Leakage Of Alkaline Phosphatase Research Articles

Trans-cinnamaldehyde inhibits Escherichia coli growth by regulating lipopolysaccharide accumulation

Colibacillosis is among the most commonly occurring and economically devastating bacterial diseases of poultry worldwide owing to the misuse and overuse of antibiotics. Trans-cinnamaldehyde exhibits effective inhibition on multidrug-resistant Escherichia coli by disrupting the integrity of its cell wall. However, the mechanism underlying this inhibition is unclear. We found that trans-cinnamaldehyde inhibited E. coli efficiently with a minimum inhibitory concentration (MIC) of 140 μg/mL. Trans-cinnamaldehyde disrupted the integrity of the E. coli cell wall due to an imbalance between lipopolysaccharide (LPS) and phospholipids. The structural change in the cell wall disrupted its stability, further causing the leakage of alkaline phosphatase (AKP) and β-galactosidase, proteins, and DNA/RNA, and altering biofilms in the E. coli CVCC 1567 and the E. coli (047). This finally resulted in the death of E. coli. Inhibition by trans-cinnamaldehyde was more significant on the wall of E. coli CVCC 1567 because of a more severe loss of high molecular weight proteins and an easier dissolution of its cells. Trans-cinnamaldehyde affected gene expression of enzymes regulating LPS synthesis (lpxC, lpxA, lpxK, lpxM, yejM, ftsH, lapB), ultimately altering (LPS) content. Indeed, this effect is particularly pronounced in the genes expression of E. coli (047). In conclusion, trans-cinnamaldehyde is a potential novel drug candidate for preventing and treating infectious diseases caused by resistant E. coli clinical strains.

Preparation and antimicrobial mechanism of Maillard reaction products derived from ε-polylysine and chitooligosaccharides

Chitooligosaccharides can be combined with amino acids or polypeptide to form Maillard reaction products (MRPs) with the antibacterial characteristics through Maillard reaction. This research aims to clarify the structure, antimicrobial effect and mechanism against Shewanella putrefaciens (S. putrefaciens) of ε-polylysine and chitooligosaccharides Maillard reaction products (LC-MRPs). The results of intrinsic fluorescence (IF) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction, proton nuclear magnetic resonance (1H NMR) spectra and scanning electron microscope (SEM) indicated Maillard reaction occurred between ε-polylysine and chitooligosaccharides. The observation of confocal laser scanning microscopy (CLSM), SEM and growth curves of S. putrefaciens evidenced that LC-MRPs have the strongest antibacterial effects. The leakage of alkaline phosphatase (AKP) and lactate dehydrogenase (LDH) implied that LC-MRPs sabotaged bacterial barrier (cell wall and cell membrane). The changes in content of nucleic acids, reactive oxygen species (ROS) level, lipid peroxidation content (LPO), succinate dehydrogenase (SDH) activity and adenosine triphosphate (ATP) content showed LC-MRPs will affect bacterial genetic gene transcription, material and energy metabolism. Therefore, the LC-MRPs were effective antibacterial agents to inhibit S. putrefaciens, which will help to preserve food with S. putrefaciens as the main spoilage bacteria.

Encapsulation of thymus vulgaris essential oil in caseinate/gelatin nanocomposite hydrogel: In vitro antibacterial activity and in vivo wound healing potential

Essential oils, derived from aromatic plants, exhibit various pharmacological properties. Nevertheless, their clinical applications are confronted by various limitations, such as chemical instability, low aqueous solubility, and poor bioavailability. Nanoencapsulation is one of the approaches that may circumvent these restraints. Accordingly, the present study encapsulated thyme essential oil (TEO) in sodium caseinate (Na CAS) nanomicelles and formulated a gelatin nanocomposite hydrogel, which was investigated as a drug delivery platform for in vitro antibacterial and in vivo wound healing potential. TEO loaded Na CAS nanomicelles showed particle size of 336 ± 17.35 nm, zeta potential of −44.0 mV and EE% of 75 ± 5%. The release profile of TEO loaded nanocomposite hydrogel revealed a sustained release pattern compared to TEO loaded micelles and free oil. The TEO loaded nanomicelles exhibited a significantly higher antibacterial effect than free TEO, as denoted by leakage of alkaline phosphatase and cell membrane disruptions. Furthermore, the TEO loaded nanocomposite hydrogel significantly promoted wound contraction, reduced interleukin-6, and increased transforming growth factor-β1and vascular endothelial growth factor levels, versus control or blank hydrogel group. Hence, the present study is putting forth the fabricated nanocomposite hydrogel as a multifunctional delivery system for TEO in wound healing applications.

Antimicrobial peptide WAM-1: a promising antibacterial and anti-inflammatory drug against carbapenem-resistant Klebsiella pneumoniae.

The emergence and spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) pose a threat to public health. Antimicrobial peptides provide a new treatment option for CRKP infections. We studied antibacterial activities of WAM-1 against CRKP in vitro and in vivo and explored its possible mechanism. We verified safety and factors affecting antibacterial effect. Furthermore, anti-inflammatory effects were investigated. We selected eight CRKP and eight carbapenem-susceptible K. pneumoniae to explore the antibacterial activity of WAM-1 by broth microdilution (BMD). The possible mechanism was investigated by alkaline phosphatase leakage and propidium iodide (PI). We evaluated safety of WAM-1 by cytotoxicity and haemolysis and effects of temperature and serum on the antibacterial activity. We investigated in vivo efficacy of WAM-1 by the Galleria mellonella infection model. We investigated the effect of WAM-1 on TNF-α. BMD showed that WAM-1 had a good antibacterial effect with MICs of 2-4 mg/L and MBCs of 4-8 mg/L. RT-qPCR showed that WAM-1 could inhibit the expression of TNF-α. The cytotoxicity and haemolysis test proved that WAM-1 had certain potential application in vivo. Alkaline phosphatase leakage and PI fluorescence showed that WAM-1 was highly likely to exert an antibacterial effect by destroying bacterial membrane. The G. mellonella infection model suggested that WAM-1 may have a good therapeutic effect in vivo. Temperature had little effect on the activity of WAM-1. Serum, however, reduced WAM-1 activity. WAM-1 has good antibacterial effect and potential anti-inflammatory effect on infection caused by CRKP.

Antibacterial Mechanism of 3-Carene against the Meat Spoilage Bacterium Pseudomonas lundensis and Its Application in Pork during Refrigerated Storage.

Pseudomonas lundensis is the main bacterium responsible for meat spoilage and its control is of great significance. 3-Carene, a natural monoterpene, has been proved to possess antimicrobial activities. This study aimed to investigate the antibacterial activity and mechanism of 3-carene against the meat spoilage bacterium P. lundensis, and explore its application on pork. After 3-carene treatment, cellular structural changes were observed. Cell walls and membranes were destroyed, resulting in the leakage of alkaline phosphatase and cellular contents. The decreased activity of Ca2+-Mg2+-ATPase and Na+-K+-ATPase showed the imbalance of intracellular ions. Subsequently, adenosine triphosphate (ATP) content and oxidative respiratory metabolism characteristics indicated that 3-carene inhibited the metabolism of the tricarboxylic acid cycle in P. lundensis. The results of binding 3-carene with the vital proteins (MurA, OmpW, and AtpD) related to the formation of the cell wall, the composition of the cell membrane, and the synthesis of ATP further suggested that 3-carene possibly affected the normal function of those proteins. In addition, the growth of P. lundensis and increase in pH were inhibited in pork during the 5 days of cold storage after the samples were pre-treated with 3-carene. These results show the anti-P. lundensis activity and mechanism of 3-carene, and its potential use in meat preservation under refrigerated conditions.

Use of Acetic Acid to Partially Replace Lactic Acid for Decontamination against Escherichia coli O157:H7 in Fresh Produce and Mechanism of Action.

Escherichia coli O157:H7 is frequently detected in ready-to-eat produce and causes serious food-borne diseases. The decontamination efficacy of lactic acid (LA) is clearly established. In this study, LA was mixed with acetic acid (AA) to reduce costs while achieving consistent or better inhibitory effects. Time-kill curves and inoculation experiments using fresh-cut spinach and arugula indicated that 0.8%LA+0.2%AA shows similar antibacterial effects to those of 1%LA. To determine whether 1%LA and 0.8%LA+0.2%AA exert antibacterial effects by similar mechanisms, proteomics analysis was used. The proteins related to macromolecule localization, cellular localization, and protein unfolding were uniquely altered after the treatment with 1%LA, and the proteins related to taxis, response to stress, catabolic process, and the regulation of molecular function were uniquely altered after the treatment with 0.8%LA+0.2%AA. Based on these findings, combined with the results of a network clustering analysis, we speculate that cell membrane damage is greater in response to LA than to 0.8%LA+0.2%AA. This prediction was supported by cell membrane permeability experiments (analyses of protein, nucleotide, ATP, and alkaline phosphatase leakage), which showed that LA causes greater membrane damage than 0.8%LA+0.2%AA. These results provide a theoretical basis for the application of an acid mixture to replace LA for produce decontamination.

Antimicrobial Activity and Proposed Action Mechanism of Linalool Against Pseudomonas fluorescens.

In this study, linalool, one of the principal components of essential oils, was used as an antibacterial agent to investigate the antibacterial activity and mechanism of linalool against Pseudomonas fluorescens. The reduction in membrane potential (MP), leakage of alkaline phosphatase (AKP) and the release of macromolecules, including DNA, RNA and protein confirmed that damage to cell wall membrane structure and leakage of cytoplasmic contents were due to the linalool treatment. Furthermore, the decrease of enzyme activity, including the succinate dehydrogenase (SDH), malate dehydrogenase (MDH), pyruvate kinase (PK), and ATPase indicated that linalool could lead to metabolic dysfunction and inhibit energy synthesis. In addition, the activity of respiratory chain dehydrogenase and metabolic activity of respiration indicated that linalool inhibits cellular respiration. These results revealed that linalool had strong antibacterial activity against P. fluorescens via membrane damage, bacterial metabolic and oxidative respiratory perturbations, interfering in cellular functions and even causing cell death. It was suggested that linalool may be a new potential source as food antiseptics in food systems.

Antibacterial Activity and Mechanism of Linalool against Shewanella putrefaciens.

The demand for reduced chemical preservative usage is currently growing, and natural preservatives are being developed to protect seafood. With its excellent antibacterial properties, linalool has been utilized widely in industries. However, its antibacterial mechanisms remain poorly studied. Here, untargeted metabolomics was applied to explore the mechanism of Shewanella putrefaciens cells treated with linalool. Results showed that linalool exhibited remarkable antibacterial activity against S. putrefaciens, with 1.5 µL/mL minimum inhibitory concentration (MIC). The growth of S. putrefaciens was suppressed completely at 1/2 MIC and 1 MIC levels. Linalool treatment reduced the membrane potential (MP); caused the leakage of alkaline phosphatase (AKP); and released the DNA, RNA, and proteins of S. putrefaciens, thus destroying the cell structure and expelling the cytoplasmic content. A total of 170 differential metabolites (DMs) were screened using metabolomics analysis, among which 81 species were upregulated and 89 species were downregulated after linalool treatment. These DMs are closely related to the tricarboxylic acid (TCA) cycle, glycolysis, amino acid metabolism, pantothenate and CoA biosynthesis, aminoacyl-tRNA biosynthesis, and glycerophospholipid metabolism. In addition, linalool substantially affected the activity of key enzymes, such as succinate dehydrogenase (SDH), pyruvate kinase (PK), ATPase, and respiratory chain dehydrogenase. The results provided some insights into the antibacterial mechanism of linalool against S. putrefaciens and are important for the development and application of linalool in seafood preservation.

Antibacterial and Antidiarrheal Activities of Houttuynia Cordata Thunb. And Portulaca Oleracea L. Extracts Against Enterotoxigenic Escherichia Coli

Houttuynia cordata Thunb. and Portulaca oleracea L. have been used as medicinal and food homologous plants. This research focused on evaluating the possibilities of the combination of H. cordata and P. oleracea extracts as alternatives to antibiotics. The antimicrobial and antidiarrheal activities of the combination of ethanol extract and essential oil from H. cordata (HEE and HEO) and ethanol extract from P. oleracea (PEE) against enterotoxigenic Escherichia coli were studied. Results showed the combination (CB-HHP) of HEE, HEO, and PEE indicated synergistic effect on E. coli , and the concentration consist when HEE, HEO, and PEE were at a concentration of 1, 0.56, and 0.5 mg/mL, respectively, was regarded as minimal inhibitory concentration (MIC) of CB-HHP. CB-HHP increased the permeability of cell wall and cell membrane and affected the integrity of cell membrane, as evidenced by the leakage of alkaline phosphatase (AKP), potassium ion (K+), and protein, and changes in cell morphology further proved the damage to the cell wall and membrane of E. coli by CB-HHP. In addition, CB-HHP treatment significantly inhibited formation of E. coli bacterial biofilm. In vivo, gavaging with CB-HHP (HEE:HEO:PEE = 1:0.56:0.5, 800 mg/kg body weight) could significantly relieve the weight loss and diarrhea rate and protect the duodenal villi of mice infected by E. coli , as well as increasing the concentration of immunoglobulin (Ig) G, IgM, and IgA in serum. Therefore, CB-HHP was demonstrated remarkable antibacterial activity, antidiarrheal activity, and intestinal protective function, with potential to be used as food-derived additives.

Gold Nanoclusters-Decorated Zeolitic Imidazolate Frameworks with Reactive Oxygen Species Generation for Photoenhanced Antibacterial Study.

The serious threat of antibiotic-resistant bacterial infections has brought an urgent need for the development of new antibacterial nanomaterials. We encapsulate glutathione (GSH)-protected gold nanoclusters (AuNCs) in zeolitic imidazolate frameworks-8 (ZIF-8) and present their potential in antibacterial capabilities. Under white light irradiation, AuNCs-embedded ZIF-8 nanocomposites show assembly-enhanced emission and reactive oxygen species (ROS) generation. AuNCs@ZIF-8 exhibit almost complete inactivation of bacterial growth within 60 min of light irradiation. Scanning electron microscopic results show that AuNCs@ZIF-8 nanocomposites are captured by bacterial cells, and the leakage of alkaline phosphatase and nucleotides from bacteria demonstrate that the photoinduced ROS can easily destroy the bacterial surface and totally kill the bacteria. Herein, our antibacterial nanocomposites have photoenhanced bactericidal capability and show promising applications for sterilization.

Study on Antibacterial Activity of Amomum tsao-ko Extracts against Bacillus subtilis and Listeria monocytogenes and Preliminary Investigation of Its Antibacterial Mchanism

The main objective of this study was to preliminarily investigate the antibacterial activity and mechanism of action of Amomum tsao-ko (A. tsaoko) extracts against Bacillus subtilis and Listeria monocytogenes. The results showed that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of A. tsaoko extracts against B. subtilis and L. monocytogenes were 1.25 mg/mL. Bacterial growth curves revealed that the A.tsaoko extracts had an obvious antibacterial activity against Bacillus subtilis and L. monocytogenes. Furthermore, the mechanism of action of A. tsaoko was evaluated by analyzing its effects on the integrity of cell membrane and cell morphology. The leakage of alkaline phosphatase (AKP) confirmed that the A. tsaoko extracts caused serious damage to the wall structure of B. subtilis and L. monocytogenes. Results of scanning electron microscopy (SEM) manifested that the destructive effect of A. tsaoko extracts on cell morphology and cell integrity of bacterial cells. The results implied that A. tsaoko extracts showed relatively good antibacterial effect on B. subtilis and L. monocytog.

Antimicrobial Activity and Proposed Action Mechanism of 3-Carene against Brochothrix thermosphacta and Pseudomonas fluorescens.

3-Carene is an antimicrobial monoterpene that occurs naturally in a variety of plants and has an ambiguous antibacterial mechanism against food-borne germs. The antibacterial effects and action mechanism of 3-carene against Gram-positive Brochothrix thermosphacta ACCC 03870 and Gram-negative Pseudomonas fluorescens ATCC 13525 were studied. Scanning electron microscopy (SEM) examination and leakage of alkaline phosphatase (AKP) verified that 3-carene caused more obvious damage to the morphology and wall structure of B. thermosphacta than P. fluorescens. The release of potassium ions and proteins, the reduction in membrane potential (MP), and fluorescein diacetate (FDA) staining further confirmed that the loss of the barrier function of the cell membrane and the leakage of cytoplasmic contents were due to the 3-carene treatment. Furthermore, the disorder of succinate dehydrogenase (SDH), malate dehydrogenase (MDH), pyruvate kinase (PK), and ATP content indicated that 3-carene could lead to metabolic dysfunction and inhibit energy synthesis. In addition, the results from the fluorescence analysis revealed that 3-carene could probably bind to bacterial DNA and affect the conformation and structure of genomic DNA. These results revealed that 3-carene had strong antibacterial activity against B. thermosphacta and P. fluorescens via membrane damage, bacterial metabolic perturbations, and genomic DNA structure disruption, interfering in cellular functions and even causing cell death.

A natural biopreservative: Antibacterial action and mechanisms of Chinese Litsea mollis Hemsl. extract against Escherichia coli DH5α and Salmonella spp.

Chemical preservatives have potential safety hazards, which may pose threats to human health. Safer biopreservatives are therefore urgently required. This study investigated the bacteriostatic activity and mechanism of Litsea mollis Hemsl. essential oil against Escherichia coli DH5α and Salmonella spp. Antibacterial activity of Litsea mollis Hemsl. essential oil 9 (LMEO9) against E. coli DH5α was observed (zone of inhibition was 5.0 ± 0.2 mm; minimum inhibitory concentration was 0.05%). Increases in electrolyte, nucleic acid, and alkaline phosphatase leakage in LMEO9-treated bacteria suggested that the cell envelope had been damaged. Scanning and transmission electron microscopy also demonstrated morphological alterations and content leakage during LMEO9 treatment. According to the kill-time analysis and propidium iodide uptake assay, LMEO9 led to cell death. These results demonstrated that LMEO9, which could affect bacterial cell envelope structural integrity, is a low-cost biopreservative that could be useful for the dairy industry and in fresh storage.

Taurine protects murine hepatocytes against oxidative stress-induced apoptosis by tert-butyl hydroperoxide via PI3K/Akt and mitochondrial-dependent pathways

The present study aims to investigate the beneficial role of taurine in tert-butyl hydroperoxide (TBHP)-induced oxidative stress, cytotoxicity and cell death using murine hepatocytes as the working model. TBHP caused a time-dependent increase in alkaline phosphatase leakage, extensive ROS formation and alteration in antioxidant machineries. Moreover, TBHP upregulated NF-κB phosphorylation, caused injury to cellular mitochondria by reciprocal regulation of Bcl2 family proteins; incapacitated cellular respiration, accelerated cytochrome c release from mitochondria and induced apoptotic death in hepatocytes. Taurine treatment prevented TBHP-induced oxidative insult and decreased the activation of NF-κB as well as apoptotic signalling pathways. Signal transduction studies with specific inhibitors PS1145 (IκB inhibitor) and LY294002 (PI3K inhibitor) showed that the mechanism of the protective action of taurine involves the upregulation of Akt and inhibition of NF-κB. Combining, our result suggest that taurine supplementation in regular diet may provide a potential therapeutic choice against TBHP-induced hepatic oxidative injury.

Adenine nucleotide and calpain inhibitor I protect against atractyloside-induced toxicity in rat renal cortical slices in vitro.

Atractyloside is a compound with a documented nephrotoxicity. It induces renal tubular necrosis at high doses and apoptosis at lower doses. This study investigates the potential protective effect of some chemical agents against atractyloside-induced nephrotoxicity in vitro using the precision-cut rat renal cortical slices obtained from kidneys of Wistar rats. For co-incubation experiments, slices were incubated for 3 h at 37 degrees C on a rocker platform with various chemical agents: ADP (5 mM), calpain inhibitor I (CPI, 1 mM), stevioside (STV, 2.5 mM) or probenecid (PRB, 2.5 mM) in the presence or absence of atractyloside (2 mM). For pre-incubation experiments, slices were incubated with the same chemical agents for 1 h before exposure to atractyloside. The nephrotoxic effects of atractyloside (2 mM) alone were manifested in several ways: by a marked increase in lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) leakage, significant inhibition of p-aminohippurate (PAH) accumulation, marked depletion of intracellular ATP and reduced glutathione (GSH), and a significant reduction in pyruvate-stimulated gluconeogenesis. Co-incubation of slices with ADP or CPI and atractyloside completely blocked atractyloside-induced increase in LDH leakage, but not ALP leakage. Atractyloside-induced depletion of ATP and reduced gluconeogenesis was prevented by co-incubation with ADP or CPI. Furthermore, co-incubation of slices with STV and atractyloside, but not PRB, completely abolished atractyloside-induced depletion of ATP and decreased gluconeogenesis in the slices. Pre-incubation of slices with either ADP or CPI protected against atractyloside-induced increase in LDH leakage, reduced ATP and decreased gluconeogenesis. PAH uptake in the slices was inhibited by atractyloside and PRB in a time-dependent manner. While ADP and CPI were found to exert complete protection against atractyloside-induced toxicity irrespective of treatment schedule, STV is effective only under certain conditions, and PRB offer no protection at all. The results of this study demonstrate the usefulness of renal cortical slices as toxicology tool for evaluating and screening compounds for their potential protective effects, and are supportive of a role of adeninine nucleotide (ADP) and protease inhibitor (CPI) in protecting against atractyloside-induced cell injury.

Optimizing preincubation conditions for precision-cut rat kidney and liver tissue slices: effect of culture media and antioxidants

Tissue slices are commonly ‘preincubated’ before use, but optimal conditions to ensure their maximal viability have not been systematically investigated. The effects of serum-free Dulbecco's minimum Eagle's medium and Ham's nutrient mixture (DMEM/F12) (1:1) culture media with and without phenol red (±PR), or RPMI-1640 and six different antioxidants on the viability of precision-cut rat kidney and liver slices (200±5 μm) were investigated. Slice viability was assessed every 30 minutes over a 2-hour preincubation period and after 24 hours of incubation in a multiwell plate culture system maintained at 37°C. In all cases, preincubation produced a time-dependent significant reduction of ethidium bromide positive nuclei stained in each medium and in both kidney and liver slices. Based on lactate dehydrogenase (LDH) leakage, there are viability differences between the media. In contrast, alkaline phosphatase (ALP) leakage and MTT reduction were less sensitive and did not differentiate between slice viability in each incubation medium. Preincubation of kidney and liver slices in DMEM/F12 medium containing antioxidants, indicated an enhanced viability which was specific for each tissue. Extension of the culture period to 24 hours after 1 hour of preincubation showed up to a further 4–13% leakage of ALP or LDH in DMEM/F12 (±PR) media for both kidney and liver slices and with a further 5–15% decline in MTT viability assay. RPMI-1640 medium on its own was not a suitable medium for maintaining the viability of either kidney or liver slices. However, kidney or liver slices preincubated with DMEM/F12 medium in the presence of some of the antioxidants were satisfactorily maintained for 24 hours. Exposure of slices to atractyloside (ATR) at concentrations of 0.2–2.0 m m in the different media for 24 hours showed a significant increase in enzyme leakage, decline of MTT reductive capacity and increased oxidative damage, with toxicity more elaborate in RPMI-1640 medium. Preincubation of kidney slices with either reduced glutathione (GSH) or α-tocopherol (TOC) and liver slices with either GSH or deferoxamine (DEF) followed by 24 hours of exposure to ATR showed a similar decline in toxicity profile. The antioxidants provided partial protection of slices from ATR toxicity. The results demonstrate the importance of slice preincubation and indicate that slices could be maintained in culture using an appropriate medium, thus providing slices that could serve as a useful alternative in vitro system for assessing novel compounds for toxicity.

A Possible Mechanism of Increase in Serum Alkaline Phosphatase Activity in Rats Given Granulocyte Colony-Stimulating Factor.

Recombinant human granulocyte colony-stimulating factor (G-CSF) at a dose of 1 to 300 micrograms/kg/day was administered intravenously to rats daily for 13 weeks. Serum alkaline phosphatase (ALP) activity increased dose-dependently with leukocytosis. Most of the increased leukocytes were segmented neutrophils, and neutrophil alkaline phosphatase (NAP) scores were elevated markedly. Serum ALP activity correlated very well with the segmented neutrophil counts, and the coefficient of correlation was more than 0.97 in both sexes. Pathological examinations revealed splenomegaly and a marked increase in neutrophils in the red pulp of the spleen. In the spleen, phagocytosis of neutrophils by macrophages was observed. These data indicate that the increased ALP was of neutrophil origin. Serum ALP activity may be increased by the direct release of ALP from the high number of neutrophils into the blood, or by the leakage of ALP into the blood mainly from the spleen where many neutrophils are pooled and destroyed by the macrophage system.

Protection effects of procaine on oxidative stress and toxicities of renal cortical slices from rats caused by cisplatin in vitro.

Incubation of rat renal cortical slices with 2 mM cisplatin (CDDP) at 37 degrees C for different periods of time (15-180 min) increased malondialdehyde (MDA) formation, decreased intracellular glutathione (GSH), and inhibited gluconeogenesis in the slices. CDDP-induced MDA formation increased by 53% after 180 min of incubation and GSH decreased by 35% after 60 min of incubation. Both depletion of GSH and inhibition of gluconeogenesis preceded MDA formation. Procaine (2 mM) completely inhibited CDDP-induced lipid peroxidation without affecting depletion of GSH, but even potentiated gluconeogenesis inhibition, while 2 mM dithiothreitol (DTT) largely reversed all of these biochemical indices. After 240 min of incubation, 2 mM CDDP produced marked cytotoxicities, characterized by an increase in leakage of alkaline phosphatase (ALP) (132%), lactate dehydrogenase (LDH) (115%) and N-acetyl-beta-glucosaminidase (NAG) (157%), decrease in intracellular K+ (64%), and change in total water contents in the slices. Procaine (2 mM) showed protection against CDDP-induced cytotoxicities to a certain extent. These results suggest that depletion of GSH might be a determinant step in the oxidative stress and subsequent cytotoxicity, and that procaine is a powerful antioxidant and would be a promising drug for ameliorating some of the adverse effects of CDDP.

Studies on the mechanism of the increase in serum alkaline phosphatase activity in cholestasis: significance of the hepatic bile acid concentration for the leakage of alkaline phosphatase from rat liver.

In experimental bile obstruction the serum activities of the membrane-bound liver enzymes, alkaline phosphatase, 5'-nucleotidase and gamma-glutamyltransferase are greatly increased, whereas in the liver only the alkaline phosphatase activity is elevated. After partial hepatectomy or tetrachloride poisoning the alkaline phosphatase activity in the regenerating live is increased to the same extent as in cholestasis without an accompanying elevation in serum activity. The following results support the hypothesis of a bile salt-mediated solubilization of membrane-bound enzymes in cholestatic liver: (1) 30 min after bile duct ligation the total bile acids in the liver were increased 5-fold, 2 h later as much as 10-fold. After 1 day, the bile acid concentration was still 4 times above normal. (2) Isolated plasma membranes from normal and obstructed livers were incubated in vitro with increasing amounts of tri- and dihydroxycholanic acids. At a final concentration of 1 mmol/l taurochenodeoxycholate significant amounts of membrane-bound enzymes were released into the 12,000-g supernatant. (3) In the regenerating liver, where tissue phsophatase activity was high and serum phosphatase activity unchanged, the bile salt concentration was not increased.