The fractionation of t-RNA on N,N'-bis(3-aminopropyl)-piperazine substituted-Sepharose

An anion exchange agarose has been prepared by modifying sepharose 6B with N,N′-bis (-3-aminopropyl) piperazine. This material (BAPP-Sepharose) has been used for the fractionation of t-RNA from E.coli by column chromatography. The results obtained with gram quantities of crude t-RNA at pH 4.6 and pH 8.0 as measured by the elution patterns of alanyl, arginyl, aspartyl, leucyl, lysyl, methionyl, phenylalanyl, prolyl, seryl, tyrosyl, and valyl t-RNA are described.     

Biochemical studies on a reovirus-like agent (rotovirus) from lambs.

Ten polypeptides were detected in double-capsid lamb rotavirus; four of these appeared to be associated with the outer capsid. Lamb rotavirus RNA, which consisted of 11 or 12 segments, differed from pig rotavirus RNA in the electrophoretic mibility of one of the genome segments.     

Novaluron (Rimon), a novel IGR: potency and cross-resistance

The potency of novaluron on laboratory susceptible and field strains of S. littoralis resembles that of chlorfluazuron and both compounds are about 20-fold more potent than teflubenzuron. No appreciable resistance to novaluron or chlorfluazuron was observed in a field strain of Spodoptera littoralis collected from cucumber field in the central part of Israel. On the other hand, the field strain showed a mild resistance of about 4-fold to teflubenzuron as compared to the laboratory susceptible strain. A very resistant colony of Bemisia tabaci to pyriproxyfen (1,200- to 2,000-fold) showed no appreciable cross-resistance to novaluron. Two field colonies of B. tabaci pressurized with acetamiprid or thiamethoxam for 22 generations, resulting in a 30- to 50-fold resistance to acetamiprid and thiamethoxam, has no cross-resistance to novaluron. The above results are of special interest, indicating a possible alternation between novaluron, pyriproxyfen, and neonicotinoids in insecticide-resistance management programs aiming at preventing resistance development to these novel groups of insecticides against important pests such as whitefly and lepidopteran species.     

N,N'-bis[4-(N-alkylamidino)phenyl]homopiperazines as anti-Pneumocystis carinii agents

The synthesis, anti-Pneumocystis carinii activity and DNA binding properties of eight new N,N'-bis[4-(N-alkylamidino)phenyl]homopiperazines are reported. Compounds 2 and 8 were the most potent and caused about 70% inhibition of Pneumocystis carinii growth in a cell culture model at 1 microM concentrations.     

A novel approach for uniform (13)C and (15)N labeling of DNA for NMR studies.

A novel method is proposed for large-scale synthesis of (13)C- and (15)N-labeled DNA for NMR studies. In this methodology, endonuclease-sensitive repeat amplification (ESRA), a modified PCR strategy, has been used to amplify tandem repeats of the target DNA sequence. The design of the template is such that restriction enzyme (RE) sites separate repeats of the target sequence. The ESRA product is then cloned into a suitable vector. The Escherichia coli cells harboring the plasmid are grown in minimal medium containing [(13)C]glucose and (15)NH(4)Cl as the sole source of carbon and nitrogen, respectively. The target sequence is released by RE digestion of the plasmid, followed by purification using PAGE. Under optimized conditions, the yield ( approximately 5 mg/liter of culture) of (13)C/(15)N-labeled DNA prepared using this approach is found to be several times higher compared to other known enzymatic methods. Successful incorporation of the isotopes has been confirmed using 2D NMR techniques.     

N,N'-bis(2-hydroxybenzyl)-1-(4-bromoacetamidobenzyl)-1,2 -ethylenediamine-N,N'-diacetic acid: a new bifunctional chelate for radiolabeling antibodies

N,N'-Bis(2-hydroxybenzyl)-1-(4-bromoacetamidobenzyl)-1,2 -ethylenediamine-N,N'-diacetic acid (Br phi HBED) was synthesized to bind trivalent metals with high stability constants and to bifunctionally link the radiometal with antibodies (Ab). This ligand has advantages over our previously reported N-(2-hydroxy-3,5-dimethylbenzyl)-N'-(2-hydroxy-5- bromoacetamidobenzyl)ethylenediamine-N,N'-diacetic acid (BrMe2HBED). Br phi HBED has the protein coupling group BrCH2CONH removed from the sterically hindered ring position with the addition of a benzyl group in the linker arm; this provides further distance between the protein and the chelate. We have also observed that the chelate was more stable than BrMe2HBED, so it can be stored longer without loss of observed chemical properties. The improved chelate design allows for more rapid radiolabeling with [111In]indium citrate (1 h at room temperature) with higher radiochemical yields. Br phi HBED was conjugated with an anticolorectal carcinoma monoclonal antibody (1A3) where radiolabeling yields of 75-90% were obtained and the antibody retained its immunoreactivity (ca. 90%) under all labeling conditions studied. Biodistribution studies in a hamster transplanted tumor (GW39) model demonstrated a high tumor uptake when compared to those of 125I-1A3 or 111In-DTPA cyclic anhydride-1A3. Blood clearance of 111In-Br phi HBED-1A3 was rapid and combined with its high target uptake has higher target to nontarget ratios in vivo at various time intervals when compared with that of 1A3 radiolabeled with either 111In-DTPA cyclic anhydride or 125I.     

Mutagenesis and repair of DNA damage caused by nitrogen mustard, N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), streptozotocin, and mitomycin C in E. coli

Cytotoxicity and mutagenesis by streptozotocin, BCNU, nitrogen mustard, and mitomycin C were evaluated in E. coli mutants deficient in SOS repair, SOS-mediated mutagenesis, the adaptive response, and mutants that engage in aberrant mismatch repair. The results demonstrate that premutagenic lesions are caused by nitrogen mustard, BCNU and streptozotocin that are not repaired by ada or recognized by umuDC. Further, recA mutants were hypomutable after exposure to nitrogen mustard, BCNU, and streptozotocin compared to wild type. With the exception of the monofunctional nitrosourea, streptozotocin, both recA and uvrA gene products contribute to the repair of DNA damage caused by the alkylating agents tested. In the case of streptozotocin, although recA mutants were more sensitive than wild type, uvrA mutants were not. Moreover, while ada and alkA E. coli mutants showed increased sensitivity to streptozotocin, they were not more sensitive to the other alkylating agents evaluated.     

The structure of 1-(16-hydroxy-phenoxymethyl)-3,4-dihydroisoquinoline hydrochloride monohydrate, a topical anti-viral agent

The crystal and molecular structure of the anti-viral agent 1-(16-hydroxy-phenoxymethyl)-3,4-dihydroisoquinoline hydrochloride monohydrate, has been determined by single-crystal X-ray diffraction analysis. A prediction, that the conformation about the C₁---C₁₁ bond should be cisoid, has been confirmed.     

Central nervous system actions of 2, 5-bis (3,4-dimethoxybenzyl) cyclopentyl amine,a peripheral dopamine blocking agent

The behavioral and brain catecholamine effects of 2,5-bis (3,4-dimethoxybenzyl) cyclopentyl amine were investigated in mice. It rapidly depleted norepinephrine. Chronic dosing also depleted dopamine, but to a lesser degree. As indicated by a lack of effect on amphetamine induced stereotypy and apomorphine induced emesis and failure to induce catalepsy, the compound does not block brain dopamine receptors. It has no effect on brain catecholamine synthesis or dopamine-β-hydroxylase activity.     

The use of DPPD (N,N'-diphenyl-P-phenylenediamine) as an anti-inflammatory agent

The anti-oxidant compound, DPPD, was found to be an effctive anti-inflammatory compound by inhibiting carrageenin rat paw edema, adjuvant athritis and serum sickness expression of vascular and renal injury. It is proposed these effects are due to the inhibition of prostaglandin synthesis or fatty acid peroxide formation.     

Physiochemical studies on achatininH, a novel sialic acid-binding lectin.

The plasmid pEAP31 contains an alkaliphilic-Bacillus penicillinase gene and a colicin E1 kil gene. Escherichia coli HB101 carrying pEAP31 grown at high temperature released outer-membrane proteins, lipopolysaccharide and phosphatidylethanolamine into the culture medium. Concurrently, penicillinase that had accumulated in the periplasm of the organism was released from the cells. Phospholipase A1-A2 in the outer membrane was not activated in the organism. The results suggest that the release of accumulated periplasmic penicillinase from the producer cells was caused by partial disruption of the outer membrane mediated by the Kil peptide.     

Synthesis, conformational studies and biological activity of N(alpha)-mono-biotinylated rat relaxin.

Biotin-avidin immobilization can be a useful tool in structure-function studies of hormone receptors. A crucial step is the preparation of a specifically biotinylated hormone that is able to bind to its receptor while leaving the biotin group free for interaction with avidin. The receptor for relaxin, an ovarian peptidic hormone produced during pregnancy, has not yet been isolated. We therefore undertook to prepare a specifically monobiotinylated rat relaxin for use in ligand-searching strategies. Rat relaxin is a convenient analogue because reliable bioassays exist, thus allowing assessment of the effect of N-biotinylation on bioactivity. To help improve the yield of the two-chain, three-disulfide bond rat relaxin, 2-hydroxy-4-methoxybenzyl (Hmb) backbone protection was used during the solid-phase assembly of the B-chain to help prevent any possible chain aggregation. As a final step, while the protected peptide was still on the resin, the biotin label was introduced at the N-terminus of the B-chain using standard coupling protocols. The chain combination with the A-chain was accomplished in reasonable yield. Secondary structural measurements demonstrated that the biotin caused the starting B-chain to adopt a more ordered conformation. The labelled synthetic relaxin exhibited similar circular dichroism spectra to native and synthetic single B-chain peptides. In addition, the biotinylated relaxin showed no significant difference in its chronotropic activity in the rat isolated heart assay compared with the native peptide. Biosensor studies showed that antibody recognition was retained upon attachment of the synthetic relaxin to the streptavidin-derivatized surface.     

Synthesis, conjugation, and radiolabeling of a novel bifunctional chelating agent for (225)Ac radioimmunotherapy applications

225Ac (t(1/2) = 10 days) is an alternative alpha-emitter that has been proposed for radioimmunotherapy (RIT) due to its many favorable properties, such as half-life and mode of decay. The factor limiting use of (225)Ac in RIT is the lack of an acceptably stable chelate for in vivo applications. Herein is described the first reported bifunctional chelate for (225)Ac that has been evaluated for stability for in vivo applications. The detailed synthesis of a bifunctional chelating agent 2-(4-isothiocyanatobenzyl)-1,4,7,10,13, 16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid (HEHA-NCS) is reported. This ligand was conjugated to three monoclonal antibodies, CC49, T101, and BL-3 with chelate-to-protein ratios between 1.4 and 2. The three conjugates were radiolabeled with (225)Ac, and serum stability study of the [(225)Ac]BL-3-HEHA conjugate was performed.     

Molecular studies of a novel dragline silk from a nursery web spider, Euprosthenops sp. (Pisauridae)

Various spider species produce dragline silks with different mechanical properties. The primary structure of silk proteins is thought to contribute to the elasticity and strength of the fibres. Previously published work has demonstrated that the dragline silk of Euprosthenops sp. is stiffer then comparable silk of Nephila edulis, Araneus diadematus and Latrodectus mactans. Our studies of Euprosthenops dragline silk at the molecular level have revealed that nursery web spider fibroin has the highest polyalanine content among previously characterised silks and this is likely to contribute to the superior qualities of pisaurid dragline.     

Synthesis, characterization and studies on DNA-binding of a new Cu(II) complex with N1,N8-bis(l-methyl-4-nitropyrrole-2-carbonyl)triethylenetetramine

A new Cu(II) complex of CuLCl(2) (here, L=N(1),N(8)-bis(1-methyl-4-nitropyrrole-2- carbonyl)triethylenetetramine) had been synthesized and characterized. The structure of the complex was investigated with density functional theory (DFT) calculations. DNA-binding of the Cu(II) complex and its effects on tumor cell viability were firstly studied. The interactions between the complex and calf thymus DNA had been investigated using UV spectra, fluorescent spectra, viscosity and CV (cyclic voltammetry). The cleavage reaction on plasmid DNA has been monitored by agarose gel electrophoresis. The experimental results show that the mode of binding of the complex to DNA is classical intercalation and the complex can cleave pBR322 DNA. The effects of the CuL on cell viability were tested using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) dye assay and the results indicate that the CuL had certain effect on cancer cells.     

In the search for new anticancer drugs. X. N,N; N',N'-bis(1,2-ethanediyl)-N'-(1-oxyl-2,2,6,6-tetramethyl- 4-piperidinylaminocarbonyl) phosphoric triamide--a new potential anticancer drug of high activity and low toxicity

A new nitroxyl labeled TEPA derivative 5 containing the urea bridge between the phosphorus and the nitroxyl moiety, and the congeners containing the NOH and NH groups instead of the nitroxyl function were synthesized, and tested in vivo on CD2F1 mice for anticancer activity against P388 and L1210. The nitroxyl compound is more active than the reduced forms. The nitroxyl compound 5 elicits 170% ILS at 90 mg/kg after 30 days and 439% ILSmax after 60 days against P388, and has a higher therapeutic ratio (26.4) than the clinically used Thio-TEPA (2.75). The LD50 of 5 is 270 mg/kg, while that of Thio-TEPA is 18 mg/kg. Consequently, the nitroxyl compound 5 is a promising new anticancer drug.