bluuebunny/crossref_metadata_2025 huggingface dataset:
Pitfalls in prenatal diagnosis: cytogenetic analysis in amniocytes fails to detect mosaic r(12)
Authors: de Pater J. M. , Scheres J. M. J. C. , Christiaens G. C. M. L. , Ausems M. G. E. M.
Published: February 2003

Objective

To review the accuracy of a prenatal diagnosis of a missed chromosomal mosaicism in amniotic fluid cell cultures and to see whether adapting the Dutch guidelines would have made any difference to the outcome in this case.

Method

Metaphases, obtained from cultured amniocytes and peripheral blood lymphocytes, were analyzed with different results. The amniocyte cultures were then reanalyzed and the risk of missing this mosaicism in prenatal analysis was assessed.

Results

The prenatal tests performed according to the Dutch guidelines showed a normal female karyotype, but more extensive postnatal analysis revealed a ring chromosome in 50% of the child's lymphocytes. Reanalysis of the original amniocytes confirmed the normal diagnosis, but when more cells from the same and other colonies were analyzed, the ring chromosome was detected.

Conclusion

The chance of missing such a supernumerary ring mosaicism is very low (about 2% in our case). Given its very rare occurrence and the low chance of it being missed if the existing Dutch guidelines are followed, adapting the number of cells or colonies to be examined for all prenatal diagnoses does not appear to be justified. Copyright © 2003 John Wiley & Sons, Ltd.
A simple and sensitive erythroblast scoring system to identify fetal cells in maternal blood
Authors: Cha Donghyun , Hogan Brad , Bohmer Ralph M. , Bianchi Diana W. , Johnson Kirby L.
Published: February 2003

Objectives

Nucleated red blood cells (NRBCs) of fetal origin appear to have distinguishable characteristics from that of maternal NRBCs in both nuclear morphology and properties of hemoglobin staining. However, these differences have yet to be quantified. Our aim was to develop an erythroblast scoring system using four distinct phenotypic parameters (nuclear roundness, nuclear morphology, gamma hemoglobin staining intensity, and peripheral brightness of the stained cytoplasm) to address this issue.

Methods

NRBCs were isolated from four maternal blood samples by density gradient separation, CD15/45 depletion, and gamma hemoglobin positive selection after elective termination of a trisomy 21 male fetus (47,XY,+21). All cells were deposited onto microscope slides and every NRBC was analyzed according to the scoring system. Each of the four individual parameters was given a value from 0 to 3 points and a combined score was obtained for each cell (range 0–12). Fluorescence in situ hybridization (FISH) using X‐ and Y‐specific probes was performed to determine, on the basis of interphase karyotype, whether the cell was maternal or fetal.

Results

The majority of maternal NRBCs were found to have a combined score of 6 or less (103/117) and the majority of fetal NRBCs were found to have a score of 7 or greater (43/53). The proportion of cells that were identified correctly as fetal increased with each ascending category of combined score. For example, 5.7% of NRBCs with a combined score of 5 points or less were found to be fetal, whereas 19.2% of NRBCs with a combined score of 6 points were fetal. At combined scores of 11 and 12 points, 100% of NRBCs were found to be fetal.

Conclusion

Fetal NRBCs have characteristic morphology and a gamma hemoglobin staining appearance that makes them distinguishable from maternal NRBCs. The scoring system presented here is a simple and sensitive method to distinguish fetal NRBCs from adult cells in maternal blood. This system may have clinical utility for noninvasive prenatal diagnosis as well as applications for basic research into the developmental biology of NRBCs. In addition, these defined parameters may serve as computational classifiers for the automated detection of fetal cells in maternal blood. Copyright © 2003 John Wiley & Sons, Ltd.
Prenatal exclusion of Leigh syndrome due to T8993C mutation in the mitochondrial DNA
Authors: Leshinsky‐Silver Esther , Perach Michal , Basilevsky Erena , Hershkovitz Eli , Yanoov‐Sharav Miri , Lerman‐Sagie Tally , Lev Dorit
Published: February 2003

Leigh syndrome (LS) is a mitochondrial encephalopathy that is caused by a mutation either in the mitochondrial DNA (mtDNA) or in the nuclear encoded genes of the mitochondrial proteins. Prenatal diagnosis of defects in the mtDNA is usually problematic because of mtDNA heteroplasmy and tissue specificity. However, the mutations T8993 G/C in the ATP synthase subunit 6 gene of the mtDNA show a more even tissue distribution and do not appear to change significantly over time. There are only few reports of prenatal diagnosis of the T8993G mutation in Leigh disease. Here we describe the first prenatal genetic testing of T8993C in a fetus of a mother whose previous child had died of Leigh syndrome due to the T8993C mutation. Mutant load in the chorionic villus sample (CVS) as well as in amniocytes was undetectable, thus predicting a very high likelihood of an unaffected outcome, indicative of a healthy baby. The diagnosis was confirmed after birth. Gathering data on the prenatal diagnosis of mtDNA mutations is of great importance so that prenatal diagnosis of both T8993G and T8993C mutations can be offered routinely. Copyright © 2002 John Wiley & Sons, Ltd.
Detection of gamma‐globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization
Authors: Larsen Rasmus Dines , Schønau Andreas , Thisted Marianne , Petersen Kenneth Heesche , Lohse Jesper , Christensen Britta , Philip John , Pluzek Karl‐Johan
Published: February 2003

Objectives

Fetal nucleated red blood cells (NRBC) that enter the peripheral blood of the mother are suitable for non‐invasive prenatal diagnosis. The application of peptide nucleic acid (PNA) probes for tyramide amplified flow fluorescence in situ hybridization (FISH) detection of gamma‐globin mRNA in fixed fetal NRBC is investigated.

Methods

Hemin‐induced K562 cells or nucleated blood cells (NBC) from male cord blood were mixed with NBC from non‐pregnant women and analysed using both slide and flow FISH protocols. Post‐chorionic villus sampling (CVS) blood samples from pregnant females carrying male fetuses were flow‐sorted (2 × 106 NBC/sample). Y chromosome–specific PNA FISH was used to confirm that the identified gamma‐globin mRNA stained cells were of fetal origin.

Results

Flow FISH isolated gamma‐globin mRNA positive NBCs showing characteristic cytoplasmic staining were all Y positive. The amplification system generated a population of false positive cells that were, however, easy to distinguish from the NRBCs in the microscope.

Conclusion

The gamma‐globin mRNA specific PNA probes can be used for detection and isolation of fetal NRBCs from maternal blood. The method has additional potential for the study of gamma‐globin mRNA levels or the frequency of adult NRBC (F cells) in patients with hemoglobinopathies. Copyright © 2003 John Wiley & Sons, Ltd.
Descriptive analyses of the integrity of a US Medicaid claims database
Authors: PharmD Sean Hennessy , Bilker Warren B. , Weber Anita , Strom Brian L.
Published: February 2003

Purpose

To examine the integrity of six Medicaid databases for use in pharmacoepidemiology research.

Methods

We performed descriptive analyses to examine four categories of potential data errors: incomplete claims for certain time periods; absence of an accurate indicator of inpatient hospitalizations; missing hospitalizations for those aged 65 years and over; and diagnostic codes in demographic groups in which those conditions should be rare.

Results

Prescription claims appeared to be missing intermittently in some states. No valid marker of inpatient hospitalizations could be found for three of six states. Hospitalizations appeared to be missing to varying degrees for those aged 65 years and over. Gross errors in diagnostic codes and demographic data did not appear to be widespread.

Conclusions

Whenever possible, investigators using administrative data should perform macro‐level descriptive analyses on the parent data set. In particular, researchers should examine the number of medical and pharmacy claims over time, looking for gaps. Validity of markers of hospitalization should be assessed. The accuracy of diagnosis and demographic data should be examined. Such a descriptive macro‐level approach should be used to supplement, and perhaps precede validation of study outcomes using clinical records. Copyright © 2002 John Wiley & Sons, Ltd.
Gas‐phase kinetics and mechanism of diallyl sulfide thermal decomposition
Authors: Gholami M. R. , Izadyar M.
Published: February 2003

The gas‐phase kinetics of diallyl sulfide pyrolysis were studied experimentally and theoretically in a static system over the temperature range 433–463 K and a total pressure of 80 Torr in a glass vessel with the free radical inhibitor cyclohexene. The experimental results show that this reaction is homogeneous, unimolecular and follows a first‐order rate law. Theoretical studies at the B3LYP/6–31G* computational level indicate that the elimination reaction proceeds through a six‐centered cyclic transition state and the reaction mechanism is concerted and non‐synchronous. Copyright © 2003 John Wiley & Sons, Ltd.
Electronic structure of N‐sulfenylimines
Authors: Bharatam Prasad V. , Amita  , Kaur Damanjit
Published: February 2003

The electronic structure of N‐sulfenylimines was studied in detail using ab initio MO and density functional methods. The S—N rotational barriers and N‐inversion barriers in HS—NCH2 at the G2MP2 level were found to be 5.60 and 21.76 kcal mol−1, respectively. There is a partial pπ–pπ bond and a relatively weak nN → σ*S—R anomeric π bond between sulfur and nitrogen in N‐sulfenylimines. NBO analysis was carried out to estimate quantitatively the above delocalizations in RS—NCH2 (R = H, Me, Cl, F, BH2) systems. Copyright © 2003 John Wiley & Sons, Ltd.
Quality of Placental Pathology Reports
Authors: Khong T. Yee , Gordijn Sanne J.
Published: February 2003

The surgical report is an important means of documenting normal and abnormal findings, and for distilling such information into a meaningful clinico-pathologic correlation. An audit of the quality of placental reports from four laboratories was performed using an arbitrary numerical scoring scheme that examined the gross, histologic, and commentary components of each report. The mean scores from the four laboratories were not statistically different from each other. Three (2%) and 48 (33%) of the 147 singleton placentas scored less than 50 and 75%, respectively, on this scoring scheme. None and 14 (41%) of the placentas from 34 multiple pregnancies scored less than 50 and 75%, respectively. Different aspects of the gross and histologic examination were reported variably by the laboratories. Commentaries on gross or histologic abnormalities, and in relation to clinical indications, were inconsistently reported. The standards of placental surgical reporting can be improved. The use of templates and checklists for reporting of placentas may be considered.
Idiopathic Hyperammonemia following an Unrelated Cord Blood Transplant for Mucopolysaccharidosis I
Authors: Snyder Matthew J. , Bradford William D. , Kishnani Priya S. , Hale Laura P.
Published: February 2003

Bone marrow transplantation (BMT) has been shown to reverse or stabilize some manifestations of mucopolysaccharidosis I (Hurler syndrome). Idiopathic hyperammonemia (IHA) is a rare complication of solid organ and BMT that is characterized by elevated serum ammonia, normal liver enzymes, and abrupt onset of neurologic deterioration. We present the case of a 14-month-old male patient with Hurler syndrome who developed fatal IHA (ammonia = 2297 μmol/L) 31 days after a cord blood transplant. A complete autopsy was performed, with examination of both frozen and formalin-fixed paraffin-embedded (FFPE) tissues using a variety of special stains and electron microscopy. Hyperammonemia was documented by analysis of antemortem serum and postmortem cerebrospinal and vitreous fluid. Other causes of hyperammonemia, including Reye syndrome, were excluded. Histologic changes included centrilobular microvesicular steatosis of the liver and storage product present in multiple organs. The highly water-soluble mucopolysaccharide (MPS) storage product was best identified by colloidal iron staining of FFPE and unfixed air-dried fresh frozen liver sections. Alcian blue stains failed to convincingly demonstrate MPS in any of the liver sections. This is the first published report, to our knowledge, of IHA in a posttransplant patient younger than 18 years old or following transplantation for Hurler syndrome. Demonstration of the hepatic centrilobular microvesicular steatosis characteristic of IHA was complicated by the diffuse storage of MPS within the liver. MPS storage can be best detected in the liver using colloidal iron staining. Oil-red-O staining may be useful to document microvesicular steatosis in cases with a clinical history of hyperammonemia following solid organ or BMT. Determining if certain subsets of children are at increased risk for IHA requires further study.
Performance Characteristics of a Reverse Transcriptase–Polymerase Chain Reaction Assay for the Detection of Tumor-specific Fusion Transcripts from Archival Tissue
Authors: Fritsch Michael K. , Bridge Julia A. , Schuster Amy E. , Perlman Elizabeth J. , Argani Pedram
Published: February 2003

Pediatric small round cell tumors still pose tremendous diagnostic problems. In difficult cases, the ability to detect tumor-specific gene fusion transcripts for several of these neoplasms, including Ewing sarcoma/peripheral primitive neuroectodermal tumor (ES/PNET), synovial sarcoma (SS), alveolar rhabdomyosarcoma (ARMS), and desmoplastic small round cell tumor (DSRCT) using reverse transcriptase–polymerase chain reaction (RT-PCR), can be extremely helpful. Few studies to date, however, have systematically examined several different tumor types for the presence of multiple different fusion transcripts in order to determine the specificity and sensitivity of the RT-PCR method, and no study has addressed this issue for formalin-fixed material. The objectives of this study were to address the specificity, sensitivity, and practicality of such an assay applied strictly to formalin-fixed tissue blocks. Our results demonstrate that, for these tumors, the overall sensitivity for detecting each fusion transcript is similar to that reported in the literature for RT-PCR on fresh or formalin-fixed tissues. The specificity of the assay is very high, being essentially 100% for each primer pair when interpreting the results from visual inspection of agarose gels. However, when these same agarose gels were examined using Southern blotting, a small number of tumors also yielded reproducibly detectable weak signals for unexpected fusion products, in addition to a strong signal for the expected fusion product. Fluorescence in situ hybridization (FISH) studies in one such case indicated that a rearrangement that would account for the unexpected fusion was not present, while another case was equivocal. The overall specificity for each primer pair used in this assay ranged from 94 to 100%. Therefore, RT-PCR using formalin-fixed paraffin-embedded tissue sections can be used to detect chimeric transcripts as a reliable, highly sensitive, and highly specific diagnostic assay. However, we strongly suggest that the final interpretation of the results from this assay be viewed in light of the other features of the case, including clinical history, histology, and immunohistochemistry, by the diagnostic pathologist. Additional studies such as FISH may be useful in clarifying the nature of equivocal or unexpected results.
Histopathological Diagnosis of Partial and Complete Hydatidiform Mole in the First Trimester of Pregnancy
Authors: Sebire Neil J. , Fisher Rosemary A. , Rees Helene C.
Published: February 2003

The diagnosis of molar pregnancy is a continuing diagnostic problem for many practicing histopathologists who are required to examine specimens of products of conception, particularly since changes in gynecological management in recent years have resulted in uterine evacuation at earlier gestations. The aim of this review is to provide practical, up-to-date, diagnostically useful information regarding the histological diagnosis of molar disease in early pregnancy. Pathophysiological issues relevant to molar pregnancies, such as genetic abnormalities, will be briefly summarized, but nonhistopathological aspects of molar disease will not be covered in detail in this review.
Renal Cell Carcinoma with t(X;17): Singular Pediatric Neoplasm with Specific Phenotype/Genotype Features
Authors: Zambrano Eduardo , Reyes-Múgica Miguel
Published: February 2003

Renal cell carcinomas in children are extremely rare and are usually associated with specific chromosomal rearrangements, different from those seen in adult patients. We present the case of a 9-year-old girl with a renal cell carcinoma with t(X;17) diagnosed at our institution. We also review the pertinent literature, with an emphasis on the genetic and molecular aspects associated with this rare neoplasm.
Child Poverty in Comparative Perspective
Authors: Bradshaw Jonathan
Published: February 2003

This paper draws on comparative evidence on child poverty in Europe. It is in four parts. The first section reviews evidence on the prevalence of child poverty, the second examines the characteristics of poor children, and the third reviews the evidence on how child poverty has changed in European countries over the last decade or so. The fourth part of the paper examines comparative evidence on the demographic, labour pre-transfer and policy factors related to the variations in child poverty observed. The article concludes with a review of the strengths and weaknesses of the comparative research on child poverty and the measures designed to relieve it.
The Potential Consequences of the Gaygusuz Judgment
Authors: Pennings Frans
Published: February 2003

On 16 September 1996 the European Court of Human Rights (ECHR) in Strasbourg delivered the Gaygusuz v. Austria judgment. This judgment may have very interesting consequences in the area of social security. The Court ruled that the Austrian emergency assistance scheme falls within the scope of the protection of property as provided for by the First Protocol of the Convention for the Protection of Human Rights and Fundamental Freedoms. Until this decision the European Commission of Human Rights consistently ruled that social security rights based on a pay-as-you-go system did not fall under this protection of property.

One consequence of this new case law is that the European Commission of Human Rights and the Court can apply the rules on discrimination in the Convention to social security cases. This is especially interesting in areas where EU law is not applicable (Section 3), and where the rules on the protection of property are applied to social security questions (Section 4 and 5).
Secretion of phosphomannosyl-deficient arylsulphatase A and cathepsin D from isolated human macrophages
Authors: MUSCHOL Nicole , MATZNER Ulrich , TIEDE Stephan , GIESELMANN Volkmar , ULLRICH Kurt , BRAULKE Thomas
Published: February 2003

The transfer of macrophage-secreted arylsulphatase A (ASA) to enzyme-deficient brain cells is part of the therapeutic concept of bone marrow transplantation in lysosomal storage diseases. Here we have investigated this transfer in vitro. The uptake of 125I-labelled recombinant human ASA purified from ASA-overexpressing mouse embryonic fibroblasts deficient for mannose 6-phosphate (M6P) receptors in a mouse ASA-deficient astroglial cell line was completely inhibited by M6P. In contrast, when ASA-deficient astroglial cells were incubated with secretions of [35S]methionine-labelled human macrophages or mouse microglia, containing various lysosomal enzymes, neither ASA nor cathepsin D (CTSD) were detected in acceptor cells. Co-culturing of metabolically labelled macrophages with ASA-deficient glial cells did not result in an M6P-dependent transfer of ASA or CTSD between these two cell types. In secretions of [33P]phosphate-labelled macrophages no or weakly phosphorylated ASA and CTSD precursor polypeptides were found, whereas both intracellular and secreted ASA from ASA-overexpressing baby hamster kidney cells displayed 33P-labelled M6P residues. Finally, the uptake of CTSD from secretions of [35S]methionine-labelled macrophages in rat hepatocytes was M6P-independent. These data indicated that lysosomal enzymes secreted by human macrophages or a mouse microglial cell line cannot be endocytosed by brain cells due to the failure to equip newly synthesized lysosomal enzymes with the M6P recognition marker efficiently. The data suggest that other mechanisms than the proposed M6P-dependent secretion/recapture of lysosomal enzymes might be responsible for therapeutic effects of bone marrow transplantation in the brain.
Site-specific phosphorylation of platelet focal adhesion kinase by low-density lipoprotein
Authors: RELOU Ingrid A.M. , BAX Liane A.B. , RIJN Herman J.M. van , AKKERMAN Jan-Willem N.
Published: February 2003

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase implicated in signalling pathways mediated by integrins and G-protein-coupled receptors (GPCRs). Upon stimulation FAK is phosphorylated on six tyrosine residues. Here we report the site-specific phosphorylation by low-density lipoprotein (LDL), which is known to induce integrin-independent FAK phosphorylation, and compare this with the effect of thrombin, which phosphorylates FAK via integrin αIIbβ3. Stimulation with LDL reveals (i) a major role for Tyr-925 phosphorylation which surpasses the phosphorylation of the other residues, including Tyr-397, in rate and extent, (ii) αIIbβ3-independent phosphorylation of Tyr-925 and Tyr-397, and (iii) complex formation between FAK and the Src-kinase Fgr but not with c-Src. These patterns differ profoundly from those induced by thrombin. LDL-induced phosphorylation of Tyr-925 and Tyr-397 was inhibited by 60—75% by receptor-associated protein, an inhibitor of members of the LDL receptor family. Thus these findings reveal a novel mechanism of FAK phosphorylation by signalling cascades involving a member of the LDL receptor family.
Impaired trafficking of human kidney anion exchanger (kAE1) caused by hetero-oligomer formation with a truncated mutant associated with distal renal tubular acidosis
Authors: QUILTY Janne A. , CORDAT Emmanuelle , REITHMEIER Reinhart A.F.
Published: February 2003

Autosomal dominant distal renal tubular acidosis (dRTA) has been associated with several mutations in the anion exchanger AE1 gene. The effect of an 11-amino-acid C-terminal dRTA truncation mutation (901 stop) on the expression of kidney AE1 (kAE1) and erythroid AE1 was examined in transiently transfected HEK-293 cells. Unlike the wild-type proteins, kAE1 901 stop and AE1 901 stop mutants exhibited impaired trafficking from the endoplasmic reticulum to the plasma membrane as determined by immunolocalization, cell-surface biotinylation, oligosaccharide processing and pulse—chase experiments. The 901 stop mutants were able to bind to an inhibitor affinity resin, suggesting that these mutant membrane proteins were not grossly misfolded. Co-expression of wild-type and mutant kAE1 or AE1 resulted in intracellular retention of the wild-type proteins in a pre-medial Golgi compartment. This dominant negative effect was due to hetero-oligomer formation of the mutant and wild-type proteins. Intracellular retention of kAE1 in the α-intercalated cells of the kidney would account for the impaired acid secretion into the urine characteristic of dRTA.
Overexpression of c-myc in diabetic mice restores altered expression of the transcription factor genes that regulate liver metabolism
Authors: RIU Efren , FERRE Tura , MAS Alex , HIDALGO Antonio , FRANCKHAUSER Sylvie , BOSCH Fatima
Published: February 2003

Overexpression of the c-Myc transcription factor in liver induces glucose uptake and utilization. Here we examined the effects of c-myc overexpression on the expression of hepatocyte-specific transcription factor genes which regulate the expression of genes controlling hepatic metabolism. At 4 months after streptozotocin (STZ) treatment, most diabetic control mice were highly hyperglycaemic and died, whereas in STZ-treated transgenic mice hyperglycaemia was markedly lower, the serum levels of β-hydroxybutyrate, triacylglycerols and non-esterified fatty acids were normal, and they had greater viability in the absence of insulin. Furthermore, long-term STZ-treated transgenic mice showed similar glucose utilization and storage to healthy controls. This was consistent with the expression of glycolytic genes becoming normalized. In addition, restoration of gene expression of the transcription factor, sterol receptor element binding protein 1c, was observed in the livers of these transgenic mice. Further, in STZ-treated transgenic mice the expression of genes involved in the control of gluconeogenesis (phosphoenolpyruvate carbokykinase), ketogenesis (3-hydroxy-3-methylglutaryl-CoA synthase) and energy metabolism (uncoupling protein 2) had returned to normal. These findings were correlated with decreased expression of genes encoding the transcription factors hepatocyte nuclear factor 3γ, peroxisome proliferator-activated receptor α and retinoid X receptor. These results indicate that c-myc overexpression may counteract diabetic changes by controlling hepatic glucose metabolism, both directly by altering the expression of metabolic genes and through the expression of key transcription factor genes.
The carp muscle-specific sub-isoenzymes of creatine kinase form distinct dimers at different temperatures
Authors: SUN Hsi-Wen , LIU Cheng-Wen , HUI Cho-Fat , WU Jen-Leih
Published: February 2003

We have previously cloned three muscle-specific sub-isoforms of creatine kinase (CK, EC 2.7.3.2) from the common carp (Cyprinus carpio), designated M1-CK, M2-CK, and M3-CK. The enzyme has a key role in maintaining the energy homoeostasis of cells with fluctuating energy requirements. In the present paper, we report that all three M-CKs in the red and white muscle of different temperature-acclimatized carp were ubiquitously distributed in the cytosol and along membranes. In addition, the expression levels of these isoforms were not significantly altered in response to the temperature acclimatization. Interestingly, our studies showed that the formation of distinct homo- or heterodimers among these three M-CKs was found at various temperatures. At higher temperature, the M1M1-CK and M2M2-CK homodimers, and the M1M3-CK heterodimer are the predominant MM-CKs, whereas the M3M3-CK achieves its homodimeric state at lower temperature. We postulated testable homology models to investigate the chemical properties of these dimeric interfaces. M1M1-CK was used as a reference to compare the structural differences with the M3M3-CK dimer. The calculated solvent accessible surface area that was buried in the contact interfaces of the M1M1-CK and M3M3-CK dimers showed an overall decrease of 12% in the M3M3-CK interface. The modelling analysis also suggested a net decrease of twelve hydrophobic residues and a Phe3→Lys substitution in the M3M3-CK interface. An increase in thermolability of the M3M3-CK homodimer might be due to the decrease in subunit ion pairs and buried surface area in this dimer. Based on our findings, we propose that the carp-muscle-specific CK isoenzymes could undergo shuffling to form distinct M-CK homo- or heterodimers in acclimatization to environmental temperatures.
Regulatable liver expression of the rabbit apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1) in mice lacking endogenous APOBEC-1 leads to aberrant hyperediting
Authors: HERSBERGER Martin , PATARROYO-WHITE Susannah , QIAN Xiaobing , ARNOLD Kay S. , ROHRER Lucia , BALESTRA Maureen E. , INNERARITY Thomas L.
Published: February 2003

Apolipoprotein (apo) B mRNA editing is the deamination of C6666 to uridine, which results in translation of the apoB-48 protein instead of the genomically encoded apoB-100. ApoB-48-containing lipoproteins are cleared more rapidly from plasma and are less atherogenic than apoB-100-containing low-density lipoproteins (LDLs). In humans, the intestine predominantly produces apoB-48 whereas the liver secretes apoB-100 only. To evaluate a potential therapeutic use for liver-induced apoB mRNA editing in humans, we investigated the efficiency and safety of transgenic expression of apoB mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1) in the absence of endogenous editing in the mouse model. Here we show that regulatable tetO-mediated APOBEC-1 expression in the livers of gene-targeted mice lacking endogenous APOBEC-1 results in 30% apoB mRNA editing. In a time-course experiment, the expression of tetO-APOBEC-1 mRNA was suppressed within 2 days after mice were fed doxycycline and apoB mRNA editing and apoB-48 formation were suppressed within 4 days. However, tetO-APOBEC-1 expression resulted in regulatable aberrant hyperediting of several cytidines downstream of C6666 in apoB mRNA and in novel APOBEC-1 target 1 (NAT1) mRNA. Several of the cytidines in apoB mRNA were hyperedited to a level similar to that of C6666, although editing at C6666 was lower than that in wild-type mice. These results demonstrate that even moderate APOBEC-1 expression can lead to hyperediting, limiting the single-gene approach for gene therapy with APOBEC-1.
Polyunsaturated fatty acids decrease the expression of sterol regulatory element-binding protein-1 in CaCo-2 cells: effect on fatty acid synthesis and triacylglycerol transport
Authors: FIELD F. Jeffrey , BORN Ella , MURTHY Shubha , MATHUR Satya N.
Published: February 2003

Regulation of sterol regulatory element-binding proteins (SREBPs) by fatty acid flux was investigated in CaCo-2 cells. Cells were incubated with 1mM taurocholate with or without 250μM 18:0, 18:1, 18:2, 20:4, 20:5 or 22:6 fatty acids. Fatty acid synthase (FAS) and acetyl-CoA carboxylase mRNA levels and gene and protein expression of SREBPs were estimated. 18:2, 20:4, 20:5 and 22:6 fatty acids decreased the amount of mature SREBP-1 and mRNA levels of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase. SREBP-2 gene or mature protein expression was not altered. Liver X receptor (LXR) activation by T0901317 increased gene expression of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase without altering SREBP-2. 20:5, but not 18:1, prevented the full expression of SREBP-1c mRNA by T0901317. T0901317 increased SREBP-1 mass without altering the mass of mature SREBP-2. Although only 18:2, 20:4, 20:5 and 22:6 suppressed SREBP-1, acetyl-CoA carboxylase and FAS expression, all fatty acids decreased the rate of fatty acid synthesis. T0901317 increased endogenous fatty acid synthesis yet did not increase secretion of triacylglycerol-rich lipoproteins. In CaCo-2 cells, polyunsaturated fatty acids decrease gene and protein expression of SREBP-1 and FAS mRNA, probably through interference with LXR activity. Since all fatty acids decreased fatty acid synthesis, mechanisms other than changes in SREBP-1c expression must be entertained. Increased endogenous fatty acid synthesis does not promote triacylglycerol-rich lipoprotein secretion.
Characterization of the human topoisomerase IIβ (TOP2B) promoter activity: essential roles of the nuclear factor-Y (NF-Y)- and specificity protein-1 (Sp1)-binding sites
Authors: LOK Chun-Nam , LANG Alexander J. , MIRSKI Shelagh E.L. , COLE Susan P.C.
Published: February 2003

Eukaryotic topoisomerase II (topo II) catalyses topological genomic changes essential for chromosome segregation, chromatin reorganization, DNA replication and transcription. Mammalian topo II exists as two isoforms, designated α and β. Human topo IIα is an important cancer drug target, and an established determinant of drug sensitivity and resistance. Human topo IIβ is also the target of anticancer drugs but its role in drug resistance is less clear. The two human topo II proteins are encoded by the TOP2A and TOP2B genes, respectively, which despite their highly conserved structural organization, are subject to distinctly different modes of regulation. In the present study, we have cloned and characterized the human TOP2B promoter containing a 1.3kb fragment of the 5′-flanking and untranslated region (-1067 to +193). We found that the promoter activity of this TOP2B fragment was constant throughout the cell cycle, in contrast to the activity of the proximal promoter of TOP2A which was low in resting cells and enhanced during proliferation. Analyses of 5′-serially and internally deleted luciferase reporter constructs revealed that 80% of the TOP2B promoter activity could be attributed to the region between −533 and −481. Mutational analyses of putative regulatory elements indicated that two inverted CCAAT boxes (ICBs) within this region were essential for TOP2B promoter activity and gel mobility-shift assays indicated these sites bound the transcription factor nuclear factor-Y (NF-Y). Co-transfection experiments using a dominant-negative form of subunit A of NF-Y suggested that TOP2B promoter activity required direct interaction of NF-Y with the ICBs. In addition, a specificity protein-1 (Sp1)-binding GC box located just upstream of the ICBs was shown to contribute to TOP2B promoter activity in a synergistic manner with the ICBs. Our results suggest that the binding sites for NF-Y and Sp1 are critical for TOP2B transcription.
Male-specific suppression of hepatic microsomal UDP-glucuronosyl transferase activities toward sex hormones in the adult male rat administered bisphenol A
Authors: SHIBATA Noriaki , MATSUMOTO Junya , NAKADA Ken , YUASA Akira , YOKOTA Hiroshi
Published: February 2003

Various adverse effects of endocrine disruptors on the reproductive organs of male animals have been reported. We found that UDP-glucuronosyltransferase (UGT) activities towards bisphenol A, testosterone and oestradiol were significantly decreased in liver microsomes prepared from adult male Wistar rats administered with the endocrine disruptor bisphenol A (1mg/2 days for 2 or 4 weeks). However, suppression of the transferase activities was not observed in female rats, even after bisphenol A treatment for 4 weeks. Diethylstilbestrol, which is well known as an endocrine disruptor, had the same effects, but p-cumylphenol had no effect on UGT activities towards sex hormones. Co-administration of an anti-oestrogen, tamoxifen, inhibited the suppression of the transferase activities by bisphenol A. Western blotting analysis showed that the amount of UGT2B1, an isoform of UGT which glucuronidates bisphenol A, was decreased in the rat liver microsomes by the treatment. Northern blotting analysis also indicated that UGT2B1 mRNA in the liver was decreased by bisphenol A treatment. The suppression of UGT activities, UGT2B1 protein and UGT2B1 mRNA expression did not occur in female rats. The results indicate that bisphenol A treatment reduces the mRNA expression of UGT2B1 and other UGT isoforms that mediate the glucuronidation of sex hormones in adult male rats, and this suggests that the endocrine balance may be disrupted by suppression of glucuronidation.
The integrin alphavbeta3 is a receptor for the latency-associated peptides of transforming growth factors beta1 and beta3
Authors: LUDBROOK Steven B. , BARRY Simon T. , DELVES Chris J. , HORGAN Carmel M.T.
Published: February 2003

The integrins αvβ1, αvβ5, αvβ6 and αvβ8 have all recently been shown to interact with the RGD motif of the latency-associated peptide (LAPβ1) of transforming growth factor β1 (TGFβ1), with binding to αvβ6 and αvβ8 leading to TGFβ1 activation. Previously it has been suggested that the remaining αv integrin, αvβ3, does not interact with LAPβ1. However, here we show clearly that αvβ3 does indeed interact with the LAPβ1 RGD motif. This interaction is similar to other αvβ3 ligands in terms of the cations required for adhesion, the concentrations of LAPβ1 required for binding and the ability of a small-molecule inhibitor of αvβ3, SB223245, to block the interaction. Using glutathione S-transferase fusion proteins we have mapped a minimal integrin-binding loop in LAPβ1 and then used this approach to probe the integrin-binding properties of the equivalent loops in LAPβ2 and LAPβ3. We show that the RGD motif of LAPβ3 also interacts with αvβ3, in addition to αvβ6, αvβ1 and αvβ5, whereas the corresponding loop in LAPβ2 does not interact with these integrins. These observations therefore correct a previously reported inaccuracy in the literature. Furthermore, they are important as they link αvβ3 and TGFβ, which may have implications in cancer and a number of inflammatory and fibrotic diseases where expression of both proteins has been documented.
Reciprocal feedback regulation of insulin receptor and insulin receptor substrate tyrosine phosphorylation by phosphoinositide 3-kinase in primary adipocytes
Authors: HERS Ingeborg , BELL Christopher J. , POOLE Alastair W. , JIANG Donyang , DENTON Richard M. , SCHAEFER Erik , TAVARÉ Jeremy M.
Published: February 2003

Signalling by the insulin receptor substrate (IRS) proteins is critically dependent on the tyrosine phosphorylation of specific binding sites that recruit Src homology 2 (SH2)-domain-containing proteins, such as the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase), the tyrosine phosphatase SHP-2 and the adapter protein Grb2. Here we show that stimulation by insulin of freshly isolated primary adipocytes resulted in the expected rapid tyrosine phosphorylation of the insulin receptor, IRS-1 and IRS-3. Inhibition of PI 3-kinase enhanced the insulin-stimulated phosphorylation of IRS-1 on (i) Tyr612 and Tyr941 (p85 binding sites), concomitant with an increased association of the p85 subunit of PI 3-kinase; (ii) Tyr896 (a Grb2 binding site); and (iii) Tyr1229 (an SHP-2 binding site), although little or no binding of SHP-2 to IRS-1 was detectable under any conditions. In contrast, inhibition of PI 3-kinase led to a decrease in insulin-stimulated p85 binding to IRS-3, but had no effect on SHP-2 binding. Furthermore, insulin-induced insulin receptor tyrosine phosphorylation, phosphorylation of Tyr1158 and insulin receptor tyrosine kinase activity were all reduced by inhibition of PI 3-kinase at later time points (20min). The results demonstrate that, in primary adipocytes, PI 3-kinase feedback control of signalling by the insulin receptor and IRS proteins is multifaceted and reciprocal, illustrating the complexity of predicting the net flux of the insulin signal(s) through the IRS proteins.
Insulin stimulation of pyruvate dehydrogenase in adipocytes involves two distinct signalling pathways
Authors: JOHNSON Sam A. , DENTON Richard M.
Published: February 2003

In isolated rat adipocytes, the insulin stimulation of pyruvate dehydrogenase can be partially inhibited by inhibitors of PI3K (phosphoinositide 3-kinase) and MEK1/2 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase). In combination, U0126 and wortmannin completely block the insulin stimulation of pyruvate dehydrogenase. It is concluded that the effect of insulin on pyruvate dehydrogenase in rat adipocytes involves two distinct signalling pathways: one is sensitive to wortmannin and the other to U0126. The synthetic phosphoinositolglycan PIG41 can activate pyruvate dehydrogenase but the activation is only approx. 30% of the maximal effect of insulin. This modest activation can be completely blocked by wortmannin alone, suggesting that PIG41 acts through only one of the pathways leading to the activation of pyruvate dehydrogenase.
Acetaldehyde stimulates the activation of latent transforming growth factor-β1 and induces expression of the type II receptor of the cytokine in rat cultured hepatic stellate cells
Authors: CHEN Anping
Published: February 2003

Acetaldehyde, the major active metabolite of alcohol, induces the activation of hepatic stellate cells (HSC), leading to over-production of α1(I) collagen and ultimately causing hepatic fibrosis. The underlying mechanisms of this process remain largely unknown. Transforming growth factor-β1 (TGF-β1) is a potent inducer of α1(I) collagen production. Accumulating evidence has shown a potential role for TGF-β1 in alcohol-induced hepatic fibrogenesis. The aims of this study were to determine the effect of acetaldehyde on TGF-β signalling, to elucidate the underlying mechanisms as well as to evaluate its role in expression of α1(I) collagen gene in cultured HSC. It was hypothesized that acetaldehyde activated TGF-β signalling by inducing the expression of elements in the TGF-β signal transduction pathway, which might contribute to α1(I) collagen gene expression in cultured HSC. Initial results revealed that acetaldehyde activated TGF-β signalling in cultured HSC. Additional studies demonstrated that acetaldehyde stimulated the secretion and activation of latent TGF-β1, and induced the expression of the type II TGF-β receptor (Tβ-RII). Further experiments found cis- and trans-activating elements responsible for Tβ-RII gene expression induced by acetaldehyde. Activation of TGF-β signalling by acetaldehyde contributed to α1(I) collagen gene expression in cultured HSC. In summary, this report demonstrated that acetaldehyde stimulated TGF-β signalling by increasing the secretion and activation of latent TGF-β1 as well as by inducing the expression of Tβ-RII in cultured HSC. Results from this report provided a novel insight into mechanisms by which acetaldehyde stimulated the expression of α1(I) collagen in HSC and a better understanding of effects of alcohol (or acetaldehyde) on hepatic fibrogenesis.
Cloning and functional characterization of the human fractalkine receptor promoter regions
Authors: GARIN Alexandre , PELLET Philippe , DETERRE Philippe , DEBRÉ Patrice , COMBADIÈRE Christophe
Published: February 2003

We have previously shown that reduced expression of the fractalkine receptor, CX3CR1, is correlated with rapid HIV disease progression and with reduced susceptibility to acute coronary events. In order to elucidate the mechanisms underlying transcriptional regulation of CX3CR1 expression, we structurally and functionally characterized the CX3CR1 gene. It consists of four exons and three introns spanning over 18kb. Three transcripts are produced by splicing the three untranslated exons with exon 4, which contains the complete open reading frame. The transcript predominantly found in leucocytes corresponds to the splicing of exon 2 with exon 4. Transcripts corresponding to splicing of exons 1 and 4 are less abundant in leucocytes and splicing of exons 3 and 4 are rare longer transcripts. A constitutive promoter activity was found in the regions extending upstream from untranslated exons 1 and 2. Interestingly, exons 1 and 2 enhanced the activity of their respective promoters in a cell-specific manner. These data show that the CX3CR1 gene is controlled by three distinct promoter regions, which are regulated by their respective untranslated exons and that lead to the transcription of three mature messengers. This highly complex regulation may allow versatile and precise expression of CX3CR1 in various cell types.
Regulation of intracellular glutathione levels in erythrocytes infected with chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum
Authors: MEIERJOHANN Svenja , WALTER Rolf D. , MÜLLER Sylke
Published: February 2003

Malaria is one of the most devastating tropical diseases despite the availability of numerous drugs acting against the protozoan parasite Plasmodium in its human host. However, the development of drug resistance renders most of the existing drugs useless. In the malaria parasite the tripeptide glutathione is not only involved in maintaining an adequate intracellular redox environment and protecting the cell against oxidative stress, but it has also been shown that it degrades non-polymerized ferriprotoporphyrin IX (FP IX) and is thus implicated in the development of chloroquine resistance. Glutathione levels in Plasmodium-infected red blood cells are regulated by glutathione synthesis, glutathione reduction and glutathione efflux. Therefore the effects of drugs that interfere with these metabolic processes were studied to establish possible differences in the regulation of the glutathione metabolism of a chloroquine-sensitive and a chloroquine-resistant strain of Plasmodiumfalciparum. Growth inhibition of P. falciparum 3D7 by d,l-buthionine-(S,R)sulphoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase (γ-GCS), and by Methylene Blue (MB), an inhibitor of gluta thione reductase (GR), was significantly more pronounced than inhibition of P.falciparum Dd2 growth by these drugs. These results correlate with the higher levels of total glutathione in P. falciparum Dd2. Short-term incubations of Percoll-enriched trophozoite-infected red blood cells in the presence of BSO, MB and N,N1-bis(2-chloroethyl)-N-nitrosourea and subsequent determinations of γ-GCS activities, GR activities and glutathione disulphide efflux revealed that maintenance of intracellular glutathione in P. falciparum Dd2 is mainly dependent on glutathione synthesis whereas in P. falciparum 3D7 it is regulated via GR. Generally, P. falciparum Dd2 appears to be able to sustain its intracellular glutathione more efficiently than P. falciparum 3D7. In agreement with these findings is the differential susceptibility to oxidative stress of both parasite strains elicited by the glucose/glucose oxidase system.
The Escherichia coli cAMP receptor protein bound at a single target can activate transcription initiation at divergent promoters: a systematic study that exploits new promoter probe plasmids
Authors: EL-ROBH Mohamed Samir , BUSBY Stephen J.W.
Published: February 2003

We report the first detailed quantitative study of divergent promoters dependent on the Escherichia coli cAMP receptor protein (CRP), a factor known to activate transcription initiation at target promoters by making direct interactions with the RNA polymerase holoenzyme. In this work, we show that CRP bound at a single target site is able to activate transcription at two divergently organized promoters. Experiments using promoter probe plasmids, designed to study divergent promoters in vivo and in vitro, show that the divergent promoters function independently. Further in vitro experiments show that two holo RNA polymerase molecules cannot be accommodated simultaneously at the divergent promoters.
Essential role of an activator protein-2 (AP-2)/specificity protein 1 (Sp1) cluster in the UVB-mediated induction of the human vascular endothelial growth factor in HaCaT keratinocytes
Authors: BRENNEISEN Peter , BLAUDSCHUN Ralf , GILLE Jens , SCHNEIDER Lars , HINRICHS Ralf , WLASCHEK Meinhard , EMING Sabine , SCHARFFETTER-KOCHANEK Karin
Published: February 2003

Chronic sun exposure of the skin has long been postulated to enhance cutaneous angiogenesis, resulting in highly vascularized skin cancers. As the UVB component of sunlight is a major contributor to photocarcinogenesis, we aimed to explore the effects of UVB radiation on vascular endothelial growth factor (VEGF) gene expression, using the immortalized keratinocyte cell line HaCaT as a model for transformed premalignant epithelial cells. In the present paper, we studied the molecular mechanism of UVB-induced VEGF providing a major angiogenic activity in tumour progression and invasion. After 12—24h of UVB irradiation, a 2.4- to 2.7-fold increase in endogenous VEGF protein level was measured, correlating with an up to 2.5-fold induction of promoter-based reporter gene constructs of VEGF. Furthermore, we identified a GC-rich UVB-responsive region between −87 and −65bp of the VEGF promoter. In electrophoretic mobility-shift assays, this region binds Sp1-dependent protein complexes constitutively and an additional UVB-inducible protein complex distinct from Sp1 protein. The transcription factor AP-2 (activator protein-2) was detected as a component of the UVB-inducible protein complex. The critical role of the AP-2/Sp1 (specificity protein 1) cluster was supported by demonstration of a significant reduction of UVB-mediated promoter activity upon deletion of this recognition site. The specificity of this region for UVB irradiation was demonstrated using PMA, which increased VEGF activity in HaCaT cells after transient transfection of the deleted promoter construct.