Genetic engineering and molecular characterization of yeast strain expressing hybrid human-yeast squalene synthase as a tool for anti-cholesterol drug assessment.

AIMS: The main objective of the study is molecular and biological characterization of the human-yeast hybrid squalene synthase (SQS), as a promising target for treatment of hypercholesterolaemia. METHODS AND RESULTS: The human-yeast hybrid SQS, with 67% amino acids, including the catalytic site derived from human enzyme, was expressed in Saccharomyces cerevisiae strain deleted of its own SQS gene. The constructed strain has a decreased level of sterols compared to the control strain. The mevalonate pathway and sterol biosynthesis genes are induced and the level of triacylglycerols is increased. Treatment of the strain with rosuvastatin or zaragozic acid, two mevalonate pathway inhibitors, decreased the amounts of squalene, lanosterol and ergosterol, and up-regulated expression of several genes encoding enzymes responsible for biosynthesis of ergosterol precursors. Conversely, expression of the majority genes implicated in the biosynthesis of other mevalonate pathway end products, ubiquinone and dolichol, was down-regulated. CONCLUSIONS: The S. cerevisiae strain constructed in this study enables to investigate the physiological and molecular effects of inhibitors on cell functioning. SIGNIFICANCE AND IMPACT OF THE STUDY: The yeast strain expressing hybrid SQS with the catalytic core of human enzyme is a convenient tool for efficient screening for novel inhibitors of cholesterol-lowering properties.

Erratum: Elastase and metalloproteinase 9 concentrations in saliva in patients with chronic periodontitis.

[This corrects the article DOI: 10.5114/ceji.2014.45948.].

MicroRNA expression profiles in liver and colon of sexually immature gilts after exposure to Fusarium mycotoxins.

To improve our knowledge of the role of microRNAs (miRs) in responses of the porcine digestive system to two Fusarium mycotoxins, zearalenone (ZEN) and deoxynivalenol (DON), we examined the expression of 7 miRs (miR-9, miR-15a, miR-21, miR-34a, miR-122, miR-125b, and miR-192), previously found to be deregulated in diseased liver and colon cells. In this study, immature gilts were exposed to NOEL doses of ZEN (40 µg/kg/d), DON (12 µg/kg/d), ZEN + DON (40 + 12 µg/kg/d), andplacebo (negative control group) for 7, 14, 21, 28, 35, and 42 days. Before the treatment, expression levels of the selected miRs were measured in the liver, the duodenum, the jejunum, and the ascending and the descending colon of the gilts. Hierarchical clustering of the tissues by their miR expression profiles was consistent with what would be expected based on the anatomical locations and the physiological functions of the organs, suggesting that functions of the miRs are related to the specificities of the tissues in which they are expressed. A subset of 2 pairs of miRs (miR-21+miR-192 and miR-15a+miR-34a), which were assigned to two distinct clusters based on their tissue abundance, was then evaluated in the liver and the ascending and the descending colon during the treatment. The most meaningful results were obtained from the ascending colon, where a significant effect of the treatment was observed, suggesting that during the exposure to mycotoxins, the pathways involved in cell proliferation and survival were disordered. Changes in miR expression in the liver and the descending colon of the treated gilts were smaller, and were associated more with treatment duration than the exposure to ZEN, DON, or ZEN + DON. Further research should focus on identification of genes whose expression is regulated by these aberrantly expressed miRs. This should facili- tate understanding of the miRNA-regulated biological effects of mycotoxins.

Assessment of temporomandibular joint disease.

The diagnosis of temporomandibular joint (TMJ) disorders consists of clinical (Reaserch Diagnostic Criteria for Temporomandibular Disorders, RDC/TMD) and additional (computer tomography, CT or magnetic resonance imaging, and MRI) examinations. Due to the growing knowledge of pathologic changes within the TMJ, the researches become more aware of the difficulty in detection the early symptoms of disorders using conventional examination. Therefore, it is now expected that the collected samples of synovial fluid, serum, or urine samples could enable easier identification of inflammatory process course, and degenerative cartilage changes state.

Energy and economic efficiency of climate-smart agriculture practices in a rice-wheat cropping system of India.

Intensive tillage operations, indiscriminate use of irrigation water, chemical fertilizers, and pesticides and crop biomass burning have made the conventional rice-wheat (RW) system highly energy-intensive and inefficient. In the recent past, portfolios of climate-smart agricultural practices (CSAP) have been promoted as a potential alternative to improve the energy efficiency in conventional RW system. Therefore, to evaluate the energy input-output relation, energy flow and economic efficiency in various combinations of crop management options, a 3-year (2014-2017) on-farm study was conducted at Karnal, India. Various portfolio of management practices; Sc1-Business as usual (BAU) or Conventional tillage (CT) without residue, Sc2-CT with residue, Sc3-Reduce tillage (RT) with residue + recommended dose of fertilizer (RDF), Sc4-RT/Zero tillage (ZT) with residue + RDF, Sc5-ZT with residue + RDF + GreenSeeker + Tensiometer, Sc6-Sc5 + Nutrient expert were investigated. Present study results revealed that net energy, energy use efficiency and energy productivity were 11-18, 31-51 and 29-53% higher under CSAP (mean of Sc4, Sc5 and Sc6) in RW system than Sc1, respectively. However, renewable and non-renewable energy inputs were 14 and 33% higher in Sc1 compared to CSAP (4028 and 49,547 MJ ha-1), respectively, it showed that BAU practices mostly dependents on non-renewable energy sources whereas CSAP dependents on renewable energy sources. Similarly, the adoption of CSAP improved the biomass yield, net farm income and economic efficiency by 6-9, 18-23 and 42-58%, respectively compared to Sc1. Overall, the adoption of CSAP could be a viable alternative for improving energy use efficiency, farm profitability and eco-efficiency in the RW system.

Scalable diversification options delivers sustainable and nutritious food in Indo-Gangetic plains.

Indo-Gangetic plains (IGP) of South Asia have supported bulk of human and bovine population in the region since ages, and a spectacular progress has been made in food production. However, malnutrition, diminishing total factor productivity, and natural resource degradation continue to plague this cereal-dominated region, which is also vulnerable to climate change. Addressing these challenges would require a transition towards diversifying cereal rotations with agroecological cropping systems. A study was, therefore, conducted at the experimental farm of ICAR-CSSRI, Karnal on crop diversification and sustainable intensification options using agro-ecological approaches such as Conservation Agriculture (CA) and diversified cropping systems to ensure food and nutritional security while sustaining the natural resources. On 2 years mean basis, CA-based cropping system management scenarios (mean of Sc2-Sc7) using diversified crop rotations; increased the system yield by 15.4%, net return by 28.7%, protein yield by 29.7%, while using 53.0% less irrigation water compared to conventional tillage (CT)-based rice-wheat system (Sc1). Maize-mustard-mungbean on permanent beds (PBs) (Sc4) recorded the highest productivity (+ 40.7%), profitability (+ 60.1%), and saved 81.8% irrigation water compared to Sc1 (11.8 Mg ha-1; 2190 USD ha-1; 2514 mm ha-1). Similarly, Sc5 (maize-wheat-mungbean on PBs) improved productivity (+ 32.2%), profitability (+ 57.4%) and saved irrigation water (75.5%) compared to Sc1. In terms of nutritional value, Sc5 was more balanced than other scenarios, and produced 43.8, 27.5 and 259.8% higher protein, carbohydrate and fat yields, respectively, compared to Sc1 (0.93, 8.55 and 0.14 Mg ha-1). Scenario 5 was able to meet the nutrient demand of 19, 23 and 32 additional persons ha-1 year-1 with respect to protein, carbohydrate and fat, respectively, compared to Sc1. The highest protein water productivity (~ 0.31 kg protein m-3 water) was recorded with CA-based soybean-wheat-mungbean (Sc6) system followed by maize-mustard-mungbean on PBs (Sc4) system (~ 0.29 kg protein m-3) and lowest under Sc1. Integration of short duration legume (mungbean) improved the system productivity by 17.2% and profitability by 32.1%, while triple gains in irrigation water productivity compared to CT-based systems. In western IGP, maize-wheat-mungbean on PBs was found most productive, profitable and nutritionally rich and efficient system compared to other systems. Therefore, diversification of water intensive cereal rotations with inclusion of legumes and CA-based management optimization can be potential option to ensure nutritious food for the dwelling communities and sustainability of natural resources in the region.

The evaluation of the efficacy of sodium carbonate as zearalenone destructor in feeding stuffs.

Contamination of feed with zearalenone (ZEA) is still a serious problem in farm animals feeding, especially in gilts, sensitive to this compound. The relative failure of current methods of decontamination and quality control lead us to look for new techniques. The commonly accepted method for breaking down ZEA was performed in controlled temperature and time conditions. Various sodium carbonate doses (0.5 - 4%) were added to feed naturally contaminated with ZEA (ZEA biosynthesis by F. graminearum isolates). These doses were found to be effective in in vitro studies. The addition of 2% sodium carbonate gave the best results in reducing the phytoestrogen in the feed.

Real-time PCR analysis of p53 mRNA levels in tissues of whitefish (Coregonus lavaretus) exposed to benzo[a]pyrene.

We developed a real-time PCR assay for measuring relative quantities (RQ) of p53 tumor suppressor mRNA in the whitefish (Coregonus lavaretus, Salmonidae, Teleostei). Real-time PCR primers for the p53 gene were designed from a region that was found to be conserved among salmonid p53 genes. To test for the usefulness of the assay we performed a treatment study, using benzo[a]pyrene (B[a]P) a putative p53-inducer. Two groups of hatchery raised whitefish, with an average body mass of 15 g and total length of 12 cm were either given an intraperitoneal injection (10 mg x kg(-1)) of B[a]P in corn oil (2 mg B[a]P ml(-1) corn oil) or corn oil alone (Control). After treatment (48 h, 7 degrees C), two random fish from each group were anesthetized and the liver, head kidney and brain were collected for mRNA isolation and analysis. In the control fish, relative quantification analysis based on the p53 mRNA levels in liver (RQ=1.00) showed higher basal levels of p53 mRNA in the head kidney (RQ= 1.69), and lower in the brain (RQ=0.41). In all three tissues sampled, p53 mRNA was affected by treatment with B[a]P. Liver tissue showed the greatest induction (RQ=1.53) from base levels (RQ=1.00), followed by brain (RQ=1.36), and head kidney (RQ=1.23). These results confirm that p53 mRNA is generally present at lower levels in differentiated tissues (liver and brain) than in those tissues with cell lines (head kidney), and demonstrate that p53 is moderately inducible by B[a]P in the whitefish. The approach presented here has the advantage of providing rapid and accurate measures of p53 induction in various tissues of fish responding to PAH contaminant exposure.

Total and high molecular weight adiponectin levels in the rat model of post-myocardial infarction heart failure.

Adiponectin is a protein secreted primarily by adipose tissue. It has been suggested that adiponectin plays a protective role in the early phase following myocardial infarction. Our primary aim was to investigate the effects of post-myocardial infarction heart failure well-characterized by left ventricular hemodynamic parameters on the total and high molecular weight adiponectin concentrations in plasma, fat and cardiac tissue. Eight weeks after myocardial infarction or sham operation, total and high molecular weight adiponectin concentrations in plasma, fat, and cardiac tissues were assayed in rats. In addition, hemodynamic parameters and expression of the genes encoding atrial natriuretic peptide and brain natriuretic peptide in left ventricle were evaluated. Atrial natriuretic peptide and brain natriuretic peptide mRNA levels in left ventricle tissue were higher in rats with myocardial infarction-induced heart failure compared with the controls. Similarly, total adiponectin concentration was increased in left ventricle (but not in right ventricle) in rats with post-myocardial infarction heart failure. In contrast, adiponectin levels in plasma and cardiac adipose tissue in rats with post-myocardial infarction heart failure were lower than in sham-operated animals. Furthermore, there were no significant differences in levels of high molecular weight adiponectin in plasma, cardiac tissue or adipose tissue between these two groups. We conclude that in the rat model of post-myocardial infarction heart failure, adiponectin level is increased in left ventricle tissue. This is accompanied by decreased adiponectin levels in plasma and cardiac adipose tissue.

Isolation and functional characterization of mutant ferrochelatases in Saccharomyces cerevisiae.

Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the incorporation of ferrous iron into protoporphyrin, the last step in protoheme biosynthesis. It is encoded by the HEM15 gene in the yeast Saccharomyces cerevisiae. Five hem15 mutants causing defective heme synthesis and protoporphyrin accumulation were investigated. The mutations were identified by sequencing the mutant hem15 alleles amplified in vitro from mutant genomic DNA. A single nucleotide change, causing an amino acid substitution, was found in each mutant. The substitution L62F caused a five-fold increase in Vmax and 32-fold and four-fold increases in the KM's for protoporphyrin and metal. Replacements of the conserved G47 by S and S102 by F increased the KM for protoporphyrin 10-fold without affecting the affinity for metal or enzyme activity. Two amino acid changes, L205P and P221L, produced a thermosensitive phenotype. In vivo heme synthesis, the amount of immunodetected protein, and ferrochelatase activity measured in vitro were more affected in cells grown at 37 degrees C than at 30 degrees C. The effects of these mutations on the enzyme function are discussed with respects to ferrochelatase structure and mechanism of action.

Suppressors of translation initiation defect in hem12 locus of Saccharomyces cerevisiae.

A system for the positive selection of transational initiation suppressors in S. cerevisiae has been developed. A mutant with an ATA initiation codon in the HEM12 gene, encoding uroporphyrinogen decarboxylase, was used to select cis- and trans-acting suppressors. These suppressors partially restore growth on nonfermentable carbon sources, such as glycerol, but still allow the accumulation of porphyrins. All extragenic suppressors are mapped to the SUI1 locus, encoding initiation factor eIF1. The effect of the hem12 mutation is also partially reversed by the known SUI3 suppressor encoding the beta subunit of eIF2. In contrast, the sui2 suppressor encoding the a subunit of eIF2 does not affect the hem12 phenotype. The intragenic suppressors are able to restore the translation of hem12 due to the generation of additional, in frame AUG codons upstream of the hem12-14 mutation. Mutational analysis of the HEM12 leader sequence was also performed to determine the role of small open reading frames (uORFs) present upstream of the HEM12 ORF. Studies on the expression of integrated hem12-1/4-lacZ fusion, devoid of all upstream ATGs, indicate a lack of regulatory effect of uORFs on HEM12 translation.

Considerations of drug therapy in patients receiving enteral nutrition.

Some basic principles to consider in giving medications to patients receiving enteral nutrition include: 1. If the patient is able to take medication by mouth, this is the preferred route. 2. Liquid medications are the preferred dosage form. 3. The use of oral medications that are not meant to be crushed for enteral tube administration should be avoided. 4. For individual doses of most medications, the tube should be flushed with at least 30 ml of water before and after administration of medications. 5. Highly concentrated solutions should be diluted with 60 ml of water. 6. When several medications are to be administered to the same patient, all medications should be delivered separately and the tube flushed with at least 5 ml of water after each dose. 7. Medications should not be added directly to the feeding formulation. 8. Drug-nutrient interactions should be considered. 9. GI side effects are the most common adverse effects that occur with enteral feedings, and treatment depends on the cause.

Meperidine in conjunction with cholescintigraphy to diagnose acute cholecystitis in a patient allergic to morphine.

Cholescintigraphy with morphine augmentation is used routinely to expedite the differential diagnosis of acute from chronic cholecystitis. A patient with hepatic dysfunction and an allergy to morphine received intravenous meperidine in conjunction with cholescintigraphy. The gallbladder was not visualized 30 minutes after administration of the drug. However, the activity accumulated in the initial photon deficient gallbladder at 4 hours after meperidine administration (6.5 hours after radiopharmaceutical administration). These findings may be explained in part by prolongation of meperidine bioavailability because of impairment of hepatic function.

Cerebral blood flow assessed by brain SPECT with 99mTc-HMPAO utilising the acetazolamide test in systemic lupus erythematosus.

BACKGROUND: Cerebrovascular diseases are one of the most important complications of systemic lupus erythematosus (SLE). The diagnostic imaging of neuropsychiatric SLE complications presents many problems. This study was undertaken to investigate cerebral blood flow char s and its reactivity to hypercapnia by means of acetazolamide test in SLE patients. METHODS: Brain SPELT studies using 99mTc-HMPAO were performed in 50 patients with SLE. Acetazolamide test was performed in 35 patients 3 days after the baseline study by means of repetitive scanning 20 min after i.v. injection of 1.0 g of acetazolamide. RESULTS: Significant interhemispheric hypoperfusion areas were shown in 76.3% of all patients, 83.8% symptomatic and 63.1 % asymptomatic. Patients with antiphospholipid syndrome showed multifocal perfusion deficits. The reaction of cerebral perfusion to acetazolamide was heterogenous and showed increase, decrease, no change or mixed reaction of baseline-study-found focal hypoperfusion. Acetazolamide test revealed hypoperfusion in two patients with normal baseline study. MRI scanning revealed cerebral lesions in 41 % of patients. CONCLUSIONS: CBF asymmetries in symptomatic and asymptomatic patients with SLE are frequent. Regional CBF alterations seem to be different in patients with and without antiphospholipid syndrome. The part of the patients with SLE shows no or paradoxically inversed reaction to acetazolamide.

Modification of zearalenone structure in model and natural conditions.

Zearalenone is a mycotoxin widely occurring in cereals and animal feed, and it is associated with hyperestrogenism and other reprodutive disorders in animals. A new method of detoxication of feedstuffs involves alkaline hydrolysis of toxic macrolactone (1) (as well as model compounds (2a, 2b)). The method caused modification of zearalenone structure under mild conditions and the toxin underwent irreversible hydrolysis with high efficiency.

Metabolic profile of pigs fed feed containing zearalenone destructor.

Zearalenone (ZEA) is a member of macrocyclic lactons family. It is a toxin--phytosteride produced by fungi of Fusarium ssp. genus. Zearalenone contaminates food and animal feeding stuffs and its destruction is difficult. It requires application of particular compounds that would bind zearalenone in the feed or feeding stuff or in the gastrointestinal tract and decrease its bio-accessibility. It should also fulfil all the safety requirements regarding the plant supplements and animals that are fed with this feed. The aim of the study was to estimate if the feed supplemented with different doses of zearalenone and zearalenone destructor causes changes of the metabolic profile in gilts. The results obtained show that applied destructor did not cause negative haematological and biochemical changes in the blood of the gilts examined. It can be suggested that it is a safe feed supplement pigs in prevention of zearalenone micotoxicosis.

Nicotine transdermal systems.

OBJECTIVE: To review the role of transdermal nicotine as an aid to smoking cessation. DATA SOURCES: A MEDLINE search was performed that included clinical studies published in English involving transdermal nicotine; references used in those articles were screened for additional published information. STUDY SELECTION: Published clinical trials were reviewed with particular emphasis on controlled trials that evaluated safety and efficacy. DATA SYNTHESIS: Transdermal nicotine therapy has been shown to be a safe and effective pharmacologic aid in a smoking cessation program when used in conjunction with a psychologic or behavior support system. Habitrol, Nicoderm, Nicotrol, and PROSTEP differ in some characteristics (i.e., delivery systems, total nicotine content and amount absorbed, rate of delivery, recommended duration of application); however, the clinical implication of these differences has not been determined. CONCLUSIONS: Transdermal nicotine is effective for patients who are motivated to quit smoking and receive concomitant behavior support.

Pleural effusions: pathophysiology and management.

OBJECTIVE: To review the pathophysiology and management of pleural effusions, including available agents for pleural sclerosis. DATA SOURCES: A MEDLINE search (1966 to present) was performed that included clinical studies in the English language involving the pathophysiology and management of pleural effusions; references used in those articles were screened for additional published information. STUDY SELECTION: All clinical trials were considered for potential inclusion in the review. DATA SYNTHESIS: Pleural effusion is an accumulation of fluid in the pleural space that results when homeostatic forces that control the flow into and out of the area are disrupted. The management of transudative pleural effusions is primarily directed at treatment of the underlying disease. There are several treatment options for pleural effusions, including chemical pleurodesis. Many of the trials that examine the use of talc, bleomycin, and doxycycline have poorly described study designs and end points, with inconsistent evaluation of patients. Each agent is considered to be generally effective and safe, with fever and pain as the most frequently reported adverse effects. The use of talc requires sterilization, and many clinicians use general anesthesia with instillation, which increases the risk associated with the procedure. Bleomycin is generally safe; however, it should not be used in doses exceeding 40 mg/m2. Only uncontrolled trials support the use of doxycycline; however, it provides an effective, safe, and relatively inexpensive alternative. CONCLUSIONS: Pleural effusions are defined as an accumulation of fluid in the pleural space. Treatment is generally palliative. Intrapleural administration of talc, bleomycin, and doxycycline are effective sclerosing agents for treatment of recurrent, symptomatic pleural effusions. Although the most cost-effective agent has not been determined, doxycycline is an inexpensive alternative to bleomycin, and may have fewer adverse effects than talc.

Methods used by pharmacy departments to identify drug interactions.

The results of a survey evaluating the methods that pharmacy departments use to manage drug interactions, and their satisfaction with those methods, are reported. A random sample of 300 hospitals with more than 200 beds was selected, and a questionnaire was mailed in January 1991 to the directors of the pharmacy departments of these hospitals. Of the 300 questionnaires mailed, 167 were completed and returned. Thirty-seven percent of the hospitals offered pharmacy services through a central location only, whereas 63% offered such services through a combination of central and decentral locations. The majority of hospitals (83%) detected drug interactions through the knowledge of the pharmacist processing orders. Nearly all central pharmacies had AHFS Drug Information and Facts and Comparisons available for drug interaction information, but these resources were available in only slightly more than half of all decentral pharmacies surveyed. Fifty-four percent of the responding institutions had a computerized drug distribution system with a drug interaction component; 56% of these classified their system as "indispensable" or "helpful," while 44% classified their system as "somewhat helpful but sometimes a hindrance." Sixty-eight percent of all respondents believed that the availability of a drug interaction computer program increased the number of drug interactions identified. Sixty-eight percent of respondents perceived that pharmacists provided proper follow-up. Overall, 66% of respondents were not satisfied with their method for detection and follow-up of interactions. Of those who were satisfied, 79% had a computerized drug interaction program. Respondents who reported departmental audits or quality assessment procedures were also more likely to be satisfied.(ABSTRACT TRUNCATED AT 250 WORDS)

Probing the active-site residues in Saccharomyces cerevisiae ferrochelatase by directed mutagenesis. In vivo and in vitro analyses.

Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the insertion of ferrous iron into protoporphyrin, the terminal step in protoheme biosynthesis. The functional/structural roles of 10 invariant amino acid residues were investigated by site-directed mutagenesis in the yeast Saccharomyces cerevisiae ferrochelatase. The mutant enzymes were expressed in a yeast strain lacking the ferrochelatase gene HEM15 and in Escherichia coli. The kinetic parameters of the mutant enzymes were determined for the enzymes associated with the yeast membranes and the enzymes in the bacterial soluble fraction. They were compared with the in vivo functioning of the mutant enzymes. The main conclusions are the following. Glu-314 is critical for catalysis, and we suggest that it is the base responsible for abstracting the N-pyrrole proton(s). His-235 is essential for metal binding. Asp-246 and Tyr-248 are also involved in metal binding in a synergistic manner. The Km for protoporphyrin was also increased in the H235L, D246A, and Y248L mutants, suggesting that the binding sites of the two substrates are not independent of each other. The R87A, Y95L, Q111E, Q273E, W282L, and F308A mutants had 1.2-2-fold increased Vm and 4-10-fold increased Km values for protoporphyrin, but the amount of heme made in vivo was 10-100% of the normal value. These mutations probably affected the geometry of the active center, resulting in improper positioning of protoporphyrin.