Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^

Expression, induction and regulation of the cytochrome P450 monooxygenase system in the rat glioma C6 cell line

The cytochrome P450 monooxygenase system consists of NADPH- cytochrome P450 reductase (P450 reductase) and cytochromes P450, which can catalyze the oxidation of a wide variety of endogenous and exogenous compounds, including steroid hormones, fatty acids, drugs, and pollutants. The functions of this system are as diverse as the substrates. P450 reductase transfers reducing equivalents from NADPH to P450, which in turn catalyzes metabolic reactions. This enzyme system has the highest level of activity in the liver. It is also present in other tissues, including brain. The functions of this enzyme system in brain seem to include: neurotransmission, neuroendocrinology, developmental and behavioral modulation, regulation of intracellular levels of cholesterol, and potential neurotoxicity.^ In this study, we have set up the rat glioma C6 cell line as an in vitro model system to examine the expression, induction, and tissue-specific regulation of P450s and P450 reductase. Rat glioma C6 cells were treated with P450 inducers phenobarbital (PB) or benzo(a)anthracene (BA). The presence of P450 reductase and of cytochrome P450 1A1, 1A2, 2A1, 2B1/2, 2C7, 2D1-5 and 2E1 was detected by reverse transcription followed by polymerase chain reaction (RT-PCR) and confirmed by restriction digestion. The induction of P450 1A1 and 2B1/2 and P450 reductase was quantified using competitive PCR. Ten- and five-fold inductions of P450 1A and 2B mRNA after BA or PB treatments, respectively, were detected. Western blot analysis of microsomal preparations of glioma C6 cells demonstrated the presence of P450 1A, 2B and P450 reductase at the protein level. ELISAs showed that BA and PB induce P450 1A and 2B proteins 7.3- and 13.5-fold, respectively. Microsomes prepared from rat glioma C6 cells showed cytochrome P450 CO difference spectra with absorption at or near 450 nm. Microsomes prepared from rat glioma C6 cells demonstrated much higher levels of ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activity, when treated with BA or PB, respectively. These experiments provide further evidence that the rat glioma C6 cell line contains an active cytochrome P450 monooxygenase system which can be induced by P450 inducers. The mRNAs of P450 1A1 and 2B1/2 can not bind to the oligo(dT) column efficiently, indicating they have very short poly(A) tails. This finding leads us to study the tissue specific regulation of P450s at post-transcriptional level. The half lives of P450 1A1 and 2B1/2 mRNA in glioma C6 cells are only 1/10 and 1/3 of that in liver. This may partly contribute to the low expression level of P450s in glial cells. The induction of P450s by BA or PB did not change their mRNA half lives, indicating the induction may be due to transcriptional regulation. In summary of this study, we believe the presence of the cytochrome P450 monooxygenase system in glial cells of the brain may be important in chemotherapy and carcinogenesis of brain tumors. ^

Genes to blood pressure: The effects of variation in genes of the renin-angiotensin system on the hormonal and renal control of blood pressure

Objective. Essential hypertension affects 25% of the US adult population and is a leading contributor to morbidity and mortality. Because BP is a multifactorial phenotype that resists simple genetic analysis, intermediate phenotypes within the complex network of BP regulatory systems may be more accessible to genetic dissection. The Renin-Angiotensin System (RAS) is known to influence intermediate and long-term blood pressure regulation through alterations in vascular tone and renal sodium and fluid resorption. This dissertation examines associations between renin (REN), angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) gene variation and interindividual differences in plasma hormone levels, renal hemodynamics, and BP homeostasis.^ Methods. A total of 150 unrelated men and 150 unrelated women, between 20.0 and 49.9 years of age and free of acute or chronic illness except for a history of hypertension (11 men and 7 women, all off medications), were studied after one week on a controlled sodium diet. RAS plasma hormone levels, renal hemodynamics and BP were determined prior to and during angiotensin II (Ang II) infusion. Individuals were genotyped by PCR for a variable number tandem repeat (VNTR) polymorphism in REN, and for the following restriction fragment length polymorphisms (RFLP): AGT M235T, ACE I/D, and AT1 A1166C. Associations between clinical measurements and allelic variation were examined using multiple linear regression statistical models.^ Results. Women homozygous for the AT1 1166C allele demonstrated higher intracellular levels of sodium (p = 0.044). Men homozygous for the AGT T235 allele demonstrated a blunted decrement in renal plasma flow in response to Ang II infusion (p = 0.0002). There were no significant associations between RAS gene variation and interindividual variation in RAS plasma hormone levels or BP.^ Conclusions. Rather than identifying new BP controlling genes or alleles, the study paradigm employed in this thesis (i.e., measured genes, controlled environments and interventions) may provide mechanistic insight into how candidate genes affect BP homeostasis. ^

Contribution of the genes of the renin-angiotensin system to interindividual differences in blood pressure levels in Caucasians from Rochester, Minnesota

The Renin-Angiotensin system (RAS) regulates blood pressure through its effects on vascular tone, renal hemodynamics, and renal sodium and fluid balance. The genes encoding the four major components of the RAS, angiotensinogen, renin, angiotensin I-converting enzyme (ACE), and angiotensin II receptor type 1 (AT1), have been investigated as candidate genes in the pathogenesis of essential hypertension. However, studies have primarily focused on small samples of diseased individuals, and, therefore, have provided little information about the determinants of interindividual variation in blood pressure (BP) in the general population.^ Using data from a large population-based sample from Rochester, MN, I have evaluated the contribution of variation in the region of the RAS genes to interindividual variation in systolic, diastolic, and mean arterial pressure in the population-at-large. Marker genotype data from four polymorphisms located within or very near these genes were first collected on 3,974 individuals from 583 randomly ascertained three-generation pedigrees. Haseman-Elston regression and variance component methods of linkage analysis were then carried out to estimate the proportion of interindividual variance in BP attributable to the effects of variation at these four measured loci.^ A significant effect of the ACE locus on interindividual variation in mean arterial pressure (MAP) was detected in a sample of siblings belonging to the youngest generation. After allowing for measured covariates, this effect accounted for 15-25% of the interindividual variance in MAP, and was even greater in a subset with a positive family history of hypertension. When gender-specific analyses were carried out, this effect was significant in males but not in females. Extended pedigree analyses also provided evidence for an effect of the ACE locus on interindividual variation in MAP, but no difference between males and females was observed. Circumstantial evidence suggests that the ACE gene itself may be responsible for the observed effects on BP, although the possibility that other genes in the region may be at play cannot be excluded.^ No definitive evidence for an effect of the renin, angiotensinogen, or AT1 loci on interindividual variation in BP was obtained in this study, suggesting that the impact of these genes on BP may not be great in the Caucasian population-at-large. However, this does not preclude a larger effect of these genes in some subsets of individuals, especially among those with clinically manifest hypertension or coronary heart disease, or in other populations. ^

Design and implementation of a multicriteria medical decision support system for diagnosis and treatment

Health care providers face the problem of trying to make decisions with inadequate information and also with an overload of (often contradictory) information. Physicians often choose treatment long before they know which disease is present. Indeed, uncertainty is intrinsic to the practice of medicine. Decision analysis can help physicians structure and work through a medical decision problem, and can provide reassurance that decisions are rational and consistent with the beliefs and preferences of other physicians and patients. ^ The primary purpose of this research project is to develop the theory, methods, techniques and tools necessary for designing and implementing a system to support solving medical decision problems. A case study involving “abdominal pain” serves as a prototype for implementing the system. The research, however, focuses on a generic class of problems and aims at covering theoretical as well as practical aspects of the system developed. ^ The main contributions of this research are: (1) bridging the gap between the statistical approach and the knowledge-based (expert) approach to medical decision making; (2) linking a collection of methods, techniques and tools together to allow for the design of a medical decision support system, based on a framework that involves the Analytic Network Process (ANP), the generalization of the Analytic Hierarchy Process (AHP) to dependence and feedback, for problems involving diagnosis and treatment; (3) enhancing the representation and manipulation of uncertainty in the ANP framework by incorporating group consensus weights; and (4) developing a computer program to assist in the implementation of the system. ^

Application of real-time fluorescent PCR for quantitative assessment of Neospora caninum infections in organotypic slice cultures of rat central nervous system tissue

The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral N. caninum infection.

A Food Recognition System for Diabetic Patients based on an Optimized Bag of Features Model

Computer vision-based food recognition could be used to estimate a meal's carbohydrate content for diabetic patients. This study proposes a methodology for automatic food recognition, based on the Bag of Features (BoF) model. An extensive technical investigation was conducted for the identification and optimization of the best performing components involved in the BoF architecture, as well as the estimation of the corresponding parameters. For the design and evaluation of the prototype system, a visual dataset with nearly 5,000 food images was created and organized into 11 classes. The optimized system computes dense local features, using the scale-invariant feature transform on the HSV color space, builds a visual dictionary of 10,000 visual words by using the hierarchical k-means clustering and finally classifies the food images with a linear support vector machine classifier. The system achieved classification accuracy of the order of 78%, thus proving the feasibility of the proposed approach in a very challenging image dataset.

The ovine cerebral venous system: comparative anatomy, visualization, and implications for translational research.

Cerebrovascular diseases are significant causes of death and disability in humans. Improvements in diagnostic and therapeutic approaches strongly rely on adequate gyrencephalic, large animal models being demanded for translational research. Ovine stroke models may represent a promising approach but are currently limited by insufficient knowledge regarding the venous system of the cerebral angioarchitecture. The present study was intended to provide a comprehensive anatomical analysis of the intracranial venous system in sheep as a reliable basis for the interpretation of experimental results in such ovine models. We used corrosion casts as well as contrast-enhanced magnetic resonance venography to scrutinize blood drainage from the brain. This combined approach yielded detailed and, to some extent, novel findings. In particular, we provide evidence for chordae Willisii and lateral venous lacunae, and report on connections between the dorsal and ventral sinuses in this species. For the first time, we also describe venous confluences in the deep cerebral venous system and an 'anterior condylar confluent' as seen in humans. This report provides a detailed reference for the interpretation of venous diagnostic imaging findings in sheep, including an assessment of structure detectability by in vivo (imaging) versus ex vivo (corrosion cast) visualization methods. Moreover, it features a comprehensive interspecies-comparison of the venous cerebral angioarchitecture in man, rodents, canines and sheep as a relevant large animal model species, and describes possible implications for translational cerebrovascular research.

The Jewish child : its history, folklore, biology, & sociology

by W. M. Feldman. With an introd. by Sir James Crichton-Browne

Developmental changes in sleep biology and potential effects on adolescent behavior and caffeine use

Adolescent development includes changes in the biological regulatory processes for the timing of sleep. Circadian rhythm changes and changes to the sleep-pressure system (sleep homeostasis) during adolescence both favor later timing of sleep. These changes, combined with prevailing social pressures, are responsible for most teens sleeping too late and too little; those who sleep least report consuming more caffeine. Although direct research findings are scarce, the likelihood of use and abuse of caffeine-laden products grows across the adolescent years due, in part, to excessive sleepiness

Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains

We describe a rational approach to simultaneously test Escherichia coli strains for the presence of known virulence genes in a reverse dot blot procedure. Specific segments of virulence genes of E. coli designed to have similar hybridization parameters were subcloned on plasmids and subsequently amplified by PCR as unlabeled probes in amounts sufficient to be bound to nylon membranes. Various pathogenic isolates and laboratory strains of E. coli were probed for the presence of virulence genes by labeling the genomic DNA of these strains with digoxigenin and then hybridizing them to the prepared nylon membranes. These hybridization results demonstrated that besides the E. coli K-12 safety strain derivatives, E. coli B and C strains are also devoid of genes encoding any of the investigated virulence factors. In contrast, pathogenic E. coli control strains, used to evaluate the method, showed typical hybridization patterns. The described probes and their easy application on a single filter were shown to provide a useful tool for the safety assessment of E. coli strains to be used as hosts in biotechnological processes. This approach might also be used for the identification and characterization of clinically significant E. coli isolates from human and animal species.

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance.