bluuebunny/crossref_metadata_2025 huggingface dataset:
The study of spontaneous GH secretion after 36‐h fasting distinguishes between GH‐deficient and normal adults
Authors: Aimaretti G. , Colao A. , Corneli G. , Pivonello R. , Maccario M. , Morrison K. , Pflaum C. D. , Strasburger C. J. , Lombardi G. , Ghigo E.
Published: May 2003

OBJECTIVE

Within an appropriate clinical context, GH deficiency (GHD) in adults can only be diagnosed biochemically by provocative testing. The evaluation of IGF‐I, IGFBP‐3 and even of spontaneous GH secretion do not establish the diagnosis of adult GHD. In fact, remarkable overlaps between normal and GHD adults have been reported for all these parameters. On the other hand, it is well known that even short‐term fasting stimulates GH secretion in normal subjects. The aim of our study was to determine the effects of 36 h fasting on 8‐h diurnal GH, insulin and glucose levels as well as on basal IGF‐I, IGFBP‐3, acid‐labile subunit (ALS), IGFBP‐1, GHBP and free fatty acid (FFA) levels.

SUBJECTS

We studied 9 GHD adults (GHD, 8 males, 1 female; age, mean ± SEM: 37.6 ± 2.3 years, body mass index (BMI): 24.5 ± 1.0 kg/m2) and 20 age‐matched normal subjects (NS) as controls (13 males, 7 females; age: 28.9 ± 0.6 years, BMI: 21.6 ± 0.4 kg/m2).

STUDY DESIGN

In all subjects we studied the effects of 36 h fasting on 8‐h daytime GH, insulin and glucose levels (assay every 30 min from 0800 h to 1600 h) as well as on basal IGF‐I, IGFBP‐3, ALS, IGFBP‐1, GHBP and FFA levels.

RESULTS

Before fasting, basal mean IGF‐I, IGFBP‐3 and ALS levels in GHD were lower (P < 0.0001) than in NS. IGFBP‐1, GHBP and FFA levels were similar in both groups. Before fasting mean GH concentration (mGHc) in GHD was lower (P < 0.05) than in NS (0.4 ± 0.2 vs. 2.2 ± 0.6 mu/l) but with a clear overlap between the 2 groups (range 0.4–0.8 vs. 0.4–6.8 mu/l). After fasting, both in GHD and NS basal IGF‐I, IGFBP‐3, ALS and GHBP levels did not change significantly. On the other hand, in both GHD and in NS IGFBP‐1 was increased (P < 0.0001) to a similar extent, while FFA increased in NS more (P < 0.01) than in GHD. Fasting significantly increased mGHc in NS (12.0 ± 1.2 mu/l, P < 0.0001) but not in GHD (0.6 ± 0.2 mu/l). After fasting, no overlap was present between GHD and NS (0.4–1.6 vs. 2.4–20.8 mu/l, respectively). Mean glucose and insulin concentrations over 8 h in GHD and NS in basal conditions were similar and were reduced to the same extent in both groups.

CONCLUSIONS

Our findings demonstrate that after short‐term fasting, the study of spontaneous GH secretion distinguishes between GH‐deficient adults and normal subjects; this phenomenon occurs before significant changes in IGF‐I and IGFBP‐3 levels. These results suggest that the assessment of spontaneous GH secretion could be useful for the diagnosis of adult GH deficiency only after short‐term fasting.
Activated Factor VIII inTherapeutic Preparations:Analysis by Ultracentrifugation
Authors: Aronson David L. , Chang Ping
Published: May 2003

Activation of factor VIII is signaled by an increase in the 1‐stage factor VIII activity and release from von Willebrand factor. Ultracentrifugation in 10–40% sucrose gradients was used to identify such activation in therapeutic concentrates. Plasma‐derived factor VIII lots were examined and factor VIII sedimenting independently of von Willebrand factor was identified in some of the preparations. In addition there was slower sedimentation of factor VIII by the 1‐stage assay than by the chromogenic assay. These results are consistent with a factor VIII cleaved at residue 1689, a site important for von Willebrand binding. This activated form leads to some of the assay discrepancies between the 1‐stage assay and the chromogenic or the 2‐stage assay. There is more rapid sedimentation of the factor VIII measured by the chromogenic assay than the von Willebrand factor in some manufacturers' samples indicating that the method of fractionation may select a low molecular weight von Willebrand factor which does not bind factor VIII. Routine comparison between the 1‐stage and chromogenic assays during fractionation may be able to identify such activated preparations. Other assay discrepancies may be due to structural differences between the standards and the tested product.
A Prospective Study of the Incidence of Red Cell Allo‐Immunisation following Transfusion
Authors: Redman Martin , Regan Fiona , Contreras Marcela
Published: May 2003

To establish the incidence and timing of red cell allo‐immunisation following transfusion, pretransfusion and serial post‐transfusion samples were screened for allo‐antibodies in a total of 452 patients who had undergone elective surgery. Antibody screening was performed by 2‐stage papain, manual polybrene and indirect antiglobulin techniques (IAT). Red cell allo‐antibodies were found in 42 patients and 38 of these (8.4% overall) demonstrated antibodies only after transfusion; 76% of them had Rh specificity. This rate of red cell allo‐immunisation is higher than what would be expected if transfused patients were tested only once post‐transfusion, as has been the case in several previous studies. For the type of patients studied, this finding may not be of clinical relevance at present because most patients undergoing elective surgery do not require further transfusion in their lifetime. However, this is changing with the longer life expectancy of the population and the increased probability of repeat surgery. Twenty‐two (58%) of the antibodies were initially detected by the 2‐stage papain and/or polybrene techniques, when the IAT was negative, although later 19 became positive by IAT; this added sensitivity of techniques other than the IAT, to detect early allo‐immunisation may be relevant in pretransfusion testing to prevent haemolytic transfusion reactions in patients requiring repeated transfusions.
Sensitivity and Specificity of Standard and Rapid HIV‐Antibody Tests Evaluated by Seroconversion and Non‐Seroconversion Low‐Titre Panels
Authors: Kuun Eben , Brashaw Marion , Heyns Anthon du P.
Published: May 2003

Background and objectives: The aim of this study is to compare the relative sensitivity and specificity of commercial HIV‐antibody assays using seroconversion, non‐seroconversion panels, and negative blood donor samples. Materials and methods: We evaluated the sensitivity of five standard ELISA HIV‐antibody assays: Vironostika HIV Uni‐Form II, Abbott recombinant HIV‐1/HIV‐2 third‐generation EIA, Biotest Anti‐HIV‐1/‐2 recombinant, Recombigen HIV‐1/HIV‐2 EIA and Wellcozyme HIV 1+2 (VK54/55), and three rapid screening tests, Capillus HIV‐1/HIV‐2, Abbott Test Pack HIV‐1/HIV‐2 third‐generation EIA, and Sensy‐Test HIV 1/2. All tests were assessed using four panels of plasma samples obtained from individuals who were seroconverting and a low‐titre HIV‐antibody panel of samples. Specificity of the standard screening tests was determined on 3,500 HIV‐antibody‐negative blood donor samples. Results: There was no statistically significant difference in sensitivity between the five standard ELISA tests. One of these tests was significantly less specific than the others. The standard ELISA tests detected all the low‐titre HIV‐antibody‐positive samples. Two of the rapid screening tests were significantly less sensitive on the seroconversion panels and all three tests failed to detect at least one of the positive samples in the low‐titre panel. Conclusions: The additional risk of using one or other of the standard ELISA tests under review of not detecting all HIV‐positive units of blood is not statistically significant. Using some of the rapid screening tests will, however, add a significant additional risk. A rapid screening test should therefore be adopted only after careful consideration of the effect of a possible lack of sensitivity on the safety of the blood supply.
Sterility Testing of Blood Products in 1994/1995 by Three Cooperating Blood Banks in The Netherlands
Authors: Soeterboek A. M. , Welle F. H. W. , Marcelis J. H. , van der Loop C. M. F.
Published: May 2003

Background and objectives: Dutch regulations require blood banks to check the sterility of random blood components to detect contamination during preparation. Materials and methods: We reviewed the results of two years' testing, using standard bacteriologic methods. Results: Of all tested components, 0.5% were contaminated, with Staphylococcus epidermidis being the most frequently detected microorganism. Platelet concentrates showed higher rates of contamination, especially when pooled. Leukocyte‐depleted red cell concentrates showed much lower contamination than red cell concentrates that had not been leuko‐depleted. Conclusions: The rate of contamination compares well with that reported by others in the literature. Since most contamination occurs from the phlebotomy site, most of the bacteria detected were derived from the skin. Leukocyte reduction lowers the rate of contamination.
Hepatitis C Virus Genotypes – Mixed Infections
Authors: Poljak M. , Seme K.
Published: May 2003

We read with interest the recent letter to the editor by Soriano et al. [1] on circulating hepatitis C virus (HCV) genotypes in Spain. We were unpleasantly surprised when we ascertained that the study published in your journal by Soriano et al. [1] was identical to the study published by the same research group in the February issue of the Journal of Infectious Diseases[2].
Hepatitis C Virus Genotypes 1a and 1b Mixed Infections
Authors: Soriano V. , Bravo R. , García‐Samaniego J.
Published: May 2003

In response to Poljak and Seme's letter, we would like to point out that the manuscript published in another journal [1], although based on the same study population, was only meant to discuss the results of Pawlotsky et al. [2], who claimed that hepatitis C virus (HCV) genotype 3a was the predominant type among injecting drug users (IDUs) in France, and most likely, in Western Europe. In light of their results, the French authors suggested that HCV‐3a might have been introduced recently in Europe, most likely through IDUs, and that a potential source of infection in Central Asia e.g. (Nepal), where HCV‐3a is prevalent, could explain the linkage, since residence in this region was in vogue in the 1970s among young people with ‘alternative’ life styles. We did not confirm this observation since in Spain HCV‐1a was the predominant subtype found in IDUs.
Morphology of the Venous System of the Heart in the Van Cat
Authors: Aksoy G. , Karadag H. , Ozudogru Z.
Published: May 2003

Morphology of the veins draining the heart was studied in the Van cat. Veins draining the heart were the great cardiac vein, the middle cardiac vein, the right cardiac veins and the smallest cardiac veins. No anastomosis was observed between the terminal branches of the paraconal interventricular vein and the subsinuosal interventricular vein. The coronary sinus was formed by the great cardiac vein and the middle cardiac vein. The origins of the right cardiac veins had significant variations. The right semicircumflex vein was observed in three (50%) cats, and it terminated at the middle cardiac vein.
Effect of dietary rare earth elements on growth performance and blood parameters of rats
Authors: He M. L. , Wang Y. Z. , Xu Z. R. , Chen M. L. , Rambeck W. A.
Published: May 2003

Rare earth elements (REE) have been shown to influence growth performance in animal production, especially in pigs. In the present study, the effect of oral administration of rare earth elements on growing rats was investigated. Pure LaCl3 or an REE mixture containing 38% of LaCl3, 52% of CeCl3, 3% of PrCl3 and 7% of chlorides of other REE were used at two different concentrations as supplements to the diets. Fifty male Wistar rats at 4 weeks of age were allotted to five experimental groups: a control group; a La‐low group and a La‐high group with 75 and 150 mg/kg LaCl3·6H2O, respectively; a REE‐low and an REE‐high group with 75 and 150 mg/kg REE mixture, respectively. The animals were housed in individual pens. Feed and water were provided ad libitum. After 18 days the oral supplementation of LaCl3·6H2O or of the REE mixture improved daily body weight gain (BWG) by up to 5 or 9% (p > 0.05), respectively. LaCl3·6H2O as well as the REE mixture had positive effects (p < 0.05) on feed conversion ratio (FCR) with a decreased ratio by up to 8 and 11%, respectively. Supplementation of REE also had clear effects on blood serum parameters. The activities of alkaline phosphatase (AP) and alanine amino transferase (ALT) increased significantly (p < 0.05). At the same time, blood glucose level decreased and blood creatine level increased significantly (p < 0.05). There was no significant difference in cholesterol, total protein, albumin and urea nitrogen among the groups. There was no significant difference in triglyceride level between the control and those REE groups, however, a significantly lower (p < 0.01) triglyceride level was found in the 150 mg/kg REE mixture group compared with that in 75 mg/kg REE mixture group and the 150 mg/kg LaCl3·6H2O group. The results suggest that oral supplementation of REE improves growth performance in rats as in pigs. In this respect, concentration and type of REE supplemented to the diets are two important factors herein.
Threshold and growth mechanism of fatigue cracks under mode II and III loadings
Authors: MURAKAMI Y. , TAKAHASHI K. , KUSUMOTO R.
Published: May 2003

ABSTRACT The fatigue crack growth behaviour of 0.47% carbon steel was studied under mode II and III loadings. Mode II fatigue crack growth tests were carried out using specially designed double cantilever (DC) type specimens in order to measure the mode II threshold stress intensity factor range, ΔKIIth. The relationship ΔKIIth > ΔKIth caused crack branching from mode II to I after a crack reached the mode II threshold. Torsion fatigue tests on circumferentially cracked specimens were carried out to study the mechanisms of both mode III crack growth and of the formation of the factory‐roof crack surface morphology. A change in microstructure occurred at a crack tip during crack growth in both mode II and mode III shear cracks. It is presumed that the crack growth mechanisms in mode II and in mode III are essentially the same. Detailed fractographic investigation showed that factory‐roofs were formed by crack branching into mode I. Crack branching started from small semi‐elliptical cracks nucleated by shear at the tip of the original circumferential crack.
Biaxial testing and analysis of bicycle‐welded components for the definition of a safety standard
Authors: PETRONE N. , SUSMEL L.
Published: May 2003

ABSTRACT This paper presents the experimental evaluation of the fatigue behaviour of welded components under non‐proportional variable amplitude biaxial loads. The study was undertaken on welded mountain bike handlebar stems, which were different in terms of geometry and technology and tested with load histories that were reconstructed and accelerated from recorded field data. Loads measured in the field were decomposed into bending and torsional components; a synchronous Peak‐Valley counting, a spectrum inflation technique, a spline interpolation and a final amplification were applied to the measured signals in order to obtain test drive signals with the correct content of biaxial non‐proportional loadings. After evaluation of the bending and torsion load‐life curves of components under constant amplitude fatigue, the resulting data from biaxial variable amplitude fatigue tests were analysed in order to evaluate the damage contribution as a result of the two load components and an equivalent simplified two‐stage constant amplitude fatigue test was proposed to the working group ISO/SC1/TC149/WG4.
Critical plane approach for the assessment of the fatigue behaviour of welded aluminium under multiaxial loading
Authors: KUEPPERS M. , SONSINO C. M.
Published: May 2003

ABSTRACT Multiaxial stress states occur in many welded constructions like chemical plants, railway carriages and frames of trucks. Those stresses can have constant and changing principal stress directions, depending on the loading mode. Latest research results on welded steel joints show a loss of fatigue life for changing principal stress directions simulated by out‐of‐phase bending and torsion compared to constant directions given by in‐phase loading. However, aluminium welds reveal no influence of changing principal directions on fatigue life compared to multiaxial loading with constant principal stress directions. This behaviour is not predictable by any conventional hypothesis. A hypothesis on the basis of local normal and shear stresses in the critical plane has been developed and applied to aluminium weldings.
An impressive therapeutic result of class III compression stockings in a patient with longstanding, extensive, combined leg ulcers
Authors: Van Geest Aj , Van Dooren‐Greebe Rj , Go Ih , Neumann Ham
Published: May 2003

Background Only a few publications are available in the literature concerning the treatment of venous leg ulcers by means of medical compression stockings.

Subject A 59‐year‐old female patient with longstanding, extensive bilateral leg ulcers due to venous insufficiency, arteriolosclerosis and secondary lymphoedema, was treated successfully by flat‐knitted medical compression stockings of compression‐class III.

Conclusion The great advantage of this treatment is the fact that it is comfortable and easy to carry out at home. Furthermore, the costs of compression therapy with medical compression stockings are only a fraction of those of conventional therapy by means of compressive non‐elastic bandages. Once or twice weekly visits of the patient to the hospital are not necessary and less specialized dermatological nurses are needed.
Family–nurse co‐construction of meaning: a central phenomenon of family caring
Authors: Meiers Sonja J. , Tomlinson Patricia S.
Published: May 2003

The purpose of the study was to understand and interpret caring in the family health experience by exploring the interactional phenomenon of family–nurse co‐construction of meaning in the paediatric intensive care unit (PICU). A hermeneutic phenomenological method within a framework of existentialism and symbolic interactionism was used in the investigation. The convenience sample for this study was four family–nurse dyads, that is four families of critically ill children (all with positive outcomes) and the four nurses assigned to their care who were participating in a larger study. Data were derived from semi‐structured interviews regarding significant interactions throughout the child's illness and subsequent significant interactions of families with other nurses and nurses with other families. Trustworthiness of the study was addressed through the criteria of credibility, dependability, transferability and confirmability.

Co‐construction of meaning in the family health experience was found to have two dimensions: interdependent and independent. Both families and nurses described being like family as an essential component of the interdependent experience. Independent dimensions for families were journeying through troubled waters of learning the meaning of the illness event and sensing family comfort through the nurse's care. Independent dimensions described by nurses were journeying through troubled waters of learning to care for families and living with another's fear.

The family–nurse interaction, the relational connection and the evolution of meanings that families and nurses construct, was affirmed as the major vehicle in the co‐construction experience. Family caring is influenced by the existential meaning constructing, process‐oriented, interactional nature of the family health experience. Caring in the family health experience is enhanced through actions the nurse performs on behalf of, and with, the family while understanding the family's unique situation. Caring enacted by nurses in participation with families holds abundant potential for enhancing the family health experience and honor the ethic of caring as central to nursing.
Resolution of persistent oral papillomatosis in a dog after treatment with a recombinant canine oral papillomavirus vaccine
Authors: Kuntsi‐Vaattovaara H. , Verstraete F. J. M. , Newsome J. T. , Yuan H.
Published: May 2003

This report describes a 16‐month‐old female, otherwise seemingly healthy, Siberian husky dog with severe oral papillomatosis that did not regress spontaneously and was refractory to surgical treatment over a 6‐month period. Regression of the papillomas was achieved by administering a series of experimental vaccinations starting at the time of the last surgery. The vaccine consisted of systemically administered canine oral papillomavirus major coat protein L1 that has been shown to self‐assemble into virus‐like particles. They cause a humoral response that has been shown to prevent the onset and development of papillomas. In this case, however, following unsuccessful surgical treatment, the vaccine acted therapeutically, causing the papillomas that had regrown to shrink. No side‐effects were noted.
Quercetin induces structural chromosomal aberrations and uncommon rearrangements in bovine cells transformed by the E7 protein of bovine papillomavirus type 4
Authors: Leal A. M. , Ferraz O. P. , Carvalho C. , Freitas A. C. , Beniston R. G. , Beçak W. , Campo M. S. , Stocco dos Santos R. C.
Published: May 2003

Bovine papillomavirus type 4 (BPV‐4) and bracken fern are cofactors in the carcinogenesis of the upper gastrointestinal (GI) tract of cattle. An experimental in vitro model system has been developed to analyse the co‐operation between the viral transforming protein E7, the cellular ras oncogene and quercetin, one of the mutagens of bracken fern, during neoplastic progression of primary bovine cells. We now report cytogenetic studies of these cells at different stages of malignant transformation: parental primary non‐transformed PalF cells; E7R cells transformed by BPV‐4 E7 and activated ras but not tumorigenic, and tumorigenic E7Q cells derived from E7R cells after treatment with quercetin. All cell lines presented increased numbers of aneuploid cells. The rate of structural chromosomal aberrations observed was increased in transformed cells. In addition, E7Q cells showed chromosomes with peculiar rearrangements, which resulted in metacentric and submetacentric marker chromosomes, with an increase in the mean chromosome arm number. These markers were the products of possible centric fusions. These aberrations and rearrangements were distributed throughout the karyotype, no specific chromosome was involved and the heterochromatic centromeric regions appeared to be preserved.
Rat Hepatocyte Aquaporin–8 Water Channels Are Down–Regulated in Extrahepatic Cholestasis
Authors: Carreras Flavia I. , Gradilone Sergio A. , Mazzone Amelia , García Fabiana , Huang Bing Q. , Ochoa Elena J. , Tietz Pamela S. , LaRusso Nicholas F. , Calamita Giuseppe , Marinelli Raúl A.
Published: May 2003

Hepatocytes express the water channel aquaporin–8 (AQP8), which is mainly localized in intracellular vesicles, and its adenosine 3′,5′–cyclic monophosphate (cAMP)–induced translocation to the plasma membrane facilitates osmotic water movement during canalicular bile secretion. Thus, defective expression of AQP8 may be associated with secretory dysfunction of hepatocytes caused by extrahepatic cholestasis. We studied the effect of 1, 3, and 7 days of bile duct ligation (BDL) on protein expression, subcellular localization, and messenger RNA (mRNA) levels of AQP8; this was determined in rat livers by immunoblotting in subcellular membranes, light immunohistochemistry, immunogold electron microscopy, and Northern blotting. One day of BDL did not affect expression or subcellular localization of AQP8. Three days of BDL reduced the amount of intracellular AQP8 (75%; P < .001) without affecting its plasma membrane expression. Seven days after BDL, AQP8 was markedly decreased in intracellular (67%; P < .05) and plasma (56%; P < .05) membranes. Dibutyryl cAMP failed to increase AQP8 in plasma membranes from liver slices, suggesting a defective translocation of AQP8 in 7–day BDL rats. Immunohistochemistry and immunoelectron microscopy in liver sections confirmed the BDL–induced decreased expression of hepatocyte AQP8 in intracellular vesicles and canalicular membranes. AQP8 mRNA expression was unaffected by 1–day BDL but was significantly increased by about 200% in 3– and 7–day BDL rats, indicating a posttranscriptional mechanism for protein level reduction. In conclusion, BDL–induced extrahepatic cholestasis caused posttranscriptional down–regulation of hepatocyte AQP8 protein expression. Defective expression of AQP8 water channels may contribute to bile secretory dysfunction of cholestatic hepatocytes.
Keratin Mutation in Transgenic Mice Predisposes to Fas But Not Tnf–Induced Apoptosis and Massive Liver Injury
Authors: Ku Nam-On , Soetikno Roy M. , Omary Bishr M.
Published: May 2003

Hepatocytes express keratins 8 and 18 (K8/18) as their only cytoskeletal intermediate filament (IF) proteins, and K8/18 mutations predispose their carriers to liver cirrhosis. Transgenic mice that overexpress mutant human K18 (Arg89→Cys [R89C]) develop mild chronic hepatitis, hepatocyte fragility, keratin filament disruption, and increased susceptibility to drug–induced liver injury. K18 is a major caspase substrate during apoptosis, and K8– or K18–null mice are significantly predisposed to Fas– and possibly tumor necrosis factor (TNF)–mediated apoptosis in the liver. Here we tested the potential role of the K18 R89C mutation on Fas– or TNF–mediated apoptotic liver injury by injecting Fas antibody (Ab) or TNF–α plus actinomycin D into mice that overexpress wild–type (WT) human K18 (with intact filament network, termed TG2 mice) or into K18 R89C mice (with disrupted filament network). K18 R89C mice are significantly more susceptible to Fas–mediated liver injury compared with nontransgenic and TG2 mice. This included differences in lethality, histology, apoptosis, and serum transaminase levels. In contrast, K18 WT and R89C mice manifest similar sensitivity to TNF–induced injury. Both Fas– and TNF–induced apoptosis in liver tissues are associated with caspase–mediated K18 degradation and increased keratin phosphorylation on several but not all sites. In conclusion, transgenic mouse K18 mutation and its consequent keratin filament disruption predispose hepatocytes to Fas– but not TNF–mediated apoptotic injury. This supports the association of keratin mutations with cirrhosis in patients with liver disease and suggests that keratins modulate apoptosis induced by Fas but not TNF.
Repopulation of Rat Liver by Fetal Hepatoblasts and Adult Hepatocytes Transduced Ex Vivo With Lentiviral Vectors
Authors: Oertel Michael , Rosencrantz Richard , Chen Yuan-Qing , Thota Prashanthi N. , Sandhu Jaswinderpal S. , Dabeva Mariana D. , Pacchia Annmarie L. , Adelson Martin E. , Dougherty Joseph P. , Shafritz David A.
Published: May 2003

Recent studies have shown that nondividing primary cells, such as hepatocytes, can be efficiently transduced in vitro by human immunodeficiency virus–based lentivirus vectors. Other studies have reported that, under certain conditions, the liver can be repopulated with transplanted hepatocytes. In the present study, we combined these procedures to develop a model system for ex vivo gene therapy by repopulating rat livers with hepatocytes and hepatoblasts transduced with a lentivirus vector expressing a reporter gene, green fluorescent protein (GFP). Long–term GFP expression in vivo (up to 4 months) was achieved when the transgene was driven by the liver–specific albumin enhancer/promoter but was silenced when the cytomegalovirus (CMV) enhancer/promoter was used. Transplanted cells were massively amplified (˜10 cell doublings) under the influence of retrorsine/partial hepatectomy, and both repopulation and continued transgene expression in individual cells were documented by dual expression of a cell transplantation marker, dipeptidyl peptidase IV (DPPIV), and GFP. In this system, maintenance or expansion of the transplanted cells did not depend on expression of the transgene, establishing that positive selection is not required to maintain transgene expression following multiple divisions of transplanted, lentivirus–transduced hepatic cells. In conclusion, fetal hepatoblasts (liver stem/progenitor cells) can serve as efficient vehicles for ex vivo gene therapy and suggest that liver–based genetic disorders that do not shorten hepatocyte longevity or cause liver damage, such as phenylketonuria, hyperbilirubinemias, familial hypercholesterolemia, primary oxalosis, and factor IX deficiency, among others, might be amenable to treatment by this approach.
Detection of Functionally Altered Hepatitis C Virus–Specific Cd4+ T Cells in Acute and Chronic Hepatitis C
Authors: Ulsenheimer Axel , Gerlach Tilman J. , Gruener Norbert H. , Jung Maria-Christina , Schirren Carl-Albrecht , Schraut Winfried , Zachoval Reinhart , Pape Gerd R. , Diepolder Helmut M.
Published: May 2003

Chronic hepatitis C is characterized by a weak or absent hepatitis C virus (HCV)–specific CD4+ T–cell response in terms of antigen–specific proliferation or interferon gamma (IFN–γ) secretion. To clarify whether this is due to the absence or functional impairment of antigen–specific CD4+ T cells we developed an assay that relies on the induced expression of the T–cell activation marker CD25 and is therefore independent from cytokine secretion or proliferation. In 10 of 20 patients with chronic hepatitis C, a significant number of antigen–specific activated CD4+ T cells (mean 1.06%/patient; range, 0% to 5.2% of CD4+ T cells) could be shown, whereas antigen–specific proliferation was present in only 1 of 20 patients. IFN–γ secretion was absent in all 13 patients tested. However, significant antigen–specific interleukin 10 (IL–10) and transforming growth factor β (TGF–β) secretion was present in 6 of 10 and 3 of 10 patients, respectively. In 8 patients with acute hepatitis C, irrespective of disease outcome, HCV–specific CD4+ T cells were detected in all patients and at a significantly higher frequency (mean 3.7%/patient; range, 1.16% to 7.17%) in the first weeks of disease. A chronic course of disease was associated either with a loss of both IFN–γ secretion and proliferation, resembling an anergic state, or a loss of T–cell proliferation followed by a rapid decline in IFN–γ–producing cells, corresponding to exhaustion of the specific immune response. In conclusion, functional changes of HCV–specific CD4+ T cells or failure to develop a long–lasting T–helper response may contribute to chronic hepatitis C viral persistence.
Overexpression of Thioredoxin Prevents Acute Hepatitis Caused by Thioacetamide or Lipopolysaccharide in Mice
Authors: Okuyama Hiroaki , Nakamura Hajime , Shimahara Yasuyuki , Araya Shinichi , Kawada Norifumi , Yamaoka Yoshio , Yodoi Yunji
Published: May 2003

Thioredoxin (Trx) is a small redox–active protein with antioxidant and antiapoptotic effects. Trx transgenic (Tg) mice are more resistant to cerebral infarction and survive longer than wild–type (WT) C57BL/6 mice. The aim of the present study was to investigate the protective role of Trx in acute hepatitis models. The expression of endogenous Trx was decreased in thioacetamide (TAA)–induced acute hepatitis. TAA (100 μg/g) was injected intraperitoneally in WT and Tg mice. Survival rate after TAA injection was higher in Tg mice than in WT mice. The level of oxidative stress was significantly less in Tg mice than in WT mice, as shown by the protein carbonylation assay and lipid peroxidation assay. Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick–end labeling (TUNEL)–positive cells were less in Tg mice than in WT mice, which was consistent with DNA laddering assay. Caspase–3 and caspase–9 activities and cytochrome c release were significantly inhibited in Tg mice compared with those in WT mice. In addition, lipopolysaccharide (LPS) plus d–galactosamine (GalN), or anti–Fas antibody (Jo2) were injected. Survival rate after LPS plus GalN injection was much higher in Tg mice than in WT mice. In contrast, there was no difference in survival rate after Jo2 injection between WT and Tg mice. In conclusion, transgene of Trx attenuated TAA– or LPS–induced acute lethal hepatitis. In addition to an antioxidant effect, Trx has the potential to protect acute liver injury via an antiapoptotic effect, which mainly inhibits mitochondria–mediated apoptosis signaling.
Materials become insensitive to flaws at nanoscale: Lessons from nature
Authors: Gao Huajian , Ji Baohua , Jäger Ingomar L. , Arzt Eduard , Fratzl Peter
Published: May 2003

Natural materials such as bone, tooth, and nacre are nanocomposites of proteins and minerals with superior strength. Why is the nanometer scale so important to such materials? Can we learn from this to produce superior nanomaterials in the laboratory? These questions motivate the present study where we show that the nanocomposites in nature exhibit a generic mechanical structure in which the nanometer size of mineral particles is selected to ensure optimum strength and maximum tolerance of flaws (robustness). We further show that the widely used engineering concept of stress concentration at flaws is no longer valid for nanomaterial design.
A conserved function of YidC in the biogenesis of respiratory chain complexes
Authors: van der Laan M. , Urbanus M. L. , ten Hagen-Jongman C. M. , Nouwen N. , Oudega B. , Harms N. , Driessen A. J. M. , Luirink J.
Published: May 2003

The
Escherichia coli
inner membrane protein (IMP) YidC is involved in the membrane integration of IMPs both in concert with and independently from the Sec translocase. YidC seems to be dispensable for the assembly of Sec-dependent IMPs, and so far it has been shown to be essential only for the proper Sec-independent integration of some phage coat proteins. Here, we studied the physiological consequences of YidC depletion in an effort to understand the essential function of YidC. The loss of YidC rapidly and specifically induced the Psp stress response, which is accompanied by a reduction of the proton-motive force. This reduction is due to defects in the functional assembly of cytochrome
o
oxidase and the F
1
F
o
ATPase complex, which is reminiscent of the effects of mutations in the
yidC
homologue
OXA1
in the yeast mitochondrial inner membrane. The integration of CyoA (subunit II of the cytochrome
o
oxidase) and F
o
c (membrane subunit of the F
1
F
o
ATPase) appeared exceptionally sensitive to depletion of YidC, suggesting that these IMPs are natural substrates of a membrane integration and assembly pathway in which YidC plays an exclusive or at least a pivotal role.
Natural variation in human membrane transporter genes reveals evolutionary and functional constraints
Authors: Leabman Maya K. , Huang Conrad C. , DeYoung Joseph , Carlson Elaine J. , Taylor Travis R. , de la Cruz Melanie , Johns Susan J. , Stryke Doug , Kawamoto Michiko , Urban Thomas J. , Kroetz Deanna L. , Ferrin Thomas E. , Clark Andrew G. , Risch Neil , Herskowitz Ira , Giacomini Kathleen M. ,   Pharmacogenetics of Membrane Transporters Investigators¶¶
Published: May 2003

Membrane transporters maintain cellular and organismal homeostasis by importing nutrients and exporting toxic compounds. Transporters also play a crucial role in drug response, serving as drug targets and setting drug levels. As part of a pharmacogenetics project, we screened exons and flanking intronic regions for variation in a set of 24 membrane transporter genes (96 kb; 57% coding) in 247 DNA samples from ethnically diverse populations. We identified 680 single nucleotide polymorphisms (SNPs), of which 175 were synonymous and 155 caused amino acid changes, and 29 small insertions and deletions. Amino acid diversity (π
NS
) in transmembrane domains (TMDs) was significantly lower than in loop domains, suggesting that TMDs have special functional constraints. This difference was especially striking in the ATP-binding cassette superfamily and did not parallel evolutionary conservation: there was little variation in the TMDs, even in evolutionarily unconserved residues. We used allele frequency distribution to evaluate different scoring systems (Grantham,
blosum
62, SIFT, and evolutionarily conserved/evolutionarily unconserved) for their ability to predict which SNPs affect function. Our underlying assumption was that alleles that are functionally deleterious will be selected against and thus under represented at high frequencies and over represented at low frequencies. We found that evolutionary conservation of orthologous sequences, as assessed by evolutionarily conserved/evolutionarily unconserved and SIFT, was the best predictor of allele frequency distribution and hence of function. European Americans had an excess of high frequency alleles in comparison to African Americans, consistent with a historic bottleneck. In addition, African Americans exhibited a much higher frequency of population specific medium-frequency alleles than did European Americans.
Evolutionary conservation predicts function of variants of the human organic cation transporter, OCT1
Authors: Shu Yan , Leabman Maya K. , Feng Bo , Mangravite Lara M. , Huang Conrad C. , Stryke Doug , Kawamoto Michiko , Johns Susan J. , DeYoung Joseph , Carlson Elaine , Ferrin Thomas E. , Herskowitz Ira , Giacomini Kathleen M. ,   Pharmacogenetics of Membrane Transporters Investigators‡‡
Published: May 2003

The organic cation transporter, OCT1, is a major hepatic transporter that mediates the uptake of many organic cations from the blood into the liver where the compounds may be metabolized or secreted into the bile. Because OCT1 interacts with a variety of structurally diverse organic cations, including clinically used drugs as well as toxic substances (e.g.,
N
-methylpyridinium, MPP
+
), it is an important determinant of systemic exposure to many xenobiotics. To understand the genetic basis of extensive interindividual differences in xenobiotic disposition, we functionally characterized 15 protein-altering variants of the human liver organic cation transporter, OCT1, in
Xenopus
oocytes. All variants that reduced or eliminated function (OCT1-R61C, OCT1-P341L, OCT1-G220V, OCT1-G401S, and OCT1-G465R) altered evolutionarily conserved amino acid residues. In general, variants with decreased function had amino acid substitutions that resulted in more radical chemical changes (higher Grantham values) and were less evolutionarily favorable (lower
blosum
62 values) than variants that maintained function. A variant with increased function (OCT1-S14F) changed an amino acid residue such that the human protein matched the consensus of the OCT1 mammalian orthologs. Our results indicate that changes at evolutionarily conserved positions of OCT1 are strong predictors of decreased function and suggest that a combination of evolutionary conservation and chemical change might be a stronger predictor of function.
The structure of nonvertebrate actin: Implications for the ATP hydrolytic mechanism
Authors: Vorobiev S. , Strokopytov B. , Drubin D. G. , Frieden C. , Ono S. , Condeelis J. , Rubenstein P. A. , Almo S. C.
Published: May 2003

The structures of
Saccharomyces cerevisiae
,
Dictyostelium
, and
Caenorhabditis elegans
actin bound to gelsolin segment-1 have been solved and refined at resolutions between 1.9 and 1.75 Å. These structures reveal several features relevant to the ATP hydrolytic mechanism, including identification of the nucleophilic water and the roles of Gln-137 and His-161 in positioning and activating the catalytic water, respectively. The involvement of these residues in the catalytic mechanism is consistent with yeast genetics studies. This work highlights both structural and mechanistic similarities with the small and trimeric G proteins and restricts the types of mechanisms responsible for the considerable enhancement of ATP hydrolysis associated with actin polymerization. The conservation of functionalities involved in nucleotide binding and catalysis also provide insights into the mechanistic features of members of the family of actin-related proteins.
Characterization of the endokinins: Human tachykinins with cardiovascular activity
Authors: Page Nigel M. , Bell Nicola J. , Gardiner Sheila M. , Manyonda Isaac T. , Brayley Kerensa J. , Strange Philip G. , Lowry Philip J.
Published: May 2003

We report four human tachykinins, endokinins A, B, C, and D (EKA–D), encoded from a single tachykinin precursor 4 gene that generates four mRNAs (α, β, γ, and δ). Tachykinin 4 gene expression was detected primarily in adrenal gland and in the placenta, where, like neurokinin B, significant amounts of EKB-like immunoreactivity were detected. EKA/B 10-mers displayed equivalent affinity for the three tachykinin receptors as substance P (SP), whereas a 32-mer N-terminal extended form of EKB was significantly more potent than EKA/B or SP. EKC/D, which possess a previously uncharacterized tachykinin motif, FQGLL-NH
2
, displayed low potency. EKA/B displayed identical hemodynamic effects to SP in rats, causing short-lived falls in mean arterial blood pressure associated with tachycardia, mesenteric vasoconstriction, and marked hindquarter vasodilatation. Thus, EKA/B could be
the
endocrine/paracrine agonists at peripheral SP receptors and there may be as yet an unidentified receptor(s) for EKC/D.
Tropical rain forest tree growth and atmospheric carbon dynamics linked to interannual temperature variation during 1984–2000
Authors: Clark D. A. , Piper S. C. , Keeling C. D. , Clark D. B.
Published: May 2003

During 1984–2000, canopy tree growth in old-growth tropical rain forest at La Selva, Costa Rica, varied >2-fold among years. The trees' annual diameter increments in this 16-yr period were negatively correlated with annual means of daily minimum temperatures. The tree growth variations also negatively covaried with the net carbon exchange of the terrestrial tropics as a whole, as inferred from nearly pole-to-pole measurements of atmospheric carbon dioxide (CO2) interpreted by an inverse tracer–transport model. Strong reductions in tree growth and large inferred tropical releases of CO2to the atmosphere occurred during the record-hot 1997–1998 El Niño. These and other recent findings are consistent with decreased net primary production in tropical forests in the warmer years of the last two decades. As has been projected by recent process model studies, such a sensitivity of tropical forest productivity to on-going climate change would accelerate the rate of atmospheric CO2accumulation.
A unifying basis of auditory thresholds based on temporal summation
Authors: Heil Peter , Neubauer Heinrich
Published: May 2003

Thresholds of auditory-nerve (AN) fibers and auditory neurons are commonly specified in terms of sound pressure only, implying that they are independent of time. At the perceptual level, however, the sound pressure required for detection decreases with increasing stimulus duration, suggesting that the auditory system integrates sound over time. The quantity commonly believed to be integrated is sound intensity, implying that the auditory system would have an energy threshold. However, leaky integrators of intensity with time constants of hundreds of milliseconds are required to fit the data. Such time constants are unknown in physiology and are also incompatible with the high temporal resolution of the auditory system, creating the resolution–integration paradox. Here we demonstrate that cortical and perceptual responses are based on integration of the pressure envelope of the sound, as we have previously shown for AN fibers, rather than on intensity. The functions relating the pressure envelope integration thresholds and time for AN fibers, cortical neurons, and perception in the same species (cat), as well as for perception in many different vertebrate species, are remarkably similar. They are well described by a power law that resolves the resolution–integration paradox. The data argue for the integrator to be located in the first synapse in the auditory pathway and we discuss its mode of operation.
Prevalence of defective DNA mismatch repair and MSH6 mutation in an unselected series of endometrial cancers
Authors: Goodfellow Paul J. , Buttin Barbara M. , Herzog Thomas J. , Rader Janet S. , Gibb Randall K. , Swisher Elizabeth , Look Katherine , Walls Ken C. , Fan Ming-Yu , Mutch David G.
Published: May 2003

Endometrial cancer is the most common gynecologic malignancy in the United States and the most frequent extracolonic tumor in hereditary nonpolyposis colorectal cancer (HNPCC). HNPCC patients have inherited defects in DNA mismatch repair and the microsatellite instability (MSI) tumor phenotype. Sporadic endometrial cancers also exhibit MSI, usually associated with methylation of the MLH1 promoter. Germ-line MSH6 mutations, which are rare in HNPCC, have been reported in several families with multiple members affected with endometrial carcinoma. We reasoned that MSH6 mutation might account for loss of mismatch repair in MSI-positive endometrial cancers in which the cause of MSI is unknown. We therefore investigated MSI and MLH1 promoter methylation in 441 endometrial cancer patients unselected for age or personal and family history of cancers. MSI and MLH1 promoter methylation status were associated with age of onset and tumor histology. One hundred cases (23% of the entire series) were evaluated for MSH6 defects. Inactivating germ-line MSH6 mutations were identified in seven women with MSI-positive, MLH1 promoter unmethylated cancers. Most of the MSI in these cases was seen with mononucleotide repeat markers. The MSH6 mutation carriers were significantly younger than the rest of the population (mean age 54.8 versus 64.6,
P
= 0.04). Somatic mutations were seen in 17 tumors, all of which had MSI. Our data suggest that inherited defects in MSH6 in women with endometrial cancer are relatively common. The minimum estimate of the prevalence of inherited MSH6 mutation in endometrial cancer is 1.6% (7 of 441), comparable with the predicted prevalence for patients with colorectal cancer.
The nuclear pore complex protein ALADIN is mislocalized in triple A syndrome
Authors: Cronshaw Janet M. , Matunis Michael J.
Published: May 2003

Triple A syndrome is a human autosomal recessive disorder characterized by an unusual array of tissue-specific defects. Triple A syndrome arises from mutations in a WD-repeat protein of unknown function called ALADIN (also termed Adracalin or AAAS). We showed previously that ALADIN localizes to nuclear pore complexes (NPCs), large multiprotein assemblies that are the sole sites of nucleocytoplasmic transport. Here, we present evidence indicating that NPC targeting is essential for the function of ALADIN. Characterization of mutant ALADIN proteins from triple A patients revealed a striking effect of these mutations on NPC targeting. A variety of disease-associated missense, nonsense, and frameshift mutations failed to localize to NPCs and were found predominantly in the cytoplasm. Microscopic analysis of cells from a triple A patient revealed no morphological abnormalities of the nuclei, nuclear envelopes, or NPCs. Importantly, these findings indicate that defects in NPC function, rather than structure, give rise to triple A syndrome. We propose that ALADIN plays a cell type-specific role in regulating nucleocytoplasmic transport and that this function is essential for the proper maintenance and/or development of certain tissues. Our findings provide a foundation for understanding the molecular basis of triple A syndrome and may lead to unique insights into the role of nucleocytoplasmic transport in adrenal function and neurodevelopment.