Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products

The genes, rpoA, rpoB and rpoC of Escherichia coli, which encode the RNA polymerase alpha-, beta- and beta'-subunits, respectively, have been individually placed on expression plasmids under the control of the bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in host cells harboring each of the three plasmids resulted in the extensive overproduction of the three polypeptides. The overproduced subunits were purified and assembled into a functional enzyme, whose specific activity and dependence on the sigma-factor were indistinguishable from native RNA polymerase purified by conventional methods.     

Water Balance in Cucumis Plants, Measured by Nuclear Magnetic Resonance, I

In this paper we demonstrate the study of plant water balanceby the non-invasive measurement of tissue water content andwater flow using proton nuclear magnetic resonance (NMR). Sapvelocity and flux were measured independently in the presenceof an excess of stationary tissue water. The instrumentationdescribed allows automated and unattended measurement of flow-and water content-variables in a well-defined region of theplant over periods of several days, with a time resolution betweensuccessive measurements of c. 5 s. Using this apparatus theeffect of changes in light intensity (day/night rhythm) andrelative humidity on stem tissue water content as well as onthe velocity and flux of xylem sap in the stem were investigatedin a cucumber plant. The results are in agreement with predictionsfrom a simple model for plant water balance, which is basedon water potential, flow rate and resistance to flow. As longas only transpiration is varied, flow rate and water content(or potential) are affected in opposite ways as demonstratedin this paper. In contrast, the model predicts that changesin uptake (resulting from changes in, for example, root resistance)will induce changes in water content and flow in the same direction.An experimental verification of this prediction is given ina subsequent paper, where, in addition, the NMR results arecompared to those obtained with a dendrometer. Key words: Water balance model, Cucumis sativus L., flow, water content, NMR, water balance measurement     

Constant-flow ventilation in pigs

Constant-flow ventilation (CFV) is a ventilatory technique in which physiological blood gases can be maintained in dogs by a constant flow of fresh gas introduced via two catheters placed in the main-stem bronchi (J. Appl. Physiol. 53: 483-489, 1982). High-velocity gas exiting from the catheters can create uneven pressure differences in adjacent lung segments, and these pressure differences could lead to gas flow through collateral channels. To examine this hypothesis, we studied CFV in pigs, animals known to have a high resistance to collateral ventilation. In three pigs we examined steady-state gas exchange, and in six others we studied unsteady gas exchange at three flow rates (20, 35, and 50 l/min) and three catheter positions (0.5, 1.5, and 2.5 cm distal to the tracheal carina). During steady-state runs we were unable to attain normocapnia; the arterial CO2 partial pressure (PaCO2) was approximately 300 Torr at all flow rates and all catheter positions, compared with 20-50 Torr at similar flows and positions in dogs studied previously. The initial unsteady gas-exchange experiments indicated no consistent effect of catheter position or flow rate on the rate of rise of PaCO2. In three other pigs, the rates of rise of PaCO2 were compared with the rates observed with apneic oxygenation (AO). At the maximum flow and deepest position, the rate of rise of PaCO2 was lower during CFV than during AO. These data suggest that flow through collateral channels might be important in producing adequate gas transport during CFV; however, other factors such as airway morphometry and the effects of cardiogenic oscillations may explain the differences between the results in pigs and dogs.     

Simulation of gas transport due to cardiogenic oscillations

We simulated gas transport due to cardiogenic oscillations (CO) using a model developed to quantify the gas mixing due to high-frequency ventilation (16). The basic components of the model are 1) gas mixing by augmented transport, 2) symmetrical lung morphometry, and 3) a Lagrangian (moving) reference frame. The theoretical predictions of the model are in general agreement with published experimental studies that have examined the effect of CO on the nitrogen concentration obtained by intrapulmonary gas sampling and the effect of CO on regional and total anatomical dead space. Further, the model predicts that augmentation of gas transport due to CO is less, nearer to the alveolar regions of the lung, and that the effect of CO during normal tidal breathing is negligible, but that CO may contribute up to approximately 10% of the alveolar ventilation in patients with severe hypoventilation. The agreement between experimental and theoretical results suggests that it may not be necessary to invoke gas transport mechanisms specific to an asymmetrical bronchial tree to explain the major proportion of gas transport due to CO.     

Contribution of cardiogenic oscillations to gas exchange in constant-flow ventilation

The contribution of cardiogenic oscillations to gas exchange during constant-flow ventilation was examined in 11 dogs. With the use of two variations of cardiopulmonary bypass to maintain the systemic and pulmonary circulation, the influence of cardiogenic oscillations was removed by arresting the heart. Cardiac arrest by ventricular fibrillation was associated with a mean decrease in alveolar ventilation of 43% in five dogs on right and left heart bypass. However, successful defibrillation and return of the prearrest level of alveolar ventilation could not be achieved; thus we studied six dogs on left heart bypass. Alveolar ventilation decreased an average of 37% with cardiac arrest, and defibrillation resulted in a return of alveolar ventilation to 81% of the prearrest value. These results are consistent with previous predictions that cardiogenic oscillations are an important mechanism of gas transport during constant-flow ventilation.     

TASTE GROUP THRESHOLDS AND SENSORY EVALUATION OF SPANISH WINE VINEGARS

Wine vinegar is a product obtained from wine acidification which contains at least 5% by wt. of acetic acid, in general without any additives or colorings.
Aspects studied in this work include: the determination of the taste group thresholds (geometric mean of the individual best-estimate thresholds "BET") of two different acids (citric and acetic acids) in aqueous solution and spanish vinegars produced from table and sherry wines. The results obtained suggest that wine vinegar can be considered something more than just an acidulant agent.
In order to evaluate differences among wine vinegars, discriminant tests for twenty-five spanish vinegars (sherry, table and flavored vinegars) were applied. Six of the twelve attributes freely chosen by assessors allowed grouping of the spanish wine vinegars according to their sensory aspects.     

Arginine deprivation in KB cells: I. Effect on cell cycle progress

When exponentially growing KB cells were deprived of arginine, cell multiplication ceased after 12 h but viability was maintained throughout the experimental period (42-48 h). Although tritiated thymidine ([(3)H]TdR) incorporation into acid-insoluble material declined to 5 percent of the initial rate, the fraction of cells engaged in DNA synthesis, determined by autoradiography, remained constant throughout the starvation period and approximately equal to the synthesizing fraction in exponentially growing controls (40 percent). Continous [(3)H]TdR-labeling indicated that 80 percent of the arginine-starved cells incorporated (3)H at some time during a 48-h deprivation period. Thus, some cells ceased DNA synthesis, whereas some initially nonsynthesizing cells initiated DNA synthesis during starvation. Flow microfluorometric profiles of distribution of cellular DNA contents at the end of the starvation period indicated that essentially no cells had a 4c or G2 complement. If arginine was restored after 30 h of starvation, cultures resumed active, largely asynchronous division after a 16-h lag. Autoradiographs of metaphase figures from cultures continuously labeled with [(3)H]TdR after restoration indicated that all cells in the culture underwent DNA synthesis before dividing. It was concluded that the majority of cells in arginine-starved cultures are arrested in neither a normal G1 nor G2. It is proposed that for an exponential culture, i.e. from most positions in the cell cycle, inhibition of cell growth after arginine with withdrawal centers on the ability of cells to complete replication of their DNA.     

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na⁺] and [K⁺] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na⁺‐selective class I‐HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single‐nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near‐isogenic lines (NILs): 157‐14 (double homozygote for the cheesmaniae alleles) and 157‐17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157‐14 in comparison with 157‐17. A significant difference between NILs was also found for [K⁺] and the [Na⁺]/[K⁺] ratio in leaf and stem but not for [Na⁺] arising a disagreement with the corresponding RIL population. Their association with leaf [Na⁺] and salt tolerance in tomato is also discussed.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.