Cloning, Expression and Regulation of CYP3A10, a Hamster Liver Cytochrome P450 Involved in Lithocholic Acid and Steroid 6β-Hydroxylation: a Dissertation

Bile acid metabolism is integrally involved in cholesterol homeostasis in mammals because it is the major means by which cholesterol is eliminated from the body. We have undertaken an effort to study the molecular mechanisms underlying the regulation of bile acid metabolism by isolating and characterizing the cDNA and gene for an enzyme that hydroxylates lithocholic acid (LCA) at position 6β, lithocholic acid 6β-hydroxylase; the first bile acid-induced gene reported. LCA is a very hydrophobic, toxic bile acid formed from chenodeoxycholic acid in the gut lumen upon reduction of the 7α-hydroxy group by microbial enzymes. The proper elimination of LCA is essential for maintenance of the bile acid pool and for prevention of cholestasis which results from LCA precipitating in the cannaculi of the liver when its concentration is high. The LCA 6β-hydroxylase cDNA was isolated by differential hybridization of hamster liver libraries prepared from animals fed either a cholic acid enriched diet or a cholestipol-rich chow and was named CYP3A10 based on its homology with other cytochrome P450s (P450) in family 3A. We found that CYP3A10 was essentially expressed only in males. A statistical analysis of RNA from young males fed with cholic acid and normal chow showed that the cholic acid induction was about 50% at the RNA level. We determined the biological nature of the protein encoded by CYP3A10 by expression of the cDNA in COS cells. Microsomes prepared from transfected cells were assayed with LCA as a substrate and found to hydroxylate LCA predominantly at position 6β. We examined whether CYP3A10 could hydroxylate other steroid compounds by assays with testosterone, progesterone and androstenedione and found that, although 6β-hydroxylase (as well as others) activity was observed with all three, LCA was the preferred substrate based on kinetic analysis. A developmental time course of CYP3A10 expression in males showed little expression before puberty, a striking induction of expression at puberty and a fourfold induction thereafter through adulthood. We then examined the male-specific expression of CYP3A10 in hamster liver. We disrupted the pattern of GH secretion in male hamsters by hypophysectomy, neonatal glutamate treatment and by continuous infusion of GH via osmotic minipumps (to mimic the female pattern of GH secretion) and found no significant effect on CYP3A10 expression. Conversely, in females, hypophysectomy and neonatal glutamate treatment significantly induced CYP3A10 expression 5- to 10-fold. Additionally, when females treated neonatally with glutamate were injected twice daily with GH as adults (to mimic the male pattern of GH secretion), the levels of CYP3A10 expression were not significantly different from those of normal males. These results led us to conclude that the pattern of GH secretion in males does not control the male-specific expression of CYP3A10 but that in females expression can be induced by altering the tonic secretion of GH. No significant effect on CYP3A10 expression was observed by castration of adult males, indicating that circulating androgens were not required for expression. We found that gonadal hormones (e.g. estrogen and progesterone) do not have a suppressive effect on CYP3A10 expression in females since ovariectomy did not induce expression. Many genes are imprinted neonatally by exposure to a given effector for developmental-, tissue- or sexually regulated expression. We investigated whether neonatal androgen exposure was required for male-specific expression of CYP3A10 by castrating hamsters neonatally and determining the level of CYP3A10 expression in adulthood. Our results indicate that androgens are required neonatally for CYP3A10 expression since no expression was observed in neonatally castrated hamsters. We were unable to induce expression in neonatally castrated hamsters by either GH or testosterone injections. These results suggest several notable points 1) that CYP3A10 expression is programmed neonatally by androgen exposure; 2) that androgens exert their effect directly on the liver and not via the hypothalamus; 3) that neither testosterone nor GH can restore CYP3A10 expression when males have not been exposed to androgens neonatally; and 4) that in experimental conditions, females can be induced to express CYP3A10, which indicates that there are two modes for regulating expression: by imprinting in males and by GH and testosterone in females. We are now studying the molecular mechanisms involved in the bile acid-mediated induction and the male-specific expression of CYP3A10. We have cloned approximately 8 kb of 5\u27 flanking DNA from a hamster genomic library and sequenced about 1 kb of proximal DNA. Primer extension and S1 digestion analyses indicate that the mRNA for CYP3A10 has multiple transcription initiation sites clustered about 90 bp from the initiator methionine codon. We have also prepared CYP3A10 promoter/lacZ chimeric constructs to begin delineating the cis-acting elements controlling CYP3A10 expression and regulation. We used H2.35 cells as recipients because they are a mouse hepatocyte cell line that has been transformed with a temperature sensitive SV40. These cells can be grown at the permissive temperature and can be induced to behave like liver cells, the differentiated condition, by switching to a nonpermissive temperature. We have found that the construct with 1 kb of proximal CYP3A10 5\u27 flanking DNA was able to express the reporter gene at higher levels under differentiated conditions, which were consistent with higher expression of an albumin promoter/lacZconstruct, upon switching the cells to the more liver phenotype. The system characterized and described here is ideally suited for dissecting the molecular details governing bile acid-mediated regulation and sexually dimorphic expression of liver genes. Very little is known about both these very important biological phenomena. Much could be learned about transcriptional regulation of liver genes by investigating the cis-elements and trans-acting factors mediating regulation of CYP3A10 expression

Synthese, Charakterisierung und katalytische Eigenschaften von metallsubstituierten Alumophosphaten

Ziel der vorliegenden Arbeit war es, Alumophosphate sowohl mit sauren Eigenschaften als auch solche mit oxidierendem Charakter herzustellen. Die katalytischen Zentren wurden durch den Einbau von Metallen in das Alumophosphat-Gitter erzeugt. Dies erfolgte durch die Zugabe von Metallsalzen zum Synthesegel der zu synthesierenden Alumophosphate. Ein besonderes Augenmerk wurde hierbei auf die Metalle Mg, Co, Zn, Mn, Cr, V und Ti gerichtet. Der Einbau der Metalle erfolgte an 10-Ring und 12-Ring Alumophosphaten. Die metallhaltigen 10 Ring Alumophosphate mit AEL und ATO Strukturen und 12-Ring Alumophosphate der AFI, ATS und AFO-Topologie sollten anschließend mittels physikalisch-chemischer Methoden charakterisiert werden. Hierzu gehörten: - Röntgen-Pulverdiffraktometrie, - Temperaturprogrammierte Desorption (TPD), - Stickstoff-Adsorption, - UV/Vis-Spektroskopie, - IR-Spektroskopie. Ein Schwerpunkt der vorliegenden Arbeit stellte die Untersuchung der katalytischen Eigenschaften der hergestellten und charakterisierten Proben dar. Ausgewählte Cr-, V- und Ti-haltige Katalysatoren wurden in der Flüssigphasenoxidation von Cyclohexen mit wässriger Wasserstoffperoxid- oder tert-Butylhydroperoxid-Lösung (TBHP) getestet. Die eingesetzten metallhaltigen Alumophosphate wiesen eine katalytische Aktivität für die Cyclohexen-Oxidation bei 80 °C auf. Während der Reaktion entstanden 1,2-Cyclohexandiol, 2-Cyclohexenol, 2-Cyclohexenon, Cyclohexenoxid und Cyclohexandion als Hauptprodukte. Die aus technischen Gesichtspunkten interessante Adipinsäure konnte zu keinem Zeitpunkt beobachtet werden. Die Aktivität der eingesetzten Katalysatoren wird durch die unterschiedlichen in die Alumophosphate eingebauten Metalle beeinflusst. Auch die Topologie der hergestellten Materialien beeinflusste den Verlauf der Reaktion. Der Einsatz von TBHP und H2O2 als Oxidationsmittel führte teilweise zu einem unterschiedlichen Produktbild. Nach mehreren Stunden wurde eine Desaktivierung der Katalysatoren beobachtet. Durch eine erneute Kalzination der in der Katalyse einsetzen Proben konnte die Aktivität fast vollständig wieder hergestellt werden. In der Disproportionierung von Ethylbenzol erwiesen sich die Co-, Mn-, Mg- und Zn-haltigen Alumophosphate als aktive Katalysatoren. Bei den Umsetzungen an Alumophosphaten mit unterschiedlichem Metallgehalt entstanden ortho-, meta- und para-Diethylbenzol als Hauptprodukte. Hierbei wiesen Mg-haltige Katalysatoren die höchste Aktivität auf. Bei einem Metallgehalt von nMg/nAl = 0,01 entstanden neben den bisher erwähnten Hauptprodukten auch Triethylbenzole. Die Aktivität der eingesetzten Alumophosphate nimmt in der Reihenfolge MgAPO > CoAPO > ZnAPO > MnAPO ab. Diese Reihenfolge steht im Einklang mit den Ergebnissen aus den Aciditäts-Untersuchungen mittels TPD. Die Porengeometrie der verwendeten Alumophosphate übt einen entscheidenden Einfluss auf das Produktbild aus. Weitporige Alumophosphate mit der AFI und ATS Topologie weisen die höchsten Umsätze an Ethylbenzol auf. Alumophosphate des AFO und AEL besitzen einen geringeren Porendurchmesser und zeigen eine geringere Aktivitat in der Disproportionierung von Ethylbenzol. Zudem treten wegen des geringeren Porendurchmessers formselektive Effekte auf. So wird das schlankere para-Diethylbenzol bevorzugt gebildet. Katalysatoren auf Basis des ATO Alumophosphates zeigten keine signifikante Aktivitat

Cost overrun in construction projects in developing countries, Gas-Oil industry of Iran as a case study

 Cost overrun in construction projects is a common issue affecting project performance, and Gas-Oil con­struction projects in Iran are no exception. This paper presents the results of a questionnaire conducted to identify and evaluate the relative importance of the significant factors contributing to the Gas-Oil construction industry of Iran as a case study for developing countries. The survey respondents included project owners, contractors and consultants in­volved in Iranian Gas-Oil construction projects. The results of the survey revealed that the main causes of cost overrun in this industry include inaccurate cost estimations, improper planning, frequent design changes, inadequate labour/skill availability, inflation of costs of machinery, labour, raw material and transportation prices. The first three factors are the project consultants’ responsibility and the appointment of qualified consultants and personnel training are strongly recommended to alleviate cost overrun. The paper also reviews and compares findings of a set of similar researches in a number of developing countries. First published online: 13 Jul 201

Effect of nacl replacement by other salts on the quality of bísaro pork sausages (Pgi chouriça de vinhais)

Concerned about the trend to reduce salt consumption, the meat industry has been increasing the strategies to produce and commercialize products where the reduction or even the replacement of NaCl is an important goal. The aim of this study was to test the effect of partial NaCl replacement by KCl and Sub4Salt® on the quality of pork sausages. Three different formulations (NaCl + KCl, NaCl + Sub4Salt®, and KCl + Sub4Salt®) were considered and compared to the control (2% NaCl). Physicochemical properties, chemical composition, and microbiological and sensory characteristics were evaluated. The replacement of NaCl did not affect pH, water activity (aw) or its chemical composition after eight or 16 days ripening time, while a significant sodium reduction was achieved. The oxidation index expressed in TBARS was also not affected by the NaCl substitution and varied between 0.01 to 0.04 of malonaldehyde (MDA) per kg of sample. Similarly, the NaCl replacement did not change the microbiological quality of the sausages, and the production of healthier meat sausages had also no significant effect on their sensory characteristics. Therefore, according to the results obtained, it is viable and a good strategy for the meat industry to produce “reduced sodium content” sausages without affecting their traditional quality.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020); to Bísaro-Salsicharia Tradicional industry and to Laboratory of Carcass and Meat Quality of Agriculture School of Polytechnic Institute of Bragança ‘Cantinho do Alfredo’. Thanks are extended to GAIN (Axencia Galega de Innovación) for supporting this work (grant number IN607A2019/01). The authors are members of the Healthy Meat network, funded by CYTED (ref. 119RT0568).info:eu-repo/semantics/publishedVersio

Physicochemical properties, fatty acid profile and sensory characteristics of sheep and goat meat sausages manufactured with different pork fat levels

The effect of three pork backfat levels (0% vs. 10% vs. 30%) on chemical composition, fatty acid profile and sensory properties on sheep and goat meat sausages was studied. All physicochemical parameters were affected by the addition of pork backfat in both types of sausages. Sausages manufactured with 30% of pork backfat showed the lowest moisture and protein contents and the highest total fat content. The lower a(w) values in sausages manufactured with higher fat content while in pH happened the reverse situation. The addition of pork backfat modified the total fatty acid profile, prompting a significant drop in the relative percentages of C14:0, C16:0, C17:0, C17:1, C18:0 and TVA (trans-vaccenic acid), together with a marked increase in oleic and linoleic acids. Finally, in goat sausages, the fat content significantly affected sensory parameters: taste, texture and overall acceptability (P<0.05). As expected, all physicochemical parameters were affected by the addition of pork backfat in both types of sausages.info:eu-repo/semantics/publishedVersio

Edible mushrooms as a natural source of food ingredient/additive replacer

Although mushrooms have been exploited since ancient times because of their particular taste and therapeutic properties, the interest in edible species as a source of ingredients and bioactive compounds is recent. Their valuable nutritional contents in protein, dietary fiber and bioactive compounds make them ideal candidates for use in foods in efforts to improve their nutritional profiles. This trend is in line with the consumer’s growing demand for more plant-based foods. The present review paper explores different studies focused on the use of common edible mushrooms as an ingredient and additive replacer by using them in fresh, dried, or even extract forms, as meat, fat, flour, salt, phosphates, and antioxidant replacers. The replacement of meat, fat, flour, and salt by mushrooms from commercial species has been successful despite sensorial and textural parameters can be affected. Moderate concentrations of mushrooms, especially in powder form, should be considered, particularly in non-familiarized consumers. In the case of antioxidant and antimicrobial properties, results are variable, and more studies are necessary to determine the chemical aspects involved.The authors thank to GAIN (Axencia Galega de Innovación) for supporting this review (grant number IN607A2019/01). Authors are members of the Healthy Meat network, funded by CYTED (ref. 119RT0568).info:eu-repo/semantics/publishedVersio

Near infrared hyperspectral imaging and spectral unmixing methods for evaluation of fiber distribution in enriched pasta

[EN] Pasta is mostly composed by wheat flour and water. Nevertheless, flour can be partially replaced by fibers to provide extra nutrients in the diet. However, fiber can affect the technological quality of pasta if not properly distributed. Usually, determinations of parameters in pasta are destructive and time-consuming. The use of Near Infrared-Hyperspectral Imaging (NIR-HSI), together with machine learning methods, is valuable to improve the efficiency in the assessment of pasta quality. This work aimed to investigate the ability of NIR-HSI and augmented Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) for the evaluation, resolution and quantification of fiber distribution in enriched pasta. Results showed R2V between 0.28 and 0.89, %LOF < 6%, variance explained over 99%, and similarity between pure and recovered spectra over 96% and 98% in models using pure flour and control as initial estimates, respectively, demonstrating the applicability of NIR-HSI and MCR-ALS in the identification of fiber in pasta.This work was supported by the Coordenaçao de Aperfeicoamento de Pessoal de Nivel Superior -Brasil (CAPES) [Finance Code 001]; Sao Paulo Research Foundation (FAPESP) [grant numbers 2008/57808-1, 2014/50951-4, 2015/24351-2, 2017/17628-3, 2019/06842-0]; and by GVA-IVIA and FEDER funds through project IVIA-51918. The authors would like to thank Nutrassim Food Ingredients company for the donation of the fiber samples, the support provided by Mrs. Cristiane Vidal during NIR-HSI system operation and data processing and Dr. Celio Pasquini for promptly receiving us in the laboratory that he coordinates (Grupo de instrumentaçao e automaçao em quimica analitica, Instituto de quimica, Universidade Estadual de Campinas, Campinas-SP, Brazil) to data acquisition.Teixeira Badaró, A.; Amigo, JM.; Blasco, J.; Aleixos Borrás, MN.; Rios Ferreira, A.; Pedrosa Silva Clerici, MT.; Fernandes Barbin, D. (2021). Near infrared hyperspectral imaging and spectral unmixing methods for evaluation of fiber distribution in enriched pasta. Food Chemistry. 343:1-9. https://doi.org/10.1016/j.foodchem.2020.128517S1934

Os anfibolitos do complexo costeiro na região de São Sebastião, SP

O Complexo Costeiro na região de São Sebastião, Estado de São Paulo, como segmento da Faixa de Dobramentos Ribeira, integra os setores paraderivados a leste e ortoderivados a oeste, organizados segundo uma estrutura de cisalhamento dúctil em flor positiva. O eixo desta estrutura, que se orienta ENE-WSW, aloja as principais ocorrências dos corpos anfibolíticos que ocorrem no interior dos predominantes granada-gnaisses paraderivados. As rochas básicas intrusivas, que deram origem a estes anfibolitos, alojaram-se em estruturas tabulares ou em câmaras magmáticas secundárias, que localmente ainda preservam características geoquímicas de toleiitos continentais. Determinações U-Pb (SHRIMP) em zircão indicam idade de 580 Ma para esse magmatismo. Idades de 570 Ma para determinações equivalentes em sobrecrescimentos de cristais detríticos de zircão, dos paragnaisses encaixantes, retratam a proximidade entre o magmatismo básico e o ápice do processo metamórfico regional, favorecendo a interpretação do ambiente tectônico de colocação como sendo uma bacia sedimentar de retroarco sobre crosta continental. As áreas-fonte envolveriam rochas diferenciadas entre o Paleoproterozoico e o Neoproterozoico. A localização dos núcleos anfibolíticos, ao longo do eixo da estrutura em flor, assim como o posicionamento das manifestações magmáticas do Mesozoico, parecem conferir a esta estrutura uma importância especial, podendo ter favorecido a recorrência de eventos geológicos.Geochronological, petrographic and lithochemical data have been used to characterize the crustal evolution and the thermochronology of the Costeiro Complex, São Paulo State, Brazil. This part of the Ribeira Fold Belt is composed of paraderived rocks to the East, and orthoderived rocks to the West, which are organized along a ductile shear zone forming a positive flower structure whose axis runs ENE-WSW. The main amphibolite occurrences are found within predominant garnet-paragneisses along this axis. The age of crystallization of the intrusive basic magmas which originated the amphibolites was 580 Ma (U-Pb SHRIMP). They formed tabular intrusions or secondary magma chambers, that locally still preserve continental tholeiitic geochemical characteristics. The close proximity of the basic magmatism and the metamorphic peak is evident from the 570 Ma, U-Pb age determination of the overgrowths on detritic zircon crystals in the paragneisses. This suggests a tectonic environment where the basic magmas intruded a back-arc sedimentary basin established over continental crust. The source areas were probably rocks differentiated from the mantle between the Paleoproterozoic and Neoproterozoic

Cocoa coproducts-based and walnut oil gelled emulsion as animal fat replacer and healthy bioactive source in beef burgers

The aim of this work was to evaluate the effects on the chemical, physic-chemical, tech-nological, and sensory properties of beef burger when replacing different quantities of fat (50 and 100%) with different levels of oil-in-water-gelled emulsion elaborated with walnut oil and cocoa bean shell flour (GECW). The chemical composition of the samples was affected by the fat replace-ment. The reformulation increased the moisture and ash content while the fat and protein content decreased with respect to the control sample. The linolenic and linolenic acid content of the beef burgers increased as the GECW replacement was augmented. The polyunsaturated fatty/saturated fatty acid ratio increased in both raw and cooked burgers, whereas the atherogenicity index and thrombogenicity index were reduced in both raw and cooked burgers with respect to the control sample. The use of GECW as a fat replacer was found to be effective in improving the cooking loss. Similarly, there were positive effects on reductions in the diameter and the increases in the thickness of the beef burgers. Regarding lipid stability, in both the raw and cooked burgers, the reformulation increased the 2-thiobarbituric acid reactive substance (TBARs) values with respect to the control sample. In both types of reformulated burgers, three bound polyphenols (mainly catechin and epicatechin) and two free polyphenols were identified, as were methylxanthines theobromine and caffeine. The sensory properties for the control and partial pork backfat replacement treatments were similar, while the sample with the total pork backfat replacement treatment showed the lowest scores. The blend of cocoa bean shell flour and walnut oil could be used as new ingredients for the development of beef burgers with a healthier nutritional profile without demeriting their sensory or cooking characteristics and physic-chemical properties.This research was funded by Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo CYTED: 119RT0568.info:eu-repo/semantics/publishedVersio