Automatic control of the extra-corporal bypass: system analysis, modelling and evaluation of different control modes.

Automatic control of the blood gas parameters during extracorporeal circulation has the potential to improve the quality of this procedure and to relieve the personnel from a time consuming task. This paper describes a model of the underlying system for a standard clinical set-up and pinpoints the major difficulties which are the variations of the process gains and the blood- and gas-flow dependent dead times and time constants. Scheduled PI-controllers both for the arterial oxygen as well as for the carbon dioxide partial pressure were designed. Scheduling was based on the blood flow rate. These controllers were tested in a simulation environment. The control systems remained stable under all tested operating condition, but if the blood flow rate was changed abruptly rather large load errors occurred. The performance was improved markedly by adding a feed-forward control path which directly influences the actuating signals based on the actual blood flow rate and the hemoglobin contents, variables which are measured anyway. The major conclusion of this study is to use such direct feed-forward compensation even if more sophisticated control algorithms are used.     

Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae. Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis.

Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic alpha1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5'-phosphate-containing subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structure-function assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg-->Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 degrees C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mm of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.     

Alteration of tobacco floral organ identity by expression of combinations of Antirrhinum MADS-box genes

Floral organ identity is largely controlled by the spatially restricted expression of several MADS-box genes. In Antirrhinum majus these organ identity genes include DEF, GLO and PLE . Single and double mutant analyses indicated that the type of organ found in a particular whorl is dependent on which combination of these genes is expressed there. This paper reports the ectopic expression of Antirrhinum organ identity genes, alone and in combinations, in transgenic tobacco. Although the phenotypes are broadly in agreement with the genetic predictions, several unexpected features are observed which provide information concerning the action of the organ identity genes. The presumed tobacco homologue of DEF, NTDEF , has been isolated and used to investigate the influence of ectopic expression of the Antirrhinum organ identity genes on the endogenous tobacco genes. Analysis of the spatial and temporal expression patterns of NTDEF and NTGLO reveals that the boundaries are not coincident and that differences exist in the regulatory mechanisms of the two genes concerning both induction and maintenance of gene expression. Evidence is provided which indicates that organ development is sensitive to the relative levels of organ identity gene expression. Expression of the organ identity genes outside the flower or inflorescence produced no effects, suggesting that additional factors are required to mediate their activity. These results demonstrate that heterologous genes can be used to predictably influence floral organ identity but also reveal the existence of unsuspected control mechanisms.     

Antigen retrieval,signal amplification and intensification in immunohistochemistry

In this overview we emphasize new methods of improving immunohistochemical results in formaldehyde-fixed tissue samples. The benefit of heat-induced antigen retrieval in demasking of concealed epitopes is demonstrated. We provide guidance on the influence of heat-induced antigen retrieval in commonly applied monoclonal and polyconal antibodies. Moreover, we show the promising methods of signal amplification using biotinylated tyramine and signal intensification of diaminobenzidine reaction products by metallic ions.     

Preparation of Insect Chromosomes for Immunolabeling

We describe a method for isolating chromosomes from testes of the desert locust, Schistocerca gregaria, and their subsequent incubation with antibodies directed against chromosomal proteins. The procedure involves hypotonic pretreatment of the germ cells, centrifugation onto coverslips in a cytocentrifuge and immunolabeling, while still unfixed, using a chromatin-stabilizing buffer. In the present case, an antibody specific for the acetylated isoforms of his tone H4 was tested. After the antibody treatment, the preparations are fixed using formaldehyde, stained with a DNA-specific fluorescent dye and mounted. Analysis of the preparations revealed good preservation of chromosome structure in prophase spermatogonia and late prophase I spermatocytes. Fully condensed chromosomes were not observed and are probably lost during preparation. The bright fluorescence of the autosomes indicates that the reaction between the antibody against acetylated histone H4 and its chromosomal antigen is not impeded. In contrast, the X univalent remained unstained with the exception of a small terminal band. Thus, cytospin preparations of locust germ cells allow high resolution immunolabeling with antibodies against chromosome-associated proteins.     

Bioenergetics of Carbon Assimilation in Intact Chloroplasts: Coupling of Proton to Electron Transport at the Ratio H+/e=3 Is Incompatible with H+/ATP=3 in ATP Synthesis

During a transition from aerobic to largely anaerobic conditionslight-saturated carbon assimilation of intact chloroplasts wasnot decreased although both the transthylakoid proton gradientand ATP levels declined. After a dark period under anaerobiosis,illumination failed to initiate carbon assimilation. ATP increasedonly transiently in the light and then returned to the darklevel. Under such conditions, the addition of electron acceptorssuch as oxygen, oxalacetate or nitrite resulted in the increaseof ATP levels and carbon assimilation was initiated. Assimilationcontinued under anaerobiosis in the presence of reduced protongradients and reduced ATP levels after electron acceptors addedin addition to bicarbonate were reduced. Cyclic electron transport was inhibited when anaerobiosis didnot permit linear electron transport. It was induced in thissituation by micromolar concentrations of oxygen or when, underanaerobiosis, DCMU decreased PSII activity. Oxygen inhibitedcyclic electron transport by draining electrons from the cyclicpathway only when electron donation from PSII was weak. Theobservations give evidence of the delicate redox balance requiredfor cyclic electron transport. Since H⁺/e=3 in linear electron transport, the observationsof effective carbon reduction under a decreased transthylakoidproton gradient and decreased levels of ATP are incompatiblewith H⁺/ATP=2 or 3. They are compatible with H⁺/ATP=4. (Received May 1, 1995; Accepted October 3, 1995)     

Purification and Properties of Manganese Superoxide Dismutase from Norway Spruce (Picea abies L. Karst)

A manganese-containing superoxide dismutase (SOD; EC 1.15.1.1 [EC] )was purified to electrophoretic homogeneity from seeds of Norwayspruce (Picea abies L.). The apparent molecular mass of thepurified enzyme was 86 kDa, as determined by gel filtration.The subunit molecular mass, estimated by SDS-polyacrylamidegel electrophoresis, was 22 kDa both in the presence and inthe absence of 2-mercaptoethanol. Thus, the native enzyme isa homotetramer with subunits that were not linked by disulfidebonds. The isoelectric point of this Mn-SOD was 5.5. The specificactivity of the Mn-SOD was strongly pH-dependent and was 400units per nmol SOD at pH 7.8 and 30 units per nmol SOD at pH10.4. The first 25 amino acid residues in the amino terminalregion of spruce Mn-SOD exhibited a high degree of sequencehomology to those of Mn-SODs from other organisms. In Mn-deficientneedles the activity of Mn-SOD was only half of that in non-deficientneedles, whereas the activity of CuZn-SOD was doubled. (Received May 20, 1994; Accepted October 31, 1994)     

Sequence analysis of the complete mitochondrial DNA molecule of the hedgehog,Erinaceus europaeus,and the phylogenetic position of the Lipotyphla

The sequence of the mitochondrial DNA (mtDNA) molecule of the European hedgehog (Erinaceus europaeus) was determined. The length of the sequence presented is 17,442 nucleotides (nt). The molecule is thus the largest eutherian mtDNA molecule so far reported. The organization of the molecule conforms with that of other eutherians, but the control region of the molecule is exceptionally long, 1,988 nt, due to the presence of repeated motifs at two different positions in the 3 part of the control region. The length of the control region is not absolute due to pronounced heteroplasmy caused by variable numbers of the motif TACGCA in one of the repetitive regions. The sequence presented includes 46 repeats of this type. The other repeated region is composed of different AT-rich repeats. This region was identical among four clones studied. Comparison of mitochondrial peptide-coding genes identified a separate position of the hedgehog among several mammalian orders. The concatenated protein sequence of the 13 peptide-coding genes was used in a phylogenetic study using the opossum as outgroup. The position of the hedgehog sequence was basal among the other eutherian sequences included: human, rat, mouse, cow, blue whale, harbor seal, and horse. The analysis did not resolve the relationship among carnivores, perissodactyls, and artiodactyls/cetaceans, suggesting a closer relationship among these orders than acknowledged by classical approaches. Correspondence to: U. Arnason