Novel type of red blood cell pyruvate kinase hyperactivity predicts a remote regulatory locus involved in PKLR gene expression.

Red blood cell pyruvate kinase (PK-R) is a key regulatory enzyme of red cell metabolism. Hereditary deficiency of PK-R is caused by mutations in the PKLR gene, leading to chronic nonspherocytic hemolytic anemia. In contrast to PK deficiency, inherited PK hyperactivity has also been described.

Quantitative erythrocyte membrane proteome analysis with Blue-native/SDS PAGE.

The erythrocyte membrane plays a pivotal role in erythrocyte functioning. Many membrane protein aberrations are known that result in hemolytic anemia, however, the origin of numerous disorders is not known to date. To extend the current set of diagnostic tools, we used a novel proteome-wide approach to quantitatively analyze membrane proteins of healthy donor and patient erythrocytes.

A family with multiple mutations and sequence variations in the alpha- and beta-globin gene clusters.

Background: Usually, laboratory diagnostics of hereditary hemoglobin disorders is fairly straightforward. Sometimes, however, correct diagnosis can be difficult. In this study, we describe a family with multiple mutations and sequence variations in the alpha- and beta-globin gene clusters.

Pyruvate kinase regulatory element 1 (PKR-RE1) mediates hexokinase gene expression in K562 cells.

We have established the functional importance of PKR-RE1, a necessary transcriptional regulatory element in the erythroid-specific promoter of the human pyruvate kinase gene (PKLR). Here, we demonstrate by electrophoretic mobility shift assay (EMSA) that the DNA-protein interaction at PKR-RE1 involves a CTGTC motif. Because the same motif is also present in the erythroid-specific promoter of the hexokinase gene (HK1), we confirmed its functional relevance by in vitro transfection in K562 cells.

Ex vivo analysis of aberrant splicing induced by two donor site mutations in PKLR of a patient with severe pyruvate kinase deficiency.

Two single-nucleotide substitutions in PKLR constituted the molecular basis underlying pyruvate kinase (PK) deficiency in a patient with severe haemolytic anaemia. One novel mutation, IVS5+1G>A, abolished the intron 5 donor splice site. The other mutation, c.

Distinct phenotypic expression of two de novo missense mutations affecting the dimer interface of glucose-6-phosphate dehydrogenase.

Mutations encoding class I glucose-6-phosphate dehydrogenase (G6PD) variants are associated with chronic nonspherocytic hemolytic anemia (CNSHA), the most severe phenotypic expression of G6PD deficiency. These mutations frequently affect the G6PD dimer interface that is essential for enzymatic activity. We detected two de novo missense mutations concerning residues located close together in the dimer interface in two patients with severe G6PD deficiency.

HK Utrecht: missense mutation in the active site of human hexokinase associated with hexokinase deficiency and severe nonspherocytic hemolytic anemia.

Hexokinase deficiency is a rare autosomal recessive disease with a clinical phenotype of severe hemolysis. We report a novel homozygous missense mutation in exon 15 (c.2039C>G, HK [hexokinase] Utrecht) of HK1, the gene that encodes red blood cell-specific hexokinase-R, in a patient previously diagnosed with hexokinase deficiency.

Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency.

We established the molecular basis for pyruvate kinase (PK) deficiency in a white male patient with severe nonspherocytic hemolytic anemia. The paternal allele exhibited the common PKLR cDNA sequence (c.) 1529G>A mutation, known to be associated with PK deficiency.

[From gene to disease; hereditary non-spherocytic hemolytic anemia caused by pyruvate kinase deficiency].

Pyruvate kinase (PK) deficiency is a common cause of hereditary non-spherocytic haemolytic anaemia. It is an autosomal recessive disorder caused by mutations in the gene coding for erythrocyte and liver-type pyruvate kinase (PKLR). So far, more than 130 mutations in this gene have been identified.

Polo-like kinase-1 is a target of the DNA damage checkpoint.

Polo-like kinases (PLKs) have an important role in several stages of mitosis. They contribute to the activation of cyclin B/Cdc2 and are involved in centrosome maturation and bipolar spindle formation at the onset of mitosis. PLKs also control mitotic exit by regulating the anaphase-promoting complex (APC) and have been implicated in the temporal and spatial coordination of cytokinesis.

p21 inhibits Thr161 phosphorylation of Cdc2 to enforce the G2 DNA damage checkpoint.

The cyclin-dependent kinase inhibitor p21 is required for a sustained G(2) arrest after activation of the DNA damage checkpoint. Here we have addressed the mechanism by which p21 can contribute to this arrest in G(2). We show that p21 blocks the activating phosphorylation of Cdc2 on Thr(161).

Negative growth regulation of SK-N-MC cells by bFGF defines a growth factor-sensitive point in G2.

Basic fibroblast growth factor (bFGF) has been shown to induce growth inhibition of the neuroepithelioma cell line SK-N-MC. Here we show that this growth inhibition occurs in G(2). We show that bFGF is active on these cells during S and early G(2) phase.

Cyclin D3 regulates proliferation and apoptosis of leukemic T cell lines.

Activation of the T cell receptor in leukemic T cell lines or T cell hybridomas causes growth inhibition. A similar growth inhibition is seen when protein kinase C is activated through addition of phorbol myristate acetate. This inhibition is due to an arrest of cell cycle progression in G(1) combined with an induction of apoptosis.

Inhibition of cell proliferation by lithium is associated with interference in cdc2 activation.

Lithium can interfere with embryonal development in a variety of organisms. We investigated the effect of lithium on the proliferation of early embryonal cells. [3H]Thymidine incorporation of non-committed mouse P19 embryonal carcinoma cells was inhibited by lithium treatment.

Overexpression of c-Src enhances cell-matrix adhesion and cell migration in PDGF-stimulated NIH3T3 fibroblasts.

c-Src is normally associated with the plasma membrane, but upon activation by tyrosine kinase receptors it translocates to the cytoskeleton. Activation of c-Src alters its conformation and induces the association of c-Src with cytoskeletal proteins. c-Src is implicated in tyrosine phosphorylation of cytoskeletal proteins, which might affect the cytoskeletal architecture.

CD28 induces cell cycle progression by IL-2-independent down-regulation of p27kip1 expression in human peripheral T lymphocytes.

CD28 is the primary T cell costimulatory receptor, and upon ligation with its ligands, it enhances T cell proliferation and IL-2 synthesis. In this study we examined the role of CD28 in the initial proliferative response and cell cycle entry of T lymphocytes. Stimulation through CD3 alone resulted in a poor proliferative response, while in the presence of CD28 costimulation a strong increase in the number of cells in S-phase could be detected after 48 h of stimulation.

Increased number of proliferating cells in oral epithelium from smokers and ex-smokers.

To analyse initial tobacco-related cellular alterations in the upper aerodigestive tract, we investigated the proliferation state in paraffin embedded samples of tumour-adjacent histologically normal mucosa from head and neck squamous cell carcinoma (HNSCC) patients and normal buccal mucosa from healthy individuals. The proliferation index (PI) was assessed by indirect immunohistochemical staining for the proliferation marker Ki-67. Only a slight rise in PI was seen in the normal epithelium from non-smoking HNSCC patients in comparison with the epithelium from non-smoking healthy individuals.

Positivity of the proliferation marker Ki-67 in noncycling cells.

Ki-67 is a proliferation marker that is often used to estimate the growth fraction of tumors and other tissues. This antigen is expressed during all phases of the cell cycle but not in quiescent G0 cells. Many studies fail to indicate that the Ki-67 antigen can be expressed even when DNA synthesis is blocked.

Increased expression of epidermal growth factor receptor in normal epithelium adjacent to head and neck carcinomas independent of tobacco and alcohol abuse.

Objective: In this study we examined if expression of the epidermal growth factor receptor (EGFR) in normal epithelium adjacent to head and neck squamous cell carcinomas (HNSCC) is increased and if this increase is due to the use of tobacco and alcohol.

Materials And Methods: Cut sections of formalin-fixed and paraffin-embedded material of histologically normal epithelium adjacent to HNSCC from 25 patients who smoke excessively and abuse alcohol, and 17 HNSCC patients who do not abuse tobacco and alcohol were compared with cut sections of normal epithelium from 27 control individuals. The sections were immunohistochemically stained for the EGFR.

Identification of connexin43 as a functional target for Wnt signalling.

Wnt mediated signal transduction is considered to regulate activity of target genes. In Xenopus embryos, ectopic Wnt1 and Wnt8 expression induces gap-junctional communication. During murine brain formation, Wnt1 and the gap-junctional protein connexin43 (Cx43) are co-expressed at the mid/hindbrain border, while interference with Wnt1 or Cx43 expression during embryogenesis leads to severe brain defects in the mid/hindbrain region.

Overexpression of c-Src in areas of hyperproliferation in head and neck cancer, premalignant lesions and benign mucosal disorders.

To examine which proteins are responsible for the elevated protein tyrosine kinase (PTK) activity in human head and neck squamous cell carcinoma (HNSCC) and adjacent histologically normal epithelium, paraffin embedded sections of these tissues were stained for PTK c-Src. Using double labeling techniques and antibodies against both the proliferation marker Ki-67 and PTK c-Src, we have shown that c-Src is overexpressed in areas of hyperproliferation in HNSCC, dysplastic epithelium, benign papillomas and inflamed normal tissue. Our data indicate that c-Src is (one of) the protein(s) responsible for the increased PTK activity in HNSCC.

Overexpression of EGFR and c-erbB2 causes enhanced cell migration in human breast cancer cells and NIH3T3 fibroblasts.

Overexpression of EGFR and c-erbB2 frequently occurs in human breast cancers, correlating with poor prognosis. Here we show that overexpression of EGFR and c-erbB2 in cell lines increases cell migration, an important step in metastasis formation. The effect of EGFR on migration is dependent on the addition of EGF to the cells.

p21waf1 can block cells at two points in the cell cycle, but does not interfere with processive DNA-replication or stress-activated kinases.

p21waf1 has been shown to mediate the p53-dependent growth arrest induced by DNA-damaging agents. Several functions have been ascribed to p21waf1 that could be involved in this growth arrest. For one, p21waf1 is an efficient inhibitor of cyclin-dependent kinases (CDKs).