Thigmotropism and stretch-activated channels in the pathogenic fungus Candida albicans.

The direction of growth of hyphae of the pathogenic fungus Candida albicans responds thigmotropically to surface contours by following scratches, ridges and grooves and by penetrating pores. Here it is shown that the thigmotropic response to ridges is attenuated by GdCl3 and verapamil [blockers of stretch-activated (SA) ion channels and L-type calcium channels, respectively]. At low concentrations, both compounds reduced the percentage of hyphae reorienting on contact with a ridge without markedly affecting hyphal extension rate, suggesting a possible role for SA or other calcium channels in the transduction of the thigmotropic response. In addition, patch-clamp recordings demonstrated SA channel activity in the plasma membrane of both yeast and hyphal cells of C. albicans. Two distinct SA channels with conductances of 54 pS and 20-25 pS in 200 mM KCl were observed in protoplasts from yeast cells and one channel of 51 pS was found in protoplasts from hyphal cells.

Altered adherence in strains of Candida albicans harbouring null mutations in secreted aspartic proteinase genes.

The aspartate proteinase inhibitor pepstatin A has been shown previously to reduce the adherence of Candida albicans yeast cells to human surfaces. This suggests that in addition to their presumed function facilitating tissue penetration, the secreted aspartate proteinases (Saps) of this fungal pathogen may have auxiliary roles as cellular adhesins. We therefore examined the relative adherence of yeast cells of a parental wild-type strain of C. albicans in relation to yeast cells of strains harbouring specific disruptions in various members of the SAP gene family in an otherwise isogenic background. The adhesiveness of delta sap1, delta sap2, delta sap3 null mutants and a triple delta sap 4-6 disruptant was examined on three surfaces--glass coated with poly-L-lysine or a commercial cell-free basement membrane preparation (Matrigel) and on human buccal epithelial cells. Pepstatin A reduced adherence to all surfaces. Adherence of the each of the single SAP null mutants to these three substrates was either reduced or not affected significantly compared to that of the parental strain. The adherence of the delta sap4-6 mutant was reduced on poly-L-lysine and Matrigel, but increased on buccal cells. The results suggest that in addition to a primary enzymatic role, various SAPs may also act singly or synergistically to enhance the adhesiveness to C. albicans cells to certain human tissues.

Real-time detection and quantification of DNA hybridization by an optical biosensor.

The use of an optical biosensor, the resonant mirror, for direct and rapid detection of DNA-DNA hybridization has been demonstrated. Biotinylated oligonucleotide probes were immobilized on the sensor surface, via streptavidin, and the hybridization of a complementary target oligonucleotide (40-mer) was monitored in real time. The interaction at the sensor surface was shown to be sequence specific under conditions of low stringency. Regeneration of the surface-immobilized probe was possible, allowing reuse without a significant loss of hybridization activity. A comparison of probes indicated that the relative position of complementary sequence and the length of probe affected the hybridization response obtained. The potential of the sensor for quantitation of a hybridized DNA target was investigated. From radiolabeling data, the lowest amount of hybridized target sequence which could be determined directly was 19.9 fmol/mm2 (263 pg/mm2) of sensor surface. The dependence of the sensor response on the concentration of probe and target oligonucleotide was established. Utilizing the assay as an end-point determination method, the lowest detectable concentration of target oligonucleotide (40-mer) was 9.2 nM. This compares favorably to other sensor methods described previously without the requirement for labels.

Optical biosensor for monitoring microbial cells.

The potential of a new optical biosensor, the resonant mirror, for detecting whole cells is demonstrated. Staphylococcus aureus (Cowan-1) cells, which express protein-A at their surface, were detected by binding to human immunoglobulin G (IgG) immobilized on an aminosilane-derivatized sensor surface at concentrations in the range 8 x 10(6)-8 x 10(7) cells/mL. A control S. aureus strain (Wood-46), which does not express protein-A, gave no significant response. Immobilization of the capture ligand on aminosilane surfaces with and without a hydrogel coating of carboxymethyl-dextran was compared. The greatest binding response was observed with non-dextran-coated surfaces. The sensitivity of the technique was increased a 1000-fold by using a human IgG-colloidal gold conjugate (30 nm) in a sandwich assay format. S. aureus (Cowan-1) cells were detected in spiked milk samples at cell concentrations from 4 x 10(3)-1.6 x 10(6) cells/mL using the sandwich assay.

Effect of a meal containing protein on lithium clearance and plasma immunoreactive atrial natriuretic peptide in man.

1. The effect of meals with a high and low protein content and of the fasting state on renal function and plasma atrial natriuretic peptide was studied in water-loaded normal volunteers. 2. Creatinine clearance increased after the high protein meal, but did not change after the low protein meal or while fasting. Observations of similar increases in urine sodium and potassium excretion and a transient decrease in urine flow after both meals suggest that the protein content of the meal is not an important contributory factor in these responses to feeding. 3. Absolute delivery of sodium and water out of the proximal tubules (assessed by the lithium clearance method) was higher after both meals than while fasting; fractional lithium clearance was higher after the low protein meal than the high protein meal and while fasting. Absolute reabsorption from proximal tubules was increased after only the high protein meal. 4. A transient decrease in the fraction of water delivered to distal nephron segments that appeared in the urine (fractional distal water excretion) was observed after both meals. Fractional distal sodium excretion and absolute distal sodium and water reabsorption increased after both meals. 5. Since plasma atrial natriuretic peptide either decreased (high protein meal) or remained unchanged (low protein meal and fasting), it is unlikely that this hormone is involved in the hyperfiltration after the high protein meal and the natriuresis after both high and low protein meals.