Biochemical Barrier Research Articles

Induction of phase-1 metabolizing enzymes by oltipraz, flavone and indole-3-carbinol enhance the formation and transport of benzo[ a]pyrene sulfate conjugates in intestinal Caco-2 cells

The small intestine is well equipped with various phase-1 and phase-2 xenobiotic metabolizing enzymes (XME), which contribute to the detoxification process of the body. Many XME are regulated via aryl hydrocarbon receptor (AhR)-dependent pathways, and numerous naturally occurring AhR agonists (e.g. flavonoids, dietary indoles) have been identified to date. In the present study we show that pretreatment of Caco-2 cells with food-associated compounds (flavone and indole-3-carbinol) and with the anticancer chemopreventive agent oltipraz enhances the formation of the major metabolites of the procarcinogen benzo[ a]pyrene (BP) formed by intestinal Caco-2 cells, namely BP-1-sulfate and BP-3-sulfate, and their transport to the apical compartment of a Transwell™ chamber. Oltipraz treatment was most effective in this regard followed by flavone and indole-3-carbinol. The effect observed here after pretreatment with oltipraz, flavone and I3C was the result of the induction of both CYP1A1 and CYP1B1, as was confirmed by analysis of CYP1A1 (protein and mRNA) and CYP1B1 (mRNA) expression. In summary, our study shows that the induction of both CYP1A1 and CYP1B1 resulted in an accelerated metabolism and an enhanced clearance of the potent procarcinogen BP, indicating that flavone, indole-3-carbinol and oltipraz have an impact on the biochemical barrier against BP in intestinal cells.

Electrochemically Assisted Microbial Production of Hydrogen from Acetate

Hydrogen production via bacterial fermentation is currently limited to a maximum of 4 moles of hydrogen per mole of glucose, and under these conditions results in a fermentation end product (acetate; 2 mol/mol glucose) that bacteria are unable to further convert to hydrogen. It is shown here that this biochemical barrier can be circumvented by generating hydrogen gas from acetate using a completely anaerobic microbial fuel cell (MFC). By augmenting the electrochemical potential achieved by bacteria in this MFC with an additional voltage of 250 mV or more, it was possible to produce hydrogen at the cathode directly from the oxidized organic matter. More than 90% of the protons and electrons produced by the bacteria from the oxidation of acetate were recovered as hydrogen gas, with an overall Coulombic efficiency (total recovery of electrons from acetate) of 60-78%. This is equivalent to an overall yield of 2.9 mol H2/mol acetate (assuming 78% Coulombic efficiency and 92% recovery of electrons as hydrogen). This bio-electrochemically assisted microbial system, if combined with hydrogen fermentation that produces 2-3 mol H2/mol glucose, has the potential to produce ca. 8-9 mol H2/mol glucose at an energy cost equivalent to 1.2 mol H2/mol glucose. Production of hydrogen by this anaerobic MFC process is not limited to carbohydrates, as in a fermentation process, as any biodegradable dissolved organic matter can theoretically be used in this process to generate hydrogen from the complete oxidation of organic matter.

Host Barriers and Responses toUncinula necatorin Developing Grape Berries

ABSTRACT Grape berries are highly susceptible to powdery mildew 1 week after bloom but acquire ontogenic resistance 2 to 3 weeks later. We recently demonstrated that germinating conidia of the grape powdery mildew pathogen (Uncinula necator) cease development before penetration of the cuticle on older resistant berries. The mechanism that halts U. necator at that particular stage was not known. Several previous studies investigated potential host barriers or cell responses to powdery mildew in berries and leaves, but none included observation of the direct effect of these factors on pathogen development. We found that cuticle thickness increased with berry age, but that ingress by the pathogen halted before formation of a visible penetration pore. Cell wall thickness remained unchanged over the first 4 weeks after bloom, the time during which berries progressed from highly susceptible to nearly immune. Autofluorescent polyphenolic compounds accumulated at a higher frequency beneath appressoria on highly susceptible berries than on highly resistant berries; and oxidation of the above phenolics, indicated by cell discoloration, developed at a significantly higher frequency on susceptible berries. Beneath the first-formed appressoria of all germinated conidia, papillae occurred at a significantly higher frequency on 2- to 5-day-old berries than on 30- to 31-day-old fruit. The relatively few papillae observed on older berries were, in most cases (82.8 to 97.3%), found beneath appressoria of conidia that had failed to produce secondary hyphae. This contrasted with the more abundantly produced papillae on younger berries, where only 35.4 to 41.0% were located beneath appressoria of conidia that had failed to produce secondary hyphae. A pathogenesis-related gene (VvPR-1) was much more highly induced in susceptible berries than in resistant berries after inoculation with U. necator. In contrast, a germin-like protein (VvGLP3) was expressed within 16 h of inoculation in resistant, but not in susceptible berries. Our results suggest that several putative barriers to infection, i.e., cuticle and cell wall thickness, antimicrobial phenolics, and two previously described pathogenesis-related proteins, are not principal causes in halting pathogen ingress on ontogenically resistant berries, but rather that infection is halted by one or more of the following: (i) a preformed physical or biochemical barrier near the cuticle surface, or (ii) the rapid synthesis of an antifungal compound in older berries during the first few hours of the infection process.

CYP3A4-transfected Caco-2 cells as a tool for understanding biochemical absorption barriers: studies with sirolimus and midazolam.

CYP3A4-transfected Caco-2 cells were used as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption. We examined the transport and metabolism of two drugs; midazolam, an anesthetic agent and CYP3A4 substrate, and sirolimus, an immunosuppressant and a dual CYP3A4/P-glycoprotein (P-gp) substrate, in the presence of cyclosporine (CsA, a CYP3A4/P-gp inhibitor) or N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918) (an inhibitor of P-gp and not CYP3A4). All major CYP3A4 metabolites were formed in the cells (1-OH > 4-OH midazolam and 39-O-desmethyl > 12-OH > 11-OH sirolimus), consistent with results from human liver microsomes. There was no bidirectional transport of midazolam across CYP3A4-transfected Caco-2 cells, whereas there was a 2.5-fold net efflux of sirolimus (1 microM) that disappeared in the presence of CsA or GG918. No change in the absorption rate or extraction ratio (ER) for midazolam was observed when P-gp was inhibited with GG918. Addition of GG918 had a modest impact on the absorption rate and ER for sirolimus (increased 58% and decreased 25%, respectively), whereas a 6.1-fold increase in the absorption rate and a 75% decrease in the ER were found when sirolimus was combined with CsA. Although both midazolam and sirolimus metabolites were preferentially excreted to the apical compartment, only sirolimus metabolites were transported by P-gp as determined from inhibition studies with GG918. Using CYP3A4-transfected Caco-2 cells we determined that, in contrast to P-gp, CYP3A4 is the major factor limiting sirolimus absorption. The integration of CYP3A4 and P-gp into a combined in vitro system was critical to unveil the relative importance of each biochemical barrier.

Intestinal absorption enhancement of the ester prodrug tenofovir disoproxil fumarate through modulation of the biochemical barrier by defined ester mixtures.

The effect of discrete esters and ester mixtures on the intestinal stability and absorption of tenofovir disoproxil fumarate (tenofovir DF, an esterase-sensitive prodrug of the antiviral tenofovir) was compared with the effect of strawberry extract, which has been shown to enhance the absorption of the prodrug across Caco-2 monolayers and in rat ileum. In addition, the mechanism of absorption enhancement was investigated. In rat intestinal homogenates, complete inhibition of the conversion of tenofovir DF (as obtained by strawberry extract) could only be obtained at relatively high concentrations of the discrete esters or by using mixtures of esters (e.g., propyl p-hydroxybenzoate 0.02%, octyl acetate 0.02%, ethyl caprylate 0.01%). Coincubation of tenofovir DF with this mixture also resulted in an enhancement of its absorption in the in vitro Caco-2 system as well as in rat ileum. As tenofovir DF is a substrate for P-glycoprotein (P-gp)-related efflux carriers in the Caco-2 model, the modulatory effect of the ester mixtures was studied on the functionality of P-gp using cyclosporin A (CsA) as a model substrate. Strawberry extract as well as the mixture of three esters interfered with the absorptive transport of CsA across Caco-2 monolayers, illustrating that both mixtures interfere with both esterase-activity and P-gp functionality. This concerted barrier was not observed in rat ileum, suggesting differential functional activities of the biochemical barrier toward tenofovir DF in different absorption systems. Overall, our results illustrate that modulation of the biochemical barrier (metabolism and efflux) of tenofovir DF by ester mixtures can be used to increase the intestinal absorption of tenofovir DF in an in vitro and an in situ absorption model; the mechanism of action appears to be a complex interplay of different systems; the differential expression of carriers and enzymes in different systems illustrates the difficulty of extrapolating observations between different systems/species.

Glial fibrillary acidic protein-positive cells of the kidney are capable of raising a protective biochemical barrier similar to astrocytes: expression of metallothionein in podocytes.

Blood-tissue exchange and homeostasis within the organs depend on various interactions between endothelial and perivascular cells (Buniatian, 2001). Podocytes possess anatomical and cellular features intermediate between those of astrocytes and hepatic stellate cells (HSCs). Podocytes, like HSCs, are associated with fenestrated capillaries and, similar to astrocytes, interact with the capillaries via the basement membrane and participate in permeability-limiting ultrafiltration. The fact that podocytes come in direct contact with xenobiotics prompted us to investigate whether they express metallothionein (MT), an anticytotoxic system characteristic of astrocytes. In comparative studies, cryosections of 1- and 3-month-old rat kidney and adult rat brain, as well as podocytes and astrocytes from early and prolonged primary cultures of glomerular explants and newborn rat brain, respectively, were investigated. The cells were double-labeled with antiserum against glial fibrillary acidic protein (GFAP) and monoclonal antibody (MAb) against the lysine-containing epitope of Cd/Zn-MT-I (MAb MT) or MAb against alpha-actin. In kidney sections, MT immunoreactivity was detected in GFAP-positive glomerular cells and in interstitial fibroblasts. The pattern of staining for MT and GFAP in glomerular cells was similar to that of astrocytes in vivo. In glomerular cell cultures, MT was expressed in cobblestone-like podocytes which contained Wilms' tumor protein and lacked desmin. MT was upregulated at later culture periods, during which podocytes acquired features typical of undifferentiated astrocytes. This study hints at the existence of common regulatory mechanisms of blood-tissue interactions by neural and non-neural perivascular cells. These mechanisms appear to be used in an organ-specific manner.

Plant Growth‐Promoting Rhizobacteria‐Elicited Alterations in Phenolic Profile of Chickpea (Cicer arietinum) Infected by Sclerotium rolfsii

AbstractEfficacy of seven strains of Pseudomonas fluorescens (Pfs1–7), plant growth‐promoting rhizobacteria (PGPR), were tested under field conditions for their ability to protect Cicer arietinum against Sclerotium rolfsii infection. Best protection was observed in strain Pfs3 where 23% seedling mortality was recorded in comparison to 44% in non‐treated control. To correlate the induction of phenolic compounds by the PGPRs with disease resistance, qualitative and quantitative alterations of phenolic compounds in different parts of C. arietinum were estimated following PGPR application as seed treatment. High performance liquid chromatographic (HPLC) analysis of the leaves, collars and roots of the PGPR‐treated and non‐treated (control) plants showed the presence of gallic, ferulic, chlorogenic and cinnamic acids with varied amounts in the PGPR‐treated as well as non‐treated (control) plants. Maximum accumulation of cinnamic acid was observed in plants treated with Pfs3 strain (1660 ng g–1 fresh wt.) which was almost 19.5 times higher than untreated control plants and also significantly high when compared to other PGPR treatments. Pfs3 also caused maximum accumulation of total phenolics and gallic acid in all chickpea plant parts as compared to other treatments and untreated control. A direct relationship between the level of total phenolics and seedling survivability was observed. PGPR‐mediated induction of phenolic compounds as a biochemical barrier in C. arietinum against S. rolfsii infection is envisaged.

Inhibition of Axon Growth by Oligodendrocyte Precursor Cells

The glial scar that forms at the site of injury is thought to be a biochemical and physical barrier to successful regeneration, although the molecules responsible for this barrier function are not well understood. Glia scars contain large numbers of oligodendrocyte precursor cells (OPCs) and these cells can produce several different growth-inhibitory chondroitin sulfate proteoglycans (CSPGs), including NG2, neurocan, and phosphacan. Here, we used membrane-based assays to show that the surface of OPCs is both nonpermissive and inhibitory for neurite outgrowth. Inhibition of growth by OPC is reversed by treatment with antibodies against the NG2 CSPG and the expression of NG2 is sufficient to change a growth-permissive cell surface to a nonpermissive surface. These result suggest that the OPCs that accumulate rapidly at sites of CNS injury can contribute to the creation of an environment that inhibits nerve regeneration and that NG2 is a necessary feature of that environment.

Return of the living dead

Current understanding dictates that the activation of caspases signifies a biochemical barrier within the apoptotic pathway beyond which a cell is destined to die. However, two recent reports in the nematode Caenorhabditis elegans have challenged this assumption, providing evidence that cells exhibiting morphological evidence of caspase-dependent events can, in the absence of efficient phagocytosis, recover.

Role of the epithelium in opposing H(2)O(2)-induced modulation of acetylcholine-induced contractions in rabbit intrapulmonary bronchiole.

1. The role played by the epithelium in H(2)O(2)-induced modulation of the mechanical responses induced by acetylcholine (ACh) in rabbit intrapulmonary bronchioles was investigated in epithelium-intact and -denuded strips. 2. When ACh (3 microM) was applied intermittently, H(2)O(2) (30 microM) enhanced the ACh-induced contractions in epithelium-intact strips. In contrast, in epithelium-denuded strips H(2)O(2) (30 microM) inhibited such contractions. At higher concentrations, H(2)O(2) concentration-dependently attenuated the ACh-induced contractions in both epithelium-intact and -denuded strips, its action being more potent in the latter strips than in the former. 3. Diclofenac (a cyclo-oxygenase inhibitor; 3 microM) reduced the H(2)O(2)-induced enhancement of ACh-contractions in epithelium-intact strips but had no effect on the H(2)O(2)-induced inhibition in epithelium-denuded strips. N(G)-nitro-L-arginine did not alter the effect of H(2)O(2) on ACh-induced contractions in epithelium-intact strips. 4. Catalase (500 u ml(-1)) completely blocked both H(2)O(2)-induced effects on ACh-contractions (enhancement and inhibition). Neither superoxide dismutase (200 u ml(-1)) nor deferoxamine (0.5 mM) had any effect on H(2)O(2)-induced inhibition in epithelium-denuded strips. 5. Aminotriazole (an inhibitor of catalase; 50 mM) significantly potentiated the H(2)O(2)-induced inhibition of ACh-contractions in epithelium-intact strips but not in epithelium-denuded strips. 6. The density ratio for catalase (epithelium-intact over -denuded strips) analysed by Western blot was about 2.1, suggesting that epithelium contains more catalase than smooth muscle. 7. It is concluded that in rabbit intrapulmonary bronchioles, H(2)O(2) has dual actions on ACh-contractions. It is suggested that the epithelium may act as a powerful biochemical barrier via both the action of catalase (scavenging H(2)O(2)) and the release of bronchoconstrictor prostaglandins, thus attenuating the H(2)O(2)-induced modulation of ACh-contractions.

Pharmacological blood-brain barrier modification for selective drug delivery.

Vasoactive agents have been identified through studies of peritumoral edema and effects on systemic capillaries. Abnormal blood-brain barrier or blood-tumor barrier can develop transient increases in permeability with the intraarterial delivery of vasoactive agents. Normal blood-brain barrier resists the effects of these compounds because of a biochemical barrier that may inactivate or become inert to vasoactive agents. Vasoactive compounds, including leukotrienes, bradykinin, and histamine appear to selectively increase permeability in abnormal brain capillaries. Intracarotid infusion of leukotrienes, bradykinin, and other vasoactive agents can increase drug delivery to diseased tissue.

Skin 2TM: An in vitro model to assess cutaneous immunotoxicity

The skin serves as a complex barrier protecting the internal environment against external factors (e.g. bacteria, viruses, environmental toxins and UV light). For the epidermis to produce the most effective toxicological, immunological and biochemical barrier, the major cell types of the epidermis (i.e. keratinocytes) and in the dermis (i.e. fibroblasts) must function together in a dynamic integrated fashion. Furthermore, epidermal-dermal intercellular biochemical signals such as interleukins (IL), cytokines and other growth factors provide the skin with local homoeostatic signals to ensure the skin's immune integrity in response to a variety of environmental insults. Other investigators have shown that exposure of the skin to long-wave ultraviolet light (UVA) and mid-range ultraviolet light (UVB) can alter epidermal immune functions, including epidermal cytokine (e.g. IL-1, IL-6, TNF-α, IL-10 and GM-CSF) levels. The studies reported here use a co-culture system of dermal fibroblasts and well differentiated epidermal layers with an attached stratum corneum to form an in vitro human skin analogue. Baseline endogenous levels of IL-1α and tumour necrosis factor-α (TNF-α) were detected by using commercially available ELISA kits. The tissue substrates were exposed to UVA/UVB light (280–400 nm). The UV light was administered by a Dermsol 3 mercury halide solar simulator configured with filters to remove energy levels below 280 nm and above 410 nm. Skin tissue irradiated at 4J/cm 2 revealed a significant increase in IL-1α and TNF-α in comparison with non-UV irradiated tissue. Additional experiments revealed that the topical administration of indomethacin (0.1 to 10mg/ml) to the skin tissue ameliorated the up-regulation of these immune cytokines following UV irradiation. The use of such an in vitro co-culture system may provide researchers with a unique method to quantify mechanistically immunotoxicological events in the skin after exposure to ultraviolet light.

Perivascular and intravenous administration of basic fibroblast growth factor: vascular and solid organ deposition.

The in vivo mitogenicity of basic fibroblast growth factor (bFGF) for arterial smooth muscle cells relies on the removal of endothelium, raising the question of whether the endothelium serves as a mechanical barrier preventing contact of circulating bFGF with underlying smooth muscle cells or as a biochemical barrier that produces a local inhibitor of bFGF activity. To better define the role of the intact endothelium in modulating the vascular and tissue deposition of bFGF, we compared the fate of intravenous injections of 125I-labeled bFGF with perivascular controlled growth factor release. Peak serum bFGF levels were detected within 1 min of injection, and the growth factor was cleared thereafter with a serum half-life of almost 3 min. Polymeric controlled release devices delivered bFGF to the extravascular space without transendothelial transport. Deposition within the blood vessel wall was rapidly distributed circumferentially and was substantially greater than that observed following intravenous injection. The amount of bFGF deposited in arteries adjacent to the release devices was 40 times that deposited in similar arteries in animals who received a single intravenous bolus of bFGF. Endothelial denudation had a minimal effect on deposition following perivascular release, and it increased deposition following intravenous delivery 2-fold. The presence of intimal hyperplasia increased deposition of perivascularly released bFGF 2.4-fold but decreased the deposition of intravenously injected bFGF by 67%. In contrast, bFGF was 5- to 30-fold more abundant in solid organs after intravenous injection than it was following perivascular release. Deposition was greatest in the kidney, liver, and spleen and was substantially lower in the heart and lung. Thus, bFGF is rapidly cleared following intravenous injection and is deposited within both solid organs and the walls of blood vessels. Unlike the mitogenic potential of bFGF within blood vessels, vascular deposition is virtually independent of the presence of endothelium. Perivascular delivery is far more efficient than intravenous delivery at depositing bFGF within the arterial wall, and an increased neointima may provide added substrate for potential bFGF deposition but has limited contact with intravascular growth factor as a result of dilutional and flow-mediated effects.

Plant Chemistry and Insect Species Richness of British Umbellifers

(1) Data on the number of insect species per host-plant, host-plant physical characteristics (geographic range, size and leaf form), the presence of biosynthetically distinct classes of secondary metabolites and intensity of biochemical investigation were compiled for the British umbellifers (Apiaceae) from the Phytophagous Insect Data Bank, previously published studies, and phytochemical literature, respectively. (2) Measures of the chemical diversity and uniqueness of each plant species were derived from the phytochemical data, and four hypotheses relating insect species richness to these chemical variables were explicitly developed and tested using single and multiple regression techniques. (3) The diverse defence and biochemical barrier hypotheses predict negative correlations between insect species richness and chemical diversity and uniqueness, respectively, because plants with diverse or unusual defences should show reduced insect colonization over evolutionary time. Alternatively, the common chemistry and enemy escape hypotheses predict positive correlations between insect richness and these two chemical variables, because plants with diverse chemistry have a higher probability of sharing chemicals common to other plants, facilitating host shifts, and because insect herbivores on biochemically unusual plants may escape from predators and parasites that use host odours to locate prey, again facilitating host shifts. (4) As in previous studies, size of plant geographic range, plant size and leaf form all influence insect species richness. Together, these variables account for 46% of the variance in insect species richness, leaving up to 54% of unexplained variance that could potentially be accounted for by host plant chemistry. (5) Insect species richness is not significantly correlated with chemical uniqueness; thus there is no support for the biochemical barrier or enemy escape hypotheses. (6) Insect species richness is positively correlated with chemical diversity, supporting the common chemistry but not the diverse defense hypothesis. However, support for the common chemistry hypothesis is tenuous, because of the confounding effects of intensity of biochemical investigation. When intensity of biochemical investigation is factored out, the effects of chemical diversity on insect species richness are still positive and significant but account for only 5% of the variation. (7) We conclude that there is no evidence from these data that biochemically diverse or unusual British umbellifers support less-species-rich assemblages of insect herbivores. While the data suggest that chemically diverse plants have more insect species, we consider this evidence to be weak, at best. (8) We discuss the need for further tests of these hypotheses and the types of phytochemical data required.

Fetal and newborn cerebral vascular responses and adaptations to hypoxia.

I n the developing fetus and newborn infant, as in the adult, regulation of the cerebral circulation is unique in several regards. First, large arteries including the internal carotid, basilar, circle of Wilhs, and immediate branches of these vessels are important resistance vessels. It has been suggested that this arrangement helps to protect cerebral microvessels, and minimizes a vascular “steal” during localized blood flow increases in the brain. Also, cerebral vessels, particularly small arterioles and capillaries, possess a morphological and biochemical barrier between the blood and brain interstitial space. This blood-brain barrier is composed of a continuous endothelium, with tight junctions between adjacent cells. This biochemical barrier consists of high levels of degradative enzymes that prevent circulating neurotransmitters from gaining access to the brain. In addition, this barrier plays a key role in regulating cerebral vascular reactivity, as it does in other vascular beds. In response to physiological stimuli, such as acute hypoxemia, cerebral blood flow increases to maintain a normal rate of oxygen consumption and metabolism. During chronic hypoxia the flow responses exhibit adaptation in direct response to environmental circumstances. To establish some background for an understanding of the role of vascular endothelium in cerebrovascular regulation, we wiI1 first detail several “facts” and the problems associated with them. Fact. Many factors can affect blood flow to the brain and other organs, including changes in 02 and substrate availability, arterial CO, tension, extracellular hydrogen ion concentration, and extrinsic neurovascular influences (see Fig 1). These influences are integrated via homeostatic regulatory mechanisms that maintain brain 02 consumption and metabolism within a given region by altering cerebral vascular resistance. Problem. The question arises how this multitude of factors, both intrinsic and extrinsic to the cerebrovascular system, is orchestrated and integrated to result in precise regulation. Specifically, in regard to the fetus and newborn, to what extent do factors such as immaturity of the receptor populations and their coupling, vascular endothelium, or adrenergic innervation affect the responses? (As an aside, the various models proposed are not simple. Regulation is complicated by interactions and interdependence among many of the variables.) Fact. In the change at birth from fetus to newborn, a large number of cardiovascular, respiratory, hormonal, and metabolic changes occur that can influence cerebral blood flow regulation (see Table 1). Problem. In the face of this virtual physiologic “storm,” the question arises how these changes interact and are orchestrated or integrated to insure optimal function with maintenance of the matching between cerebral blood flow and cerebral metabolic rates. Fact. In response to acute hypoxia, a number of physiologic changes of the cardiovascular, respiratory, and endocrine systems occur. In response to chronic or long-term hypoxia, many different acclimatization responses take place (see Fig 2). Problem. The question arises how the fetus and newborn adjust and/or regulate cerebral blood flow, 02 consumption, etc, to compensate for acute and/or chronic hypoxia, and what role is played by the vascular endothelium in these adaptations. Fact. A number of clinical problems in the fetus and newborn infant occur as sequelae of dysregulation of blood flow to the brain, lungs, and other organs. Problem. A further question regards the means and to what extent the various regulatory factors, including the vascular endothelium, play a roIe in the genesis of these disorders.

Blood-brain barrier monoamine oxidase: enzyme characterization in cerebral microvessels and other tissues from six mammalian species, including human.

We studied the enzyme monoamine oxidase (MAO) in isolated cerebral microvessels, and in mitochondria-enriched brain and liver preparations from six mammalian species, including human. We also studied MAO distribution in various tissues and in discrete brain regions of the rat. MAO was assessed by measuring the specific binding of [3H]pargyline, an irreversible MAO inhibitor, and the rates of oxidation of known MAO substrates: benzylamine, tyramine, tryptamine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Molecular forms of MAO were examined by using specific MAO inhibitors, and by polyacrylamide gel electrophoresis after [3H]pargyline binding. In general, the liver from all species had higher MAO levels than the brain, with minor variation among species in their brain and liver MAO content. However, there were remarkable species differences in brain microvessel MAO, with rat microvessels having one of the highest MAO activity among all tissues, whereas MAO activities in brain microvessels from humans, mice, and guinea pigs were very low. In most rat tissues, including the brain, there was a preponderance of MAO-B over MAO-A. The only exceptions were the heart and skeletal muscle. Estimates of MAO half-life in rat brain microvessels, rat brain, and rat liver indicated that microvessel MAO had a higher turnover rate. The reasons underlying the remarkable enrichment of rat cerebral microvessels with MAO-B are unknown, but it is evident that there are marked species differences in brain capillary endothelium MAO activity. The biological significance of these findings vis a vis the role of MAO as a "biochemical blood-brain barrier" that protects the brain from circulating neurotoxins and biogenic amines should be investigated.

3 Molybdenum Hydroxylases: Biological Distribution and Substrate-Inhibitor Specificity

This chapter discusses the biological distribution and substrate-inhibitor specificity of molybdenum hydroxylases. Aldehyde oxidase and xanthine oxidase are molybdenum-containing enzymes that catalyze the oxidation of many aldehydes and nitrogen heterocycles. They have also been shown to catalyze, under in vitro conditions, a number of reductions. Although aldehyde oxidase is known to oxidize physiological compounds, such as N1-methylnicotinamide and pyridoxal, there does not yet appear to be a specific endogenous role for this enzyme. Xanthine oxidase is involved in endogenous purine catabolism, catalyzing the oxidation of hypoxanthine and xanthine to uric acid. This enzyme also has wide substrate specificity, and different functions have been assigned to it. One of the most plausible proposals is that both molybdenum hydroxylases may provide a biochemical barrier against ingested purines and pyrimidines, rendering them harmless. This is similar to the view put forward in a recent review on the role of the molybdenum hydroxylases as drug-metabolizing enzymes, except that the substrate specificity is not restricted to purines and pyrimidines.

Catechol-O-methyltransferase in rat placenta, human placenta and choriocarcinoma grown in culture.

The present report is the first in a projected series of investigations on biogenic amine-synthesizing and -degrading enzymes in trophoblast. The work is being undertaken to gain information relating to two questions. First, do the placental enzymes which degrade biogenic amines and related compounds provide an important biochemical barrier which protects the fetus against undesirable stimulation by physiological and pharmacological compounds in the maternal circulation? Secondly, do critical changes in biogenic amine metabolism in the placenta precede or accompany parturition, and might they be involved in the actual mechanism underlying the initiation of parturition? Catechol-O-methyltransferase (COMT, E.C.2.1.1.6) is an enzyme which inactivates certain catecholamines and O-methylates 2-hydroxyoestradiol-17 β (Ball, Knuppen, Haupt & Breuer, 1971); it has been purified 66-fold from human placenta (Gugler, Knuppen & Breuer, 1970).

Evidence for semiquinone-metal interaction in metal-flavoproteins

This chapter discusses the biological distribution and substrate-inhibitor specificity of molybdenum hydroxylases. Aldehyde oxidase and xanthine oxidase are molybdenum-containing enzymes that catalyze the oxidation of many aldehydes and nitrogen heterocycles. They have also been shown to catalyze, under in vitro conditions, a number of reductions. Although aldehyde oxidase is known to oxidize physiological compounds, such as N1-methylnicotinamide and pyridoxal, there does not yet appear to be a specific endogenous role for this enzyme. Xanthine oxidase is involved in endogenous purine catabolism, catalyzing the oxidation of hypoxanthine and xanthine to uric acid. This enzyme also has wide substrate specificity, and different functions have been assigned to it. One of the most plausible proposals is that both molybdenum hydroxylases may provide a biochemical barrier against ingested purines and pyrimidines, rendering them harmless. This is similar to the view put forward in a recent review on the role of the molybdenum hydroxylases as drug-metabolizing enzymes, except that the substrate specificity is not restricted to purines and pyrimidines.