RNA-binding protein PTB in spermatogenesis: Progress in studies / 中华男科学杂志

RNA binding proteins (RBPs) regulate the function of cells by interacting with nascent transcripts and therefore are receiving increasing attention from researchers for their roles in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. Further investigations on the post-transcriptional regulation mechanisms and isoforms of PTB proteins in the spermatogenesis show that PTB protein 1 (Ptbp1) is a predominant isoform in mitotic cells (spermatogonia), while Ptbp2 predominates in meiotic spermatocytes and postmeiotic spermatids and binds to the specific 3' untranslated region (3' UTR) of the phosphoglycerate kinase 2 (Pgk-2) mRNA, which helps to stabilize Pgk-2 mRNA in male mouse germ cells. In case of Ptbp2 inactivation in the testis, the differentiation of germ cells arrests in the stage of round spermatids, with proliferation of multinucleated cells in the seminiferous tubule, increased apoptosis of spermatocytes, atrophy of seminiferous tubules, and lack of elongating spermatids, which consequently affects male fertility. This article presents an overview on the structure of the PTB protein and its role in regulating mammalian spermatogenesis.

Fibrous dysplasia: molecular clonality analysis of 21 cases / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the neoplastic nature of fibrous dysplasia by molecular clonality approaches.</p><p><b>METHODS</b>Twenty-one cases of fibrous dysplasia were examined by clonality assays based on X-chromosomal inactivation mosiacism. Lesional and non-lesional tissues were microdissected from paraffin sections followed by DNA extraction. The DNA was predigested by HpaII or HhaI, and then amplified by nested PCR targeting phosphoglycerate kinase (PGK) and androgen receptor (AR) genes. Single nucleotide polymorphism (SNP) at the PGK locus was identified by incubation of the PCR products with Bst XI and agarose gel electrophoresis, and CAG repeat length polymorphism at AR locus was determined by denaturing polyacrylamide gel electrophoresis and visualized by silver staining.</p><p><b>RESULTS</b>Microscopically, all 21 cases showed characteristic features of fibrous dysplasia, including spindle fibrous cell proliferation and immature bone trabeculae at various proportions. DNA polymorphisms at AR locus and SNP of PGK gene were found in 15 of 21, and 4 of 21 cases, respectively. All 19 cases were monoclonal in nature. Two cases showed no polymorphism at either AR or PGK gene locus.</p><p><b>CONCLUSIONS</b>Fibrous dysplasia is likely a clonal, neoplastic process. Additional studies of larger number of cases are needed for a definitive conclusion.</p>

Downregulation of Peroxisome Proliferator-Activated Receptor (PPAR)alpha, PPARgamma, and Phosphoglycerate Mutase 2 in Prostate Cancer / 대한비뇨기과학회지

PURPOSE: To evaluate whether factors related to lipid and glucose metabolism have a potential role in the progression of prostate cancer, we measured the mRNA levels of the peroxisome proliferator-activated receptor (PPAR), fatty acid elongase (ELOVL), and two glycolytic enzymes in prostate cancer (CaP) tissues. MATERIALS AND METHODS: Prostate tissues, obtained from radical prostatectomy (n=10) and transurethral resection of prostate (n=18), were quickly frozen in liquid nitrogen for RNA measurements. Transcript signals of PPAR alpha, PPAR gamma, ELOVL2, ELOVL5, phosphoglycerate kinase 1 (PgK1) and phosphoglycerate mutase 2 (PgM2) were measured using a reverse-transcription polymerase chain reaction. RESULTS: The transcript signals of PPAR alpha and PPAR gamma were down-regulated in CaP tissues. In addition, the mRNA level of PgM2 in CaP tissues was lower than that in benign prostatic hyperplasia (BPH) tissues. However, the messages for ELOVL2, ELOVL5, and PgK1 were not significantly changed. CONCLUSIONS: These results suggest that lowering of the PPARalpha, PPARgamma and PgM2 messages may be involved in aberrant and uncontrolled prostate cell growth and differentiation.

Study of androgen receptor and phosphoglycerate kinase gene polymorphism in major cellular components of the so-called pulmonary sclerosing hemangioma / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clonality of polygonal cells and surface cuboidal cells in the so-called pulmonary sclerosing hemangioma (PSH).</p><p><b>METHODS</b>17 female surgically resected PSH were found. The polygonal cells and surface cuboidal cells of the 17 PSH cases were microdissected from routine hematoxylin and eosin-stained sections. Genomic DNA was extracted, pretreated through incubation with methylation-sensitive restrictive endonuclease HhaI or HpaII, and amplified by nested polymerase chain reaction for X chromosome-linked androgen receptor (AR) and phosphoglycerate kinase (PGK) genes. The length polymorphism of AR gene was demonstrated by denaturing polyacrylamide gel electrophoresis and silver staining. The PGK gene products were treated with Bst XI and resolved on agarose gel.</p><p><b>RESULTS</b>Amongst the 17 female cases of PSH, 15 samples were successfully amplified for AR and PGK genes. The rates of polymorphism were 53% (8/15) and 27% (4/15) for AR and PGK genes respectively. Polygonal cells and surface cuboidal cells of 10 cases which were suitable for clonality study, showed the same loss of alleles (clonality ratio = 0) or unbalanced methylation pattern (clonality ratio < 0.25).</p><p><b>CONCLUSIONS</b>The polygonal cells and surface cuboidal cells in PSH demonstrate patterns of monoclonal proliferation, indicating that both represent true neoplastic cells.</p>

Dynamic change of metabolism related protein in liver tissue of rats' model of hepatic fibrosis and regulatory effect of fuzheng huayu decoction on it / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the effect of Fuzheng Huayu decoction (FHD) intervention on hepatic fibrosis.</p><p><b>METHODS</b>Wistar rats were randomly divided into 3 groups: rats in the normal group only treated with subcutaneous injection of saline, rats in the model group and the FHD group were made into hepatic fibrosis by subcutaneous injection of 40% carbon tetrachloride (CCl4)-olive solution and then those in the FHD group were treated with FHD by gastric perfusion after modeling. Liver samples of the rats were obtained for routine pathological observation, hydroxyproline determination and proteome quantitative determination. After then, the proteome profile was obtained through 2-dimensional electrophoresis and silver staining, and analyzed. More than 30 proteins with different expression were identified by MALDI-TOF-MS.</p><p><b>RESULTS</b>(1) The integral response of vital movement such as body weight and activity of hepatic fibrosis declined in the CCl4 induced liver fibrosis rats; (2) Liver fibrosis were associated with abnormal metabolism; (3) There were four material metabolism-related protein showed by hepatic proteome mass spectrography, which expressed different between the normal and the fibrotic rats, i. e. the perchloric acid soluble protein, the phosphatidylinositol transferase, the phosphoglycerate kinase and the endoplasmic reticulum-60 protease; (4) The expressions of the above-mentioned four proteins in the FHD group were nearly the same as those of normal level.</p><p><b>CONCLUSION</b>(1) Liver fibrosis is accompanied with abnormal protein synthesis and decomposition, as well as the enhanced activity of glycolysis; (2) The existence of metabolism-related proteins is one of the elements for the liver in regulating metabolism; (3) The regulation on the expressions of metablism-related proteins is one of the pathways for FHD to exert its anti-hepatic fibrosis effect.</p>

X-chromosome inactivation patterns of different tumor nodules in multiple leiomyomas of uterus / 中华病理学杂志

<p><b>OBJECTIVE</b>To describe the relationship among different tumor nodules in multiple leiomyomas of uterus.</p><p><b>METHODS</b>Genomic DNA was extracted from fresh tissue samples, digested through incubation with methylation-sensitive Hha I or Hpa II, and amplified via PCR for androgen receptor (AR) and phosphoglycerate kinase (PGK) genes. The length polymorphism on AR gene was demonstrated by denaturing polyacrylamide gel electrophoresis and silver staining, the PGK gene products were treated with Bst XI and resolved on agarose gels.</p><p><b>RESULTS</b>112 cases of leiomyomas and one case of leiomyosarcoma were examined, 89% showing the length polymorphism for AR gene and 30% carrying the polymorphic Bst XI site at PGK locus. Loss of X-chromosome inactivation mosaicism was observed in all the 321 tumor nodules examined from 77 cases, reflecting their clonal cellular composition. The relationship between different nodules was evaluated by their X-chromosome inactivation patterns in the 295 tumor nodules taken from 57 multiple leiomyomas. Similar inactivated alleles were found in all nodules in 30, in most nodules in 7 cases, similar to a multi-nodular leiomyosarcoma, while 20 cases showed near-random distribution of the inactivated alleles in different nodules, indicating their multicentric origins. No relevance was found between this difference and any histopathological parameters including number of mitotic figures and occurrence of bizarre nuclei and degeneration. In addition, an identical mutation and loss of heterozygosity were found at the AR locus in two apparently discrete tumor nodules in one case, providing further evidence for the unicellular origin of these lesions.</p><p><b>CONCLUSIONS</b>The multi-nodular leiomyomas may be classified into multicentric, unicentric types, as well as a mixed type. It remains to be clarified whether different nodules in the unicentric cases originate from a parent tumor by migration or by spreading.</p>

Protein folding: a perspective for biology, medicine and biotechnology

At the present time, protein folding is an extremely active field of research including aspects of biology, chemistry, biochemistry, computer science and physics. The fundamental principles have practical applications in the exploitation of the advances in genome research, in the understanding of different pathologies and in the design of novel proteins with special functions. Although the detailed mechanisms of folding are not completely known, significant advances have been made in the understanding of this complex process through both experimental and theoretical approaches. In this review, the evolution of concepts from Anfinsen's postulate to the "new view" emphasizing the concept of the energy landscape of folding is presented. The main rules of protein folding have been established from in vitro experiments. It has been long accepted that the in vitro refolding process is a good model for understanding the mechanisms by which a nascent polypeptide chain reaches its native conformation in the cellular environment. Indeed, many denatured proteins, even those whose disulfide bridges have been disrupted, are able to refold spontaneously. Although this assumption was challenged by the discovery of molecular chaperones, from the amount of both structural and functional information now available, it has been clearly established that the main rules of protein folding deduced from in vitro experiments are also valid in the cellular environment. This modern view of protein folding permits a better understanding of the aggregation processes that play a role in several pathologies, including those induced by prions and Alzheimer's disease. Drug design and de novo protein design with the aim of creating proteins with novel functions by application of protein folding rules are making significant progress and offer perspectives for practical applications in the development of pharmaceuticals and medical diagnostics

Phosphoglycerate kinase--glyceraldehyde-3-phosphate dehydrogenase interaction: reaction rate studies.

Rate studies using phosphoglycerate kinase (PGK)--glyceraldehyde-3-phosphate dehydrogenase (GPDH) enzyme pair have been carried out to distinguish between the two mechanisms of intermediate metabolite transfer, namely diffusion through the solvent versus "substrate channelling" within an enzyme-enzyme complex. A procedure has been described for the assay of the rates of PGK-catalysed and the PGK-GPDH coupled reactions at high (saturating) GPDH concentration. With PGKs of rabbit muscle and yeast, the coupled reaction proceeded faster than the PGK-catalysed reaction. At a high salt concentration (0.5 M KCl), where a PGK-GPDH complex is known to dissociate, the two reactions proceeded at almost equal rates. At fixed PGK concentration, the rate of the coupled reaction at high (saturating) GPDH concentration varied with the nature (biological origin) of the latter enzyme. In the presence of 0.5 M KCl, the saturating rate values with different GPDHs were almost equal. The PGK-catalysed reaction exhibited typical Michaelian behaviour on varying the substrate concentrations (linear double reciprocal plots). The Km values for 3-PGA (0.51 mM) and ATP (0.40 mM) were independent of the concentration of the second substrate. The double reciprocal plots for the coupled reaction showed downward curvature, i.e. activation at higher substrate concentrations. The ratio of the rate of the coupled reaction: the rate of the PGK catalysed reaction was found to be a function of the nature of PGK, nature of GPDH, nature of buffer, pH, salt concentration and substrate concentrations. The ratio varied between close to unity at low substrate concentrations, to three when the Vmax values of the two reactions were compared. At low substrate concentrations, the rate of the coupled reaction became independent of the nature of GPDH. It has been suggested that in the PGK-GPDH pair, the intermediate metabolite (BPG) is transferred directly from one enzyme to the other within an enzyme-enzyme complex, except at high salt or low substrate concentrations. Under the latter conditions, data were consistent with metabolite transfer by diffusion. Implications of these results for coupled enzyme assays have been discussed.

Chromosomal localization of phosphoglycerate kinase (PGK) gene in Candida albicans using a heterologous probe.

The glycolytic enzyme phosphoglycerate kinase (PGK) is highly conserved throughout the evolutionary ladder. Using a previously cloned Saccharomyces cerevisiae PGK gene as hybridization probe, chromosomal localization and strain-specific variation of PGK gene in C. albicans have been examined. Separation of chromosome-size DNA fragments by pulse field gel electrophoresis followed by DNA- DNA hybridization analysis revealed that the PGK is encoded by a single copy gene located on the large chromosome of the commonly used laboratory strain ATCC 32354, as well as of several clinical isolates of C. albicans. Restriction fragment length analysis indicated that the PGK gene locus is highly polymorphic, and this strain-specific variation could be exploited to study intra-specific relatedness of clinical isolates.

Steady-state kinetic properties of phosphoglycerate kinase of mung beans.

Steady-state kinetics of the action of mung bean phosphoglycerate kinase have been investigated using 3-phosphoglycerate and ATP as substrates in the presence of Mg2+ ions. Keeping a constant and high Mg2+ concentration and varying the concentration of one of the substrates (ATP or 3-phosphoglycerates) at several fixed concentrations of the other substrate (3-phosphoglycerate or ATP), the Km values of Mg.ATP2- and 3-phosphoglycerate were found to be 0.42 and 0.60 mM, respectively. These values are independent of the concentration of the other substrate. A limiting value of Vmax, where the enzyme is saturated with both the substrates, was found to be 39.4 mumoles product formed per min per mg enzyme protein. This corresponds to a turnover number equal to 31.5 sec-1 (for molecular weight of the enzyme equal to 48,000). If [Mg2+] and [ATP4-] are held equal and varied together at several fixed concentrations of 3-phosphoglycerate, deviations from Michaelis-Menten kinetics (non-linear Lineweaver-Burk plots) are observed at lower values of ATP4- and Mg2+ (less than 0.1 mM), giving rise to apparent sigmoidicity in the rate versus [ATP4-] plots. It has been suggested that the real substrate for this enzyme is the Mg.ATP2- complex (and not free ATP4-). The complex dissociates at lower values of [Mg2+] and [ATP4-]. The resulting disproportionate decrease in the concentration of the complex brings about a steeper fall in the rate of reaction than is required by the Michaelis-Menten equation, giving rise to an apparent sigmoidicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Red cell phosphoglycerate kinase in insulin-dependent diabetes mellitus.

Red cell phosphoglycerate kinase (PGK) activity was determined in normal individuals and patients with, type I (insulin-dependent) diabetes and insulin treated diabetes. The PGK activity was significantly (P less than 0.001) elevated in diabetes, however it is restored to normalcy after insulin treatment (normal 282.54 +/- 9.46, type I diabetic 342.06 +/- 6.24, insulin treated diabetic 292.66 +/- 7.12 IU/g haemoglobin at 37 degrees C). No significant alteration was observed in the percentage of PGK bound to the membrane fraction of red cells in all the three conditions. The results indicate that the increased PGK activity is a result of a regulatory mechanism induced by the fluctuation of ATP level in response to elevated Na:K pump rate of erythrocytes in type I diabetes mellitus.

Alteraçöes enzimáticas eritrocitárias na hemofilia A / Erythrocyte enzymatic changes in hemophilia A

Cinco quinases eritrocitárias foram estudadas em pacientes portadores de hemofilia A; foi encontrado um aumento das atividades da fosfogliceratoquinase e da adenilatoquinase. Este achado sugere que há alteraçöes metabólicas eritrocitárias na hemofilia