Quantitative interactions between cryptdin-4 amino terminal variants and membranes

Paneth cells secrete alpha-defensins into the lumen from the base of small intestinal crypts, and cryptdin-4 (Crp4) is the most potent mouse alpha-defensin in vitro. Purified recombinant Crp4 and Crp4 variants with (des-Gly)-, (Gly1Val)-, (Gly1Asp)-, and (Gly1Arg)-substitutions were all bactericidal with Crp4 and (Gly1Arg)-Crp4 being slightly more active than other variants. Bactericidal activities correlated directly with permeabilization of live Escherichia coli, with equilibrium binding to E. coli membrane phospholipid bilayers and vesicles, and with induced graded fluorophore leakage from phospholipid vesicles. The Crp4 peptide N-terminus affects bactericidal activity modestly, apparently by influencing peptide binding to phospholipid bilayers and subsequent permeabilization of target cell membranes.     

Differential effects of stretch and compression on membrane currents and [Na+]c in ventricular myocytes

Mechano-electrical feedback was studied in the single ventricular myocytes. A small fraction (approximately 10%) of the cell surface could be stretched or compressed by a glass stylus. Stretch depolarised, shortened the action potential and induced extra systoles. Stretch activated non-selective cation currents (I_ns) showed a linear voltage dependence, a reversal potential of 0 mV, a pure cation selectivity, and were blocked by 8 μM Gd³⁺ or 30 μM streptomycin. Stretch reduced Ca²⁺ and K⁺ (I_K) currents. Local compression of broadwise attached cells activated I_K but not I_ns. Cytochalasin D or colchicin, thought to disrupt the cytoskeleton, suppressed the mechanosensitivity of I_ns and I_K. During stretch, the cytosolic sodium concentration increased with spatial heterogeneities, local hotspots with [Na⁺]_c>24 mM appeared close to surface membrane and t-tubules (pseudoratiometric imaging using Sodium Green fluorescence). Electronprobe microanalysis confirmed this result and indicated that stretch increased total sodium [Na] in cell compartments such as mitochondria, nuclear envelope and nucleus. Our results obtained by local stretch differ from those obtained by end-to-end stretch (literature). We speculate that channels may be activated not only by axial but also by shear stress, and, that stretch can activate channels outside the deformed sarcomeres via second messenger.     

Sol-gel immobilisation of Saccharomyces cerevisiae enhances viability in organic media

The polyhydroxylated silane network of a sol-gel protected immobilised Saccharomyces cerevisiae against the effects of five organic solvents. The viability of immobilised yeast directly correlated with the logarithm of the partition coefficient of the solvent in an octanol/water two phase system increasing the decimal reduction time (D) and reaching the maximum with octanol, the most hydrophobic solvent assayed. The D value increased from 0.16 min for free yeast to 1.9 and to 22 min for immobilised yeast exposed to ethanol and 1-octanol respectively.     

Development,validation, and applications of a new laboratory-scale indirect impedancemeter for rapid microbial control

We introduce a new laboratory-scale impedance-meter which is specially intended for indirect technique. It consists of a software system enabling data acquisition via a connected bus which is wired to the measuring cells. These measuring cells are individual impedance-meters that can be activated independently of one another. In the current configuration, the device is slightly affected by temperature, but it can register as little as 10.9 micromol of CO(2). With Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae cultures, the conductance responses were highly replicable and repeatable for inocula concentrations of 1-10(8) colony-forming units (CFU) ml(-1). The main use for such devices could be the detection of contamination in foodstuffs. Several of these foodstuffs, when incubated at 37 degrees C, spontaneously release quite large amounts of CO(2). Our impedance meter, however, was able to detect an E. coli presence in canned French beans at 2.35 x 10(-2) CFU ml(-1) and a S. cerevisiae contamination of apple purée in glass jars at 6.1 x 10(-3) CFU ml(-1). The conductance response and the detection time (the time needed for a significant change in conductance) were correlated to the concentration of ampicillin (an antibiotic added to E. coli cultures). The device is thus able to detect the presence of inhibitory compounds in milk or other foodstuffs. Some industrial assays are in process to complement these laboratory tests. Compared with other available techniques for CO(2) measurement (manometry, infrared, radioactive labeling), the technique put forward here appears to be the best compromise between sensitivity, technical constraints, and cost. A commercial version of the impedance meter would enable routine measurements in the quality control of foodstuffs, pharmaceuticals, cosmetics and in R&D laboratories.     

A critical appraisal of vitiligo etiologic theories. Is melanocyte loss a melanocytorrhagy?

Common generalized vitiligo is an acquired depigmenting disorder characterized by a chronic and progressive loss of melanocytes from the epidermis and follicular reservoir. However, the mechanism of melanocyte disappearance has never been clearly understood, and the intervention of cellular and humoral autoimmune phenomena as primary events remains unproven. In this review, is discussed the data supporting the major theories of vitiligo, namely melanocyte destruction (autoimmune, neural and impaired redox status) and melanocyte inhibition or defective adhesion. Based on recent morphologic findings in vivo supporting a chronic detachment and transepidermal loss of melanocytes in common generalized vitiligo, a new theory is suggested proposing melanocytorrhagy as the primary defect underlying melanocyte loss, integrating most of the possible triggering/precipitating/enhancing effects of other known factors.     

Regeneration of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) by a cyclic organogenic system

In a five-step procedure, plants were regenerated from meristematic tissue initiated from nodal tissue in four pea cultivars (Espace, Classic, Solara, and Puget). In step 1, stem tissue with one node (1-cm size) was subcultured on medium containing thidiazuron. As a result multiple shoots were produced, appearing normal or swollen at their bases. The multiple shoots were subcultured in the same medium, resulting in the formation of a green hyperhydric tissue in the swollen bases of the multiple shoots, which is fully covered with small buds [bud-containing tissue (BCT)]. In step 2, BCT fragments were isolated and subcultured in the same medium and, as a result, they were able to reproduce themselves in a cyclic fashion. In step 3, subculture of BCT on medium supplemented with a combination of gibberelic acid, 6-benzyladenine and -naphthalene acetic acid (NAA), resulted in the formation of shoots, which were rooted in step 4 on medium supplemented with 0.5 mg/l NAA, indole-3-acetic acid (IAA) or indole-3-butyric acid. In step 5, in vitro plants were transferred to the greenhouse for acclimatisation and further development. The four varieties tested were all able to produce meristematic tissue, suggesting that its production is genotype independent.     

Sensitivity and variability of visual scoring in the comet assay. Results of an inter-laboratory scoring exercise with the use of silver staining

Nineteen scorers from seven Cuban laboratories participated in this slide exercise designed to test the influence of the scorer on the accuracy, sensitivity and variability of the comet assay when a visual method of DNA damage evaluation is used. The assay was performed using human lymphocytes from a single donor exposed in vitro for 5 min at 0 degrees C to doses of 0, 5, 10, 25, 50, 100 and 200 microM of hydrogen peroxide. Each participant scored the same set of 14 coded slides with silver stained comets. The comets were classified visually into five categories according to the appearance resulting from the relative proportion of DNA in the tail. The extent of DNA damage was expressed in arbitrary units. At zero dose the median values of 12 scorers out of 19 were included between the values of the overall 25 and 75 per thousand. This proportion remains practically the same as the dose increases. The lowest dose detected by this method for the majority of scorers (11) was 10 microM. The coefficient of variation at the control dose was the highest (median value 26%), progressively declined to 20%, and starting from 25 microM, values are around 10%. The results of the exercise show the reliability of the silver staining and visual scoring for the comet method.     

Reclassification of "atypical" diagnoses in endoscopic retrograde cholangiopancreaticography-guided biliary brushings

OBJECTIVE: To evaluate the usefulness of reclassifying "atypical" diagnoses in reporting biliary cytology using strict morphologic criteria. STUDY DESIGN: Cytologic specimens from 139 patients (direct, alcohol-fixed smears or cytocentrifuge preparations) were evaluated. Diagnoses were benign (70), atypical (36) and malignant (33). Using strict criteria--major (nuclear contour, chromatin pattern) and minor (polarity, cell types, nuclear size, nuclear grooves, nucleoli, mitosis, nuclear/cytoplasmic [N/C] ratio)--atypical cases were reevaluated and reclassified. Follow-up (F/U) was available on all cases. RESULTS: Atypical cases, (36) were reclassified as malignant (26), atypical favor benign (2)/reactive (3) and atypical, not otherwise specified (NOS) (5). Cases reclassified as malignant showed irregular nuclear contours, chromatin irregularities and rare mitosis. Nuclear enlargement, nucleoli and cellularity varied widely in all groups. N/C ratio was increased in most reclassified malignant cases. All 26 malignant reclassifications correlated with F/U of malignancy. Benign and reactive cases (5) were negative for malignancy on F/U (4), and in 1 case a metastatic carcinoma involving the biliary tree was found. In the 5 atypical (NOS) cases, F/U showed malignancy (3) and pancreatitis (2). Cytocentrifuge preparations made in our laboratory were of superior quality when compared to other methods of cell preparation. CONCLUSION: Irregularities in nuclear membrane and abnormal chromatin pattern were the most consistently useful features correlating with malignancy. The sensitivity and specificity of biliary brush cytology can be enhanced by using strict cytomorphologic criteria and proper collection and fixation, all of which decrease atypical diagnoses.