Production and purification of Escherichia coli fimbrial antigen F165

Abstract The production of fimbrial antigen F165 by Escherichia coli strains was found to be dependent on the composition of the culture medium and was repressed in the presence of alanine or high levels of glucose, in anaerobic conditions or at growth temperatures of lower than 37°C. Optimal F165 production was found on a minimal medium containing 1% (w/v) casamino acids (MD-1). F165 antigen was isolated from bacteria by mechanical shearing, precipitated with ammonium sulfate, and purified by deoxycholate treatment and gel filtration on Superose 12. The purified fimbriae retained their native morphology as observed by electron microscopy and consisted of two separate protein subunits with apparent molecular weights of 17 500 and 19 000 on sodium dodecyl sulfate-polyacrylamide gels.     

Gravitropism in Higher Plant Shoots : V. Changing Sensitivity to Auxin

An alternative to the Cholodny-Went, auxin-transport hypothesis of gravitropic stem bending was proposed as early as 1958, suggesting that gravistimulation induces changes in sensitivity to auxin, accounting for differential growth and bending. To test the sensitivity hypothesis, we immersed marked, decapitated sunflower (Helianthus annuus L.) hypocotyl sections in buffered auxin solutions over a wide concentration range (0, 10⁻⁸ to 10⁻² molar IAA), photographed them at half-hour intervals, analyzed the negatives with a digitizer/computer, and evaluated surface-length changes in terms of Michaelis-Menten enzyme kinetics. Bending decreases with increasing auxin concentration; above about 10⁻⁴ molar IAA the hypocotyls bend down; increasing auxin inhibits elongation growth of lower surfaces (which is high at zero or relatively low auxin levels) but promotes upper-surface growth (which is low at low auxin levels). Thus, lower surfaces have a greater K_m sensitivity to applied auxin than upper surfaces. At optimum auxin levels (maximum growth), growth of bottom surfaces exceeds that of top surfaces, so bottom tissues have a greater V_max sensitivity. V_max sensitivity of vertical controls is slightly lower than it is for either horizontal surface; K_m sensitivity is intermediate. Clearly, gravistimulation leads to significant changes in tissue sensitivity to applied auxin. Perhaps these changes are also important in normal gravitropism.     

Recent Advances in Geriatric Dentistry: Implications for Clinical Practice1

The clinical applications of recent research advances in oral health are discussed using a modified case presentation. Conversely, clinical care often highlights the research questions that must be answered to adequately address the oral health problems of the elderly. It is stressed that science transfer is and will continue to be necessary to take the findings from the basic and clinical research arena to the practice arena.     

Intercalation of anthracyclines into living cell DNA analyzed by flow cytometry.

Anthracyclines (ANT) are used in the treatment of leukemia and other cancers. These drugs have been shown to intercalate between the strands of DNA. In the present study, we show that the amount of ANT intercalated into DNA can be determined by measuring the fluorescence resonance energy transfer (FRET) between Hoechst 33342 (H33342) and ANT bound to DNA. The transfer efficiency was found to depend on the amount of disposable ANT but was independent of the amount of H33342 bound to DNA over a wide range of H33342 concentrations. The method was adapted for flow cytometric measurement of FRET in whole living cells and was used to evaluate the degree of intercalation of daunorubicin (DAU) and idarubicine (IDA) into DAU-sensitive and DAU-resistant leukemic cell lines. ANT intercalation into DNA was affected by factors which modify the intracytoplasmic concentration of ANT, and it was shown that the action of ANT and the resistance to ANT could not be attributed solely to the intercalative effect of the drugs. The method has advantages over previously described methods and represents a useful complementary tool in studies on the mode of action of ANT and the mechanisms of chemoresistance.     

Insertional inactivation of a gene which controls expression of vancomycin resistance on plasmid pHKK100

Expression of inducible high level vancomycin resistance (Vmr) in enterococci appears to require other plasmid-encoded genes in addition to the previously described structural genes vanA and vanH. Tn917 mutagenesis was used to identify such a region in the Vmr plasmid pHKK100. Insertional inactivation of a 693-bp open reading frame upstream from vanH resulted in complete loss of Vmr. This putative 26,642-Da protein has been designated VanR.     

The limits of the cellular capacity to mediate an estrogen response.

While steroid response is generally restricted by the availability of steroid receptors, the theoretical limits of the response are not known. We have constructed a series of cell lines that stably express the estrogen receptor (ER) at levels up to 5,000,000 ERs per cell and employed these cells to explore the limits of the estrogen response. Several reporter genes with estrogen response elements upstream of the herpes thymidine kinase promoter showed hyperbolic saturation kinetics with increasing ER. Maximum response was 10 times that seen in cell lines with receptor titers comparable to physiological levels. Half-maximal responses required 500,000 receptors per cell, and cells with 5,000,000 ERs showed greater than 90% maximum induction. Estradiol dose-response studies indicated that the receptors are limiting below 500,000 ERs per cell, but at higher ER titers there are spare receptors. In contrast to most reporters, the widely used reporter pA2-CAT, which has 200 base pairs of Xenopus vitellogenin DNA between the response element and the promoter, showed squelching at ER levels beyond 500,000 per cell. Cell lines that expressed ER above this level activated pA2-CAT with a distorted hormone dependence, where saturating ligand concentrations were inhibitory. All reporters displayed squelching when the ER was provided by transient transfection at a level that we judge is 20,000,000 per cell by extrapolation from the behavior of stable cell lines. These findings suggest that saturation of the cellular capacity to mediate an estrogen response and ER-dependent squelching occur at receptor titers well above those encountered in nature. If current models of steroid hormone action are correct, the findings also imply that estrogen response elements are occupied to very small extents under normal conditions.     

The problem of the acts duration measurement in relation to the “focal-animal” technique of behavioral recording

The focal-animal technique assumes the continuous recording of the behavior of an individual during a certain time interval. The length of this interval (sampling unit) can be problematic when estimating the duration of behavioral acts. Two acts from the behavioral repertoire of the ant Leptothorax fuenteiwere focused on in this work at different ranges of temporal scales. Analyzing these acts we show the possibility of existence of a sampling artifact, in such a way that the estimates of the durations of the acts would be forced to depend upon the length of the sampling unit that is being used.     

Effect of diisopropylfluorophosphate on muscarinic and gamma-aminobutyric acid receptors in visual cortex of cats.

Administration of diisopropylfluorophosphate (DFP), an organophosphorus (OP) compound, irreversibly inhibits acetylcholinesterase (AChE) and results in cholinergic hyperactivity. This study investigated muscarinic and gamma-aminobutyric acid (GABA) receptor changes in visual cortex of cats following an acute exposure to DFP. A single acute administration of DFP (4 mg/kg) decreased the number of muscarinic receptors at 2, 10, and 20 hours after treatment. GABA receptors were elevated at 2 and 10 hours but returned to within control levels at 20 hours. No significant alteration in muscarinic or GABA receptor affinity was noted. In all cases cortical AChE activity was inhibited 60-90%. These findings show a down regulation of muscarinic receptors after DFP associated with low AChE activity. GABA receptors also are altered, and may be part of a compensatory mechanism to counteract excess cholinergic stimulation.     

Action of endogenous prostaglandins on postprandial pyloric motility: a possible modulation by fats.

The involvement of endogenous prostaglandins (PGs) and the effect of exogenous PGs on the myoelectrical activity of the pylorus were examined for 6 hours after a meal in dogs chronically fitted with intraparietal electrodes on the gastroduodenal junction. The animals received either a standard meal or a fat meal which consisted of canned food added or not (standard meal) with arachis oil. The cyclooxygenase inhibitors, indomethacin (1 mg/kg) and piroxicam (0.2 mg/kg) given prior a fat meal significantly increased the frequency of pyloric spike bursts but did not modify the pyloric motility associated with a standard meal. Synthetic derivatives of PGE1 (misoprostol, 5-10 micrograms/kg) or PGE2 (enprostil 0.5-1 micrograms/kg) reduced the frequency of pyloric contractions after a fat but not a standard meal. It is suggested that both endogenous and exogenous prostaglandins may modulate postprandial pyloric motility when fats are present in sufficient amount into the meal.