bluuebunny/crossref_metadata_2025 huggingface dataset:
Heme Oxygenase-1 Protects Pancreatic β Cells from Apoptosis Caused by Various Stimuli
Authors: Tobiasch Edda , Günther Lukas , Bach Fritz H.
Published: August 2007

Background

Several problems can occur after allogeneic islet transplantation: primary nonfunction, rejection, and the recurrence of autoimmune disease, which involve attack by the recipient's cytokines, T cells, natural killer cells, and monocytes on the donor's β cells, which leads to β-cell destruction. Recent studies have revealed that loss of transplanted islets is caused mainly by apoptosis. Heme oxygenase-1 (HO-1) is one of the antiapoptotic genes up-regulated under stress conditions. The aim of this work was to investigate any mechanisms of HO-1-mediated protection of β cells from apoptosis.

Methods

Apoptosis was assessed by comparison of viable transfected cells with and without apoptotic stimuli, and with and without HO-1 overexpression. Activation and function of p38 mitogen-activated protein kinase were determined using the specific inhibitor SB203580.

Results

We have shown that HO-1 mediates antiapoptotic effects in β cells. The percentage of apoptotic cells after stimulation with tumor necrosis factor α decreased from 75% without HO-1 to 5% when HO-1 was overexpressed. Our data indicate that HO-1 acts as a signal terminator of tumor necrosis factor α-induced apoptosis by modulation of the p38 mitogen-activated protein kinase pathway.

Conclusions

Profound cell stress that occurs in islets after transplantation, as well as at the onset of diabetes, results in β-cell loss through apoptosis. Protection of β cells by HO-1 improves their survival in vitro after various proapoptotic stimuli, suggesting that HO-1 suppresses one or several signaling pathways leading to apoptosis. We hypothesize that our in vitro findings can be extrapolated to the in vivo situation, and we propose that expression of HO-1 in islets may illuminate a valuable new approach to improving diabetes treatment.
Increased Soluble P-Selectin Levels in Hepatitis C Virus-Related Chronic Hepatitis: Correlation with Viral Load
Authors: Ferroni Patrizia , Mammarella Antonio , Martini Francesca , Paoletti Vincenzo , Cardarello Cristiano M. , Labbadia Giancarlo , Donnarumma Lucia , De Matteis Antonio , Gazzaniga Pier Paolo , Musca Antonino , Basili Stefania
Published: August 2007

Background

Platelet functional abnormalities are commonly found in patients with chronic liver disease; however, their nature and clinical significance are still a matter of discussion.

Methods

Soluble P-selectin (sP-selectin, a marker of in vivo platelet activation) levels, lipid pattern, and clotting activity were investigated in 39 patients with histologically confirmed chronic C hepatitis.

Results

Serum factor VIIc ( P<0.01), total cholesterol ( P<0.005), high density lipoprotein ( P<0.001), and low density lipoprotein ( P<0.05) levels were lower in patients compared with healthy subjects, whereas triglyceride and fibrinogen levels were similar in both groups. Platelet counts were lower in chronic hepatitis patients compared with controls ( P<0.0001), and approximately 20% of patients had thrombocytopenia (platelet counts <110x103/μL). Platelet-associated immunoglobulin G (PAIgG) was present in 30.8% of patients. Plasma sP-selectin levels were higher in hepatitis C patients compared with controls ( P<0.0001), and significant differences were observed with respect to the Scheuer score ( P<0.01). The analysis of the distribution of plasma sP-selectin showed the presence of higher levels in patients with low platelet counts compared with patients with normal platelet counts and controls ( P<0.0001); moreover, sP-selectin levels did not correlate with the presence of PAIgG. On the other hand, sP-selectin levels directly correlated with serum hepatitis C virus (HCV)-RNA ( P<0.05) and inversely correlated with platelet count, blood lipids, and factor VIIc.

Conclusions

The results obtained in this study support the hypothesis that HCV infection might be directly responsible for a condition of in vivo platelet activation in patients with chronic C hepatitis.
Tacrolimus and Cyclosporine a Inhibit Allostimulatory Capacity and Cytokine Production of Human Myeloid Dendritic Cells
Authors: Szabo Gyongyi , Gavala Cristina , Mandrekar Pranoti
Published: August 2007

Myeloid dendritic cells (DCs) are pivotal in the recognition of alloantigens and, therefore, in the induction of allograft rejection. Induction of alloreactive T cell proliferation by myeloid DCs depends on the maturation of DCs, the expression of costimulatory molecules, and the cytokine environment. This study investigated the effects of tacrolimus and cyclosporine A (CsA) on DC maturation and allostimulatory capacity. Myeloid DCs were propagated from normal blood monocytes with interleukin (IL) 4 and GM-CSF for 7 days in the presence or absence of tacrolimus (FK506; 10 nM) or CsA (1 μg/mL). Exposure of DCs during maturation to tacrolimus or CsA resulted in no significant change in the expression of DC phenotypic markers, including CD80, CD86, and HLA Class I and II antigens determined by flow cytometry. T cell proliferation in one-way, mixed-leukocyte reaction experiments revealed a decreased allostimulatory capacity of DCs that matured in the presence of tacrolimus or CsA compared with untreated controls ( P<0.02). Production of inflammatory cytokines, tumor necrosis factor α ( P<0.04) and IL-12 ( P<0.04) in response to lipopolysaccharide (1 μg/mL) or staphylococcal enterotoxin B (1 μg/mL) induction was significantly reduced in DCs exposed to tacrolimus or CsA during maturation. In contrast, production of the immuninhibitory cytokine IL-10 was not decreased in tacrolimus- or CsA-treated DCs. These results suggest that tacrolimus and CsA inhibit the allostimulatory capacity of in vitro-generated myeloid DCs without significant effects on DC phenotypic maturation. Decreased production of IL-12 and tumor necrosis factor α, but not of IL-10, is likely to contribute to the impaired accessory-cell function of tacrolimus- and CsA-treated DCs. Thus, tacrolimus and CsA can inhibit recognition of alloantigens by decreasing the accessory-cell capacity of monocyte-derived myeloid DCs.
Early Onset of Lipid Peroxidation after Human Traumatic Brain Injury: A Fatal Limitation for the Free Radical Scavenger Pharmacological Therapy?
Authors: Cristofori Luciano , Tavazzi Barbara , Gambin Roberta , Vagnozzi Roberto , Vivenza Carlo , Amorini Angela Maria , Di Pierro Donato , Fazzina Giovanna , Lazzarino Giuseppe
Published: August 2007

Background

On the basis of the contradiction between data on experimental head trauma showing oxidative stress-mediated cerebral tissue damage and failure of the majority of clinical trials using free radical scavenger drugs, we monitored the time-course changes of malondialdehyde (MDA, an index of cell lipid peroxidation), ascorbate, and dephosphorylated ATP catabolites in cerebrospinal fluid (CSF) of traumatic brain-injured patients.

Methods

CSF samples were obtained from 20 consecutive patients suffering from severe brain injury. All patients were comatose, with a Glasgow Coma Scale on admission of 6±1. The first CSF sample for each patient was collected within a mean value of 2.95 hours from trauma (SD=1.98), after the insertion of a ventriculostomy catheter for the continuous monitoring of intracranial pressure. During the next 48 hours, CSF was withdrawn from each patient once every 6 hours. All samples were analyzed by an ion-pairing high-performance liquid chromatographic method for the simultaneous determination of MDA, ascorbic acid, hypoxanthine, xanthine, uric acid, inosine, and adenosine.

Results

In comparison with values recorded in 10 herniated-lumbar-disk, noncerebral control patients, data showed that all CSF samples of brain-injured patients had high values (0.226 μmol/L; SD=0.196) of MDA (undetectable in samples of control patients) and decreased ascorbate levels (96.25 μmol/L; SD=31.74), already at the time of first withdrawal at the time of hospital admission. MDA was almost constant in the next two withdrawals and tended to decrease thereafter, although 48 hours after hospital admission, a mean level of 0.072 μmol/L CSF (SD=0.026) was still recorded. The ascorbate level was normalized 42 hours after hospital admission. Changes in the CSF values of ATP degradation products (oxypurines and nucleosides) suggested a dramatic alteration of neuronal energy metabolism after traumatic brain injury.

Conclusions

On the whole, these data demonstrate the early onset of oxygen radical-mediated oxidative stress, proposing a valid explanation for the failure of clinical trials based on the administration of oxygen free radical scavenger drugs and suggesting a possible rationale for testing the efficacy of lipid peroxidation “chain breakers” in future clinical trials.
Ontogeny of P-Glycoprotein in Mouse Intestine, Liver, and Kidney
Authors: Mahmood Burhan , Daood Monica J. , Hart Claudia , Hansen Thor W.R. , Watchko Jon F.
Published: August 2007

Background

P-glycoprotein (Pgp) is an ATP-dependent, integral plasma-membrane efflux pump that is constitutively expressed on (i) adult apical brush-border epithelial cells of the intestine, (ii) the bile canalicular face of hepatocytes, and (iii) the brush border epithelium of renal proximal tubules. This Pgp tissue distribution and localization affects the absorption, distribution, metabolism, and excretion of Pgp substrates. Little is known regarding the ontogeny of Pgp expression in these tissues.

Methods

Postnatal expression of Pgp on brush border membranes of small intestine, liver, and kidney as a function of maturity from birth through adulthood was determined using Western immunoblotting and immunohistochemical techniques. Tissue was isolated from FVB mice at four different ages: day of life 0 (D0), day of life 7 (D7), day of life 21 (D21), and adult (Ad). The relative expression of Pgp protein on Western immunoblots was assessed by scanning densitometry and indexed as a percentage (mean±SEM) of the adult levels.

Results

On Western immunoblots, Pgp expression was limited at birth (19±6% of Ad) and increased significantly with maturation in intestine (ANOVA, P <0.005). In contrast, hepatic (113±12% of Ad) and renal (96±15% of Ad) Pgp expression were at adult levels at birth. The tissue-specific developmental pattern of Pgp expression was confirmed by immunohistochemistry.

Conclusions

We conclude that Pgp is expressed in a tissue-specific and developmentally regulated fashion and speculate that developmental modulation of intestine-Pgp expression may affect the oral bioavailability of Pgp substrates.
Gamma-Irradiation Markedly Inhibits the Hydrated Collagen Gel Contraction by Arterial Smooth Muscle Cells
Authors: Keller Pierre-Frederic , Verin Vitali , Ziegler Thierry , Mermillod Bernadette , Popowski Youri , Delafontaine Patrice
Published: August 2007

Background

Vessel wall responses to percutaneous transluminal coronary angioplasty include neointimal proliferation and arterial remodeling. The contraction of a collagen gel is a good in vitro model of wound repair and vascular remodeling. Because irradiation is an important new therapeutic modality capable of preventing restenosis, the purpose of this study was to evaluate the effect of irradiation on the contraction of a collagen gel by smooth muscle cells (SMCs), on SMCs viability, and on DNA synthesis.

Methods

We studied the effect of different doses of gamma-irradiation (0 [control], 6, 12, and 18 Gy) on the contraction of a collagen gel seeded with SMCs (calf carotid arteries) during a period of 15 days.

Results

Maximal gel diameter reduction (from 35 to 6.8 mm, ±0.5 mm in control) was markedly inhibited in the 6-, 12-, and 18-Gy groups (35 to 13.7 mm, ±0.8 mm; 35 to 15.5 mm, ±0.9 mm; and 35 to 16.1 mm, ±0.9 mm, respectively; P<0.0001). The irradiated gels showed a dose-dependent reduction in the SMC proliferation rate ( P<0.0001) and an increase in the number of nonviable SMCs ( P<0.002) 15 days after irradiation.

Conclusions

Gamma-irradiation produces a significant dose-dependent inhibition of the contraction of collagen gels seeded with arterial SMCs. This effect is related to a significant decrease in SMC viability and a decrease in SMC proliferation rate. These findings shed light on mechanisms whereby irradiation may positively affect arterial remodeling after percutaneous transluminal coronary angioplasties.
Synapsin I Identified as a Novel Brain-Specific Autoantigen
Authors: Gitlits Veronika M. , Sentry John W. , Matthew Mary L.S.M. , Smith Ian A. , Toh Ban Hock
Published: August 2007

Background

We report the identification and characterization of a novel 74-kd brain-specific autoantigen that is reactive with serum from a patient with discoid lupus erythematosus and chronic lymphocytic leukemia.

Methods

We determined the molecular weight, tissue distribution and subcellular distribution of the autoantigen and obtained limited amino acid sequence after purification by ion-exchange chromatography and trypsin digestion.

Results

We identified the 74-kd autoantigen as synapsin I on the basis of the following observations. First, the autoantigen has properties consistent with synapsin I: molecular weight of ≈74 kd, brain-specific distribution, presence in cytosol and on synaptosomes, and association with taxol-stabilized microtubules. Second, limited amino acid sequence determination after trypsin digestion of the autoantigen shows identity with synapsin I. Third, the autoimmune serum immunoblots fusion proteins that incorporate rat synapsin Ia. The autoantibodies reactive to synapsin Ia are of immunoglobulin (Ig) G and IgM class.

Conclusions

This is the first report of autoantibodies that are reactive to synapsin Ia. Autoantibodies that are reactive to synapsin Ia are not restricted to discoid lupus erythematosus patients, because we found identical reactivity in two of 18 sera from dsDNA-positive systemic lupus erythematosus patients and in two of 14 rheumatoid factor-positive sera. Whether autoantibodies to synapsin I are associated with neuropsychiatric manifestations is currently unknown.
Endstage Renal Disease Owing to Diabetic Nephropathy in Mississippi: An Examination of Factors Influencing Renal Survival in a Population Prone to Late Referral
Authors: Crook Errol D. , Harris Jonathan , Oliver Bonnie , Fleischman Erwin , Crenshaw Gregory , Taylor Robert
Published: August 2007

Background

Diabetic nephropathy (DN) is the leading cause of endstage renal disease (ESRD) in the United States. We reviewed our experience with DN as a cause of ESRD in a predominantly poor, African American (AA) population.

Methods

Charts of patients who entered the ESRD program through the University of Mississippi Medical Center with a primary diagnosis of DN from 1993 through 1998 were reviewed for factors that may affect renal survival. Time from initial clinic visit to entry into the ESRD program, or time to ESRD (TTE), was the primary end point.

Results

Five hundred sixty-two patients entered the ESRD program (85% AA), and 210 of them had DN as their primary ESRD diagnosis. DN accounted for 50.5% of ESRD cases among AA females, but for less than 20% among AA males. In contrast, hypertension was the ESRD diagnosis in 48% of AA males. Patients observed in our nephrology clinic were analyzed further (n=171). At presentation, patients had advanced disease (serum creatinine [Cr]=5.92 mg/dL), were hypertensive, obese, and not likely to be on an angiotensin-converting enzyme (ACE) inhibitor. Determinants of TTE in univariate analysis were race (AA did better), initial blood urea nitrogen and plasma serum Cr levels, starting an ACE inhibitor at the University of Mississippi Medical Center, and the level of mean arterial pressure (MAP) during the course of follow-up. On multivariate analysis only initial Cr and race remained significant. The 142 AA diabetics (111 female) were analyzed separately. The only significant sex difference was body mass index (female, 33.6 vs male, 28.4 kg/m2; P=0.0069), but females tended to have relatively shorter TTE and higher blood pressure (BP). Univariate and multivariate analyses revealed the same factors as above as determinants of TTE; however, among AAs, presenting on a calcium channel blocker was negatively correlated with TTE in univariate analysis. Among the entire cohort and the AAs, patients who had MAP between 100 and 110 mm Hg during the course of follow-up did better in terms of renal survival than those who fell outside of that range.

Conclusions

We conclude that AA females in Mississippi are significantly more predisposed to DN as a cause of ESRD than are AA males. Patients with DN in our population had poor BP control, presented to nephrologists with advanced disease, and often were not on an ACE inhibitor. The optimal level of BP control and which BP agents are best for this population need to be determined.
Use of Potassium Tellurite for Rapid-Drug Susceptibility Testing of Mycobacterium avium Complex
Authors: Afghani Behnoosh , Fujiyama Dave
Published: August 2007

Background

Mycobacterium avium complex (MAC) is a major cause of infection in immunocompromised patients. MAC possesses an enzyme that reduces potassium tellurite in less than 3 days and results in formation of a black precipitate. The objective of this study was to determine whether reduction of potassium tellurite by mycobacteria can be used as a means of testing the susceptibility of MAC to clarithromycin.

Methods

Minimum inhibitory concentrations (MICs) for 104 clinical isolates of MAC were determined by the tellurite method and compared with those tested by a recommended microdilution method. Microdilution breakpoints were used for interpretation of susceptibility. MIC of less than 8 μg/mL was considered as susceptible, and MIC of greater than or equal to 8 μg/mL was resistant.

Results

There was agreement within a 2-fold dilution between MICs for 89% of isolates. Of the 53 isolates that had discrepant MICs by the two methods, 70% had higher MICs by the tellurite method. When the MICs were classified into interpretive categories, there was 100% agreement by the two methods. The MIC tested by the tellurite method was available within 5 days.

Conclusions

These data suggest that use of potassium tellurite is a more rapid, reliable, and inexpensive method of testing the susceptibility of MAC to clarithromycin.
Silica and its Antagonistic Effects on Transforming Growth Factor-β in Lung Fibroblast Extracellular Matrix Production
Authors: Baroni Tiziano , Bodo Maria , D'Alessandro Alessandra , Conte Carmela , Calvitti Mario , Muzi Giacomo , Lumare Alessandro , Bellocchio Silvia , Abbritti Giuseppe
Published: August 2007

Background

Silicosis, a pneumoconiosis marked by interstitial pulmonary fibrosis, is caused by inhalation of free crystalline silica particles. When silica particles are injected into the lower lung, they are translocated across the epithelium into the interstitial space, where macrophage-derived growth factors affect lung fibroblast proliferation and collagen deposition. We hypothesized that silica may act directly on pulmonary fibroblasts modifying extracellular matrix (ECM) synthesis and that the effects of silica may be mediated by transforming growth factor-β (TGFβ) overproduction.

Methods

To test this hypothesis, we studied a human lung fibroblast cell line (WI-1003) exposed to silica in vitro. We investigated cell morphology by electron microscopic procedure, cell growth, collagen production, and glycosaminoglycans (GAG) composition by radiolabeled precursors. Cytokine and growth factor synthesis were evaluated by specific enzyme-linked immunoadsorbent assay kits and Northern blotting analysis.

Results

Pulmonary fibroblasts internalized silica particles without detectable cell damage. Silica directly stimulated collagen synthesis and decreased the amount of 3H-glucosamine-labeled GAG. Silica-treated fibroblasts secreted less TGFβ than untreated controls, antagonized the stimulatory effect of TGFβ on ECM synthesis, and reversed TGFβ-induced inhibition of cell proliferation. Northern blotting analysis showed increased interleukin-1α (IL-1α) mRNA after silica treatment. IL-1α had no influence on collagen synthesis but increased the number of WI-1003 fibroblasts.

Conclusions

These results support our hypothesis that lung fibroblasts are direct silica targets. However, contradicting our hypothesis, silica antagonized TGFβ activities through a TGFβ downregulation and an IL-1α upregulation. The complex pattern of TGFβ and IL-1α regulation in pulmonary fibroblasts is imbalanced by silica exposure and might play a key role in silica-mediated pulmonary fibrosis.
How Low can you Go? Chronic Hypoglycemia versus Normal Glucose Homeostasis
Authors: Sood Vidushi , Costello Brian A. , Burge Mark R.
Published: August 2007

Background

Set point errors in glucose homeostasis that result in chronic, mild hyperglycemia in the setting of maturity onset diabetes of the young have been described. Similar set point errors may exist that result in chronic, asymptomatic glucopenia.

Case

A healthy 39-year-old female was referred for evaluation of chronic, persistent, and asymptomatic glucopenia that persisted over the prior several years with a record of numerous random plasma glucose concentrations between 35 and 45 mg/dL. She denied ethanol intake and family history of hypoglycemia or diabetes. She was not taking any medications known to cause hypoglycemia, and a urine sulfonylurea screen was negative. Fasting insulin and C-peptide levels were not elevated, and pancreatic imaging studies were normal. We hypothesized that this patient possessed an error in glucose metabolism that resulted in chronic, asymptomatic glucopenia.

Results

In a series of clinical studies, we demonstrated a nadir plasma glucose concentration of 35 mg/dL in the absence of symptoms during a 60-hour fast. C-peptide secretion was appropriately suppressed during symptomatic hypoglycemia with exogenous insulin infusion, and counterregulatory hormone secretion was intact during insulin-induced symptomatic hypoglycemia. Finally, the patient demonstrated an incremental increase in insulin concentration in response to minimal increases in plasma glucose during a sequential, stepped infusion of 10% dextrose.

Conclusions

We conclude that this patient exhibits features of a set point error in glucose homeostasis that manifests as chronic, asymptomatic glucopenia. Although the mechanism for this condition remains to be elucidated, such set point errors do exist and should be considered in the differential diagnosis of chronic hypoglycemia.
Relationship of Lipoprotein(a) to Variables of Coagulation in Hypertensive Subjects
Authors: Sechi Leonardo A. , De Marchi Sergio
Published: August 2007

Background

Coagulation factors are independent predictors of cardiovascular damage in the general population. The purpose of this study was to investigate the relationships between general cardiovascular risk factors, lipoprotein(a) (Lp(a)), and some hemostatic variables, and to characterize the isoforms of apolipoprotein(a) (apo(a)) in hypertensive subjects.

Methods

Plasma lipids, apolipoproteins, Lp(a), apo(a) isoforms, fibrinogen, and parameters that directly reflect the coagulation activation were measured in 389 untreated essential hypertensive patients recruited at a hypertension clinic. Hypertensive patients were compared with 323 normotensive controls.

Results

In normotensive subjects, Lp(a) concentrations were significantly correlated with fibrinogen ( r=0.138; P<0.02) but not D-dimer ( r=0.074; not significant). In hypertensive subjects, log Lp(a) concentrations were significantly correlated with age ( r=0.127; P<0.02), apo-B ( r=0.128; P<0.02), plasma fibrinogen ( r=0.193; P<0.001), and fibrin D-dimer ( r=0.200; P<0.001) levels, but not with body mass index, blood pressure, cholesterol, triglycerides, apo-AI, prothrombin fragment 1+2, and antithrombin III. The relationship of Lp(a) with fibrinogen (male: r=0.198, P<0.002; female: r=0.177, P<0.01) and D-dimer (male: r=0.211, P<0.002; female: r=0.188, P<0.01) was significant in both sexes, whereas the relationship of Lp(a) with age and apo-B was found only in males. Multivariate analysis showed that both fibrinogen and D-dimer were independently related with Lp(a). Elevated fibrinogen, D-dimer, and Lp(a) levels were significantly and independently associated with clinical evidence of atherosclerotic disease. Apo(a) phenotypes were analyzed to investigate the genetic background of the relationships between Lp(a) and coagulation parameters. In both hypertensive and normotensive subjects, Lp(a) levels were inversely correlated with apo(a) isoform protein size, whereas fibrinogen and D-dimer concentrations were comparable in patients with apo(a) isoforms of different size.

Conclusions

The relationship between Lp(a) and clotting variables is significantly stronger in hypertensive than in normotensive subjects, providing a compelling argument for accelerated progression of atherothrombosis in these patients.
Superoxide Dismutase SOD1, Encoded on Chromosome 21, but Not SOD2 is Overexpressed in Brains of Patients with down Syndrome
Authors: Gulesserian Talin , Seidl Rainer , Hardmeier Rosmarie , Cairns Nigel , Lubec Gert
Published: August 2007

Background

The antioxidant enzyme Cu/Zn-superoxide dismutase-1 ( SOD1) gene is localized to chromosome 21q22.1 and catalyzes the dismutation of superoxide anions to hydrogen peroxide, which may lead to the increased production of active oxygen species in Down Syndrome (DS), trisomy 21. Although a number of studies have addressed this question, proposing the overexpression hypothesis, no specific protein-chemical data on SOD protein levels in the brains of patients with DS are available.

Methods

We therefore determined the protein SOD-1 and SOD-2 levels in the brains of controls (n=9) and adult patients with DS (n=9) and Alzheimer disease (AD; n=9). Two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization-mass spectroscopy detection and identification was used for the analyses.

Results

We found significantly increased SOD-1 levels in DS temporal, parietal, and occipital cortex, whereas SOD-1 was decreased in the AD temporal cortex and SOD-2 was comparable between all groups.

Conclusions

Increased SOD-1 levels in patients with DS may reflect the overexpression by the trisomic state, as a response to the oxidative stress, as has been proposed in DS by several authors. However, it well may be that glial proliferation, which is markedly increased in DS brain, may underlie the increased brain levels of this ubiquitous protein. The decrease of SOD1 in the temporal cortex of patients with AD may reflect an antiapoptotic mechanism or simply cell loss in the brain.
Primate Mammary Gland Insulin-Like Growth Factor System: Cellular Localization and Regulation by Sex Steroids
Authors: Zhou Jian , Anderson Kristin , Bievre Marsha , Ng Sui , Bondy Carolyn A.
Published: August 2007

Background

Insulin-like growth factor 1 (IGF1) plays a critical role in estrogen-induced uterine proliferation, but it is unclear whether this estromedin function occurs in other estrogen-sensitive tissues such as the mammary gland. To elucidate this possibility, we investigated the cellular localization and hormonal regulation of mRNAs for IGF1 and 2, their cognate receptors (IGF1R, IGF2R), and IGF binding proteins 2-5 (BPs 2-5) in the rhesus monkey mammary gland.

Methods

Ovariectomized monkeys were treated with placebo, estradiol (E2), and E2 plus progesterone (E2/P4) for 3 days, after which mammary tissue was harvested for in situ hybridization and immunohistochemical analyses.

Results

IGF1 and IGF2 mRNA levels were significantly increased and BP2 mRNA decreased by E2 and by E2/P4 treatment. IGF1R mRNA was increased by combined E2/P treatment but not by E2 alone. BP5 mRNA was decreased by E2/P4. No differences in IGF2R, BP3, and BP4 mRNA levels were detected in any treatment group. Mammary IGF1 and IGF2 mRNA levels were both positively correlated with local epithelial proliferation, assessed by immunodetection of the proliferation-specific antigen, Ki67. IGF1 and IGF1R expression were negatively correlated with local programmed cell death, as assessed by the in situ TUNEL method. In contrast, BP2 expression was negatively correlated with epithelial proliferation and positively correlated with programmed cell death. IGF2R, BP3, BP4, and BP5 levels were not significantly correlated with either proliferation or death.

Conclusions

Thus, E2-induced proliferation is associated with upregulation of both IGF1 and IGF2 expression and downregulation of BP2 expression. These data suggest that the local mammary IGF system is involved in sex steroid-induced mammary epithelial cell hyperplasia.
Cigarette Smoke and Asbestos Activate Poly-ADP-Ribose Polymerase in Alveolar Epithelial Cells
Authors: Kamp David W. , Srinivasan Malathi , Weitzman Sigmund A.
Published: August 2007

Background

Cigarette smoke augments asbestos-induced bronchogenic carcinoma in a synergistic manner by mechanisms that are not established. One important mechanism may involve alveolar epithelial cell (AEC) injury resulting from oxidant-induced DNA damage that subsequently activates poly (ADP-ribose) polymerase (PARP), an enzyme involved in DNA repair that can deplete cellular energy stores. We previously showed that whole aqueous cigarette smoke extracts (CSE) augment amosite asbestos-induced DNA damage and cytotoxicity to cultured AEC in part by generating iron-induced free radicals. We hypothesized that CSE increase asbestos-induced AEC injury by triggering PARP activation resulting from DNA damage caused by iron-induced free radicals.

Methods

Aqueous CSE were prepared fresh on the day of each experiment. PARP activity in WI-26 (a type I-like cell line) and A549 (a type II-like cell line) cells was assessed by the uptake of labeled NAD over 4 hours and confirmed on the basis of the reduction of PARP levels in the presence of a PARP inhibitor, 3-aminobenzamide (3-ABA). Cell survival was assessed by trypan blue dye exclusion.

Results

Hydrogen peroxide (H2O2; 1-250 μM), CSE (0.4-10 vol%), and amosite asbestos (5-250 μg/cm2) each caused PARP activation in WI-26 and A549 cells. The combination of asbestos (5 μg/cm2) and CSE (0.04-10%) induced WI-26 and A549 cell PARP activation without evidence of synergism. 3-ABA significantly attenuated WI-26 and A549 cell PARP activity and cell death after exposure to H2O2, CSE, and asbestos. Phytic acid, an iron chelator, catalase, and superoxide dismutase each decreased WI-26 cell PARP activation caused by asbestos and CSE.

Conclusions

CSE and asbestos induce PARP activation in cultured AEC in a nonsynergistic manner. These data provide further support that asbestos and cigarette smoke are genotoxic to relevant lung target cells and that iron-induced free radicals in part cause these effects.
Hepatic Lipase as a Focal Point for the Development and Treatment of Coronary Artery Disease
Authors: Zambon Alberto , Brown B. Greg , Deeb Samir S. , Brunzell John D.
Published: August 2007

Recent epidemiological evidence suggests that although lowering low-density lipoprotein (LDL) cholesterol is important in decreasing cardiovascular disease morbidity and mortality, it accounts only for part of the coronary artery disease (CAD) improvement with lipid-lowering therapy. In the last decade, it has become evident that the atherogenicity of LDL particles is associated not only with their plasma levels, but also with their size and density. The presence of small, dense LDL particles is associated with a three fold increase in CAD risk. Hepatic lipase (HL), a key enzyme in the formation of small, dense LDL particles, modulates their phospholipid and triglyceride contents. The higher the HL activity, the smaller, denser, and more atherogenic the resulting lipoprotein particle. It is, therefore, plausible to hypothesize that at least part of the CAD benefits observed in the recent CAD-prevention pharmacological trials, which are not accounted for by the decrease in LDL-C (LDL-cholesterol), might be explained by a pharmacological effect on LDL size and density, possibly mediated by changes in hepatic lipase activity. By studying patients with dyslipidemia and CAD, we have been able to provide strong evidence that regression of coronary atherosclerosis results from at least two independent effects of lipid-lowering therapy on lipoprotein metabolism: the well known one that leads to changes in LDL-C and apo B levels, and a new pathway of HL-mediated improvement in LDL buoyancy. Finally, HL activity and LDL density appear to be significantly affected by the presence of a common C>T substitution at position -514 with respect to the transcription start site of the HL gene, raising the possibility that the -514 C>T polymorphism may significantly contribute to differences in individual CAD response to lipid-lowering treatment, as seen in the recent major primary and secondary CAD-prevention clinical trials.
Stimulatory Effects of Hyperprolactinemia on Aldosterone Secretion in Ovariectomized Rats
Authors: Kau Mei-Mei , Chang Ling-Ling , Kan Shu-Fen , Ho Low-Tone , Wang Paulus S.
Published: August 2007

Background

To evaluate the effects of hyperprolactinemia on aldosterone secretion and its mechanisms of action in ovariectomized (OVX) rats.

Methods

Hyperprolactinemia was induced by the transplantation of rat anterior pituitary (AP) glands under the kidney capsule for 6 weeks in female rats. Control rats underwent cerebral cortex (CX) transplantation. Four weeks after transplantation, the rats were OVX 2 weeks before decapitation. After decapitation, the trunk blood was collected, and the adrenal glands of CX- and AP-grafted rats were prepared as zona glomerulosa (ZG) cells for in vitro study.

Results

Plasma prolactin and aldosterone in the rats were increased by AP gland transplantation. In the in vitro study, the basal aldosterone secretion by the adrenal ZG cells was higher in AP-grafted rats than in CX-grafted rats. The AP-grafted group showed increased responsiveness to angiotensin II (10−8 M), KCl (8x10−3 M), or 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-br-cAMP; 10−4 M, a membrane-permeable analogue of cAMP) with regard to aldosterone secretion as compared with the CX-grafted group. N-(2-[p-Bromocinnamylamine]ethyl)-5-isoquinolinesulfonamide (H89; 10−6, 10−5 M, a protein kinase A inhibitor) or tetrandrine (10−5 M, a blocker for both L-type and T-type Ca2+ channels) induced a greater suppression of aldosterone secretion in the AP-grafted group than in the CX-grafted group. No significant differences between the CX- and AP-grafted groups were observed, however, with regard to the adrenocorticotropic hormone (10−9 M)-, forskolin (10−5 M, an adenylyl cyclase activator)-, or nifedipine (10−5 M, an L-type Ca2+ channel blocker)-induced responsiveness of aldosterone secretion. In addition, there was no difference in the expression of desmolase (i.e., cytochrome P450 side-chain cleavage enzyme) in ZG cells between AP- and CX-grafted rats. The conversions of 25-OH-cholesterol into pregnenolone in the presence of trilostane (an inhibitor of 3β-hydroxysteroid dehydrogenase) and corticosterone into aldosterone, as well as the expression of the steroidogenic acute regulatory protein in ZG cells, were greater in AP-grafted rats than in CX-grafted rats.

Conclusions

These results suggest that hyperprolactinemia increases basal, angiotensin II- and KCl-stimulated aldosterone secretion by ZG cells in OVX rats through activation of T-type Ca2+ channels, the post-cAMP and protein kinase A pathway, cytochrome P450 side-chain cleavage enzyme, and aldosterone synthase, as well as by causing increased expression of steroidogenic acute regulatory protein in ZG cells.
Impaired Fibrinolysis in Premenopausal Women and Age-matched Men with Type 2 Diabetes Mellitus: A Pilot Study
Authors: Brandenburg Suzanne L. , Reusch Jane E.B. , Felder Kimberly K. , Nelson-Wong Erica , Lindenfeld JoAnn , Manco-Johnson Marilyn , Regensteiner Judith G.
Published: August 2007

Background

The presence of Type 2 diabetes mellitus (DM) is one of the strongest predictors of cardiovascular disease (CVD) in women. Although the specific mechanisms underlying this increased risk are unknown, one factor that may contribute to CVD in women with Type 2 DM is impaired fibrinolysis. Healthy premenopausal women have a low rate of CVD and excellent fibrinolytic potential. Impairment in fibrinolysis in people with DM has been demonstrated mainly in men, whereas the fibrinolytic potential of women with Type 2 DM has not been characterized well. This pilot study compared fibrinolytic measures in premenopausal women and men with DM with those of healthy age-matched control women and men to help determine whether fibrinolysis is abnormal in women with DM.

Methods

Fibrinolytic measurements included euglobulin clot lysis time (ELT), fibrinogen, plasminogen activator inhibitor 1, and tissue-type plasminogen activator.

Results

Poststasis ELT was significantly impaired in the women with DM as compared with the control women. The men with DM had a tendency toward slower poststasis ELT than did the control men, but the differences between the men's groups were not significant. In the women's groups only, we observed a trend toward increased plasminogen activator inhibitor 1 among the women with DM.

Conclusions

Women with DM have a more significant abnormality in poststasis ELT than do men with DM as compared with sex-specific counterparts without DM.
Pathogenic Mechanism, Prophylaxis, and Therapy of Symptomatic Acidosis Induced by Acetazolamide
Authors: Filippi Luca , Bagnoli Franco , Margollicci Maria , Zammarchi Enrico , Tronchin Michele , Rubaltelli Firmino F.
Published: August 2007

Background

Acetazolamide, a noncompetitive carbonic anhydrase inhibitor, can produce symptomatic acidosis and bone marrow suppression by a mechanism that is still unknown. This presentation occurs in the elderly, patients with renal or liver failure, people with diabetes, and newborns. The objective of this study was to understand the pathogenic mechanism of these adverse effects and to propose a possible prophylaxis and therapy.

Methods

Four human clinical cases were studied, and one animal experiment was performed. Four preterm newborns with posthemorrhagic ventricular dilation developed severe metabolic acidosis after treatment with acetazolamide. The acidosis suddenly disappeared after a packed red blood cell transfusion. Metabolic studies were performed in one patient and in newborn guinea pigs treated with 200 mg/kg acetazolamide.

Results

Acetazolamide can produce severe lactic acidosis with an increased lactate-to-pyruvate ratio, ketosis with a low β-hydroxybutyrate-to-acetoacetate ratio, and a urinary organic acid profile typical of pyruvate carboxylase deficiency. The acquired enzymatic injury resulting from the inhibition of mitochondrial carbonic anhydrase V that provides bicarbonate to pyruvate carboxylase can produce tricarboxylic acid cycle damage. We demonstrate that the dramatic disappearance of metabolic acidosis and normalizing metabolism after blood transfusion were due to the citrate contained in the packed red blood cell bag. This hypothesis was confirmed by animal experimentation. We argue that the metabolic disorder and bone marrow suppression may be related.

Conclusion

We demonstrate how acetazolamide can lead to symptomatic metabolic acidosis and probably to bone marrow suppression. We suggest citrate as a possible prophylaxis and treatment for these adverse reactions.
Safety Evaluation of Routine Intracoronary Acetylcholine Infusion in Patients Undergoing a First Diagnostic Coronary Angiogram
Authors: Tio R. A. , Monnink S. H. J. , Amoroso G. , Jessurun G. A. J. , Veeger N. , Volkers C. , Hautvast R. , Tan E. S. , Van Gilst W. H. , Van Boven A. J.
Published: August 2007

Background

Recent findings imply prognostic significance of intracoronary acetylcholine infusion for endothelial function testing. We evaluated whether routine use of this test in coronary angiography patients is safe.

Methods

Patients undergoing a first diagnostic coronary angiography were selected to receive intracoronary acetylcholine for endothelial function evaluation. The relation between adverse reactions during infusion and risk factors was analyzed with a logistic regression model. Included in the multiple logistic regression model were the variables with a univariate P value < 0.20.

Results

Adverse reactions occurred in 16% (49/299) of the patients. This included two life-threatening events caused by occlusive spasm and flow limitation in the left coronary artery. Other adverse events were chest pain (n = 38), AV block or sinus bradycardia (n = 10), dyspnea (n = 3). Adverse reactions were more likely to occur in patients younger than 60 years of age (relative risk, 5.6 [2.2–14.3]).

Conclusion

Intracoronary acetylcholine infusion is safe, but may lead to serious adverse reactions. Care should be taken especially in patients younger than 60 years of age. Routine use of acetylcholine infusion can thus only be justified if it has important prognostic significance. This has to be proven further in large prospective studies.
Isolation of Primary Rat Liver Fibroblasts
Authors: Kruglov Emma A. , Jain Dhanpat , Dranoff Jonathan A.
Published: August 2007

Introduction

One of the major advances in liver research in the past decade was the ability to isolate distinct liver cell populations. Although there are established methods of isolating hepatocytes, cholangiocytes, and stellate cells, before this study no technique for liver fibroblast isolation had been devised. Consequently, we developed a technique to isolate primary rat liver fibroblasts.

Methods

Fibroblasts were isolated from a freshly perfused rat liver with a modification of the procedure for isolation of rat cholangiocytes. Cell markers were assessed with the use of confocal immunofluorescence. Cell morphology was assessed with transmission electron microscopy. Expression of procollagen-1 was assessed by reverse transcription polymerase chain reaction.

Results

The appearance of cells with fibroblast morphology was first noted at 48 hours, and almost all cells in culture had fibroblast morphology at 96 hours. Putative fibroblasts stained for vimentin, but not for smooth muscle actin, von Willebrand factor, or cytokeratins. Cell morphology was consistent with that of fibroblasts and showed no features of epithelial, endothelial, or smooth muscle cells. Liver fibroblasts expressed procollagen-1 mRNA.

Conclusion

Primary isolated rat fibroblasts can be produced from a freshly perfused rat liver with a modification of standard cell culture methods. The role of fibroblasts in liver physiology can now be studied directly.
High Overexpression of the Human α-Galactosidase a Gene Driven by Its Promoter in Transgenic Mice: Implications for the Treatment of Fabry Disease
Authors: Ashley Grace A. , Desnick Robert J. , Gordon Ronald E. , Gordon Jon W.
Published: August 2007

Background

Human α-galactosidase A (α-Gal A) is the lysosomal enzyme that cleaves α-galactosyl residues from glycoconjugates and is the deficient enzyme in Fabry disease. To date, there have been no studies on the regulation of this “housekeeping” gene.

Methods

Transgenic mice were established with either 1) a 13.3-kilobase (kb) human genomic fragment that contained 246 bp of 5′- and approximately 2.8 kb of 3′- untranslated sequences, or 2) an “intronless” construct derived from the genomic sequence with the 5′ and 3′ flanking regions intact. Tissues that expressed high levels of α-Gal A activity were examined by light and electron microscopy.

Results

Transgenic mice were generated with 2 and 12 copies of the genomic sequence (Lines 1 and 2) or about 60 copies of the intronless construct (Lines 3 and 4). In mice hemizygous for the genomic transgene (Lines 1 and 2), tissue α-Gal A activities were 12 to 155 times higher than those in the respective wild-type tissue, depending on tissue and transgene copy number. Of note, the high overexpression did not alter the cellular or subcellular cytoarchitecture. In contrast, α-Gal A activities expressed by mice that carried the intronless construct were only two- to sixfold more than in wild-type tissues in which the genomic transgene was highly expressed.

Conclusions

The remarkably high levels of α-Gal A expression in transgenic mice carrying the intact genomic sequence versus the intronless construct suggested that the genomic sequence contained a strong intronic enhancer element. Identification of this regulatory element or elements may be useful in efforts to overexpress human α-Gal A for gene therapy endeavors. In addition, overexpression of human α-Gal A did not affect cellular morphology, which indicates that its overexpression in gene therapy endeavors should be safe.
Disruption of Mitochondrial Calcium Homeostasis after Chronic α-Naphthylisothiocyanate Administration: Relevance for Cholestasis
Authors: Rolo Anabela P. , Oliveira Paulo J. , Seiça Raquel , Santos Maria S. , Moreno António J. , Palmeira Carlos M.
Published: August 2007

Background

Hepatocyte dysfunction caused by impaired mitochondrial function has been pointed out as a probable leading cause of cholestatic liver injury. The aim of this study was to evaluate liver mitochondrial bioenergetics that followed repeated in vivo administration of α-naphthylisothiocyanate, a known cholestatic agent.

Methods

Serum markers of liver injury and endogenous adenine nucleotides were measured in α-naphthylisothiocyanate-treated rats (intraperitoneally, 100 mg/Kg/wkx6 wk). Changes in membrane potential, mitochondrial respiration, as well as alterations in mitochondrial calcium homeostasis were monitored.

Results

In rats injected with α-naphthylisothiocyanate, liver injury with cholestasis developed within 48 hours, as indicated by both serum enzyme activities and total bilirubin concentration. However, 1 week after the last injection, serum enzyme activity returned to control levels. In addition, after chronic α-naphthylisothiocyanate administration, no alterations in mitochondrial respiratory function and membrane potential were observed. Associated with these parameters, mitochondria from treated animals exhibited increased susceptibility to disruption of mitochondrial calcium homeostasis by calcium phosphate and by bile acids, which was probably caused by induction of permeability transition pore.

Conclusions

Our data suggest that chronic cholestasis in rats leads to impaired mitochondrial function due to the disruption of mitochondrial calcium homeostasis. The initiating event is the induction of a cyclosporine A-sensitive release of calcium. This event may be an important determinant of the progression of cholestatic liver injury and associated liver cirrhosis. In addition, in the present study we observed that impairment of mitochondrial function is potentiated by chenodeoxycholate, a bile acid that is known to be toxic. Ursodeoxycholate (the β- epimer of chenodeoxycholate) is approved for the treatment of chronic cholestatic liver disease. Interestingly, we show that the susceptibility to the cyclosporine A-sensitive release of calcium was increased by the combination of both bile acids. These results indicate that the reported improvement of biochemical parameters in cholestatic patients treated with ursodeoxycholate would not prevent the associated mitochondrial dysfunction. This may explain the progression of the histological stage and the maintenance of symptoms during cholestasis.