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At least 37 records · Page 2Linked to original sources
PubMed1988

Benzothiopyranoindazoles, a new class of chromophore modified anthracenedione anticancer agents. Synthesis and activity against murine leukemias.

The synthesis of the benzothiopyranoindazoles, a new class of chromophore modified anthracenediones related to mitoxantrone, is described. In this structural class the quinone moiety, which is believed to be responsible for the cardiotoxicity of the anthracyclines, has been designed out. The synthesis of the benzothiopyranoindazoles was carried out by a multistep sequence from requisite 1-chloro-4-nitro-9H-thioxanthen-9-one precursors. Reaction with a monoalkylhydrazine gave a 5-nitrobenzothiopyranoindazole adduct, which was catalytically reduced to a corresponding C-5 anilino intermediate. Alkylation of 7 with a requisite X(CH2)nNR1R2 (X = Cl, Br; R1, R2 = H, alkyl, acyl; n = 2,3) provided target "two-armed" benzothiopyranoindazoles or A-ring methoxy and/or side chain acyl intermediates, which could be converted to 3 by appropriate deprotection methodologies. Alternatively, certain target compounds 3 were synthesized by reaction of 7 with appropriately functionalized glycine precursors under Schotten-Bauman or BOP chloride condensation conditions to provide C-5 acylamino intermediates, followed by Red-Al reduction and deprotection steps. Described also is the synthesis of selected benzothiopyranoindazole congeners with proximal acylamino side chains at C-5 and B-ring sulfone functionality at S-6. Potent activity was demonstrated against murine L1210 leukemia in vitro (IC50 = 10(-7)-10(-9) M) as well as against P388 leukemia in vivo over a wide range of structural variants. In general, activity against the P388 line was maximized by (a) a basic side chain at N-2 and a dibasic side chain at C-5 with primary or secondary distal amine substitution, (b) certain patterns of A-ring hydroxylation with 8-OH and 9-OH most favorable, and (c) sulfide oxidation state at S-6. Besides having curative activity against the P388 line, the more active compounds were curative against murine B-16 melanoma in vivo. On the basis of their exceptional broad-spectrum in vivo anticancer activity, selected compounds in this series have been chosen for development toward clinical trials.

Animals↗
PubMed1992

Extrathymic positive selection of alpha beta T-cell precursors in nude mice.

T lymphocytes expressing alpha beta T-cell receptors with sufficient affinity to major histocompatibility complex (MHC) molecules expressed on thymus epithelial cells are positively selected and mature to functional T cells. But several studies have demonstrated that athymic nude mice grafted with MHC-incompatible thymuses developed T cells specific for nude host rather than thymic MHC. We examined this paradox by analysing the specificity of T lymphocytes derived from nude mice. We report here that nude T lymphocyte precursors transferred to allogeneic SCID (severe combined immunodeficiency) mice with a functioning thymus (but lacking T or B cells) generated host MHC-restricted effector T cells but also contained T cells restricted to donor MHC. If nude T cells were depleted from nude lymphohaemopoietic donor cells before or after transfer, only host MHC-specific T cells matured. The results may explain the unusual MHC specificities of nude T lymphocytes described in earlier studies and demonstrate two separate differentiation steps: in nude mice, T cells may be positively selected for self-MHC restriction specificity extrathymically; then a functional thymus is required for efficient T cell maturation.

Animals↗
PubMed1991

Complete inhibition of glucose-induced desensitization of the glucose transport system by inhibitors of mRNA synthesis. Evidence for rapid turnover of glutamine:fructose-6-phosphate amidotransferase.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) plays a key role in desensitizing the insulin-responsive glucose transport system (GTS), and recent studies have revealed that loss of GFAT activity accompanies desensitization. To gain insights into the mechanisms underlying loss of enzyme activity, we have used primary cultured adipocytes and two well established inhibitors of mRNA synthesis to estimate GFAT turnover. Both actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) caused a rapid and extensive loss in GFAT activity (greater than 70% loss, t1/2 of 45 min) indicating that GFAT has a relatively short half-life. Since induction of insulin resistance requires GFAT, we next examined the ability of mRNA inhibitors to block glucose-induced desensitization. When adipocytes were cultured for 18 h with 20 mM glucose, amino acids, and 25 ng/ml insulin, maximal insulin responsiveness of the GTS was reduced by greater than 70%. Both actinomycin D and DRB rapidly and completely prevented desensitization in a dose-dependent manner (ED50 of 16 nM and 15 microM, respectively). These findings are the predicted functional consequence of diminished GFAT activity. Evidence that actinomycin D acts selectively on GFAT without influencing other steps within the desensitization pathway was obtained using glucosamine, an agent that enters the hexosamine biosynthesis pathway at a point distal to the action of GFAT. Actinomycin D inhibited glucose-induced desensitization but failed to block glucosamine-induced desensitization. From these studies we conclude that 1) glucose-induced desensitization of the GTS can be completely prevented by actinomycin D and DRB, two potent and diverse inhibitors of mRNA synthesis; 2) the functional integrity of the desensitization pathway is maintained by a short-lived protein; and 3) the identity of this short-lived protein is most likely GFAT, the first and rate-limiting enzyme of the hexosamine biosynthesis pathway.

Animals↗
PubMed1990

Nucleoplasmin: the archetypal molecular chaperone.

Nucleoplasmin was the first protein to be described as a molecular chaperone. Studies of nucleoplasmin have resulted in advances in two areas of cell biology. Firstly, the pathway of nucleosome assembly in Xenopus oocytes and eggs has been elucidated and is the only assembly pathway known in detail. Nucleosome assembly represents the major chaperoning function of nucleoplasmin. Secondly, nucleoplasmin has been used to elucidate the transport of proteins into the nucleus, revealing a selective entry mechanism for nuclear proteins, passage through the nuclear pore complex, and a two-step mechanism of transport. The properties and functions of nucleoplasmin are reviewed, together with other proteins which are related either structurally or functionally to nucleoplasmin.

Animals↗
PubMed1979

Regulatory mutants at the his1 locus of yeast.

The his1 gene in Saccharomyces cerevisiae codes for phosphoribosyl transferase, an allosteric enzyme that catalyzes the initial step in histidine biosynthesis. Mutants that specifically alter the feedback regulatory function were isolated by selecting his1 prototrophic revertants that overproduce and excrete histidine. The prototrophs were obtained from diploids homoallelic for his1--7 and heterozygous for the flanking markers thr3 and arg6. Among six independently derived mutant isolates, three distinct levels of histidine excretion were detected. The mutants were shown to be second-site alterations mapping at the his1 locus by recovery of the original auxotrophic parental alleles. The double mutants, HIS1--7e, are dominant with respect to catalytic function but recessive in regulatory function. When removed from this his1--7 background, the mutant regulatory site (HIS1-e) still confers prototrophy but not histidine excretion. To yield the excretion phenotype, the primary and altered secondary sites are required in cis array. Differences in histidine excretion levels correlate with resistance to the histidine analogue, triazoalanine.

Alleles↗
PubMed1992

The use of pharmacologic agents to study mechanisms of intestinal calcium transport.

The mechanism of vitamin D-dependent intestinal calcium transport has been explored in experimental animals in vivo and in vitro with the aid of pharmacologic agents that inhibit steps in the translocation process. Glucocorticoids in vivo, but not in vitro, inhibit the mucosal-to-serosal flux (Jms) of calcium and thus reduce net calcium absorption. Chronic metabolic acidosis inhibits calcium transport in vivo through inhibition of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] production and by a direct effect in vitro on the enterocyte to decrease calcium Jms. Cellular functions that may be involved in the transport process have been inhibited in vitro, including brush border calcium uptake by calcium channel blockers; calmodulin-dependent Ca-activated ATPase by trifluoperazine; calcium binding to vitamin D-dependent calcium-binding protein (CaBP, calbindin) by theophylline and acidic lysosomal vesicle function by quinacrine, chloroquine and ammonium chloride. The results of these studies demonstrate the consequences of selectively inhibiting steps thought to be involved in calcium transport and suggest new directions for further research in elucidating mechanisms of cellular calcium transport.

Animals↗
PubMed1989

Determination of the partial benzodiazepine receptor agonist Ro 16-6028 in plasma by capillary gas chromatography with nitrogen-selective detection after conversion into the ethyl ester derivative.

A highly sensitive capillary gas chromatographic method was developed to determine plasma levels of a novel partial benzodiazepine receptor agonist in man following the very low therapeutic doses required for anxiolysis. The compound was isolated from plasma by liquid-liquid extraction at basic pH, converted into the ethyl ester analogue by a two-step procedure, separated from plasma constituents by capillary gas chromatography and quantified by means of nitrogen-selective detection. Because of the thermolabile tert.-butyl ester function, the agonist could not be gas chromatographed without degradation. Formation of the far more stable ethyl ester analogue was achieved by treatment with hydrogen chloride in ethanol, followed by an ethylation step with diazoethane. The high sensitivity of the new method (about 100 pg/ml, using 1-ml plasma specimens) allowed the monitoring of plasma levels of the agonist for up to 8 h (about three elimination half-lives) after a single 0.1-mg oral dose to human volunteers. The practicability of the procedure was demonstrated by the analysis of more than 600 plasma samples from clinical studies performed with human volunteers.

Anti-Anxiety Agents↗
PubMed1992

Molecular bases for hereditary cancer-prone diseases.

Constitutional loss or inactivation of one copy of a tumor-suppressor gene, as exemplified by hereditary retinoblastoma, increases the propensity for malignancies by reducing the number of events necessary for the complete loss of the negative regulatory function. We developed a selectable mutation assay employing a human lymphoblastoid cell line (LCL) derived from a heterozygous carrier of 2,8-dihydroxyadenine urolithiasis, adenine phosphoribosyltransferase (APRT) deficiency, for dissecting the second step in loss-of-function mutations and for determining the potential of physical and chemical agents for producing such mutations. The mode of mutational events arising in the wild-type allele of the functionally heterozygous APRT gene resembled that reported for tumor-suppressor genes in malignancies in that mitotic non-disjunctions or recombinations as well as deletions prevailed. Ultraviolet light (UV) was much less efficient in inducing these types of mutations than ionizing radiation. A group of autosomal recessive cancer-prone diseases, including xeroderma pigmentosum (XP), has been characterized as being more susceptible to genomic insults, owing to some defects in DNA processing, such as replication, repair, or recombination. This increased genomic instability may accelerate the gain-of-function mutation at a proto-oncogene and/or the loss-of-function mutation at a tumor-suppressor gene. XP complementation group A (XP-A) LCLs were extremely sensitive to UV-mutagenesis at the hypoxanthine phosphoribosyltransferase (HPRT) locus even at equicytotoxic doses. Some unique mechanism may operate in UV-mutagenesis in XP-A. We have succeeded for the first time in rendering XP-A cells tumorigenic in athymic mice by applying multiple exposures to UV and subsequent treatment with TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

AMP deaminase and thymidine kinase deficiencies in a mutant mouse S49 cell clone.

From a mutagenized population of wild type S49 cells, a clone was isolated in a single step that possessed functional and biochemical deficiencies in both AMP deaminase and thymidine kinase activities. This mutant cell line, DTB6, was selected in semi-solid medium containing 1mM thymidine and 1mM dibutyryl cyclic AMP. In comparative growth rate experiments, DTB6 cells were considerably less sensitive than parental cells to the growth inhibitory effects of thymidine. In contrast, DTB6 cells were much more sensitive to the cytotoxic effects of adenine and adenosine. The supersensitivity of DTB6 cells toward adenine could be ameliorated by the addition of hypoxanthine to the culture medium. The growth phenotype of the mutant cells could be attributed to deficiencies in two enzyme activities. First, DTB6 cells possessed a 60-70% deficiency in AMP deaminase activity, although the residual activity appeared kinetically similar to the wild type enzyme. Second, DTB6 cells possessed a virtual complete deficiency in thymidine kinase activity. Both enzyme deficiencies behaved in a recessive fashion in intraspecies hybrids. Revertants of DTB6 cells possessed wild type levels of AMP deaminase activity but remained deficient in thymidine kinase activity, while another revertant of DTB6 cells expressed 11% of the wild type thymidine kinase level but did not perceptibly change its AMP deaminase activity. The ability to isolate single step mutants with two seemingly independent biochemical abnormalities raises the speculation that there may be some link between cellular functions responsible for purine nucleotide and thymidine metabolism.

AMP Deaminase↗
PubMed1990

Effects of a novel immunosuppressive agent, FK506, on human B cell activation.

We examined the effect of new immunosuppressive agent, FK506, on the human B cell function, in comparison with that of cyclosporin A (CyA) and tried to define the discrete activation step(s) which is selectively affected by FK506 and CyA. We used polyclonal B cell activators, Staphylococcus aureus Cowan I (SAC) and pokeweed mitogen (PWM). We found that (i) the initial B cell activation process by PWM, which is on the basis of T cell-dependent manner, is susceptible to the inhibitory effects of FK506 and CyA, while initial B cell activation on the basis of T cell-independent manner by SAC is resistant to these drugs; (ii) they also inhibit helper factor production by T cells; (iii) once they are activated, the B cells become resistant to inhibition by the drugs; and (iv) on an equimolar basis, FK506 exhibits 100-fold greater inhibitory activity than does CyA. Thus FK506 mainly interferes with interactions between T cells and other cells which are essential for B cell activation process, resulting in inhibition of B cell function.

Anti-Bacterial Agents↗
PubMed1979

Cellular and humoral requirements for T-cell development.

T-cells develop as a consequence of intrathymic and postthymic events, in which hemopoietic progenitors are differentiated into precursors and effector cells. We are proposing that such process includes three integrated steps: (1) T-cell differentiation; (2) selection of the T-cell repertoire and (3) specialization into functional subsets of T-cells. Although there is evidence of specific and nonspecific humoral factors (i.e. thymic extracts, etc.) affecting T-cell differentiation, it is also proposed that the most critical component in this integrated process is the consequence of direct cell to cell interactions between precursor and inducer cells. Thus, the three processes are the consequence of the appropriate or condordant matching of precursor-inducer populations, both at intra- and extrathymic sites. It is also proposed that MHC determinants are critical in permitting the appropriate matching. The model can thus account for nonfunctional differentiation when the appropraite matching is not available and with intrathymic selection by excess cell production favoring the appropriate matching.

Cell Communication↗
PubMed1976

Sudden potential drop in bullfrog gastric mucosa.

The previously reported sudden potential drop (SPD), which occurs under anoxia in 10% CO2, has been further explored. We find several conditions necessary for this effect: 1) anoxia; 2) serosal pH less than 7.1;3) presence of chloride; and 4) a PD, either spontaneous or voltage clamped, which changes across the region of 10 mV, serosal positive. With the first three conditions satisfied, a reversible decrease in measured resistance can be produced at will by changing the clamp voltage. In the anoxic, low-resistance state, changes in K+ or C1- concentration give little change in voltage, showing that the increased conductivity is not selective for either ion. A model is proposed containing a shunt-resistance element whose resistance is a step function of PD. This model can mimic the responses of the tissue and provides a working model for the SPD.

Animals↗
PubMed1986

Physiological determination of methicillin resistance in Staphylococcus aureus: comparison of clinical and genetically derived isolates.

The expression of methicillin resistance in 10 clinical and 5 genetically constructed strains of Staphylococcus aureus has been measured in relation to temperature of incubation (37 degrees C versus 30 degrees C), the presence of additional sodium chloride in the medium (5.5% vs. 0.5%), and pH (7.4 vs. 5.4). Resistance was quantitatively measured by disc diffusion and agar dilution assays. In disc assays of methicillin resistance, all but one clinical isolate and both transduced strains displayed increased the resistance at the lower temperature. Increased salt had little effect, or decreased the resistance of these strains. At pH 5.4 resistance decreased substantially. Methicillin resistance in three step-selected strains, by contrast, was variably affected by changes in temperature or salt concentration, though the effects were never great. The most distinctive feature of these strains was that pH change did not affect their resistance. In agar dilution assays, where surviving fractions within the population were measured as a function of antibiotic concentration, the temperature and salt effects seen in disc assays of resistance were again evident for the two clinical isolates tested, but not for the transduced strains, where added salt increased resistance at the higher concentrations of methicillin tested. For two step-selected strains, the effects of lowering temperature and increasing salt on resistance were variable. By contrast with the disc assays, these strains were less resistant at the lower pH, though not to the same relative extent as that shown by the clinical isolates and transduced strains. Resistance heterogeneity was not significantly affected by higher salt and lower incubation temperature in the two clinical strains tested, but elevated salt consistently increased heterogeneity in transduced and step-selected strains at the higher concentrations of methicillin tested. In the absence of salt, step-selected strains exhibited little heterogeneity, unlike the clinical and transduced strains. We conclude that the similarities and differences seen in clinical isolates and laboratory strains favour the idea that methicillin resistance arises clinically in S. aureus as a result of transduction (or other genetic transfer) of existing gene(s) determining resistance, rather than as a result of selection of mutants among sensitive or less resistant strains. Our findings also indicate that inclusion of salt in media containing methicillin may attenuate resistance to the antibiotic and thus decrease the sensitivity of detection of marginally resistant strains of S. aureus.

Culture Media↗
PubMed1989

Heparin-binding growth factor 1 induces the formation of organoid neovascular structures in vivo.

One of the promises of modern molecular biology has been the opportunity to use genetically modified human cells in a patient to permanently restore inborn errors of metabolism. Although it has been possible to introduce genes into mammalian cells and to control their expression, it has proven difficult to introduce mammalian cells as carriers of the modified genetic information into hosts. The successful implantation of selective cells cannot be achieved without adequate vascular support, an essential step toward integration and reconstitution of a new biological function. Although a partial solution to this problem has been found by inducing specific site-directed neovessel formation using heparin-binding growth factor 1 (HBGF-1) adsorbed to a collagen matrix, these implants function for only a short period (weeks). We now report the formation of organoid neovascular structures using polytetrafluoroethylene fibers coated with collagen and HBGF-1 implanted in the peritoneal cavity of the rat. The organoid structures contained readily visible vascular lumina and nonvascular structures that resemble nerve tissue. It was also possible to demonstrate that the vascular system on the implant is continuous with the vascular tree of the host. This feature was used to demonstrate that the organoid structures are capable of sustaining the biological function of implanted normal rat hepatocytes over long periods of time (months) in the homozygous Gunn rat, thereby facilitating future applications involving the delivery of new genetic information.

Animals↗
PubMed1990

The 5-HT1B receptors.

The 5-HT1B receptors have been identified by radioligand binding techniques predominantly in the basal ganglia of the rat and mouse brain. A number of 5-HT receptor agonists have been shown to display high affinity but limited selectivity for the 5-HT1B recognition site. These include 5-CT, 5-HT, RU 24969, TFMPP, MCPP, and CGS 12066B. Antagonists at the 5-HT1B site include the drugs metitepin, metergoline, cyanopindolol, isamoltane, and propranolol but none of these drugs are selective for this receptor. Functional correlates of 5-HT1B receptor activation have been most closely defined in vitro. These include inhibition of transmitter release, inhibition of forskolin-stimulated adenylate cyclase and actions on the mouse urinary bladder strip and the rat vena cava. Many functional correlates of 5-HT1B receptor activation in vivo have been proposed, but convincing evidence from antagonist studies is generally lacking. The development of selective 5-HT1B receptor agonists and antagonists will be a key step in defining the physiological role of this receptor site in the brain and periphery of the mouse and rat although it must be realized that these compounds, if they are developed, are unlikely to have functional effects in man since the 5-HT1B recognition site is absent in the human CNS. Nevertheless many of these studies on the 5-HT1B receptor may aid the development of drugs acting at the 5-HT1D site since this receptor has been identified as being the equivalent of the 5-HT1B site in species other than the rat and mouse.

Adenylyl Cyclases↗
PubMed1990

A 3' splice site-binding sequence in the catalytic core of a group I intron.

Ribozymes use specific RNA-RNA interactions for substrate binding and active-site formation. Self-splicing group I introns have approximately 70 nucleotides constituting the core, a region containing sequences and structures indispensable for catalytic function. The catalytic core must interact with the substrates used for the two steps of the self-splicing reaction, that is, guanosine, the 5'-splice-site helix (P1) and the 3' splice site. Mutational evidence suggests that core sequences near segment J6/7 that joins the base-paired stems P6 and P7, and the bulged base of P7(5'), participate in binding guanosine substrate, but nothing is known about the interactions between the core, the 5'-splice-site helix and the 3' splice site. On the basis of comparative sequence data, it has been suggested that two specific bases in the catalytic core of group I introns might form a binding sequence for the 3' splice site. Here we present genetic evidence that such a binding site exists in the core of the Tetrahymena large subunit ribosomal RNA intron. We demonstrate that this pairing, termed P9.0, is functionally important in the exon ligation step of self-splicing, but is not itself responsible for 3'-splice-site selection.

Animals↗
PubMed1988

Cytoplasmic domain affects membrane expression and function of an Ia molecule.

The association of foreign antigen with Ia molecules on the surface of antigen-presenting cells is necessary for the interaction with the clonally distributed antigen receptor on T cells and is therefore critical in the initiation and regulation of immune responses. Ia polypeptides (alpha and beta) are composed of two extracellular domains, a transmembrane domain and a cytoplasmic domain. Although exon-shuffling experiments have demonstrated that antigen associates with the NH2-terminal alpha 1 and beta 1 domains, the roles that the other domains play in Ia function are still poorly understood. The B-hybridoma cell line 2B1 was selected in a series of positive and negative immunoselection steps for a mutation in the Ek alpha polypeptide. It was found to fortuitously contain a mutation in the Ak alpha polypeptide as well. Sequence analysis of the Ak alpha gene showed that a single base transition (C----T) resulted in a stop codon at amino acid residue 222. This caused the loss of 12 amino acids from the cytoplasmic domain of the mature polypeptide. This mutation results in a decreased level of Ak alpha polypeptide expression on the cell surface (50% of wild-type levels), an increased half-life of Ak alpha polypeptide in the cell, and a specific limited defect in antigen presentation.

Amino Acid Sequence↗
PubMed1988

Novel assay for pancreatic cellular damage: 1. Characterization of protein profiles in human pancreatic cytosol and purification and characterization of a pancreatic specific protein.

The protein patterns of cytosols from normal human pancreas and pancreatic carcinoma were studied by a two-dimensional separation technique using high-performance liquid chromatography followed by isoelectric focusing on polyacrylamide gels and visualization of the focused proteins by Coomassie Blue staining. Almost identical protein patterns were obtained for 20 different specimens from normal pancreas, whereas quite different protein patterns were found in 12 samples of pancreatic carcinoma. A major protein in normal pancreatic cytosol, not identical to any macromolecule previously tested as a marker for pancreatic function, was selected for further studies. The protein was not found in specimens of pancreatic carcinoma. It was purified by a single step chromatofocusing procedure, focused at pH 6.9, and moved as one single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 44,500 daltons. Total amino acid analysis revealed a high concentration of glutamic acid, leucine, and lysine. The purified protein had no amylase activity or lipase immunoactivity. It constituted approximately 2% of the total normal pancreatic cytosol protein. Later immunological studies have shown the protein to be highly specific for normal human pancreas, indicating a possible future use as a marker for pancreatic cell damage.

Adenocarcinoma↗