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Biomedical subjects

R L Noble

Publications and source records attributed to R L Noble.

At least 19 recordsLinked to original sources
PubMed1992

Sequencing of peptides and proteins from the carboxy terminus.

A new chemical method for carboxy-terminal (C-terminal) protein sequencing has been developed. This approach has been successfully used to sequence 5 residues of standard proteins and 5 to 10 residues of synthetic peptides at low nanomole levels. The sequencing procedure consists of converting the C-terminal amino acid into a thiohydantoin (TH) derivative, followed by transformation of the TH into a good leaving group by alkylation. Next, the alkylated TH is cleaved mildly and efficiently with (N = C V S)- anion, which simultaneously forms a TH on the newly truncated protein or peptide. Thus, after the initial TH derivatization, there is no return to a free carboxyl group at the C-terminus. An additional benefit of this method is that the alkylating moiety can be chosen with a variety of properties allowing for variation in the detection method. This chemistry has been adapted to automated protein sequencers with a cycle time of about 1 h.

Amino Acid Sequence↗
PubMed1992

A long synthetic peptide containing a nuclear localization signal and its flanking sequences of SV40 T-antigen directs the transport of IgM into the nucleus efficiently.

Synthetic short peptides containing only the nuclear localization signal (NLS) direct the transport of nonnuclear proteins into the nucleus. As a conjugate of the synthetic peptide with immunoglobulin M (IgM) did not enter the nucleus, there was believed to be a size limit for nuclear transport of NLS-conjugated proteins. However, we found that IgM conjugated with purified nucleoplasmin, a nuclear protein of Xenopus oocytes, rapidly accumulated in the nucleus. For direct comparison with the short peptide, we prepared a long peptide containing the NLS and its flanking sequences of SV40 large T-antigen and its mutated long peptide, in which possible phosphorylation sites located at the amino terminal of the NLS were changed to alanine. Kinetic experiments showed that wild-type long peptide-IgM conjugates were almost entirely taken up into the nucleus within 30 min after their injection, whereas almost 60 min was required for the mutated long peptide-IgM conjugates to enter the nucleus of all the cells examined, and there was no apparent accumulation of short peptide-IgM conjugates in the nucleus within 60 min. These results indicate that even when the kinetics of transport are affected by amino acid substitutions, the long peptide directs the transport of large molecules such as IgM into the nucleus.

Amino Acid Sequence↗
PubMed1990

Dissociation of tissue destruction induced by cytolytic T cells in vivo and cytotoxicity as measured in vitro.

Two forms of local cutaneous graft-versus-host reactions were used to examine the in vivo activity of cytolytic T cells in a large number of antigen systems and mouse strain combinations. In immune lymphocyte transfer reactions (TrRs), CTL were injected intradermally into allogeneic hosts to which they were sensitized; in bystander reactions (ByRs), CTL were mixed with target cells and the mixture injected into hosts syngeneic to the CTL. Both reactions frequently culminate in full-thickness skin destruction. However, CTL highly active in cell-mediated lympholysis assays in vitro sometimes failed to induce significant reactions in vivo, and CTL with negligible CML activity often induced severe, necrotizing lesions. In addition, Clone 58, a non-MHC-specific CD8+ clone that originated from cells extracted from a sponge matrix allograft, lost its CML activity but continued to induce necrotizing TrRs and ByRs. Insofar as these reactions may exemplify the specific (TrR) and nonspecific (ByR) tissue injury that occurs in the rejection process, these findings question the reliability of CML for predicting the ability of CTL to induce the tissue destruction seen in allograft rejection.

Animals↗
PubMed1990

Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids.

9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly "orthogonal" scheme, and thus offers many unique opportunities for bioorganic chemistry.

Amino Acids↗
PubMed1990

The discovery of the vinca alkaloids--chemotherapeutic agents against cancer.

In folklore medicine, extracts of the leaves of the subtropical plant Catharanthus roseus (L.) G. Don (sometimes known as Madagascar periwinkle) were reputed to be useful in the treatment of diabetes. This review describes how attempts to verify the antidiabetic properties of the extracts led instead to the discovery and isolation of two complex indole alkaloids, vinblastine and vincristine, which are used in the clinical treatment of a variety of cancers. The two alkaloids, although structurally almost identical, nevertheless differ markedly in the type of tumors that they affect and in their toxic properties. These and related alkaloids have been the subject of many pharmacological and biochemical investigations both in vivo and in vitro in the search for improved cancer treatments. A model system used in these studies, a transplantable lymphoma in Noble strain rats designated Nb2 node, has serendipitously led to the development of a highly sensitive and specific bioassay for lactogenic hormones.

Animals↗
PubMed1989

Blocking of interleukin-2 production, but not the tissue destruction induced by cytotoxic T cells, by cyclosporine.

Cytotoxic T lymphocytes generated against epidermal alloantigen-1 (Epa-1), a tissue-restricted, non-H-2 alloantigen that is a target-cell determinant of both skin allograft rejection and cutaneous graft-versus-host reactions, directly produce full-thickness ulcerative skin lesions in Epa-1+ mice. Anti-Epa-1 CTL also indirectly cause extensive damage of "innocent bystander" tissue when injected admixed with Epa-1+ target cells into the skin of Epa-1- hosts. Unlike the direct destruction of host tissue by CTL in "immune lymphocyte transfer reactions" (TrR), "bystander reactions" (ByR) apparently are initiated by the release of lymphokines that recruit host inflammatory cells to the injection site. Treatment of CTL with cyclosporine in vitro prevents their production of lymphokines like interleukin 2 but has no effect on cell-mediated cytotoxicity. However, little is known about the function of CsA-treated CTL in vivo. We confirmed the differential effect of CsA on CTL function in vitro with bulk-culture anti-Epa-1 CTL-CsA pretreatment of CTL abrogated IL-2 production but did not affect CMC. Moreover, we found that CsA pretreatment did not affect the ability of CTL to evoke TrR, nor did it significantly impair their ability to mediate ByR. Therefore, when CTL are treated with CsA in such a way that they lose their capacity to produce IL-2, their cytotoxic activity in vitro as well as their ability to directly and indirectly mediate tissue destruction in vivo are left intact. These results suggest that the ability of CTL to mediate allograft rejection is not dependent on their ability to produce IL-2 and that CMC plays a role in the rejection process.

Animals↗
PubMed1989

Solid phase peptide synthesis of 15N-gramicidins A, B, and C and high performance liquid chromatographic purification.

Four single-site 15N-labeled molecules of gramicidin have been synthesized using the 9-fluorenylmethoxycarbonyl method of solid phase peptide synthesis. Formylvaline was coupled as the N-terminal amino acid, and the peptide was cleaved from the resin with ethanolamine. Each synthesized gramicidin was purified in one step by semipreparative reverse phase high performance liquid chromatography and obtained in overall yields as high as 86%. The peptide was characterized by comparison with natural gramicidin using amino acid analysis, u.v. spectroscopy, and analytical high performance liquid chromatography.

Chromatography, High Pressure Liquid↗
PubMed1988

Analysis of blood cell populations, plasma zinc and natural killer cell activity in young children with Down's syndrome.

Down's syndrome (DS) is associated with respiratory infections, lymphoblastic leukemia and a mortality rate that is elevated particularly during the first 5 years of life. Unfortunately, most studies on immune function in DS have been performed on adults or older children, the results of which may be not relevant to a primary immune defect in DS since maturation of the immune system and compensatory mechanisms may mask to a certain degree primary immune defects which are associated with this syndrome. In the present study, several immune and hematologic parameters were studied in DS children under 6 years of age and in age-matched controls. The children with DS exhibited a reduced number of lymphocytes while the total number of white blood cells was normal. In addition, the children with DS had reduced numbers of circulating OKT4+ cells and a significantly depressed ratio of OKT4+ to OKT8+ cells, but their overall proportion of T-cells was normal. Normal NK activity was observed and cells from DS children were no more sensitive to the augmenting effects of alpha interferon than cells from control children. Hematocrit levels were slightly elevated in the DS group while plasma zinc levels appeared to be normal in this group.

Child, Preschool↗
PubMed1988

Tissue destruction resulting from the interaction of cytotoxic T cells and their targets.

In vitro and in vivo-generated cytotoxic T lymphocytes (CTL) specific for major and minor histocompatibility antigens evoked antigen-specific full-thickness skin necrosis when injected intradermally into allogeneic mice in a variety of strain combinations. In addition, CTL-target-cell mixtures injected intradermally into hosts syngeneic to the CTL also evoked destruction of host tissue. These "innocent bystander" reactions were evoked with alloreactive CTL as well as with CTL directed against hapten (TNP)-modified and virus (influenza A)-infected target cells. Unlike the direct reactions, the bystander reactions in histocompatibility-antigen systems occurred in spite of H-2 incompatibility of the CTL, admixed target cells, and the hosts. One explanation for these results, currently under investigation, is that some bystander reactions may occur without MHC restriction. In aggregate, our findings indicate that nonspecific as well as antigen-specific reactions initiated by CTL-target-cell interactions may contribute to tissue destruction in allograft rejection, in severe forms of delayed-type hypersensitivity, and in certain viral infections.

Animals↗
PubMed1987

Dietary fat in relation to hormonal induction of mammary and prostatic carcinoma in Nb rats.

Mammary carcinoma was induced in female Nb rats by implanting pellets of estrone at 25-28 days of age, and prostatic cancer was induced in male rats of the same strain by repeatedly implanting pellets of testosterone propionate. Feeding semipurified diets containing either 3% or 20% corn oil did not significantly affect the incidence or yield of mammary tumors, although tumors tended to appear somewhat earlier in animals on the high-fat diet, confirming the report of Dunning et al. published nearly 40 years ago. The yield of prostatic tumors was too low to draw any conclusions with respect to dietary fat. Hormonally induced mammary tumors appear to be less responsive to dietary fat than those induced by commonly used chemical carcinogens.

Animals↗
PubMed1987

Altered T-cell subsets and defective T-cell function in young children with Down syndrome (trisomy-21).

Children with Down syndrome (DS) suffer an increased incidence of severe viral and bacterial infections particularly during the first 5 years of life. Unfortunately, few studies have been performed on the immune systems of young children with Down syndrome. Peripheral blood mononuclear cells (PBMC) from a group of non-institutionalized children less than 6 years of age were studied and compared with PBMC from age-matched controls. The children with DS had reduced numbers of circulating OKT4+ (helper/inducer) T cells and a significantly depressed ratio of OKT4+ to OKT8+ (suppressor/cytotoxic) T cells. PBMC from the DS subjects exhibited reduced proliferative responses to phytohemagglutinin and to an optimal concentration of concanavalin A (Con A), but normal responses to suboptimal doses of Con A and pokeweek mitogen. PBMC from young children with DS appeared to produce normal levels of interleukin-2 (IL-2). These findings provide evidence that the primary immune defect in DS is in part a depressed number and function of helper T cells. They also indicate that IL-2 production may not be defective in DS, but rather that the mechanism for response to IL-2 may be faulty.

Child, Preschool↗
PubMed1986

Cultured Nb rat lymphoma cells in endocrine and cancer research.

This review outlines the establishment, properties, and use of two lines of cultured Nb rat lymphoma cells. The cultured cells have retained important properties of the cancers of origin, such as dependency on prolactin for growth and a high sensitivity to antineoplastic Vinca alkaloids. The cultures have been useful for defining the hormonal dependency of the lymphomas in the animal and for studying the progression of the lymphomas from hormonal dependency to autonomy. A new, specific and highly sensitive in vitro bioassay for lactogenic hormones has been developed from one of the cultures. The use of the lymphoma cell cultures has revealed unsuspected pharmacological differences between the closely related, clinically useful antineoplastic Vinca alkaloids, vinblastine and vincristine. The lymphoma cell cultures are also useful tools for studying biochemical, cell cycle related events which follow the mitogenic stimulation of cells by a polypeptide growth factor.

Animals↗
PubMed1985

Rat hepatic cytosolic phosphoenolpyruvate carboxykinase (GTP). Structures of the protein, messenger RNA, and gene.

The primary structure of the messenger RNA coding for cytosolic phosphoenolpyruvate carboxykinase was determined by sequencing cDNA and genomic DNA and by primer extension of the mRNA. The molecule is 2624 nucleotides in length; this includes 143 nontranslated nucleotides at the 5' end and 615 nontranslated nucleotides at the 3' end. The 3' nontranslated sequence contains a 102-base pair region of alternating purine-pyrimidine nucleotides (the majority of which are UpG dinucleotides), several direct repeats and palindromic sequences, and 8 CpG dinucleotides. The corresponding segment of the phosphoenolpyruvate carboxykinase gene thus has characteristics which favor the formation of Z-DNA. The amino acid sequence of phosphoenolpyruvate carboxykinase was deduced from the mRNA sequence and confirmed by fast atom bombardment mass spectrometric analysis of peptides generated with trypsin and Staphylococcus aureus V8 protease. The protein consists of 621 amino acids and has a molecular weight of 69,289. Charon 4A lambda bacteriophage clones containing genomic DNA coding for phosphoenolpyruvate carboxykinase were isolated from a library of partial HaeIII digests of rat liver DNA. Two clones, lambda PC112 and lambda PC103, contained the entire coding region in 15-kilobase inserts and were used to subclone the gene into pBR322 as EcoRI, BamHI, or SstI-KpnI fragments. Using these subclones, the structure of the phosphoenolpyruvate carboxykinase gene was determined by S1 nuclease mapping, R-loop analysis, and DNA sequencing. The gene is composed of 10 exons and 9 introns with a total length of 6.0 kilobases. The transcription initiation site of the gene was determined by a combination of in vitro transcription in a HeLa cell lysate system, primer extension of mRNAPEPCK, and S1 nuclease mapping. In vitro transcription of purified DNA templates revealed three RNA polymerase II-dependent start sites. Two sites were separated by 600 base pairs on the coding strand and the third site was on the noncoding strand. The products of S1 nuclease mapping and primer extension from a BglII site were compared in order to determine which of the coding strand initiation sites was expressed in vivo. In both cases a 69-base pair fragment was generated and the 5' end of this corresponded to a thymidine residue identified in a sequence ladder of the genomic DNA coding strand. We conclude that mRNAPEPCK synthesis initiates with an adenine residue 69 base pairs 5' of the BglII site; this corresponds to the 3' most transcription initiation site determined in vitro.

Amino Acid Sequence↗
PubMed1984

Vinblastine and vincristine--growth-inhibitory effects correlate with their retention by cultured Nb 2 node lymphoma cells.

Vinblastine (VLB) and vincristine (VCR) have been compared with respect to their inhibitory effects on the growth of cultured Nb2 node rat lymphoma cells and their binding and release by these cells. When examined over a wide concentration range, the two alkaloids were found to be almost equally effective in inhibiting culture growth, providing they were continuously present in the incubation medium. A striking difference was found, however, when the cells were incubated for short periods with the alkaloids and then restored to alkaloid-free medium for further incubation. Cells treated with VCR (5 X 10(-9) g/ml) for 3 h and washed free of extracellular alkaloid failed to resume normal proliferation during a subsequent 48 h incubation period in alkaloid-free medium. In contrast, cells treated with VLB at a higher concentration (5 X 10(-8) g/ml) and for a longer time (7 h), rapidly resumed growth in alkaloid-free medium. Binding studies, using (3H)VLB and (3H)VCR, showed that when cells were incubated continuously with the alkaloids at a given concentration, they bound equal amounts of the drugs. When labelled cells were resuspended in alkaloid-free medium, VLB was released very readily by the cells, whereas VCR was tenaciously retained. The findings indicate that the growth-inhibitory effects of VLB and VCR are related to the amount of alkaloid associated with the cells and that the long-lasting effects of VCR, relative to VLB, are very likely due to the retention of VCR by the cells when the extracellular alkaloid levels decline. The study also suggests that, in the treatment of malignancies, VLB could be more effective if its extracellular levels were maintained, e.g. by continuous administration.

Animals↗
PubMed1984

Effects of folic acid malnutrition on rotaviral infection in mice.

A study was undertaken to determine if dietary deficiencies of folic acid would influence rotaviral diarrheal disease in infant mice. Female mice were fed diets containing essentially no folic acid, 25% of a normal quantity of folic acid, or a normally recommended quantity of folic acid, beginning at time of breeding and continuing through periods of gestation and lactation. Two-day-old infants from these dams were exposed to purified murine rotavirus or to sterile virus diluent and the severity of the rotaviral infection monitored. Infants from the low folic acid group had significantly lower folate levels in their livers, indicating a deficiency was achieved, and developed more severe disease manifestations than those infants from the dams receiving the normal folic acid levels in their diet. The infection enhancement was seen as increased incidences of diarrhea and a significantly greater number of mice exhibiting high intestinal rotaviral antigen titers. Serum rotavirus antibody titers were below detectable levels in a significant number of these same infants.

Animals↗