Biochemical and molecular medicine最新文献

{"title":"Characterization of Human Proximal Tubular Cells after Hypoxic Preconditioning: Constitutive and Hypoxia-Induced Expression of Heat Shock Proteins HSP70 (A, B, and C), HSC70, and HSP90","authors":"Martin A. Turman ,&nbsp;Daniel A. Kahn ,&nbsp;Scott L. Rosenfeld ,&nbsp;Courtney A. Apple ,&nbsp;Carlton M. Bates","doi":"10.1006/bmme.1996.2556","DOIUrl":"10.1006/bmme.1996.2556","url":null,"abstract":"<div><p>In animal models of cardiac or cerebral ischemic preconditioning, induction of heat shock proteins (HSPs), especially HSP70, correlates with protection from subsequent injury. The extent of HSP70 induction after stress correlates inversely with initial HSP70 levels. Primate cells, unlike nonprimate cells, express high basal levels of HSP70; thus, primate cells may respond differently to preconditioning than nonprimate cells. We have demonstrated that exposing cultured human proximal tubular epithelial cells (PTEC) to 12 h of hypoxia followed by a 24-h recovery period (hypoxic preconditioning) induces resistance to subsequent hypoxic injury. Herein, we characterize the expression of HSP70, HSP90, and heat shock cognate-70 (HSC70) in PTEC under basal conditions and after hypoxic preconditioning. By Northern blot analysis, we demonstrate that hypoxic preconditioning of PTEC increases mRNA for HSP70 &gt; HSP90 &gt; HSC70. With reverse transcription and polymerase chain reaction, mRNA transcripts from three different HSP70 genes (HSP70 A, B, and C) were detected in unstressed PTEC. Transcripts from these genes were also detected in freshly isolated human renal cortex, indicating that all three genes are expressed<em>in vivo.</em>By Western blot analysis, we demonstrate that PTEC express high basal levels of HSP70, HSC70, and HSP90. Hypoxic preconditioning did not lead to a significant increase in protein content of any of these HSPs, despite increased mRNA levels. This suggests that HSP accumulation cannot account for the development of cytoresistance after hypoxic preconditioning in PTEC. However, high basal expression of HSP70 in human PTEC may contribute to their innate resistance for hypoxia.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"60 1","pages":"Pages 49-58"},"PeriodicalIF":0.0,"publicationDate":"1997-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.2556","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20021550","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Growth Factor Control of Growth and Epithelial Differentiation in Embryonic Lungs","authors":"Heber C. Nielsen,&nbsp;Ana Martin,&nbsp;MaryAnn V. Volpe,&nbsp;Dimitrios Hatzis,&nbsp;Robert J. Vosatka","doi":"10.1006/bmme.1996.2560","DOIUrl":"10.1006/bmme.1996.2560","url":null,"abstract":"<div><p>Branching morphogenesis and epithelial differentiation occur simultaneously during lung development, hence controlling factors may affect both aspects of development simultaneously. We hypothesized that in the embryonic lung EGF and TGFβ1 alter both epithelial differentiation and developmental morphogenesis. Day 10<span><math><mtext>1</mtext><mtext>2</mtext></math></span>embryonic lung cultures were exposed to either EGF (10 ng/ml) or TGFβ1 (2 ng/ml) for 72 h, and branching morphogenesis, cell proliferation, and epithelial differentiation (the expression of DSPC synthesis and of surfactant protein C (SP-C) mRNA) were studied. EGF treatment stimulated branching morphogenesis (measured as the number of terminal left lung buds), epithelial differentiation, and cell proliferation. Branching morphogenesis was increased compared to controls after 48 h of culture by 47% and after 72 h by 34% (<em>P</em>&lt; 0.0005). Choline incorporation into DSPC was stimulated by 343% (<em>P</em>= 0.05). SP-C expression was increased sixfold. Thymidine incorporation was stimulated by 49% (<em>P</em>&lt; 0.05). The effects of EGF on thymidine labeling were distributed among epithelial cells of the airway walls and of the branching tips, and also the mesenchyme (<em>P</em>&lt; 0.01 for each area compared to controls). In contrast, TGFβ1 did not alter the number of terminal left lung buds, inhibited choline incorporation into DSPC by 35% (<em>P</em>&lt; 0.05), and had no effect on thymidine incorporation (87% of control). There was increased thymidine labeling at the branching tips (<em>P</em>&lt; 0.01), while other areas were not different from controls. We conclude that both EGF and TGFβ1 affect the development of branching morphogenesis and of epithelial differentiation in the embryonic lung.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"60 1","pages":"Pages 38-48"},"PeriodicalIF":0.0,"publicationDate":"1997-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.2560","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20021549","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Role of Nitric Oxide in the Regulation of Cellular Iron Metabolism","authors":"Joseph B. Domachowske","doi":"10.1006/bmme.1996.2557","DOIUrl":"10.1006/bmme.1996.2557","url":null,"abstract":"<div><p>Eukaryotic cellular iron homeostasis becomes impaired during inflammation, manifesting itself most dramatically as the anemia of chronic disease. This alteration in cellular iron metabolism is the result of a complex network of events, acting at the transciptional and translational levels to alter the expression of proteins involved in the uptake, storage, and utilization of iron. With the discovery of nitric oxide (NO), its role in host defense, and its interactions with a number of different iron-containing proteins, investigators have begun unravelling the connection between iron metabolism and NO. Following a brief discussion of normal cellular iron metabolism, this review focuses on alterations in iron homeostasis observed during inflammation with an emphasis on the role of NO. A working model involving NO in the pathogenesis of the anemia of chronic disease is proposed.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"60 1","pages":"Pages 1-7"},"PeriodicalIF":0.0,"publicationDate":"1997-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.2557","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20023600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-Resolution Detection of PCR Products from a Microsatellite Marker Using a Nonradioisotopic Technique","authors":"Debomoy K. Lahiri ,&nbsp;Aiwu Zhang ,&nbsp;John I. Nurnberger Jr.","doi":"10.1006/bmme.1996.2558","DOIUrl":"10.1006/bmme.1996.2558","url":null,"abstract":"<div><p>We report a safe, rapid, and economical method for polymerase chain reaction (PCR)-based genotype analysis using a microsatellite marker specific for the human chromosome 18 locus, D18S53. This method does not involve radioisotopes and makes use of ethidium bromide fluorescence to detect PCR products. Our method enables direct analysis and easy detection of PCR products on nondenaturing polyacrylamide gels. The genotyping using this method can be scaled up to 100 samples at one time by adding a step of “double loading” of samples in a single sequencing size gel. We could resolve PCR products and DNA fragments, differing in size by only 2 bp, in the range of 150–200 bp by a 7% nondenaturing polyacrylamide gel. This technique can be applied for population-based genomic screening and linkage analysis.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"60 1","pages":"Pages 70-75"},"PeriodicalIF":0.0,"publicationDate":"1997-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.2558","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20021552","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Association of an Exon 3 Mutation (Trp66 → Gly) of the LDL Receptor with Variable Expression of Familial Hypercholesterolemia in a French Canadian Family","authors":"Emile Levy ,&nbsp;Anne Minnich ,&nbsp;Suzanne Lussier Cacan ,&nbsp;Louise Thibault ,&nbsp;Louise-Marie Giroux ,&nbsp;Jean Davignon ,&nbsp;Marie Lambert","doi":"10.1006/bmme.1996.2549","DOIUrl":"10.1006/bmme.1996.2549","url":null,"abstract":"<div><p>The ligand-binding domain of low-density lipoprotein (LDL) is composed of seven 40-amino-acid repeats encoded by exons 2–6. Previous studies identified a missense mutation in codon 66 of exon 3, which resulted in the production of LDL receptor protein that is not processed to its mature form. In the current investigation, we documented the presence of two identical mutant LDL receptor alleles (Trp₆₆→ Gly) in two familial hypercholesterolemia (FH) probands, II-1 and II-2, associated with markedly elevated plasma LDL cholesterol (17.22 ± 0.78 and 11.95 ± 0.24 mmol/liter, respectively). Functional assays of their fibroblast LDL receptor showed inefficient binding (39 and 50%), internalization (33 and 37%), and degradation (32 and 37%) compared with controls. The contribution of the apo B gene to variation in LDL levels was virtually eliminated given the normal ligand interaction with cell surface receptors and the absence of the mutation occurring in codon 3500 of the apo B gene. Similarly, the homozygous apo E₃/E₃wildtype phenotype excluded any genetic contribution of apo E to the lipoprotein abnormalities. Furthermore, the LPL mutations commonly observed in French Canadians could not account for the observed lipid alterations. Several alterations in lipoprotein composition characterized VLDL, IDL, LDL, HDL₂, and HDL₃fractions. Moreover, defective intestinal fat transport was observed in both probands (II-1 and II-2). Thus, the disturbance of lipoprotein concentration, composition, size, and metabolism may in part be related to the exon 3 mutation (Trp₆₆→ Gly) of the LDL receptor gene. The biochemical phenotype was more severe in the father (I-1) than in the mother (I-2), and in the younger homozygous proband (II-1) than in the older (II-2). The greater severity was associated with a higher LDL cholesterol/HDL cholesterol ratio. Whether the differences between the two probands are due to polygenic factors or to a metabolic consequence of a major nonallelic trait is unknown. Nevertheless, the present biochemical findings stress the extent of the lipid abnormalities associated with homozygous FH and the importance of the phenotypic variability encountered even among subjects carrying the same mutation.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"60 1","pages":"Pages 59-69"},"PeriodicalIF":0.0,"publicationDate":"1997-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.2549","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20021551","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exclusion of the Nuclear Factor-κB3 (REL A) Gene as Candidate for the Multiple Endocrine Neoplasia Type 1 (MEN 1) Gene","authors":"Rudy M. Landsvater ,&nbsp;Mireille J. de Wit ,&nbsp;Luke F. Peterson ,&nbsp;Richard J. Sinke ,&nbsp;Ad Geurts van Kessel ,&nbsp;Cornelis J.M. Lips ,&nbsp;Jo W.M. Höppener","doi":"10.1006/bmme.1996.2561","DOIUrl":"10.1006/bmme.1996.2561","url":null,"abstract":"<div><p>Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by neoplasia and hyperplasia in specific endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a region of approximately 900 kb on chromosome 11q13. The nuclear factor-κB (NF-κB) is a transcription factor with pleiotropic expression, which is involved in the regulation of expression of many cellular genes. The p50/p65 heterodimer is the most abundant form of NF-κB. The gene encoding the p65 subunit (NF-κB3/REL A) was recently localized in the 900-kb MEN 1 region and was considered a good candidate gene for MEN 1. The structure and nucleotide sequence of the NF-κB3 coding region in MEN 1 patients were compared with those of non-MEN 1 subjects, to determine the potential role of this gene in MEN 1 tumorigenesis. Southern blot analysis with constitutional DNA from probands of 14 independent MEN 1 families and DNA from four MEN 1 tumor specimens did not reveal any structural abnormality of the NF-κB3 gene. Direct sequencing of cDNAs from two affected subjects from 2 different MEN 1 families, as well as nucleotide sequence analysis of exon/intron boundaries in these patients, did not reveal MEN 1-specific point mutations or other small structural aberrations in the NF-κB3 gene. These results make it very unlikely that the NF-κB3 gene is the gene responsible for the development of MEN 1.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"60 1","pages":"Pages 76-79"},"PeriodicalIF":0.0,"publicationDate":"1997-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.2561","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20021553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular Cloning and Characterization of Rab27a and Rab27b, Novel Human Rab Proteins Shared by Melanocytes and Platelets","authors":"David Chen ,&nbsp;Juanru Guo ,&nbsp;Toru Miki ,&nbsp;Masayoshi Tachibana ,&nbsp;William A. Gahl","doi":"10.1006/bmme.1996.2559","DOIUrl":"10.1006/bmme.1996.2559","url":null,"abstract":"<div><p>Rabs are prenylated, membrane-bound proteins involved in vesicular fusion and trafficking. We isolated the complete cDNAs of two rab isoforms, rab27a and rab27b, from human melanoma cells and melanocytes. Rab27a is the human homolog of a rat megakaryocyte rab called ram p25. Rab27b corresponds to a small GTP-binding protein, c25KG, which was previously purified from platelets but whose cDNA had not been cloned. Sequence comparisons with known rabs indicate that rab27a and rab27b comprise a melanocyte/platelet subfamily within the rab family. In addition, rab27a was expressed in a large variety of cell and tissue types, excluding brain, and rab27b manifested itself primarily in testis. Bacterially expressed and purified rab27a and rab27b exhibited GTP-binding activity and can now be used for antibody production and studies of the substrate specificities of geranylgeranyl transferase. In addition, the expression of rab27a and rab27b in both melanocytes and platelets makes them candidates for involvement in mouse and human disorders characterized by the combination of pigment dilution and a platelet storage pool defect.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"60 1","pages":"Pages 27-37"},"PeriodicalIF":0.0,"publicationDate":"1997-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.2559","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20023602","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"8th International Center for Biotechnology Symposium on Cytoplasmic Protein Tyrosine Kinases","authors":"","doi":"10.1006/bmme.1996.2555","DOIUrl":"https://doi.org/10.1006/bmme.1996.2555","url":null,"abstract":"","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"60 1","pages":"Page 80"},"PeriodicalIF":0.0,"publicationDate":"1997-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.2555","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"137415303","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Apoptosis and Free Radicals","authors":"Irina Stoian ,&nbsp;Andra Oros ,&nbsp;Elena Moldoveanu","doi":"10.1006/bmme.1996.0072","DOIUrl":"10.1006/bmme.1996.0072","url":null,"abstract":"<div><p>Free radicals that appear during physiological processes may lead to apoptosis in some pathological conditions when antioxidant capacity of the tissue is surpassed. Additionally, free radicals are involved in the control of apoptosis; antioxidant agents suppress apoptosis induced by a variety of stimuli. The possibility that apoptosis is regulated by modulation of the levels of free radicals is discussed.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 93-97"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0072","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19947590","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Measurement of GLUT mRNA in Liver of Fetal and Neonatal Rats Using a Novel Method of Quantitative Polymerase Chain Reaction","authors":"Robert H. Lane ,&nbsp;Annette S. Flozak,&nbsp;Rebecca A. Simmons","doi":"10.1006/bmme.1996.0087","DOIUrl":"10.1006/bmme.1996.0087","url":null,"abstract":"<div><p>Transfer of glucose into the hepatocyte is mediated by glucose transporters (GLUTs). GLUT mRNA levels are usually measured by Northern blot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) is often used to measure RNA abundance. However, this method is only semiquantitative and has no internal control during first-strand synthesis. We designed a method of coreverse transcription and PCR amplification using bovine rhodopsin as an internal control for both cDNA synthesis and amplification. As part of the validation of this technique, we determined that there was no nonspecific amplification of bovine GLUTs by rhodopsin primers, that there were no differences in amplification due to different regions of the Glut gene amplified, and that there were no secondary structure effects on amplification. We applied our modified method of RT-PCR to measure the ontogeny of GLUT expression in liver of fetal and postnatal rats (d20 fetuses and d1, d4, d14, and d21 juvenile rat pups). GLUT 1 mRNA quantity decreased whereas GLUT 2 increased with age. We were able to detect small quantities of GLUT 3 in fetal liver and of GLUT 5 in postnatal liver. This method of RT-PCR provides an internal control and allows measurement of mRNA levels in small quantities of tissue, making it ideal for use in the fetus and any system in which mRNA levels are low.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 192-199"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0087","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19948039","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}