Verification of the role of spiperone in the treatment of COPD through bioinformatics analysis.

BACKGROUND: Aim of this study is investigates the influence of spiperone on hydrolase activity pathway in chronic obstructive pulmonary disease (COPD). PATIENTS AND METHODS: Differentially expressed genes (DEGs) were calculated by the limma package from microarray data GSE20257, and analysed via gene set enrichment analysis (GSEA) for identifying COPD related pathways. The regulation of hydrolase activity pathway related drugs was predicted by connectivity Map analysis (CMap). Western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were used to investigate the effect of spiperone on regulation of hydrolase activity pathway in vitro experiment. RESULTS: A total of 378 DEGs were identified by the limma package. GSEA suggested that the regulation of hydrolase activity pathway was involved in the development of COPD. CMap of hub genes of regulation of hydrolase activity pathwayshown the most significant compound was spiperone. Results of vitro experiment verify that cigarette smoke extract (CSE) can increase the expression of fibronectin 1 (FN1) and epidermal growth factor (EGF), coinsided with decrease the expression of chemokine (C-X3-C motif) ligand 1 (CX3CL1), chemokoine (C-C motif) ligand 20 (CCL20), complement component 3 (C3) and slithomolog 2 (SLIT2) in BESA-2B cells and U937 cells. Spiperone can reverse the effect of CSE in BESA-2B cells and U937 cells. CONCLUSION: Regulation of hydrolase activity pathway was involved in the occurrence of COPD, spiperone was a potential drug for the treatment of COPD by affecting the regulation of hydrolase activity pathway. This study had provided new insights into the potential pathogenesis and treatment of COPD.

Multilevel Approach for the Treatment of Giardiasis by Targeting Arginine Deiminase.

Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme's structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman's method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis.

Multi-targeted Effect of Nicotinamide Mononucleotide on Brain Bioenergetic Metabolism.

Dysfunctions in NAD+ metabolism are associated with neurodegenerative diseases, acute brain injury, diabetes, and aging. Loss of NAD+ levels results in impairment of mitochondria function, which leads to failure of essential metabolic processes. Strategies to replenish depleted NAD+ pools can offer significant improvements of pathologic states. NAD+ levels are maintained by two opposing enzymatic reactions, one is the consumption of NAD+ while the other is the re-synthesis of NAD+. Inhibition of NAD+ degrading enzymes, poly-ADP-ribose polymerase 1 (PARP1) and ectoenzyme CD38, following brain ischemic insult can provide neuroprotection. Preservation of NAD+ pools by administration of NAD+ precursors, such as nicotinamide (Nam) or nicotinamide mononucleotide (NMN), also offers neuroprotection. However, NMN treatment demonstrates to be a promising candidate as a therapeutic approach due to its multi-targeted effect acting as PARP1 and CD38 inhibitor, sirtuins activator, mitochondrial fission inhibitor, and NAD+ supplement. Many neurodegenerative diseases or acute brain injury activate several cellular death pathways requiring a treatment strategy that will target these mechanisms. Since NMN demonstrated the ability to exert its effect on several cellular metabolic pathways involved in brain pathophysiology it seems to be one of the most promising candidates to be used for successful neuroprotection.

Fluoxetine-induced androgenic failure impairs the seminiferous tubules integrity and increases ubiquitin carboxyl-terminal hydrolase L1 (UCHL1): Possible androgenic control of UCHL1 in germ cell death?

The selective serotonin reuptake inhibitor fluoxetine has been used for the treatment of depression. Although sexual disorders have been reported in male patients, few studies have demonstrated the fluoxetine effect on the reproductive histophysiology, and the target of this antidepressant in testes is unknown. We evaluated the impact of short-term treatment with fluoxetine on the adult rat testes, focusing on steroidogenesis by Leydig cells (LC) and androgen-dependent testicular parameters, including Sertoli cells (SC) and peritubular myoid cells (PMC). Since UCHL1 (ubiquitincarboxyl-terminal hydrolase L1) seems to control spermatogenesis, the immunoexpression of this hydrolase was also analyzed. Adult male rats received 20 mg/kg BW of fluoxetine (FG) or saline (CG) for eleven days. In historesin-embedded testis sections, the seminiferous tubule (ST) and epithelial (Ep) areas, and the LC nuclear diameter (LCnu) were measured. The number of abnormal ST, androgen-dependent ST, SC and PMC was quantified. Testicular ß-tubulin levels and peritubular actin immunofluorescence were evaluated. Serum testosterone levels (STL) and steroidogenesis by 17ß-HSD6 immunofluorescence were analyzed, and either UCHL1-immunolabeled or TUNEL-positive germ cells were quantified. In FG, abnormal ST frequency increased whereas ST and Ep areas, androgen-dependent ST number, LCnu, 17ß-HSD6 activity and STL reduced significantly. TUNEL-positive PMC and SC was related to decreased number of these cells and reduction in peritubular actin and ß-tubulin levels. In FG, uncommon UCHL1-immunoexpression was found in spermatocytes and spermatids, and the number of UCHL1-immunolabeled and TUNEL-positive germ cells increased in this group. These findings indicate that LC may be a fluoxetine target in testes, impairing PMC-SC integrity and disturbing spermatogenesis. The increase of UCHL1 in the damaged tubules associated with high incidence of cell death confirms that this hydrolase regulates germ cell death and may be controlled by androgens. The fertility in association with the androgenic status of patients treated with fluoxetine should be carefully evaluated.

Improved hydrolase activity in barley and reduced malting time by adding phytase as an activator during malting steeping.

OBJECTIVES: Exogenous phytase improved the activity of hydrolases to decrease the malting time. RESULTS: Treatment with phytase during barley steeping increased activity of hydrolases (α-/ß-amylase, proteinase, ß-glucanase and xylanase) in green malt. Maximal activity was observed for α-/ß-amylase, ß-glucanase and xylanase with 0.8 U phytase/g and proteinase with 1.2 U phytase/g. Phytase promoted acrospire growth of green malt and reduced malting process with 0.8 U phytase/g in 96 h, which is 24 h less than the control. No significant variation of malt quality was found between control malt and malt treated with 0.8 U/g or 1.2 U phytase/g (P > 0.05), which makes application of exogenous phytase during steeping process as a good option for reducing malting time. CONCLUSION: Adding phytase during steeping process increases the activity of hydrolases, which reduces malting time without impacting on malt quality.

Expression and Characterization of Serotype 2 Streptococcus suis Arginine Deiminase.

BACKGROUND: Arginine deiminase (ArcA) has been speculated to facilitate the intracellular survival of Streptococcus suis under acidic conditions. However, the physical and biological properties and function of SS2-ArcA have not yet been elucidated. METHODS: Recombinant SS2-ArcA (rSS2-ArcA) was expressed and purified using Ni-NTA affinity chromatography. Under various pH and temperature conditions, the enzymatic properties of purified rSS2-ArcA and crude native SS2-ArcA were determined. RESULTS: The SS2-arcA-deduced amino acid sequence contained a conserved catalytic triad (Cys399-His273-Glu218). The optimum temperature and pH of 47-kDa rSS2-ArcA and crude native SS2-ArcA were 42°C and pH 7.2. The rSS2-ArcA and crude native SS2-ArcA were stable for 3 h at 4 and 25°C, respectively. The pH stability and dependency tests suggested that rSS2-ArcA and crude native SS2-ArcA were functionally active in acidic conditions. The L-arginine substrate binding affinity (Km) values of rSS2-ArcA (specific activity 16.00 U/mg) and crude native SS2-ArcA (specific activity 0.23 U/mg) were 0.058 and 0.157 mM, respectively. rSS2-ArcA exhibited a weak binding affinity with the common ArcA inhibitors L-canavanine and L-NIO. Furthermore, the partial inactivation of SS2-ArcA significantly impaired the viability and growth of SS2 at pH 4.0, 6.0, and 7.5. CONCLUSIONS: This study profoundly demonstrated the involvement of ArcA enzymatic activity in S. suis survival under acidic conditions.

The ATP-Dependent Protease ClpP Inhibits Biofilm Formation by Regulating Agr and Cell Wall Hydrolase Sle1 in Staphylococcus aureus.

Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA). The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection.

Comparative Effects of Alkaloid Extracts from Aframomum melegueta (Alligator Pepper) and Aframomum danielli (Bastered Melegueta) on Enzymes Relevant to Erectile Dysfunction.

Aframomum melegueta (alligator pepper (AP)) and Aframomum danielli (bastered melegueta (BM)) seeds have been known to improve sexual function in folkloric medicine. This study investigates the effects of AP and BM seeds' alkaloid extracts on the activities of enzymes (acetylcholinesterase (AChE), angiotensin-1-converting enzyme (ACE), phosphodiesterase-5 (PDE-5), and arginase) relevant to erectile dysfunction (ED). Alkaloids from the seeds were prepared by the solvent extraction method and their interactions with AChE, ACE, PDE-5, and arginase were assessed. Gas chromatographic (GC) analyses of the extracts were also performed. The results revealed that the extracts inhibited the enzymes in a concentration-dependent manner. However, alkaloid extract from AP seed had higher AChE (IC50 = 5.42 µg/mL) and ACE (IC50 = 12.57 µg/mL) but lower PDE-5 (IC50 = 33.80 µg/mL) and arginase (IC50 = 31.36 µg/mL) inhibitory effects when compared to that of BM extract (AChE, IC50 = 42.00; ACE, IC50 = 60.67, PDE-5, IC50 = 7.24; and arginase, IC50 = 2.53 µg/mL). The GC analyses revealed the presence of senkirkine, angustifoline, undulatine, myristicin, safrole, lupanine, powelle, and indicine-N-oxide, among others. The inhibition of these enzymes could be the possible mechanisms by which the studied seeds were being used in managing ED in folklores. Nevertheless, the seed of AP exhibited higher potentials.

Elevation of Kynurenine Metabolites in Rat Liver and Serum: A Potential Additional Mechanism of the Alcohol Aversive and Anti-cancer Effects of Disulfiram?

AIMS: The tryptophan metabolites 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HAA) inhibit the liver mitochondrial low Km aldehyde dehydrogenase and possess alcohol-aversive and immunosuppressant properties. As the disulfiram (DS) metabolite carbon disulphide activates enzymes forming 3-HK and 3-HAA, we investigated if repeated disulfiram treatment increases the hepatic and serum levels of these 2 metabolites. METHODS: Livers and sera of male Wistar rats were analysed for tryptophan and kynurenine metabolites after repeated DS treatment for 7 days. RESULTS: DS increased liver and serum [3-HK] and [3-HAA] possibly by increasing the flux of tryptophan down the hepatic kynurenine pathway and activation of kynurenine hydroxylase and kynureninase. CONCLUSIONS: We provisionally suggest that elevation of some kynurenine metabolites may be an additional mechanism of the alcohol-aversive and anticancer effects of disulfiram.

The applications of binuclear metallohydrolases in medicine: recent advances in the design and development of novel drug leads for purple acid phosphatases, metallo-ß-lactamases and arginases.

Binuclear metallohydrolases are a family of proteins that can be targeted for drug discovery. The common feature of these enzymes is the presence of two closely spaced metal ions (i.e. less than 4 Å apart) that capture a water molecule that is used as a nucleophile in highly specific hydrolytic reactions. In this mini-review we describe what is known about the biological and catalytic activity, three-dimensional structure and inhibition for three prominent drug targets in this family of enzymes, (i) purple acid phosphatases, (ii) metallo-ß-lactamases and (iii) arginases. These enzymes are targets for the development of chemotherapeutics to treat a range of disorders including osteoporosis, cardiovascular disease and erectile dysfunctions, but also to stem the spread of antibiotic resistance, a major threat to global health care.

Identification of shikimate kinase inhibitors among anti-Mycobacterium tuberculosis compounds by LC-MS.

Increasing drug resistance has challenged the control and treatment of tuberculosis, sparking recent interest in finding new antitubercular agents with different chemical scaffolds and mechanisms of action. Mycobacterium tuberculosis shikimate kinase (MtSK), an enzyme present in the shikimate pathway in bacteria, is essential for the survival of the tubercle bacillus, representing an ideal target for therapeutic intervention given its absence in mammals. In this study, a small library of 404 synthetic antimycobacterial compounds identified and supplied through the NIH Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) high throughput screening program against whole cell M. tuberculosis H37Rv was further screened using a mass spectrometry-based functional assay in order to identify a potential enzymatic target. Fourteen compounds containing an oxadiazole-amide or a 2-aminobenzothiazole core scaffold showed MtSK inhibitory activity at 50 µM, with the lowest giving an IC50 of 1.94 µM. Induced fit docking studies suggested that the scaffolds shared by these compounds fit well in the shikimate binding pocket of MtSK. In summary, we report new early discovery stage lead scaffolds targeting the essential protein MtSK that can be further pursued in a rational drug design program for the discovery of more selective antitubercular drugs.

Molecular characteristics of MDR Mycobacterium tuberculosis strains isolated in Fujian, China.

Of 75 MDR isolates from Fujian Province, the sensitivity of RIF, INH, EMB, SM, OFLX and KAN resistance by DNA sequencing was 96.0%, 96.0%, 66.7%, 66.0%, 84.2% and 75.0%, respectively. We also identified that minority mutations in the mixed Mycobacterium tuberculosis population may be responsible for two "false-negative" results. In addition, Beijing genotype is still the predominant sublineage in the MDR TB cases from Fujian.

Effects of cold storage and 1-methylcyclopropene treatments on ripening and cell wall degrading in rabbiteye blueberry (Vaccinium ashei) fruit.

The effect of postharvest 1-methylcyclopropene and/or cold storage application on texture quality parameters during storage was determined. The changes in fruit quality (including weight loss, firmness, total soluble solids content, and ethylene production), cell wall material (including water-soluble fraction, ethylenediaminetetraacetic acid-soluble fraction, Na2CO3-soluble fraction, 4% KOH-soluble fraction, and 14% KOH-soluble fraction), and cell wall hydrolase activities (including polygalacturonase, endo-1,4-beta-D-glucanase, pectinesterase, alpha-L-arabinofuranosidase, and beta-galactosidase) were periodically measured up to 25 days after postharvest treatments. The application of cold storage reduced weight loss, ethylene production, and delayed ripening of blueberry fruit. The inhibition of senescence was associated with suppressed increase in cell wall hydrolase activities and retarded solubilization of pectins and hemicelluloses. Furthermore, no obvious differences in firmness, weight loss, ethylene production, and cell wall hydrolase activities between fruits with or without 1-methylcyclopropene application were observed, while significant lower levels of the detected parameters were found in cold storage fruit compared with fruit stored in room temperature. Thus, cold storage can be viewed as an effective means to extend the shelf life of blueberry fruit.

Pseudomonas aeruginosa lipopolysaccharide inhibits Candida albicans hyphae formation and alters gene expression during biofilm development.

Elucidation of bacterial and fungal interactions in multispecies biofilms will have major impacts on understanding the pathophysiology of infections. The objectives of this study were to (i) evaluate the effect of Pseudomonas aeruginosa lipopolysaccharide (LPS) on Candida albicans hyphal development and transcriptional regulation, (ii) investigate protein expression during biofilm formation, and (iii) propose likely molecular mechanisms for these interactions. The effect of LPS on C. albicans biofilms was assessed by XTT-reduction and growth curve assays, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Changes in candidal hypha-specific genes (HSGs) and transcription factor EFG1 expression were assessed by real-time polymerase chain reaction and two-dimensional gel electrophoresis, respectively. Proteome changes were examined by mass spectrometry. Both metabolic activities and growth rates of LPS-treated C. albicans biofilms were significantly lower (P < 0.05). There were higher proportions of budding yeasts in test biofilms compared with the controls. SEM and CLSM further confirmed these data. Significantly upregulated HSGs (at 48 h) and EFG1 (up to 48 h) were noted in the test biofilms (P < 0.05) but cAMP levels remained unaffected. Proteomic analysis showed suppression of candidal septicolysin-like protein, potential reductase-flavodoxin fragment, serine hydroxymethyltransferase, hypothetical proteins Cao19.10301(ATP7), CaO19.4716(GDH1), CaO19.11135(PGK1), CaO19.9877(HNT1) by P. aeruginosa LPS. Our data imply that bacterial LPS inhibit C. albicans biofilm formation and hyphal development. The P. aeruginosa LPS likely target glycolysis-associated mechanisms during candidal filamentation.

PLP-dependent enzymes as potential drug targets for protozoan diseases.

The chemical properties of the B(6) vitamers are uniquely suited for wide use as cofactors in essential reactions, such as decarboxylations and transaminations. This review addresses current efforts to explore vitamin B(6) dependent enzymatic reactions as drug targets. Several current targets are described that are found amongst these enzymes. The focus is set on diseases caused by protozoan parasites. Comparison across a range of these organisms allows insight into the distribution of potential targets, many of which may be of interest in the development of broad range anti-protozoan drugs. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

Ameliorative prospective of alpha-mangostin, a xanthone derivative from Garcinia mangostana against beta-adrenergic cathecolamine-induced myocardial toxicity and anomalous cardiac TNF-alpha and COX-2 expressions in rats.

Altered membrane integrity and inflammation play a key role in cardiovascular damage. We investigated the salubrious effect of exogenously administered alpha-mangostin against beta-adrenergic cathecolamine-induced cardiovascular toxicity with special reference to membrane ATPases, lysosomal hydrolases and inflammatory mediators TNF-alpha and Cyclooxygenase-2 (COX-2) expressions in albino rats. Induction of rats with isoproterenol (150mg/kg body wt, i.p.) for 2 days resulted in a significant increase in the activities of serum and cardiac lysosomal hydrolases (beta-d-glucuronidase, beta-d-galactosidase, beta-d-N-acetylglucosaminidase, acid phosphatase and cathepsin-D). A significant increase in cardiac levels of sodium, calcium with a decrease in the level of potassium paralleled by abnormal activities of membrane-bound phosphatases (Na(+)-K(+) ATPase, Ca(2+) ATPase and Mg(2+) ATPase) were observed in the heart of ISO-administered rats. Cardiac TNF-alpha and COX-2 expressions were assessed by Western blotting. Cardiac TNF-alpha and COX-2 expressions were significantly elevated in ISO-intoxicated rats. Pre-co-treatment with alpha-mangostin (200mg/kg body wt.) orally for 8 days significantly attenuated these abnormalities and restored the levels to near normalcy when compared to ISO intoxicated group of rats. In conclusion, alpha-mangostin preserves the myocardial membrane integrity and extenuates anomalous TNF-alpha and COX-2 expressions by mitigating ISO-induced oxidative stress and cellular damage effectively. Restoration of cellular normalcy accredits the cytoprotective role of alpha-mangostin.

Alteraciones clínicas y bioquímicas en ratas tratadas con dosis altas de vitamina A / Clinical and biochemical alterations in rats treated with high doses of vitamin A

En el presente trabajo se estudió el efecto de la administración intramuscular de 30.000, 50.000 y 100.000 UI de palmitato de vitamina A/día, durante 7 días, respectivamente, sobre la actividad enzimática hepática en 45 ratas Wistar machos, de 12 semanas de edad, con pesos entre 180 y 200 gramos. El grupo control estuvo integrado por 15 ratas Wistar sanas, con género, edad y peso similares a los animales tratados. El consumo de alimentos y de agua, y el peso de las ratas se determinó al finalizar el período experimental. Las ratas se examinaron en busca de manifestaciones clínicas de toxicidad. Al final el estudio, las ratas se sacrificaron bajo anestesia con éter y se tomaron muestras de tejido hepático para la determinación de la actividad enzimática. La administración de vitamina A en exceso incrementó de manera significativa (p menor que 0,05) el contenido hepático del retinol, determinó diversos y variados signos clínicos (tales como: anorexia, pérdida de peso, alopecia, conjuntivitis, hemorragias internas y externas, alteraciones cutáneas y muerte de los animales) e incrementó (p menor que 0,05) la actividad de las siguientes enzimas: alanina aminotransferasa, aspartato aminotransferasa, maltasa ácida (alfa-1,4-glucosidasa ácida), proteasas ácidas, lactato dehidrogenasa y fosfatasa alcalina mientras que las actividades de la glucosa-6-fosfatasa, glucógeno fosforilasa, alfa-amilasa, colinesterasa y arginasa disminuyeron (p menor que 0,05) al comparar con los controles no tratados. Estos cambios son proporcionales a las dosis inyectadas de vitamina A. En conclusión, nuestros resultados proporcionan evidencias que la administración de dosis altas de vitamina A a corto plazo determina diversos y variados signos clínicos y produce una marcada alteración de la actividad enzimática hepática.

In the present work the effect of intramuscular administration of 30.000, 50.000 and 100.000 IU of vitamin A palmitate daily for seven days, respectively, on the liver enzyme activity in 45 white male Wistar rats, aged 12 weeks and weighing 180-200 g, have been studied. The group control was integrated by 15 healthy rats with similar characteristics (strain, gender, age and weight) to treated animals. Food and water consumption and body weights were recorded at the end of the experimental period. Rats were observed for clinical signs of toxicity. At the end of the study, rats were sacrificed under ether anesthesia. Liver samples were taken for the determination of enzyme activity. Administration of excess of vitamin A produced a significant (p menor 0.05) increase in the content of liver vitamin A, determined diverse and variable clinical signs (such as, anorexia, loss of body weight, alopecia, conjunctivitis, external and internal hemorrhages, skin abnormalities and death) and increased (p menor que 0.05) the activity of the following enzymes: alanine aminotransferase, aspartate aminotransferase, acid maltase (acid alfa-1,4-glucosidase), acid proteases, lactate dehydrogenase and alkaline phosphatase while glucose-6-phosphatase, glycogen phosphorylase, alfa-amylase, cholinesterase and arginase decreased (p menor que 0.05) as compared with untreated controls. These changes depend on the doses given of vitamin A. In conclusion, our results provide evidence that short-term administration of high doses of vitamin A determined diverse and variable clinical signs and produces a marked alteration of activity of liver enzymes.

A study on the inactivation of micro-organisms and enzymes by high pressure CO2.

This study addresses some microbial inactivation phenomena induced by high pressure CO2 over micro-organisms and enzymes. The activity of four selected enzymes was measured before and after treatment with CO2 under pressure in both buffer solutions and natural cellular environment (E. coli cells and tomato paste). Results are reported for acid phosphatase, alkaline phosphatase, ATPase, and pectinase at different conditions of temperature, CO2 pressure, and treatment time (32-40 degrees C, 85-150 bar, 30-70 min). The results obtained show that the high pressure CO2 treatment induces an inactivation of cellular enzymatic activity higher than the one caused on the same enzymes in solution. However, the measured activity difference is not caused by a damage at the enzymes molecular level but is a consequence of the permeabilization of the cellular envelopes which leads to a release of unmodified enzymes from the cells with simultaneous drop of enzymatic cellular activity. The reported data suggest that the bacterial cell death is probably due not to a selective effect of high pressure CO2 treatment but to simultaneous detrimental action of CO2 on cellular membrane and cell wall.

Gene expression profiling in spleens of deoxynivalenol-exposed mice: immediate early genes as primary targets.

Exposure to the trichothecene mycotoxin deoxynivalenol (DON) alters immune functions in vitro and in vivo. To gain further insight into DON's immunotoxic effects, microarrays were used to determine how acute exposure to this mycotoxin modulates gene expression profiles in murine spleen. B6C3F1 mice were treated orally with 25mg/kg body weight DON, and 2h later spleens were collected for macroarray analysis. Following normalization using a local linear regression model, expression of 116 out of 1176 genes was significantly altered compared to average expression levels in all treatment groups. When genes were arranged into an ontology tree to facilitate comparison of expression profiles between treatment groups, DON was found primarily to modulate genes associated with immunity, inflammation, and chemotaxis. Real-time polymerase chain reaction was used to confirm modulation for selected genes. DON was found to induce the cytokines interleukin (IL)-1alpha, IL-1beta, IL-6 and IL-11. In analogous fashion, DON upregulated expression of the chemokines macrophage inhibitory protein-2 (MIP-2), cytokine-induced chemoattractant protein-1 (CINC-1), monocyte chemoattractant protein (MCP)-1, MCP-3, and cytokine-responsive gene-2 (CRG-2). c-Fos, Fra-, c-Jun, and JunB, components of the activator protein-1 (AP-1) transcription factor complex, were induced by DON as well as another transcription factor, NR4A1. Four hydrolases were found to be upregulated by DON, including mitogen-activated protein kinase phosphatase 1 (MKP1), catalytic subunit beta isoform (CnAbeta), protein tyrosine phosphatase receptor type J (Ptprj), and protein tyrosine phosphatase nonreceptor type 8 (Ptpn8), whereas three other hydrolases, microsomal epoxide hydrolase (Eph) 1, histidine triad nucleotide binding protein (Hint), and proteosome subunit beta type 8 (Psmb8) were significantly decreased by the toxin. Finally, cysteine-rich protein 61 (CRP61) and heat-shock protein 40 (Hsp40), genes associated with signaling, were increased, while Jun kinase 2 (JNK2) was decreased. Taken together, data suggest that DON upregulated the expression of multiple immediate early genes, many of which are likely to contribute to the complex immunological effects reported for this and other trichothecenes.

Effect of L-arginine and L-lysine on lysosomal hydrolases and membrane bound phosphatases in experimentally induced myocardial infarction in rats.

The protective effect of L-arginine and L-lysine on lysosomal enzymes and membrane bound ATPases was examined on isoproterenol induced myocardial infarction in rats. Lysosomal enzymes play an important role in the inflammatory process. The rats given isoproterenol (150 mg kg(-1) daily) intraperitoneally for 2 days showed significant changes in the marker enzymes, lysosomal enzymes and membrane bound phosphatases. Histopathological studies also confirmed the induction of myocardial infarction in isoproterenol administered rats. Prior oral treatment with L-arginine (250 mg kg(-1) daily) and L-lysine (5 mg kg(-1) daily) for 5 days significantly prevented these alterations and restored the enzyme activities to near normal. These findings demonstrate the protective effect of L-arginine and L-lysine in combination against isoproterenol induced cardiac damage.