Exposure of Staphylococcus aureus to Targocil Blocks Translocation of the Major Autolysin Atl across the Membrane, Resulting in a Significant Decrease in Autolysis.

Peptidoglycan (PG) and wall teichoic acid (WTA) are the major staphylococcal cell wall components, and WTA biosynthesis has recently been explored for drug development. Targocil is a novel agent that targets the TarG subunit of the WTA translocase (TarGH) that transports WTA across the membrane to the wall. Previously we showed that targocil treatment of a methicillin-susceptible Staphylococcus aureus strain led to a rapid shut down of cellular autolysis. Targocil II, which targets the TarH subunit of TarGH, also resulted in a drastic decrease in autolysis. Here, we address the mechanism of targocil-mediated decreased autolysis. The mechanism is WTA dependent since targocil treatment decreased autolysis in methicillin-resistant strains but not in a WTA-deficient mutant. Similar to cellular autolysis, autolysin-retaining crude cell walls isolated from targocil-treated cells had vastly decreased autolytic activity compared to those from untreated cells. Purified cell walls from control and targocil-treated cells, which lack autolytic activity, were similarly susceptible to lysozyme and lysostaphin and had similar O-acetyl contents, indicating that targocil treatment did not grossly alter PG structure and chemistry. Purified cell walls from targocil-treated cells were highly susceptible to autolysin extracts, supporting the notion that targocil treatment led to decreased autolysin in the crude cell walls. Quantitative real-time PCR analysis revealed that the decrease in autolysis in the targocil-exposed cells was not due to transcriptional repression of the autolysin genes atl, lytM, lytN, and sle1 Zymographic analysis of peptidoglycan hydrolase profiles showed a deficiency of cell surface autolysins in targocil-treated cells but higher activity in cell membrane fractions. Here, we propose that the untranslocated WTA molecules in the targocil-exposed cells sequester Atl at the membrane, resulting in significantly decreased autolysis.

The effect of temperature on the activity of µ- and m-calpain and calpastatin during post-mortem storage of porcine longissimus muscle.

The experiment was conducted to determine the effect of temperature during post-mortem muscle storage on the activity of the calpain system, the myofibril fragmentation and the free calcium concentration. Porcine longissimus muscle were incubated from 2h post-mortem at temperatures of 2, 15, 25 and 30 °C and sampling times were at 2, 6, 24, 48 and 120 h post-mortem. After 120 h at 30 °C the free calcium concentration increased to 530 µM from 440 µM at 2 °C. Incubation at temperatures higher than 2 °C resulted in the appearance of autolyzed m-calpain activity and a decrease of native m-calpain activity. Native m-calpain decreased more slowly than native µ-calpain, and the autolysis process started later. Myofibril fragmentation increased with storage time and incubation temperature, while calpastatin activity decreased. The study showed that high temperature incubation not only rapidly activated µ-calpain but at higher temperatures and later time points also m-calpain.

Comparison of different tissue sampling methods for protein extraction from formalin-fixed and paraffin-embedded tissue specimens.

AIMS: Protein extracts from formalin-fixed and paraffin-embedded (FFPE) tissue for proteomic analysis has recently gained attention. In this study, we explored the possibility to standardize tissue sampling from paraffin blocks and compared the protein extracts with those obtained from fresh frozen material. MATERIALS AND METHODS: Fresh frozen and FFPE material was obtained from five patients with pancreatic ductal adenocarcinoma either by cutting sections with a microtome or by stamping a cylinder with tissue micro-array technology. All samples were weighed, forwarded to protein extraction and analyzed by polyacrylamide gel electrophoresis and Western blotting. Immunohistochemistry allocated proteins in tissue sections. RESULTS: Sampling of tissue was highly reproducible, as assessed by sample weight. While protein concentrations were significantly higher in fresh frozen material compared to FFPE material, equal amounts of protein were extracted from FFPE using either paraffin sections or core cylinders in SDS-PAGE, all three procedures showed comparable protein patterns. In Western blotting, annexin I had the same molecular weight independent of the sample source and sampling procedure. CONCLUSIONS: The sampling of FFPE specimens for protein extraction and analysis can be standardized, uncovering equal amounts of tissue and protein. In addition, the proteins extracted from FFPE tissue seem to be the same compared with those extracted from fresh frozen tissue.

A three-dimensional analysis of the lymphatics of a bilateral breast specimen: a human cadaveric study.

BACKGROUND: There is an increasing clinical need for evaluation of the lymphatic anatomy of the breast. Because of tissue putrefaction, previous studies have not been able to achieve radiographic analyses of bilateral breasts in cadaver specimens. The use of improved preservation techniques with computed tomographic lymphangiography (CT; CTL) has now allowed this analysis to be undertaken. MATERIALS AND METHODS: The bilateral breasts and anterior upper torso from a female unembalmed human cadaver was studied over an 8-week period. Using microsurgical techniques, lymphatic vessels were identified with hydrogen peroxide, injected with lead oxide mixture, and radiographed to demonstrate lymphatic vessels in both breasts. Multiple frozen domestic ice bricks were used to cover the contralateral side of tissues to keep them partially frozen during this lengthy process. The specimen was radiographed, CT scanned, cross-sectioned, and radiographed again, with images digitalized for analysis. RESULTS: A three-dimensional analysis of lymph collecting vessels in the breasts, anterior upper torso and the internal mammary vascular bundles was achieved using both plain radiography and CTL. The lymphatics of the breast and anterior upper torso drain radially into the axillary lymph nodes. A predominance of superficial lymphatics are noted. Importantly, lymphatic vessel patterns of the breast are asymmetric between breasts of each side. CONCLUSION: Three-dimensional images of the lymphatic drainage of the breasts using advanced imaging technologies are described, with lymphangiography achieved in bilateral cadaveric breasts and anterior upper torso for the first time. This has significant future application for clinical practice.

A novel approach to slow down putrefaction of unembalmed cadaveric tissue during lymphatic dissection: a preliminary study.

BACKGROUND: The putrefaction factor is one of the biggest problems when undertaking unembalmed cadaveric tissue dissections for lymphatic vessel mapping. METHODS: One female bilateral breast and anterior upper torso from an unembalmed human cadaver was studied over an 8-week period. Multiple prefrozen domestic ice bricks were used to cover in turn and keep constantly cold or partially frozen, the contralateral side of the tissue during the dissections. Using hydrogen peroxide to identify the lymphatics under the surgical microscope, the vessels were injected with a lead oxide mixture, and then radiographed to demonstrate lymphatic vessels in both breasts. The final results transferred to the computer for analysis. RESULTS: Lymph collecting vessels were found in breasts, anterior upper torso, and the internal mammary vascular bundles. The lymphatics of the breast and the anterior upper torso drain radially into the axillary lymph nodes. Unexpectedly, the lymphatic vessel drainage patterns of each breast are asymmetrical in this specimen. CONCLUSION: This study provides a new method to slow down putrefaction of unembalmed cadaveric tissue, thus enabling lymphatic dissection of a bilateral breast and torso specimen.

A simple method for the prevention of endometrial autolysis in hysterectomy specimens.

AIMS: Uteri are among the most common surgical pathology specimens. Assessment of the endometrium is often difficult because of pronounced tissue autolysis. This study describes a simple method to prevent endometrial autolysis and aid in interpretation of the endometrium. METHODS: Sixty uteri were injected with formalin using a needle and syringe directed alongside a probe, which was inserted through the external cervical os into the endometrial cavity. Injection was performed on the same day as removal of the uterus. As controls, 60 uteri that were not injected with formalin were examined. The degree of endometrial autolysis was assessed on a four point scale (0-3), with a score of 0 representing no or minimal autolysis and a score of 3 representing extensive autolysis, such that histological interpretation of the endometrium was impossible. RESULTS: In the injected group, the number of cases with scores of 0, 1, 2, and 3 was 42, 13, four, and one, respectively. The corresponding values for the control group were 17, 23, eight, and 12, respectively. This was highly significant (p < 0.001) CONCLUSIONS: There was significantly less endometrial autolysis in uteri injected with formalin. The use of this simple procedure should be encouraged in hysterectomy specimens.

A possible zymogen self-destruct mechanism preventing pancreatic autodigestion.

Incubation at 37 degrees C of human cationic trypsinogen purified by PAGE electrophoresis, results in development of proteolytic activity (enzyme Y) capable of rapidly degrading cationic and anionic trypsinogens to inert products. Enzyme Y appears to be a serine protease with a molecular weight of about 20,000 daltons and is different from any of the known pancreatic enzymes. The active enzyme may be derived from trypsinogen itself or a hitherto unrecognized precursor contaminating the trypsinogen fraction used in this work. Appearance of enzyme Y activity seems to be associated with the presence of traces of free trypsin. Enzyme Y possesses insignificant or no activity when tested with a variety of synthetic trypsin, chymotrypsin and other protease substrates. It is not inactivated by the specific trypsin and chymotrypsin inhibitors TLCK and TPCK, but its activity is reduced gradually by increasing concentrations of pancreatic secretory trypsin inhibitor. Ca2+ concentrations greater than 3 mM strongly inhibit enzyme Y, and diisopropylfluorophosphate completely inactivates it. The enzyme is stable when incubated at pH 1.9 and 37 degrees C for 30 min and its activity is not abolished by treatment with Hg2+. When added to pancreatic juice with low inhibitor content it causes rapid inactivation of zymogens without significant release of active enzymes or reduction of pancreatic trypsin inhibitor. Its physiological role may be perceived as a second line of defense against premature intrapancreatic activation of zymogens. Enzyme Y activity may be generated when trypsin inhibitor, the first line of defense, is sufficiently depleted by complex formation with inappropriately released trypsin to permit dissociation of a small amount of trypsin from this complex. This in turn may lead to activation of enzyme Y and inactivation of the zymogens of pancreatic proteases.

Scanning electron microscopy of the human cochlea--postmortem autolysis artefacts.

Changes in the ultrastructure of the cochlea due to postmortem autolysis make the assessment of the normal or damaged anatomy difficult. Three methods of preserving the human cochlea were compared on the basis of the state of preservation of the sensory cell hairs of the organ of Corti as seen in the scanning electron microscope. Perfusion of the perilymphatic space with a glutaraldehyde-formaldehyde fixative within 40 min of death gave preservation as good as that seen in animal studies. Injecting formalin into the middle ear within 40 min of death allowed artefacts to develop when compared with the control ear which had been perfused with fixative. Refrigeration and early removal of the temporal bone gave poor preservation of surface structures.

The prevention of autolysis in stored corneas by lysosome stabilization. A histochemical study.

We wished to determine if dexamethasome, acting as a lysosome stabilizer, could reduce the release and activation of the lysosomal acid hydrolases, and thus retard autolysis of stored corneas. One cornea (experimental) of a rabbit was soaked in a 2 per cent steroid solution and the other cornea (control) in physiological saline for 3 hours at 23 degrees C. The experimental and control corneas were then processed histochemically to show the localization of the lysosomal marker enzymes beta-glucuronidase and acid phosphatase. Compared to the controls the steroid treated corneas showed reduced enzyme activity suggesting that autolysis during storage had been retarded.