Search PubMed⌕ Search

Biomedical subjects

A Smith

Publications and source records attributed to A Smith.

At least 631 records · Page 35Linked to original sources
PubMed1996

A case for the use of aminopterin in treatment of patients with leukemia based on metabolic studies of blasts in vitro.

Clinical and laboratory investigations during the past four decades have resulted in numerous schedules, doses, and routes of delivery for methotrexate (MTX). It remains as an important drug for the treatment of children with acute lymphoblastic leukemia (ALL). Aminopterin (AMT) was the initial antifolate showing promise as an anticancer drug. It is more potent than MTX and also is known to be accumulated more efficiently than MTX in model systems. Because Whitehead et al. (Blood, 76: 44-49, 1990) have shown that MTX accumulation by blasts at diagnosis is of prognostic significance in children with ALL, we reasoned that if accumulation of a "stoichiometric inhibitor" of dihydrofolate reductase by leukemic blasts was of prognostic importance, then whether it was AMT or MTX may be relevant only with respect to the absolute dose. To compare MTX and AMT metabolism, we incubated lymphoblasts with 1 microM radiolabeled drug in vitro. MTX and AMT accumulation by ALL cells (n = 24) was 0.7 +/- 0.7 and 1.47 +/- 0.9 pmol/10(6) cells, respectively. Based on the data of Whitehead et al., this predicts pharmacological success in 59 and 84% of the MTX and AMT groups, respectively. Moreover, 5 of 10 patients considered poor risks based on MTX accumulation would be "cures" based on AMT uptake. Even at only 0.1 microM AMT, a concentration at which there is little accumulation of MTX, 5 of 11 patients studied would be "pharmacological cures" based on AMT uptake. Accumulation of AMT by blasts from 11 patients with T-cell-lineage ALL and 5 patients with acute myelogenous leukemia was also found to be twice the uptake of MTX. These data allow the suggestion that AMT, despite increased potential for toxicity, may be useful in children who are identified as poor risks with respect to MTX uptake.

Aminopterin↗
PubMed1996

Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in vivo tyrosine kinase inhibition.

We have performed a Phase I clinical trial with the naturally occurring flavonoid quercetin (3,3',4',5,7-pentahydroxyflavone). Quercetin has antiproliferative activity in vitro and is known to inhibit signal transduction targets including tyrosine kinases, protein kinase C, and phosphatidyl inositol-3 kinase. Quercetin was administered by short i.v. infusion at escalating doses initially at 3-week intervals. The first dose level was 60 mg/m2; at the 10th dose level of 1700 mg/m2, dose-limiting nephrotoxicity was encountered, but no myelosuppression. At the preceding dose level of 1400 mg/m2, five patients were treated at 3-week intervals, and another eight patients were treated on a once-weekly schedule; overall, 2 of 10 evaluable patients had renal toxicity, 1 at grade 2 and 1 at grade 4. We therefore treated other patients at 945 mg/m2 (eight at 3-week intervals and six at weekly intervals); 3 of 14 patients had clinically significant renal toxicity, 2 patients with grade 2 and 1 patient with grade 3. Patients treated on the weekly schedule did not have cumulative renal impairment but did have a fall in the glomerular filtration rate of 19 +/- 8% in the 24 h after drug administration. We recommend 1400 mg/m2 as the bolus dose, which may be given either in 3-week or weekly intervals, for Phase II trials. Quercetin pharmacokinetics were described by a first-order two-compartment model with a median t(1/2)alpha of 6 min and median t(1/2)beta of 43 min. The median estimated clearance was 0.28 liter/min/m2, and median volume of distribution at steady state was 3.7 liter/m2. In 9 of 11 patients, lymphocyte protein tyrosine phosphorylation was inhibited following administration of quercetin at 1 h, which persisted to 16 h. In one patient with ovarian cancer refractory to cisplatin, following two courses of quercetin (420 mg/m2), the CA 125 had fallen from 295 to 55 units/ml, and in another patient with hepatoma, the serum alpha-fetoprotein fell. In conclusion, quercetin can be safely administered by i.v. bolus at a dose injection. The plasma levels achieved inhibited lymphocyte tyrosine kinase activity, and evidence of antitumor activity was seen.

Adult↗
PubMed1995

Essential function of LIF receptor in motor neurons.

Development and maintenance of the mammalian nervous system is dependent upon neurotrophic cytokines. One class of neurotrophic factor acts through receptor complexes involving the low-affinity leukaemia inhibitory factor receptor subunit (LIF-R). Members of this family of cytokines, such as ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF), have profound effects on the survival and maintenance of motor neurons. Recently it was reported that mice lacking LIF-R die shortly after birth unlike mice lacking CNTF or LIF which are viable. Here we describe histopathological analyses of lifr mutants that reveal a loss > 35% of facial motor neurons, 40% of spinal motor neurons and 50% of neurons in the nucleus ambiguus. These findings point to the existence of a ligand for LIF-R that is required for the normal development of motor neurons in both brainstem nuclei and spinal cord.

Animals↗
PubMed1995

MCD, EPR and NMR spectroscopic studies of rabbit hemopexin and its heme binding domain.

Heme binding to rabbit hemopexin and its domain I, obtained by proteolytic cleavage of intact hemopexin, was studied by EPR, MCD and 1H-NMR spectroscopies. The data obtained support the proposal that the heme Fe(III) is coordinated by two histidine ligands (Morgan et al. (1988) J. Biol. Chem. 263, 8220-8225; Muster et al. (1991) J. Protein Chem. 10, 123-128) and are inconsistent with recently reported mutagenesis studies indicating that bis-histidine ligation is unlikely (Satoh et al. (1994) Proc. Natl. Acad. Sci. USA 91, 8423-8427). Although the MCD data are consistent with both bis-histidine and histidine/lysine ligation, the EPR spectra are typical of bis-histidine ligation. Overall the magneto-optical spectra are characteristic for bis-histidine ligation. The EPR and NMR data indicate that there is a difference in the heme environments of the intact hemopexin and its domain I but overall the spectroscopic information suggests heme bound to domain I has the same ligands as intact hemopexin. The 1H-NMR studies indicate that heme binding to domain I perturbs at least 4 of the 5 histidines. This is consistent with axial ligation of the heme by two histidines, and a conformational change induced by heme binding affecting two more. Interestingly, resonances of the carbohydrate bound to intact hemopexin and domain I were also perturbed by heme binding. pH dependence studies showed that heme remained bound to intact hemopexin over the pH range 6.5-10.0 without any major change in the ligation or environment of the heme.

Amino Acid Sequence↗
PubMed1995

Mechanism of metallothionein gene regulation by heme-hemopexin. Roles of protein kinase C, reactive oxygen species, and cis-acting elements.

Heme-hemopexin or cobalt protoporphyrin (CoPP)-hemopexin (a model ligand for hemopexin receptor occupancy) is shown to increase transcription of the metallothionein-1 (MT-1) gene by activation of a signaling pathway. Promoter deletion analysis followed by transient transfection assays show that 110 base pairs (-153 to -43) of 5'-flanking region of the murine MT-1 promoter are sufficient for increasing transcription in response to heme-hemopexin or to CoPP-hemopexin in mouse hepatoma cells. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), prevented the increase in MT-1 transcription by heme-hemopexin, CoPP-hemopexin, or phorbol 12-myristate 13-acetate, but the protein kinase A inhibitor, HA1004, was without effect. N-Acetylcysteine (NAC) and glutathione, as well as superoxide dismutase and catalase, inhibited both the increase in endogenous MT-1 mRNA and the activation of reporter gene activity by heme-hemopexin, CoPP-hemopexin, and phorbol 12-myristate 13-acetate. In sum, these data suggest that reactive oxygen intermediates are generated by heme-hemopexin via events associated with receptor binding, including protein kinase C activation. Induction of heme oxygenase-1 expression, in contrast to MT-1, is significantly less sensitive to NAC. Deletion and mutation analyses of the MT-1 proximal promoter revealed that the sequence 5'-GTGACTATGC-3' (from -98 to -89 base pairs) is, in part, responsible for the hemopexin-mediated regulation of MT-1 which is inhibited by H7. Regulation via this element is also induced by H2O2 showing that it is an antioxidant response element. Heme itself acts via more distal elements on the MT-1 promoter. In contrast to NAC and glutathione, diethyl dithiocarbamate and pyrrolidine dithiocarbamate, which inactivate reactive oxygen intermediates and chelate Zn(II), synergistically augment the induction of MT-1 mRNA levels and reporter gene activity in response to heme-hemopexin via the antioxidant response element by both metal-responsive element-dependent and -independent mechanisms.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes.

MHC class I antigen expression is necessary for CD8+ T-cell-mediated recognition of tumors. Recently, several mechanisms leading to loss or decreased expression of MHC antigens on the tumor cell surface have been described that may account for tumor escape from immune recognition. It is yet unknown whether tumor recognition by CTL occurs at a threshold amount of MHC molecules or correlates with the level of HLA-allele expression. In this study, a model was developed in which clones derived from the 624-MEL melanoma cell line and expressing varying amounts of HLA-A2 molecules were lysed in a standard 51Cr release assay by an HLA-A2-restricted CTL clone (A42) or a bulk culture of tumor-infiltrating lymphocytes. The A42 clone and the tumor-infiltrating lymphocyte culture were characterized previously as specifically recognizing the melanoma antigen MART-1(27-35) peptide. A marked heterogeneity in the susceptibility to lysis by A42 was observed in tumor clones and was not due to heterogeneous expression of MART-1 by the clones or loss of accessory molecules involved in the lymphocyte-target interaction. Lysis by A42 and by the tumor-infiltrating lymphocyte culture significantly correlated with the level of HLA-A2 expression, evaluated as mean channel number of fluorescence by flow cytometry (P < 0.001). Transfection of an HLA-A2-negative clone (624.28) with the HLA-A2.1 gene produced a panel of clones expressing different levels of HLA-A2, the lysis of which was highly correlated with the expression of HLA-A2 (P < 0.001). The addition of exogenous MART-1(27-35) peptide enhanced lysis of clones expressing intermediate amounts of HLA-A2 but did not affect clones with high expression. These data suggest that the number of HLA molecules present on the surface of tumor cells can quantitatively affect their lysis by CTL in situations with borderline amounts of peptide and/or MHC.

Alleles↗
PubMed1995

1.8 A crystal structure of the C-terminal domain of rabbit serum haemopexin.

BACKGROUND: Haemopexin is a serum glycoprotein that binds haem reversibly and delivers it to the liver where it is taken up by receptor-mediated endocytosis. Haemopexin has two homologous domains, each having a characteristic fourfold internal sequence repeat. Haemopexin-type domains are also found in other proteins, including the serum adhesion protein vitronectin and various collagenases, in which they mediate protein-protein interactions. RESULTS: We have determined the crystal structure of the C-terminal domain of haemopexin at 1.8 A resolution. The domain is folded into four beta-leaflet modules, arranged in succession around a central pseudo-fourfold axis. A funnel-shaped tunnel through the centre of this disc-shaped domain serves as an ion-binding site. CONCLUSIONS: A model for haem binding by haemopexin is proposed, utilizing an anion-binding site at the wider end of the central tunnel, together with an associated cleft. This parallels the active-site location in other beta-propeller structures. The capacity to bind both cations and anions, together with the disc shape of the domain, suggests that such domains may be used widely for macromolecular recognition.

Amino Acid Sequence↗
PubMed1995

Altered immune responses in mice lacking inducible nitric oxide synthase.

Nitric oxide (NO) is important in many biological functions. It is generated from L-arginine by the enzyme NO synthase (NOS). The cytokine-inducible NOS (iNOS) is activated by several immunological stimuli, leading to the production of large quantities of NO which can be cytotoxic. To define the biological role of iNOS further, we generated iNOS mutant mice. These are viable, fertile and without evident histopathological abnormalities. However, in contrast to wild-type and heterozygous mice, which are highly resistant to the protozoa parasite Leishmania major infection, mutant mice are uniformly susceptible. The infected mutant mice developed a significantly stronger Th1 type of immune response than the wild-type or heterozygous mice. The mutant mice showed reduced nonspecific inflammatory response to carrageenin, and were resistant to lipopolysaccharide-induced mortality.

Amino Acid Oxidoreductases↗
PubMed1995

46,XY/47,XYY/48,XYYY karyotype in a 3-year-old boy ascertained because of radioulnar synostosis.

Chromosome analysis was performed on a 3-year-old boy because of bilateral radioulnar synostosis and demonstrated a mosaic karyotype 46,XY/47,XYY/48,XYYY. He had minor facial anomalies and mild intellectual delay. He appears to be the youngest patient reported with this rare chromosome complement. His father, mother, and brother had normal chromosomes. Fluorescence in situ hybridization (FISH) was performed on the propositus and his father with the Y chromosome heterochromatic probe (pHY3.4) to add to the evaluation of mosaicism.

Aneuploidy↗
PubMed1995

Analysis of randomly amplified flow-sorted chromosomes using the polymerase chain reaction.

Bivariate fluorescence-activated sorting is a method for obtaining relatively pure fractions of chromosomal DNA. Unfortunately, the yields (< 0.25 microgram/day) frequently limit the types of molecular analysis that can be performed. The polymerase chain reaction (PCR) is capable of amplifying unique sequences from scant amounts of template DNA. The purpose of this study was to determine whether the sensitivity of the PCR could be used to detect sequences specific to chromosomes discriminated and purified by flow cytometry. Flow-sorted chromosomal DNA was prepared by collecting approximately 10(5) chromosomes onto a nitrocellulose filter and eluting the DNA by boiling. Amplification products were not detected when different amounts of chromosomal DNA were used in a single 30 to 40-cycle PCR assay. However, when the eluted DNA was primed with degenerate 15-bp oligonucleotides and randomly amplified prior to performing the PCR assay, sequence-tagged sites (STSs) were detected after gel electrophoresis and ethidium bromide staining. This random amplification step eliminated the need for both reamplification with nested primers and detection by DNA hybridization. Furthermore, the random amplification scheme provided enough template DNA from a single sort (10(5) chromosomes) to perform > 1000 PCR assays. Representational analysis of one chromosome type revealed that > 74% of 70 STSs were detected. Moreover, the technology could be used to identify and delineate the breakpoint region of a marker chromosome. This amplification scheme should simplify greatly the molecular analysis of normal and aberrant chromosomes.

Base Sequence↗
PubMed1995

Interaction of histidine-proline-rich glycoprotein with plasminogen: effect of ligands, pH, ionic strength, and chemical modification.

The association of plasma histidine-proline-rich glycoprotein (HPRG) with plasminogen (PLG) was examined using a sucrose density gradient assay in order to evaluate the effects of several relevant conditions on complex formation. Addition of PLG shifts the S-value of 125I-labeled HPRG from 4.8S to 6.8S, providing the first direct evidence that HPRG associates with the zymogen form of plasmin in solution. Complex formation is not sensitive to pH in the range of pH 6.5-8.5, but is abolished at high ionic strength (1 M NaCl). No species differences were found, as either rabbit or human HPRG bound readily to rabbit or human PLG. Of the ligands of HPRG tested, mesoheme (20 microM) but not heparin (M(r) 10,000, 10 microM) inhibits the formation of the HPRG-PLG complex. Modification of lysine residues of HPRG or competitive binding by lysine and anti-fibrinolytic agents containing primary amino groups also inhibits association. Conversely, modification of arginine or histidine residues of HPRG has no effect on complex formation. These results indicate that HPRG has independent binding sites for heparin and PLG and confirm that one or more lysine residues of HPRG are involved in its recognition by PLG. The protein-protein interaction was also quantitatively characterized at thermodynamic equilibrium by analytical ultracentrifugation. The stoichiometry and dissociation constant (KD) of the complex were determined from the equilibrium distribution of fluorescein-isothiocyanate-labeled PLG in the presence of HPRG. The experimental data were analyzed by nonlinear least-squares curve fitting and indicated that a heterodimer is formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The proto-oncogene/translation factor eIF4E: a survey of its expression in breast carcinomas.

The eukaryotic translation initiation factor eIF-4E binds to the cap structure of mRNAs as one component of the eIF-4 translation initiation complex, which mediates the recruitment of mRNA to the ribosomes. Overexpression of eIF-4E can result in oncogenic transformation and uncontrolled growth of mammalian cells, presumably by facilitating the expression of growth-control gene products which are normally translationally repressed. Whereas the mechanism of eIF-4E-mediated transformation is being actively pursued, clinical investigations into the expression of eIF-4E in prevalent human cancers are lacking. We have recently initiated a screen of breast carcinomas by probing with eIF-4E antiserum. Using Western blots, we have analyzed the level of eIF-4E in 38 carcinomas, 7 normal samples and 3 fibroadenomas. We found that eIF-4E was elevated 3- to 10-fold in virtually all the carcinomas we analyzed, but not in fibroadenomas. This analysis was also confirmed by immunohistological staining in situ, showing that overexpression of eIF-4E can be readily identified at the single-cell level. Our results suggest that an elevation of eIF-4E may be an essential component in the development of breast cancer.

Adenoma↗