A silent, neutral substitution detected by reverse-phase high-performance liquid chromatography: hemoglobin Beirut.

A substitution of alanine for valine at position 126 in the beta-chain of hemoglobin was discovered in a hematologically normal adult male of Lebanese extraction. The variant beta-globin was initially observed and subsequently purified by reverse-phase high-performance liquid chromatography (HPLC). Reverse-phase HPLC was also used to isolate the variant tryptic peptide of beta-T13 that has alanine replacing valine at residue 126. The discovery of hemoglobin Beirut illustrates the usefulness of reverse-phase HPLC for the detection of neutral amino acid substitutions in proteins. The ability to detect neutral substitutions in undigested proteins is pertinent to the monitoring of genetic variation in human populations.

Differential expression of Op18 phosphoprotein during human thymocyte maturation.

Op18 (also termed prosolin/stathmin) is a highly conserved 18-kD cytosolic phosphoprotein expressed in low levels in mature resting G0 lymphocytes, but induced in late G1 and S phases after entry into the cell cycle. In addition to its induction in normal proliferating lymphocytes, Op18 has been found to occur at high levels in acute leukemias and in neuroendocrine tissue. The presence and rapid phosphorylation of Op18 after stimulation of proliferating cells correlates with subsequent functional responses of the cells, and, therefore, Op18 has been suggested to play a key role in signal transduction. The pattern of expression of Op18 during lymphoid development is of interest in view of its high levels of expression in acute leukemias, representing cells arrested at an immature stage, thus raising the possibility that Op18 may be regulated differently in mature and immature lymphoid cells. We report here that immature human thymocytes bearing the cortical double positive phenotype (CD4+CD8+) constitutively express high levels of Op18 protein. In contrast, in mature single positive thymocytes (CD3+CD4+ or CD3+CD8+), Op18 protein is expressed at a lower level, comparable to that seen in peripheral blood T cells. Cell cycle analysis demonstrated that most of the cells in the double positive thymocyte population expressing high levels of Op18 were noncycling and arrested in G0. Furthermore, there was no correlation between Op18 levels and the proportion of cycling cells in double positive thymocyte populations isolated from different thymuses. Interestingly, although Op18 protein levels did not increase any further after mitogenic stimulation of double positive thymocytes, an increase in Op18 phosphorylation was observed, thus coupling of Op18 phosphorylation to cell activation remained intact. Our results show that during lymphoid maturation Op18 expression is uncoupled from cell proliferation. These data also suggest that the ordered expression of proliferation-associated genes seen in mature T cells may be disrupted during T cell maturation.

Identification of two related markers for common acute lymphoblastic leukemia as heat shock proteins.

By direct analysis of the polypeptide constituents of leukemic cells, we have previously detected several polypeptides that are restricted in their expression to acute lymphoblastic leukemia (ALL). In this study, we provide evidence that two polypeptides designated L2 and L4 are structurally related and represent novel markers for common ALL. Partial amino acid sequence analysis did not uncover differences between L2 and L4. The sequences obtained correspond to a previously cloned human gene designated hsp 27 that is expressed, following heat shock treatment, in a variety of cells. 32Pi incorporation studies indicate that L4 is an unphosphorylated form and L2 is a phosphorylated form of hsp27. The two forms were inducible by heat shock in leukemic and nonleukemic lymphoid cells. Thus, in acute leukemia, the common ALL subtype is uniquely characterized by the constitutive expression of a polypeptide that represents a major cellular phosphoprotein.

High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplification.

The gene encoding a novel protein designated nm23-H1, which was recently identified as identical to the A subunit of nucleotide diphosphate kinase from human erythrocytes, has been proposed to play a role in tumor metastasis suppression. We report that untreated neuroblastoma tumors contain a cellular polypeptide (Mr = 19,000) designated p19, identified in two-dimensional electrophoretic gels, which occurs at significantly higher levels (P = 0.0001) in primary tumors containing amplified N-myc gene. The partial amino acid sequence obtained for p19 is identical to the sequence of the human nm23-H1 protein. An antibody to the A subunit of erythrocyte nucleotide diphosphate kinase reacted exclusively with p19. In this study, significantly higher levels of p19/nm23 occurred in primary neuroblastoma tumors from patients with advanced stages (III and IV) relative to tumors from patients with limited stages (I and II) of the disease. Even among patients with a single copy of the N-myc gene, tumors from patients with stages III and IV had statistically significantly higher levels of p19/nm23 than tumors from patients with stages I and II. Our findings indicate that, in contrast to a proposed role for nm23-H1 as a tumor metastasis suppressor, increased p19/nm23 protein in neuroblastoma is correlated with features of the disease that are associated with aggressive tumors. Therefore, nm23-H1 may have distinct if not opposite roles in different tumors.

Hsp27 expression in neuroblastoma: correlation with disease stage.

BACKGROUND: The 27-kd heat shock protein (Hsp27) is differentially expressed in some malignancies, including breast carcinoma, leukemia, and malignant fibrous histiocytoma. In breast carcinoma, a high-level expression of Hsp27 has been associated with shorter disease-free survival in patients with localized disease. PURPOSE: We have observed variable levels of Hsp27 among neuroblastoma tumors. Our aim in this study was to investigate the relationship between Hsp27 expression and stage of the disease and N-myc gene copy number. METHODS: We determined Hsp27 protein levels in 53 neuroblastoma tumors representing different stages of the disease and in 17 neuroblastoma cell lines by quantitative two-dimensional polyacrylamide gel electrophoresis (PAGE). We also performed statistical analysis of Hsp27 levels in relation to stage of the disease and to N-myc gene copy number. RESULTS: Increased Hsp27 expression in neuroblastomas was associated with limited stage disease and inversely correlated with N-myc gene amplification, a feature known to predict poor clinical outcome. An inverse correlation was also observed between N-myc gene amplification and Hsp27 protein levels among the neuroblastoma cell lines analyzed. Immunohistochemical staining of sections of neuroblastomas showed that Hsp27 was most prominently expressed in the cytoplasm of large ganglionic tumor cells present in neuronally differentiated areas of the tumors. Induction of neuronal differentiation in SMS-KCNR neuroblastoma cells using retinoic acid resulted in an increase in Hsp27. CONCLUSION: High level expression of Hsp27 in neuroblastoma is a feature of limited stage, differentiated tumors. IMPLICATION: Hsp27 may play a part in the biology of neuroblastomas with a favorable outcome.

A nucleoside diphosphate kinase A (nm23-H1) serine 120-->glycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interaction.

A high level of nucleoside diphosphate kinase A (NDPK A/nm23-H1) in neuroblastoma is associated with advanced stage disease. We have also found a serine 120-->glycine substitution in NDPK A and/or amplification of the nm23-H1 gene in advanced stage neuroblastomas. Serine 120, a highly conserved residue, is located in proximity to histidine 118 which forms a phosphorylated intermediate essential for NDPK activity. The effect of Ser120-->Gly substitution on the biochemical properties of NDPK A was investigated. Phosphate-transferase activity was lower in the recombinant mutant NDPK A and in the immunoprecipitated complex consisting of NDPK A and NDPK B prepared from a neuroblastoma tumor containing the mutation, relative to the wild-type. There was a significant decrease in the enzyme stability toward urea- or temperature-induced denaturation for the recombinant mutant NDPK A and in an immunoprecipitate from a tumor containing the mutation. Recombinant NDPK A containing the Ser120-->Gly mutation exhibited reduced hexameric and increased dimeric oligomerization relative to the wild-type. Moreover a 28 kDa cellular protein was detected, that co-precipitated with the mutant but not wild-type NDPK A. The altered properties of the mutant protein may have relevance to a role for NDPK A in neuroblastoma progression.

Two distinct pancreatic amylase genes are active in YBR mice.

The genetic determinants of pancreatic amylase expression in YBR mice differ in two respects from those of other inbred strains. First, there are two nonallelic amylase isozymes present in YBR pancreas, while most mouse strains express a single pancreatic amylase protein. In addition, the in vivo rate of total pancreatic amylase synthesis is 50% of that in other strains. Both these traits are determined by genetic sites in the region of the Amy-2 locus on mouse chromosome 3. To determine the molecular basis for the presence of two isozymes in this strain, we have compared portions of their amino acid sequences. Two differences between isozymes A1 and B1 were identified among the 77 residues compared. This result demonstrates that two distinct amylase genes are expressed in YBR pancreas.

Identification of novel Escherichia coli ribosome-associated proteins using isobaric tags and multidimensional protein identification techniques.

Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16 degrees C and 37 degrees C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles.

Advances in two-dimensional electrophoresis.

New developments in the first dimension step of two-dimensional electrophoresis have expanded the utility of the technique in cell and molecular biology.

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.

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.

Identification of lamin B and histones as 1,25-dihydroxyvitamin D3-regulated nuclear phosphoproteins in HL-60 cells.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3)-induced differentiation of HL-60 leukemia cells is accompanied by a number of cellular changes including regulation of oncogene expression and induction of terminal differentiation. We investigated the mechanism by which 1,25-(OH)2D3 induces these changes. We detected 10 nuclear phosphoproteins, designated p66, p45, p36, p33, p32, p27, p22, p19, p18 and p17, that show alterations in phosphorylation within 6-40 h of 1,25-(OH)2D3 treatment. When phosphorylation reactions were performed with isolated nuclei (in vitro), three of these proteins were phosphorylated in a calcium and phospholipid dependent manner: p66, p36, and p19 P66 was phosphorylated in response to 1,25-(OH)2D3 and purified in a manner similar to that used for nuclear lamins. Western blot analysis of 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels confirmed its identity as lamin B. Phosphorylation of p17 and p18 decreased following 1,25-(OH)2D3 treatment. We separated p17 and p18 by SDS-PAGE and obtained N-terminal amino acid sequence to identify these phosphorproteins as histones H2b and H3, respectively. P19 and p22 were both DNA-cellulose binding proteins whose phosphorylation was altered by 1,25-(OH)2D3 treatment. Increased phosphorylation of p27 was detected using 2-dimensional SDS-PAGE. Phosphorylation of nuclear proteins in the intact cell (in vivo), revealed increases in p66, p45, p36, and p33 phosphorylation and a decrease in p17 phosphorylation following 1,25-(OH)2D3 treatment. We detected an increase in phosphorylation of p32, which was extracted with salt from nuclei and migrated on SDS-PAGE similar to histone H1. Thus, we have identified 1,25-(OH)2D3-sensitive nuclear phosphoproteins, including lamin B and several histones. We have also detected and characterized several less abundant nuclear DNA binding phosphoproteins whose phosphorylation was affected by 1,25-(OH)2D3.

Neither HMG-14a nor HMG-17 gene function is required for growth of chicken DT40 cells or maintenance of DNaseI-hypersensitive sites.

HMG-14 and HMG-17 form a family of ubiquitous non-histone chromosomal proteins and have been reported to bind preferentially to regions of active chromatin structure. Our previous studies demonstrated that the chicken HMG-17 gene is dispensable for normal growth of the DT40 chicken lymphoid cell line. Here it is shown that the major chicken HMG-14 gene,HMG-14a, is also dispensable and, moreover, that DT40-derived cells lacking both HMG-17 and HMG-14a proteins show no obvious change in phenotype with respect to the parental DT40 cells. Furthermore, no compensatory changes in HMG-14b or histone protein levels were observed in cells lacking both HMG-14a and HMG-17, nor were any alterations detected in such hallmarks of chromatin structure as DNaseI-hypersensitive sites or micrococcal nuclease digestion patterns. It is concluded that the HMG-14a and HMG-17 proteins are not required for normal growth of avian cell linesin vitro, nor for the maintenance of DNaseI-hypersensitive sites in chromatin.

Diminished phosphorylation of a heat shock protein (HSP 27) in infant acute lymphoblastic leukemia.

We have previously reported lack of expression of a polypeptide designated L3 in infant acute lymphoblastic leukemia (ALL). Expression of L3 occurred predominantly in older children with pre-B ALL. We have recently reported the expression during B cell ontogeny of two other polypeptides, designated L2 and L4 with a similar Mr as L3, which were identified as phosphorylated and non-phosphorylated forms respectively of the low Mr heat shock protein. hsp27. In this study we have characterized L3 and identified it as another phosphorylated form of hsp27. The two phosphorylated forms appear to be differentially expressed in acute leukemia. L3 levels in infants who expressed hsp27 isoforms L2 and L4 were significantly diminished compared to levels in older children with an equivalent amount of hsp27. We conclude that leukemic cells in infant ALL exhibit a unique pattern of phosphorylation of hsp27 expressed at a pre-B cell stage of differentiation.

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.

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)

Separation of transferrin types in human plasma by anion-exchange high-performance liquid chromatography.

An anion-exchange high-performance liquid chromatographic procedure was developed for the separation of transferrin in human plasma. The procedure allowed the four molecular forms of transferrin, which differ with respect to bound iron, to be separated from each other and from other plasma proteins. Transferrin variants, including B and D types, could also be identified using anion-exchange high-performance liquid chromatography. The approach followed for optimizing the separation of transferrin included identification of the peaks in the chromatogram by two-dimensional polyacrylamide gel electrophoresis. This approach could be extended to other proteins in plasma or biological fluids in order to optimize their separation.

Temperature-dependent spot positional variability in two-dimensional polypeptide patterns.

The effect of temperature, at which isoelectric focusing with immobilized pH gradients is performed, on spot positions and pattern quality in two-dimensional (2-D) electrophoresis was examined. Increased temperatures revealed improved 2-D patterns with respect to sample entry, resolution, and background staining. Focusing at 20 degrees C was superior to focusing at 10 and 15 degrees C. Even at 30 degrees C, a pattern of well-resolved polypeptide spots with a minimum amount of horizontal streaking at the basic end was observed. A computer-based analysis showed that a substantial proportion of polypeptides assumed altered positions in the 2-D pattern in relation to temperature. Mobility shifts of polypeptides were more variable on the neutral part than on the acidic or basic end. The mobility shifts were not restricted to one direction for all the spots whose migration was altered. However, for any given spot, the direction was the same with subsequent increments of temperature. The results clearly demonstrate that a defined temperature for first-dimensional isoelectric focusing is a requirement for the reproducibility of 2-D electrophoresis. After elimination of the cathodic drift, a major source of variability in 2-D patterns, associated with carrier ampholytes, temperature control becomes a critical parameter.

Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin.

We have identified the low MW 27 kD heat shock protein as a major phosphoprotein constituent of smooth muscle and have investigated its potential role in agonist induced smooth muscle contraction. The neuropeptides bombesin and substance P, which are present in neurons of the anorectal region, induce contraction of isolated smooth muscle cells from this region by activating different intracellular pathways. Substance P-induced contraction is 1,4,5-inositol trisphosphate (IP3)/calmodulin dependent, while contraction induced by bombesin is mediated by a protein kinase C (PKC)-dependent pathway. The sustained contraction induced by bombesin or exogenous PKC was blocked by preincubation of cells with monoclonal antibodies to hsp27, while the transient contraction induced by substance P or IP3 was unaffected by the antibodies. Preincubation with isotype matched control antibodies had no inhibitory effect on contraction induced in response to the agents used. These data support a novel role for hsp27 in the non calmodulin mediated sustained contraction induced by bombesin or PKC.

Expression of a novel nuclear protein in activated and in tat-I expressing T cells.

The intracellular events that occur in T lymphoid cells after activation or after infection with HIV-1 are not well defined. In the case of HIV-1 infection, it is unknown whether the tat-I gene, an essential gene for viral replication, affects host cell nuclear factors. Using two-dimensional PAGE, we have identified a novel nuclear protein, designated nuclear protein-28,000 (NP-28), which is induced in Jurkat T cells by stimulation with PMA and/or PHA or ionomycin. This nuclear protein has an apparent molecular mass of 28,000 Da and an isoelectric point of 4.6. Interestingly, Jurkat cells transfected with tat-I express higher levels of NP-28 constitutively, without added stimulation. Incubation of Jurkat cells expressing tat-I with PMA and/or PHA or ionomycin causes superinduction of NP-28. We have therefore identified a novel lymphoid nuclear protein induced by T cell activation that occurs in tat-I expressing cells in the absence of activating agents.