Characterization of the best anatomical sites in screening for methicillin-resistant Staphylococcus aureus colonization.

The purpose of this study was to identify differences in the sensitivity of anatomical sites sampling for methicillin-resistant Staphylococcus aureus (MRSA) colonization related to age, gender, clinical situation, and acquisition source as a base for screening protocols. We used a database that included all MRSA-positive cultures (Carmel Medical Center, 2003-2006) taken from nares, throat, perineum, and infection sites. The study population of 597 patients was divided into: "screening sample" (SS), which were cases of routine screening, and "clinical diagnostic sample" (CDS), which were patients with concurrent MRSA infection. MRSA acquisition sources were classified as internal medicine, surgical, referral patients, or intensive care unit (ICU). CDS patients were older than SS patients (median age 78 vs. 74 years, p = 0.0002), more commonly throat colonized (47.5% vs. 31.8%, p = 0.0001), and colonized in more multiple sites (65.7% vs. 43.3% were colonized in three sites in the CDS and SS groups, respectively, p < 0.001) than SS patients. In the SS, group throat colonization was higher in internal medicine wards than in the ICU (odds ratio [OR] = 3.98, p < 0.0001). In the CDS group, perineal colonization was more common in referral patients than in the ICU (OR = 4.52, p < 0.05). Patient age was the most influential factor on nares and multiple sites colonization in the SS and CDS groups, respectively. Our data support multiple sites sampling. Throat cultures are crucial in MRSA-infected patients and internal medicine ward patients. Multiple body sites colonization is more likely in older or MRSA-infected patients, affecting decisions regarding eradication using topical antibiotics.

Evidence for the presence of a trypsin inhibitor within rabbit and mouse tumour cells.

Post-granule supernatant fractions obtained from induced tumour cells in rabbits and from Ehrlich ascites tumour cells in mice have been shown to contain a protease inhibitor, inhibiting trypsin (EC 3.4.21.4) and neutral proteases located in the cytoplasm of the cells. This inhibition was found to be irreversible over the time period studied, independent of the time of enzyme incubation and independent of the extent of trypsin digestion within an insoluble substrate (within the limits of linear enzyme kinetics).

The interaction of a trypsin-dependent neutral protease and its inhibitor found in tumour cells. Analysis of complex kinetic data involved in a thiol-disulphide exchange mechanism.

Ehrlich ascites tumour cells contain a granule-derived zymogen which on trypsin activation yields a collegenolytic neutral protease. The preparation of the granule fraction by subcellular fractionation procedure results in the preparation of a second fraction referred to as the post-granule supernatant fraction. The post-granule supernatant fraction contains a latent form of the granule-derived neutral protease and an excess of cytoplasmic inhibitor for this enzyme. The inhibitor of neutral protease is also capable of inhibiting trypsin and in each case the chemical mechanism of enzyme.inhibitor complex formation has been shown to be a reversible thiol-disulphide exchange. The post-granule supernatant fraction exhibited complex kinetic data when the interactions between the inhibitor, the latent enzymes and trypsin were examined simultaneously by incremental analysis. The data were interpreted and quantitatively analysed by computer analysis. It was demonstrated that the conventional types of analysis could not have provided meaningful interpretations of the experimental data provided by these complex-interacting systems.

Evidence for the carriage of silver by sulphadimidine: inhibition of proteolytic enzymes.

1 Trypsin in free solution and trypsin-sepharose were shown to be inhibited by Ag+ in the absence of Cl-. 2 In the presence of Cl- and absence of a suitable carrier, Ag+ has no inhibitory action on trypsin or chymotrypsin. 3 Sulphadimidine bound Ag+ in the presence of Cl-, and carried the Ag+ to both trypsin and chymotrypsin in free solution as well as to trypsin-sepharose leading to the inhibition of all these enzyme systems. 4 The neutral protease of tumour cell surfaces was inhibited by Ag+ transported by sulphadimidine in the presence of Cl-. 5 Kinetic data demonstrated the requirements for both Ag+ and carrier to effect inhibition, the degree of inhibition being directly related to the molarity of each of these reagents. 6 The known inhibition of trypsin by Ag+ binding to histidine in the active site has been defined in mechanistic terms employing the sulphonamide drug, sulphadimidine, to illustrate this exchange mechanism.

Reverse intermetamorphosis--a rare misidentification phenomenon.

The authors present a case including two variants of delusional misidentification: Fregoli variant and intermetamorphosis. The present case has two interesting aspects: a) intermetamorphosis occurred in the patient himself, rather than in another person; b) the intermetamorphosis was only psychological and not physical.

Studies in vitro of the effects of adenosine 3':5'-cyclic monophosphate on the phosphorylation of nuclear proteins in isolated rat heart nuclei.

Isolated rat heart nuclei were prepared by homogenization and sucrose-density-gradient centrifugation. The protein/DNA ratio of these nuclei was 3.1:1 (w/w), and the histones/non-histone proteins/DNA proportions were 1.4:1.6:1 (by wt.). Non-histone proteins were fractionated into six major groups by elution on a quaternized anion-exchanger (QAE-Sephadex A-50 column with increasing concentrations of NaCl in 5M-urea/0.01 M-Tris/HCl buffer (pH8.3). When isolated nuclei were incubated in a medium containing [gamma-32P]ATP, a differential distribution of 32P was observed in the six fractions of nonhistone proteins. The fractions eluted from the Sephadex column with 0.35M- and 0.6M-NaCl contained contained 80% of the total radioactivity incorporated into the non-histone proteins. This incorporation into the 0.35M- and 0.6M-NaCl fractions was increased by 66 and 112% respectively in the presence of cyclic AMP. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these two particular fractions showed a selective increase in labelling of five protein bands in the presence of cyclic AMP.

Inhibition of free and bound trypsin-like enzymes.

1. The inhibition of beta-naphthylamidase activity of free trypsin, trypsin-Sepharose and a trypsin-like neutral protease on the surface of tumour cells have been studied in independent systems and with mixtures of free trypsin plus surface-bound trypsin. 2. Kinetic data have demonstrated that high-molecular-weight (protein) inhibitors of free trypsin are less effective inhibitors of trypsin-Sepharose and fail to inhibit the cell surface neutral protease. 3. Inhibition of mixtures of free trypsin plus trypsin-Sepharose follows independent kinetic plots for each component. The free trypsin is reacted before any Sepharose-bound trypsin reacts with high-molecular-weight inhibitors. 4. Low-molecular-weight inhibitors of trypsin also inhibit bound trypsin equally well. 5. Papain-derived peptides from high-molecular-weight inhibitors of trypsin inhibit free trypsin, trypsin-Sepharose and the cell-surface neutral protease almost equally well. 6. Fluorescence microscopy has shown that a high-molecular-weight inhibitor of trypsin does not bind to the tumour cell-surface neutral protease, but it does bind to trypsin-Sepharose. 7. The cell-surface neutral protease has been shown to be capable of activation of latent beta-naphthylamidase activity in the presence of excess extracellular inhibitors of free trypsin. 8. The mechanism by which trypsin-Sepharose remains partially active in the presence of excess inhibitor necessary to inhibit an equivalent quantity of free trypsin has been discussed. 9. These studies indicate that a search for inhibitors which are selectively active against the cell-surface neutral protease and have no action on trypsin-like enzymes in free solution must take into account the modifying effects of the cell surface on neutral protease activity.

Evidence for inhibitors of the cell surface protease guanidinobenzoatase.

Guanidinobenzoatase is a protease present on the surface of tumour cells. The present study describes the isolation of a protein inhibitor of guanidinobenzoatase obtained from extracts of liver and pancreas and purified by affinity techniques. Pancreatic acinar cells have been shown to possess a latent form of guanidinobenzoatase and this latency is due to complex formation with the inhibitor. A fluorescent marker has been employed to demonstrate the presence or absence of the inhibitor on sections of pancreatic tissue. The inhibitor has been shown to be exchangeable with liver and pancreatic inhibitors obtained from different species. It is postulated that these inhibitors may play a role in enzyme control.

Evidence for metal inhibition of tumour membrane-bound neutral protease and the control of tumour-induced target cell cytolysis.

Previous studies have characterized the enzymatic properties and inhibition of a trypsin-like neutral protease on the surface of Ehrlich ascites cells by means of kinetic analysis. The present study links these kinetic studies with the recently reported role of a tumour-cell membrane-bound serine protease in tumour-induced target cell lysis. Low-mol.-wt inhibitors of this cell-surface trypsin-like neutral protease exhibited a corresponding ability to prevent tumour-induced haemolysis. High-mol.-wt inhibitors of trypsin in free solution had no inhibitory action either on the tumour-bound enzyme or on the ability of tumour cells to lyse erythrocytes. Fragments of tumour-cell membrane retain both the trypsin-like neutral protease activity and the ability for haemolysis. This study represents a correlation between an easily assayed membrane-bound enzyme on tumour cells and a function of possible biological relevance.

Studies on ehrlich ascites tumour cells: DNA synthesis, and template activity of chromatin for DNA polymerase.

Chromatin obtained from Ehrlich ascites cells on different days after cell inoculation has been assayed for its template activity with added DNA polymerase I. We have found that the template activity is 2 times higher in 7-8 day cell chromatin than in 4-day chromatin. Studies with added polylysine indicate that this increase reflects an increase in initiation sites rather than in accessibility to the enzyme. We have measured the growth fraction, mitotic index and rate of DNA chain growth in the intact cells. The results show that there is a large decrease in growth fraction with age of tumour, the number of cells dropping out of cycle approximately doubling over the period studied. The overall rate of chain growth decreases in the later stages of growth but in a small proportion of cells there is an increase in rate with fewer replicons involved in DNA synthesis. We suggest that in the ascites cells there is a decrease in level of repair and replicative enzymes with age of tumour; this would account both for the increase in initiation sites in the chromatin DNA, for the decrease in number of cells in cycle and for the overall decreased rate of chain growth.

A trypsin-like neutral protease on Ehrlich ascites cell surfaces: its role in the activation of tumour-cell zymogen of collagenase.

Ehrlich ascites cells in mice have been shown to have a cell-surface trypsin-like neutral protease (TLNP) with proteolytic and beta-naphthylamidase activity. This activity is inhibited by low-mol.-wt inhibitors of trypsin but not by 11 high-mol.-wt inhibitors of trypsin in free solution. We believe that lack of inhibition is due to protection given to the enzyme by the chemical environment of the cell surface. These cells were demonstrated to export a collagenase zymogen which has been shown to be activated by the cell-surface TLNP. When this protease was completely inhibited by low-mol.-wt inhibitors of trypsin, chymotrypsin was used to activate the collagenase zymogen exported by Ehrlich ascites cells. Examination of the products of collagenolysis at 15 degrees C demonstrated the expected 3/4- and 1/4-length alpha-chain fragments derived from monomeric collagen, confirming that collagenase was one of the enzymes responsible for lysis of the collagen fibrils in the test system.

beta-Naphthylamidase activity of the cell surface of Ehrlich ascites cells. Reversible control of enzyme activity by metal ions and thiols.

Ehrlich ascites tumour cells grown in mice have a cell-surface trypsin-like neutral protease (TLNP) which is not inhibited by high-mol.-wt inhibitors of trypsin. This enzyme is inhibited by low concentrations of zinc, which may be removed by chelating agents, with the consequent return of enzymic activity. Gold, provided as the drugs aurothiomalate or auranofin, also inhibits TLNP. The gold can be removed from the enzyme by incremental addition of thiols. The mechanisms of gold transfer to the active site to cause inhibition and subsequent removal of gold with reactivation of TLNP, have been shown to be controlled by reversible thiol-exchange reactions.

Inhibition of cell-surface neutral protease of Ehrlich ascites tumor cells by potassium p-(3,3-dimethyl-1-triazeno) benzoate.

Ehrlich ascites tumour (EAT) cells possess a trypsin-like neutral protease on the cell surface. The antimetastatic triazene drug potassium p-(3,3-dimethyl-1-triazeno) benzoate (DM-COOK) inhibits this neutral protease, and also trypsin. Incubation of EAT cells with human erythrocytes (ratio of 1 to 5) at 37 degrees C for 18 h caused haemolysis of 28.8% of the erythrocytes. Addition of millimolar concentrations of DM-COOK to a fixed quantity (2.5 X 10(8)) of EAT cells caused a dose-related inhibition of haemolysis. DM-COOK was strongly bound to EAT cells and could not be removed by repeated washing.