Serum and Synovial Fluid Serum Amyloid A Response in Equine Models of Synovitis and Septic Arthritis.

OBJECTIVE: To investigate the serum and synovial fluid serum amyloid A (SAA) response in equine models of synovitis and septic arthritis and to compare handheld and validated immunoturbidometric assays for SAA quantification. STUDY DESIGN: Controlled, experimental study. ANIMALS: Healthy adult horses (n = 9). METHODS: Synovitis (n = 4) and septic arthritis (n = 5) were induced using lipopolysaccharide and Staphylococcus aureus, respectively, and serial serum and synovial fluid samples were collected. Serial synovial fluid cytology was performed for both models and synovial fluid from the septic arthritis model was submitted for bacterial culture. Serum and synovial fluid SAA were quantified by handheld test and immunoturbidometric assay. Cytologic and SAA data were compared within and between models (mixed model ANOVA) and results of SAA assays were compared using category-by-category analysis (weighted kappa coefficient). RESULTS: Synovial fluid total nucleated cell counts and total protein increased significantly following induction of both models. Serum and synovial fluid SAA remained normal in synovitis horses and increased significantly in septic arthritis horses. Serum SAA increased more rapidly than synovial fluid SAA. Agreement was 98% when SAA concentrations were low (<50 µg/mL) but the assays diverged when concentrations were greater than ∼100 µg/mL. Overall, there was good category-by-category agreement between SAA assays (weighted kappa = 0.824). CONCLUSION: Serum and synovial fluid SAA may be useful adjuncts in diagnosing septic arthritis in horses. SAA concentrations for the assays diverged and examination using a larger sample size is needed before direct numeric comparisons between the assays can be made.

The urban stormwater farm.

Currently more than 3 billion people live in urban areas. The urban population is predicted to increase by a further 3 billion by 2050. Rising oil prices, unreliable rainfall and natural disasters have all contributed to a rise in global food prices. Food security is becoming an increasingly important issue for many nations. There is also a growing awareness of both 'food miles' and 'virtual water'. Food miles and virtual water are concepts that describe the amount of embodied energy and water that is inherent in the food and other goods we consume. Growing urban agglomerations have been widely shown to consume vast quantities of energy and water whilst emitting harmful quantities of wastewater and stormwater runoff through the creation of massive impervious areas. In this paper it is proposed that there is an efficient way of simultaneously addressing the problems of food security, carbon emissions and stormwater pollution. Through a case study we demonstrate how it is possible to harvest and store stormwater from densely populated urban areas and use it to produce food at relatively low costs. This reduces food miles (carbon emissions) and virtual water consumption and serves to highlight the need for more sustainable land-use planning.

[A survey of labour pain management in Germany]. / Eine Umfrage zur geburtshilflichen Schmerztherapie in Deutschland.

BACKGROUND: In a survey of German hospitals with obstetric units, data on anaesthesia and analgesia in obstetric pain management were collected. METHODS: At each of 918 hospitals with obstetric units, the head of the anaesthetic department received a questionnaire on obstetric pain management. RESULTS: The response rate was 47.1%. On average, there were 748+/-407 (median 663;1st/3rd quartiles 309/1,303) births per year and hospital, 69.4% with spontaneous vaginal delivery. Opioids were the systemic analgesics most frequently administered in the delivery rooms. Epidural analgesia (EA) was given to 17.5+/-12.6% of the parturients. The number of deliveries per annum had a significant influence on the frequency of EA (<500 deliveries/year: 12.7%, 500-1000/year: 18.6%, >1,000/year: 21.6%). The preferred local anaesthetics were ropivacaine und bupivacaine. When an opioid was given this was almost always sufentanil. In 16% of the responding hospitals adrenaline was added to the epidural test bolus. CONCLUSION: EA is the mainstream method of relieving labour pains in almost all German hospitals, but is used significantly more often in hospitals with rather high numbers of yearly deliveries than in hospitals in which there are few deliveries per year.

Contraindications to regional anaesthesia in obstetrics: a survey of German practice.

BACKGROUND: We assessed current practice regarding indications and contraindications to regional analgesia and anaesthesia for labour and delivery in Germany. METHODS: Questionnaires were mailed to the directors of 918 German departments of anaesthesiology. RESULTS: A total of 397 completed replies were received representing 41.3% of all deliveries in Germany. More than half of the respondents never perform spinal or epidural anaesthesia when the platelet count falls below 65x10(9)/L. Preeclampsia, which was not graded for severity, was considered an absolute contraindication to regional block by 15% and placenta praevia by 30% of respondents. If a woman had taken aspirin three days before, the numbers of respondents considering epidural anaesthesia contraindicated (40.2%) were nearly double those considering spinal anaesthesia contraindicated (21.7%) (P<0.001). For a platelet count of 79x10(9)/L, epidural anaesthesia was thought to be contraindicated by 37% and spinal anaesthesia by 22.2% (P=0.001). In departments with <500 deliveries/year, reluctance to use regional blockade was more pronounced than in departments with >1000 deliveries/year. CONCLUSION: Clinical practice varies considerably in Germany. Concerns regarding the use of regional blockade were more prevalent in hospitals with small delivery units. Indications and contraindications are not consistent in Germany and some recommendations or guidelines are needed.

Change in anaesthetic practice for Caesarean section in Germany.

BACKGROUND: Initial data from 1996 revealed that in contrast to several other countries general anaesthesia was the preferred anaesthetic technique for Caesarean section in Germany. However, anaesthetic practice for Caesarean section has changed during the last decades world-wide. This investigation was performed to obtain more actual data on anaesthetic procedures in obstetric patients in German hospitals. METHODS: Questionnaires on the practice of anaesthesia for Caesarean section were mailed to 918 German departments of anaesthesiology. Furthermore, the survey evaluated severe perioperative complications in obstetric patients. RESULTS: The 397 completed replies in this survey represent 41.3% of all German deliveries in 2002. Spinal anaesthesia is now the most common technique (50.5%) for elective Caesarean section. In case of urgent and emergency Caesarean, delivery figures decrease to 34.6% and 4.8%, respectively. Epidural anaesthesia is performed in 21.6% of scheduled and 13.2% and 1.0% of non-scheduled urgent or emergency Caesarean sections, respectively. Four maternal deaths and several non-fatal episodes of gastric content aspiration were reported by the respondents. CONCLUSIONS: Compared to data obtained 6 years ago a significant increase in regional anaesthesia for Caesarean section has developed, with spinal anaesthesia being the preferred technique. Surveys can help to initiate discussion and improve current practice of anaesthetic care.

The transcription factor GATA4 is activated by extracellular signal-regulated kinase 1- and 2-mediated phosphorylation of serine 105 in cardiomyocytes.

The zinc finger-containing transcription factor GATA4 has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Here we demonstrate that GATA4 is itself regulated by the mitogen-activated protein kinase signaling cascade through direct phosphorylation. Site-directed mutagenesis and phospho-specific GATA4 antiserum revealed serine 105 as the primary site involved in agonist-induced phosphorylation of GATA4. Infection of cultured cardiomyocytes with an activated MEK1-expressing adenovirus induced robust phosphorylation of serine 105 in GATA4, while a dominant-negative MEK1-expressing adenovirus blocked agonist-induced phosphorylation of serine 105, implicating extracellular signal-regulated kinase (ERK) as a GATA4 kinase. Indeed, bacterially purified ERK2 protein directly phosphorylated purified GATA4 at serine 105 in vitro. Phosphorylation of serine 105 enhanced the transcriptional potency of GATA4, which was sensitive to U0126 (MEK1 inhibitor) but not SB202190 (p38 inhibitor). Phosphorylation of serine 105 also modestly enhanced the DNA binding activity of bacterially purified GATA4. Finally, induction of cardiomyocyte hypertrophy with an activated MEK1-expressing adenovirus was blocked with a dominant-negative GATA4-engrailed-expressing adenovirus. These results suggest a molecular pathway whereby MEK1-ERK1/2 signaling regulates cardiomyocyte hypertrophic growth through the transcription factor GATA4 by direct phosphorylation of serine 105, which enhances DNA binding and transcriptional activation.

High-throughput genomic and proteomic analysis using microarray technology.

BACKGROUND: High-density microarrays are ideally suited for analyzing thousands of genes against a small number of samples. The next step in the discovery process is to take the resulting genes of interest and rapidly screen them against thousands of patient samples, tissues, or cell lines to further investigate their involvement in disease risk or the response to medication. METHODS: We used a microarray technology platform for both single-nucleotide polymorphisms (SNPs) and protein expression. Each microarray contains up to 250 elements that can be customized for each application. Slides contained either a 16- or 96-microarray format (4000-24,000 elements per slide), allowing the corresponding number of samples to be rapidly processed in parallel. RESULTS: Results for SNP genotyping and protein profiling agreed with results of restriction fragment length polymorphism (RFLP) analysis or ELISA, respectively. Genotyping analyses, using the microarray technology, on large sample sets over multiple polymorphisms in the NAT2 gene were in full agreement with traditional methodologies, such as sequencing and RFLP analysis. The multiplexed protein microarray had correlation coefficients of 0.82-0.99 (depending on analyte) compared with ELISAs. CONCLUSIONS: The integrated microarray technology platform is adaptable and versatile, while offering the high-throughput capabilities needed for drug development and discovery applications.

Simultaneous multianalyte ELISA performed on a microarray platform.

BACKGROUND: A logical progression of the widely used microtiter plate ELISA is toward a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. Here we describe the construction and use of such a multiplex ELISA to measure prostate-specific antigen (PSA), alpha1-antichymotrypsin-bound PSA (PSA-ACT), and interleukin-6 (IL-6). METHODS: We silanized glass plates and printed the appropriate capture antibodies to allow for the construction of "sandwich" ELISA quantification assays. We examined specificity of the assay for appropriate antigen, assembled calibration curves, and obtained PSA concentrations for 14 human serum samples. We compared the serum PSA concentrations derived through the use of our array with values obtained independently using a standard ELISA method. RESULTS: R2 values generated by our microarray for the PSA and PSA-ACT calibration curves were 0.989 and 0.979, respectively. Analyte concentrations used for the construction of these curves were 0.31-20 microg of protein/L of diluent. IL-6 calibration curve concentrations were 4.9-300 ng of IL-6/L of diluent. The R2 value for the IL-6 calibration curve was 0.983. The 14 human serum samples screened by this micro-ELISA technique for PSA concentrations generated a regression equation (linear) with a slope of 0.83 +/- 0.10 and intercept of 0.74 +/- 0.70 (R2 = 0.88). CONCLUSIONS: Multiplexed ELISA arrays are a feasible option for analyte quantification in complex biologic samples.

DNA microarrays based on noncovalent oligonucleotide attachment and hybridization in two dimensions.

Short oligonucleotide probes have been linked to a solid support by simple electrostatic adsorption onto a positively charged surface film. Attachment was obtained by microfluidic application of unmodified oligonucleotides in distilled water onto amino-silanized glass. It has been demonstrated that an extremely stable monolayer of oligonucleotide is obtained by this method, at a density of about 10(11) molecules/mm(2), which approaches the limit expected for a two-dimensional closest-packed array. Application of oligonucleotide by adsorption is followed by capping with acetic anhydride in the vapor phase, and then capping with succinic anhydride in solution to form a surface with weak negative charge. The capping method has been successfully employed for microarray fabrication and for the analysis of single nucleotide polymorphisms in the k-ras gene. The data reveal that, subsequent to capping, the adsorptive association of oligonucleotide to the surface yields a probe layer which is capable of single nucleotide base mismatch discrimination and high apparent binding affinity.

Nearly instantaneous, cation-independent, high selectivity nucleic acid hybridization to DNA microarrays.

Hybridization rate enhancement has been demonstrated for high molecular weight DNA target binding to a microarray. Microarrays were fabricated using biotin-modified oligonucleotides complexed with streptavidin (SA), which serves as an attachment to the underlying surface. It is shown that at low salt and pH 5, where SA develops a positive charge, duplex formation becomes at least 80-fold faster than seen under standard conditions, where SA is neutral or anionic. Duplex formation becomes independent of solution state cation concentration in the low pH state, under conditions where specificity remains high. The utility of such applied surface science is discussed.

Managing farming systems for nitrate control: a research review from management systems evaluation areas.

The U.S. Department of Agriculture funded the Management Systems Evaluation Area (MSEA) research project in 1990 to evaluate effectiveness of present farming systems in controlling nitrate N in water resources and to develop improved technologies for farming systems. This paper summarizes published research results of a five-year effort. Most research is focused on evaluating the effectiveness of farming system components (fertilizer, tillage, water control, cropping systems, and soil and weather variability). The research results show that current soil nitrate tests reliably predict fertilizer N needed to control environmental and economic risks for crop production. A corn (Zea mays L.)-soybean [Glycine max (L.) Merr.] rotation usually controls risk better than continuous corn, but both may result in unacceptable nitrate leaching. Reduced tillage, especially ridge-till, is better than clean tillage in reducing risk. Tile drainage controls nitrate in ground water, but discharge may increase nitrate in surface waters. Sprinkler irrigation systems provide better water control than furrow irrigation because quantity and spatial variability of applied water is reduced. Present farming systems have two major deficiencies: (i) entire fields are managed uniformly, ignoring inherent soil variability within a field; and (ii) N fertilizer rates and many field practices are selected assuming normal weather for the coming season. Both deficiencies can contribute to nitrate leaching in parts of most fields.

The cardiac tissue-restricted homeobox protein Csx/Nkx2.5 physically associates with the zinc finger protein GATA4 and cooperatively activates atrial natriuretic factor gene expression.

Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, we tested whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein-protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways.

German inflection: the exception that proves the rule.

Language is often explained as the product of generative rules and a memorized lexicon. For example, most English verbs take a regular past tense suffix (ask-asked), which is applied to new verbs (faxed, wugged), suggesting the mental rule "add -ed to a Verb." Irregular verbs (break-broke, go-went) would be listed in memory. Alternatively, a pattern associator memory (such as a connectionist network) might record all past tense forms and generalize to new ones by similarity; irregular and regular patterns would differ only because of their different numbers of verbs. We present evidence that mental rules are indispensible. A rule concatenates a suffix to a symbol for verbs, so it does not require access to memorized verbs or their sound patterns, but applies as the "default," whenever memory access fails. We find 21 such circumstances for regular past tense formation, including novel, unusual-sounding, and rootless and headless derived words; in every case, people inflect them regularly (explaining quirks like flied out, sabre-tooths, walkmans). Contrary to the connectionist account, these effects are not due to regular words constituting a large majority of vocabulary. The German participle -t applies to a much smaller percentage of verbs than its English counterpart, and the German plural -s applies to a small minority of nouns. But the affixes behave in the language like their English counterparts, as defaults. We corroborate this effect in two experiments eliciting ratings of participle and plural forms of novel German words. Thus default suffixation is not due to numerous regular words reinforcing a pattern in associative memory. Because default cases do not occupy a cohesive similarity space, but do correspond to the range of a symbol, they are evidence for a memory-independent, symbol-concatenating mental operation.

Mitogen-activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin.

Insulin stimulates the activity of mitogen-activated protein kinase (MAPK) via its upstream activator, MAPK kinase (MEK), a dual specificity kinase that phosphorylates MAPK on threonine and tyrosine. The potential role of MAPK activation in insulin action was investigated with the specific MEK inhibitor PD98059. Insulin stimulation of MAPK activity in 3T3-L1 adipocytes (2.7-fold) and L6 myotubes (1.4-fold) was completely abolished by pretreatment of cells with the MEK inhibitor, as was the phosphorylation of MAPK and pp90Rsk, and the transcriptional activation of c-fos. Insulin receptor autophosphorylation on tyrosine residues and activation of phosphatidylinositol 3'-kinase were unaffected. Pretreatment of cells with PD98059 had no effect on basal and insulin-stimulated glucose uptake, lipogenesis, and glycogen synthesis. Glycogen synthase activity in extracts from 3T3-L1 adipocytes and L6 myotubes was increased 3-fold and 1.7-fold, respectively, by insulin. Pretreatment with 10 microM PD98059 was without effect. Similarly, the 2-fold activation of protein phosphatase 1 by insulin was insensitive to PD98059. These results indicate that stimulation of the MAPK pathway by insulin is not required for many of the metabolic activities of the hormone in cultured fat and muscle cells.

Detection of a 60 kDa tyrosine-phosphorylated protein in insulin-stimulated hepatoma cells that associates with the SH2 domain of phosphatidylinositol 3-kinase.

Activation of the tyrosine kinase activity of the insulin receptor by autophosphorylation leads to phosphorylation of cellular substrates on tyrosine. Thus far, the best characterized is the insulin receptor substrate (IRS) 1, which has been proposed to serve as a docking protein for other molecules involved in signal transduction. A number of other proteins that become phosphorylated in response to insulin have been identified, some of which are reported to be tissue-specific. A 60 kDa phosphoprotein has been detected in adipocytes after insulin stimulation [Lavan and Lienhard (1993) J. Biol. Chem. 268, 5921-5928]. We have identified a protein of similar molecular mass in rat hepatoma cells transfected with the human insulin receptor. The 60 kDa protein in hepatoma cells is tyrosine-phosphorylated in response to insulin in a dose-dependent manner, with maximal phosphorylation occurring at 50 nM insulin. Although the dose-response of p60 phosphorylation mirrors that of IRS-1, the time course is slightly slower, with maximal phosphorylation observed 5 min after addition of insulin. Like the adipocyte protein, the 60 kDa protein detected in liver cells binds to the SH2 domain of the p85 regulatory subunit of phosphatidylinositol 3-kinase, but not to other SH2 domains. Binding of p60 to p85 is similar to the interaction between p85 and IRS-1 in that a tyrosine-phosphorylated peptide containing the YVXM motif can inhibit the association. The presence of this 60 kDa tyrosine-phosphorylated protein in adipocytes and hepatoma cells suggests that it represents another important intermediate in the insulin-receptor signal-transduction pathway.

Activation of mitogen-activated protein kinase and phosphatidylinositol 3'-kinase is not sufficient for the hormonal stimulation of glucose uptake, lipogenesis, or glycogen synthesis in 3T3-L1 adipocytes.

The precise mechanism by which insulin regulates glucose metabolism is not fully understood. However, it is known that insulin activates two enzymes, phosphatidylinositol 3'-kinase (PI 3'-K) and mitogen-activated protein kinase (MAPK), which may be involved in stimulating the metabolic effects of insulin. The role of these enzymes in glucose metabolism was examined by comparing the effects of insulin, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in 3T3-L1 adipocytes. Treatment of the cells with PDGF or EGF for 5 min increased the MAPK activity 3-5-fold, while insulin treatment produced a 2.5-fold increase. The MAPK activity remained elevated for 1 h after either PDGF or insulin treatment. PDGF and insulin, but not EGF, caused a transient increase in the amount PI 3'-K activity coprecipitated with tyrosine phosphorylated proteins. Although PDGF and insulin caused a similar increase in the activities of these two enzymes, only insulin caused substantial increases in glucose utilization. Insulin increased the transport of glucose and the synthesis of lipid 4- and 17-fold, respectively, while PDGF did not affect these processes significantly. Glycogen synthesis was increased 15-fold in response to insulin and only 3-fold in response to PDGF. Thus, the activation of MAPK and PI 3'-K are not sufficient for the complete stimulation of glucose transport, lipid synthesis, or glycogen synthesis by hormones in 3T3-L1 adipocytes, suggesting a requirement for other signaling mechanisms that may be uniquely responsive to insulin.

Glycosylation sites encoded by exon 2 of the human insulin receptor gene are not required for the oligomerization, ligand binding, or kinase activity of the insulin receptor.

Asparagine-linked glycosylation of the insulin receptor is required for complete biosynthesis and acquisition of function. However, the relative role of each individual glycosylation site has not been elucidated. Previously, it has been shown that removal, by site-directed mutagenesis, of the four amino terminal glycosylation sites (N16,N25,N78, and N111) results in a mutant insulin receptor that remained in the endoplasmic reticulum as an unprocessed proreceptor (Collier E., Carpentier J.-L., Beitz L., Caro L. H. P., Taylor S. I., and Gorden P. [1993] Biochemistry 32, 7818-7823). In the present study, the contribution of these independent glycosylation sites to dimerization and insulin binding has been evaluated. Chinese hamster ovary cells were transfected with the wild-type human insulin receptor cDNA, or cDNA that had Q substituted for N at one, two, or all four of these glycosylation sites. Electrophoretic characterization of the proteins immunoprecipitated from 35S-labeled cells showed that both the wild-type and the quadruple mutant receptor had similar profiles, indicating that the mutant receptor is capable of undergoing dimerization. Analysis of the biochemical properties of this mutant showed that this receptor binds insulin, but ligand binding does not result in kinase stimulation. We demonstrated that the absence of kinase activation is not a property of the mutated receptor since the wild-type proreceptor behaves in a similar manner. Only partial glycosylation in this region of the receptor is required for its targeting to the cell membrane since single and double glycosylation mutants were found processed to their alpha and beta subunits on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

[Shock and coagulation disorders in systemic mastocytosis]. / Schock und Gerinnungsstörungen bei systemischer Mastozytose.

A 41-year-old man was admitted in circulatory shock of unknown aetiology (systolic pressure 50 mm Hg) and marked reddening of the upper part of the body as well as maculopapular rash over the whole body. After 1500 ml of colloidal solution had been infused the blood pressure rose to a level at which the patient's condition was no longer at risk. He reported having had a similar attack of flushing and circulatory collapse during the last few years, each time after drinking 3-41 of beer. Laboratory tests showed thromboplastin time 56%, partial thromboplastin time 130 s and thrombin time > 180 s. Three hours after admission the coagulation times had further deteriorated, but had become normal within 20 hours. After rest and after a provocation (hot bath) the serum concentrations were: heparin 0.21 U/ml and 0.85 U/ml; histamine 1.9 micrograms/ml and 2.3 micrograms/ml; serotonin 23 micrograms/ml and 38 micrograms/ml. Histological examination of an iliac crest bone marrow biopsy revealed dense collections of mast cells, as seen in systemic mastocytosis. A skin biopsy was diagnostic of urticaria pigmentosa.