Biomedical subjects

S M Hanash

Publications and source records attributed to S M Hanash.

At least 91 records · Page 5Linked to original sources

PubMed1989

Identification of a cellular polypeptide that distinguishes between acute lymphoblastic leukemia in infants and in older children.

We analyzed the polypeptide pattern of leukemic cells of infants and older children with acute lymphoblastic leukemia (ALL), using two-dimensional polyacrylamide gel electrophoresis (PAGE). Patterns were analyzed for the occurrence of a previously detected cytosolic polypeptide, designated L3. Quantitative analysis of L3 in 12 infants and 91 older children with non-T ALL indicated lack of expression of polypeptide L3 in leukemic cells of infants which, in most cases, expressed HLA-DR and CD19 and lacked CD10. Quantitative analysis of L3 in relation to cell surface marker expression revealed that L3 was limited in its occurrence to non-T ALL and was not coordinately expressed with any of the surface markers included in the study. Among patients in the HLA-DR-positive, CD19-positive, and CD10-negative group, different levels of polypeptide L3 were observed between infants and older children. These results indicate differences in leukemic cell constituents between infants and older children with ALL and an otherwise similar cell surface marker phenotype.

Adolescent↗

PubMed1988

Identification of a polypeptide associated with the malignant phenotype in acute leukemia.

A novel polypeptide, designated p18, was detected in a variety of hematopoietic cells by two-dimensional polyacrylamide gel electrophoresis. Quantitative analysis of p18 indicated its occurrence in a substantially greater amount in acute leukemia relative to nonleukemic cells. The increased amount of p18 in leukemia could not be explained on the basis of specific lineage, differentiation stage, or cell proliferation and thus appears to be a part of the malignant phenotype of the leukemic cells.

Acute Disease↗

PubMed1988

Stroke associated with obstructive sleep apnea in a child with sickle cell anemia.

We describe a child with sickle cell anemia and multiple ischemic infarctions who was found to have severe obstructive sleep apnea and hypoxemia, secondary to adenotonsillar enlargement. The apnea-associated hypoxemia likely contributed to the development of the strokes in this child. Moreover, because stroke in patients with sickle cell anemia, and maximal tonsillar enlargement (the most common cause of obstructive apnea in children) both have peak incidence at the same age (6-7 years), obstructive sleep apnea may be an important factor in the development of stroke in other children with sickle cell anemia.

Adenoidectomy↗

PubMed1988

Approach to stationary two-dimensional pattern: influence of focusing time and immobiline/carrier ampholytes concentrations.

Horizontal two-dimensional (2-D) electrophoresis with immobilized pH gradients (IPG) in the first dimension for buffer soluble proteins and for complex proteins solubilized in the presence of Nonidet P-40 (Görg et al., Electrophoresis 1987, 8, 45-51), has been extended to analyze basic proteins of yeast cells focused under non-equilibrium and equilibrium conditions. Transient state isoelectric focusing (IEF) in IPG gels revealed sample smearing and background staining, displaying horizontal streaks in the resultant 2-D patterns. Inclusion of 0.5% carrier ampholytes (CA) to the IPG gel (IPG-CA), resulted in the formation of many sharp protein bands after transient state IEF with resultant distinct spots in the 2-D patterns; however, resolution was poor and the gel contained heavy background staining. With prolonged focusing time, background staining disappeared and there was less difference in the final steady state IEF patterns obtained with IPG and IPG-CA. Reduction of the Immobiline concentration to one third the manufacturer's recommended amount did not improve IEF resolution with respect to streaking and background staining under either transient state or equilibrium conditions. In general, spot intensities were less on 2-D gels using diluted IPG gels than with "standard" IPG gels. Optimization of 2-D electrophoresis with IPGs in the first dimension was strongly related to IEF conditions. The use of IPG gels focused to equilibrium should not only improve inter-gel reproducibility and resolution but also the quality of the final 2-D patterns with respect to background staining and horizontal streaking.

Ampholyte Mixtures↗

PubMed1988

Effect of salt on the performance of immobilized pH gradient isoelectric focusing gels.

The effect of salt and buffer ions in the sample or in an immobilized pH gradient (IPG) on sample entry into the gel and on the final focused pattern are presented. During the initial phase of electrofocusing, ions present in the gel, either as counter ions to the immobilized charge groups of the IPG gel or added to the gel matrix during the rehydration process, are transported toward the electrodes. For ions present at a concentration exceeding approximately 1 mM the transport can be followed by the refractile line marking the trailing edge of an ion-containing zone. Gradual sample entry may be achieved by applying the sample at a site (near the anode or cathode) opposite to that from which the sharpest refractile line, marking the ion present in the highest concentration, approaches the sample. Additionally, lateral band spreading of the sample is avoided. Thus, sample applied at the cathode for IPG gels rehydrated with 1-2 mM Tris base, or at the anode for gels rehydrated with 1-2 mM acetic acid or sodium acetate, enters the gel matrix gradually without lateral band spreading. In contrast, sample applied at the anode, for Tris-containing gels, or at the cathode, for acetate-containing gels, enters rapidly in a sharp zone when the refractile line reaches the sample zone. This results in a high local protein concentration in the zone immediately behind the boundary with lateral band spreading.(ABSTRACT TRUNCATED AT 250 WORDS)

Gels↗

PubMed1988

Somatic mutation studies in human lymphoid cells: the detectability of quantitative genetic variants in two-dimensional gels.

The feasibility of detecting quantitative genetic variants based on a decrease in the integrated intensity of polypeptide spots in two-dimensional polyacrylamide gels of human lymphoblastoid cell clones was investigated. A battery of 65 spots on 115 gels was studied to determine the distribution of quantitative measures for spots where no mutation had occurred. The corresponding distribution for spots which have decreased integrated intensity as a result of a mutation at one of two alleles coding for the spot was investigated by quantitating spots for which mutations were known to have occurred. These two distributions allowed the estimation of the rates of false positive and false negative errors for any particular strategy aimed at detecting null mutations, and thus provides a basis for the design of efficient strategies. Our silver stained gels have sufficient reproducibility of spot integrated intensities so that, for situations in which the mutation rate is relatively high, it is practical to monitor a subset of spots for null variants using the same digitized images as are used to detect structural variants.

Clone Cells↗

PubMed1988

Nonrandom distribution of structural mutants in ethylnitrosourea-treated cultured human lymphoblastoid cells.

Two-dimensional polyacrylamide gel electrophoresis has been used to detect somatic cell gene mutations altering protein structure, following ethylnitrosourea treatment of cultured human lymphoblastoid cells. A total of 267 polypeptides encoded by 263 loci were scored in a series of 1143 lymphoblastoid clones. Sixty-five electrophoretic mutants were detected at a total of 49 loci. Sixteen of the 65 mutations were phenotypically repeat mutations, occurring at 11 loci. Furthermore, structural mutations occurred more frequently at loci known to be polymorphic. These results provide evidence that the mutations that are detectable at the protein level by two-dimensional polyacrylamide gel electrophoresis do not occur at random and that their frequency is greater among polymorphic loci.

Cell Line↗

PubMed1988

Estimation of mutation rates based on the analysis of polypeptide constituents of cultured human lymphoblastoid cells.

A subclone of a human diploid lymphoblastoid cell line, TK-6, with consistently high cloning efficiency has been used to estimate the rates of somatic mutations on the basis of protein variation detected by two-dimensional polyacrylamide gel electrophoresis. A panel of 267 polypeptide spots per gel was screened, representing the products of approximately 263 unselected loci. The rate of human somatic mutation in vitro was estimated by measuring the proportion of protein variants among cell clones isolated at various times during continuous exponential growth of a TK-6 cell population. Three mutants of spontaneous origin were observed, giving an estimated spontaneous rate of 6 x 10(-8) electrophoretic mutations per allele per cell generation (i.e., 1.2 x 10(-7) per locus per cell generation). Following treatment of cells with N-ethyl-N-nitrosourea, a total of 74 confirmed variants at 54 loci were identified among 1143 clones analyzed (approximately 601,000 allele tests). The induced variants include 65 electromorphs which exhibit altered isoelectric charge and/or apparent molecular weight and nine nullimorphs for each of which a gene product was not detected at its usual location on the gel. The induced frequency for these 65 structural gene mutants is 1.1 x 10(-4) per allele. An excess of structural gene mutations at ten known polymorphic loci and repeat mutations at these and other loci suggest nonrandomness of mutation in human somatic cells. Nullimorphs occurring at three heterozygous loci in TK-6 cells may be caused by genetic processes other than structural gene mutation.

Cell Line↗

PubMed1987

Identification and characterization of a human transthyretin variant.

An apparent Mr variant of plasma transthyretin (TTR), previously detected using 2-D PAGE, is the first reported occurrence of this type of human TTR variant. We characterized the variant TTR to determine the nature of this difference. Comparative tryptic peptide maps of variant and normal TTR and sequencing of peptides which differed indicated the variant contained a single amino acid substitution of valine for tyrosine at position 116. Because such a change requires two nucleotide substitutions, we postulate the variant arose through mutation in codon 116 of a heretofore unrecognized polymorphic or rare variant allele of TTR.

Amino Acid Sequence↗

PubMed1987

The use of measured genotype information in the analysis of quantitative phenotypes in man. II. The role of the apolipoprotein E polymorphism in determining levels, variability, and covariability of cholesterol, betalipoprotein, and triglycerides in a sample of unrelated individuals.

Recent advances in molecular biology provide measures of genotypes at loci involved in lipid metabolism. Genotypes for apolipoprotein E (apo E) and quantitative levels of total plasma cholesterol, betalipoprotein, and triglycerides were measured in a sample of 223 unrelated individuals from Nancy, France. The frequencies of the epsilon 2, epsilon 3, and epsilon 4 alleles are 0.13, 0.74, and 0.13, respectively, in this sample. Significant differences among apo E genotypes were detected for these lipoprotein phenotypes. The average effect of the epsilon 2 allele was to reduce total plasma cholesterol and betalipoprotein levels by 0.52 mmol/L and 0.98, respectively, while the epsilon 4 allele raised these levels by 0.26 mmol/L and 0.61, respectively. Apo E genotype specific correlations suggest that this locus also has an effect on the coordinated metabolism between cholesterol and triglycerides. We infer that approximately 17% of the genetic variability in total plasma cholesterol may be attributable to this apo E polymorphism. No other single locus has been identified with such a large contribution to cardiovascular disease risk factors in the general population.

Alleles↗

PubMed1987

Detection of human somatic cell structural gene mutations by two-dimensional electrophoresis.

The feasibility of detecting human somatic structural gene mutations by two dimensional electrophoresis has been investigated. A lymphoblastoid cell line was grown as a mass culture in the presence of ethylnitrosourea, after which cells were regrown as single cell clones. A total of 257 polypeptide spots were analyzed in gels derived from 186 clones. Four structural mutations were detected by visual analysis of the gels. Computer analysis of gels corresponding to the mutant clones was also undertaken. At a spot size threshold of 200 spots to be matched using a computer algorithm, all four mutant polypeptides were detected. These results indicate the usefulness of the two-dimensional approach for mutagenesis studies at the protein level.

Clone Cells↗

PubMed1986

Detection of electrophoretically silent mutations by immobilized pH gradients.

The detection of neutral amino acid mutants, by isoelectric focusing in immobilized pH gradients (IPGs), is exemplified by the separation of hemoglobin (Hb) Beirut (126 beta Val----Ala) from Hb A in a shallow pH 7.2-7.6 IPG gradient with 2% Ampholine pH 6-8. The mechanism of these separations appears to involve minute alterations in the pK values of ionizable groups bordering the mutation site, which are in turn reflected in tiny alterations in the net surface charge, delta pI. The delta pI values are of the order of 0.01 to 0.001 pH units, outside the resolving limits of conventional isoelectric focusing, and correspond to changes of the order of 0.1-0.01 unit charge (a proton or an electron).

Amino Acids↗

PubMed1986

Two-dimensional gel electrophoresis of cellular proteins reveals myeloid origin of blasts in two children with otherwise undifferentiated leukemia.

In previous studies, two-dimensional polyacrylamide gel electrophoresis allowed the detection of 11 polypeptide markers that distinguished between subtypes of acute lymphoblastic leukemia and between acute lymphoblastic and acute myelocytic leukemia. This report describes the occurrence of polypeptide markers in two-dimensional gels that indicate a myeloid origin of blasts obtained from two children who presented with acute leukemia, the cells of origin of which could not be determined, at the time of diagnosis, by morphologic, cytochemical, or immune marker analysis. The authors conclude that two-dimensional electrophoresis provides a new tool for the delineation of the cell of origin in acute leukemia.

Acute Disease↗

PubMed1986

A statistical analysis of spot variation using the two-dimensional polyacrylamide gel electrophoresis.

For the development of valid algorithms for matching protein spots between two-dimensional gels, it is essential that one has an understanding of the relative roles of the many sources of variability affecting the location of spots. We first consider the contribution of observers to the measurement variability of spot location, arriving at a simple model for these effects. Next we present an analysis of the variability in spot locations for a sample of gels containing duplicate gels for each sample. Our data indicate that both differences between duplicates and between samples are considerable, and that the size of the effects depends on the region of the gel being considered. In the discussion we examine several matching strategies that match large groups of gels based on algorithms which match two gels at a time.

Analysis of Variance↗

PubMed1986

Lineage-related polypeptide markers in acute lymphoblastic leukemia detected by two-dimensional gel electrophoresis.

We have utilized two-dimensional polyacrylamide gel electrophoresis, coupled with ultrasensitive silver staining, to identify lineage-related polypeptide markers in lymphoblasts from children with acute lymphoblastic leukemia. Twelve polypeptides were detected that could distinguish between the major subgroups of acute lymphoblastic leukemia. These included a new marker for common acute lymphoblastic leukemia and markers for cells of B and T lineages. Analysis of the two-dimensional patterns also allowed the tentative identification of T-cell lineage in two cases with an otherwise undifferentiated non-T-cell non-B-cell phenotype. Two-dimensional electrophoresis thus provides a powerful tool for the delineation of the cell of origin in leukemia.

B-Lymphocytes↗

PubMed1986

Genetic variants detected among 106 lymphocyte polypeptides observed in two-dimensional gels.

Peripheral blood lymphocytes from 40 children have been examined for genetic variation in their protein constituents by two-dimensional polyacrylamide gel electrophoresis. One hundred six polypeptides chosen without respect to genetic variability were scored in gels from the 40 children. For each child, gels from both parents were also examined to substantiate the genetic basis of variants observed. Of the total of 4,240 polypeptides, 23 could not be scored unambiguously. Fourteen of the polypeptides showed genetic variants in one or more of the children. One hundred twenty-nine of 4,217 polypeptides scored exhibited the combination of a normal and a variant polypeptide. All variants were present in at least one of the parents of the subject. The index of heterozygosity observed (3.05% +/- .23%) indicates substantial genetic variation in cellular protein constituents.

Adult↗

PubMed1986

Genetic analysis of thirty-three platelet polypeptides detected in two-dimensional polyacrylamide gels.

Two-dimensional gel electrophoresis followed by silver-staining was utilized to visualize platelet polypeptides for genetic analysis. A subset of 33 polypeptides that were most suited for scoring was selected. Families consisting of father-mother-child trios were studied. Thirty-six polypeptides of a total of 1,413 scored in children's gels exhibited the combination of a normal and a variant polypeptide. The observed index of heterozygosity of 2.55% is comparable to our previously reported findings for red cell proteins.

Blood Platelets↗

PubMed1985

Peripheral blood erythroid progenitors from patients with sickle cell anemia: HPLC separation of hemoglobins and the effect of a HbF switching factor.

We describe the use of HPLC for the separation and quantitation of hemoglobin and globin chains. Utilization of this sensitive technique allows the analysis of hemoglobin and globin chains in the total accumulated and newly synthesized hemoglobin. Using this methodology, we have studied the effect of a previously described HbF factor in fetal calf serum on peripheral blood BFUe-derived erythroblasts from patients with sickle cell anemia. As in previous studies of peripheral blood BFUe-derived erythroblasts from hematologically normal adults, this HbF factor promoted the increased synthesis of HbF in the BFUe-derived erythroblasts from the sickle cell anemia patients.

Anemia, Sickle Cell↗