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PubMedDate not supplied

K-Ras-independent effects of the farnesyl transferase inhibitor L-744,832 on cyclin B1/Cdc2 kinase activity, G2/M cell cycle progression and apoptosis in human pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma is a highly lethal malignancy that is resistant to traditional cytotoxic therapy. High rates of activating codon 12 K-Ras mutations in this disease have generated considerable interest in the therapeutic application of novel farnesyl transferase inhibitors (FTIs). However, a comprehensive analysis of the effects of FTI treatment on pancreatic cancer cells has not been performed. Treatment of five different human pancreatic cancer cell lines with FTI L-744,832 resulted in inhibition of anchorage-dependent growth, with wide variation in sensitivity among different lines. Effective growth inhibition by L-744,832 correlated with accumulation of cells with a tetraploid (4N) DNA content and high levels of cyclin B1/cdc2 kinase activity, implying cell cycle arrest downstream from the DNA damage-inducible G2/M cell cycle checkpoint. In addition, sensitive cell lines underwent apoptosis as evidenced by changes in nuclear morphology and internucleosomal DNA fragmentation. L-744,832 at a concentration of 1 microM additively enhanced the cytotoxic effect of ionizing radiation, apparently by overriding G2/M checkpoint activation. The effects of FTI treatment on cell growth and cell cycle regulation were associated with changes in posttranslational processing of H-Ras and N-Ras, but not K-Ras. The results confirm the potential therapeutic efficacy of FTI treatment in pancreatic cancer, and suggest that farnesylated proteins other than K-Ras may act as important regulators of G2/M cell cycle kinetics.

Antineoplastic Agents↗
PubMed1997

ErbB kinases and NDF signaling in human prostate cancer cells.

Prostate carcinoma (PCA) is the most commonly diagnosed malignancy in American men. Our knowledge of PCA growth regulation lags behind that of other cancers, such as breast and colon carcinomas. Among receptor tyrosine kinases, the ErbB family is most frequently implicated in neoplasia. We report here the expression of ErbB family kinases and their ligands in PCA cell lines and a xenograft. While ErbB1/EGFR, ErbB2/NEU, and ErbB3 were always observed in a distinct pattern, ErbB4 was not observed. Interestingly, while TGF-alpha was expressed in the majority of PCA lines, the ligand Neu Differentiation Factor/Heregulin (NDF) was expressed only in an immortalized, non-transformed prostate epithelial line. Concomitantly, there was a significant difference in biological response to these ligands. NDF inhibited LNCaP growth and induced an epithelial-like morphological change, in contrast to TGF-alpha, which accelerated cell growth. We also performed the first comprehensive analysis of NDF signaling in a prostate line. LNCaP stimulated with NDF demonstrated crosstalk between ErbB3 and ErbB2 which did not involve ErbB1. NDF also turned on several cascades, including those of PI3-K, ERK/MAPK, mHOG/p38 and JNK/SAPK, but not those of PLCgamma or the STAT family. This signaling pattern is distinct from that of TGF-alpha. The activation of mHOG by ErbB2 or ErbB3 has not been reported, and may contribute to the unusual phenotype. PI3-K activation is characterized by the formation of a striking 'activation complex' with multiple tyrosine-phosphorylated species, including ErbB3. Our studies provide a framework in which to dissect the growth and differentiation signals of prostate cancer cells.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Allelic deletion at 11q23 is common in MYCN single copy neuroblastomas.

Deletions of the long arm of chromosome 11 (11q) have been noted in primary neuroblastomas, but a comprehensive analysis has not been performed. Therefore, we analysed 331 neuroblastomas (295 sporadic, 15 familial and 21 tumor-derived cell lines) to determine the prevalence of 11q allelic deletions, to map the location of a putative tumor suppressor gene and to perform clinical correlative studies. Assays for loss of heterozygosity (LOH) were performed at 24 microsatellite loci spanning 11q. LOH was observed at multiple 11q loci in 129/295 (44%) sporadic neuroblastomas, 5/15 (33%) familial neuroblastomas, and 5/21 (24%) neuroblastoma cell lines. A single region of 2.1 cM within 11q23.3, flanked by markers D11S1340 and D11S1299, was deleted in all specimens with 11q LOH. Allelic loss at 11q23 was inversely related to MYCN amplification (P<0.001). Within the subset of cases with a single copy of MYCN, 11q LOH was associated with advanced stage disease (P=0.008), unfavorable histopathology (P=0.042), and decreased overall survival probability (P=0.008). However, 11q LOH was not independently prognostic in multivariate analyses. These data support the hypothesis that a tumor suppressor gene mapping within 11q23.3 is commonly inactivated during the malignant evolution of a large subset of neuroblastomas, especially those with unamplified MYCN.

Alleles↗
PubMed2006

Dual role of LOH at MMR loci in hereditary non-polyposis colorectal cancer?

hMLH1 and hMSH2 can be considered tumor suppressor genes, as both alleles must be inactivated in order to lose the mismatch repair (MMR) function. In this regard, it has been proposed that LOH at MMR loci is a common Knudson's second-hit mechanism in HNPCC patients. However, experimental evidence supporting this view is scarcely found in the literature. We have performed a comprehensive analysis of LOH in 45 HNPCC tumors carrying a germline alteration in MMR loci. Overall, we have detected LOH at MMR loci in 56% of the cases. However, up to 40% of the LOH events targeted the mutant allele, arguing against a second-hit role in these tumors. Interestingly, the age at diagnosis was significantly older in these patients. To explain this and previous data, we propose a dual role for LOH at MMR loci in HNPCC.

Adult↗
PubMed2006

Erasure of methylation imprint at the promoter and CTCF-binding site upstream of H19 in human testicular germ cell tumors of adolescents indicate their fetal germ cell origin.

Genome-wide epigenetic modification plays a crucial role in regulating genome functions at critical stages of development. In particular, DNA methylation is known to be reprogrammed on a genome-wide level in germ cells and in preimplantation embryos, although it is relatively stable in somatic cells. In this reprogramming process, the genome becomes demethylated, and methylated de novo during later stages of development. Reprogramming of DNA methylation in male germ cells has not been fully investigated. Testicular germ cell tumors (TGCTs) possess a pluripotential nature and display protean histology from germ cells to embryonal and somatic cell differentiation. These properties make TGCT a unique model for studying germ cell development and gametogenesis in respect of DNA reprogramming. In order to obtain an insight into the epigenetic dynamics of TGCTs, we conducted a comprehensive analysis of differential methylated regions (DMRs) on H19 and IGF2 in TGCTs compared with testicular malignant lymphomas. In the present study, we show that methylation imprint at the promoter and CTCF-binding site upstream of H19 was completely erased in both seiminomatous and non-seminomatous TGCTs, whereas differential methylation was observed in testicular lymphomas. The erasure of methylation imprint was also observed in TGCTs with malignant transformation. We found biallelic unmethylation at the promoter and the CTCF-binding site upstream of H19 is required, but not sufficient for the biallelic expression of H19 in TGCTs. These data suggest that factors other than methylation contribute to transcriptional regulation of imprinted genes in TGCTs. The present data have shown that TGCTs carry distinctive epigenetic profiles at the core-imprinting domain of H19/IGF2 from other neoplasms of somatic cell origin. The data also suggest that both seminomatous and non-seminomatous TGCTs carry methylation profiles similar to fetal germ cells, but not adult germ cells, indicating the origin of TGCTs as fetal germ cells.

Adolescent↗
PubMed2026

Multi-omic analyses of the same sample using metabolomics, lipidomics, proteomics, phosphoproteomics, and glycoproteomics.

Mass spectrometry (MS)-based multi-omics offers powerful tools to comprehensively characterize proteins, post-translational modifications, metabolites, and lipids. However, these measurements are typically performed using separate sample preparation workflows and modality-specific liquid chromatography mass spectrometry (LC-MS) platforms, limiting integration and constraining applications to small amounts of sample materials, especially scarce clinical specimens. Here, we describe a unified nano-LC-MS framework that enables metabolomic, lipidomic, proteomic, phosphoproteomic, and glycoproteomic analyses from the same starting material using a single nano-LC-MS platform, with only the chromatographic conditions, acquisition methods, and enrichment procedures tailored to each omics. This integrated strategy reduces workflow complexity and sample consumption while improves analytical continuity across molecular layers. By enabling deep multi-omics characterization from the same sample, this platform provides a practical foundation for comprehensive analysis of precious clinical samples.

Proteomics↗
PubMed2006

Identification of Rho GTPases implicated in terminal differentiation of muscle cells in ascidia.

BACKGROUND INFORMATION: Members of the Rho GTPase family mediate changes in the actin cytoskeleton and are also implicated in developmental processes, including myogenesis. Nevertheless, a comprehensive analysis of these proteins during myofibrillogenesis has never been performed in any organism. RESULTS: Using the ascidian model to identify the role of Rho GTPases on myofibrillogenesis, we show that transcripts for all Rho GTPases are detected in muscle cells of the embryo. We find that activation of RhoA, TC10 and Cdc42 (cell division cycle 42) disturbs the polarity of muscle cells, whereas that of other Rho GTPases induced cell positioning defects. Moreover, dominant negative version of five Rho GTPases, RhoA, Rac2, RCL2 (Rac- and Cdc42-like 2), TC10 and WRCH (Wnt-1 responsive Cdc42 homologue), impaired the formation of mature myofibrils. CONCLUSIONS: Taken together, our results show that several Rho GTPase-dependent pathways are required to control the spatial localization of muscle cells in the embryo and to coordinate myofibril assembly. This stresses the importance of analysing the entire Rho family when studying a new biological process.

Animals↗
PubMed1981

Calcium uptake in isolated brush-border vesicles from rat small intestine.

Ca2+ uptake in brush-border vesicles isolated from rat duodena was studied by a rapid-filtration technique. Ca2+ uptake showed saturation kinetics, was dependent on the pH and ionic strength of the medium and was independent of metabolic energy. Uptake activity was readily inhibited by Ruthenium Red, La3+, tetracaine, EGTA, choline chloride and Na+ or K+. The effect of variations in medium osmolarity on Ca2+ uptake and the ionophore A23187-induced efflux of the cation from preloaded vesicles indicated that the Ca2+-uptake process involved binding to membrane components, as well as transport into an osmotically active space. Scatchard-plot analyses of the binding data suggested at least two classes of Ca2+-binding sites. The high-affinity sites, Ka = (2.7 +/- 1.1) x 10(4) M-1 (mean +/- S.D.) bound 3.2 +/- 0.8 nmol of Ca2+/mg of protein, whereas the low-affinity sites (Ka = 60 +/- 6 M-1) bound 110 +/- 17 nmol of Ca2+/mg of protein. In the presence of 100 mM-NaCl, 1.7 and 53 nmol of Ca2+/mg of protein were bound to the high- and low-affinity sites respectively. Decreased Ca2+-uptake activity was observed in vesicles isolated from vitamin D-deficient as compared with vitamin D-replete animals and intraperitoneal administration of 1,25-dihydroxycholecalciferol to vitamin D-deficient rats 16 h before membrane isolation stimulated the initial rate of Ca2+ uptake significantly. The data indicated that Ca2+ entry and/or binding was passive and may involve a carrier-mediated Ca2+-uptake component that is associated with the brush-border membrane. Altering the electrochemical potential difference across the membrane by using anions of various permeability and selected ionophores appeared to increase primarily binding to the membrane rather than transport into the intravesicular space. Since there is considerable binding of Ca2+ to the vesicle interior, a comprehensive analysis of the transport properties of the brush-border membrane remains difficult at present.

Animals↗
PubMed1982

Characterization of calcium binding to brush-border membranes from rat duodenum.

The Ca2+-binding properties of isolated brush-border membranes at physiological ionic strength and pH were examined by rapid Millipore filtration. A comprehensive analysis of the binding data suggested the presence of two types of Ca2+-binding sites. The high-affinity sites, Ka = (6.3 +/- 3.3) X 10(5) M-1 (mean +/- S.E.M.), bound 0.8 +/- 0.1 nmol of Ca2+/mg of protein and the low-affinity sites, Ka = (2.8 +/- 0.3) X 10(2) M-1, bound 33 +/- 3.5 nmol of Ca2+/mg of protein. The high-affinity site exhibited a selectivity for Ca2+, since high concentrations of competing bivalent cations were required to inhibit Ca2+ binding. The relative effectiveness of the competing cations (1 and 10 mM) for the high-affinity site was Mn2+ approximately equal to Sr2+ greater than Ba2+ greater than Mg2+. Data from the pH studies, treatment of the membranes with carbodi-imide and extraction of phospholipids with aqueous acetone and NH3 provided evidence that the low-affinity sites were primarily phospholipids and the high-affinity sites were either phosphoprotein or protein with associated phospholipid. Two possible roles for the high-affinity binding sites are suggested. Either high-affinity Ca2+ binding is involved with specific enzyme activities or Ca2+ transport across the luminal membrane occurs via a Ca2+ channel which contains a high-affinity Ca2+-specific binding site that may regulate the intracellular Ca2+ concentration and gating of the channel.

Animals↗
PubMed2001

Phosphate-binding loop and Rab GTPase function: mutations at Ser29 and Ala30 of Rab5 lead to loss-of-function as well as gain-of-function phenotype.

Ras-like GTPases contain a structurally conserved GTP-binding domain. An important element of the GTP-binding domain is the phosphate-binding loop, which contains two Gly residues (Gly(12) and Gly(13)) in Ras. Because the two Gly residues are crucial for normal Ras function, it is intriguing that they are not conserved in other Ras-like GTPases, including the Rab GTPases; for example, the equivalent residues in Rab5 are Ser(29) and Ala(30). The present study builds on earlier biochemical characterizations of the Rab5 mutants containing substitutions at Ala(30) and provides a comprehensive analysis of the structure-function relationship of the Rab5 phosphate-binding loop. We have generated 19 new mutants containing amino acid substitutions at Ser(29) and determined whether these Ser(29) mutants, as well as the Ala(30) mutants, remain able to stimulate the endocytosis of horseradish peroxidase in baby hamster kidney cells. A total of 11 mutants lose the activity of stimulating endocytosis. Of these 11 mutants, 9 are defective in membrane association. In contrast, 27 mutants remain able to stimulate endocytosis. Five of them induce a novel cellular phenotype: cell rounding and detachment from culture dishes. They also induce super-large early endosomes such as the constitutively activated Rab5:Q79L mutant. Biochemical results suggest that the constitutive activation of Rab5 requires an increased nucleotide exchange rate and/or decreased GTPase activity. This study establishes functional significance for the phosphate-binding loop of Rab5 and shows that mutations in this region lead to either a loss-of-function or a gain-of-function phenotype, indicating a structure-function relationship distinct from that of Ras.

Alanine↗
PubMed1988

Immunoblotting and the immune response to leprosy.

Immunoblotting has provided a powerful and effective approach to dissection of the immune response to mycobacterial antigens in a situation in which the availability of isolated antigenic components is severely limited. The basic approach of blotting on to a solid-phase support has been used in combination with SDS/polyacrylamide-gel electrophoresis, t.l.c., recombinant DNA technology and T-cell cloning in order to carry out a comprehensive analysis of glycolipid and protein antigens involved in the immune response to mycobacterial infection.

Antigens, Bacterial↗
PubMed1983

Phonological proficiency of two cleft palate toddlers with school-age follow-up.

This study provides a comprehensive analysis of the speech-sound production of two children with repaired bilateral cleft lip and palate. Speech samples were obtained during videotaping of spontaneous interactions between the children and their parent(s) when the subjects were between 29 and 37 months of age. Re-evaluation of both language and phonological abilities was accomplished when the children were between 5 and 7 years of age. Based on phonemic transcription of these data, error matrices were prepared illustrating word-initial and word-final consonants produced. Place and manner of consonant production were analyzed and the individual phonological processes employed by each child were discussed. Results indicated individual differences between the subjects in that one subject's emerging phonological system was more characteristic of developmental delay while the other was more characteristic of structural inadequacy. These differences have implication for the management of preschool cleft palate children.

Child↗
PubMed1993

Facilitating prelinguistic communication skills in young children with developmental delay.

Very little research has focused on the development and evaluation of intervention strategies designed to facilitate the acquisition of prelinguistic communication skills. We conducted two experiments to determine the effects of a milieu teaching approach on the acquisition and generalization of specific prelinguistic communication skills. In the first experiment, we utilized this intervention approach within a multiple baseline design to teach prelinguistic requesting, commenting, and vocal imitation to a single subject with Down syndrome and language delay. The results indicated that the intervention approach was effective at facilitating the child's use of these skills within the treatment setting. Therefore, in the second experiment we conducted a more comprehensive analysis of this approach with 4 subjects with mental retardation. Three of these subjects were taught to request, and 1 subject was taught both to request and to comment. The effects were experimentally evaluated with multiple baseline across subjects design. The results indicated that the intervention was effective in eliciting the intervention targets within the training setting for all 4 subjects. All 4 subjects showed evidence of generalization across stimulus materials, setting, teachers, and interaction style. There was also evidence of reciprocal effects on how classroom teachers in the generalization setting interacted with the subjects as a result of changes in the child's communication behavior.

Child, Preschool↗
PubMed2003

Molecular pathology of haemophilia B in Turkish patients: identification of a large deletion and 33 independent point mutations.

Heterogeneous mutations in the coagulation factor IX (FIX) gene result in a bleeding tendency known as haemophilia B. The haemophilia B mutation database has a total of 2353 patient entries, including 10 of the estimated 1000 Turkish patients. In this study, a more comprehensive analysis of the molecular pathology of haemophilia B in Turkey revealed one large deletion and 33 point mutations in the FIX gene of 34 unrelated patients. Haplotype analysis using six polymorphic sites showed that the mutations identified in a total of 45 patients occurred on 13 different haplotypes and that each mutation was family specific.

Base Sequence↗
PubMed2001

Clinical safety and tolerability of montelukast, a leukotriene receptor antagonist, in controlled clinical trials in patients aged > or = 6 years.

OBJECTIVE: Montelukast is a leukotriene receptor antagonist administered orally once daily for treatment of chronic asthma in adults and children. A comprehensive analysis of safety data from double-blind, randomized, placebo-controlled trials with montelukast has not been previously reported. PATIENTS AND METHODS: A pooled analysis of safety data from 11 multicentre, randomized, controlled montelukast Phase IIb and III trials and five long-term extension studies was performed. A total of 3386 adult patients (aged 15-85 years) and 336 paediatric patients (aged 6-14 years) were enrolled in the trials; 2031 adults received montelukast for up to 4.1 years, and 257 children received montelukast for up to 1.8 years. Summary statistics comparing incidences of adverse events among treatment groups were calculated. RESULTS: The overall incidence of clinical and laboratory adverse events among montelukast-treated patients, both adult and paediatric, was similar to that among patients receiving placebo. There were no clinically relevant differences in individual adverse events, including infectious upper respiratory conditions and transaminase elevations, between montelukast and placebo groups. Discontinuations due to adverse events occurred with similar frequencies during placebo, montelukast and inhaled beclomethasone therapy. No dose-related adverse effects of montelukast were observed in adults treated with dosages as high as 200 mg per day (20 times the recommended dose) for 5 months. This tolerability profile montelukast observed in clinical trials has been generally reflected in the post-marketing safety experience seen to date. CONCLUSIONS: These data indicate a tolerability profile for montelukast similar to placebo during both short-term and long-term administration, even at doses substantially higher than the recommended clinical dose of 10 mg once daily for adults and 5 mg once daily for children aged 6-14 years.

Acetates↗
PubMed1999

The effect of G-CSF on the composition of human bone marrow.

The effects on bone marrow cellularity and morphology of 6 days' treatment with granulocyte colony-stimulating factor (G-CSF) in 35 patients were studied. Examination of trephine biopsies showed a highly significant increase in cellularity (P < 10-13). Assessment of aspirates revealed an increase in the myeloid to erythroid (M : E) ratio (P = 0.00006), the proportion of myeloid cells (P < 10-8), myelocytes (P = 0.00007), metamyelocytes (P = 0.04), band forms (P = 0.0005) and neutrophils (P = 0.02). This study presents a comprehensive analysis of the effects of six days' administration of G-CSF on human bone marrow.

Adult↗
PubMed1996

Polymorphism of the apolipoprotein A-IV gene and its significance in lipid metabolism and coronary heart disease in a Japanese population.

Apolipoprotein A-IV (apo A-IV) is involved in the metabolism of both triglycerides and high-density lipoproteins (HDLs). Apo A-IV has been suggested as participating in several stages of reverse cholesterol transport. Uncertainty about the exact biochemical function of apo A-IV has made the use of genetic apo A-IV polymorphism (variants) attractive in evaluating its physiological role. To date, although some reports indicate that DNA polymorphisms at this locus play an important role in the metabolism of lipids and lipoproteins in western (Caucasian) populations, no similar comprehensive analysis has been performed in a distinct Japanese population. Using DNA sequencing and a restriction fragment length polymorphism (RFLP) study with polymerase chain reaction (PCR), the following allele frequencies were established: (a) codon -8 (G-->A, non-synonymous) allele 2 = 0 (n = 105); (b) codon 9 (A-->G, synonymous) allele 2 = 0.388 (n = 152); (c) codon 347 (A-->T, non-synonymous) allele 2 = 0 (n = 900); (d) codon 360 (T-->G, non-synonymous) allele 2 = 0 (n = 800); (e) VNTR exon 3 [(CTGT)3 and (CTGT)4] (CTGT)3 = 0.262 (n = 105); and (f) MspI (newly detected polymorphic site) polymorphism (C C/T GG) within intron 2, allele 2 = 0.096 (n = 193). The frequencies of these polymorphisms, except for that of the newly identified MspI site, are completely different from those reported in western populations. Among the 900 subjects examined, we found one ACT (Thr) to ACG (Thr) synonymous mutation at codon 347, which does not change the primary structure of apo A-IV. The apo A-IV allele frequency in patients (166 men and 56 women) with angiographically proven coronary heart disease (CHD) was also studied [codon 9 allele 2 = 0.329 (n = 217); VNTR exon 3 (CTGT)3 = 0.262 (n = 84); MspI within intron 2, allele 2 = 0.092 (n = 222)]. Furthermore, we evaluated serum lipid and lipoprotein levels quantitatively in control subjects and Japanese CHD patients. These polymorphisms did not show any consistent and significant association with lipid and lipoprotein parameters. In addition, no gender-specific effects of apo A-IV polymorphisms on lipid parameters adjusted for confounding factors were observed in either CHD patients or control subjects. Our results indicate that the apo A-IV gene is not a major determinant of the risk for CHD in Japanese.

Adult↗
PubMed1997

Compilation of distinct HLA-A, -B and -C transmembrane and cytoplasmic domain-encoding sequences.

Exons 5-8 of HLA class I genes encode the transmembrane and cytoplasmic domains of HLA class I proteins. Of the more than 300 HLA-A, -B and -C alleles recognized by the WHO Nomenclature Committee for Factors of the HLA System, the number of alleles for which exon 5-8 sequence information is available and the extent of genetic variability in this region are undocumented. Thus, a comprehensive analysis of HLA-A, -B and -C exon 5-8 sequence variability was undertaken. Of 219 HLA-A, -B and -C alleles for which exon 5-8 sequences are known, 40 unique HLA-A, -B and -C exon 5-8 sequences were identified. At the amino acid level, these encode 28 distinct HLA-A, -B and -C transmembrane and cytoplasmic domains.

Alleles↗