Rac1 modulates acute and subacute genotoxin-induced hepatic stress responses, fibrosis and liver aging.

To investigate the importance of the Ras-homologous GTPase Rac1 for the hepatic response to genotoxic insults and liver aging, rac1 was deleted in liver of mice by Mx1-Cre-based recombination. Knockout of rac1 caused complex changes in basal as well as doxorubicin and ionizing radiation-induced mRNA expression of various genotoxic stress response-related genes, including hspa1b, rad51, wrn and xpc. Rac1 deletion protected the liver from acute toxicity following doxorubicin treatment. Moreover, the level of S139 phosphorylated histone H2AX (γH2AX), which is indicative of DNA damage, and mRNA expression of pro-inflammatory (IL-6) and pro-fibrotic (CTGF, TGFß, αSMA) factors were mitigated in rac1 knockout animals. By contrast, lack of rac1 promoted subacute hepatotoxicity, which was determined 3 weeks after injection of multiple low doses of doxorubicin by assaying the γH2AX level, mitotic index and pro-fibrotic gene expression. Regarding ionizing radiation, rac1 deficiency had no major effects on DNA damage induction or acute pro-inflammatory and pro-fibrotic stress responses. Mice lacking hepatic rac1 for extended period of time (15 months) revealed increased mRNA expression of fibrosis-related factors (CTGF, TGFß, collagen, MMP1) and fibrotic tissue remodeling. In addition, protein expression of the senescence marker p16 was enhanced in the absence of rac1. Taken together, the data provide evidence that Rac1 is required for doxorubicin-induced DNA damage induction. It is also involved in both the acute and delayed inflammatory and fibrotic stress response in the liver following doxorubicin, but not ionizing radiation, treatment and, furthermore, protects against endogenous liver aging.

Syzygium cumini inhibits adenosine deaminase activity and reduces glucose levels in hyperglycemic patients.

Syzigium cumini (L.) Skeels from the Myrtaceae family is among the most common medicinal plants used to treat diabetes in Brazil. Leaves, fruits, and barks of S. cumini have been used for their hypoglycemic activity. Adenosine deaminase (ADA) is an important enzyme that plays a relevant role in purine and DNA metabolism, immune responses, and peptidase activity. ADA is suggested to be an important enzyme for modulating the bioactivity of insulin, but its clinical significance in diabetes mellitus (DM) has not yet been proven. In this study, we examined the effect of aqueous leaf extracts of S. cumini (L.) (ASC) on ADA activity of hyperglycemic subjects and the activity of total ADA, and its isoenzymes in serum and erythrocytes. The present study indicates that: (i) the ADA activity in hyperglycemic serum was higher than normoglycemic serum and ADA activity was higher when the blood glucose level was more elevated; (ii) ASC (60-1000 microg/mL) in vitro caused a concentration-dependent inhibition of total ADA activity and a decrease in the blood glucose level in serum; (iii) ADA1 and 2 were reduced both in erythrocytes and in hyperglycemic serum. These results suggest that the decrease of ADA activity provoked by ASC may contribute to control adenosine levels and the antioxidant defense system of red cells and could be related to the complex ADA/DPP-IV-CD26 and the properties of dipeptidyl peptidase IV (DPP-IV) inhibitors which serve as important regulators of blood glucose.

Detection of human neutrophil elastase with peptide-bound cross-linked ethoxylate acrylate resin analogs.

An assessment of elastase-substrate kinetics and adsorption at the solid-liquid interface of peptide-bound resin was made in an approach to the solid-phase detection of human neutrophil elastase (HNE), which is found in high concentration in chronic wound fluid. N-succinyl-alanine-alanine-proline-valine-p-nitroanilide (suc-Ala-Ala-Pro-Val-pNA), a chromogenic HNE substrate, was attached to glycine-cross-linked ethoxylate acrylate resins (Gly-CLEAR) by a carbodiimide reaction. To assess the enzyme-substrate reaction in a two-phase system, the kinetic profile of resin-bound peptide substrate hydrolysis by HNE was obtained. A glycine and di-glycine spacer was placed between the resin polymer and substrate to assess the steric and spatial requirements of resin to substrate with enzyme hydrolysis. The enzymatic activities of suc-Ala-Ala-Pro-Val-pNA and suc-Ala-Ala-Pro-Ala-pNA on the solid-phase resin were compared with similar analogs in solution. An increase in visible wavelength absorbance was observed with increasing amounts of substrate-resin and enzyme concentration. Enzyme hydrolysis of the resin-bound substrate was also demonstrated on a polypropylene surface, which was employed for visible absorbance of released chromophore. A soluble active substrate analog was released from the resin through saponification of the ethoxylate ester linkages in the resin polymer. The resin-released conjugate of the HNE substrate demonstrated an increased dose response with increasing enzyme concentration. The synthesis and assay of elastase substrates bound to CLEAR resin gives an understanding of substrate-elastase adsorption and activity at the resin's solid-liquid interface for HNE detection with a solid-phase peptide.

Molecular analysis of the Pi*Z allele in patients with liver disease.

Alpha1-antitrypsin (AAT) is the main protease inhibitor in human plasma. There are more than 75 variants of this protein that differ from each other by their isoelectric point. Most of these alleles cause a reduction in AAT levels; the most common allele is Pi*Z. The main complications related to the Pi*Z allele are obstructive pulmonary disease and liver disease. Some Pi*Z allele carriers present cholestatic jaundice and cirrhosis. The Z type is associated with a secretion defect, which leads to deficiency of AAT and to the formation of intrahepatocytic inclusions in affected subjects. The diagnosis of AAT deficiency can be made by different techniques, including molecular analysis, although the final diagnosis should be done in conjunction with demonstration of the periodic acid-Schiff-positive globules on liver biopsy. In this study, specimens of 29 patients with cryptogenic cirrhosis between age 1 month and 18 years, and of 100 controls were submitted to polymerase chain reaction followed by digestion with TaqI enzyme. Five of the 29 patients had undergone liver transplantation. Three patients were heterozygous for the Pi*Z allele, and two were homozygous (allele frequency = 12.07%; 7/58). Among the controls, who represented the population of Porto Alegre, 1 in 100 individuals was heterozygous for the Pi*Z allele, resulting in an allele frequency of 0.5% (1/200). The high frequency of Pi*Z alleles among the patients indicates the usefulness of AAT molecular testing in children with cholestatic jaundice and cirrhosis.

Modified cotton gauze dressings that selectively absorb neutrophil elastase activity in solution.

Dressings for chronic human wounds have been aimed at protection, removal of exudate, and improved appearance. However since the time of ancient Greece wound care and dressing strategies have primarily relied on empiricism. Recent studies have shown that chronic wounds contain high levels of tissue and cytokine destroying proteases including collagenase and neutrophil elastase. Therefore we sought to develop an effective wound dressing that could absorb elastase through affinity sequestration. Cotton gauze was modified by oxidation, phosphorylation, and sulfonation to enhance elastase affinity by ionic or active site uptake. Type VII absorbent cotton gauze was oxidized to dialdehyde cotton which was subsequently converted in part to the bisulfite addition product. Gauze preparations were also phosphorylated and carboxymethylated. Modified cotton gauzes were compared with untreated gauze for reduction of elastase activity in buffered saline. Solutions of elastase that were soaked in oxidized, sulfonated, and phosphorylated cotton gauze showed reduced elastase activity. The initial velocities (v(o)) and turnover rates of elastase showed significant decreases compared with solutions taken from untreated gauze. The reduction in enzyme activity with dialdehyde cotton gauze was confirmed in solution by determining elastase inhibition with dialdehyde starch. The dialdehyde cotton gauze also decreased elastase activity in human wound fluid in a dose response relation based on weight of gauze per volume of wound fluid. Absorbency, pH, air permeability and strength properties of the modified gauze were also compared with untreated cotton gauze. This report shows the effect of reducing elastase activity in solution with cotton containing aldehydic or negatively charged cellulose fibers that may be applicable to treatment modalities in chronic wounds.

Inhibition of elastase by a synthetic cotton-bound serine protease inhibitor: in vitro kinetics and inhibitor release.

A cotton-bound serine protease inhibitor of elastase (fiber-inhibitor) has been formulated for in vitro evaluation in chronic wound fluid. As a model to understand the properties of the inhibitor in wound dressings, the kinetic profile and in vitro release of the fiber-inhibitor formulation have been examined. The elastase inhibitor N-Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone was modified onto cotton cellulose fibers and assayed as a colloidal system. Amino acid analysis and reversed phase high performance liquid chromatography were compared as semiquantitative methods to assess elastase inhibitor release from the cotton fibers. The kinetics of inhibition was assessed on treated fibers of synthetic dressings such that a colloidal suspension of the fiber-inhibitor and elastase was employed as an assay. A dose-response relationship was observed in the kinetics of substrate hydrolysis catalyzed by three elastases: porcine pancreatic elastase, which was employed to model this approach; human leukocyte elastase; and elastase in human chronic wound fluid. Both freely dissolved and fiber-bound inhibitors were studied. The initial rates of substrate hydrolysis were inversely linear with freely dissolved inhibitor dose. The apparent first order rate constants, kobs, for the elastase-inhibitor complex were calculated from the kinetic profiles. The kobs for inhibitor bound enzyme varied as a function of inhibitor vs. enzyme concentration and based on the order of mixing of substrate, inhibitor and enzyme in the assay. Enzyme inhibition by the fiber-inhibitor was measured as inhibitor concentration at 50% inhibition (I50). I50 values measured from the colloidal assay with fiber-released inhibitor were within the same range to those for freely dissolved inhibitor. Inhibition of elastase activity in chronic wound fluid was observed with 1-5 mg of fiber-inhibitor formulation. This approach constitutes an in vitro assessment of synthetic serine protease inhibitors on fibers and may be employed to evaluate structure vs. function of elastase inhibition in the modified fibers of wound dressing composites.

Changing associate degree nursing curricula to meet evolving healthcare delivery system needs.

Curricular change for evolving healthcare delivery: everyone says to do it, but how, and what do you do? The authors explore the need for curricular change, a process to use in making the changes, essential elements to explore, and what changes one community college made. Included in the curricular revision is the hardest part-what was deleted-as well as what was added, and how nurse educators can continue to evolve.

An innovative model for clinical teaching.

Using traditional approaches, nursing faculty members may find clinical teaching stressful in today's fast-paced hospital settings. The Clinical Teaching Associate Model, pilot tested by an associate and a baccalaureate degree program in three hospitals, enabled staff nurses to assist faculty members in the direct clinical supervision of students. The benefits for students and faculty members and the potential use of the model to design varied clinical experiences for students are discussed.

Defining scholarly activity in nursing education.

In an attempt to address the question of what activities should be considered scholarly, thereby warranting their inclusion in a nursing faculty evaluation model, a study was undertaken which surveyed all National League for Nursing accredited baccalaureate educational programs. The items in the instrument were generated utilizing the Delphi method and a pilot study established inter-respondent reliability. A response rate of 73% was attained. Scholarly activity was considered highly important in evaluation for promotion and tenure in over 50% of the schools. There were distinct differences in the activities deemed scholarly when respondents were broken down into categories such as size and type of institution and the existence or non-existence of graduate nursing programs in the same institution.

Endonuclease activities in extracts of Micrococcus luteus that act on gemma-irradiated DNA.

Several protein fractions containing endonuclease activity against gemma-irradiated DNA (gamma-endonuclease) were isolated from M. luteus. The crude extract was eluted on a phosphocellulose column and chromatographed on TEAE cellulose and subsequently on hydroxyapatite. Five peaks of gamma-endonuclease were obtained from each preparation. Repeated experiments showed comparable chromatographic behavior of the fractions. There was no detectable activity of U.V.-endonuclease in the fractions with gamma-endonuclease but a small contamination of endonuclease against unirradiated DNA and against DNA with apurinic sites. The gamma-endonuclease is stimulated by, but is not dependent on, magnesium. Several tests for endonuclease activity have been used: the analysis of strand breaks in calf-thymus DNA or in PM2 DNA, and the determination of end-groups formed by endonuclease, either 3'OH end-groups or phosphomonoester end groups. From the results obtained it can be assumed that the strand breaks induced by the gamma-endonuclease carry 3'OH and 5' phosphate end groups.

Oxygen--effect on strand breaks and specific end-groups in DNA of irradiated thymocytes.

Thymocytes were irradiated with fast electrons up to 6 Mrad in the presence and absence of oxygen. The cells were treated before irradiation with a cold shock to prevent any repair rejection during irradiation. The DNA isolated subsequently was analysed for double-strand breaks (dsb), actual single-strand breaks (ssb) and alkali-induced strand breaks (alisb). We observed a linear increase of all types of lesions with dose and an o.e.r. for dsb of 3-6, for ssb of 4-9 and for alisb of 2-1. The data do not deviate significantly from those, measured on thymocytes irradiated without cold shock. In DNA of irradiated thymocytes, the frequency of 3' and 5' hydroxyl and 5' phosphate end-groups was analysed enzymatically. In both the ssb and alisb, about 11 per cent of the terminals carry 5'OH end-groups and 20-40 per cent 5' phosphate groups. On the 3' terminals, 60-80 per cent of the ssb are identified as 3'OH end-groups, whereas on the alisb only a small amount of 3'OH end-groups if found. The frequency of characterized end-groups shows the same oxygen effect as the corresponding strand breaks. Therefore, in the presence and absence of oxygen, the same mechanism may be responsible for formation of DNA strand breaks in vivo.