bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporary, but Essential Requirement of CD8+ T Cells Early in the Pathogenesis of Diabetes in BB Rats as Revealed by Thymectomy and CD8 Depletion
Authors: Groen Herman , Klatter Flip , Pater Jennie , Nieuwenhuis Paul , Rozing Jan
Published: December 2003

Autoimmunity‐prone BB rats demonstrate a T lymphocytopenia and abnormal T cell subset distribution. To test whether the life span of all T cells or only of certain subsets is reduced in BB rats, we thymectomised 8‐week‐old BB and PVG rats and subsequently assessed size and composition of the T cell population over a 6‐week‐period. In both strains, thymectomy (Tx) was followed by a decrease in peripheral T cell numbers, which was proportionally larger in BB rats. The decline of the Thy‐1+ recent thymic migrant (RTM) T cell phenotype was similar in both strains. BB rats showed a rapid preferential loss of CD8+ and CD45RC+ T cells, whereas the relative loss of RT6+ T cells was proportional to that of all T cells and not significantly different from that in PVG rats. Tx at 8‐week did not prevent diabetes. Tx of 4‐week‐old BB rats revealed essentially the same changes in peripheral T cell subset distribution as in 8‐week‐old animals. However, Tx at week 4 did prevent diabetes. Since this raised the possibility of a temporary requirement of CD8+ T cells for the development of diabetes, we performed CD8 depletions during different pre‐diabetic intervals. We found that CD8 depletion from 4 to 8 and 4 to 14 weeks, but not from 8 to 14 weeks of age prevented diabetes. We conclude that the protective effect of early adult Tx is, at least in part, due to the rapid loss of CD8+ T cells, and that these cells are only required between 4 and 8 weeks of age for diabetes to develop in BB rats.
Role of Peritoneal Macrophages in Cytomegalovirus‐induced Acceleration of Autoimmune Diabetes in BB‐rats
Authors: Hillebrands Jan-Luuk , van der Werf Nienke , Klatter Flip A. , Bruggeman Cathrien A. , Rozing Jan
Published: December 2003

Background: As one of the natural perturbants, infection with cytomegalovirus (CMV) is believed to play a role in the development of Type I diabetes. Using the DP‐BB rat model for autoimmune diabetes, we here report about possible mechanisms responsible for R(at)CMV‐induced accelerated onset of diabetes.

Methods: Rats were i.p. infected with 2 × 106 plaque forming units (pfu) RCMV and followed for diabetes development. Presence of RCMV antigens and DNA was analyzed by immunohistochemistry and PCR on pancreatic tissue and isolated islets. The effect of viral infection on peritoneal macrophages (pMΦ) and diabetes development was studied by analyzing numbers of pMΦ, virus permissiveness and by depletion of this subset by peritoneal lavage.

Results: RCMV accelerated onset of diabetes without infecting pancreatic islets. Immunohistochemistry and PCR on pancreas and isolated islets indicated that islets are non‐permissive for RCMV. Infection results in an influx of pMΦ 1 day p.i. of which ~0.05% showed signs of reproductive infection. Depletion of pMΦ on days 1‐3 p.i. completely counteracted the accelerating effect of RCMV.

Interpretation: RCMV accelerates onset of diabetes without infecting pancreatic islets. pMΦ might function as an carriage to disseminate virus to the pancreas where they enhance activation of autoreactive T cells resulting in accelerated onset of diabetes.
Geographic Clusters of Primary Biliary Cirrhosis
Authors: Abu-Mouch Saif , Selmi Carlo , Benson Gordon D. , Kenny Thomas P. , Invernizzi Pietro , Zuin Massimo , Podda Mauro , Rossaro Lorenzo , Gershwin M. Eric
Published: December 2003

Genetic and environmental factors have been widely suggested to contribute to the pathogenesis of primary biliary cirrhosis (PBC), an autoimmune disease of unknown etiology leading to destruction of small bile ducts. Interestingly, epidemiologic data indicate a variable prevalence of the disease in different geographical areas. The study of clusters of PBC may provide clues as to possible triggers in the induction of immunopathology. We report herein four such unique PBC clusters that suggest the presence of both genetic and environmental factors in the induction of PBC. The first cluster is represented by a family of ten siblings of Palestinian origin that have an extraordinary frequency of PBC (with 5/8 sisters having the disease). Second, we describe the cases of a husband and wife, both having PBC. A family in which PBC was diagnosed in two genetically unrelated individuals, who lived in the same household, represents the third cluster. Fourth, we report a high prevalence of PBC cases in a very small area in Alaska. Although these data are anedoctal, the study of a large number of such clusters may provide a tool to estimate the roles of genetics and environment in the induction of autoimmunity.
New ELISA Kits using C3 Binding Glycoprotein from Cuscuta europea Detect Mainly IgM CIC in Rheumatoid Arthritis and Progressive Systemic Sclerosis, but not in Systemic Lupus Erythematosus
Authors: Stanilova Spaska Angelova , Slavov Emil Slavov
Published: December 2003

Elevated levels of circulating immune complexes (CIC), containing IgG, IgM or IgA antibodies were detected in the sera of patients with autoimmune diseases. This might indicate a different biological meaning of the three isotypes of immunoglobulin (Ig) in the CIC. Each CIC assay detected only certain classes and subclasses of Ig in CIC material or fixed complement protein. In this study, a new method based on C3binding glycoprotein named CIF‐ELISA and a well‐known method ANTI‐C3 ELISA, were used for quantitative assessment of IgM‐CIC, IgG‐CIC and IgA‐CIC levels in human sera. A modified CIF‐ELISA and ANTI‐C3 ELISA for simultaneous detection of CIC, containing IgG, IgM and IgA, (stCIC), were also performed. The assays were evaluated on the same specially prepared samples: 55 normal sera, 99 sera from rheumatoid arthritis (RA), 88 sera from systemic lupus erythematosus (SLE), and 27 sera from progressive systemic sclerosis (PSS). We found that the sensitivity of the tests used varied depending on the diseases studied. CIF‐ELISA displayed higher sensitivity of IgM‐CIC when compared to ANTI‐C3 ELISA in RA patients (40.0 and 20.95%, respectively) and PSS (44.43 and 37.04%, respectively). Results for the sensitivity of IgA‐CIC were in adverse direction in the RA group (14.28 and 19.05%) and PSS (14.81 and 25.93%) by both methods. It was also established that the concordance of IgM‐CIC positives by both methods was 48.84% in RA and 46.67% in PSS, while in SLE it was 18.78%. These results are most probably due to the different assay abilities to detect antibody isotype of the CIC material and help to explain what specific role each Ig isotype in CIC has in the course of the disease.
Lack of Association between Anti‐Phospholipid Antibodies (APLA) and Attention Deficit/Hyperactivity Disorder (ADHD) in Children
Authors: Bujanover Shay , Levy Yair , Katz Miriam , Leitner Yael , Vinograd Isaac , Shoenfeld Yehuda
Published: December 2003

Numerous studies have shown the pathological influence anti‐phospholipid antibodies (APLA) have on the physiology of the single neuron as well as the function of the entire human nervous system. The influence is well demonstrated in the antiphospholipid syndrome (APS). This syndrome is characterized by a triad of arterial or venous thrombotic events, recurrent fetal loss and thrombocytopenic purpura. The syndrome exhibits different neurological pathologies such as: chorea, seizures, transverse myelopathy, migraine, cerebral ataxia, hemiballismus and transient global amnesia, which are not fully explained by the procoagulopathic trait of APLA. A study on mice induced with APS demonstrated hyperactive behavior when compared to the control group. The information gathered from these different studies raised the question whether APLA has any part in the etiology of Attention Deficit/Hyperactive Disorder (ADHD) in children.

We compared 41 children diagnosed with ADHD to a control of 28 healthy children. Blood drawn from the two groups was screened using ELISA for the presence of anti‐cardiolipin antibodies, antiβ2GP antibodies, anti‐phosphatidyleserine antibodies and anti‐ethanolamine antibodies. The results show no significant difference in the level of antiphospholipid antibodies (APLA) measured between the children diagnosed with ADHD and the control group.
DNA Double Strand Breaks Occur Independent of AID in Hypermutating Ig Genes
Authors: Bross Linda , Jacobs Heinz
Published: December 2003

Somatic hypermutation (SHM) and class switch recombination (CSR) take place in B cells of the germinal center (GC) and are associated with DNA double‐strand breaks (DNA‐DSBs). Transcription favors the generation of DNA‐DSBs in the V‐regions and switch regions of Ig genes. Both SHM and CSR are controlled by the Activation Induced Cytidine Deaminase (AID), an enzyme exclusively expressed in B cells of the GC. Because AID is capable of deaminating deoxy‐cytidine (dC) to deoxy‐uracil (dU), it might directly induce nicks (single strand DNA breaks) and also DNA‐DSBs via a U‐DNA glycosylase mediated base excision repair pathway (′DNA‐substrate model′). Alternatively, AID could function like its closest homologue Apobec‐1 as a catalytic subunit of a RNA editing holoenzyme (′RNA‐substrate model′). To determine whether AID lies upstream or downstream of the DNA lesions found in hypermutating Ig genes, we have analysed the Vλ locus of AID proficient and AID deficient GC B cells for the presence of DNA‐DSBs. Although rearranged Vλ genes are preferred targets of SHM we find that AID‐proficient and ‐deficient Vλ1/2‐expressing GC B cells display a similar frequency, distribution and sequence preference of DNA‐DSBs in rearranged and germline Vλ genes, favoring the idea that AID acts downstream of the DNA lesions to mediate error prone processing.
IgG Autoantibodies against β2‐Glycoprotein I Complexed with a Lipid Ligand Derived from Oxidized Low‐Density Lipoprotein are Associated with Arterial Thrombosis in Antiphospholipid Syndrome
Authors: Lopez Daniel , Kobayashi Kazuko , Merrill Joan T. , Matsuura E. , Lopez Luis R.
Published: December 2003

We recently reported [J. Lipid Res. 42 (2001), 697; 43 (2002), 1486; 44 (2003), 716] that β2‐glycoprotein I (β2GPI) forms complexes with oxidized LDL (oxLDL) and autoantibodies against these complexes are present in patients with SLE and antiphospholipid syndrome (APS). The relationship of β2GPI/oxLDL complexes and IgG autoantibodies against β2GPI complexed with oxLig‐1 (an oxLDL‐derived ligand) with clinical manifestations of APS was studied in 150 APS and SLE patients. The β2GPI/oxLDL levels of APS patients were similar to those of SLE patients without APS, but they were significantly higher than healthy individuals. There was no difference in the complex levels among the patients with arterial, venous thrombosis, or pregnancy morbidity. IgG anti‐β2GPI/oxLig‐1 levels of APS were significantly higher than those of SLE without APS and healthy individuals. Further, antibody levels of APS patients with arterial thrombosis were significantly higher than those patients with venous thrombosis and pregnancy morbidity. Thus, oxidation of LDL leads the complex formation with β2GPI in SLE and APS patients. In contrast, anti‐β2GPI/oxLig‐1 autoantibodies were generated only in APS and were strongly associated with arterial thrombosis. These results suggest that autoantibodies against β2GPI/oxLDL complexes are etiologically important in the development of atherosclerosis in APS.
Pseudo Alopecia Areata Caused by Skull‐caps with Metal Pin Fasteners used by Orthodox Jews in Israel
Authors: Yosefy Chaim , Ronnen Meir , Edelstein Dennis
Published: December 2003

Background: Alopecia Areata (AA) is a disease characterized by hair loss that is widely believed to be autoimmune in origin. Thus treatment is generally aimed in this direction using immune inhibitors such as steroids and PUVA.

Objective: To describe a variant of AA, Pseudo Alopecia Areata, caused by a particular cupola pin holder (tic‐tac) and to offer a non‐pharmacological treatment option (NPT).

Methods: A prospective open label study in 37 Jewish religious patients (34 males, 3 females, mean 35 ± 2 years), previously diagnosed and treated for scalp AA were randomly referred to one of the three NPT intervention methods: small cupola held by two pins, large cupola held by one pin and similar cupola held by a different pin.

Results: Three of the ten patients (33.3%) from the first group developed secondary AA from the additional pin. No changes were seen in the second group. Ten of the seventeen patients (58.8%) from the third group achieved immediate improvement subsequent to replacing the original pin with a new one on a larger cupola.

Conclusions: Conservative pharmacological treatment failed to repair the lesions. The addition of a second pin caused an additional lesion. In contrast, replacing the cupola with a larger one and the original pin‐fastener with a different type, successfully reduced the lesions.
Models for Natural Killer Cell Repertoire Formation
Authors: Salmon-Divon Mali , Höglund Petter , Mehr Ramit
Published: December 2003

Natural killer (NK) cells lyse only cells that do not express sufficient levels of self class I MHC molecules. Inhibition of lysis is mediated by inhibitory receptors expressed by NK cells, such as the murine Ly49 receptors, that bind to MHC class I molecules. Since inhibitory receptor genes and MHC class I genes are located on different chromosomes, and are hence not automatically co‐inherited, NK cells apparently adapt to the MHC environment during their development. Two models have been proposed to account for this “education” process of NK cells. The two-step selection model postulates that developing NK cells initiate the stable expression of a random set of Ly49 genes, and then undergo two selection steps, one for cells that express a sufficient number of self‐MHC receptors, and one against cells that express too many inhibitory receptors. The sequential model postulates that a cell keeps initiating the stable expression of additional inhibitory receptors until a sufficient expression level of self‐MHC specific receptors is reached, and the cell matures. In this study we implement both models in computer simulations, and compare simulation results to experimental data, in order to evaluate the relative plausibility of the two models.
Is there a Relation between Chlamydia Infection and Primary Biliary Cirrhosis?
Authors: Leung Patrick S. C. , Park Ogyi , Matsumura Shuji , Ansari Aftab A. , Coppel Ross L. , Gershwin M. Eric
Published: December 2003

Over the past two decades, a number of studies have failed to provide direct evidence of specific microbial chronic infection in primary biliary cirrhosis (PBC). However, a recent report suggests that there is a specific association of Chlamydia pneumoniae in patients with PBC and that C. pneumoniae or similar antigens might play a role in the pathogenesis of disease. To determine if Chlamydia infection is associated with PBC, we applied a combination of immunological and molecular approaches to investigate (a) the serological reactivity against two common Chlamydia human pathogens, C. pneumoniae and C. trachomatis, by immunoblotting, (b) the presence of Chlamydia in liver samples of patients with PBC and controls by PCR amplification of Chlamydia specific 16S rRNA and (c) the presence of Chlamydia proteins in liver samples of patients with PBC and controls by immunohistochemical staining. By immunoblotting, C. trachomatis and C. pneumoniae specific serological antibodies were found in 52/57 (91.2%) AMA positive PBC, 7/33 (21/2%) of AMA negative PBC, 1/25 (4%) PSC, 0/15 (0%) Sjorgen′s syndrome and 0/20 (0%) systemic lupus erythematosus patients and 0/20 (0%) healthy volunteers at 1:200 sera dilution. PBC sera reacted to Chlamydia and E. coli lysates in western blots up to a maximum of 10-4 dilution. However, PCR amplification of the Chlamydia specific 16S rRNA gene was negative in 25/25 PBC livers but positive in 1/4 PSC liver, 3/6 in other liver disease controls and 1/4 normal liver samples. While two commercially available specific monoclonal antibodies stained positive controls (Chlamydia infected HEp‐2 cells) they failed to detect Chlamydia antigens in PBC livers. The detection of Chlamydia specific antibodies but not Chlamydia rRNA gene and Chlamydia antigens in PBC suggests that Chlamydia infection is not involved in PBC.
Treatment of Epithelioid Sarcoma at the Royal Marsden Hospital
Authors: Livi L. , Shah N. , Paiar F. , Fisher C. , Judson I. , Moskovic E. , Thomas M. , Harmer C.
Published: December 2003

Purpose: The aim of this study was to assess treatment and outcome with respect to clinical and pathological features.

Patients and methods: Thirty‐nine patients were identified (range 7–66 years, mean 23). Initial treatment comprised local
excision in 11 patients and wide excision in 14. Post‐operative external beam radiotherapy was prescribed in 22 patients
with a total dose of 60 Gy, delivered in two phases.

Results: The cause‐specific survival for the entire group was 79, 63, 56 and 45% at 1, 3, 5 and 10 years, respectively. A distal
limb location was associated with a better prognosis than proximal limb location (P = 0.04).

Conclusions: Our data favour treatment with wide functional excision followed by radical dose radiotherapy in attempt to
minimize risk of local recurrence, especially when primary tumours are bigger than 3 cm. Our data also suggest the same
treatment for local recurrence, when technically possible, to avoid amputation.
Low Dose Methotrexate and Vinblastine, Given Weekly to PatientsWith Desmoid Tumours, is Associated With Major Toxicity
Authors: van der Hul René L. , Seynaeve Caroline , van Geel Bert N. , Verweij Jaap
Published: December 2003

Purpose: To evaluate the tolerance of a low dose chemotherapy regimen for desmoid tumours.

Patients and methods: Patients with desmoids for whom radical resection was impossible or related to extensive mutilation
were treated with chemotherapy. Treatment consisted of intravenous methotrexate at a dose of 50mg and vinblastine at a
dose of 10mg weekly, scheduled to be given for a total period of 1 year. Doses were reduced and/or delayed on an individual
basis, depending on the observed type of toxicity.

Results: Ten patients (six males; four females), median age 43 years (range17–75), median WHO performance score 1
(range 0–1), were treated. None of them was able to complete the treatment as scheduled, due to observed side effects,
while in two patients treatment was also discontinued because of progressive disease. In six patients, less than 50% of the
projected administrations and dose could be given. Severe organ toxicity was noted in three patients (one interstitial
pneumonitis, two toxic hepatitis), which, however, was reversible in all cases.

Discussion: Methotrexate and vinblastine given at this dose and schedule lead to an unacceptable level of toxicity for a
long‐term treatment, and cannot be recommended for standard use.
Splenic Angiosarcoma Presenting With Jaundice, Ascites and Bone Marrow Fibrosis
Authors: Vaiphei Kim , Singh Virinder , Varma Subhash
Published: December 2003

A middle aged chronic alcoholic presented with deep jaundice, markedly enlarged and tender spleen with leukoerythroblastic
blood picture and bone marrow biopsy showing mild fibrosis. He was tested negative for HIV, hepatitis B and C viruses.
Besides very high serum bilirubin, alkaline phosphatase was raised four times the normal value. Contrast enhanced CT
showed enlarged spleen and liver with multiple heterogenous lesions in spleen and tiny hypo‐dense lesions in liver. In
hospital, he developed haemolytic uraemic syndrome and succumed to his illness. At autopsy spleen weighed 5200 gms and
variegated in appearance due to large areas of necrosis and whitish tumour nodules. Histology revealed morphology of an
angiosarcoma. Liver was also infiltrated by the tumour mainly in and around portal tract areas.
Factors Influencing Prognosis After Initial Inadequate Excision(IIE) for Soft Tissue Sarcoma
Authors: Van Geel Albert N. , Eggermont Alexander M. M. , Hanssens Patrick E. J. , Schmitz Paul I. M.
Published: December 2003

Purpose. The influence of initial inadequate excision (IIE) of soft tissue sarcoma (STS) on local control and overall survival
is not well established. It is generally believed that an IIE may have a negative impact on both, despite subsequent treatment
by radical surgery and radiotherapy. However, data on local recurrence‐free survival/overall survival are conflicting and there
are no data on the effect of IIE on overall survival.

Patients and methods. A retrospective analysis was made of 86 patients with soft tissue sarcoma of the extremities and trunk
after an IIE had been performed due to inappropriate work‐up. The minimal follow‐up was 7 years. Specimens of the
subsequent radical resection were evaluated for residual tumor, grade of tumor and complications of IIE. Endpoints were
recurrence‐free survival and overall survival.

Results. Specimens of the subsequent radical resection showed residual tumor in 66 patients (77%). The most common
complication after IIE was hematoma. In both univariate and multivariate analyses, grade II/III tumors and complications
after IIE are significant negative prognostic factors for local recurrence‐free survival (P = 0.008 and P = 0.002, respectively,
in the Cox model). For this survival, three prognostic groups could be formed based on grade, or presence or absence of
complications. Adjuvant radiotherapy did not change the rate of local recurrence‐free survival. For overall survival, only
tumor grade is a significant factor (log‐rank test).

Conclusion. This retrospective study shows that complications associated with an IIE have a significant negative effect on
local control, but not on overall survival, because IIE is often the result of inappropriate work‐up before surgery. For better
diagnosis and therapy STS should be treated in specialized centers.
Abortion rate and contraceptive practices in immigrant and native women in Sweden
Authors: Helström Lotti , Odlind Viveca , Zätterström Catharina , Johansson Monica , Granath Fredrik , Correia Nestor , Ekbom Anders
Published: December 2003

Aim: The aim of this study was to analyse whether immigrant women request induced abortion more frequently than Swedish-born women and, if so, to study possible explanations, including contraceptive practices and attitudes. Methods: All women who requested induced abortion during a period of one year were included in the study. The 1,289 women, of whom 36% were born outside Sweden, were interviewed by a nurse-midwife who, using a structured protocol, gathered information on socioeconomic factors, reasons for abortion, experience of contraceptive methods, and family planning counselling. The proportion of women with non-Swedish origin in the study population was compared with the official demographic statistics of the corresponding area. Results: The number of women born outside Sweden who requested induced abortion was larger than expected from their proportion in the population. The immigrant women originated from 77 countries and four continents, the largest subgroup, 11%, coming from Iran. Immigrant women had less experience of contraceptive use, more previous pregnancies and more induced abortions than women born in Sweden. In a multivariate analysis, immigrant status and educational level were found to be independent risk factors for repeat abortion. Conclusion: Immigrant status seems to be an independent risk factor for induced abortion. The immigrant women originated from a wide range of cultures. There is no reason to believe that the women in this heterogeneous group should have any cultural factor in common that could explain their higher proneness to seek induced abortion. The most probable cause is that immigrant status is associated more often with low education, weak social network, poverty, unemployment, and being outside common pathways to healthcare.
Dying for the union?: Military officers and the creation of a European defence force
Authors: Mérand Frédéric
Published: December 2003

In spite of the vagaries of the common foreign and security policy and the enduring domination of NATO, there now exist a fairly large number of West European military structures. Recently, European countries pledged substantial units towards the creation of a 60,000-strong EU Rapid Reaction Capability, due to be fully operational by the mid-2000s. What did military officers in France, the United Kingdom and Germany think of ‘European defence’ before recent political developments took place? This paper attempts to assess the level of support for military integration among officers as well as the reasons and causes underlying their opinion. In particular, I demonstrate that, beyond cross-national variation, speaking foreign languages, having few soldiers in one’s family, belonging to a ‘support’ unit, being highly ranked and believing in the virtues of international missions are significant predictors of support for European armed forces.
The ‘politics of institutionalization’: The inner discourse of German churches after radical political change
Authors: Thériault Barbara
Published: December 2003

The Protestant and Catholic churches in Germany embodied continuity in a country marked by discontinuity. During the forty years of division, they were the only organizations in East Germany to retain strong ties and organizational structures with their West German counterparts. Yet, the continuity that characterized the churches as organizations does not corroborate the stability of their values and self-definitions. Drawing on conceptual tools such as guiding metaphors and organizing principles, this paper looks at the debates that accompanied the re-establishment of the churches’ unity after radical political change in order to delineate the conflicting conceptions of the churches’ public role. The focus is on the work carried out by religious agents in either maintaining or changing these conceptions, a process we refer to as the ‘politics of institutionalization’. Hirschman's framework on rhetoric enables us to reconstruct the debates to identify and compare the strategies and patterns of argumentation advanced by the actors in this process. Special attention is paid to the ‘East German metaphors’, or what is generally termed the ‘positive experiences of the GDR and the “Wende”’, and their defenders following the remarkable events that paved the way for the collapse of the German Democratic Republic and for German unification.
NO Production by Pseudomonas aeruginosa cd1 Nitrite Reductase
Authors: Cutruzzolà Francesca , Rinaldo Serena , Centola Fabio , Brunori Maurizio
Published: December 2003

The structural and catalytic properties of Pseudomonas aeruginosa cd1 nitrite reductase, a key enzyme in bacterial denitrification, are reviewed in this paper. The mechanism of reduction of nitrite to NO is discussed in detail with special attention to the structural interpretation of function. The ability to stabilize negatively charged molecules, such as the substrate (nitrite) and other ligands (hydroxide and cyanide), is a key feature of catalysis in cd1NIRs. The positive potential in the active site is largely due to the presence of the two conserved distal histidines, which are involved in both substrate binding and product release. IUBMB Life, 55: 617‐621, 2003
Interplay of Cu,Zn Superoxide Dismutase and Nitric Oxide Synthase in Neurodegenerative Processes
Authors: Rotilio Giuseppe , Aquilano Karia , Ciriolo Maria Rosa
Published: December 2003

Reactive oxygen and nitrogen species (ROS and RNS) have been extensively recognized as important signaling molecules implicated in physiological processes such as gene expression, cell differentiation and immune activation. Nevertheless, continuous production of these species may produce oxidative and/or nitrosative stress resulting in cell damage and ultimately leading to cell death. Due to the high oxygen consumption and relative poor antioxidant defense, the central nervous system is highly susceptible to ROS‐ and RNS‐mediated toxicity. Actually, the oxidative and nitrosative stress have been implicated in the pathogenesis of neurodegeneration of a large variety of neurological disorders. This review will cover some aspects of the involvement of ROS‐ and RNS‐mediated apoptotic processes occurring in cellular models of familial amyotrophic lateral sclerosis (FALS), in particular the cases associated with mutations in SOD1, the gene encoding Cu,Zn superoxide dismutase (Cu,Zn SOD). A possible role for proteasome in the inhibition of neurodegenerative process by balancing ROS and RNS species is envisaged on the basis of evidence provided by results obtained from studies on this experimental model. IUBMB Life, 55: 629‐634, 2003
Nitric Oxide and Mitochondrial Complex IV
Authors: Sarti Paolo , Arese Marzia , Bacchi Antonella , Barone Maria Cecilia , Forte Elena , Mastronicola Daniela , Brunori Maurizio , Giuffrè Alessandro
Published: December 2003

Micromolar nitric oxide (NO) rapidly (ms) inhibits cytochrome c oxidase in turnover with physiological substrates. Two reaction mechanisms have been identified leading, respectively, to formation of a nitrosyl‐ [a32+‐NO] or a nitrite‐ [a33+‐NO2‐] derivative of the enzyme. In the presence of O2, the nitrosyl adduct recovers activity slowly, following NO displacement at k'≈0.01 s‐1 (37°C); the recovery of the nitrite adduct is much faster. Relevant to patho‐physiology, the enzyme does not degrade NO by following the first mechanism, whereas by following the second one it promotes NO oxidation and disposal as nitrite/nitrate. The reaction between NO and cytochrome c oxidase has been investigated at different integration levels of the enzyme, including the in situ state, such as in mouse liver mitochondria or cultured human SY5Y neuroblastoma cells. The respiratory chain is inhibited by NO, either supplied exogenously or produced endogenously via the NO synthase activation. Inhibition of respiration is reversible, although it remains to be clarified whether reversibility is always full and how it depends on concentration of and time of exposure to NO. Oxygraphic measurements show that cultured cells or isolated state 4 mitochondria exposed to micromolar (or less) NO recover from NO inhibition rapidly, as if the nitrite reaction was predominant. Mitochondria in state 3 display a slightly more persistent inhibition than in state 4, possibly due to a higher accumulation of the nitrosyl adduct. Among a number of parameters that appear to control the switch over between the two mechanisms, the concentration of reductants (reduced cytochrome c) at the cytochrome c oxidase site has been proved to be the most relevant one. IUBMB Life, 55: 605‐611, 2003
SMC Proteins at the Crossroads of Diverse Chromosomal Processes
Authors: Jessberger Rolf
Published: December 2003

How should a protein be designed to serve in processes as diverse as chromosome condensation, sister chromatid cohesion, DNA recombination, gene dosage regulation, and perhaps even gene silencing or transcriptional regulation ‐ which occur in both mitosis and meiosis? Such a protein or protein complex needs to bear DNA interaction domains, it needs the capacity to use energy to move DNA, it needs to enter into highly specific protein interactions, it needs to be large enough to link two DNA molecules, it needs to be of sufficient flexibility to cope with different types of chromatin structure, yet it also needs to be rigid enough to pull, push or enclose DNA. SMC proteins fulfill these requirements and form the core units of high molecular weight complexes that act in all those processes, and are essential for some of them. SMC stands for 'Structural Maintenance of Chromosomes', although SMC proteins are not static scaffold proteins merely providing support for a particular chromosome structure. SMC proteins are rather highly dynamic actors, that generate and modulate chromosome structures, affecting a plethora of biological processes. IUBMB Life, 55: 643‐652, 2003
Small Heat‐shock Proteins and Clusterin: Intra‐ and Extracellular Molecular Chaperones with a Common Mechanism of Action and Function?
Authors: Carver John A. , Rekas Agata , Thorn David C. , Wilson Mark R.
Published: December 2003

Small heat‐shock proteins (sHsps) and clusterin are molecular chaperones that share many functional similarities despite their lack of significant sequence similarity. These functional similarities, and some differences, are discussed. sHsps are ubiquitous intracellular proteins whereas clusterin is generally found extracellularly. Both chaperones potently prevent the amorphous aggregation and precipitation of target proteins under stress conditions such as elevated temperature, reduction and oxidation. In doing so, they act on the slow, off‐folding protein pathway. The conformational dynamism and aggregated state of both proteins may be crucial for their chaperone function. Subunit exchange is likely to be important in regulating chaperone action; the dissociated form of the protein is probably the chaperone‐active species rather than the aggregated state. They both exert their chaperone action without the need for hydrolysis of ATP and have little ability to refold target proteins. Increased expression of sHsps and clusterin accompanies a range of diseases that arise from protein misfolding and deposition of highly structured protein aggregates known as amyloid fibrils, e.g., Alzheimer's, Creutzfeldt‐Jakob and Parkinson's diseases. The interaction of sHsps and clusterin with fibril‐forming species is discussed along with their ability to prevent fibril formation. IUBMB Life, 55: 661‐668, 2003
The HIV‐1 Tat Transactivator Protein: a Therapeutic Target?
Authors: Fulcher Alex J. , Jans David A.
Published: December 2003

The human immunodeficiency virus‐1 (HIV‐1), the causative agent of autoimmune deficiency syndrome (AIDS) is a major health problem world‐wide. Central to HIV infection is the transactivator protein Tat, that plays a critical role in the nucleus during the HIV infectious cycle, by binding the transactivation‐responsive region (TAR) and thereby enhancing transcriptional elongation. Tat appears to gain nuclear entry through a novel mechanism, independent of the normal cellular importin/Ran‐dependent pathways, and regulated by a cytoplasmic retention mechanism. Since blocking Tat nuclear import is likely to prevent HIV infection, detailed delineation of Tat's nuclear import pathway is critical to assessing its viability as a therapeutic target. Other feasible anti‐HIV therapies include approaches to inhibit Tat‐TAR interaction. IUBMB Life, 55: 669‐680, 2003
The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria
Authors: Gentle Ian , Gabriel Kipros , Beech Peter , Waller Ross , Lithgow Trevor
Published: December 2003

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.
Two Motifs Target Batten Disease Protein CLN3 to Lysosomes in Transfected Nonneuronal and Neuronal Cells
Authors: Kyttälä Aija , Ihrke Gudrun , Vesa Jouni , Schell Michael J. , Luzio J. Paul
Published: December 2003

Batten disease is a neurodegenerative disorder resulting from mutations in CLN3, a polytopic membrane protein, whose predominant intracellular destination in nonneuronal cells is the lysosome. The topology of CLN3 protein, its lysosomal targeting mechanism, and the development of Batten disease are poorly understood. We provide experimental evidence that both the N and C termini and one large loop domain of CLN3 face the cytoplasm. We have identified two lysosomal targeting motifs that mediate the sorting of CLN3 in transfected nonneuronal and neuronal cells: an unconventional motif in the long C-terminal cytosolic tail consisting of a methionine and a glycine separated by nine amino acids [M(X)9G], and a more conventional dileucine motif, located in the large cytosolic loop domain and preceded by an acidic patch. Each motif on its own was sufficient to mediate lysosomal targeting, but optimal efficiency required both. Interestingly, in primary neurons, CLN3 was prominently seen both in lysosomes in the cell body and in endosomes, containing early endosomal antigen-1 along neuronal processes. Because there are few lysosomes in axons and peripheral parts of dendrites, the presence of CLN3 in endosomes of neurons may be functionally important. Endosomal association of the protein was independent of the two lysosomal targeting motifs.
Routing ofHansenula polymorphaAlcohol Oxidase: An Alternative Peroxisomal Protein-sorting Machinery
Authors: Gunkel Katja , van Dijk Ralf , Veenhuis Marten , van der Klei Ida J.
Published: December 2003

Import of Hansenula polymorpha alcohol oxidase (AO) into peroxisomes is dependent on the PTS1 receptor, HpPex5p. The PTS1 of AO (-LARF) is sufficient to direct reporter proteins to peroxisomes. To study AO sorting in more detail, strains producing mutant AO proteins were constructed. AO containing a mutation in the FAD binding fold was mislocalized to the cytosol. This indicates that the PTS1 of AO is not sufficient for import of AO. AO protein in which the PTS1 was destroyed (-LARA) was normally sorted to peroxisomes. Moreover, C-terminal deletions of up to 16 amino acids did not significantly affect AO import, indicating that the PTS1 was not necessary for targeting. Consistent with these observations we found that AO import occurred independent from the C-terminal TPR-domain of HpPex5p, known to bind PTS1 peptides. Synthesis of the N-terminal domain (amino acids 1-272) of HpPex5p in pex5 cells restored AO import, whereas other PTS1 proteins were mislocalized to the cytosol. These data indicate that AO is imported via a novel HpPex5p-dependent protein translocation pathway, which does not require the PTS1 of AO and the C-terminal TPR domains of HpPex5p, but involves FAD binding and the N-terminus of HpPex5p.
A Novel Role of Nectins in Inhibition of the E-Cadherin–induced Activation of Rac and Formation of Cell-Cell Adherens Junctions
Authors: Hoshino Takashi , Shimizu Kazuya , Honda Tomoyuki , Kawakatsu Tomomi , Fukuyama Taihei , Nakamura Takeshi , Matsuda Michiyuki , Takai Yoshimi
Published: December 2003

Nectins are Ca2+-independent immunoglobulin (Ig)-like cell-cell adhesion molecules. The trans-interactions of nectins recruit cadherins to the nectin-based cell-cell adhesion, resulting in formation of cell-cell adherens junctions (AJs) in epithelial cells and fibroblasts. The trans-interaction of E-cadherin induces activation of Rac small G protein, whereas the trans-interactions of nectins induce activation of not only Rac but also Cdc42 small G protein. We showed by the fluorescent resonance energy transfer (FRET) imaging that the trans-interaction of E-cadherin induced dynamic activation and inactivation of Rac, which led to dynamic formation and retraction of lamellipodia. Moreover, we found here that the nectins, which did not trans-interact with other nectins (non–trans-interacting nectins), inhibited the E-cadherin–induced activation of Rac and reduced the velocity of the formation of the E-cadherin-based cell-cell AJs. The inhibitory effect of non–trans-interacting nectins was suppressed by the activation of Cdc42 induced by the trans-interactions of nectins. These results indicate a novel role of nectins in regulation of the E-cadherin–induced activation of Rac and formation of cell-cell AJs.
X-ray Tomography Generates 3-D Reconstructions of the Yeast, Saccharomyces cerevisiae, at 60-nm Resolution
Authors: Larabell Carolyn A. , Le Gros Mark A.
Published: December 2003

We examined the yeast, Saccharomyces cerevisiae, using X-ray tomography and demonstrate unique views of the internal structural organization of these cells at 60-nm resolution. Cryo X-ray tomography is a new imaging technique that generates three-dimensional (3-D) information of whole cells. In the energy range of X-rays used to examine cells, organic material absorbs approximately an order of magnitude more strongly than water. This produces a quantifiable natural contrast in fully hydrated cells and eliminates the need for chemical fixatives or contrast enhancement reagents to visualize cellular structures. Because proteins can be localized in the X-ray microscope using immunogold labeling protocols (Meyer-Ilse et al., 2001. J. Microsc. 201, 395–403), tomography enables 3-D molecular localization. The time required to collect the data for each cell shown here was <15 min and has recently been reduced to 3 min, making it possible to examine numerous yeast and to collect statistically significant high-resolution data. In this video essay, we show examples of 3-D tomographic reconstructions of whole yeast and demonstrate the power of this technology to obtain quantifiable information from whole, hydrated cells.
Depletion of Centromeric MCAK Leads to Chromosome Congression and Segregation Defects Due to Improper Kinetochore Attachments
Authors: Kline-Smith Susan L. , Khodjakov Alexey , Hergert Polla , Walczak Claire E.
Published: December 2003

The complex behavior of chromosomes during mitosis is accomplished by precise binding and highly regulated polymerization dynamics of kinetochore microtubules. Previous studies have implicated Kin Is, unique kinesins that depolymerize microtubules, in regulating chromosome positioning. We have characterized the immunofluorescence localization of centromere-bound MCAK and found that MCAK localized to inner kinetochores during prophase but was predominantly centromeric by metaphase. Interestingly, MCAK accumulated at leading kinetochores during congression but not during segregation. We tested the consequences of MCAK disruption by injecting a centromere dominant-negative protein into prophase cells. Depletion of centromeric MCAK led to reduced centromere stretch, delayed chromosome congression, alignment defects, and severe missegregation of chromosomes. Rates of chromosome movement were unchanged, suggesting that the primary role of MCAK is not to move chromosomes. Furthermore, we found that disruption of MCAK leads to multiple kinetochore–microtubule attachment defects, including merotelic, syntelic, and combined merotelic-syntelic attachments. These findings reveal an essential role for Kin Is in prevention and/or correction of improper kinetochore–microtubule attachments.
Endoplasmic Reticulum Quality Control of Unassembled Iron Transporter Depends on Rer1p-mediated Retrieval from the Golgi
Authors: Sato Miyuki , Sato Ken , Nakano Akihiko
Published: December 2003

Endoplasmic reticulum (ER) quality control is a conserved process by which misfolded or unassembled proteins are selectively retained in the endoplasmic reticulum (ER). Failure in oligomerization of multisubunit membrane proteins is one of the events that triggers ER quality control. The transmembrane domains (TMDs) of unassembled subunits are determinants of ER retention in many cases, although the mechanism of the TMD-mediated sorting of unassembled subunits remains elusive. We studied a yeast iron transporter complex on the cell surface as a new model system for ER quality control. When Fet3p, a transmembrane subunit, is not assembled with the other membrane subunit, Ftr1p, unassembled Fet3p is exclusively localized to the ER at steady state. The TMD of Fet3p contains a determinant for this process. However, pulse-chase analysis and in vitro budding assays indicate that unassembled Fet3p rapidly escapes from the ER. Furthermore, Rer1p, a retrieval receptor for ER-resident membrane proteins in the Golgi, is responsible for the TMD-dependent ER retrieval of unassembled Fet3p. These findings provide clear evidence that the ER quality control of unassembled membrane proteins can be achieved by retrieval from the Golgi and that Rer1p serves as a specific sorting receptor in this process.
Comparative Diagnostic Performances of Auscultation, Chest Radiography, and Lung Ultrasonography in Acute Respiratory Distress Syndrome
Authors: Lichtenstein Daniel , Goldstein Ivan , Mourgeon Eric , Cluzel Philippe , Grenier Philippe , Rouby Jean-Jacques
Published: December 2003

Background

Lung auscultation and bedside chest radiography are routinely used to assess the respiratory condition of ventilated patients with acute respiratory distress syndrome (ARDS). Clinical experience suggests that the diagnostic accuracy of these procedures is poor.

Methods

This prospective study of 32 patients with ARDS and 10 healthy volunteers was performed to compare the diagnostic accuracy of auscultation, bedside chest radiography, and lung ultrasonography with that of thoracic computed tomography. Three pathologic entities were evaluated in 384 lung regions (12 per patient): pleural effusion, alveolar consolidation, and alveolar-interstitial syndrome.

Results

Auscultation had a diagnostic accuracy of 61% for pleural effusion, 36% for alveolar consolidation, and 55% for alveolar-interstitial syndrome. Bedside chest radiography had a diagnostic accuracy of 47% for pleural effusion, 75% for alveolar consolidation, and 72% for alveolar-interstitial syndrome. Lung ultrasonography had a diagnostic accuracy of 93% for pleural effusion, 97% for alveolar consolidation, and 95% for alveolar-interstitial syndrome. Lung ultrasonography, in contrast to auscultation and chest radiography, could quantify the extent of lung injury. Interobserver agreement for the ultrasound findings as assessed by the kappa statistic was satisfactory: 0.74, 0.77, and 0.73 for detection of alveolar-interstitial syndrome, alveolar consolidation, and pleural effusion, respectively.

Conclusions

At the bedside, lung ultrasonography is highly sensitive, specific, and reproducible for diagnosing the main lung pathologic entities in patients with ARDS and can be considered an attractive alternative to bedside chest radiography and thoracic computed tomography.