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The aim of this study was to assess post-trial treatment costs, clinical outcomes [decayed, missing or filled surfaces (DMFS) scores], and utilization of dental services among adolescents who had participated in a randomized clinical trial (RCT) in Pori, Finland, in 2001-2005. At baseline the children were 11-12 yr of age and had had at least one active initial caries lesion. The children in the experimental group (n = 250) had been exposed to multiple measures for caries control, while those in the control group (n = 247) had received standard dental care. During the post-trial period (2005-2008), all participants received the standard dental care offered in public dental clinics in Pori. In both groups the costs of treatment procedures and outcomes for the post-trial period were calculated for each adolescent. Information from patient records was available for 487 adolescents (former experimental n = 246, control n = 241). The mean total costs per adolescent were lower and the clinical outcome was better among the former experimental-group participants. The differences in mean costs between the groups were statistically significant for preventive and restorative procedures. The utilization of dental services was significantly more regular among the former experimental-group participants.

Objective: To assess the readiness of the Western Region Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) states to implement participant-centered nutrition education (PCE) and to develop a PCE model for WIC service delivery., Design: Formative research including on-line survey, qualitative in-depth interviews, focus groups, and observational assessments., Setting: WIC clinics within the Western Region WIC states., Participants: State and local staff and WIC clients within 8 states, 2 tribal organizations, and 2 territories., Phenomenon of Interest: Readiness indicators of states to implement and expand PCE elements to include in PCE model development., Analysis: On-line surveys were collected and analyzed. On-site assessment forms, interviews, and focus group findings were collected, coded, and summarized by themes., Results: Key themes from state and local findings guided the model development for PCE implementation in the Western Region WIC states. The PCE model must be flexible and systems oriented, contain strong training and mentoring components, and integrate cultural sensitivity to best reach program participants., Conclusions and Implications: The PCE model has the potential to improve WIC nutrition services and enable participants to make positive health-related behavior changes that will influence long-term health issues. Further outcome studies are needed to determine the success of PCE implementation in the Western Region WIC states., (Copyright 2010 Society for Nutrition Education. Published by Elsevier Inc. All rights reserved.)

Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C-O dimer were observed, whereas a C-C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.

Both household food insecurity and childhood overweight are serious public health problems that appear to be paradoxically correlated. This study examines the relationship between overweight and household food insecurity with/without hunger in low-income children participating in the Special Supplemental Nutrition Program for Women, Infants, and Children. Weight, height, and household food insecurity data were collected on 8,493 children ages 1 month to 5 years and analyzed by sex and age groups using logistic regression to model the odds of being overweight (weight for length or body mass index [calculated as kg/m(2)] for age > or =95th percentile) given household food insecurity status, controlling for race/ethnicity and maternal education. Analyses were stratified by age and sex because interaction terms with household food insecurity were significant (P<0.10). In this sample, prevalence of household food insecurity was 30.7% (8.3% with hunger) and 18.4% were overweight. Among girls younger than 2 years of age, household food insecurity was associated with reduced odds of overweight compared with food-secure households (odds ratio=0.65; 95% confidence interval: 0.47 to 0.88); hunger status did not alter this association. Among 2- to 5-year-old girls, there was no overall significant association between household food insecurity and overweight; however, household food insecurity with hunger was positively associated with overweight compared with those from food-secure households (odds ratio=1.49; 95% confidence interval: 1.06 to 2.10). No association between household food insecurity and overweight was found among boys. These findings suggest an association between household food insecurity and overweight prevalence in this low-income population. However, sex and age appear to modify both the magnitude and direction of the association.

Neuropeptide Y (NPY) is a universally expressed neuropeptide involved in the regulation of several physiological functions. The rather common leucine7 to proline7 (L7P) polymorphism in the signal peptide of preproNPY is a functional substitution, which changes the processing and release of NPY in cells. The mutation is associated with altered lipid levels and accelerated atherosclerosis in humans. Based on previous studies, we investigated the effect of the Pro7 allele in endothelial cells, which are known to play a role in the development of atherosclerosis. Cell proliferation and apoptosis were studied in primary cultured, genotyped human umbilical vein endothelial cells (HUVECs). Our results indicate that cells with the [p.L7]+[p.P7] genotype seem to have a tendency to be more sensitive to the growth stimulating effect of NPY and less sensitive to the effect of vascular endothelial growth factor compared to cells with the [p.L7]+[p.L7] genotype. Additionally, cells with the [p.L7]+[p.P7] genotype seem to be more sensitive to apoptosis than [p.L7]+[p.L7] cells. We speculate that the L7P substitution in preproNPY might cause a state of cellular pre-senescence, leading to endothelial dysfunction. This might be one reason for the associations of the L7P polymorphism with atherosclerosis and type II diabetes found in clinical studies.

The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene cre1. In this study, strains were constructed in which the cre1 gene was either completely removed or replaced by a truncated mutant variant, cre1-1, found previously in the Rut-C30 mutant strain with enhanced enzyme production capability. The T. reesei transformants with either deletion or truncation of cre1 had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores. Liquid cultures in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production. Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes. This suggests that cre1 acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing conditions. There was no phenotypic difference between the Deltacre1 and cre1-1 mutant strains in any of the experiments done, indicating that the cre1-1 gene is practically a null allele. The results of this work indicate that cre1 is a valid target gene in strain engineering for improved enzyme production in T. reesei.

Deteriorated male reproductive health has been connected to overexposure to estrogens or to imbalanced androgen-estrogen ratio. Transgenic male mice expressing human aromatase (AROM(+) mice) serve as an apt model for the study of the consequences of an altered androgen-estrogen ratio. Our previous studies with AROM(+) mice showed that low androgen levels together with high estrogen levels result in cryptorchidism and infertility. In the present study, the AROM(+) mice were shown to have severe abnormalities in the structure and function of Leydig cells before the appearance of spermatogenic failure. Decreased expression of adult-type Leydig cell markers (Ptgds, Vcam1, Insl3, Klk21, -24 and -27, Star, Cyp17a1, and Hsd17b3) indicated an immature developmental stage of the Leydig cells, which appears to be the first estrogen-dependent alteration. Genes involved in steroidogenesis (Star, Cyp17a1, and Hsd17b3) were suppressed despite normal LH levels. The low expression level of kallikreins 21, 24, and 27 potentially further inhibited Leydig cell function via remodeling extracellular matrix composition. In connection with disrupted steroidogenesis, Leydig cells showed enlarged mitochondria, a reduced amount of smooth endoplasmic reticulum, and an accumulation of cholesterol and precursors for cholesterol synthesis. The results of studies with AROM(+) mice crossed with estrogen receptor alpha or beta (ERalpha and ERbeta, respectively) knockout mice lead to the conclusion that the structural and functional disorders caused by estrogen exposure were mediated via ERalpha, whereas ERbeta was not involved.

We conducted a controlled investigation to examine whether a combination of computer imagery and tactile tools helps introductory cell biology laboratory undergraduate students better learn about protein structure/function relationships as compared with computer imagery alone. In all five laboratory sections, students used the molecular imaging program, Protein Explorer (PE). In the three experimental sections, three-dimensional physical models were made available to the students, in addition to PE. Student learning was assessed via oral and written research summaries and videotaped interviews. Differences between the experimental and control group students were not found in our typical course assessments such as research papers, but rather were revealed during one-on-one interviews with students at the end of the semester. A subset of students in the experimental group produced superior answers to some higher-order interview questions as compared with students in the control group. During the interview, students in both groups preferred to use either the hand-held models alone or in combination with the PE imaging program. Students typically did not use any tools when answering knowledge (lower-level thinking) questions, but when challenged with higher-level thinking questions, students in both the control and experimental groups elected to use the models.

Childhood cancer treatment can lead to infertility. Organ culture of early postnatal testicular tissue might provide a valuable approach to the study of acute testicular toxicity. The aim of the present study was to develop a functional in vitro organ culture method, in order to identify sensitive target cells to doxorubicin-induced cytotoxicity in immature rat testis during germ cell migration prior initiation of the first wave of spermatogenesis. Testicular tissue fragments from 5-day-old Sprague-Dawley rats were cultured in the absence or presence of doxorubicin (40 and 100ng/ml) and morphology, apoptosis, proliferation and testosterone secretion was analyzed. Postnatal testicular development proceeded normally in control samples for 48h in vitro. In these untreated culture conditions germ and Sertoli cell numbers and germ cell migration were comparable to in vivo. Germ cells were the primary, most sensitive targets for in vitro-induced doxorubicin (100ng/ml) toxicity and their death was not associated with any morphological defects in the Sertoli cells. Organ culture which reduces the need of animal experimentation can be used to study the cytotoxic effects of doxorubicin on the immature testis.

The right-ear advantage (REA) in Dichotic listening (DL) reflects stimulus-driven bottom-up asymmetry in speech processing. The REA can be modified by top-down attentional control. We investigated attentional control in DL task as a function of age. A total of 186 participants between the ages of 5 and 79 years were tested. The youngest children demonstrated a REA that was not modified by attention, suggesting that bottom-up functional asymmetry was present. The 10-11-year-olds began to show ability to voluntarily modify DL, but only young adults were fully capable of doing so. In 59-79-year-olds, this top-down attentional control was lost again.

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-10 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70 degrees C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes., ((c) 2008 Wiley Periodicals, Inc.)

Imatinib mesylate (Glivec, STI 571; Novartis), a small-molecular analog of ATP that potently inhibits the tyrosine kinase activities of Bcr-Abl, PDGFR-alpha, PDGFR-beta, c-Fms, Arg and c-kit, is one of the novel molecularly targeted agents being introduced into cancer therapy. Stem cell factor (SCF)/c-kit and platelet-derived growth factor (PDGF) signaling pathways regulate postnatal formation of the pools of spermatogonial stem cells and Leydig cells in the rat testis. The effect of short postnatal imatinib exposure on fertility of the male rats and offspring of these animals were investigated. Imatinib significantly reduced the litter size sired by the treated animals and led to permanently slightly elevated serum levels of the gonadotropins. Testicular morphology and mRNA levels of ligands and receptors involved in stem cell factor/c-kit and PDGF signaling returned to control levels, and the offsprings were born healthy. Our findings indicate that treatment of cancer with certain molecularly targeted drugs may have latent effects on testicular development by inhibiting specific physiological signaling pathways.

NF-kappaB transcription factors are involved in evolutionarily conserved signaling pathways controlling multiple cellular processes including apoptosis and immune and inflammatory responses. Immune response of the fruit fly Drosophila melanogaster to Gram-negative bacteria is primarily mediated via the Imd (immune deficiency) pathway, which closely resembles the mammalian TNFR signaling pathway. Instead of cytokines, the main outcome of Imd signaling is the production of antimicrobial peptides. The pathway activity is delicately regulated. Although many of the Imd pathway components are known, the mechanisms of negative regulation are more elusive. In this study we report that a previously uncharacterized gene, pirk, is highly induced upon Gram-negative bacterial infection in Drosophila in vitro and in vivo. pirk encodes a cytoplasmic protein that coimmunoprecipitates with Imd and the cytoplasmic tail of peptidoglycan recognition protein LC (PGRP-LC). RNA interference-mediated down-regulation of Pirk caused Imd pathway hyperactivation upon infection with Gram-negative bacteria, while overexpression of pirk reduced the Imd pathway response both in vitro and in vivo. Furthermore, pirk-overexpressing flies were more susceptible to Gram-negative bacterial infection than wild-type flies. We conclude that Pirk is a negative regulator of the Imd pathway.

The aryl hydrocarbon receptor (AhR) is an ancient protein that is conserved in vertebrates and invertebrates, indicating its important function throughout evolution. AhR has been studied largely because of its role in toxicology-gene expression via AhR is induced by many aromatic hydrocarbons in mammals. Recently, however, it has become clear that AhR is involved in various aspects of development such as cell proliferation and differentiation, and cell motility and migration. The mechanisms by which AhR regulates these various functions remain poorly understood. Across-species comparative studies of AhR in invertebrates, non-mammalian vertebrates and mammals may help to reveal the multiple functions of AhR. Here, we have studied AhR during larval development of Baltic salmon (Salmon salar). Our results indicate that AhR protein is expressed in nervous system, liver and muscle tissues. We also present putative regulatory modules and module-matching genes, produced by chromatin immunoprecipitation (ChIP) cloning and in silico analysis, which may be associated with evolutionarily conserved functions of AhR during development. For example, the module NFKB-AHRR-CREB found from salmon ChIP sequences is present in human ULK3 (regulating formation of granule cell axons in mouse and axon outgrowth in Caernohabditis elegans) and SRGAP1 (GTPase-activating protein involved in the Slit/Robo pathway) promoters. We suggest that AhR may have an evolutionarily conserved role in neuronal development and nerve cell targeting, and in Wnt signaling pathway.

Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5-7). It was the most suitable for industrial bleaching of kraft pulp at high temperature.

We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3' to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter-1 of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.

The extracellular bga1-encoded beta-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 +/- 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl- and alkyl-beta-D-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-beta-D-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is approximately 5.0, and the optimum temperature was found to be 60 degrees C. Bga1 is also capable of releasing D-galactose from beta-galactans and is thus actually a galacto-beta-D-galactanase. beta-Galactosidase is inhibited by its reaction product D-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides.

Introduction/purpose: Recovery of force and stretch reflex from exhaustive stretch-shortening cycle (SSC) exercise is usually bimodal, characterized as immediate exercise-induced performance reduction, with its quick recovery followed by a longer-lasting reduction in performance. A clear parallel exists between the respective changes in performance, neural activation, and metabolic or structural exercise-induced changes. This implies the existence of potential coupling between muscle failure and the induced neural adjustments that take place along its recovery. The present study was designed to explore the evidence of this coupling more thoroughly., Methods: H- and stretch reflexes were measured before and periodically after exhaustive SSC exercise in human subjects. Several markers of muscle damage and inflammation were also measured during the 8-d postexercise follow-up period., Results: The results indicate that acute changes of H- and stretch reflex patterns and maximal isometric force are associated with significant increases in lactate, interleukin 6, and prostaglandin E2 concentrations. The delayed changes in reflexes and isometric force occurred concomitantly with increases in muscle thickness, C reactive protein, and substance P concentrations and also in serum creatine kinase activity., Conclusion: The immediate postexercise decreases in H- and stretch reflexes are probably partially caused by activation of group III and IV afferent fibers by high lactate concentration in combination with possible increases in potassium outflow. Both of these parameters recovered quickly (i.e., 2 h after exercise). The events after the 2-h postexercise point are very likely to be related to muscle damage and associated inflammation. Group III and IV afferent fibers are probably reactivated during this period by mechanical factors.

Purpose: Because of the regulatory role of neuropeptide Y (NPY) in angiogenesis, we set out to determine the presence of the leucine 7-proline 7 (Leu7Pro) polymorphism in exudative age-related macular degeneration (AMD) patients and to analyse its implications., Methods: Genotype analysis of the Leu7Pro polymorphism in the signal peptide region of the human prepro-NPY was performed in blood samples from exudative AMD patients (n = 240) and control subjects (n = 79)., Results: In all, 11% of exudative AMD patients and 14% of control subjects exhibited the NPY signal peptide Leu7Pro polymorphism. There were no statistically significant differences in Leu7Pro polymorphism frequency between the exudative AMD and control cases, as analysed by Fisher's exact two-sided test., Conclusions: Leu7Pro polymorphism in the signal peptide region of the human prepro-NPY is not a risk factor for exudative AMD.

Background and Objectives: Several studies have shown that treatment of coronary heart disease (CHD) does not meet the goals set in recommendations. The aim of this study was to investigate the adequacy of CHD drug treatment and secondary prevention measures, particularly with respect to age and gender biases, in a Finnish university hospital setting., Methods: The participant pool consisted of patients in FINCAVAS (Finnish Cardiovascular Study), which is a cohort study recruiting consecutive patients performing a clinical exercise test at Tampere University Hospital, Tampere, Finland. 802 patients (581 men, 221 women) with a prior diagnosis of CHD recruited between October 2001 and December 2004 were included in the analysis., Results: Only roughly 12% of both men and women had an optimal risk factor profile. High blood pressure and hypercholesterolaemia were more common in women than in men, whereas smoking was more frequent among men. Men used ACE inhibitors (32.9% vs 20.4%, respectively), beta-adrenoceptor antagonists (80.8% vs 68.3%, respectively) and aspirin (acetylsalicylic acid) [69.7% vs 58.8%, respectively] more frequently than women, but the frequency of use of these medications was also not at the recommended levels in men. Risk factor control is poorer in older than younger age groups., Conclusions: CHD patients, particularly women, who performed an exercise stress test in a university hospital are suboptimally treated.

Background: The purpose of the Finnish Cardiovascular Study (FINCAVAS) is to construct a risk profile--using genetic, haemodynamic and electrocardiographic (ECG) markers--of individuals at high risk of cardiovascular diseases, events and deaths., Methods and Design: All patients scheduled for an exercise stress test at Tampere University Hospital and willing to participate have been and will be recruited between October 2001 and December 2007. The final number of participants is estimated to reach 5,000. Technically successful data on exercise tests using a bicycle ergometer have been collected of 2,212 patients (1,400 men and 812 women) by the end of 2004. In addition to repeated measurement of heart rate and blood pressure, digital high-resolution ECG at 500 Hz is recorded continuously during the entire exercise test, including the resting and recovery phases. About 20% of the patients are examined with coronary angiography. Genetic variations known or suspected to alter cardiovascular function or pathophysiology are analysed to elucidate the effects and interactions of these candidate genes, exercise and commonly used cardiovascular medications., Discussion: FINCAVAS compiles an extensive set of data on patient history, genetic variation, cardiovascular parameters, ECG markers as well as follow-up data on clinical events, hospitalisations and deaths. The data enables the development of new diagnostic and prognostic tools as well as assessments of the importance of existing markers.

The aim of the study was to estimate the significance of oxidative/nitrosative damage and expression of antioxidant enzymes in renal cell carcinomas (RCC). For this we investigated immunohistochemically six antioxidant enzymes (AOEs) including MnSOD, ECSOD, thioredoxin, thioredoxin reductase, and gammaglutamyl cysteine synthetase heavy and light chain in 138 RCCs. As an indicator of oxidative/nitrosative damage, sections were stained with an antibody to nitrotyrosine. The extent of apoptosis was evaluated by TUNEL method and proliferation by immunohistochemistry to Ki67. Variable expression of all AOEs could be seen in RCC with expression of MnSOD being strongest. Nitrotyrosine was significantly associated with high grade tumors. MnSOD was associated with tumors of a lower stage. Cases showing ECSOD reactivity had higher and cases expressing thioredoxin lower apoptotic index than other tumors. No association with patient prognosis was observed. According to the results renal cell carcinomas show oxidative/nitrosative damage which, according to nitrotyrosine staining, was higher in high grade tumors. Of AOEs, MnSOD was more abundantly expressed in low stage tumors suggesting that its antioxidant function could play a main role to prevent development of oxidative damage leading to more aggressive tumors.

Poly(dimethylsiloxane) also known as PDMS is used in a wide range of biomedical applications. These range from implants through catheters to soft contact lenses. Therefore, it is understandable that PDMS has been extensively tested for these purposes. In past years, the microfluidics has moved from predominantly silicon and glass structures towards polymers due to their ease of manufacturing and moderate cost. PDMS has gained a lot of attention in various analytical applications. However, the testing of its suitability for such applications has not been as thorough as in the biomedical applications, perhaps relying on the experiments from that field. Microfluidic PDMS structures are more and more popular in various analytical devices. Such devices consume less reagents and can work with lower sample volumes. On the other hand, the surface-to-sample-volume ratio becomes larger. That increases the influence of material properties on the actual measurement. Some of the challenges include adsorption, diffusion, surface roughness, permeability and elasticity of PDMS, which are discussed in this paper.

Neuropeptide Y (NPY) is universally expressed in many different neuronal and non-neuronal cells. Human NPY gene has two in-frame kozak sequences and thus, has potentially two translation initiation sites producing two NPY peptides with different molecular weights. In the present study, the intracellular location of NPY was studied in endothelial cells endogenously expressing NPY, and in neuronal (SK-N-BE) and non-neuronal (CHO-K1) cells transfected with NPY-GFP-constructs. By mutating kozak sequences we discovered that kozak-1 directs the NPY peptide to secretory vesicles, and kozak-2 is a prerequisite for mitochondrial targeting. If both kozak sequences are present, non-neuronal cells seem to benefit leaky scanning to initiate translation at both initiation sites, in contrast to neuronal cells, which prefer the kozak-1. This finding suggests that both the kozak sequences of NPY mRNA can be used in the translation depending on the cell type. The size and the function of the novel NPY fragment routed to mitochondria remains to be determined.

The Imd signaling cascade, similar to the mammalian TNF-receptor pathway, controls antimicrobial peptide expression in Drosophila. We performed a large-scale RNAi screen to identify novel components of the Imd pathway in Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell-derived cDNA library were analyzed for their effect on Attacin promoter activity in response to Escherichia coli. We identified seven gene products required for the Attacin response in vitro, including two novel Imd pathway components: inhibitor of apoptosis 2 (Iap2) and transforming growth factor-activated kinase 1 (TAK1)-binding protein (TAB). Iap2 is required for antimicrobial peptide response also by the fat body in vivo. Both these factors function downstream of Imd. Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in Relish nuclear localization in vitro, suggesting a novel mode of regulation of the Imd pathway. Our results show that an RNAi-based approach is suitable to identify genes in conserved signaling cascades.

Objective: We investigated the replicability of the magnetically measured mismatch negativity (MMNm)., Methods: The MMNm was recorded twice by using a 122-channel whole-head magnetometer in 15 healthy young adults. The MMNm responses for duration, intensity and frequency deviants were analyzed separately in left and right hemispheres for the response strength, latency, dipole moment, and generator loci., Results: In the right hemisphere, the test-retest correlations were statistically significant for all MMNm parameters (r = 0.49-0.89). In the left hemisphere, the majority of the MMNm parameters also demonstrated statistically significant test-retest correlations (r = 0.61-0.82). In addition, the MMNm generator loci were stable for all deviants., Conclusions and Significance: The present results are encouraging in terms of both research and clinical use of MMNm in studying human auditory discrimination in its normal and deteriorated states.

Context: Neuropeptide Y (NPY) plays a role in angiogenesis, cardiovascular regulation, and hormone secretion. The leucine7 to proline7 (Leu7Pro) polymorphism of preproNPY is associated with vascular diseases and has an impact on hormone levels in healthy subjects., Objective: The current study investigated the role of the Leu7Pro polymorphism in metabolic and cardiovascular autonomic regulation., Design and Subjects: A 5-h oral glucose tolerance test was performed on 27 healthy volunteers representing two preproNPY genotypes (Leu7/Pro7 and Leu7/Leu7) matched for age, sex, body mass index and physical activity., Main Outcome Measures: Simultaneously we performed cardiovascular autonomic function tests and plasma measurements of sympathetic transmitters, glucose, insulin, and ghrelin., Results: The subjects with Leu7/Pro7 genotype had decreased plasma NPY, norepinephrine (NE), and insulin concentrations and insulin to glucose ratios. The suppression of ghrelin concentrations after glucose ingestion was delayed in these subjects. They also had increased heart rate variability indices and baroreflex sensitivity. However, they displayed significant negative association of NE concentration with variability of low-frequency R-R-intervals and with baroreflex sensitivity., Conclusions: The Leu7Pro polymorphism of preproNPY is related to decreased level of basal sympathetic activity, decreased insulin secretion, and delayed ghrelin suppression during oral glucose tolerance test. The increased responsiveness of autonomic functions to NE associated with the polymorphism may be connected to increased cardiovascular vulnerability.

The templates of innate immunity have ancient origins. Thus, such model animals as the fruit fly, Drosophila melanogaster, can be used to identify gene products that also play a key role in the innate immunity in mammals. We have used oligonucleotide microarrays to identify genes that are responsive to gram-negative bacteria in Drosophila macrophage-like S2 cells. In total, 53 genes were induced by greater than threefold in response to Escherichia coli. The induction of all these genes was peptidoglycan recognition protein LC (PGRP-LC) dependent. Twenty-two genes including 10 of the most strongly induced genes are also known to be up-regulated by septic injury in vivo. Importantly, we identified 31 genes that are not known to respond to bacterial challenge. We carried out targeted dsRNA treatments to assess the functional importance of these gene products for microbial recognition, phagocytosis and antimicrobial peptide release in Drosophila S2 cells in vitro. RNAi targeting three of these genes, CG7097, CG15678 and beta-Tubulin 60D, caused altered antimicrobial peptide release in vitro. Our results indicate that the JNK pathway is essential for normal antimicrobial peptide release in Drosophila in vitro.

Most G protein-coupled receptors are desensitized by a uniform two-step mechanism: phosphorylation followed by arrestin binding and internalization. In this study we explored the time-, ligand-, and concentration dependence of alpha2-adrenoceptor internalization in human embryonal kidney (HEK-293) cells expressing alpha2A- and alpha2B-adrenoceptors. We also explored the relationship between ligand-induced receptor internalization and agonist efficacy, determined with a [35S]GTPgammaS binding assay. The results showed rapid dose-dependent internalization of both alpha2A- and alpha2B-receptors; the extent of internalization was directly proportional to agonist efficacy. The agonist UK 14,304 had a subtype-specific high efficacy at alpha2A-AR and dexmedetomidine at alpha2B-AR. Agonist-induced [35S]GTPgammaS binding was totally blocked by pretreatment with pertussis toxin (PTX) for both receptor subtypes, while only about 50% of the internalization was blocked by PTX. The results indicate that the extent of internalization of alpha2A-AR and alpha2B-AR is proportional to agonist efficacy, but only partly dependent on Gi protein coupling.

Objective: Neuropeptide Y is a potent vasoconstrictor thought to enhance the development of atherosclerosis. The leucine 7 to proline 7 (Leu7Pro) polymorphism, located in the signal peptide part of the human preproneuropeptide Y, has been associated with serum lipid levels, intima-media thickness of the common carotid arteries, and diabetic retinopathy in type 2 diabetic patients. Therefore, we investigated the impact of the Leu7Pro polymorphism on diabetic nephropathy, cardiovascular risk factors, and cardiovascular disease in type 1 diabetic patients., Research Design and Methods: A total of 996 patients from the Finnish Diabetic Nephropathy study were studied in a case-control, cross-sectional study. The carrier frequency of the Pro7 substitution was 13% in the entire study population., Results: The Pro7 substitution was more common in patients with proteinuria than in those with a normal albumin excretion rate (16 vs. 11%, P < 0.05). Patients with the Pro7 allele had worse glycemic control (HbA(1c) 8.8 vs. 8.5%, P < 0.005), more coronary heart disease (CHD) (14 vs. 8%, P < 0.05), and higher serum triglycerides (1.65 vs. 1.35 mmol/l, P < 0.005) than patients with the wild-type genotype. There were no differences in the plasma neuropeptide Y levels between the patients with Pro7 compared with those with the wild-type genotype. The Leu7Pro polymorphism was independently associated with HbA(1c) (P < 0.001), proteinuria (P < 0.01), and CHD (P < 0.01) in multiple regression analyses., Conclusions: We conclude that the Leu7Pro polymorphism may contribute to the genetic susceptibility to diabetic nephropathy and CHD in type 1 diabetic patients, possibly by influencing glycemic control and triglycerides.

Background: Neuropeptide Y is a sympathetic neurotransmitter, a potent endothelium-derived angiogenic factor and a vascular mitogen. We have studied the role of the functional leucine7 to proline7 polymorphism of the signal peptide region of preproneuropeptide Y (prepro-NPY) as a genetic susceptibility factor for diabetic retinopathy. In addition, we investigated the role of the NPY Y2-receptor as a putative mediator of angiogenic NPY signaling in the retina., Methods: Frequencies of proline7 (Pro7) carriers in the prepro-NPY were determined in type 1 and type 2 diabetes patients having retinopathy, in type 2 diabetes patients without retinopathy and in healthy control subjects. The role of Y2-receptor in hyperoxemia-induced retinal neovascularization was investigated in Y2-receptor knockout mice (Y2-/-) and in rats administered Y2-receptor mRNA antisense oligonucleotide., Results: The carriers having Pro7 in the preproNPY are markedly over-represented among type 2 diabetes patients with retinopathy compared to type 2 diabetes patients without retinopathy and to the population control. Neonatal exposure to hyperoxia resulted in development of retinal neovascularization that was prevented in Y2(-1-) -mice, and significantly inhibited in rats treated with the Y2-receptor antisense oligonucleotide., Conclusions: NPY and Y2-receptor play important roles in diabetic retinopathy and retinal neovascularization and are thus potential new targets for drug molecules for treatment of retinopathy.

A bacterial xylanase gene, Nonomuraea flexuosa xyn11A, was expressed in the filamentous fungus Trichoderma reesei from the strong cellobiohydrolase 1 promoter as fusions to a variety of carrier polypeptides. By using single-copy isogenic transformants, it was shown that production of this xylanase was clearly increased (up to 820 mg/liter) when it was produced as a fusion protein with a carrier polypeptide having an intact domain structure compared to the production (150 to 300 mg/liter) of fusions to the signal sequence alone or to carriers having incomplete domain structures. The carriers tested were the T. reesei mannanase I (Man5A, or MANI) core-hinge and a fragment thereof and the cellulose binding domain of T. reesei cellobiohydrolase II (Cel6A, or CBHII) with and without the hinge region(s) and a fragment thereof. The flexible hinge region was shown to have a positive effect on both the production of Xyn11A and the efficiency of cleavage of the fusion polypeptide. The recombinant Xyn11A produced had properties similar to those of the native xylanase. It constituted 6 to 10% of the total proteins secreted by the transformants. About three times more of the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A was observed. Even in the best Xyn11A producers, the levels of the fusion mRNAs were only approximately 10% of the level of cel7A (cbh1) mRNA in the untransformed host strain.

Receptor density is an important determinant of cellular effector responses to receptor activation. We analysed cytosolic Ca(2+) responses to alpha(2)-adrenergic agents in PC12 cells expressing human alpha(2B)-adrenergic receptors (AR) at two densities (3.8 and 1.3 pmol/mg protein). The efficacy (E(max)) of agonists was greater in cells with higher receptor expression; while the potency (EC(50)) of norepinephrine and oxymetazoline was independent of alpha(2B)-AR levels. Several classical alpha(2)-AR antagonists behaved as either partial or inverse agonists in a receptor density-dependent fashion. No apparent structural similarities were found among the inverse agonists, precluding simple predictions of inverse agonist activity. Transfected PC12 cells expressing alpha(2B)-AR at relatively high density would be a useful approach to screen inverse agonists for this class of receptors. Our results further indicate that receptor density significantly influences the properties of ligands, not only of partial agonists as predicted by classical receptor theory, but also of antagonists and full agonists.

The human alpha(2B)-adrenoceptor (alpha(2B)-AR) was mutated by substituting the D(3.49) aspartate in position 109 with an alanine (alpha(2B)-D109A) in the conserved DRY sequence at the cytoplasmic face of TM3. We studied the effects of the mutation on agonist binding and on receptor activation in CHO cells, including possible inverse agonism monitored by measuring intracellular Ca(2+) concentrations ([Ca(2+)](i)). The mutated receptor had increased binding affinity for agonists, especially dexmedetomidine (3.8-fold). The increased affinity was abolished by pretreatment of the cells with pertussis toxin. The mutation produced constitutive receptor activity evidenced as increased basal [Ca(2+)](i) and increased potency and efficacy of agonists to elicit Ca(2+) responses. The imidazoline derivative RX821002 functioned as an inverse agonist only through the alpha(2B)-D109A, reducing [Ca(2+)](i). The results thus indicate that this mutation causes constitutive receptor-G(i)-protein precoupling, and that the D(3.49) aspartate residue of the DRY motif is involved in controlling coupled and uncoupled conformations of alpha(2B)-AR.

Neuropeptide Y (NPY) is an important neurotransmitter in the central and peripheral nervous systems. It has a regulatory role in cardiovascular and metabolic functions and control of hormone release. The leucine 7 to proline 7 (Leu7Pro) polymorphism in the signal peptide of prepro-NPY is associated with increased blood lipid levels, accelerated atherosclerosis, and diabetic retinopathy. This study elucidated the role of this polymorphism in diurnal cardiovascular, metabolic, and hormonal functions of healthy subjects during rest. The two study groups comprised individuals with different genotype, but they were matched for age and body mass index. Subjects with the Leu7Pro polymorphism had significantly lower plasma NPY and norepinephrine concentrations, lower insulin concentrations, higher glucose concentrations, and lower insulin-glucose ratio in plasma than the controls. Heart rate was significantly higher during daytime in the subjects with Leu7Pro polymorphism. Furthermore, these subjects had significantly lower prolactin concentrations in plasma. Systolic and diastolic blood pressure, serum free fatty acid and plasma leptin, ACTH, cortisol, LH, FSH, TSH, free thyroxin, and melatonin concentrations were similar during the 24-h period, compared with controls. These results show that genetically determined changes in NPY levels lead to widespread consequences in the control of sympathoadrenal, metabolic, and hormonal balance in healthy subjects.

Many G protein-coupled receptors (GPCRs) are internalized from the plasma membrane after agonist exposure. Previously, marked agonist-induced internalization of human alpha2A- and alpha2B-adrenergic receptors (AR) was observed in transfected neuronal rat pheochromocytoma (PC12) cells; alpha2A- and alpha2B-AR were internalized into partly distinct intracellular vesicles (Olli-Lähdesmäki et al., J. Neurosci. 19, 9281-9288, 1999). In this paper, the extent of alpha2-AR internalization was quantitated in human embryonic kidney (HEK-293) and PC12 cells by combined application of cell surface biotinylation and ELISA methods, which allow measurement of protein trafficking in intact, differentiated and undifferentiated cells. Significant subtype-specific (but not cell type-dependent) trafficking of human alpha2-AR was observed by quantitation and immunocytochemistry. Agonist-induced sequestration of alpha2B-AR was markedly reduced after blocking the formation of clathrin-coated vesicles by hyperosmotic sucrose pretreatment. The sequestration of alpha2A-AR was partly inhibited after sucrose pretreatment but could be further reduced after inhibiting the formation of both clathrin-coated and caveolin vesicles by combined pretreatment with hyperosmotic sucrose and filipin. Differences were also observed in the recycling of alpha2A- and alpha2B-AR. The extent of maximal agonist-induced sequestration in PC12 cells was not directly dependent on relative agonist efficacy.

The Leu7Pro polymorphism in the signal peptide of the preproneuropeptide Y (NPY) has been associated with dyslipidemias and free fatty acid (FFA) levels during exercise. The association of this polymorphism with insulin sensitivity has not been studied. In this study, the Leu7Pro polymorphism was determined in 2 groups of nondiabetic middle-aged subjects (n = 266 and n = 295). Insulin sensitivity was measured with the hyperinsulinemic euglycemic clamp (n = 266) or with an intravenous glucose tolerance test (IVGTT, n = 295). First-phase insulin secretion was determined as insulin area under the curve (AUC) during the first 10 minutes of the IVGTT. FFAs were measured both in the fasting state and during the hyperinsulinemic clamp. The Leu7Pro polymorphism of the NPY gene was not associated with the rates of whole body glucose uptake, insulin sensitivity index, insulin secretion during the IVGTT, or insulin AUC during the oral glucose tolerance test. However, the Pro7 allele was associated with low FFA levels both in the fasting state (P =.043) and during the hyperinsulinemic clamp (P =.003). In conclusion, the Leu7Pro polymorphism of the NPY gene associates with alterations in FFA metabolism but does not have an impact on insulin sensitivity, insulin secretion, or glucose metabolism., (Copyright 2003 Elsevier Inc. All rights reserved.)

Objective: One prerequisite for performing multicentre studies is that the clinical handling of the patients must be uniform. We therefore evaluated possible differences in pretreatment evaluation, surgical treatment and follow-up regimes between the Nordic countries and between the different departments that performed nephrectomy due to renal cell carcinoma., Material and Methods: A questionnaire comprising 21 different questions was sent to all hospitals in the five Nordic countries performing nephrectomy. The questionnaires were returned by 195/226 (86%) departments., Results: In total, 24% of the departments performed fewer than five tumour nephrectomies per year. The main differences were as follows. I.v. pyelography was never used in Finland in clinics with urologists while preoperative CT scans were performed by most departments and in most countries. Cytology/biopsy examinations were never used in urological clinics in Finland and Iceland in contrast to 31% of urological clinics in Denmark. In Finland, 69% of the departments performed nephrectomy in patients with multiple distant metastases, compared to only 15% in the other Nordic countries. Follow-up after nephrectomy was done in 38% of Danish departments and in 96% of departments in the other Nordic countries., Conclusion: There were evident differences between the urological/surgical departments in the five Nordic countries, especially concerning radiological evaluation, treatment of patients with metastases and postoperative follow-up.

In the mammalian ovary cell growth and differentiation is regulated by several members of the transforming growth factor beta (TGF beta) superfamily including activins, inhibins, growth differentiation factors and bone morphogenetic proteins (BMPs). The effects of TGF beta family members are mediated to the target cells via heteromeric complexes of type I and II serine/threonine kinase receptors which activate Smad signaling protein pathways in various cell types. We have previously shown that inhibin B, a hormonally important product from human granulosa cells, is up regulated by activin and BMPs. Here, we report the use of adenoviral gene transfer methodology to manipulate the TGF beta growth factor signaling system in primary cultures of human granulosa cells. These cells are exceedingly difficult to transfect by conventional transfection methods, but were virtually 100% infected with recombinant adenoviruses expressing green fluorescent protein (GFP). Adenoviruses expressing constitutively active forms of the seven known mammalian type I activin receptor-like kinase receptors (Ad-caALK1 through Ad-caALK7) cause activation of endogenous and adenovirally transferred Smad signaling proteins so that Ad-caALK1/2/3/6 and Ad-caALK4/5/7 induced phosphorylation of the Smad1 and Smad2 pathways, respectively. Activin A and BMP-2 activated the Smad1 and Smad2 pathways as well as inhibin B production as did all the Ad-caALKs. Furthermore, overexpression of adenoviral Smad1 and Smad2 proteins without exogenously added ligands induced inhibin B production. The inhibitory Smad7 protein suppressed BMP-2 and activin induced inhibin B production. Collectively, the present data demonstrate that adenoviral gene transfer provides an effective approach for dissecting the TGF beta signaling pathways in primary ovarian cells in vitro and more specifically indicate that the Smad1 and Smad2 pathways are involved in the regulation of inhibin B production by TGF beta family ligands in the ovary.

Cells of the PC12 rat pheochromocytoma cell line acquire characteristics of sympathetic neurons under appropriate treatment. Stably transfected PC12 cells expressing individual alpha2-adrenergic receptor (alpha2-AR) subtypes were used to assess the role of alpha2-ARs in neuronal differentiation and to characterise the signalling pathways activated by the alpha2-AR agonist epinephrine in these cells. The effects of alpha2-AR activation were compared with the differentiating action and the signalling mechanisms of nerve growth factor (NGF). Epinephrine induced neuronal differentiation of PC12alpha2 cells through alpha2-AR activation in a subtype-dependent manner, internalization of all human alpha2-AR subtypes, and activation of mitogen-activated protein kinase (MAPK) and the serine-threonine protein kinase Akt. Epinephrine and NGF showed synergism in their differentiating effects. The MAPK kinase (MEK-1) inhibitor PD 98059 abolished the differentiating effect of epinephrine indicating that the differentiation is dependent on MAPK activation. Activating protein-1 (AP-1) DNA-binding activity was increased after epinephrine treatment in all three PC12alpha2 subtype clones. Evaluation of the potential physiological consequences of these findings requires further studies on endogenously expressed alpha2-ARs in neuronal cells.

Objectives: The objective of the study is to evaluate the agreement of local and review urinary cytology in patients with newly diagnosed bladder cancer and in those being followed for their disease. In addition, the effect of the type of institution on agreement was determined., Methods: A total of 652 consecutive patients with bladder cancer from 19 institutions were evaluated; 575 (88.2%) of the patients had cytopathological sample available for central review and were eligible for analysis. One hundred and twenty nine (22.4%) of the patients had newly diagnosed bladder cancer, whereas the remaining 446 (77.6%) patients were under follow-up. A voided urine sample was obtained prior to transurethral resection of the bladder (TURB) or routine follow-up cystoscopy and split for culture and cytology. The cytopathological samples were first analysed by a local pathologist, and then re-analysed by a central reviewer. The agreement of cytological readings was determined by Kappa coefficient., Results: The sensitivities of local and review cytology in detection of primary bladder cancer were 38.8 and 31.0%, respectively. Recurrence was observed in 119 of the 446 (26.7%) patients under follow-up, of which both local and review cytology detected 21 (17.6%) cases. Specificities of local and review cytology were 97.6 and 96.6%, respectively. Overall agreement of urine cytology was good in patients with primary bladder cancer and moderate in those being followed for their disease as Kappa coefficients were 0.70 and 0.60, respectively. However, some disagreement was found when results were analysed according to type of institution, to type of primary tumour, and to result of follow-up cystoscopy. In patients with primary bladder cancer the Kappa coefficient was 0.86 (very good) in university hospitals and 0.36 (fair) in city hospitals. Accordingly, in patients under follow-up the Kappa coefficient was 0.65 (good) in university hospitals and 0.39 (fair) in district hospitals. Although the stage of primary tumour had no effect on agreement, agreement was moderate (Kappa coefficient 0.45) in those with low grade tumour and good (Kappa coefficient 0.67) in those with high grade tumour. In addition, Kappa coefficients were 0.65 (good) and 0.40 (fair) in those with and without recurrence at follow-up cystoscopy., Conclusions: Although overall agreement of routine cytology was from moderate to good in both diagnosis and monitoring of bladder cancer, there is some variation in agreement according to the type of institution. Accordingly, grade of primary tumour and the result of follow-up cystoscopy had effect on agreement reflecting subjectiveness and weak reproducibility of this test. This not only emphasises the need for continuing education and quality control for urine cytologic analysis, but also inspires the development of more objective tests.

Pilot-scale tests to enhance phosphorus removal with ferrous sulphate in a biological phosphorus and nitrogen removal process (modified UCT) for treating municipal wastewater were performed. The results indicated that Fe(II) competes with the BioP organisms and will inhibit the biological phosphorus removal process completely at doses exceeding 9 g m(-3) of Fe(II). The goal of an effluent P level of 0.5 mg l(-1) can be attained with 5 g m(-3) of Fe(II). A consistent effluent concentration of 0.3 mg l(-1) could not be achieved with this method. A centrifugation method to evaluate the dewatering properties of sludge was developed. Comparison of the settling and dewatering properties between activated sludge from the pilot plant and a full-scale simultaneous precipitation process indicated no consistent differences between them. The poor settling properties are due to the long sludge retention time needed by nitrification and not to the biological phosphorus removal process.

In local or metastatic cancer, a prognostic tumour marker could be a valuable tool in the selection of different treatments. In renal cell cancer (RCC) no such markers have been available. We therefore evaluated the association between several pretreatment serum markers, tumour classification and short term survival in RCC patients. Serum samples were collected before surgery and three months thereafter from 24 RCC patients. Interleukin-6 (IL-6), IL- 12, soluble IL-2 receptor (sIL-2R) and intercellular adhesion molecule-1 (sICAM-1) were measured in serum samples using specific commercial enzyme immunoassay kits. Serum IL-6, sIL-2R and sICAM-1 levels before nephrectomy were significantly higher in non-local tumours than in local ones (mean IL-6 53 pg/ml versus 6.3 pg/ml, and sICAM-1 443 ng/ml versus 290 ng/ml, sIL-2R 3779 pg/ml versus 1796 pg/ml). In contrast, IL-12 levels were higher in local tumours (148 versus 102 pg/ml) and the levels increased significantly (P < 0.005) after removal of the primary tumour in patients with local disease. All patients with local tumours had normal IL-6 values, while only one with a non-local tumour had IL-6 levels below 10 pg/ml. In addition, IL-6 and sICAM-1 levels before operation were significantly higher in patients with short (less than one year) survival (p=0.007 to IL-6 and p=0.006 to sICAM-1). In contrast, patients with shorter survival had significantly lower IL-12 (p=0.03) levels. Our findings suggest that RCC induces changes in several immunological parameters. These soluble immunological factors, IL-6, IL-12, sIL-2R and sICAM-1, might have a role as prognostic factors in RCC.

The leucine 7 to proline 7 (Leu7Pro) polymorphism in the signal peptide of NPY is associated with high blood lipid concentrations and accelerated rate of atherosclerosis as well as diabetic retinopathy. Also, healthy subjects with this polymorphism have increased NPY secretion during sympathetic stimulation. Because NPY may regulate GH release and ghrelin may regulate NPY formation, we studied the effects of the Leu7/Pro7 genotype on GH, ghrelin, and IGF-I secretion during standardized cycle-ergometer exercise. Furthermore, we studied the effect of the Leu7/Pro7 genotype on diurnal GH secretion in rest in a separate study. The subjects with Leu7/Pro7 genotype had 54% higher maximal increases in the plasma GH concentrations than the controls during exercise. There were no significant differences in the ghrelin or IGF-I concentrations during exercise among the groups. Furthermore, there were no differences in diurnal GH secretion between the genotypes. The results indicate that the prepro-NPY genotype has an influence on GH response during exercise in humans. The clinical significance of this finding is not known, and further studies are needed to evaluate whether the observed change in GH secretion during exercise could play a role in promoting diseases.

Objectives: The relation of the mismatch negativity (MMN) elicitation with behavioral stimulus discrimination as well as the replicability of the MMN was evaluated for intervals between paired tones., Methods: The MMN, obtained in a passive oddball paradigm in two sessions separated by 4-21 days and behavioral responses (button presses to target stimuli) in a separate session were recorded from 10 adult healthy subjects. The standard stimulus (P=0.79) was a tone pair separated by a 120 ms silent inter-stimulus interval (ISI) and the deviant stimuli were tone pairs with an ISI of 100, 60, and 20 ms (P=0.07 for each)., Results: The 20 and 60 ms ISI deviant tone pairs elicited a significant MMN during both recording sessions and they were also behaviorally discriminated, whereas neither did the 100 ms ISI deviant pair elicit significant MMN nor was it behaviorally discriminated. Furthermore, there was a significant correlation between the MMN and reaction times to the 20 and 60 ms ISI deviant pairs. The 20 ms ISI deviant stimulus elicited highly replicable MMNs (r=0.75), whereas the less well discriminated 60 ms ISI deviant pair did not (r=0.60)., Conclusions: The MMN reflects discrimination accuracy of temporal sound intervals. Furthermore, when the physical difference between the standard and deviant tone pair in the temporal domain is large, it is elicited with high reliability.

Background and Aims: Urine cytology is gold standard for clinical tests used in the diagnosis and follow-up of bladder cancer. Cytology, however, exhibits variable sensitivity depending on tumour grade and interpretation of urine specimens is highly dependent on the skill of the examiner. Furthermore, a "suspicious" cytology report (class III) makes clinicians uncomfortable. In these cases, a more objective test, such as the BTA stat Test, may be useful in providing clarification. The aim of this study was to evaluate the dilemma of suspicious routine urine cytology and to determine whether the BTA stat Test provides diagnostic aid in this rare but controversial category., Material and Methods: 506 consecutive patients who were being followed for bladder cancer were included in the study. A voided urine sample was obtained prior to routine follow-up cystoscopy and split for culture and testing with the BTA stat Test. Clinical status of the disease was evaluated in patients with suspicious urine cytology, and the diagnostic aid of the BTA stat Test in these patients was determined., Results: A total of 57 patients (11.3%) had urine cytology classified as suspicious. The BTA stat Test was positive in 29 (50.9%) and negative in 28 (49.1%) patients. Nineteen (33.3%) patients had recurrence at routine cystoscopy. Of the remaining 38 patients, 10 were further investigated due to a positive BTA stat Test. Two additional recurrences were detected bringing the total number of recurrences to 21 (36.8%), 48.3% (14/29) of the patients with positive and 25.0% (7/28) of the patients with negative BTA stat Test had recurrence (p = 0.069). Overall, 65.5% (19/29) of the patients with a positive BTA stat Test were found to have recurrence either at routine cystoscopy, at further investigations, or at the next cystoscopy compared to that of 35.7% (19/28) in those with negative testing (p = 0.024). The overall sensitivity of the BTA stat Test was 66.7%, and the specificity was 58.3%., Conclusions: At least a third of the patients under follow-up for bladder cancer with suspicious cytology had a recurrence, indicating that these patients are a risk group for recurrence. More importantly, a BTA stat Test result seems to provide some help in distinguishing those patients with very high risk for recurrence, for whom invasive further investigations should be conducted and a close follow-up policy maintained.

Transforming growth factor-beta (TGF-beta) superfamily related growth factors signal by binding to transmembrane type I and type II receptor serine/threonine kinases (RSTK), which phosphorylate intracellular Smad transcription factors in response to ligand binding. Here we describe the cloning of the human type I RSTK activin receptor-like kinase 7 (ALK7), an orthologue of the previously identified rat ALK7. Nodal, a TGF-beta member expressed during embryonic development and implicated in developmental events like mesoderm formation and left-right axis specification, was recently shown to signal through ALK7. We found ALK7 mRNA to be most abundantly expressed in human brain, pancreas and colon. A cDNA encoding the open reading frame of ALK7 was obtained from a human brain cDNA library. Furthermore, a P1 artificial chromosome (PAC) clone containing the human ALK7 gene was isolated and fluorescent in situ hybridization (FISH) on metaphase chromosomes identified the gene locus as chromosome 2q24.1-->q3. To test the functionality of the ALK7 signaling, we generated recombinant adenoviruses containing a constitutively active form of ALK7 (Ad-caALK7), which is capable of activating downstream targets in a ligand independent manner. Infection with Ad-caALK7 of MIN6 insulinoma cells, in which ALK7 has previously been shown to be endogenously expressed, led to a marked increase in the phosphorylation of Smad2, a signaling molecule also used by TGF-betas and activins., (Copyright 2002 S. Karger AG, Basel)

The short ACTH test is used in evaluating the hypothalamo-pituitary-adrenal axis (HPA-axis) in preterm neonates after dexamethasone treatment. This test mainly examines primary adrenal suppression but is also used as a method to test secondary adrenal insufficiency because long-term deprivation of ACTH causes atrophy of the adrenal cortex. The CRH test, on the other hand, directly examines the function of the pituitary. We tested 18 infants in the neonatal intensive care unit with both the ACTH test and the CRH test to determine which of these two tests more reliably demonstrates HPA-axis suppression. One patient had normal responses both in the ACTH test and in the CRH test when the limit of 360 nmol/L was used as a sign of proper cortisol secretion. In four cases the patients' cortisol secretion would have been regarded as normal by the low-dose ACTH test, whereas the CRH test did not show an adequate cortisol response. In conclusion, the ACTH test did not reliably indicate HPA-axis suppression after a short (<2 weeks) course of dexamethasone therapy in this study. Therefore, whether the infant is or will be under acute stress after short glucocorticoid treatment, ensuring adequate cortisol secretion with the CRH test should be considered.