bluuebunny/crossref_metadata_2025 huggingface dataset:
Catecholamines, hypoxia and high altitude
Authors: ROSTRUP M.
Published: May 2003

Hypoxia is a potent activator of the sympathetic nervous system by stimulating arterial chemoreceptors. However, out of 15laboratorystudies on the effects of acute and prolonged hypoxia on catecholamines, 14 failed to show any changes in plasma or urinary noradrenaline and only four studies showed significant increases in plasma or urinary adrenaline. By contrast, six out of eight studies on MSNA showed increased sympathetic nerve activity to the leg. An increased clearance of plasma catecholamines during hypoxia may be a possible explanation. Furthermore, many of the studies had limitations in a number of subjects and catecholamine assays used. Emotional aspects of the study protocols, which could contribute to the increase in adrenaline, was only assessed by sham runs in one chamber study. However, 13 out of 14 reviewedfieldstudies on subjects staying for more than 1 week at high altitude, reported increased plasma or urinary excretion of noradrenaline which may be compatible with increased sympathetic activity. Adrenaline changed to a lesser degree. Out of seven studies on more short‐term (4 h to 3 days) exposure to high altitude, only one demonstrated significantly increased plasma noradrenaline. In this study, however, several subjects had been exposed to high altitude less than 1 week before the experiment. In a new study on 12 climbers reported in this paper, a temporary reduction in plasma catecholamines was found 2 days after arrival at 4200 m. There was a steady increase towards normal levels after 1 week. Plasma vasopressin (AVP) increased suggesting a compensatory mechanism. Both plasma noradrenaline and adrenaline were positively correlated with oxygen saturation in these subjects. Thus, in previously unacclimatized subjects, short‐term exposure to high altitude does not increase plasma catecholamines, rather plasma levels decreased. In addition to increased clearance, there is some evidence of reduced synthesis of catecholamines during short‐term hypoxia. The oxygen sensitivity of tyrosine hydroxylase (TH) activity, may be one possible mechanism.
Muscle blood flow and oxygen uptake in recovery from exercise
Authors: BANGSBO J. , HELLSTEN Y.
Published: May 2003

The metabolic and muscle blood flow response in recovery from exercise is dependent on the type and the duration of the exercise. Immediately after both intense static and dynamical exercise blood flow to the exercised muscles increases suggesting that blood flow is mechanically hindered by muscle contraction. After the initial rise (seconds) muscle blood flow decreases at a moderate rate and the time to reach resting flow levels varies from seconds to more than 30 min. It is unclear as to what causes the elevated blood flow during recovery. A mismatch between the time course of changes in blood flow and oxygen uptake suggests that the blood flow is not directly regulated by the need of oxygen in the exercised muscles. The hyperaemic response may be linked to locally released factors, such as ions and metabolites. However, the signal by which the blood flow is elevated remains unknown. After exercise both pulmonary and muscle oxygen uptake decrease rapidly, but can remain above resting levels for several hours. Resynthesis of substrates such as CP, ATP and glycogen cannot account for the entire excessive post‐exercise oxygen uptake (EPOC) in the exercised muscles and the cause of the elevated muscle oxygen uptake in recovery from exercise remains to be assessed.
Hepatitis Virus Infections in Spouses of Blood Donors
Authors: Zimmermann Robert , Glaser Anke , Eckstein Reinhold
Published: May 2003

An important purpose of the medical history in selecting individuals for blood donation is to protect the recipient against the transmission of viral diseases. Over many years hepatitis was the main cause of transfusion‐associated chronic disease and death. Three steps led to a large reduction of the frequency of transfusion‐associated hepatitis: Systematic exclusion of donors reporting defined risk factors and the introduction of tests for hepatitis B surface antigen (HBsAg) and antibodies to hepatitis C virus (HCV) [1]. However, despite the development of sensitive and specific screening tests there is no doubt that a detailed medical history is the basis of all progress in providing safe blood products because any screening test is tainted with the problem of the window period between infection and seroconversion.
Blood Components – Transfusion Support in Post‐Transfusion Purpura Due to HPA‐1a Immunization
Authors: Win Nay , Matthey Francis , Slater Nicolas G. P.
Published: May 2003

Post‐transfusion purpura (PTP) is usually a self‐limiting disease. Nevertheless, haemorrhage in these patients may be life‐threatening and hence red cell transfusions may be clinically urgent. It is widely recommended that such blood be from donors who are negative for the HPA‐1a platelet antigen. However, these donors are uncommon and such blood is often in short supply. We report 2 cases of PTP in which either filtered leucodepleted red cells or washed red cells were transfused in combination with high‐dose intravenous immunoglobulin (both cases) and HPA‐1a negative platelets (1 case) to good effect.
Interaction of Platelet Fc and Complement Receptors with Circulating Immune Complexes Involving the AB0 System
Authors: Heal J. M. , Masel D. , Blumberg N.
Published: May 2003

When platelets that are AB0‐nonidentical are transfused, circulating immune complexes (CIC) are formed. In the present study we examined the ability of polyclonal antibodies to two C1q receptors on platelets, cC1q‐R and gC1q‐R, and a monoclonal IgG FcγRII (CD32) antibody directed against the platelet Fc receptor to inhibit the uptake of CIC involving the AB0 blood group system by normal platelets. Four types of immune complexes of varying purity were made in vitro. In addition serum from 5 refractory group A patients who had demonstrable AB0 CIC, 5 patients who had received only AB0‐identical platelets and had no AB0 CIC and 6 normal donors were evaluated. After exposure of normal platelets to serum of patients with demonstrable AB0 CIC there were increased levels of platelet‐associated IgG. This binding was partially inhibited by preincubation of the platelets with either anti‐cC1q‐R (3/5 patients), gC1q‐R (3/5 patients) or IgG FcγRII in 4/5 patients. However, the pattern of inhibition by the three antibodies was variable. Using the artificial immune complexes a more consistent pattern was obtained. The binding of four types of artificial immune complexes to platelets was reduced by 67–99% after preincubation of the platelets with antibodies to the complement and Fc receptors. The present work supports the hypothesis that AB0 CIC bind to Fc and complement receptors on the platelet and if confirmed would suggest a pathophysiological mechanism for the clinical observations of the important role of AB0 in platelet transfusion.
Clumps Present in Platelet Concentrates Are Formed from Fibronectin
Authors: Rácz Z.
Published: May 2003

Appearance of clumps in stored platelet concentrates (PCs) due to intensive agitation presents a serious practical problem in the quality control of platelet products. It has previously been demonstrated that the appearance of clumps is strongly correlated with the final pH of PCs [1].
Human Parvovirus B19 and Blood Products
Authors: Prowse C. , Ludlam C. A. , Yap P. L.
Published: May 2003

Background and objectives: Human B19 parvovirus (B19), identified in 1975, was only recognised as the causative agent of fifth disease in 1983. The incidence of viraemia is low, around 1 in 1,000, but is sufficient to ensure that most plasma pools for fractionation contain some virus. While infection usually occurs in childhood and is benign, chronic infection sometimes occurs and may be of concern in certain patient groups. Materials and methods: This review is based on a meeting held in March 1995, and addresses recent concerns regarding the potential transmission of B19 infection by pooled plasma products. Results: Recent data on the pathophysiology and assay of this virus are summarised along with possible approaches to donor screening, product screening, and virus removal. Only five cases of symptomatic infection have been reported in persons with haemophilia, but no technology for virus removal is established, and infection may be of concern in pregnant women, and in patients with enhanced red cell turnover or who are immunosuppressed, including those infected with human immunodeficiency virus, but only rarely in immunocompetent patients. Conclusions: For the future, well‐validated assays relevant to virus infectivity are required if blood donations, plasma pools, or plasma products are to be screened, and an in‐process virus inactivation step for B19 would be highly desirable. In the interim, non‐plasma or recombinant products or a selective transfusion policy might be used in patient groups in which B19 infection is of particular concern. Further clinical data on the prognosis and impact of B19 infection are needed to justify both such policies and the future adoption of new technologies designed to reduce any excess B19 infectivity arising from transfused products.
A Comparative Study on Cardiac Ganglia in Midday Gerbil, Egyptian Spiny Mouse, Chinchilla Laniger and Pigeon
Authors: Kuder T. , Nowak E. , Szczurkowski A. , Kuchinka J.
Published: May 2003

Using the thiocholine method and histological techniques, the topography and morphology of cardiac ganglia in midday gerbil, Egyptian spiny mouse, chinchilla laniger and pigeon were studied. The results demonstrated that cardiac ganglia in all investigated species are embedded in epicardial fat. They formed plexo‐ganglionic structures. Each of them composed of many ganglia (from seven up to 36) different in size and shape, and interconnected by fascicles of nerve fibres. Comparative analysis showed that the density of neural network and cell aggregations was different in individual species. The richest plexo‐ganglionic structure was in pigeon. It was organized in three plexo‐ganglia with an average of 30 ganglia. The largest one was located along the anterior interventricular sulcus. The cardiac ganglia of investigated mammals were localized mainly on the epicardium of atria; in Egyptian spiny mouse and chinchilla laniger on the ventral surface of right atrium, but in midday gerbil on the dorsal surface of left atrium. Moreover, in midday gerbil and Egyptian spiny mouse the little plexo‐ganglionic structure on the ventricle were noticed. Additionally, in midday gerbil the single nerve cells might be observed between cardiac muscle of atria. It can be said that, the strongly developed cardiac plexus in pigeon is probably connected with his behaviour and functional properties of the heart. The arrangement of neurones in cardiac ganglia of all examined mammals was uniform over the whole surface of the sections, while in the pigeon, neurones were located mainly in the peripheral part of the ganglion.
Immunohistochemical Evaluation of the Muscularis Mucosae in the Ruminant Forestomach
Authors: Kitamura N. , Yoshiki A. , Sasaki M. , Baltazar E. T. , Hondo E. , Yamamoto Y. , Agungpriyono S. , Yamada J.
Published: May 2003

The muscularis mucosae and condensed fibrous layer of the ruminant forestomach were studied by immunohistochemistry using specific antibodies against α‐smooth muscle actin (αSMA) and γ‐smooth muscle actin (γSMA). The specimens were collected from the rumen, reticulum and omasum of cattle, water buffalo, sheep, goat, Barbary sheep, Japanese serow, sika deer and mouse deer.

The muscularis mucosae showed immunoreactivity for both αSMA and γSMA. On the other hand, the condensed fibrous layer appearing between the propria mucosa and tela submucosa was immunoreactive only for αSMA except for that in the goat and Barbary sheep reticulum which is intermingled with γSMA immunoreactivity. The distribution of muscularis mucosae and/or condensed fibrous layer varied among the compartments of forestomach and ruminant species. In the rumen, only the condensed fibrous layer was detected. On the other hand, the omasum contained only the muscularis mucosae. In the reticulum, both were detected. The amount of the condensed fibrous layer in the reticulum varied among different species in the following order of abundance: goat > Barbary sheep > sika deer> sheep > water buffalo > cattle and Japanese serow. Smooth muscle cells of external muscle layer were immunoreactive for αSMA and γSMA whereas those of blood vessels and pericytes were immunoreactive only for αSMA. The present findings on the actin immunoreactivity and distribution profile of muscularis mucosae and the condensed fibrous layer provide additional knowledge to further understand the histophysiological specialization of the different compartments of the ruminant forestomach.
Assessment of the minimum protein requirement of adult ponies
Authors: Olsman A. F. S. , Jansen W. L. , Sloet van Oldruitenborgh‐Oosterbaan M. M. , Beynen A. C.
Published: May 2003

The aim of the present study was to determine the minimum protein requirement in adult ponies. Literature data from nitrogen‐balance trials in adult ponies were used. In addition, nitrogen balance was measured in three mature ponies that were fed a low‐ and a high‐protein diet according to a cross‐over design. On the low‐protein ration, which provided 1.1 g digestible crude protein (DCP)/kg0.75 day, plasma albumin and total protein concentrations were non‐significantly decreased by about 6% and group‐mean nitrogen balance was negative. Using data from the present experiment and from six other studies, the sum of endogenous faecal and urinary nitrogen losses was calculated to be 166 ± 37 mg/kg0.75 day (mean ± SE, n = 13). Subsequently, a minimum protein requirement of 1.7 g DCP/kg0.75 day was calculated. The value for minimum protein requirement has low precision, which should be taken into account when setting the protein allowance of adult ponies.
Nutrition studies on protein and energy in domestic cats
Authors: Riond J.‐L. , Stiefel M. , Wenk C. , Wanner M.
Published: May 2003

The effects of three diets varying in their protein, fat and carbohydrate contents and of physical activity on energy and protein metabolism were investigated in eight domestic cats. The diets were based on canned food with addition either of minced cattle heart, melted swine fat or polenta. The activity‐induced heat production was measured in a respiration chamber using the Doppler effect of radio‐waves reflected from moving bodies. In the group receiving the high‐protein diet, urinary nitrogen concentration was higher which was reflected in higher urinary loss of energy without affecting the amount of energy retained. Nitrogen utilization was not significantly influenced by nitrogen intake. Mean daily total heat production ranged between 163 and 187 kJ/kg BW/day or 215 and 270 kJ/kg BW0.75/day. The daily crude protein requirement of adult cats was determined to be 2.7 g/kg BW/day. The daily requirement of metabolizable energy for maintenance was estimated to be 153 kJ/kg BW/day or 226 kJ/kg BW0.75/day. The activity‐induced heat production was 25.1 kJ/kg BW/day which corresponded to 13.5% of the total daily heat production. The daily requirement for metabolizable energy was thus reduced by 20.9 kJ/kg BW/day or 13.7%. The activity pattern of the cats was not characterized by a circadian rhythm.
Application of multiaxial fatigue criteria to materials containing defects
Authors: BERETTA S.
Published: May 2003

ABSTRACT Estimation of fatigue strength under biaxial and multiaxial loading is usually based on the assumption that fatigue limit is the limit stress for crack nucleation. However, in presence of defects and inhomogeneities the fatigue limit is determined by the threshold condition of small cracks. Nevertheless, multiaxial criteria, especially the Dang Van's criterion, could be successfully applied to defective materials. The scope of this paper is to discuss the differences in the two approaches (multiaxial criteria versus mechanics of the small cracks) for fatigue design of components containing defects subjected to biaxial and multiaxial in‐phase loading. In particular it will be discussed, on the basis of the fatigue properties of large springs, the need of a correct description of the fatigue mechanism in order to determine the lower bound of fatigue strength in presence of defects.
In vivo spectroscopy in dermatology: methods and new fields of application
Authors: Liebold K , Faßler D , Schmidt W‐D , Kühn T , Wollina U
Published: May 2003

Background The human skin is an easily accessible organ on which non‐invasive examination methods can be applied. Remittance spectroscopy uses registration of the proportion of reflected light from the skin surface. Optical characteristics of skin are determined by scattering, reflection and absorption. The remittance is influenced by various test conditions and topography. Spectroscopic methods are applied for determination of microcirculation, measuring of erythema and pigmentation and in pharmacology and toxicology of applied drugs.

Conclusion The standardization of spectroscopic measurement of human skin is essential to reduce error sources. In future a better investigation of skin structures and molecules, which are responsible for optical characteristics of skin and a more precise evaluation of spectroscopic signals are necessary to get more information about the state of skin and to develop new fields of applications, such as early diagnosis of skin diseases or monitoring of metabolic processes.
Extensive and deep dermatophytosis caused by Trichophyton mentagrophytes var. Interdigitalis in an HIV‐1 positive patient
Authors: Muñoz‐Pèrez Ma , Rodriguez‐Pichardo A , Camacho F , Ríos Jj
Published: May 2003

Background Cutaneous infections are common in HIV‐1 positive patients and are usually severe, recurrent, and caused by microorganisms that are unusual in immunocompetent patients.

Objective We report a case of an HIV‐1‐positive 23‐year‐old male, with a history of intravenous drug use, in stage C‐II (CDC ‘86), with a CD4 lymphocyte count of 335 cells/mm3. He had multiple, large erythematous, circinate and pustular plaques on his abdomen, back, arms and legs.

Results We isolated Trichophyton mentagrophytes var. interdigitalis from the lesions. The biopsy showed suppurative deep dermatophytosis and folliculitis. The patient satisfactorily responded to itraconazole (100 mg/d for 14 days).

Conclusion This is the first reported case of deep dermatophytosis caused by T. mentagrophytes in an HIV‐positive patient.
Recombinant Human Interleukin–11 Improves Thrombocytopenia in Patients With Cirrhosis
Authors: Ghalib Reem , Levine Cheryl , Hassan Manal , McClelland Tricia , Goss John , Stribling Risë , Seu Philip , Patt Yehuda Z.
Published: May 2003

To elucidate the hematopoietic activity of recombinant human interleukin–11 (rhIL–11, [Neumega, Cambridge, MA]) in patients with cirrhosis and thrombocytopenia, we administered rhIL–11 at 50 μg/kg/d subcutaneously to 10 patients for 10 days with a 30–day follow–up period. All treated patients (n = 9) experienced a gradual, yet significant increase in their platelet count above the baseline value (P ≤ .01) reaching the peak value (median, 93,000/μL; range, 60,000–206,000/μL) at a median of 13 days (range, 6–23 days). Eight patients (89%) had a significant increase of ≥50% over the baseline value (P < .05). Moreover, further increases to ≥60,000/μL, ≥80,000/μL, and ≥100,000/μL were observed in 100%, 78%, and 33% of the patients, respectively. A subsequent decline in platelet count was observed at a median of 19 days (range, 7–26 days) after the occurrence of peak concentration. A significant increase in neutrophil count was also demonstrated starting on the third day of treatment (P ≤ .01). Concurrent with an increase in the serum level of fibrinogen, transaminase levels declined significantly during treatment period, while bilirubin levels continued to drop for up to 20 days after the initiation of treatment (P < .05). The most frequent effects were due to plasma volume expansion, including conjunctival redness and edema. In conclusion, rhIL–11 can improve platelet counts in patients with early cirrhosis and these patients could benefit from rhIL–11 treatment. However, given the high frequency of regimen–related toxicity, the use of rhIL–11 in patients with cirrhosis should be administered with caution.
Gene Expression Associated With Interferon Alfa Antiviral Activity in An Hcv Replicon Cell Line
Authors: Zhu Haizhen , Zhao Hongshan , Collins Christin D. , Eckenrode Sarah E. , Run Qingguo , McIndoe Richard A. , Crawford James M. , Nelson David R. , She Jin-Xiong , Liu Chen
Published: May 2003

Interferon alfa (IFN–α)–based treatment is the only therapeutic option for chronic hepatitis C viral infection. However, the molecular mechanisms of IFN–α antiviral activity are not completely understood. The recent development of an HCV replicon cell culture system provides a feasible experimental model to investigate the molecular details of IFN–induced direct antiviral activity in hepatocytes. In this report, we show that IFN–α can effectively inhibit HCV subgenomic RNA replication and suppress viral nonstructural protein synthesis. Using cDNA microarray analysis, we also show that the replicon cells have different gene expression profile compared with the parental hepatoma cells (Huh7). IFN–α can induce a number of responsive genes in the replicon cells. One of the genes, 6–16 (G1P3), can enhance IFN–α antiviral efficacy. In addition, we demonstrate that IFN–α can significantly activate STAT3 in hepatoma cells, suggesting that this pathway plays a role in IFN–α signaling. In conclusion, our results indicate that IFN–α antiviral activity is associated with activation of STAT3–signaling pathway and intracellular gene activation. Our results also suggest that IFN–α–induced target genes may play an important role in IFN–α anti–HCV activity.
Alterations in Bronchoalveolar Lavage Fluid During Ischemia–Induced Acute Hepatic Failure in the Pig
Authors: Kostopanagiotou Georgia , Routsi Christina , Smyrniotis Vassilios , Lekka Marilena E. , Kitsiouli Eirini , Arkadopoulos Nikolaos , Nakos George
Published: May 2003

The objective of this controlled experimental animal study was to evaluate whether acute hepatic failure (AHF) can cause acute lung injury (ALI) and to investigate possible pathophysiologic mechanisms. Seventeen domestic pigs were randomly assigned to AHF and sham groups. AHF was induced by surgical devascularization of liver in 10 animals. Seven animals were sham operated. Hemodynamics, lung mechanics, extravascular lung water (EVLW), and intracranial pressure, blood gas, liver function tests, and serum endotoxin levels were measured. Cells count, total protein, and phospholipids and phospholipases A2 were determined in the bronchoalveolar lavage (BAL) fluid. Measurements were obtained after the insertion of central lines and 4 hours and 7 hours after the completion of the surgical procedure. Hemodynamic, biochemical, neuromonitoring, and histologic data confirmed the development of liver failure. Seven hours after devascularization, EVLW was higher in AHF (13.7 ± 1.8 mL/kg) compared with the sham group (5.9 ± 0.7 mL/kg) (P < .05); in AHF, increase of neutrophils (5% ± 8% to 25% ± 8%, P < .001), total protein (6.2 ± 3.7 to 11.2 ± 6.5 μg/mL, P < .048), and phospholipase A2 (1.43 ± 0.56 to 2.38 ± 1.38 nmoL/mL/h, P < .03) and decrease in PAF–acetylhydrolase (0.114 ± 0.128 to 0.039 ± 0.038 nmol/mL/h, P < .01) compared with baseline were observed; total phospholipids decreased in AHF and increased in the sham model. Histologic examination confirmed lesions compatible with acute lung injury. In conclusion, AHF due to hepatic devascularization induced acute lung injury, confirmed by the increase of inflammatory cells in the alveoli as well as by histologic findings. The decreased PAF–AcH and the increased phospholipase A2 may play a significant role in the perpetuation of inflammation accompanied by surfactant disorders.
Human Wisp1v, A Member of the Ccn Family, Is Associated With Invasive Cholangiocarcinoma
Authors: Tanaka Shinji , Sugimachi Keishi , Kameyama Toshifumi , Maehara Shin-ichiro , Shirabe Ken , Shimada Mitsuo , Wands Jack R. , Maehara Yoshihiko
Published: May 2003

Family members of the connective tissue growth factor, cysteine–rich 61, nephroblastoma over–expressed gene (CCN) encode cysteine–rich secreted proteins with roles in human fibrotic disorders and tumor progression. In this study, we identified a CCN family member, WISP1v, as over–expressed in human cholangiocarcinomas. Genetic analysis of WISP1v was performed on surgically resected specimens of cholangiocarcinoma. The WISP1v biological effects were analyzed using the HuCCT1 human cholangiocarcinoma cell line. The WISP1v gene was expressed in 19 of 39 cholangiocarcinoma tissues (49%) but not in normal livers. Expression of WISP1v was significantly associated with lymphatic and perineural invasion of tumor cells (P < .05), as well as a poor clinical prognosis (P < .01). In the intraductal papillary cholangiocarcinomas, WISP1v was detected only in the cases with duct wall invasion but not in the cases without duct wall invasion (P < .05). No mutation of WISP1v gene was detected in the examined samples. In vitro analysis revealed that WISP1v stimulated the invasive phenotype of cholangiocarcinoma cells with activation of both p38 and p42/p44 mitogen–activated protein kinases (MAPKs). Furthermore, WISP1v–induced cholangiocarcinoma invasion was significantly suppressed by the p38 MAPK inhibitor SB203580 but not by the p42/p44 MAPK kinase (MEK) inhibitor PD98059. Our findings suggest that WISP1v–mediated signaling is involved in the generation of invasive cellular properties and leads to progression of cholangiocarcinoma.
Conditional Tetracycline–Regulated Expression of Tgf–β1 in Liver of Transgenic Mice Leads to Reversible Intermediary Fibrosis
Authors: Ueberham Elke , Löw Rainer , Ueberham Uwe , Schönig Kai , Bujard Hermann , Gebhardt Rolf
Published: May 2003

Based on the tetracycline–regulated gene expression system, a double–transgenic mouse model for liver fibrosis was established in which the expression of transforming growth factor β1 (TGF–β1) can be regulated deliberately by addition or removal of doxycycline hydrochloride to the drinking water. TGF–β1 plasma levels in induced double–transgenic mice reached values ranging from 250 to 1,200 ng/mL, being 10 to 30 times above the normal plasma levels. By applying a cyclic induction–deinduction protocol, deleterious effects of the high plasma TGF–β1 levels were overcome. By using this protocol, liver fibrosis occurred within a few cycles and progressed further to an intermediary fibrosis when cyclic induction was continued. On histochemical staining, a marked perisinusoidal deposition of extracellular matrix was detected accompanied by the activation of hepatic stellate cells as shown by alpha–smooth muscle actin (α–SMA) expression. Apoptosis of hepatocytes was prominent in TGF–β1 high producers, leading to a decreasing number of TGF–β1–expressing cells with time. No compensatory proliferation of hepatocytes could be detected. In advanced stages, fibrogenesis could be stopped by switching off TGF–β1 production and reversal of fibrosis could be shown by (immuno)histochemistry within 6 to 21 days. Determination of messenger RNA (mRNA) levels of procollagen I and III, laminin (B1), matrix metalloproteinase (MMP)–2, –9, and –13, and tissue inhibitor of matrix metalloproteinase (TIMP)–1 and –2 by real–time reverse–transcription polymerase chain reaction (RT–PCR) provided insight into some mechanistic details of the fibrogenic process and its reversal. In conclusion, this model will enable the analysis of fibrogenesis at progressive stages and help in elucidating the cellular changes during development and regression of liver fibrosis caused by elevated TGF–β1 expression.
Mimicking natural evolution
in vitro
: An
N
-acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity
Authors: Joerger Andreas C. , Mayer Sebastian , Fersht Alan R.
Published: May 2003

N
-acetylneuraminate lyase (NAL) and dihydrodipicolinate synthase (DHDPS) belong to the NAL subfamily of (β/α)
8
-barrels. They share a common catalytic step but catalyze reactions in different biological pathways. By rational design, we have introduced various mutations into the NAL scaffold from
Escherichia coli
to switch the activity toward DHDPS. These mutants were tested with respect to their catalytic properties
in vivo
and
in vitro
as well as their stability. One point mutation (L142R) was sufficient to create an enzyme that could complement a bacterial auxotroph lacking the gene for DHDPS as efficiently as DHDPS itself.
In vitro
, this mutant had an increased DHDPS activity of up to 19-fold as defined by the specificity constant
k
cat
/
K
M
for the new substrate
l
-aspartate-β-semialdehyde when compared with the residual activity of NAL wild-type, mainly because of an increased turnover rate. At the same time, mutant L142R maintained much of its original NAL activity. We have solved the crystal structure of mutant L142R at 1.8 Å resolution in complex with the inhibitor β-hydroxypyruvate. This structure reveals that the conformations of neighboring active site residues are left virtually unchanged by the mutation. The high flexibility of R142 may favor its role in assisting in catalysis. Perhaps, nature has exploited the catalytic promiscuity of many enzymes to evolve novel enzymes or biological pathways during the course of evolution.
Resistance of cell membranes to different detergents
Authors: Schuck Sebastian , Honsho Masanori , Ekroos Kim , Shevchenko Andrej , Simons Kai
Published: May 2003

Partial resistance of cell membranes to solubilization with mild detergents and the analysis of isolated detergent-resistant membranes (DRMs) have been used operationally to define membrane domains. Given the multitude of detergents used for this purpose, we sought to investigate whether extraction with different detergents might reflect the same underlying principle of domain formation. We therefore compared the protein and lipid content of DRMs prepared with a variety of detergents from two cell lines. We found that the detergents differ considerably in their ability to selectively solubilize membrane proteins and to enrich sphingolipids and cholesterol over glycerophospholipids as well as saturated over unsaturated phosphatidylcholine. In addition, we observed cell type-dependent variations of the molecular characteristics of DRMs and the effectiveness of particular detergents. These results make it unlikely that different detergents reflect the same aspects of membrane organization and underscore both the structural complexity of cell membranes and the need for more sophisticated analytical tools to understand their architecture.
Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors
Authors: Foster Leonard J. , de Hoog Carmen L. , Mann Matthias
Published: May 2003

Membrane lipids were once thought to be homogenously distributed in the 2D surface of a membrane, but the lipid raft theory suggests that cholesterol and sphingolipids partition away from other membrane lipids. Lipid raft theory further implicates these cholesterol-rich domains in many processes such as signaling and vesicle traffic. However, direct characterization of rafts has been difficult, because they cannot be isolated in pure form. In the first functional proteomic analysis of rafts, we use quantitative high-resolution MS to specifically detect proteins depleted from rafts by cholesterol-disrupting drugs, resulting in a set of 241 authentic lipid raft components. We detect a large proportion of signaling molecules, highly enriched versus total membranes and detergent-resistant fractions, which thus far biochemically defined rafts. Our results provide the first large-scale and unbiased evidence, to our knowledge, for the connection of rafts with signaling and place limits on the fraction of plasma membrane composed by rafts.
An estrogen-dependent four-gene micronet regulating social recognition: A study with oxytocin and estrogen receptor-α and -β knockout mice
Authors: Choleris Elena , Gustafsson Jan-Åke , Korach Kenneth S. , Muglia Louis J. , Pfaff Donald W. , Ogawa Sonoko
Published: May 2003

Estrogens control many physiological and behavioral processes, some of which are connected to reproduction. These include sexual and other social behaviors. Here we implicate four gene products in a micronet required for mammalian social recognition, through which an individual learns to recognize other individuals. Female mice whose genes for the neuropeptide oxytocin (OT) or the estrogen receptor (ER)-β or ER-α had been selectively “knocked out” were deficient specifically in social recognition and social anxiety. There was a remarkable parallelism among results from three separate gene knockouts. The data strongly suggest the involvement in social recognition of the four genes coding for ER-α, ER-β, OT, and the OT receptor. We thus propose here a four-gene micronet, which links hypothalamic and limbic forebrain neurons in the estrogen control over the OT regulation of social recognition. In our model, estrogens act on the OT system at two levels: through ER-β, they regulate the production of OT in the hypothalamic paraventricular nucleus, and through ER-α, they drive the transcription of the OT receptor in the amygdala. The proper operation of a social recognition mechanism allows for the expression of appropriate social behaviors, aggressive or affiliative.
Mammalian linker-histone subtypes differentially affect gene expressioninvivo
Authors: Alami Raouf , Fan Yuhong , Pack Stephanie , Sonbuchner Timothy M. , Besse Arnaud , Lin Qingcong , Greally John M. , Skoultchi Arthur I. , Bouhassira Eric E.
Published: May 2003

Posttranslational modifications and remodeling of nucleosomes are critical factors in the regulation of transcription. Higher-order folding of chromatin also is likely to contribute to the control of gene expression, but the absence of a detailed description of the structure of the chromatin fiber has impaired progress in this area. Mammalian somatic cells contain a set of H1 linker-histone subtypes, H1 (0) and H1a to H1e, that bind to nucleosome core particles and to the linker DNA between nucleosomes. To determine whether the H1 histone subtypes play differential roles in the regulation of gene expression, we combined mice lacking specific H1 histone subtypes with mice carrying transgenes subject to position effects. Because position effects result from the unique chromatin structure created by the juxtaposition of regulatory elements in the transgene and at the site of integration, transgenes can serve as exquisitely sensitive indicators of chromatin structure. We report that some, but not all, linker histones can attenuate or accentuate position effects. The results suggest that the linker-histone subtypes play differential roles in the control of gene expression and that the sequential arrangement of the linker histones on the chromatin fiber might regulate higher-order chromatin structure and fine-tune expression levels.
A novel class of gene controlling virulence in plant pathogenic ascomycete fungi
Authors: Lu Shun-Wen , Kroken Scott , Lee Bee-Na , Robbertse Barbara , Churchill Alice C. L. , Yoder O. C. , Turgeon B. Gillian
Published: May 2003

Insertional mutants of the fungal maize pathogen
Cochliobolus heterostrophus
were screened for altered virulence. One mutant had 60% reduction in lesion size relative to WT but no other detectable change in phenotype. Analysis of sequence at the insertion site revealed a gene (
CPS1
) encoding a protein with two AMP-binding domains.
CPS1
orthologs were detected in all
Cochliobolus
spp. examined, in several other classes of ascomycete fungi, and in animals but not in basidiomycete fungi, bacteria, or plants. Phylogenetic analysis suggested that
CPS1
represents a previously undescribed subset of adenylate-forming enzymes that have diverged from certain acyl-CoA ligases, which in bacteria are involved in biosynthesis of nonribosomal peptides or polyketide/peptide hybrids. Disruption of
CPS1
caused reduced virulence of both race T and race O of
C. heterostrophus
on maize, of
Cochliobolus victoriae
on oats, and of
Gibberella zeae
on wheat. These results suggest that
CPS1
functions as a general fungal virulence factor in plant pathogenic ascomycetes.
An unusual mechanism of bacterial gene expression revealed for the RNase P protein of
Thermus
strains
Authors: Feltens Ralph , Gößringer Markus , Willkomm Dagmar K. , Urlaub Henning , Hartmann Roland K.
Published: May 2003

The RNase P protein gene (
rnpA
) completely overlaps the
rpmH
gene (encoding ribosomal protein L34) out of frame in the thermophilic bacterium
Thermus thermophilus
. This results in the synthesis of an extended RNase P protein (C5) of 163 aa and, by inference, of 240 aa in the related strain
Thermus filiformis
. Start codons of
rnpA
and
rpmH
, apparently governed by the same ribosome binding site, are separated by only 4 nt, which suggests a regulatory linkage between L34 and C5 translation and, accordingly, between ribosome and RNase P biosynthesis. Within the sequence encoding the N-terminal extensions and downstream of
rpmH
, several
Thermus
species exhibit in-frame deletions/insertions, suggesting relaxed constraints for sequence conservation in this region. Roughly the N-terminal third of
T. thermophilus
C5 was further shown to be dispensable for RNase P function
in vitro
by using a precursor tRNA
Gly
substrate from the same organism. Taken together, these data reveal a mode of gene expression that is to our knowledge unprecedented in bacteria.
Rhythmic binding of a WHITE COLLAR-containing complex to the
frequency
promoter is inhibited by FREQUENCY
Authors: Froehlich Allan C. , Loros Jennifer J. , Dunlap Jay C.
Published: May 2003

The biological clock of
Neurospora crassa
includes interconnected transcriptional and translational feedback loops that cause both the transcript and protein encoded by the
frequency
gene (
frq
) to undergo the robust daily oscillations in abundance, which are essential for clock function. To understand better the mechanism generating rhythmic
frq
transcript, reporter constructs were used to show that the oscillation in
frq
message is transcriptionally regulated, and a single cis-acting element in the
frq
promoter, the Clock Box (C box), is both necessary and sufficient for this rhythmic transcription. Nuclear protein extracts used in binding assays revealed that a White Collar (WC)-1- and WC-2-containing complex (WCC) binds to the C box in a time-of-day-specific manner. Overexpression of an ectopic copy of FRQ or addition of
in vitro
-generated FRQ resulted in reduced WCC binding to the C box. These data suggest that oscillations in
frq
transcript result from WCC binding to the
frq
promoter and activating transcription with subsequent changes in FRQ levels having an inverse effect on WCC binding. In this way rhythmic expression and turnover of FRQ drives the rhythm in its own transcription.
The mechanism of ferrichrome transport through Arn1p and its metabolism in
Saccharomyces cerevisiae
Authors: Moore Robert E. , Kim Youngwoo , Philpott Caroline C.
Published: May 2003

Siderophores are low molecular weight compounds, synthesized and secreted by microorganisms, that specifically bind ferric iron with exceptionally high affinity. Microbes capture these compounds and take up the bound iron through specific, high-affinity systems.
Saccharomyces cerevisiae
can take up iron bound to siderophores through the transporters of the
ARN
family; however, the mechanism by which the siderophore-bound iron enters the cell via these transporters is not known. Here we describe how ferrichrome, a siderophore of the hydroxamate class, is taken up by Arn1p. Arn1p exhibits two surface binding sites for ferrichrome, one that is similar in affinity to the
K
T
for uptake and one of a much higher affinity that is specific for the metallated form of ferrichrome. Ferrichrome may gain access to the higher-affinity site through endocytosis. Tracer studies using
14
C-labeled ferrichrome bound to either iron(III) or aluminum(III), a nonreducible ligand for ferrichrome, indicate that ferrichrome enters the cell as the intact metallosiderophore and accumulates in the cytosol. Both ferrichrome chelates were relatively stable within the cell, and metal-free ferrichrome did not accumulate, indicating a role for ferrichrome in intracellular iron storage. Iron stored as ferrichrome was readily mobilized to meet the metabolic needs of the cell.
Targets of the transcriptional repressor oncoprotein Gfi-1
Authors: Duan Zhijun , Horwitz Marshall
Published: May 2003

Gfi-1 is a zinc finger transcriptional repressor originally recognized for its role in T cell differentiation and lymphomas. Recent experiments reveal that gene-targeted
Gfi-1
-deficient mice are neutropenic and that
Gfi-1
mutations cause human neutropenia. In both cases, myeloid progenitor cells lose the ability to distinctly differentiate granulocytes from monocytes. The molecular mechanism of the hematopoietic abnormalities caused by
Gfi-1
deficiency remains undetermined because of a lack of known Gfi-1 target genes. To identify Gfi-1 targets
in vivo
, we performed large-scale chromatin immunoprecipitation analysis on a set of 34 candidate genes in myeloblast (KG-1 and HL-60), monoblast (U937), and T lymphocyte cell lines (Jurkat), in concert with RT-PCR-based expression profiling. We identified 32 Gfi-1 binding sites in a functionally variable set of 16 genes, including complements of cell-cycle regulators, transcription factors, and granulocyte-specific markers. Cluster analysis of expression patterns and chromatin immunoprecipitation data reveals that Gfi-1 targets a subset of genes differentiating hematopoietic lineages and therefore plays a relatively superior role in the hierarchy of factors governing stem cell differentiation.
Effect of the anorectic fatty acid synthase inhibitor C75 on neuronal activity in the hypothalamus and brainstem
Authors: Gao Su , Lane M. Daniel
Published: May 2003

Intraperitoneal (i.p.) injection of C75, a fatty acid synthase inhibitor, causes a rapid (≤2-h) and persistent (to at least 24-h) ≈95% decrease in food intake. The persistent effect seems to be due to inhibition of the fasting-induced up-regulation of expression of hypothalamic orexigenic neuropeptides neuropeptide Y and agouti-related protein and down-regulation of expression of anorexigenic neuropeptides pro-opiomelanocortin/α-melanocyte-stimulating hormone and cocaine-amphetamine-related transcript. The effect of C75 on neuronal activity in the hypothalamus and brainstem was assessed by c-Fos expression. Consistent with its effect on neuropeptide expression, C75 blocked fasting-induced c-Fos expression in the arcuate nucleus (Arc), lateral hypothalamic area (LHA), and paraventricular nucleus (PVN) 10–24 h after i.p. injection. However, i.p. C75 induced a rapid (≤2-h) c-Fos expression in the nucleus of the solitary tract (NTS) and area postrema of the brainstem but not in the Arc or LHA. Intracerebroventricular administration of C75 rapidly induced c-Fos expression in the Arc, PVN, and NTS, supporting a central role of C75 in the regulation of food intake. Thus, suppression of food intake by C75 administered i.p. seems to be mediated in two phases, a rapid initial phase via the NTS/area postrema of the brainstem and a delayed phase via the Arc, LHA, and PVN of the hypothalamus. The delayed effect of C75 on the Arc, LHA, and PVN correlates well with its ability to interfere with the fasting-induced effects on the expression of key orexigenic (neuropeptide Y and agouti-related protein) and anorexigenic (pro-opiomelanocortin/α-melanocyte-stimulating hormone and cocaine-amphetamine-related transcript) messages in the hypothalamus.
Ultra-high-pressure inactivation of prion infectivity in processed meat: A practical method to prevent human infection
Authors: Brown Paul , Meyer Richard , Cardone Franco , Pocchiari Maurizio
Published: May 2003

Bovine spongiform encephalopathy contamination of the human food chain most likely resulted from nervous system tissue in mechanically recovered meat used in the manufacture of processed meats. We spiked hot dogs with 263K hamster-adapted scrapie brain (10% wt/wt) to produce an infectivity level of ≈9 log
10
mean lethal doses (LD
50
) per g of paste homogenate. Aliquots were subjected to short pressure pulses of 690, 1,000, and 1,200 MPa at running temperatures of 121–137°C. Western blots of PrPres were found to be useful indicators of infectivity levels, which at all tested pressures were significantly reduced as compared with untreated controls: from ≈10
3
LD
50
per g at 690 MPa to ≈10
6
LD
50
per g at 1,200 MPa. The application of commercially practical conditions of temperature and pressure could ensure the safety of processed meats from bovine spongiform encephalopathy contamination, and could also be used to study phase transitions of the prion protein from its normal to misfolded state.
A quantitative model for transforming reflectance spectra into the Munsell color space using cone sensitivity functions and opponent process weights
Authors: D'Andrade Roy G. , Romney A. Kimball
Published: May 2003

This article presents a computational model of the process through which the human visual system transforms reflectance spectra into perceptions of color. Using physical reflectance spectra data and standard human cone sensitivity functions we describe the transformations necessary for predicting the location of colors in the Munsell color space. These transformations include quantitative estimates of the opponent process weights needed to transform cone activations into Munsell color space coordinates. Using these opponent process weights, the Munsell position of specific colors can be predicted from their physical spectra with a mean correlation of 0.989.
Identification of genes involved in cell invasion by using a library of randomized hybrid ribozymes
Authors: Suyama Eigo , Kawasaki Hiroaki , Nakajima Motowo , Taira Kazunari
Published: May 2003

Libraries of RNA helicase-coupled randomized ribozymes are a powerful tool for the identification of functional genes. We have demonstrated the usefulness of this functional gene-discovery system by identifying genes involved in tumor invasion, a process that is an essential feature of tumor metastasis: the spread of cancer cells from the original tumor to other sites in the body that imposes serious problems in the prognosis and treatment of cancer. Using a filter-based invasion assay
in vitro
, we isolated ribozymes that enhanced the invasive properties of NIH 3T3 fibroblasts. Sequence analysis of selected clones and a database search revealed that genes such as the gene for Gem GTPase and uncharacterized genes that resemble genes for myosin phosphatase and protein-tyrosine-phosphatase are involved in cell invasion. Our system for gene identification by using ribozymes and the functional analysis of target genes should help to clarify the complex mechanisms of invasion and metastasis and might provide information that is relevant to cancer therapy.