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Biomedical subjects

L M Rose

Publications and source records attributed to L M Rose.

At least 37 records · Page 2Linked to original sources
PubMed1994

Bcl-2 and Fas, molecules which influence apoptosis. A possible role in systemic lupus erythematosus?

Polyclonal B cell activation and the production of antibodies against a variety of autoantigens are features of systemic lupus erythematosus (SLE). Autoreactive B cells are found in healthy individuals but their numbers are probably regulated by cell death, after a few days, in the absence of proliferative stimuli. The process which achieves this regulation is known as apoptosis or programmed cell death. It has been postulated that in SLE patients dysfunction of apoptosis could result in the inappropriate longevity of autoreactive B cells, allowing autoantibody levels to reach pathogenic thresholds. This hypothesis has arisen as a result of studies revealing links between autoimmunity and two molecules which influence apoptosis. These are bcl-2 which enhances cell survival by inhibiting or delaying apoptosis and Fas, a cell surface molecule involved in the induction of apoptosis. Transgenic mice over expressing bcl-2 in their B cells showed polyclonal B cell expansion and their B cells showed extended survival in vitro. After a few months these mice developed an autoimmune syndrome resembling SLE. Mice that carry the lpr disorder have defects in the Fas gene. These mice, which do not express functional Fas molecules, suffer from an SLE-like autoimmune syndrome. Thus inappropriate expression of both bcl-2 and Fas can result in SLE-like autoimmune disease in mice. Research is now in progress to ascertain whether quantitative or functional abnormalities in these molecules exist in human SLE patients and contribute to the pathogenesis of the disease in some or all cases.

Animals↗
PubMed1993

Diffusion imaging of experimental allergic encephalomyelitis.

Diffusion-weighted magnetic resonance imaging (MRI) was compared with T2-weighted MRI in longitudinal studies of experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis, in five monkeys (Macaca fascicularis). In a region of the brain that had highly directional myelinated fibers (internal capsule) sequential changes were identified on diffusion-weighted images on and before the day these changes were detected on conventional T2-weighted images. Changes were also identified on diffusion-weighted images in brain areas that did not develop T2-weighted abnormalities. This result suggests that diffusion-weighted image intensities are sensitive to pathologic conditions of the brain that can not be seen on T2-weighted images.

Animals↗
PubMed1993

Metabolism of carbovir, a potent inhibitor of human immunodeficiency virus type 1, and its effects on cellular metabolism.

Carbovir (CBV) [the (--)-enantiomer of the carbocyclic analog of 2',3'-dideoxy-2',3'-didehydroguanosine] is a potent inhibitor of human immunodeficiency virus type 1 (HIV) replication in vitro. We have characterized the metabolism of CBV and its effect on cellular metabolism in an effort to better understand its mechanism of action. CBV was primarily metabolized to the 5'-triphosphate of CBV (CBV-TP) to concentrations sufficient to inhibit HIV reverse transcriptase. Infection of CEM cells with HIV did not affect the metabolism of CBV. In CEM cells, there was no evidence of the degradation of CBV by purine nucleoside phosphorylase. The half-life of CBV-TP in CEM cells was 2.5 h, similar to that of the 5'-triphosphate of zidovudine (AZT). However, unlike the levels of the 5'-triphosphate of AZT, CBV-TP levels declined without evidence of a plateau. CBV did not affect the metabolism of AZT, and AZT did not affect the metabolism of CBV. A small amount of CBV was incorporated into DNA in intact CEM cells, and this incorporation was increased by incubation with mycophenolic acid, an inhibitor of IMP dehydrogenase. CBV specifically inhibited the incorporation of nucleic acid precursors into DNA but had no effect on the incorporation of radiolabeled precursors into RNA or protein. CBV did not decrease the level of TTP, dGTP, dCTP, or dATP. These results suggested that the cytotoxicity of CBV was due to the inhibition of DNA synthesis. Further studies are necessary to identify the target(s) responsible for growth inhibition.

Antiviral Agents↗
PubMed1993

Phosphorylation of the enantiomers of the carbocyclic analog of 2'-deoxyguanosine in cells infected with herpes simplex virus type 1 and in uninfected cells. Lack of enantiomeric selectivity with the viral thymidine kinase.

CdG, the carbocyclic analog of 2'-deoxyguanosine, is active against herpes, hepatitis B, and human cytomegaloviruses. We have studied the interaction of the tritiated enantiomers of CdG with the herpes simplex virus type 1-specific thymidine kinase (HSV-1 TK) and have examined their metabolism in uninfected and HSV-1-infected cells. D- and L-CdG were equally effective competitive inhibitors of the phosphorylation of thymidine (dThd) by the partially purified HSV-1 TK (Ki values were 2.1 and 3.4 microM, respectively) and were also equal as substrates (Km values were 17 and 26 microM, respectively, and Vmax values of the enantiomers were equal and about 50% greater than the Vmax for dThd). The partially purified enzyme preparation, which contained cellular nucleotide kinase activities (pyruvate kinase also was present in the assay medium), converted D-CdG almost exclusively to the triphosphate and L-CdG almost exclusively to the monophosphate. Similarly, in virus-infected cells the D-enantiomer was converted predominantly to the triphosphate and the L-enantiomer predominantly to the monophosphate. In uninfected cells the results were qualitatively similar. In CEM cells deoxycytidine (dCyd) kinase (EC 2.7.1.74) seemed to be the enzyme principally responsible for the phosphorylation of both enantiomers, as shown by competition studies. Thus, both the HSV-1 TK and cellular dCyd kinase (of CEM cells) showed no selectivity for the enantiomers of CdG. This lack of enantiomeric specificity has obvious implications for the design of inhibitors of both viral proliferation and cellular metabolism.

Antiviral Agents↗
PubMed1993

Purine nucleoside phosphorylase inhibitors: biochemical and pharmacological studies with 9-benzyl-9-deazaguanine and related compounds.

Certain derivatives of 9-deazaguanine that contain arylmethyl, heteroarylmethyl or cycloalkylmethyl groups at the 9-position are potent inhibitors of purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1). To determine whether these agents can produce metabolically significant inhibition of PNP in cells and in animals, the authors performed pharmacological studies with a representative member of the series, 9-benzyl-9-deazaguanine (BzDAG). BzDAG was a potent inhibitor of PNP from calf spleen (Ki = 12 nM). It was also an effective inhibitor of PNP in cells and in animals as shown by the findings that it 1) inhibited the conversion of inosine to nucleotides in L1210 cells in culture at concentrations that had little effect on the utilization of hypoxanthine; 2) potentiated the toxicity of deoxyguanosine to CCRF-CEM cells in culture; 3) increased the pools of deoxy GTP in CCRF-CEM, Molt-3 and Molt-4 cells that had been treated with deoxyguanosine; 4) prevented the toxicity of 6-thioguanosine to HEp-2 cells in culture; 5) increased the plasma levels of endogenous inosine in rats; and 6) increased the plasma levels of 2',3'-dideoxyinosine in rats that had received BzDAG and dideoxyinosine in combination. Pharmacokinetic analysis of BzDAG in the rat showed it to be 48% orally bioavailable (at a dose of 5 mg/kg). About 95% of BzDAG was protein bound. After i.v. administration of BzDAG (5 mg/kg), more than 50% of the erythrocyte PNP was inhibited for 40 min. These results indicate that the 9-substituted-9-deazaguanines are potent orally active PNP inhibitors and are therefore of potential clinical interest as immunosuppressive and anti-inflammatory agents.

Animals↗
PubMed1992

Determination of absolute phosphate metabolite concentrations in RIF-1 tumors in vivo by 31P-1H-2H NMR spectroscopy using water as an internal intensity reference.

The absolute metabolite quantification method of Thulborn and Ackerman [J. Magn. Reson. 55, 357 (1983)] in which the tissue water proton signal is used as an internal intensity standard and its more recent variation in which NMR peak intensities are referenced to that of the natural abundance deuterium signal of water [Li et al., SMRM Abstr. 2, 825 (1988); Song et al., Magn. Reson. Med. 25, 45 (1992) have been implemented to obtain absolute phosphate metabolite concentrations in subcutaneous RIF-1 tumors during untreated growth and following treatment with 5-fluorouracil. The equivalence of these two hydrogen isotopes as intensity standards and the validity of their use in the determination of absolute metabolite concentrations in vivo by NMR has been demonstrated. On matched in vivo and extract tumor samples (n = 5), excellent agreement has been obtained between nucleoside triphosphate concentrations determined by NMR and those derived by HPLC analysis for the control tumors. Following 3 days of untreated growth, absolute concentrations of phosphate metabolites in RIF-1 tumors (n = 10) decreased significantly, except for the Pi concentration which did not vary. For the treated tumors (n = 10) there were no changes in metabolite concentrations except for a decrease in the PCr and, possibly, Pi concentrations. The PCr/Pi ratio in the latter tumors did not change. These observations suggest that changes in absolute metabolite concentrations may be more sensitive indices of response to therapy than changes in metabolite peak amplitude ratios, a parameter commonly used to express in vivo NMR data.

Animals↗
PubMed1992

Cross-reactive maternal autoantibodies and congenital heart block.

Epitopes with linear sequences recognized by anti-La autoantibodies from seven mothers of children with congenital heart block were recently defined. Eight of these epitopes share sequence identity with three other proteins in addition to the original autoantigen, La. The three proteins are human cardiac myosin beta heavy chain, laminin B1 chain and the M6 protein of Streptococcus pyogenes. Affinity purified anti-La antibodies from a further three mothers bound to the La antigen and also to human cardiac myosin and mouse laminin. Affinity purified antibodies from three mothers of healthy children bound to the La antigen but showed minimal binding to either human cardiac myosin or mouse laminin. Cardiac myosin inhibited the binding of CHB-related anti-La antibodies to both La and myosin. These data support a role for maternal autoantibodies crossing the placenta and recognizing foetal cardiac antigens accessible at a critical developmental stage during gestation. We suggest that this would lead to complement fixation, inflammation and the subsequent pathology associated with congenital heart block.

Amino Acid Sequence↗
PubMed1992

Incorporation of the carbocyclic analog of 2'-deoxyguanosine into the DNA of herpes simplex virus and of HEp-2 cells infected with herpes simplex virus.

The carbocyclic analog of 2'-deoxyguanosine (CdG) is active against herpes simplex virus (HSV), human cytomegalovirus, and human hepatitis-B virus. In order to understand the mechanism of action of this compound against HSV, we have evaluated (a) the incorporation of [3H]CdG into viral and host DNA in HEp-2 cells infected with HSV and (b) the interaction of the 5'-triphosphate of CdG (CdG-TP) with the HSV DNA polymerase and human DNA polymerases alpha, beta, and gamma (EC 2.7.7.7). Incubation of HSV-1-infected HEp-2 cells with [3H]CdG resulted in the incorporation of CdG into both the HSV and the host cell DNA. These results indicated that CdG-TP was used as a substrate for HSV DNA polymerase and for at least one of the cellular DNA polymerases. Degradation of both viral and host DNA with micrococcal nuclease and spleen phosphodiesterase indicated that CdG was incorporated primarily into internal positions in both DNAs. The viral DNA containing CdG sedimented in neutral and alkaline sucrose gradients in the same way as did viral DNA labeled with [3H]thymidine, indicating that the HSV DNA containing CdG was similar in size to untreated HSV DNA. CdG-TP was a competitive inhibitor of the incorporation of dGTP into DNA by the HSV DNA polymerase (Ki of 0.35 microM) and the human DNA polymerase alpha (Ki of 1 microM). CdG-TP was not a potent inhibitor of either DNA polymerase beta or gamma. Using DNA-sequencing technology, CdG-TP was found to be an efficient substrate for HSV DNA polymerase. Incorporation of CdG monophosphate (CdG-MP) into the DNA by HSV DNA polymerase did not interfere with subsequent chain extension. These results suggested that the antiviral activity of CdG was due to its incorporation into the DNA and subsequent disruption of viral functions. In contrast, CdG-TP was not as good as dGTP as a substrate for DNA synthesis by DNA polymerase alpha, and incorporation of CdG-MP by DNA polymerase alpha inhibited further DNA chain elongation.

Base Sequence↗
PubMed1991

Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5'-triphosphate.

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5'-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗
PubMed1991

Identification of 7-(2-hydroxyethyl)guanine as a product of alkylation of calf thymus DNA with clomesone.

Evidence at the molecular level is presented in support of alkylation of O6-guanine moieties of DNA as the mechanism of cytotoxicity of Clomesone to HT-29 cells and consists in the isolation and identification of a product resulting from alkylation of calf thymus DNA with Clomesone, followed by depurination to yield 7-(2-hydroxyethyl)guanine, whose formation is reasonably explained by O6-guanine chloroethylation followed by intramolecular alkylation at N7 of guanine and subsequent hydrolysis to the hydroxyethylguanine.

Alkylation↗
PubMed1990

Phosphorylation of the carbocyclic analog of 2'-deoxyguanosine in cells infected with herpes viruses.

The carbocyclic analog of 2'-deoxyguanosine [(+-)-2-amino-1,9-dihydro-9-[(1 alpha,3 beta,4 alpha)-3-hydroxy-4-(hydroxymethyl)cyclopentyl]-6H-purine-6-one] (2'-CDG) is highly active in cell culture against strains S148 and E377 of herpes simplex virus type 1 (HSV-1), both of which code for thymidine kinase, and much less active against strain BW10168 which is deficient in this enzyme activity. Antiviral activity is associated primarily with the D-enantiomer; the L-enantiomer has much lower but significant activity. The metabolism of racemic 2'-CDG and its D- and L-enantiomers was studied in uninfected HEp-2 cells and in HEp-2 cells infected with the S148 or BW10168 strains of HSV-1. Nucleotides were separated by HPLC, and their elution was monitored by spectrophotometry. The chromatograms of extracts of cells infected with the S148 strain and treated with (+/-)-2'-CDG or D-2'-CDG included a new peak which appeared in the triphosphate region. This peak, the area of which exceeded that of the GTP peak, was shown to be due to the triphosphate of 2'-CDG. The new peak was not observed by HPLC of extracts of uninfected cells treated with (+/-)-2'-CDG or either of its enantiomers, cells infected with the S148 strain and treated with L-2'-CDG, or cells infected with the BW10168 strain and treated with (+/-)-2'-CDG or either of its enantiomers. The results were similar when these studies were performed with uninfected Vero cells and with Vero cells infected with strain S148 of HSV-1. In experiments with D-[8-3H]-2'-CDG, small amounts of phosphates of 2'-CDG could also be detected in uninfected HEp-2 cells and in cells infected with the BW10168 strain of HSV-1. Thus, 2'-CDG apparently is a good substrate for the virus-coded kinase and a very poor substrate for cellular phosphorylating enzymes. The selective phosphorylation of 2'-CDG by the virus-specific kinase presumably is critical for its antiviral activity as it is for that of acyclovir and other acyclic derivatives of guanine.

Animals↗
PubMed1990

Zidovudine in combination with alpha interferon and interleukin-2 as prophylactic therapy for FeLV-induced immunodeficiency syndrome (FeLV-FAIDS).

Zidovudine (3'-azido-3'-deoxythymidine; AZT) inhibited replication of an immunodeficiency-inducing strain of feline leukemia virus (FeLV-FAIDS) in vitro at concentrations of 0.5-0.005 micrograms/ml. A 25-30% additional antiviral effect was achieved in vitro when AZT was combined with human recombinant alpha interferon 2a (IFN alpha). Oral administration of AZT (20 mg/kg three times daily) to cats resulted in plasma concentrations of 3 micrograms/ml at 2 h post-administration with a T1/2 of approximately 1.60 h. Administration of AZT alone or in combination with IFN alpha or interleukin-2 (IL-2) throughout a 6-week treatment period enabled cats to resist challenge with FeLV-FAIDS. In contrast, those cats treated with IFN alpha or IL-2 alone became persistently antigenemic (core protein p27) in parallel with placebo-treated controls. Antigenemia remained undetectable in AZT-treated cats throughout an 80-day period post-inoculation (38 days after treatment was withdrawn). However, latent FeLV-FAIDS in bone marrow was detectable by in vitro culture of progenitor cells in the presence of hydrocortisone. Serial analysis of circulating p27 antigen, neutralizing antibody, and quantification of latent, reactivatable virus indicated that those animals receiving AZT in combination with IFN alpha were most able to resist FeLV-FAIDS challenge. This work provides additional evidence that early presymptomatic treatment employing combination chemoimmunotherapy can be effective in medical intervention of retroviral infection.

Acquired Immunodeficiency Syndrome↗
PubMed1989

T-cell alterations in late postpoliomyelitis.

The immunologic status of patients with prior poliomyelitis was studied using two-color flow cytometric analyses. Ten lymphocyte subsets including subsets of CD4+ T cells, CD8+ T cells, B cells, and natural killer cells were examined. Eighteen patients presented with clinical symptoms compatible with the postpolio syndrome. This group was compared with 18 asymptomatic postpolio survivors and 22 age-matched healthy controls. The results demonstrated significant alterations in CD4+ subsets in the postpolio group as a whole when compared with normal controls. These findings reveal definite alterations in the immune status of postpolio survivors and raise the possibility that immunologic factors may contribute to late disease progression.

Adult↗
PubMed1989

Magnetic resonance imaging and peripheral blood abnormalities in experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) was induced in twelve cynomologous macaques (Macaca fascicularis) by sensitization to autologous myelin basic protein (BP) in complete Freund's adjuvant (CFA). The white blood cell (WBC) count, absolute number of lymphocytes and absolute numbers of CD4+ and CD8+ T-cell subsets were measured weekly. Using magnetic resonance imaging (MRI), the animals were monitored twice weekly for the development of central nervous system (CNS) lesions. Conventional spin-warp imaging was performed using a General Electric CSI-II NMR imager/spectrometer (2 Tesla magnet). CNS lesions were detected by MRI in all of the animals sensitized to myelin BP. Longitudinal analysis of their peripheral blood leukocytes revealed a progressive leukocytosis and lymphopenia, which always preceded the onset of clinical signs and almost always also preceded the formation of detectable CNS lesions. These results suggest that frequent analysis of T-cell subsets may provide a more accurate means of predicting episodes of disease activity than clinical or MRI evaluation.

Animals↗
PubMed1989

Autoantibody synthesis in salivary glands of Sjögren's syndrome patients.

We investigated the possibility that autoantibodies are locally synthesized and secreted within salivary glands of patients with Sjögren's syndrome by measuring specific autoantibody as a proportion of the total immunoglobulin present both in serum and saliva. Of 25 patients studied, 21 showed salivary enrichment of IgA anti-La, in three cases IgA anti-La being detected in saliva when IgG anti-La was negative (by ELISA) in serum. Twenty-four showed enrichment of salivary rheumatoid factors and IgA and/or IgM carrying the 17-109 idiotype, a marker of kappa IIIb light chains. These data suggest that autoantibodies, especially of the IgA class, are synthesized primarily in salivary gland and that they can be detected in saliva before they become apparent in the peripheral circulation. The subsequent deposition of these antibodies within salivary glands may be a contributory factor to the pathogenesis of Sjögren's syndrome.

Antibodies↗
PubMed1988

Alterations in nucleotide pools induced by 3-deazaadenosine and related compounds. Role of adenylate deaminase.

3-Deazaadenine, 3-deazaadenosine, and the carbocyclic analog of 3-deazaadenosine produced similar effects on nucleotide pools of L1210 cells in culture: each caused an increase in IMP and a decrease in adenine nucleotides and had no effect on nucleotides of uracil and cytosine. Concentrations of 50-100 microM were required to produce these effects. Although 3-deazaadenosine and carbocyclic 3-deazaadenosine are known to be potent inhibitors of adenosylhomocysteine hydrolase, the effects on nucleotide pools apparently are not mediated via this inhibition because they are also produced by the base, 3-deazaadenine, and because the concentrations required are higher than those required to inhibit the hydrolase. Cells grown in the presence of 3-deazaadenine or 3-deazaadenosine contained phosphates of 3-deazaadenosine (the mono- and triphosphates were isolated); from cells grown in the presence of the carbocyclic analog of 3-deazaadenosine, the monophosphate was isolated, but evidence for the presence of the triphosphate was not obtained. A cell-free supernatant fraction from L1210 cells supplemented with ATP catalyzed the formation of monophosphates from 3-deazaadenosine or carbocyclic 3-deazaadenosine, and a cell-free supernatant fraction supplemented with 5-phosphoribosyl 1-pyrophosphate (PRPP) catalyzed the formation of 3-deaza-AMP from 3-deazaadenine. Adenosine kinase apparently was not solely responsible for the phosphorylation of the nucleosides because a cell line that lacked this enzyme converted 3-deazaadenosine to phosphates. No evidence was obtained that the effects on nucleotide pools resulted from a block of the IMP-AMP conversion, but the results could be rationalized as a consequence of increased AMP deaminase activity. This explanation is supported by two observations: (a) coformycin, an inhibitor of AMP deaminase, prevented the effects on nucleotide pools, and (b) 3-deazaadenine decreased the conversion of carbocyclic adenosine to carbocyclic ATP and increased its conversion to carbocyclic GTP. The latter conversion requires the action of AMP deaminase and the observed effects can be rationalized by a nucleoside analog-mediated increase in AMP deaminase activity. Because these effects on nucleotide pools are produced only by concentrations higher than those required to inhibit adenosylhomocysteine hydrolase, they may not contribute significantly to the biological effects of 3-deazaadenosine or carbocyclic 3-deazaadenosine.(ABSTRACT TRUNCATED AT 400 WORDS)

AMP Deaminase↗