Lysis

About: Lysis is a research topic. Over the lifetime, 6072 publications have been published within this topic receiving 216978 citations.

Intracellular localization of the dermonecrotic toxin of Bordetella pertussis.

Abstract: Localization of the heat-labile dermonecrotic toxin of Bordetella pertussis strain 114 grown in chemically defined Stainer-Scholte medium was studied by using skin reaction in 4-day-old suckling mice as the assay for toxin. Through log phase and into stationary phase of growth the toxin was cell associated and not detected in the culture supernatant. Only about 4% of the activity present in a suspension of lysed cells was detected in a suspension of whole cells, and the dermonecrotic activity was not released by subjecting whole cells to osmotic shock, a procedure that releases proteins from the periplasmic space of many gram-negative bacteria. After cell lysis and preparation of soluble and membrane fractions, 73 to 80% of the activity in the cell lysate was recovered in the soluble fraction, with only 3 to 6% present in a membrane fraction. Further evidence for the intracellular cytoplasmic localization of the dermonecrotic toxin was the insensitivity of the toxin to trypsin treatment of whole cells. Treatment of whole cells with trypsin (80 micrograms/ml) for 20 min at 37 degrees C did not decrease dermonecrotic or malate dehydrogenase activities, but did inhibit more than 95% of the extra-cytoplasmic adenylate cyclase activity. Identical trypsin treatment of a cell lysate decreased all the above activities by more than 90%.

Single-Cell Chemical Lysis on Microfluidic Chips with Arrays of Microwells

Abstract: Many conventional biochemical assays are performed using populations of cells to determine their quantitative biomolecular profiles. However, population averages do not reflect actual physiological processes in individual cells, which occur either on short time scales or nonsynchronously. Therefore, accurate analysis at the single-cell level has become a highly attractive tool for investigating cellular content. Microfluidic chips with arrays of microwells were developed for single-cell chemical lysis in the present study. The cellular occupancy in 30-μm-diameter microwells (91.45%) was higher than that in 20-μm-diameter microwells (83.19%) at an injection flow rate of 2.8 μL/min. However, most of the occupied 20-μm-diameter microwells contained individual cells. The results of chemical lysis experiments at the single-cell level indicate that cell membranes were gradually lysed as the lysis buffer was injected; they were fully lysed after 12 s. Single-cell chemical lysis was demonstrated in the proposed microfluidic chip, which is suitable for high-throughput cell lysis.

Spontaneous transformation in Bacillus subtilis

Abstract: Cultures of Bacillus subtilis release transforming DNA during the early exponential and stationary phases of growth. The pattern of release of transforming DNA was followed by measuring transformation in a system consisting of a non-transformable DNA donor and a differently marked transformable recipient. Transformation in this system seems to be at least as efficient as that induced by purified DNA. Fluorescence microscopy revealed that released DNA remained bound extracellular to intact cells. The release of DNA during early exponential growth seemed to be correlated with the cells' proneness to lysis; both DNA release and cell lysis were inhibited by chloramphenicol. In stationary cells, the release of DNA was neither correlated with a similar proneness to lysis nor inhibited by chloramphenicol.

Sidedness of native membrane vesicles of Escherichia coli and orientation of the reconstituted lactose :H+ carrier

Abstract: The orientation of the lactose:H+ carrier of Escherichia coli in various preparations of native and reconstituted vesicles is determined with two impermeant, macromolecular probes: antibodies directed against the C-terminal decapeptide of the carrier and carboxypeptidase A (EC 3.4.17.1). Two methods are employed. Method I is based upon the digestion of all accessible and, therefore, presumably external, C termini of the carrier with carboxypeptidase A and detection of the remaining, internal C termini with 125I-labelled anti-(C-terminus) antibody after electrophoresis of the carrier in the presence of sodium dodecyl sulfate and transfer to nitrocellulose filters. Method II is based upon the binding of 125I-labelled anti-(C-terminus) antibody to the external C termini of the carrier in vesicles and the subsequent isolation of bound antibody by centrifugation. The labelled antibodies are calibrated using a preparation of inside-out vesicles prepared by high-pressure lysis of strain T206. The carrier content is determined by substrate binding. Because the C terminus of the carrier is known to reside on the cytoplasmic side of the membrane, these methods can also be used to determine the sidedness of various preparations of membrane vesicles. Spheroplasts are confirmed to contain carrier molecules of a single orientation, corresponding to that in right-side-out vesicles. In contrast, in purified cytoplasmic membrane vesicles and in crude membrane preparations obtained by sonication or by high-pressure lysis, 96% of the C termini are accessible to carboxypeptidase A, even after repeated sonication. This implies that nearly all carrier molecules in these preparations possess an orientation opposite to that in the cell or in right-side-out vesicles. In proteoliposomes containing carrier reconstituted or purified and reconstituted by two different methods, only 48% of the carrier molecules are oriented in the same way as in the cell. Subjecting such proteoliposomes to cycles of freezing and thawing or to sonication results in a reshuffling of carrier molecules between the inside-out and right-side-out populations while maintaining 41% in the right-side-out orientation. Digestion of the C terminus of the carrier with carboxypeptidase A does not alter either galactoside binding or countertransport. Thus carrier molecules of the inside-out orientation cannot be selectively inactivated. Additionally, an antiserum directed against the purified carrier is demonstrated to contain nearly exclusively anti-(C-terminus) antibodies, which can, in principle, be used in Method I.(ABSTRACT TRUNCATED AT 400 WORDS)