To fully understand the function of the Cu- and Zn-containing superoxide dismutases in normal and disordered cells, it is essential to study protein variants with full metal contents. We describe the use of an Escherichia coli-based expression system for the overproduction of human intracellular wild type CuZn-superoxide dismutase (SOD), the CuZnSOD variant F50E/G51E (monomeric), two amyotrophic lateral sclerosis-related mutant CuZnSOD variants (D90A and G93A), and PseudoEC-SOD, all with high Cu contents. This system is based on coexpression of the SOD variants with the yeast copper chaperone yCCS during growth in a medium supplemented with Cu2+ and Zn2+. The recombinant SOD enzymes were all found in the cytosol and represented 30-50% of the total bacterial protein. The enzymes were purified to homogeneity and active enzymes were obtained in high yield. The resulting proteins were characterized through immunochemical reactivity and specific activity analyses, in conjunction with mass-, photo-, and atomic absorption-spectroscopy. © 2004 Elsevier Inc. All rights reserved.

ZnO nanorods with 80 nm diameter and 700 nm length and grown on the tip of a borosilicate glass capillary (0.7 μm in diameter) were used to create a highly sensitive pH sensor for monitoring in vivo biological process within single cells. The ZnO nanorods, functionalized by proton H3 O+ and hydroxyl O H- groups, exhibit a pH -dependent electrochemical potential difference versus a AgAgCl microelectrode. The potential difference was linear over a large dynamic range (4-11), which could be understood in terms of the change in surface charge during protonation and deprotonation. These nanoelectrode devices have the ability to enable analytical measurements in single living cells and have the capability to sense individual chemical species in specific locations within a cell. © 2007 American Institute of Physics.

Background: Carbohydrate-deficient transferrin (CDT) is elevated during prolonged overconsumption of alcohol and CDT is considered to be the most specific biochemical marker for alcohol overconsumption. However, an accurate method for analysing CDT is necessary because the test is frequently used for example in legal matters. Methods: Patient serum samples were analysed with the Axis-Shield %CDT and eluates were pooled together. Transferrin was purified from the pool by affinity chromatography and further analysed with HPLC to determine the ratios of different transferrin isoforms. Results: In the eluates using the Axis-Shield %CDT method, a substantial amount of trisialo transferrin was found, which is generally not considered a CDT isoform. Conclusions: The fact that trisialo transferrin is present may generate falsely elevated CDT results and it could at least partly explain the discrepancy between results of the Axis-Shield %CDT assay and HPLC in routine analysis. © 2005 Elsevier B.V. All rights reserved.

The transcription factor PAX5 is a critical regulator of B-cell commitment and development. Although normally not expressed in myeloid progenitors, PAX5 has recently been shown to be frequently expressed in myeloid malignancies and to suppress expression of myeloid differentiation genes, compatible with an effect on the differentiation or maintenance of myeloid progenitors. However, previous studies in which PAX5 was ectopically expressed in normal myeloid progenitors in vivo and in vitro provided conflicting results as to the effect of PAX5 on myeloid development. Herein, we demonstrate that on ectopic expression of PAX5 in bone marrow multipotent stem/progenitor cells, cells with a biphenotypic B220+GR-1/MAC-1+ phenotype are produced. These remain cytokine-dependent, but unlike control-transduced cells they sustain long-term generation of myeloid progenitors in vitro and remain capable of myeloid differentiation. Notably, PAX5+B220+GR-1/MAC- 1+ myeloid progenitors coexpress, at the single-cell level, myeloid genes and otherwise B-cell-specific PAX5 target genes. These findings establish that ectopic expression of PAX5 introduces extensive self-renewal properties in otherwise short-lived myeloid progenitors. Along with the established ectopic expression of PAX5 in acute myeloid leukemia, this motivates a careful investigation of the potential involvement of ectopic PAX5 expression in myeloid and biphenotypic leukemias. © 2007 by The American Society of Hematology.

Interaction with the environment has a key role in refining the neuronal circuitry required for normal brain function throughout life. Profound effects of enriched environment have been shown on neuronal structure and chemistry in experimental animals. Epidemiological studies imply that this is true also in man, thus cognitive stimulation has a protective effect on neurodegeneration, e.g., in Alzheimer's disease. Glutamatergic pathways are imperative for cognitive functions, such as memory, learning and long-term potentiation, and relies on the AMPA and NMDA glutamate receptors and the hippocampus, with its specific subregions, is an important anatomical substrate in this. The glutamate signalling is also dependent on a fine-tuned transport system, in the hippocampus primarily achieved by the glutamate transporter EAAC1. In this study we show how environmental enrichment modulates these parts of the glutamatergic system using quantitative in situ hybridisation. This work demonstrates for the first time that environmental enrichment modulates the mRNA expression of EAAC1 which is significantly and region specifically decreased in the hippocampus. We also provide evidence for regional and hemisphere-specific upregulation of NMDA mRNA in the hippocampus after environmental enrichment. The current work also shows that AMPA mRNA of the hippocampus is not per se changed by environmental enrichment in adult animals. Taken together, our results extend the knowledge of the glutamatergic system of specific regions of the hippocampus and its modulation by environmental enrichment and could contribute to the development of strategies aimed at limiting pathological changes associated with glutamatergic dysfunctions. © 2007.

For almost 30 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.  These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, all protocols from the series are indexed in Pub Med, comprehensive and reliable.

Aims/hypothesis: The World Health Organization considers an aetiological classification of diabetes to be essential. The aim of this study was to evaluate whether HLA-DQB1 genotypes facilitate the classification of diabetes as compared with assessment of islet antibodies by investigating young adult diabetic patients. Subjects and methods: Blood samples were available at diagnosis for 1,872 (90%) of the 2,077 young adult patients (aged 15-34 years old) over a 5-year period in the nationwide Diabetes Incidence Study in Sweden. Islet antibodies were measured at diagnosis in 1,869 patients, fasting plasma C-peptide (fpC-peptide) after diagnosis in 1,522, while HLA-DQB1 genotypes were determined in 1,743. Results: Islet antibodies were found in 83% of patients clinically considered to have type 1 diabetes, 23% with type 2 diabetes and 45% with unclassifiable diabetes. After diagnosis, median fpC-peptide concentrations were markedly lower in patients with islet antibodies than in those without (0.24 vs 0.69 nmol/l, p<0.0001). Irrespective of clinical classification, patients with islet antibodies showed increased frequencies of at least one of the risk-associated HLA-DQB1 genotypes compared with patients without. Antibody-negative patients with risk-associated HLA-DQB1 genotypes had significantly lower median fpC-peptide concentrations than those without risk-associated genotypes (0.51 vs 0.74 nmol/l, p=0.0003). Conclusions/ interpretation: Assessment of islet antibodies is necessary for the aetiological classification of diabetic patients. HLA-DQB1 genotyping does not improve the classification in patients with islet antibodies. However, in patients without islet antibodies, HLA-DQB1 genotyping together with C-peptide measurement may be of value in differentiating between idiopathic type 1 diabetes and type 2 diabetes. © Springer-Verlag 2006.

Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS-PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls (P<0.05). Conversely, auranofin failed to protect cells from the toxicity of pre-reduced ricin which does not require intracellular reduction of disulfide between the two ricin subunits. We conclude that TrxR, by activating disulfide reductase activity of PDI, can ultimately lead to reduction/activation of ricin and immunotoxin in the cell.

Proliferation of fibroblasts is vital for adequate wound healing but is probably also involved in different hyperproliferative disorders such as atherosclerosis and cancer. The regeneration of tissue usually starts with coagulation, involving release of mitogenic and inflammatory factors from activated platelets. This study focuses on the role of eicosanoids in the proliferative effects of platelets on human fibroblasts. We show that the phospholipase A2 inhibitor 7,7-dimethyl-5,8-eicosadienoic acid (DMDA), the combined cyclooxygenase (COX) and lipoxygenase (LOX) inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) and the LOX inhibitor 5,8,11-eicosatriynoic acid (ETI) block the platelet-induced proliferation of serum starved subconfluent human fibroblasts. Anti-proliferative effects were also obtained by specific inhibition of 5-LOX with 5,6-dehydro arachidonic acid (5,6-dAA), whereas the 12-LOX inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) did not affect the platelet-stimulated growth of fibroblasts. The expression of 5-LOX was analyzed by reverse-transcriptase-mediated PCR (RT-PCR), Western blotting and HPLC. 5-LOX message and protein was detected in fibroblasts but not in platelets. Incubation with platelets markedly increased, already after one hour, the expression of 5-LOX in the fibroblast culture. The increased 5-LOX activity was associated with an elevated level of the 5-LOX metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) reaching its maximum after 1-2 hours of co-incubation of fibroblasts and platelets. The 5-HETE production was reduced by the inhibitors DMDA, ETYA and ETI. In conclusion, this study suggests that platelet-stimulated proliferation of fibroblasts is mediated by an increased 5-LOX activity, which supports recent findings indicating a crucial role for this enzyme in proliferative disorders such as atherosclerosis. © 2006 Schattauer GmbH, Stuttgart.

A strategic selection of instruction and assessment methods geared to higher cognition levels was used to foster increasingly complex learning. Assessment results from the redesigned course show that students are capable of mastering higher-order cognitive skills relative to these objectives, however the synthesis category constitutes a threshold in students' cognitive development. No single assessment method was found to be clearly the best, yet careful selection of the method that allows higher-order thinking made it possible to develop a combination of assessments that meet objectives.

The study of protein glycosylation and its significance in biological interactions is a field of growing interest. This work demonstrates a lectin-based separation of protein glycoforms of α1-acid glycoprotein (AGP or orosomucoid) with capillary electrophoresis. Glycoform analysis was performed with a "partial filling technique" with the lectin Concanavalin A (Con A) as affinity ligand. Con A separated human AGP into two peaks, the first peak included AGP glycoforms without biantennary glycans, and the second peak represented the fraction that had one or more biantennary glycans. The applicability of the method was demonstrated with the analysis of AGP from clinical samples and AGP treated with N-glycosidase F. The AGP separation was also used as a reporter system to estimate the dissociation constant (KD) between Con A and a competing sugar. © 2004 Elsevier B.V. All rights reserved.

In this paper we present an in situ evaluation of a haptic system, with a representative test population, we aim to determine what, if any, benefit haptics can have in a biomolecular education context. We have developed a haptic application for conveying concepts of molecular interactions, specifically in protein-ligand docking. Utilizing a semi-immersive environment with stereo graphics, users are able to manipulate the ligand and feel its interactions in the docking process. The evaluation used cognitive knowledge tests and interviews focused on learning gains. Compared with using time efficiency as the single quality measure this gives a better indication of a system's applicability in an educational environment. Surveys were used to gather opinions and suggestions for improvements. Students do gain from using the application in the learning process but the learning appears to be independent of the addition of haptic feedback. However the addition of force feedback did decrease time requirements and improved the students understanding of the docking process in terms of the forces involved, as is apparent from the students' descriptions of the experience. The students also indicated a number of features which could be improved in future development.

Within the molecular life sciences extensive use is made of visual representations, ranging from sketches to advanced computer graphics, often used to convey abstract knowledge that is difficult for the student to grasp. This work evaluates a new visual and haptic (tactile/kinetic) tool for protein docking in an in situ learning situation by combining qualitative and quantitative methods, performing tests and interviews with students; all aiming at a proper inclusion of visualization tools into biomolecular education. Preliminary results indicate time gains, strong positive affective responses and learning gains from the tasks, however the influence of haptics needs further investigation.

Objective: To assess the usefulness of measuring plasma cortisol profiles in growth hormone-treated hypopituitary adults and to compare these with cortisol levels in healthy controls. Methods: Eleven ACTH-deficient adult patients received 12.5 mg cortisone-acetate orally at 16.00 h and 25 mg at 07.00 h. The patients arrived in the ward at 12.00 h. After tablet intake at 16.00 h, samples for serum cortisol were taken at hourly intervals for the next 24 h, except between 07.00 and 12.00 h when samples were drawn every half hour, 24-h urinary free cortisol (24-h-UFC) excretion was collected simultaneously. For comparison, 8 healthy controls were investigated. Results: The patients had circulating cortisol levels with very low plasma cortisol at 07.00 h before their morning dose of cortisone acetate. At the same time period, controls had their highest plasma cortisol levels. After tablet intake the patients had a rapid initial absorption of cortisol, but a marked variability in the morning peak levels (Cmax), and the Cmax was in general higher and occurred 90 min later than the Cmax in the controls. The 24-h-UFC excretion and 24-h area under the curve (24-h-AUC) did not differ between patients and controls. The female patients had higher 24-h-AUC for plasma cortisol (p=0.032) and tended to have higher plasma cortisol peaks in the morning, but had levels of 24-h-UFC similar to those of the male patients. Conclusions: Conventional cortisone substitution with a twice-daily replacement regimen in hypopituitary adults results in abnormal circulating cortisol profiles with low or non-measurable morning values and variable individual peaks. This suggests that the present dosing schemes have to be improved and that cortisone substitution should be individualized.

Objective. To investigate whether measurements of proinsulin and/or intermediate proinsulin degradation products could be used to differentiate between autoimmune (type 1) and non-autoimmune (type 2) diabetes in young adults. Material and methods. Total proinsulin, intact proinsulin and 32,33 split proinsulin concentrations were measured in 25 patients aged 15-34 years with type 1 diabetes, as defined by the presence of at least two positive islet autoantibodies, and in 23 antibody-negative patients of similar age with type 2 diabetes, at the time of clinical onset of diabetes and at 3-4 months thereafter. Comparisons were made with data from 25 healthy subjects matched for gender and age. Results. Plasma levels of total proinsulin, intact proinsulin and 32,33 split proinsulin were significantly increased 2-3-fold in the patients with newly diagnosed type 2 diabetes as compared with the controls, both in absolute terms (p<0.0001) and when related to circulating insulin (p<0.01-0.0002). In contrast, absolute proinsulin and 32,33 split proinsulin concentrations were significantly lower in patients with onset of type 1 diabetes than in controls. When proinsulin and split proinsulin release were related to plasma insulin, however, similar ratios were found in the type 1 diabetes patients and in controls. Using the 90th percentile for total proinsulin in the control group as the cut-off, the sensitivity and specificity for differentiation between autoimmune and non-autoimmune diabetes were 87% and 92%, respectively. At 3-4 months after clinical onset of diabetes, proinsulin secretion was still 2-3 times higher in type 2 than in type 1 diabetes patients (p<0.001). Conclusions. Young adult patients with newly diagnosed type 2 diabetes display disproportionate hyperproinsulinemia, whereas proinsulin secretion appears to be normal in patients with clinical onset of type 1 diabetes. Evaluation of proinsulin and 32,33 split proinsulin concentrations may be useful as a diagnostic tool in differentiating between autoimmune and non-autoimmune diabetes in young adults, particularly in those lacking islet autoantibodies at diagnosis. © 2005 Taylor & Francis.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarizations that cause an inward movement of the positive charges in the fourth transmembrane domain (S4), which triggers channel opening. The mechanism of how the motion of S4 charges triggers channel opening is unknown. Here, we used voltage clamp fluorometry (VCF) to detect S4 conformational changes and to correlate these to the different activation steps in spHCN channels. We show that S4 undergoes two distinct conformational changes during voltage activation. Analysis of the fluorescence signals suggests that the N-terminal region of S4 undergoes conformational changes during a previously characterized mode shift in HCN channel voltage dependence, while a more C-terminal region undergoes an additional conformational change during gating charge movements. We fit our fluorescence and ionic current data to a previously proposed 10-state allosteric model for HCN channels. Our results are not compatible with a fast S4 motion and rate-limiting channel opening. Instead, our data and modeling suggest that spHCN channels open after only two S4s have moved and that S4 motion is rate limiting during voltage activation of spHCN channels. © The Rockefeller University Press.

Background and Objectives: The redox-regulatory protein thioredoxin has several functions including transcriptional regulation, and antioxidant, cytokine, and chemokine activities. We have previously shown that extracellular thioredoxin protects B-cell chronic lymphocytic leukemia (CLL) cells from apoptosis in vitro. In this study we were interested to determine whether thioredoxin is produced by cells surrounding the CLL cells in the in vivo microenvironment and whether this cell-derived thioredoxin has any leukemia growth-promoting effect in vitro. Design and Methods: Lymph nodes from CLL patients (n=25) were analyzed for thioredoxin expression by immunohistology. Stromal cells purified from the lymph nodes were analyzed for thioredoxin secretion at the single cell level using an ELIspot assay. The survival effect of the stromal-derived thioredoxin was tested by co-culturing stromal- and CLL cells with and without Fab-fragments of an anti-thioredoxin antibody. Results: The results indicated that the thioredoxin production correlated with the amount of proliferating cells and was mainly localized to the proliferation centers (pseudofollicles) in the CLL lymph nodes. The leukemia cells per se showed minimal thioredoxin levels, in contrast, stromal cells strongly expressed thioredoxin. Purified primary stromal cells, which secreted extracellular thioredoxin, significantly protected the CLL cells from undergoing apoptosis in 72 h co-cultures. Interestingly, this anti-apoptotic effect could be abrogated by addition of Fab-fragments of an anti- thioredoxin antibody. Interpretation and Conclusions: In conclusion, we have shown that stromal cells in the lymph node microenvironment produce thioredoxin and that the thioredoxin production is localized to the proliferation centers of the CLL lymph nodes. In addition, thioredoxin produced by purified stromal cells rescued CLL cells from apoptosis in vitro. ©2007 Ferrata Storti Foundation.

Background: Iatrogenic nerve injury caused by heat from dissection instruments is a significant problem in many areas of surgery. The aim of the present study was to compare the risk of nerve injury for three different dissection instruments: monopolar and bipolar electrosurgery (ES) and an ultrasonically activated (US) instrument. Methods: The biceps femoris muscle was cut in a standard manner just adjacent to the sciatic nerve using monopolar ES, bipolar ES or US shears. A total of 73 functional experiments were conducted in which the nerve was isolated, divided proximally, and stimulated supramaximally in 37 anaesthetized rats. The electromyographic (EMG) potential was recorded distally before and after each experiment. Nerve dysfunction was defined as more than 10 per cent loss of the evoked EMG potential. Fifty-nine nerves were examined histologically after dissection with the different instruments. The extent of heat damage was determined in four nerves that were divided with ES bipolar scissors and five that were divided with US shears. Results: Reduction in the EMG potential was significantly more frequent in the monopolar ES group than in the US group. Morphological examination also showed significantly less nerve damage in the US group. Conclusion: US instruments may be safer than ES for dissection close to nerves. Copyright © 2005 British Journal of Surgery Society Ltd.

Hematopoietic differentiation is a complex process involving many genes inducing functional changes and characteristics of different cell lineages. To understand this process, it is important to identify genes involved in lineage commitment and maturation of hematopoietic progenitor cells. Recently we isolated the novel gene MYADM which is strongly up-regulated as multipotent progenitor cells differentiate towards myeloid cells. Because it is not expressed in lymphocytes, understanding the transcriptional control of MYADM could further explain differences in gene expression between myeloid and lymphoid cells. To identify regulatory elements controlling its restricted expression, we have analyzed the 5′-flanking region of the MYADM gene. The proximal promoter was found to lack both TATA and CCAAT boxes, but contained several potential binding sites for both ubiquitous and myeloid-specific transcription factors. Maximal promoter activity was contained within 800∈bp from the tentative transcription initiation site, which was reduced as portions of the 5′-end were deleted, and completely abolished when the transcription initiation site was deleted. This promoter sequence had higher activity in myeloid cells compared to B cells, and activity was enhanced during myeloid differentiation, suggesting that we have identified the MYADM core promoter. Computer predictions had suggested MYADM to encode a protein with multiple transmembrane domains. By immunofluorescence and confocal microscopy we demonstrate that the protein is localized to the nuclear envelope and to intracytoplasmic membranes, indicating that MYADM constitutes an integral membrane protein. © Springer 2005.

Objectives: To investigate associations of Parkinson's disease (PD) and parkinsonian syndromes with polymorphic genes that influence metabolism of either foreign chemical substances or dopamine and to seek evidence of gene-environment interaction effects that modify risk. Methods: A case-control study of 959 prevalent cases of parkinsonism (767 with PD) and 1989 controls across five European centres. Occupational hygienists estimated the average annual intensity of exposure to solvents, pesticides and metals, (iron, copper, manganese), blind to disease status. CYP2D6, PON1, GSTM1, GSTT1, GSTM3, GSTP1, NQO1, CYP1B1, MAO-A, MAO-B, SOD 2, EPHX, DATl, DRD2 and NAT2 were genotyped. Results were analysed using multiple logistic regression adjusting for key confounders. Results: There was a modest but significant association between MAO-A polymorphism in males and disease risk (G vs T, OR 1.30, 95% C1 1.02 to 1.66, adjusted). The majority of gene-environment analyses did not show significant interaction effects. There were possible interaction effects between GSTM1 null genotype and solvent exposure (which were stronger when limited to PD cases only). Conclusions: Many small studies have reported associations between genetic polymorphisms and PD. Fewer have examined gene-environment interactions. This large study was sufficiently powered to examine these aspects. GSTM1 null subjects heavily exposed to solvents appear to be at increased risk of PD. There was insufficient evidence that the other gene-environment combinations investigated modified disease risk, suggesting they contribute little to the burden of PD.

Objective: To investigate the associations between Parkinson's disease and other degenerative parkinsonian syndromes and environmental factors in five European countries. Methods: A case-control study of 959 prevalent cases of parkinsonism (767 with Parkinson's disease) and 1989 controls in Scotland, Italy, Sweden, Romania and Malta was carried out. Cases were defined using the United Kingdom Parkinson's Disease Society Brain Bank criteria, and those with drug-induced or vascular parkinsonism or dementia were excluded. Subjects completed an interviewer-administered questionnaire about lifetime occupational and hobby exposure to solvents, pesticides, iron, copper and manganese. Lifetime and average annual exposures were estimated blind to disease status using a job-exposure matrix modified by subjective exposure modelling. Results were analysed using multiple logistic regression, adjusting for age, sex, country, tobacco use, ever knocked unconscious and family history of Parkinson's disease. Results: Adjusted logistic regression analyses showed significantly increased odds ratios for Parkinson's disease/parkinsonism with an exposure-response relationship for pesticides (low vs no exposure, odds ratio (OR) =1.13, 95% Cl 0.82 to 1.57, high vs no exposure, OR =1.41, 95% Cl 1.06 to 1.88) and ever knocked unconscious (once vs never, OR= 1.35, 95% Cl 1.09 to 1.68, more than once vs never, OR = 2.53, 95% Cl 1.78 to 3.59). Hypnotic, anxiolytic or antidepressant drug use for more than 1 year and a family history of Parkinson's disease showed significantly increased odds ratios. Tobacco use was protective (OR = 0.50, 95% Cl 0.42 to 0.60). Analyses confined to subjects with Parkinson's disease gave similar results. Conclusions: The association of pesticide exposure with Parkinson's disease suggests a causative role. Repeated traumatic loss of consciousness is associated with increased risk.

The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates. © 2007 American Chemical Society.

OBJECTIVE - C-peptide replacement in animals results in amelioration of diabetes-induced functional and structural abnormalities in peripheral nerves. The present study was undertaken to examine whether C-peptide administration to patients with type 1 diabetes and peripheral neuropathy improves sensory nerve function. RESEARCH DESIGN AND METHODS - This was an exploratory, double-blinded, randomized, and placebo-controlled study with three study groups that was carried out at five centers in Sweden. C-peptide was given as a replacement dose (1.5 mg/day, divided into four subcutaneous doses) or a dose three times higher (4.5 mg/day) during 6 months. Neurological examination and neurophysiological measurements were performed before and after 6 months of treatment with C-peptide or placebo. RESULTS - The age of the 139 patients who completed the protocol was 44.2 ± 0.6 (mean ± SE) years and their duration of diabetes was 30.6 ± 0.8 years. Clinical neurological impairment (NIA) (score >7 points) of the lower extremities was present in 86% of the patients at baseline. Sensory nerve conduction velocity (SCV) was 2.6 ± 0.08 SD below body height-corrected normal values at baseline and improved similarly within the two C-peptide groups (P < 0.007). The number of patients responding with a SCV peak potential improvement >1.0 m/s was greater in C-peptide-treated patients than in those receiving placebo (P < 0.03). In the least severely affected patients (SCV < 2.5 SD below normal at baseline, n = 70) SCV improved by 1.0 m/s (P < 0.014 vs. placebo). NIA score and vibration perception both improved within the C-peptide-treated groups (P < 0.011 and P < 0.002). A1C levels (7.6 ± 0.1% at baseline) decreased slightly but similarly in C-peptide- and placebo-treated patients during the study. CONCLUSIONS - C-peptide treatment for 6 months improves sensory nerve function in early-stage type 1 diabetic neuropathy. © 2007 by the American Diabetes Association.

Mast cells play critical roles in the regulation of inflammation. One characteristic feature of mast cells is their relatively long lifespan in vivo. Members of the Bcl-2 protein family are regulators of cell survival and apoptosis, where the BH3-only proteins are critical proapoptotic proteins. In this study we investigated the role of the BH3-only proteins Noxa, Bad, Bim, Bmf, Bid, and Puma in apoptosis of mucosal-like mast cells (MLMCs) and connective tissue-like mast cells (CTLMCs). We demonstrate that Puma is critical for the induction of mast-cell death following cytokine deprivation and treatment with the DNA-damaging agent etoposide in MLMCs and CTLMCs. Using p53-/- mast cells, we found that cytokine deprivation-induced apoptosis, in contrast to that elicited by etoposide, is p53-independent. Interestingly, mast cells deficient in FOXO3a, previously proposed as a transcription factor for Puma induction in response to growth factor deprivation, were markedly resistant to cytokine withdrawal compared with wildtype cells. Moreover, overexpression of phosphorylation-deficient, constitutively active FOXO3a caused an up-regulation of Puma. In conclusion, our data demonstrate a pivotal role for Puma in the regulation of cytokine deprivation-induced mast-cell apoptosis and suggest a plausible role for Puma in the regulation of mast cell numbers in vivo. © 2007 by The American Society of Hematology.

Recent work demonstrated that the febrile response to peripheral immune stimulation with proinflammatory cytokine IL-1β or bacterial wall lipopolysaccharide (LPS) is mediated by induced synthesis of prostaglandin E2 by the terminal enzyme microsomal prostaglandin E synthase-1 (mPGES-1). The present study examined whether a similar mechanism might also mediate the anorexia induced by these inflammatory agents. Transgenic mice with a deletion of the Ptges gene, which encodes mPGES-1, and wild-type controls were injected intraperitoneally with IL-1β, LPS, or saline. Mice were free fed, and food intake was continuously monitored with an automated system for 12 h. Body weight was recorded every 24 h for 4 days. The IL-1β induced anorexia in wild-type but not knock-out mice, and so it was almost completely dependent on mPGES-1. In contrast, LPS induced anorexia of the same magnitude in both phenotypes, and hence it was independent of mPGES-1. However, when the mice were prestarved for 22 h, LPS induced anorexia and concomitant body weight loss in the knock-out animals that was attenuated compared with the wildtype controls. These data suggest that IL-1β and LPS induce anorexia by distinct immune-to-brain signaling pathways and that the anorexia induced by LPS is mediated by a mechanism different from the fever induced by LPS. However, nutritional state and/or motivational factors also seem to influence the pathways for immune signaling to the brain. Furthermore, both IL-1β and LPS caused reduced meal size but not meal frequency, suggesting that both agents exerted an anhedonic effect during these experimental conditions. Copyright © 2007 the American Physiological Society.

Background: Matrix metalloproteinases (MMPs) are a group of matrix-degrading proteins implicated in several pathological processes, e.g., invasion and metastasis in malignant diseases such as colorectal cancer (CRC). Materials and Methods: One hundred and twenty-seven CRC patients and 208 controls were genotyped for MMP-1, -2, -3 and -9 promoter polymorphisms. The genotyping was performed with PCR/primer-extension/DHPLC or PCR/RFLP. Results: The MMP-1 2G allele was significantly associated with CRC (p=0.037). No significant association between CRC and MMP-2, -3 or -9 polymorphisms was evident. The analysis of polymorphisms in the clinicopathological subgroups displayed no significant associations. Conclusion: The MMP-1 promoter polymorphism seems to affect the susceptibility to CRC, while MMP-2, -3 and -9 polymorphisms appear less likely to have any impact on CRC.

[No abstract available]

Hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channels regulate pacemaker activity in the heart and the brain. Previously, we showed that spHCN and HCN1 channels undergo mode shifts in their voltage dependences, shifting the conductance versus voltage curves by more than +50 mV when measured from a hyperpolarized potential compared to a depolarized potential. In addition, the kinetics of the ionic currents changed in parallel to these voltage shifts. In the studies reported here, we tested whether slower cardiac HCN channels also display similar mode shifts. We found that HCN2 and HCN4 channels expressed in oocytes from the frog Xenopus laevis do not display the activation kinetic changes that we observed in spHCN and HCN1. However, HCN2 and HCN4 channels display changes in their tail currents, suggesting that these channels also undergo mode shifts and that the conformational changes underlying the mode shifts are due to conserved aspects of HCN channels. With computer modelling, we show that in channels with relatively slow opening kinetics and fast mode-shift transitions, such as HCN2 and HCN4 channels, the mode shift effects are not readily observable, except in the tail kinetics. Computer simulations of sino-atrial node action potentials suggest that the HCN2 channel, together with the HCN1 channel, are important regulators of the heart firing frequency and that the mode shift is an important property to prevent arrhythmic firing. We conclude that although all HCN channels appear to undergo mode shifts -and thus may serve to prevent arrhythmic firing -it is mainly observable in ionic currents from HCN channels with faster kinetics. 2006 The Authors. Journal compilation © 2006 The Physiological Society.

Major histocompatibility complex (MHC) class I presentation of exogenous antigens is the mechanism enabling professional antigen-presenting cells (APCs) to induce CD8⁺ T-cell responses against viruses and tumors that do not have access to the classical MHC class I pathway. We have characterized the uptake, processing, and MHC class I cross-presentation by human dendritic cells (DCs) of cell-associated antigens derived from physiologically relevant sources, namely, vaccinia virus-infected apoptotic and necrotic cells. We show that cross-presentation is a rapid process, detectable within 2 to 4 hours after uptake of dead cells, and that proteolysis by cathepsin D in an acidic endosomal compartment is essential for cross-presentation. The presentation is abolished when the phagocytic or macropinocytic functions of the cells are inhibited and is dependent on transporter associated with antigen processing, sensitive to brefeldin A, and requires functional proteasomes. Altogether, these data suggest that antigens derived from apoptotic and necrotic cells require access to the cytosol to intersect with the conventional MHC class I pathway for presentation of cytosolic proteins.

Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27%). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P = 0.815), nor between Dukes' classes A/B and C/D (P = 0.262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens. © 2005 SGM.

[No abstract available]

LMO2, a member of the LIM-only protein family, is essential for the regulation of hematopoietic stem cells and formation of erythroid cells. It is found in a transcriptional complex comprising LMO2, TAL1, E47, GATA-1, and LDB1 which regulates erythroid genes. While TAL1 has been shown to induce erythroid differentiation, LMO2 appears to suppress fetal erythropoiesis. In addition to LMO2, the closely related LMO4 gene is expressed in hematopoietic cells, but has unknown functions. Here we demonstrate that LMO2 and LMO4 are expressed at the same level in erythroid colonies from mouse bone marrow, implying a function in erythroid differentiation. However, while LMO2 induced erythroid differentiation, LMO4 had no such effect. Interestingly, both LMO2 and TAL1 were able to partially suppress myeloid differentiation, implying that they activate erythroid differentiation in uncommitted bone marrow progenitors. Both LMO2 and LMO4 interacted strongly to LDB1, which was required for their localization to the nucleus. © 2007 Elsevier Inc. All rights reserved.

This book examines the role of neurons in multiple sclerosis (MS) and the changes that occur in neurons as a result of MS. It places MS in a new and important perspective that not only explains the basis for symptom production, remission, and progress in MS, but also promises to open up new therapeutic possibilities. * Brings together the latest information from clinical, pathological, imaging, molecular, and pharmacological realms to explore the neurobiology of Multiple Sclerosis* Places MS in a new and important perspective that promises to open up new therapeutic avenues* Superbly illustrated and referenced

Background: Firm recommendations about the way thiopurine drugs are introduced and the use of thiopurine methyltransferase (TPMT) and metabolite measurements during treatment in inflammatory bowel disease (IBD) are lacking. Aim: To evaluate pharmacokinetics and tolerance after initiation of thiopurine treatment with a fixed dosing schedule in patients with IBD. Patients: 60 consecutive patients with Crohn's disease (n = 33) or ulcerative colitis (n = 27) were included in a 20 week open, prospective study. Methods: Thiopurine treatment was introduced using a predefined dose escalation schedule, reaching a daily target dose at week 3 of 2.5 mg azathioprine or 1.25 mg 6-mercaptopurine per kg body weight. TPMT and ITPA genotypes, TPMT activity, TPMT gene expression, and thiopurine metabolites were determined. Clinical outcome and occurrence of adverse events were monitored. Results: 27 patients completed the study per protocol, while 33 were withdrawn (early protocol violation (n = 5), TPMT deficiency (n = 1), thiopurine related adverse events (n = 27)), 67% of patients with adverse events tolerated long term treatment on a lower dose (median 1.32 mg azathioprine/kg body weight). TPMT activity did not change during the 20 week course of the study but a significant decrease in TPMT gene expression was found (TPMT/huCYC ratio, p = 0.02). Patients with meTIMP concentrations > 11 450 pmol/8 × 108 red blood cells during steady state at week 5 had an increased risk of developing myelotoxicity (odds ratio = 45.0, p = 0.015). Conclusions: After initiation of thiopurine treatment using a fixed dosing schedule, no general induction of TPMT enzyme activity occurred, though TPMT gene expression decreased. The development of different types of toxicity was unpredictable, but we found that measurement of meTIMP early in the steady state phase helped to identify patients at risk of developing myelotoxicity.

Islet amyloid deposition is a pathogenic feature of type 2 diabetes, and these deposits contain the unique amyloidogenic peptide islet amyloid polypeptide. Autopsy studies in humans have demonstrated that islet amyloid is associated with loss of β-cell mass, but a direct role for amyloid in the pathogenesis of type 2 diabetes cannot be inferred from such studies. Animal studies in both spontaneous and transgenic models of islet amyloid formation have shown that amyloid forms in islets before fasting hyperglycemia and therefore does not arise merely as a result of the diabetic state. Furthermore, the extent of amyloid deposition is associated with both loss of β-cell mass and impairment in insulin secretion and glucose metabolism, suggesting a causative role for islet amyloid in the islet lesion of type 2 diabetes. These animal studies have also shown that β-cell dysfunction seems to be an important prerequisite for islet amyloid formation, with increased secretory demand from obesity and/or insulin resistance acting to further increase islet amyloid deposition. Recent in vitro studies suggest that the cytotoxic species responsible for islet amyloid-induced β-cell death are formed during the very early stages of islet amyloid formation, when islet amyloid polypeptide aggregation commences. Interventions to prevent islet amyloid formation are emerging, with peptide and small molecule inhibitors being developed. These agents could thus lead to a preservation of β-cell mass and amelioration of the islet lesion in type 2 diabetes.

Background: The aim of this study was to ascertain whether dysglycemic episodes occur in institutionalized elderly persons and, if that is the case, to determine whether such episodes are related to meal patterns. Another objective was to investigate the feasibility of subcutaneous (s.c.) glucose measurements in the elderly using a Medtronic MiniMed (Sylmar, CA) continuous glucose monitoring system (CGMS®). Methods: Nine nursing home residents (74-95 years old) without known diabetes or other metabolic disorders were included. The s.c. glucose level was measured for 3 days with the Medtronic MiniMed CGMS. Capillary blood glucose was measured four times daily with a Glucometer Elite® device (Bayer, Leverkusen, Germany). Body mass index and basal metabolic rate were calculated, and food intake was recorded. Results: The s.c. glucose level fluctuated noticeably over time, 22.5% of the values recorded during the 3-day period were ≥8 mmol/L, and values <3.5 mmol/L were rarely seen. A marked (>5 mmol/L) and short-term (2-4 h) increase in s.c. glucose was seen after a meal. The mean capillary blood glucose concentration was 7.5 ± 1.8 mmol/L. Capillary blood glucose ≥8 mmol/L was recorded on 32.5% of the measurement occasions, and no values were <3.5 mmol/L. The s.c. glucose values agreed with corresponding capillary blood glucose levels (mean r = 0.75, range 0.43-0.86). Five participants consumed less energy than recommended according to their age, weight, and physical activity level. Conclusions: Postprandial hyperglycemia frequently occurs in elderly people living in nursing homes. The CGMS is convenient to use to detect hyperglycemia in this age group.

The detoxification enzyme sulfotransferase1A1 (SULT1A1) is implicated in the inactivation of estrogens and the activation of promutagens andprocarcinogens. SULT1A1 activity varies among individuals, and this difference in phenotype is, in part, controlled by genetic polymorphism (Arg→His in codon 213). It is hypothesized that the His allele contributes to the risk of postmenopausal breast cancer. Frequencies of the Arg/His alleles were estimated in 229 postmenopausal breast cancer patients and 227 age-matched controls using a PCR-RFLP assay. Allele frequencies and genotype distributions were not statistically different between postmenopausal breast cancer patients and the population-based controls, i.e. neither of the alleles is associated with an increased risk of breast cancer in the present study.

Although visceral innocuous cold receptors have been documented, the central termination of their afferents is unknown. We used menthol solution (0. 6 m M) to obtain selective activation of cold receptors in the urinary bladder of rats. Innocuous cold stimulation induced Fos expression in a population of neurons in the superficial dorsal horn of L6-SI segments of the spinal cord. Neurons in other regions of the spinal cord, e.g. the lumbar parasympathetic nucleus or the dorsal commissure region, were activated to a similar degree by menthol and control infusions, indicating a response to bladder filling. Our results are consistent with the proposal that subsets of modality-specific dorsal horn neurons convey specific information regarding the exteroceptive and interoceptive state of the animal. © 2004 Lippincott Williams & Wilkins.

[No abstract available]

Proliferation, differentiation, and survival of hematopoietic cells are regulated by cytokines, acting through specific receptors. FLT3 ligand (FL) is one of the most important cytokines for regulation of the hematopoietic system, and its receptor FLT3 is expressed on both stem cells and progenitors. Regulation of Forkhead transcription factors has been described as an important mechanism to control apoptosis and cell cycle progression in hematopoietic progenitors. Here we report that FL induces AKT/PKB activation, which in turn phosphorylates and thereby inactivates the Forkhead protein FoxO3 in the progenitor cell line FDC-P1 stably expressing murine FLT3 receptor. Phosphorylation of AKT and FoxO3 was blocked by the PI-3 kinase inhibitor LY294002 but not by the MAP kinase inhibitor PD98059. Expression of a mutated FoxO3, in which all three inhibitory phosphorylation sites were mutated to alanine, led to rapid increase of apoptotic cells in the presence of FL. These results suggest that FL-induced regulation of apoptosis is executed by FoxO3.

By using in situ hybridization histochemistry the distribution of growth hormone (GH) receptor mRNA was examined in the rat brain stem and spinal cord. Dense labeling was seen in the arcuate nucleus of the hypothalamus, as reported previously, but also in several other areas, including the locus coeruleus, the area postrema, and the commissural part of the nucleus of the solitary tract. Other labeled structures included the superior lateral parabrachial nucleus, the facial, hypoglossal and trigeminal motor nuclei, the nucleus incertus, the dorsal tegmental nucleus, the dorsal raphe nucleus, the nucleus of the trapezoid body, and the superficial layers of the dorsal horn of the spinal cord. These findings provide support for a direct action of GH on brain regions involved in various aspects of homeostatic control. Thus, the distribution of GH receptor mRNA to visceral sensory and motor structures is consonant with a role of GH in the regulation of food intake and energy homeostasis. Its presence in the superficial dorsal horn of the spinal cord indicates a role for GH in the initial processing of fine afferent input, and may help explain the beneficial effects of GH replacement in certain unclear pain conditions. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

Objective. Capsaicin, which acts by binding to the vanilloid receptor-1 (VR1), has been shown to give protection against gastric mucosal injury and to enhance healing of gastric ulcers. Although VR1 has recently been reported to be present in non-neural tissues, it is primarily considered to be expressed in nociceptor sensory neurons of small diameter. The aim of the present study was to evaluate the distribution of VR1 immunoreactivity in the normal human gastric mucosa. Material and methods. Ten volunteers underwent gastroscopy and biopsies were obtained from the corpus and the antrum. The specimens were labelled immunohistochemically using polyclonal goat anti-VR1 and evaluated at the light- and electronmicroscopic level. Moreover, post-embedding immunogold labelling was performed and subsequently analysed at the electronmicroscopic level. Results. In the antrum, VR1 immunoreactivity was located in epithelial cells that fulfilled the criteria of endocrine cells of the "open type". These cells were located primarily in the neck region of the antral glands and the labelling was concentrated on the microvilli of these cells. At the ultrastructural level, round granulae with differences in electron density were identified in the basal compartment of the labelled cells. VR1 immunoreactivity was also identified in axon-like structures that were located in the lamina propria, often in close vicinity of vessels, in the corpus as well as in the antrum. Conclusions. VR1-immunoreactivity was evident in antral epithelial cells exhibiting characteristics of endocrine-like cells. This may indicate that the gastroprotective effects of capsaicin, which hitherto have been attributed to primary afferent neurons, at least partly may be explained by an action on specific epithelial cells in the antrum. © 2005 Taylor & Francis.