Exclusion mapping of 12 X-linked disease loci and 10 DNA probes from the long arm of the X-chromosome

Specific chromosome rearrangements associated with disease entities are invaluable resources for physical mapping. A deletion on the X chromosome of a male leads to the nullisomy for X-linked genes, resulting in the onset of genetic diseases and/or the absence of the DNA probe detectable sequences. This permits the localization of these loci within the deleted area. On the other hand, the region for some other X-linked loci can be excluded from the deleted area according to the absence of the characteristic symptoms of the disease and/or the presence of the hybridization signals. An interstitial deletion on the long arm of the X chromosome of a male has been characterized by high resolution banding. The karyotype of the proband is 46,Y,del(X)(pter----q21.1::q21.33----qter). The regions for 12 X-linked disease loci as well as 10 DNA probes are excluded from the deleted area, and localized either proximally or distally to the deletion. The results also reveal a controversy in the present linkage data concerning the assignment of these loci.     

Pharmacologically inhibiting phosphoglycerate kinase 1 for glioma with NG52

Inhibition of glycolysis process has been an attractive approach for cancer treatment due to the evidence that tumor cells are more dependent on glycolysis rather than oxidative phosphorylation pathway.Preliminary evidence shows that inhibition of phosphoglycerate kinase 1(PGK1)kinase activity would reverse the Warburg effect and make tumor cells lose the metabolic advantage for fueling the proliferation through restoration of the pyruvate dehydrogenase(PDH)activity and subsequently promotion of pyruvic acid to enter the Krebs cycle in glioma.However,due to the lack of small molecule inhibitors of PGK1 kinase activity to treat glioma,whether PGK1 could be a therapeutic target of glioma has not been pharmacologically verified yet.In this study we developed a high-throughput screening and discovered that NG52,previously known as a yeast cell cycle-regulating kinase inhibitor,could inhibit the kinase activity of PGK1(the IC50=2.5±0.2μM).We showed that NG52 dose-dependently inhibited the proliferation of glioma U87 and U251 cell lines with IC50 values of 7.8±1.1 and 5.2±0.2μM,respectively,meanwhile it potently inhibited the proliferation of primary glioma cells.We further revealed that NG52(12.5–50μM)effectively inhibited the phosphorylation of PDHK1 at Thr338 site and the phosphorylation of PDH at Ser293 site in U87 and U251 cells,resulting in more pyruvic acid entering the Krebs cycle with increased production of ATP and ROS.Therefore,NG52 could reverse the Warburg effect by inhibiting PGK1 kinase activity,and switched cellular glucose metabolism from anaerobic mode to aerobic mode.In nude mice bearing patient-derived glioma xenograft,oral administration of NG52(50,100,150 mg·kg?1·d?1,for 13 days)dose-dependently suppressed the growth of glioma xenograft.Together,our results demonstrate that targeting PGK1 kinase activity might be a potential strategy for glioma treatment.     

Trilineage Response to rhG-CSF with Subsequent Clonal Hematopoiesis in a Patient with Severe Bone Marrow Aplasia

We treated a patient with severe aplastic anemia with long-term administration of recombinant human granulocyte-colony stimulating factor (rhG-CSF). When a trilineage response of hematopoiesis was obtained after the first treatment, a chromosomal change [45XX, -7] was observed in 20 of the 20 metaphases examined. Later, we were able to show a monoclonal X inactivation pattern in the phosphoglycerate kinase (PGK) gene in the peripheral blood polymorphonuclear leukocytes and mononuclear cells, indicating the presence of clonal hematopoiesis regardless of the disappearance of the karyotype abnormality. We suggest that it is important to pay close attention to the appearance of clonal hematopoiesis during the administration of G-CSF to patients with idiopathic severe bone marrow aplasia.     

Correlation Studies of Morphological and Biochemical Parameters in Human Ejaculate in Various Andrological Diagnoses. 3rd Report: Correlations between Morphological and Biochemical Parameters

Untersuchungen zur Korrelation morphologischer und biochemischer Meßgrößen im menschlichen Ejakulat bei verschiedenen andrologischen Diagnosen. III. Mitteilung: Beziehungen zwischen morphologischen und biochemischen Parametern Als Abschluß einer umfangreichen Studie über morphologische und biochemische Meßgrößen im menschlichen Ejakulat wird das Ergebnis einer Untersuchung von Korrelationen zwischen diesen beiden Parametem mitgeteilt. Alle Untersuchungen der I.–III. Mitteilung zu diesem Problemkomplex sind am gleichen Untersuchungsmaterial vorgenommen worden. Alle Untersuchungen sind jeweils 10 Minuten nach der Ejakulation durchgeführt worden, nachdem das Ejakulat sich verflüssigt hatte; das bedeutet, daß diese Intervallzeit für alle vorgelegten Untersuchungen in gleicher Weise zutrifft und daß die Untersuchungen Einblick in die aktuelle Situation des Stoffwechsels zu diesem Zeitpunkt gestatten. Es kann unter Berücksichtigung der Ergebnisse, für die eine statistische Signifikanz von p < 0,05 ermittelt werden konnte, festgestellt werden, daß höhere Fruktosekonzentrationen mit einer niedrigeren Spermatozoendichte einhergehen; gleichzeitig ergibt sich, daß die höheren Fruktosekonzentrationen mit einem höheren Prozentsatz an unbeweglichen und an pathologisch veränderten Spermatozoen korrelieren. Ähnliche interessante Beziehungen lassen sich für einige Substrate und Enzyme herstellen. Hier ist die wesentlichste Feststellung, daß bei den z.T. erhöhten Aktivitäten für PGK, PGI und ATPase bei höherer Spermatozoendichte die Möglichkeit einer Diffusion der Enzyme aus den zellulären Bestandteilen zu diskutieren ist, da die Analysen im Spermaplasma vorgenommen worden sind. Daraus wird die Schlußfolgerung gezogen, in einer weiteren Versuchsreihe an einem neuen Untersuchungsmaterial entsprechende Messungen in den zellulären Elementen des Ejakulates (Spermatozoen, Rundzellen) vorzunehmen.     

The majority of T lymphocytes are polyclonal during the chronic phase of chronic myelogenous leukemia

To clarify the extent of cell lineage involvement in chronic myelogenous leukemia (CML), we investigated the bcr gene rearrangement and clonality using the X-chromosome-linked restriction fragment length polymorphism (RFLP) methylation method in T lymphocytes and granulocytes. We examined the granulocyte and T-cell fractions from the peripheral blood of seven female patients with CML during the chronic phase; patients were heterozygous for RFLPs at the phosphoglycerate kinase (PGK) or the hypoxanthine phosphoribosyltransferase (HPRT) gene. RFLP-methylation analysis of granulocytes demonstrated a monoclonal pattern in six of the seven patients and a rearrangedbcr gene in all seven patients. In contrast, T lymphocytes exhibited a polyclonal pattern in six cases; in one case, a faint band was observed following methyl-sensitive enzyme cleavage. Thebcr gene analysis in T lymphocytes showed the germline in every case. Our results indicate that the majority of T lymphocytes are polyclonal during the chronic phase of CML and confirm previous reports based on glucose-6-phosphate dehydrogenase, cytogenetic, andbcr rearrangement analyses.     

Carrier Detection in Agammaglobulinemia by X Chromosome Inactivation Analysis

Using a recently developed strategy to analyze patterns of X chromosome inactivation in cell populations, we found that two mothers and a sister were carriers in three atypical or sporadic cases of patients with agammaglobulinemia, two of whom were brothers. In this study, a phosphogiycerate kinase 1 (PGK1) gene probe was used to detect patterns of methylation of X-chromosome genes. A random pattern of X inactivation was observed in isolated peripheral blood granulocytes. In contrast, one of the two X chromosomes was preferentially active in the Epstein-Barr virus (EBV)-transformed peripheral B cells of the family members of these patients. The volume of the blood specimen could be significantly reduced using EBV-transfomed B cell lines which contained multiple clones. The analysis described here can be used to distinguish between X-linked agammaglobulinemia (XLA) and other forms of a- or hypogammaglobulinemia as well as to detect the carrier state.     

Cloning and sequencing of the 3-phosphoglycerate kinase (PGK) gene from Penicillium citrinum and its application to heterologous gene expression

The gene coding for 3-phosphoglycerate kinase (PGK) in ML-236B (compactin)-producing Penicillium citrinum was isolated from the recombinant phage lambda library using the corresponding Aspergillus nidulans pgk gene as a probe. The P. citrinum pgk gene has an open reading frame of 1,254 bp, encoding a protein of 417 amino acids with a predicted molecular weight of 44,079 daltons. The position of the two introns, 59 and 60 bp respectively, was deduced from an homology comparison with the sequence of the A. nidulans pgk gene. The PGK protein of P. citrinum shows extensive high homology to the PGKs of four other fungi: P. chrysogenum (93%), A. nidulans (84%), Trichoderma reesei (78%) and Saccharomyces cerevisiae (68%). Almost total conservation is found in P. citrinum of residues thought to be important for the structure and function of the yeast enzyme. The strong codon preference found has greater similarity to that in other filamentous fungi than in yeast. A DNA fragment encompassing the pgk gene was shown to hybridize a 1.35-kb poly(A)⁺RNA, sufficient to encode the PGK polypeptide. A fused gene, pgk-hpt, containing the putative pgk promoter and the open reading frame of the Escherichia coli hygromycin B phospho-transferase (hpt) gene was constructed, and was successfully used to transform P. citrinum to a hygromycin B (HmB)-resistant phenotype.     

In vivo effect of nitrogen dioxide on the activities of glycolytic enzymes in red blood cells of rats

The effects of short-term exposure to NO2 on the glycolytic enzymes of rat red cells were examined. Exposure to 10 ppm NO2 resulted in decrease in activities of pyruvate kinase (PK) and phosphofructokinase (PFK) at day 1 and then increased progressively. Exposure to 4 ppm NO2 caused progressive increases in these enzyme activities from the first day. At day 5, the PK and PFK activities of exposed animals became higher than those of controls followed by decreasing to the control level at day 10. Red blood cells were fractionated according to their ages. The specific activities of PK and PFK decreased as red cells became older. 7-day exposure to 4 ppm NO2 resulted in elevation of PK and PFK activities in all red cell subfractions. In addition, the amount of youngest cells decreased concomitant with increases in those of older cells. These findings suggest that NO2 inhalation enhances red cell turnover.     

A novel combined approach to detect androgenic activities with yeast based assays in Schizosaccharomyces pombe and Saccharomyces cerevisiae

We describe the construction and validation of novel test systems for detecting androgenic activities using a combination of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. By applying the reporter enhanced Green Fluorescent Protein (EGFP) the incubation time could be reduced to only 24h if compared to the classical β-galactosidase reporter (48 h). Both yeast systems were validated by analyzing the effects of seven androgens as well as five anti-androgens. One androgen (stanozolol) could be detected ten times more sensitive in S. cerevisiae than in S. pombe. Three of the five anti-androgenic substances showed no or only a slight effect in both yeast assays. The other two anti-androgens could be detected much better in S. pombe. Additionally, we could show that both yeast assays tolerated 10% urine within the media and still were capable to detect dihydrotestosterone at a concentration of 10(-8)M suggesting the use of the assays for applied doping pre-screening. In summary, the novel androgen-sensitive yeast assays have a large potential for various applications, e.g. as pre-screening in doping analysis or cattle feeding. A combination of both assays, exploiting these two phylogenetic very different yeasts, allows detection of the activity of a wide range of androgenic substances.     

PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

Objective: Glycogen synthase kinase-3β(GSK3β) has been recognized as a suppressor of Wnt/β-catenin signaling, which is critical for the stemness maintenance of breast cancer stem cells. However, the regulatory mechanisms of GSK3β protein expression remain elusive.Methods: Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β, and to characterize the interactions of GSK3β, heat shock protein 90(Hsp90), and co-chaperones. The role of PGK1 in Hsp90 ch...