(-7) M and d-tubocurarine 7 x 10(-6) M, respectively.     

Inhibition of purified rabbit muscle phosphorylase a and phosphorylase b by polychlorinated biphenyls, polychlorinated biphenylols and polybrominated biphenyls

Polychlorinated biphenyls, polychlorinated biphenylols and polybrominated biphenyls inhibited both rabbit muscle phosphorylase a and phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-d-glucosyltransferase, EC 2.4.1.1). The degree of inhibition was dependent upon the relative hydrophobicity of the compounds and steric hinderance. 2,4,5,2',4',5'-Hexabromobiphenyl and Firemaster BP-6 were the most effective inhibitors (Ki, 15 . 10(-6) M). Phosphorylase b was inhibited by compounds of all three groups. 2,4,5,2',4',5'-Hexachlorobiphenyl and 2,4,5,2',4',5'-hexabromobiphenyl did not significantly inhibit phosphorylase a. All of the compounds inhibited phosphorylase a less than phosphorylase b, except 2',3',4',5,5'-pentachloro-2-biphenylol, which was equally effective on each enzyme. Kinetic analysis showed the inhibition was non-competitive and mixed. The results indicate that the compounds bind to hydrophobic site(s) on phosphorylase, access to which is limited by phosphorylation of serine 24.