bluuebunny/crossref_metadata_2025 huggingface dataset:
TE2 and TE1 sub‐elements of the testis‐specific histone H1t promoter are functionally different
Authors: Wilkerson Donald C. , Wolfe Steven A. , Grimes Sidney R.
Published: March 2003

The testis‐specific linker histone H1t gene is transcribed exclusively in pachytene primary spermatocytes. Tissue specific expression of the gene is mediated in part by transcriptional factors that bind elements located within the proximal and distal promoter. A 40 bp promoter element, designated H1t/TE, that is located within the proximal promoter between the CCAAT‐box and AC‐box, is known to be essential for H1t gene transcription in transgenic animals. In the present study, we show by SDS–PAGE analysis of UV crosslinked protein and DNA and by electrophoretic mobility shift assays (EMSA) of testis nuclear proteins separated on a non‐denaturing glycerol gradient that the TE1 sub‐element is bound by a protein complex. Mutation of TE1 leads to a drop in H1t promoter activity in germinal GC‐2spd cells as well as in nongerminal Leydig, NIH3T3, and C127I cell lines. Although TE1 and TE2 sub‐elements have similar sequences, mutation of the TE2 sub‐element causes an increase in promoter activity in C127I and Leydig cells. The rat TE1 but not TE2 contains a CpG dinucleotide and this cytosine is methylated in liver but not in primary spermatocytes. Methylation of the cytosine at this site almost eliminates nuclear protein binding. Thus, there are significant functional differences in the TE2 and TE1 sub‐elements of the H1t promoter with TE1 serving as a transcriptional activator binding site and TE2 serving as a repressor binding site in some cell lines. J. Cell. Biochem. 88: 1177–1187, 2003. © 2003 Wiley‐Liss, Inc.
Characterization of PTH/PTHrP receptor in rat duodenum: Effects of ageing
Authors: Gentili Claudia , Morelli Susana , de Boland Ana Russo
Published: March 2003

In rat enterocytes, signaling through the paratyroid hormone (PTH)/PTH‐related peptide receptor type 1(PTHR1) includes stimulation of adenylyl cyclase, increases of intracellular calcium, activation of phospholipase C, and the MAP kinase pathway, mechanisms that suffer alterations with ageing. The purpose of this study was to evaluate whether an alteration at the level of the PTH receptor (PTHR1) is the basis for impaired PTH signaling in aged rat enterocytes. Western Blot analysis with a specific monoclonal anti‐PTHR1 antibody revealed that a 85 kDa PTH binding component, the size expected for the mature PTH/PTHrP receptor, localizes in the basolateral (BLM) and brush border (BBM) membranes of the enterocyte, being the protein expression about 7‐fold higher in the BLM. Two other bands of 105 kDa (corresponding to highly glycosylated, incompletely processed receptor form) and 65 kDa (proteolytic fragment) were also seen. BLM PTHR1 protein expression significantly decreases with ageing, while no substantial decrease was observed in the BBM from old rats. PTHR1 immunoreactivity was also present in the nucleus where PTHR1 protein levels were similar in enterocytes from young and aged rats. Immunohistochemical analysis of rat duodenal sections showed localization of PTHR1 in epithelial cells all along the villus with intense staining of BBM, BLM, and cytoplasm. The nuclei of these cells were reactive to the PTHR1 antiserum, but not all cells showed the same nuclear staining. The receptor was also detected in the mucosae lamina propria cells, but was absent in globets cells from epithelia. In aged rats, PTHR1 immunoreactivity was diffused in both membranes and cytoplasm and again, PTH receptor expression was lower than in young animals, while the cell nuclei showed a similar staining pattern than in young rats. Ligand binding to PTHR1 was performed in purified BLM. rPTH(1‐34) displaced [I125]PTH(1‐34) binding to PTHR1 in a concentration‐dependent fashion. In both, aged (24 months) and young (3 months) rats, binding of [I125]PTH was characterized by a single class of high‐affinity binding sites. The affinity of the receptor for PTH was not affected by age. The maximum number of specific PTHR1 binding sites was decreased by 30% in old animals. The results of this study suggest that age‐related declines in PTH regulation of signal transduction pathways in rat enterocytes may be due, in part, to the loss of hormone receptors. J. Cell. Biochem. 88: 1157–1167, 2003. © 2003 Wiley‐Liss, Inc.
Nuclear trafficking of FGFR1: A role for the transmembrane domain
Authors: Myers Jason M. , Martins Gabriel G. , Ostrowski Jacek , Stachowiak Michal K.
Published: March 2003

Several members of the fibroblast growth factor (FGF) family lack signal peptide (SP) sequences and are present only in trace amounts outside the cell. However, these proteins contain nuclear localization signals (NLS) and accumulate in the cell nucleus. Our studies have shown that full length FGF receptor 1 (FGFR1) accumulates within the nuclear interior in parallel with FGF‐2. We tested the hypothesis that an atypical transmembrane domain (TM) plays a role in FGFR1 trafficking into the nuclear interior. With FGFR1 destined for constitutive fusion with the plasma membrane due to its SP, how the receptor may enter the nucleus is unclear. Sequence analysis identified that FGFR1 has an atypical TM containing short stretches of hydrophobic amino acids (a.a.) interrupted by polar a.a. The β‐sheet is the predicted conformation of the FGFR1 TM, in contrast to the α‐helical conformation of other single TM tyrosine kinase receptors, including FGFR4. Receptor trafficking in live cells was studied by confocal microscopy via C‐terminal FGFR1 fusions to enhanced green fluorescent protein (EGFP) and confirmed by subcellular fractionation and Western immunoblotting. Nuclear entry of FGFR1–EGFP was independent of karyokinessis, and was observed in rapidly proliferating human TE671 cells, in slower proliferating glioma SF763 and post‐mitotic bovine adrenal medullary cells (BAMC). In contrast, a chimeric FGFR1/R4‐EGFP, where the TM of FGFR1 was replaced with that of FGFR4, was associated with membranes (golgi‐ER, plasma, and nuclear), but was absent from the nucleus and cytosol. FGFR1Δ‐EGFP mutants, with hydrophobic TM a.a. replaced with polar a.a., showed reduced association with membranes and increased cytosolic/nuclear accumulation with an increase in TM hydrophilicity. FGFR1(TM−)‐EGFP (TM deleted), was detected in the golgi‐ER vesicles, cytosol, and nuclear interior; thus demonstrating that the FGFR1 TM does not function as a NLS. To test whether cytosolic FGFR1 provides a source of nuclear FGFR1, cells were transfected with FGFR1(SP−) (SP was deleted), resulting in cytosolic, non‐membrane, protein accumulation in the cytosol and the cell nucleus. Our results indicate that an unstable association with cellular membranes is responsible for the release of FGFR1 into the cytosol and cytosolic FGFR1 constitutes the source of the nuclear receptor. J. Cell. Biochem. 88: 1273–1291, 2003. © 2003 Wiley‐Liss, Inc.
Vascular Anatomy of the Palmar Surfaces of the Distal Radius And Ulna: Its Relevance to Pedicled Bone Grafts at the Distal Palmar Forearm
Authors: HAERLE M. , SCHALLER H. E. , MATHOULIN C.
Published: March 2003

The potential for harvesting vascularized bone grafts from the palmar surface of the distal radius has been studied in 40 arms of fresh cadavers which had previously been injected with coloured latex solution. It was found that vascularized grafts can be pedicled on the radial part of the palmar carpal arterial arch. If a longer pedicle is required, the bone graft can be pedicled on the anterior branch of the anterior interosseous artery with retrograde flow occurring from the palmar carpal arch.
The Dorsal Flap of the First Web
Authors: PAGLIEI A. , ROCCHI L. , TULLI A.
Published: March 2003

In the first web space region, there are communications between the dorsal arteries which arise from the dorsal branch of the radial artery and the digital arteries of the thumb and the index finger. These allow a distally based flap to be raised in the first dorsal intermetacarpal area. This flap has been used in 15 cases of soft-tissue loss from the thumb and index finger. The donor sites were closed primarily in all but two patients. There were no complications, and the results show that this flap is useful for soft-tissue defects on the tip and the palmar and dorsal surfaces of the thumb. Moreover, it may be used as a “cross-finger” flap.
Italian Version of the Disability of the Arm, Shoulder and Hand (dash) Questionnaire. Cross-Cultural Adaptation and Validation
Authors: PADUA R. , PADUA L. , CECCARELLI E. , ROMANINI E. , ZANOLI G. , AMADIO P.C. , CAMPI A.
Published: March 2003

An Italian version of the Disability of the Arm, Shoulder and Hand (DASH) questionnaire has been devised and its reliability and validity have been assessed in a cross-sectional study of 108 consecutive patients with upper extremity pathologies. A sub-sample of 30 patients was used to assess re-test reliability. The principal DASH scale showed a high correlation with other patient-oriented measures and demonstrated good reproducibility, consistency and validity, which were similar to those for other languages’ versions of DASH. These findings suggest that the evaluation capacities of the Italian DASH are equivalent to those of other language versions of the DASH.
Relationship Between Joint Laxity and Radio-Ulno-Carpal Joint Morphology
Authors: GARCIA-ELIAS M. , PITÁGORAS T. , GILABERT-SENAR A.
Published: March 2003

The relationships between wrist laxity, ulnar variance, sigmoid notch inclination, and lunotriquetral motion were analysed in 60 normal volunteers. A strong correlation between ulnar length and sigmoid notch inclination was found for the entire group. Joint laxity was found to correlate with ulnar variance and lunotriquetral mobility in women, but not in men. The greater the laxity, the shorter the ulna and the greater the lunotriquetral motion during radial to ulnar deviation. These results support the concept that laxity increases the vulnerability of the wrist to injury.
The anatomy of the posterolateral aspect of the rabbit knee
Authors: Crum Joshua A. , Laprade Robert F. , Wentorf Fred A.
Published: March 2003

The purpose of this study was to determine the anatomy of the posterolateral aspect of the rabbit knee to serve as a basis for future in vitro and in vivo posterolateral knee biomechanical and injury studies. Twelve nonpaired fresh‐frozen New Zealand white rabbit knees were dissected to determine the anatomy of the posterolateral corner.

The following main structures were consistently identified in the rabbit posterolateral knee: the gastrocnemius muscles, biceps femoris muscle, popliteus muscle and tendon, fibular collateral ligament, posterior capsule, ligament of Wrisberg, and posterior meniscotibial ligament. The fibular collateral ligament was within the joint capsule and attached to the femur at the lateral epicondyle and to the fibula at the midportion of the fibular head. The popliteus muscle attached to the medial edge of the posterior tibia and ascended proximally to give rise to the popliteus tendon, which inserted on the proximal aspect of the popliteal sulcus just anterior to the fibular collateral ligament. The biceps femoris had no attachment to the fibula and attached to the anterior compartment fascia of the leg.

This study increased our understanding of these structures and their relationships to comparative anatomy in the human knee. This knowledge of the rabbit's posterolateral knee anatomy is important to understand for biomechanical and surgical studies which utilize the rabbit knee as a model for human posterolateral knee injuries. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Subadditivity of Entropy for Stochastic Matrices
Authors: Słomczyński Wojciech
Published: March 2003

The entropy of an irreducible stochastic matrix measures its mixing properties. We show that this quantity is subadditive and strongly subadditive.
Pamidronate treatment improves bone mineral density in children with Menkes disease
Authors: Kanumakala S. , Boneh A. , Zacharin M.
Published: March 2003

Menkes disease is a severe multisystem disorder due to defective bioavailability and transport of copper at the cellular level. Deficient activity of lysyl oxidase, a copper‐dependent enzyme, causes defective collagen cross‐linking leading to osteoporosis and pathological fractures in these children. The objective of the study was to evaluate the changes in bone mineral density following pamidronate treatment in children with Menkes disease. The study design was an open observational study of three children with Menkes disease and significant osteoporosis with or without pathological fractures, all of whom received pamidronate treatment for 1 year. There were 34–55% and 16–36% increases in lumbar spine bone mineral content and areal bone mineral density, respectively, following 1 year of treatment with pamidronate. There were no further fractures in two of the three children treated. No adverse effects of pamidronate treatment were noted. Pamidronate treatment was associated with an increase in bone mineral density and may be an effective treatment modality for the management of osteoporosis in children with Menkes disease.
Propranolol for reemergent posttraumatic stress disorder following an event of retraumatization: A case study
Authors: Taylor Fletcher , Cahill Larry
Published: March 2003

This case report concerns a 44‐year‐old woman who experienced 5 similar motor vehicle accidents, the last 3 causing severe PTSD episodes of over 6 months each, despite multiple pharmacotherapies. Following a 6th accident, severe PTSD symptoms reemergcd. Forty‐eight hours after this trauma, propranolol (60 mg) orally, twice a day (1.75 mg/kg/day) was begun, and the PTSD symptoms were rapidly and markedly reduced. The Clinician‐Administered PTSD Rating Scale score was reduced from an initial 86 to 56 by 11 days posttrauma. To our knowledge, this is the first report of the effects of propranolol treatment on reemergent PTSD symptoms. Propranolol may be particularly efficacious in the prevention of initial or reemergent PTSD symptoms.
Identification of novel mutations in the NPC1 gene in German patients with Niemann–Pick C disease
Authors: Kaminski W. E. , Klünemann H. H. , Ibach B. , Aslanidis C. , Klein H. E. , Schmitz G.
Published: March 2003

Niemann–Pick disease type C (NPC) is an inherited neuro degenerative disorder associated with intracellular cholesterol trafficking defects. Mutations in two distinct genes, NPC1 and HE1, have recently been shown to cause this disease. We have analysed the NPC1 gene in five German patients with NPC from four unrelated families. We identified a total of five novel mutations in the coding region of the NPC1 gene (G231V, D874V, I642M, I1094T and R116stop). All affected individuals displayed compound heterozygosity. The mutated alleles were transmitted by the nonaffected parents with the exception of one patient, in whom a de novo mutation (G231V) had occurred. Interestingly, the G231V/P237S NPC1 genotype in this individual is associated with an early‐onset form of NPC. In contrast, we found that the D874V/D948N genotype, observed in another NPC patient, is characterized by a late onset of clinical symptoms that presents with a pronounced white‐matter disease. Our results will contribute to defining the association between the clinical phenotypes and the genetic abnormalities in Niemann–Pick C disease.
Fluorimetric Analysis of Phospholipase Activity in Tetrahymena pyriformis GL.
Authors: Kovács Péter , Csaba György
Published: March 2003

The unicellular Tetrahymena enzymatically split the synthetic phosphodiester, 4-methylum-belliferyl phosphocoline substrate. The enzyme activity was completely blocked in vitro and drastically inhibited in vivo by G-protein activating fluorides (NaF; AlF4– and BeF3–). The phospholipase A2 inhibitor, quinacrine, and the protein phosphatase inhibitor, neomycin, inhibited the enzyme activity in vitro and activated it in vivo. Another phospholipase A2 inhibitor 4-bromo phenacyl bromide was ineffective in vivo and in vitro alike, as well as the cyclooxygenase inhibitor indomethacin. Results of these experiments indicate that some treatments could be specific for a well defined activity (e.g., phospholipase A2, G-protein) but subject to influence by other enzymes (e.g., phospholipase C, sphingomyelinase). The experiments call attention to the differences in the results of the in vivo and in vitro studies.
Fear activation and habituation patterns as early process predictors of response to prolonged exposure treatment in PTSD
Authors: van Minnen Agnes , Hagenaars Muriel
Published: March 2003

Improved (n = 21) and nonimproved (n = 13) PTSD patients (a mixed trauma population) were compared for fear activation and habituation patterns during and between the 1st and 2nd prolonged exposure sessions. Drop‐outs (n = 11) were also evaluated. Nonimproved patients had significantly higher ratings of anxiety at the start of the first exposure session. Improved patients showed more within‐session habituation during the self‐exposures at home and more between‐session habituation. Even after controlling for initial PTSD and depression symptom severity, habituation between the first and second exposure sessions was significantly related to treatment outcome. Patients who dropped out of the treatment were found not to differ from completers on fear activation and within‐session habituation during the first exposure session.
Ceramide Glycanase Activities in Human Cancer Cells
Authors: Basu Manju , Kelly Patrick , O'Donnell Peter , Miguel Maria , Bradley Mathew , Sonnino Sandro , Banerjee Sipra , Basu Subhash
Published: March 2003

Ceramide glycanase (CGase) activities have been detected in different human tumor cells (colon, carcinoma Colo-205; neuroblastoma, IMR-32; breast cancer lines, SKBr3 and MCF7). However, the level of enzymatic activity is lower in these cells compared to that present in other mammalian tissues reported before (Basu, M., Kelly, P., Girzadas, M. A., Li, Z., and Basu, S. Methods Enzymol. (in press)). The majority of CGase activity was found in the 100,000g soluble supernatant fraction isolated from all these cell lines and tissues. Using the soluble enzyme, the requirement for optimum CGase activity was found to be consistent with previous observations found for rat and rabbit tissues (Basu, M., Dastgheib, S., Girzadas, M. A., O'Donnell, P. H., Westervelt, C. W., Li, Z., Inokuchi, J. I., and Basu, S. (1998) Acta Pol. Biochim. 42:327). The CGase activities from both Colo-205 and IMR-32 cells are optimum at a protein to detergent ratio of one. All the mammalian CGases, including human cancer cells, show an optimum pH between 5.5 and 5.8 in sodium acetate buffer. The CGase activities from cancer cells are found to be cation-independent; however, mercury, zinc, and copper ions seem to inhibit the enzyme activity substantially in both tumor cells lines. The mercury ion inhibition of CGase activities from all different sources indicates a possible structural homology in the CGase proteins.

Radiolabeled substrates, labeled at the sphingosine double bond or at the 3-position of sphingosine without modifying double bond of sphingosine were used in this investigation. Both were active substrates with all enzyme preparations isolated from different cancer cells (apparent Km, 500 μM for nLcOse5[3H-DT]Cer and 350 μM for GgOse4[sph-3-3H]Cer with Colo-205 enzyme). Structural analogues of ceramide and sphingosine (L-PPMP, L-PDMP, alkylamines, and Tamoxifen) inhibited cancer cell CGase activities in vitro.
Thermodynamic Analysis of Chitooligosaccharide Binding to Urtica dioica agglutinin by Isothermal Titration Calorimetry
Authors: Katiyar Samiksha , Van Damme E. J. M. , Peumans Willy J. , Surolia Avadhesha
Published: March 2003

UDA (Urtica dioica agglutinin) contains two hevein like domains with two non-identical interacting sites and is specific for chitooligosaccharides. The binding of chitooligosaccharides to UDA was studied by Isothermal Titration Calorimetry. Each site is composed of three subsites, each binding to a sugar residue. Thermodynamic parameters obtained show that while chitobiose has two independent non-interacting sites, chitotriose, chitotetrose and chitopentose have two interacting sites on each monomer of UDA. Values of binding enthalpy (ΔH) increase almost by a factor of 7 in going from chitobiose to chitotriose indicating the existence of three subsites in the combining site of UDA. The binding constant for chitotetrose and chitopentose increase without any further enhancement in the values of ΔH indicating that for oligomers larger than chitotriose interaction is favoured entropically.
Overexpression of Orphan G–Protein-Coupled Receptor, Gpr49 , in Human Hepatocellular Carcinomas With β–Catenin Mutations
Authors: Yamamoto Yoshiya , Sakamoto Michiie , Fujii Gen , Tsuiji Hitomi , Kenetaka Kengo , Asaka Masahiro , Hirohashi Setsuo
Published: March 2003

To identify the genes responsible for carcinogenesis and progression of hepatocellular carcinoma (HCC), we screened differentially expressed genes in several human HCC cell lines. Among these genes, Gpr49 was up–regulated in PLC/PRF/5 and HepG2. Gpr49 is a member of the glycoprotein hormone receptor subfamily, which includes the thyroid–stimulating hormone receptor (TSHR). However, Gpr49 remains to be an orphan G–protein–coupled receptor. By real–time quantitative reverse transcriptase polymerase chain reaction (RT–PCR) analysis, overexpression (>3–fold increase compared with the corresponding noncancerous liver tissue) of Gpr49 mRNA was observed in 18 of 38 (47%) HCCs compared with corresponding noncancerous livers. Clinicopathologically, overexpression of Gpr49 was frequently observed in HCC with mutation in β–catenin exon 3 (14 of 16 cases, 87.5%). Moreover, introduction of mutant β–catenin into mouse hepatocytes in culture caused up–regulation of the Gpr49 mouse homologue. Therefore, Gpr49 is likely to be a target gene activated by Wnt–signaling in HCC. In conclusion, although much is still unknown, Gpr49 may be critically involved in the development of HCCs with β–catenin mutations and has the potential to be a new therapeutic target in the treatment of HCC.
Proangiogenic Role of Tumor–Activated Hepatic Stellate Cells in Experimental Melanoma Metastasis
Authors: Olaso Elvira , Salado Clarisa , Egilegor Eider , Gutierrez Virginia , Santisteban Aitor , Sancho-Bru Pau , Friedman Scott L. , Vidal-Vanaclocha Fernando
Published: March 2003

Myofibroblasts infiltrate malignant liver tumors, although their pathogenic implications are unclear. Immunohistochemical detection of α–smooth muscle actin, glial fibrillary acidic protein (GFAP), and CD31 and CD34 expression was used to analyze the contribution of myofibroblasts to angiogenesis in hepatic metastasis produced by intrasplenically–injected B16 melanoma (B16M). Because activated hepatic stellate cells (HSCs) are oxygen–sensing myofibroblasts producing vascular endothelial growth factor (VEGF), the effect of B16M and human A375 melanoma supernatants on VEGF production by immortalized rat HSC line T6 and primary cultured human HSCs also was studied under an hypoxic atmosphere mimicking a tumor microenvironment. Myofibroblast infiltration preceded endothelium recruitment in avascular micrometastasis and generated specific stroma for sinusoidal–type and portal–type angiogeneses. Thereafter, myofibroblasts and endothelial cells colocalized within both angiogenic patterns and their numerical densities correlated with metastasis development. Myofibroblasts often were GFAP–positive, suggesting an HSC origin. Melanoma supernatants stimulated VEGF messenger RNA and protein synthesis by HSCs. These effects were potentiated by hypoxia. VEGF up–regulation was accompanied by increased expression of cyclooxygenase type 2 (COX–2) and PGE2 synthesis. HSC production of VEGF decreased under COX–2 inhibition, whereas it was increased by exogenous PGE2. The high VEGF expression in HSCs induced by melanoma factors and hypoxia resulted in mitogenic, antiapoptotic, and motogenic stimulation of both murine hepatic sinusoidal endothelium and human umbilical vein endothelium. In conclusion, temporal and positional relationships evolve between myofibroblast and endothelium recruitment during metastasis development. Mechanistically, hypoxic induction of VEGF in tumor–activated HSCs may create a proangiogenic microenvironment, facilitating endothelial cell recruitment and survival during hepatic metastasis transition from an avascular to a vascular stage.
Antiangiogenic Gene Therapy for Hepatocellular Carcinoma Using Angiostatin Gene
Authors: Ishikawa Hiroki , Nakao Kazuhiko , Matsumoto Kojiro , Ichikawa Tatsuki , Hamasaki Keisuke , Nakata Keisuke , Eguchi Katsumi
Published: March 2003

Recent studies have reported that antiangiogenic gene delivery into cancer cells inhibits growth of certain tumors in vivo. Hepatocellular carcinoma (HCC) is a hypervascular cancer, and antiangiogenic gene therapy might be suitable for HCC. In the present study, we investigated the antiangiogenic effects of angiostatin gene transduction into HCC both in vitro and in vivo. Angiostatin gene was cloned into a pSecTag2B mammalian expression vector to construct pSecTag2B–ANG. pSecTag2B or pSecTag2B–ANG were transfected into an HCC cell line, PLC/PRF/5, and then stable transfectants were obtained by Zeocin selection. pSecTag2B or pSecTag2B–ANG transfection did not alter the expression of vascular endothelial growth factor (VEGF), a potent angiogenic stimulator, or pigment epithelium–derived factor (PEDF), an angiogenic inhibitor, in PLC/PRF/5 cells. However, conditioned media (CM) derived from pSecTag2B–ANG-transfected PLC/PRF/5 cells (CM–ANG) suppressed the proliferation and migration of human umbilical vein endothelial cells (HUVEC) by 35% and 50%, respectively, relative to their effects on nontransfected cells. In in vivo experiments, pSecTag2B–ANG stable transfected (CM–Mock) and nontransfected cells (CM–N) were mixed at various proportions and the mixed cells were subcutaneously implanted into athymic mice. Suppression of tumor growth was noted in mice implanted with angiostatin gene-transfected cells, and such suppression was proportional with the percentage of transfected cells. Analysis of the vascular density in these tumors showed that the tumor growth suppression effect of angiostatin gene correlated with suppression of tumor vascularity. In conclusion, antiangiogenic gene therapy using angiostatin gene is potentially suitable for the treatment of patients with HCC.
Replicative Senescence of Activated Human Hepatic Stellate Cells Is Accompanied by A Pronounced Inflammatory But Less Fibrogenic Phenotype
Authors: Schnabl Bernd , Purbeck Carrie A. , Choi Youkyung Hwang , Hagedorn Curt H. , Brenner David
Published: March 2003

Limited proliferative capacity is a characteristic of most normal human cells and results in a growth–arrested state, called replicative senescence. Functional expression of the telomerase catalytic subunit (human telomerase reverse transcriptase; hTERT) in human activated hepatic stellate cells (HSCs) rescues them from death with immortalization and maintains an activated HSC phenotype. The aim of this study was to evaluate alterations in gene and protein expression of in vitro aged human activated HSCs and to define the pathway by which senescent–activated HSCs are eliminated in culture. Altered patterns of gene expression in senescent human HSCs were assessed using DNA microarray analysis and compared with early passage HSCs or hTERT immortalized HSCs. Senescent HSCs showed higher expression of inflammation and stress–associated genes as compared with early passage HSCs. Senescent HSCs expressed reduced levels of extracellular matrix proteins, including collagens, tenascin, and fibronectin. TUNEL staining of senescent HSCs showed approximately 21% positive cells, indicating DNA fragmentation and apoptosis. Apoptosis involved the mitochondrial pathway with decreased levels of Bcl–2 and Bcl–xL protein, release of cytochrome c, and increased caspase–3 activity. In contrast, 4% to 5% of early activated HSCs or telomerase positive HSCs were TUNEL positive. In conclusion, cultured human HSCs undergo a switch from a fibrogenic to an inflammatory phenotype, suggesting that senescent human HSCs might modulate chronic wound healing processes. Maintenance of telomere length represents an important survival factor for activated human HSCs.
Platelet Activation and the Formation of the Platelet Plug
Authors: Tsai Han-Mou
Published: March 2003

In the circulation, a plasma metalloprotease, ADAMTS13, cleaves von Willebrand factor (vWF) in a shear-dependent manner. This article reviews the role of this cleavage in regulating vWF-platelet interaction and proposes a scheme for understanding how a deficiency of ADAMTS13 results in the development of microthrombi in patients with thrombotic thrombocytopenic purpura.
Obesity and Systemic Oxidative Stress
Authors: Keaney John F. , Larson Martin G. , Vasan Ramachandran S. , Wilson Peter W.F. , Lipinska Izabella , Corey Diane , Massaro Joseph M. , Sutherland Patrice , Vita Joseph A. , Benjamin Emelia J.
Published: March 2003

Objective—

To determine the clinical conditions associated with systemic oxidative stress in a community-based cohort. Information regarding cardiovascular risk factors associated with systemic oxidative stress has largely been derived from highly selected samples with advanced stages of vascular disease. Thus, it has been difficult to evaluate the relative contribution of each cardiovascular risk factor to systemic oxidative stress and to determine whether such risk factors act independently and are applicable to the general population.

Methods and Results—

We examined 2828 subjects from the Framingham Heart Study and measured urinary creatinine–indexed levels of 8-epi-PGF
2α
as a marker of systemic oxidative stress. Age- and sex-adjusted multivariable regression models were used to assess clinical correlates of oxidative stress. In age- and sex-adjusted models, increased urinary creatinine–indexed 8-epi-PGF
2α
levels were positively associated with female sex, hypertension treatment, smoking, diabetes, blood glucose, body mass index, and a history of cardiovascular disease. In contrast, age and total cholesterol were negatively correlated with urinary creatinine–indexed 8-epi-PGF
2α
levels. After adjustment for several covariates, decreasing age and total/HDL cholesterol ratio, sex, smoking, body mass index, blood glucose, and cardiovascular disease remained associated with urinary 8-epi-PGF
2α
levels.

Conclusions—

Smoking, diabetes, and body mass index were highly associated with systemic oxidative stress as determined by creatinine-indexed urinary 8-epi-PGF
2α
levels. The effect of body mass index was minimally affected by blood glucose, and diabetes and may suggest an important role of oxidative stress in the deleterious impact of obesity on cardiovascular disease.
Gonadal Steroids Differentially Regulate the Messenger Ribonucleic Acid Expression of Pituitary Orexin Type 1 Receptors and Adrenal Orexin Type 2 Receptors
Authors: Jöhren Olaf , Brüggemann Norbert , Dendorfer Andreas , Dominiak Peter
Published: March 2003

Hypothalamic prepro-orexin as well as pituitary and adrenal orexin receptors are gender-specifically expressed. To assess the regulation by gonadal steroids, we investigated the effect of 17β-estradiol in female and of testosterone in male rats on prepro-orexin and orexin receptor mRNA expression. Rats were either sham-operated or gonadectomized and subsequently treated with placebo, 17β-estradiol, or testosterone for 21 d. Tissue mRNA levels of prepro-orexin, orexin type-1 (OX1), and orexin type-2 (OX2) receptors were measured using quantitative real-time RT-PCR.

In female rats, pituitary OX1 receptor mRNA levels were increased 12-fold after ovariectomy compared with sham- operated rats. The increase of pituitary OX1 receptor mRNA was inhibited by treatment with 17β-estradiol. Adrenal mRNA levels of OX2 receptors in ovariectomized rats were increased 2-fold compared with sham-operated rats and were also reduced by treatment with 17β-estradiol. In male rats, orchidectomy increased the mRNA levels of pituitary OX1 receptors compared with sham-operated rats. In contrast, adrenal OX2 receptor mRNA was reduced after orchidectomy. Testosterone treatment reversed the effect of orchidectomy on pituitary OX1 and adrenal OX2 receptors. In the hypothalamus, no differences were found in the mRNA levels of prepro-orexin, OX1, and OX2 receptors between sham-operated, placebo-treated, and steroid-treated female or male rats.

Our results indicate that gonadal steroids differentially regulate pituitary OX1 receptors and adrenal OX2 receptors in male and female rats and may contribute to specific sex- dependent neuroendocrine and endocrine actions of orexins.
Bone morphogenetic protein‐2 enhances osterix gene expression in chondrocytes
Authors: Yagi K. , Tsuji K. , Nifuji A. , Shinomiya K. , Nakashima K. , deCrombrugghe B. , Noda Masaki
Published: March 2003

Osterix is a recently identified zinc‐finger‐containing transcription factor, which is required for skeletogenesis as no bone formation was observed in osterix‐deficient mice. Osterix was first cloned as a gene whose expression was enhanced by BMP in C2C12 cells. As BMP induces ectopic bone formation in vivo via a pathway reminiscent to endochondral bone formation, BMP may also regulate osterix gene expression in chondrocytes. However, no information was available regarding the BMP actions on osterix gene expression in chondrocytes. We therefore examined the effects of BMP‐2 on osterix gene expression in chondrocytes in culture. RT‐PCR analysis indicated that osterix mRNA was expressed in the primary cultures of chondrocytes derived from mouse rib cartilage. The treatment with BMP‐2 enhanced the levels of osterix transcripts within 24 h and the enhancement was still observed at 48 h based on RT‐PCR analysis. This BMP effect was specific to this cytokine, as TGF‐β did not alter osterix gene expression. BMP effects on the osterix mRNA levels were also confirmed by Northern blot analysis. The enhancing effect of BMP on osterix gene expression was observed in a dose‐dependent manner starting at 200 ng/ml. The BMP enhancement of the osterix gene expression in chondrocytes was blocked in the presence of a protein synthesis inhibitor, cycloheximide, while it was still observed in the presence of 5,6‐dichloro‐1‐β D‐ribofuranosylbenzimidazol (DRB) suggesting the involvement of post‐transcriptional events, which require new protein synthesis. These results indicated that osterix gene is expressed in the primary cultures of chondrocytes and its expression is under the control of BMP‐2. J. Cell. Biochem. 88: 1077–1083, 2003. © 2003 Wiley‐Liss, Inc.
Resonant States and Classical Damping
Authors: Chruściński Dariusz
Published: March 2003

Using Koopman's approach to classical dynamical systems we show that classical damping may be interpreted as the appearance of resonant states of the corresponding Koopman's operator. It turns out that simple classical damped systems give rise to discrete complex spectra. Therefore, the corresponding generalized eigenvectors may be interpreted as classical resonant states.
Structural Study of the Interaction Between the Mitochondrial Presequence of Cytochrome c Oxidase Subunit IV and Model Membranes
Authors: Colotto A. , Martin I. , Ruysschaert J.-M. , Sen A. , Epand R. M.
Published: March 2003

The structural effect of the presequence of cytochrome oxidase subunit IV (p25) on multilamellar liposomes with different lipid compositions has been investigated using X-ray diffraction and electron microscopy. The presequence causes the disordering of the liposomes containing negatively charged lipids, without destabilizing the bilayer structure or destroying the multilamellar nature of the liposomes. In the systems containing only zwitterionic lipids, a small increase in the d-spacing (lamellar stacking spacing) is observed without any disorder effect suggesting a weaker interaction of the peptide and lipid. Circular Dichroism measurements of the peptide, in the presence and absence of the different lipid systems studied, show that the secondary structure of the peptide is modulated by the lipid environment. Considerable amounts of α-helix in the presequence is only observed in the systems containing negatively charged lipids. These are the same systems for which the disordering effect is observed with X-ray diffraction. It is proposed that p25 disorders the bilayer stacking by corrugating the membranes. The results are discussed in terms of the relevance of the specific lipid properties (e.g., electric charge and ability to form inverted phases) in determining how the peptide interacts with the lipid and affects its structural organization. It is suggested that the lipid properties relevant for the disordering effect induced by the peptide are the same as those involved in the formation of contact sites between mitochondrial membranes during the import of nuclear coded proteins.
Phosphorylation of H,K-ATPase α-Subunit in Microsomes from Rabbit Gastric Mucosa by cAMP-Dependent Protein Kinase
Authors: Murtazina Dilyara A. , Petukhov Sergei P. , Storey Kenneth B. , Rubtsov Alexander M. , Lopina Olga D.
Published: March 2003

A 100-kDa protein that is a main component of the microsomal fraction from rabbit gastric mucosa is phosphorylated by cAMP-dependent protein kinase (PKA) in the presence of 0.2% Triton X-100. Microsomes from rabbit gastric mucosa possess activity of H,K-ATPase but not activity of Na,K-ATPase. Incubation of microsomes with 5 μM fluorescein 5′-isothiocyanate (FITC) results in both an inhibition of H,K-ATPase and labeling of a protein with an electrophoretic mobility corresponding to the mobility of the protein phosphorylated by PKA. The data suggest that the α-subunit of H,K-ATPase can be a potential target for PKA phosphorylation.
Effect of Palmitic and Lauric Acids on the Phospholipid Bilayer Membrane Conductivity
Authors: Tsofina L. M. , Mokhova E. N.
Published: March 2003

Palmitic acid increased the conductivity of BLM from mitochondrial phospholipids when they were dissolved in a mixture of decane and chlorodecane, and was ineffective when phospholipids were dissolved in decane. Lauric acid produced an increase in the membrane conductivity independently of the phospholipid type in the membrane-forming solutions (mitochondrial phospholipids, asolectin, lecithin with cholesterol) and their solvents (decane or decane with chlorodecane). The results show that discrepancies between published data concerning fatty acid effects on the BLM conductivity may be explained by differences in phospholipids, their solvents and fatty acid used.
Inhibition of Both Na/H and Bicarbonate-Dependent Exchange is Required to Prevent Recovery of Intracellular pH in Single Cardiomyocytes Exposed to Metabolic Stress
Authors: Serrano Oscar K. , Jovanovic Aleksandar , Terzic Andre
Published: March 2003

Although tight regulation of intracellular pH (pHi) is critical for the survival under stress, paradoxically a slowed recovery of pHi under hypoxic injury may be cardioprotective. In this study, we investigated the recovery of pHi after hypoxia-induced intracellular acidosis in cardiomyocytes loaded with the H+-sensitive dye SNARF-1. Exposure of single cardiomyocytes to 2,4-dinitrophenol (DNP), an inhibitor of mitochondrial oxidative phosphorylation, induced significant intracellular acidification. However, within 10–12 min upon removal of DNP, cardiomyocytes restituted their intracellular H+ concentration. The presence either of 5-N-ethyl-N-isopropylamiloride (EIPA) an inhibitor of Na/H antiporter, or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of bicarbonate-dependent exchange, did not modify the cellular response to DNP. But, combined use of EIPA and DIDS prevented the restitution of intracellular pH following removal of DNP. This study, thus, demonstrated, for the first time, that blockade of both Na/H and bicarbonate-dependent exchange is necessary and sufficient to maintain the hypoxia-induced intracellular acidification. Therefore, concomitant blockade of both pH-regulating mechanisms deserves to be further considered as a novel strategy against hypoxia-reoxygenation injury in the heart.
A Folate Binding Protein in Ascitic Fluid, Serum and Ovarian Tissue of Patients with Ovarian Adenocarcinoma Immunoreacts with Antibodies Against Human Milk Folate Binding Protein
Authors: Holm Jan , Hansen Steen Ingemann , Høier-Madsen Mimi , Helkjær Poul-Erik
Published: March 2003

The presence of a folate binding protein which immunoreacts with antibodies against human milk folate binding protein was demonstrated in ascitic fluids from seven patients with ovarian adenocarcinoma. Ascitic fluids collected from two patients with other malignancies contained non-immunoreactive FBP. Tumor tissue specimens from five patients with ovarian carcinoma contained immunoreactive FBP. By contrast to normal ovaries ovarian carcinoma tissue showed positive immunostaining on immunohistochemistry. Ascitic fluids from two patients with ovarian carcinoma exhibited single distinct bands on SDS-PAGE immunoblotting. The gel filtration profile of ovarian carcinoma tissue homogenate from two patients contained 25 and 100 kDa peaks of radioligand-bound and immunoreactive folate binding protein, while ascitic fluid from one of the patients exhibited a large 100 kDa immunoreactive peak with no radioligand binding activity. The immunoreactive non-functional 100 kDa FBP could represent unprocessed precursor FBP. Future studies are necessary to evaluate whether determination of immunoreactive FBP in ovarian adenocarcinomatosis is of any diagnostic value.
Adherence of Pathogenic Mycoplasmas to Host Cells
Authors: Razin Shmuel
Published: March 2003

The significant genome compaction in mycoplasmas was made possible by adoption of a parasitic lifestyle. During their evolution and adaptation to a parasitic mode of life the mycoplasmas have developed various genetic systems enabling their attachment to host tissues as well as a highly plastic set of variable surface proteins. The generation of a versatile surface coat through high-frequency phase and size variation provides the organism with a useful tool for immune system avoidance, allowing the mycoplasmas to escape antibody attack, explaining why these minute organisms are such successful parasites.
Retention and Degradation of N-Glycoproteins in the Rough Endoplasmic Reticulum
Authors: Duvet Sandrine , Dubuisson Jean , Ermonval Myriam , Cacan René , Verbert André
Published: March 2003

Recent studies have shown that newly synthesized proteins and glycoproteins are submitted to a quality control mechanism in the rough endoplasmic reticulum (ER). In this report we present two models: One model will illustrate a transient retention in rough ER leading to a further degradation of glycoproteins in the cytosol, (soluble alkaline phosphatase expressed in Man-P-Dol deficient CHO cells lines). The second model will illustrate a strict retention of glycoproteins in rough ER without degradation nor recycling through the Golgi (E1, E2 glycoproteins of Hepatitis C virus in stably transfected UHCV-11.4 cells and in infected Hep G2 cells).

In both cases, oligomannoside structures are markers of these phenomena, either as free soluble released oligomannosides in the case of degradation, or as N-linked oligomannosides for strict retention in rough ER.
Conformational Analysis of the Phosphorylated Epidermal Growth Factor Receptor
Authors: Bishayee Anupam , Beguinot Laura , Bishayee Subal
Published: March 2003

Phosphorylation-induced conformational changes have been well documented with different receptor tyrosine kinases. However, the susceptible epitopes and the tyrosine residue(s) involved in particular structural alteration mostly remain to be determined. Using a conformation-specific anti-peptide antibody, we have not only identified one such domain in the C-terminal tail of the EGF receptor but also identified the phosphate acceptor sites that are involved in the conformational change.
Validation of the Dutch EDI-2 in One Clinical and Two Nonclinical Populations1
Authors: van Strien Tatjana , Ouwens Machteld
Published: March 2003

Summary The present study establishes the validity of the Dutch translation of the EDI-2 by determining its factor structure, construct validity and its effectiveness for screening using a clinical and two nonclinical populations. An earlier study had shown that item transformation damaged the validity of the EDI-1 in a nonclinical population of female high-school students. Therefore, the issue of the use of transformed (0-3) versus untransformed (1-6) item responses was addressed first. The present study replicated the earlier finding for the EDI-2 in the same population and similar results were also found in another nonclinical population of female college students. Furthermore, untransformed responses were found to also work well in a clinical population of female patients suffering from eating disorders. Accordingly, further analyses were based on untransformed responses. The factorial integrity of the original 64-item EDI-1 was best supported in the clinical group and the nonclinical group of college students, but the three provisional scales performed poorly in terms of internal consistency and factorial integrity. In all three populations, the EDI-2 was shown to be specific for eating-disorder pathology, and to have good construct validity. It is concluded that the EDI-2 is a suitable screening tool to identify those cases in large populations that warrant more thorough examination.
Paraventricular Nucleus Administration of Calcitonin Gene-Related Peptide Inhibits Food Intake and Stimulates the Hypothalamo-Pituitary-Adrenal Axis
Authors: Dhillo Waljit S. , Small Caroline J. , Jethwa Preeti H. , Russell Sabina H. , Gardiner James V. , Bewick Gavin A. , Seth Asha , Murphy Kevin G. , Ghatei Mohammad A. , Bloom Stephen R.
Published: March 2003

Calcitonin gene-related protein (CGRP) inhibits food intake and stimulates the hypothalamo-pituitary-adrenal (HPA) axis after intracerebroventricular injection in rats. However, the hypothalamic site and mechanism of action are unknown. We investigated the effects of intraparaventricular nucleus administration (iPVN) of CGRP on food intake and the HPA axis in rats and the effect of CGRP on the release of hypothalamic neuropeptides in vitro. In addition, we investigated the effects of food deprivation on hypothalamic CGRP expression. CGRP dose-dependently reduced food intake in the first hour after iPVN injection in fasted male rats (saline, 5.1 ± 0.8 g; 0.3 nmol CGRP, 1.1 ± 0.5 g; P < 0.001 vs. saline). iPVN injection of CGRP8–37 (a CGRP1 receptor antagonist) alone had no effect on food intake. However, the reduction in food intake by iPVN CGRP was attenuated by prior administration of CGRP8–37 [CGRP8–37 (10 nmol)/CGRP (0.3 nmol), 3.0 ± 0.8 g; P < 0.05 vs. 0.3 nmol CGRP]. CGRP (100 nm) stimulated the release of α-melanocyte stimulating hormone, cocaine- and amphetamine-related transcript, corticotropin-releasing hormone, and arginine vasopressin from hypothalamic explants to 127 ± 19%, 148 ± 10%, 158 ± 17%, and 198 ± 21% of basal levels, respectively (P < 0.05 vs. basal), but did not alter the release of either neuropeptide Y or agouti-related protein. Hypothalamic CGRP mRNA levels in 24-h fasted rats were increased to 130 ± 8% of control levels [CGRP mRNA (arbitrary units), 4.75 ± 0.4; controls, 3.65 ± 0.34; P < 0.05]. Our data suggest that CGRP administered to the PVN inhibits food intake and stimulates the HPA axis.