Advances in Experimental Medicine and Biology

Published by Kluwer
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Protein C (PC) is an essential blood factor in the human blood coagulation cascade. PC can help achieve blood hemostasis in many deadly disease conditions such as sepsis, cancer, HIV, etc.; reduced oxygen transport due to blood agglutination within the body can cause tissue death and organ failure as a result of low oxygen transport. Our goal is to produce large quantities of low cost zymogen PC for the treatment and prevention of blood clotting resulting from many disease states, as well as provide an effective therapy for PC deficiency. Current studies show that Immobilized Metal Affinity Chromatography (IMAC) has high specificity and can be used for difficult separations among homologous proteins at relatively low cost compared to current methods, such as Immunoaffinity Chromatography. Thus, we are investigating the optimization of IMAC for the separation and purification of PC from Cohn fraction IV-I. Molecular interactions within the chromatography column involve many parameters that include: the use and type of chromatographic gel and buffer solution, the pH, temperature, metal ion, chelator, and the sequence and structure of the protein itself. These parameters all influence the protein’s interaction with the column. Experimental equilibrium isotherms show that PC has primary and secondary binding characteristics, demonstrating that the interaction is not just a simple process of one protein binding to one metal ion. Understanding the thermodynamics of interfacial interaction between proteins and surface-bound Cu2+ is essential to optimizing IMAC for PC purification, as well as for separation of other proteins in general. Hence we are undertaking theoretical and experimental studies of IDA-Cu/PC adsorption. The differences in structures of PC and other critical homologous blood factors are examined using the protein visualization program Cn3D. A better understanding of the interfacial phenomena will help determine the most effective conditions to achieve our goal.
 
Temporal control is considered the fourth dimension in embryonic development and it sets the pace to attain the correct molecular patterning of the developing embryo. In this chapter we review one of the best-studied time dependent events in embryogenesis, which is the formation ofsomites. Somites are the basis of the future segmented framework of the vertebrate adult body and their reiterated appearance during the early stages of embryo development establishes the proper temporal and physical template from where other structures will develop and consequently shape the segmentation pattern of the embryo. Several models have been proposed over the last few decades to explain the mechanism(s) regulating somite periodicity, but no molecular evidence seemed to back up any of the postulated models. Remarkably, in 1997 the first evidence that the formation of the somites depended on an intrinsic molecular clock was at last provided through the description of oscillating gene expression in the tissue from which somites are generated. Since then, a huge amount of data has been and continues to be provided that is gradually revealing the ever more complex molecular mechanism underlying this segmentation clock. We are also beginning to learn about embryonic structures other than the somites which exhibit oscillations of gene expression suggesting they too are dependent upon a segmentation-like clock. This is in itself the clearest evidence that there is still a long way to go before we unveil the myriad of molecular mechanisms that lead to the time control of embryonic development.
 
One of the major advances in the treatment of cancer with lipid A is the possibility of chemically synthesizing, lipid A analogs that are both biologically active in cancer patients and very well tolerated. Biotechnological production of different lipid A forms is also possible but the chemical route is preferable as it avoids LPS contamination. This significant step was somewhat unexpected as in the 1980s the debate had centered on the question “Is it possible to separate the toxic activity of lipid A from its anti-tumor activity?” and I will come back to this question here.
 
Now we have come to the conclusion of this book.
 
Common ophthalmic preservatives can be divided into five classes: quaternary ammonium compounds (benzalkonium chloride), mercurials (thimerisol), alcohols (chlorobutanol), esters of parahydroxybenzoic acid, and other compounds (polymyxin and chlorhexidine).1
 
It is well-established that volatile anesthetic agents at doses of < 0.2 MAC depress the acute hypoxic ventilatory response (AHVR) by ∼50–70%1,2. However, the effect of anesthetic agents can be very variable3,4.
 
Interstitial Cystitis (IC) is an inflammatory disease of the bladder wall and its etiology is unknown. It is characterized by frequency, urgency, suprapubic pain, and pressure. Although there is no single etiology confirmed, evidence suggests that various etiologies may be implicated in this syndrome. Treatment modalities have been elusive and unpredictable in effectiveness. It has recently been shown that approximately 70% of IC patients have damaged or defective glycosaminoglycan (GAG) layers,1,2,5 a mucus lining made of GAGs, such as chondroitin, heparin, dermatin, and hyaluronate. This damage causes epithelial leaks, allowing urinary solutes to gain access to subepithelial tissues, inducing an inflammatory response. This has lead to replenishing this mucus lining with GAG products such as heparin, hyaluronate, and pentosan polysulfate,3,4,7 again with varying results.
 
In summary, we can come to a number of meaningful conclusions regarding chronic exposure to hypobaric hypoxia in rats (refer to Figure 1). First, despite an increased hematocrit, and thus increased oxygen carrying capacity, regional cerebral blood flow is elevated after 4 weeks of chronic hypobaric hypoxia. This elevation in blood flow occurs even though the rat hyperventilates to lower than normal arterial CO2 content which would ordinarily decrease cerebral blood flow. Second, although blood flow is increased in both chronic and acute hypoxia, the increases can not be through similar mechanisms since in the acute hypoxic condition there is also an increase in local blood volume that is absent in the chronic response. Third, the effect of chronic hypoxic exposure on cerebral blood flow persists for at least 4 hours after the animal is returned to normobaric normoxia. Fourth, sometime between 4 and 24 hours of recovery is necessary to reverse the effect of chronic hypoxia on cerebral blood flow. One day after having been returned to normobaric normoxia cerebral blood flow had returned to control. On the other hand, hematocrit was still elevated in these rats. Thus, the change in hematocrit does not seem to be associated in any mechanistic manner with the blood flow response.
 
Terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been purified 4365-fold from pig thymus. It was further separated into two molecular forms of 57,000 and 45,000 dalton by Sephadex G-100 gel-filtration. The former sedimented at 4.2s through a sucrose gradient, while the latter, at 3.6s. By a SDS-polyacrylamide gel-electrophoresis, their molecular weights were estimated as 57,000 and 42,000 dalton, respectively. Thus each of the large and small pig TdT consists of a single polypeptide of 57,000 or 42,000 dalton and has no subunit structure. These two forms were indistinguishable in antigenicity by a neutralization assay of 42,000 dalton-TdT antibody. The enzymological properties of 42,000 dalton-TdT from pig thymus were very similar to those of calf TdT which has a two-subunits structure.
 
The 3'-portion of the genome from a Taiwan isolate of porcine reproductive and respiratory syndrome (PRRS) virus, strain MD-001, was cloned and sequenced. The resultant 549 nucleotides contained an open reading frame with a coding capacity of 123 amino acids (predicted Mr 13,600). The predicted protein corresponds to the nucleocapsid protein, the gene product of ORF7. Comparative sequence analysis of several known PRRSV strains indicated that this protein showed the highest degree of amino acid similarity to the US VR2332 and the Canadian IAF-Exp91 strains (92.7%) and the least to the Dutch Lelystad strain (56.5%). The phylogenic trees constructed on the basis of the known PRRSV nucleotide sequences indicated that MD-001 strain belongs to the North American strain cluster and that it is distinct from the European virus.
 
An immunoreactive role of lactic acid bacteria established in animals has seldom been investigated in humans. In a large-scale clinical study, children from day-care centers received either yoghurt (Y), milk fermented by yoghurt symbiosis and Lactobacillus casei (DN 114 001) (YC), or gelified milk (GM) as diet supplements during two 30-day supplementation periods separated by one 30-day period without supplementation. Feces samples were collected before, during, and after the 2nd supplementation period. Proteins were extracted in a buffer containing enzymatic inhibitors. IgA levels were assessed and adjusted to the weight of feces samples. Specific IgA to lactic acid bacteria strains (Streptococcus thermophilus 8901A, 8902A; Lactobacillus bulgaricus; Lactobacillus casei) present in Y and YC were assayed in ELISA and adjusted to individual IgA levels. Mean levels of fecal IgA were within reported ranges for pediatric populations of similar age. IgA levels decreased significantly but transiently in children receiving Y, and increased significantly in children receiving GM, but did not vary in the group of children who were given YC. Specific IgA to the 4 strains tested increased significantly during the supplementation period only in the group of children receiving GM, while it was transient and not significant in children receiving YC. No variation was noted in children given Y Specific IgA to lactic acid bacteria can be assayed in feces. Supplementation with fermented milks might induce a mucosal tolerance to environmental flora.
 
Changes in kininogen, prostaglandin E and hemodynamics were studied during 2 hours after left anterior descending artery ligation in 19 anesthetized dogs with and without FOY-007, an inhibitor of kinin forming enzyme. Significant decrease in kininogen in aorta and great cardiac vein (aorta greater than great cardiac vein) and increase in prostaglandin E in great cardiac vein were observed after ligation, indicating release of kinin and prostaglandin E from ischemic area. Both kininogen and prostaglandin E changes were inhibited by FOY-007 but further decrease in cardiac output and increase in systemic vascular resistance were observed. In summary, myocardial release of both kinin and prostaglandin E was inhibited by FOY-007, with increase of afterload of the heart.
 
The therapeutic efficacy of 5-fluorouracil (5-FU) can be enhanced by the addition of compounds which increase reduced folate pools in tumor cells, thereby increasing the inhibition of thymidylate synthase by 5-FU. In vitro studies have suggested that maximal thymidylate synthase inhibition is achieved when the extracellular folate concentration is 10 mmol/L, which can be achieved in vivo by intravenous doses of 500 mg/M2 of body surface area of leucovorin.1 However, controversy exists concerning the optimal necessary dose of leucovorin, as well as the optimal schedule, when combined with 5-FU. In addition to any theoretical or academic concerns about basing treatment on laboratory-derived, rather than empiric regimens, there are also practical issues involved in choosing an acceptable regimen for clinical practice. In addition to the cost of the leucovorin to the patient, there are also choices to be made concerning the impact of the treatment on nursing facilities, patient travel and patient tolerance.
 
01 Vibrio cholerae is divided into two major serological types, Ogawa and Inaba, on the basis of their heat stable somatic antigen (or 0 antigen). These two serotypes share group somatic antigen A. but can be separated by Ogawa factor B and Inaba factor C. This is generally accepted as so-called ABC concept for the O-antigenic structure of 01 V. cholerae (4, 11). Recently, three groups of non-cholera vibrios possessing common antigen factors of 01 V. cholerae have been isolated in Japan. First, a marine vibrio, Vibrio bio-serogroup 1875 Original was isolated from sea water in Osaka Bay. Besides its Inaba antigenic variant, so called Vibrio bio-serogroup 1875 Variant was isolated (13). Second, Vibrio fluvialis Kobe was isolated from imported squid in Kobe (14). Third, Vibrio cholerae bio-serogroup Hakata was isolated from sea water in Hakata Bay, Kyushu island (12). The last group, Hakata strain, has now become of current importance in Japan from the public health point of view, in particular, in quarantine administration. At least 31 strains of this group have already been isolated in Japan; 9 of them from imported squid and shrimp, and 22 from the environment. This will probably become a more important public health problem soon, because they may be misdiagnosed as 01 V. cholerae unless a polyclonal absorbed antiserum or monoclonal antibody for the single factor A of 01 V. cholerae is used. As long as present commercial diagnostic sera for 01 V. cholerae are used in practical bacteriological examination, it is impossible to distinguish 01 V. cholerae from this group. Antigenic formulae for their 0-antigenic structures were recently elucidated by Shimada and Sakazaki, National Institute of Health, Tokyo, Japan, by cross-agglutination and cross- agglutinin absorption test as shown in Fig 1.
 
The chemical composition of lipopolysaccharides isolated from respective Proteus vulgaris and P. mirabilis strains belonging to 49 different serotypes (Kauffmann and Perch classification) allowed us to divide the investigated LPS’s into 16 chemotypes. The major part of LPS’s is clustered in two chemotypes — IX and XV (4). The structure of the 0 specific part of LPS, isolated from P. vulgaris serotype 019 strain, from the chemotype IX, together with some serological data, is presented in this paper.
 
Numerous studies have shown that exposure to hypobaric hypoxia induces altered respiration as well as circulatory activities. It has been suggested that acclimatization to high altitude leads to increased capillary density1, red blood cell (RBC) counts and Hemoglobin (Hb) concentration2,3, which may play a beneficial role in maintaining energy production of the muscles with low Po2. Previous studies4 have indicated that elevation of respiratory enzyme activity occurs in some muscles with a high oxidative capacity. Although many studies have reported changes of skeletal muscle metabolism from sea level5 to hypobaric hypoxia, there are few studies that address the opposite process, i.e. from hypobaric hypoxia to sea level. Furthermore, respiratory alkalosis is initially induced by exposure to hypoxia, while the 02 dissociation curve is shifted to the right due to an increased level of 2,3-diphosphoglycerate (DPG) in RBC resulting from the hypoxemia and alkalosis6. This right shift of the 02 dissociation curve is assumed to facilitate a better 02 unloading in venous blood at a given Po2.
 
Among the brain imaging techniques, X-ray computed tomography (CT) and magnetic resonance imaging (MRI) give morphological images, but positron emission tomography (PET), which gives functional images, allows monitoring non-invasively of energy metabolism, local blood flow, some enzyme reactions and several types of receptor bindings1. The developments of PET technique and the selective radioligands has potentiated to improve our knowledge of the physiology of neurotransmitter systems and to monitor drug action by directly measuring changes in energy metabolism and receptor occupancy as results of altered storage pool of neurotransmitter, neurotransmitter release, re-uptake, etc. in human and non human primates2. Especially, the dopaminergic system has been well studied by PET, since the dopaminergic terminals are highly concentrated in the striatum and the size of the striatum is suitable for spatial resolution of PET. Within the components of dopaminergic system, measurable postsynaptic components by PET are D1 and D2 receptors,3 and measurable presynaptic components are turnover of dopamine4 and dopamine re-uptake site.5 Furthermore, in this paper, we attempt to analyse the dopamine D3 receptor by PET using the selective ligand (+)[11C]UH232 (cis-(+)-5-methoxy-l-methyl-2-(di-n-propylamino)tetralin), which was characterized as an acting dopamine autoreceptor antagonist with the biochemical and behavioral profile.6 The aim of the present study is to clarify the effects of peripherally administered R-THBP (SUN 0588, the chemically synthesized dihydrochlorides salt of R-THBP, Suntory Limited) on the dopamine D1, D2 and D3 receptor systems in the living rhesus monkey brain using PET technique.
 
6R-L-erythro-5,6,7,8-tetrahydrobiopterin (R-THBP) is the natural cofactor for phenylalanine, tyrosine and tryptophan monooxygenases, the latter two enzymes catalyses the rate-limiting reactions in the synthesis of catecholamines and serotonin, respectively. Among various types of pterins, R-THBP has attracted researcher’s interest in recent years, since R-THBP regulates not only the biosynthesis but also the release of biogenic amines such as dopamine and serotonin1,2 and other neurotransmitters, glutamate2 and acetylcholine (ACh)3. Ohue et al.3 reported that intracerebroventricular injection of R-THBP dose-dependently increased extracellular ACh levels in the rat hippocampus as monitored by microdialysis technique. Further, they demonstrated that the ACh releasing action of R-THBP was evoked through excitation of monoaminergic system4. Therefore, experimental studies have been planned on the cooperation of the monoaminergic with cholinergic functions to explain the pharmacological role of R-THBP.
 
The large bowel of the newborn is colonized during the first days of life with enterobacteria1, and in most cases Escherichia coli soon becomes the dominant enterobacterial species.2,3 E. coli commonly express type 1 fimbriae that mediate adherence to human colonic epithelial cells.4 Thus, fimbrial expression may be of importance for the ability of E. coli to colonize and persist in the large bowel of the neonate. On the other hand, secretory IgA (S-IgA) carries oligosaccharide chains that function as receptors for type 1 fimbriae.5 The S-IgA in milk may provide extra binding sites for type 1-fimbriated strains, or block their adherence to colonic epithelial cells. In either case, breast feeding may influence the early enterobacterial colonization of the neonate.
 
The rfb gene encoding the proteins responsible for the synthesis of the repeating units (O side-chain) of Escherichia coli 09 lipopolysaccharide was cloned into a conjugative plasmid RP4::miniMu and was expressed in E. coli K-12.
 
2’,6’-dimethyl substitution of the Tyr1 residue of opioid agonist peptides and deletion of the N-terminal amino group have been shown to represent a general structural modification to convert opioid peptide agonists into antagonists [1]. This conversion required the syntheses of opioid peptide analogues containing 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) in place of Tyr1. The cyclic enkephalin analogue Dhp-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 is a potent µ opioid antagonist and a less potent δ and κ antagonist [2]. An analogue of this peptide with β-methylated Dhp, (3S)-3-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(3S)-Mdp], in place of Dhp showed increased antagonist activity at all three receptors, whereas an analogue containing β-isopropyl-substituted Dhp [(3R)-Idp] turned out to be a mixed κ agonist/µ antagonist [3]. To further investigate the SAR at the β position of Dhp in the cyclic analogue, we examined the effect of substituting β-hydroxylated Dhp on the opioid activity profile. This required the development of stereoselective syntheses of (3S)- and (3R)-3-hydroxy-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(3S)-and (3R)-Hdp].
 
Gout disease in our country as both exogenous factors (dietary habits, professions, etc.) and endogenous factors (sex, inheritance, etc.) play a polygenic role in its incidence. Those conditions resulting in hyperuricemia (diseases, drugs, etc.) facilitate the clinical manifestations. But a genetic determinant in the articular reaction in front of an accumulation of uric acid crystals or in the development of such crystallization is a fundamental factor in the manifestation of the disease. In our series we were able to confirm the high incidence of arterial hypertension, renal lithiasis and renal participation in gout. The role of lead and of certain professions in favouring the hyperuricemia is suggested. A classification of gout, from a functional point of view, must include both the clinical form and the analysis of the whole renal function, and the renal handling of uric acid. This is mandatory not only for the diagnosis and prognosis of the disease but to set the basis of its adequate management.
 
1,1-Dicholorethylene (DCE), a widely used compound in the manufacture of plastics, has been shown to be toxic to kidney, liver and lung in laboratory animals1–5, and to exhibit some mutagenic and carcinogenic potential6–8. Its toxicity has been suggested to result from covalent binding of reactive metabolites to cellular macromolecules3,9,10; these metabolites are nprm jly detoxified by conjugation with reduced glutathione (GSH)11–16. However the exact mechanisms of its expressed toxicity and its application to man are not understood. Species differences among animals in toxicity of DCE are thought to be due to the relative amount of bioactivated and covalently bound DCE produced in each species We present in this paper the in vivo tissue distribution and covalent binding as well as the in vitro metabolism and covalent binding of DCE in the mouse, the species Wight to be most suscep-tible to its toxicity9,10, with particular reference to the kidney, liver and lung.
 
Metabolic activation by glutathione conjugation of the dihaloethanes ethylene dichloride (EDC) and ethylene dibromide (EDB), continues to be important as these compounds are used commercially in enormous quantities and the mechanisms by which they exert toxicity and mutagenicity are not yet fully understood. The mutagenicity of EDC has been demonstrated to be dependent on the presence of cytosol and glutathione (Rannug et al., 1978). In line with this, is the observation of covalent association of EDC-derived radioactivity with polynucleotides in incubation mixtures containing cytosol, glutathione (GSH), and EDC (Guengerich et al., 1980). In the case of EDB, an alkylated purine has been isolated from similar incubation mixtures (Ozawa and Guengerich, 1983). These and other results with cofactors and inhibitors indicate that the GSH-transferases are initially responsible for the formation of important reactive intermediate(s) from these compounds.
 
Recent evidence has implicated lipid-mediated second messengers such as 1,2diacylgjycerol (DAG)-protein protein kinase C (PKC) pathway as an important mediator underling multiple aspects of myocardial function (1). For example, PKC is involved in Ca2+ -induced inotropy, in mediating myocardial preconditioning by diverse stimuli both in animals and humans, and in the signaling processes which lead to the production of proinflammatory mediators. Hyperglycemia and diabetes have been shown to be associated with PKC activities (2). The changes in DAG-PKC pathway are significant in causing cardiovascular dysfunctions and pathologies.
 
Transforming growth factor beta 1 (TGF beta 1) increases the phosphorylation of the epidermal growth factor (EGF) receptor and inhibits the growth of A431 cells, but the mechanism of TGF beta 1 signaling is unknown. Recent studies from this and other laboratories suggest a novel sphingomyelin signal transduction pathway (1-4). Ceramide, which is generated by sphingomyelinase action, can be deacylated to sphingoid bases, which are potential inhibitors of protein kinase C (PKC). Ceramide appears to have bioeffector properties. Cell-permeable ceramide analogs stimulate monocytic differentiation of human leukemia (HL60) cells (1), as well as the phosphorylation of the EGF receptor at Thr669 in A431 human epidermoid carcinoma cells (2). Further studies (3,4) demonstrate the existence of a ceramide-activated protein kinase (CAPK) that may mediate some of these aspects. The present studies aim to investigate the mechanism of TGF beta 1 signaling and to explore whether TGF beta 1's pathway involves activation of PKC by 1,2-Diacylglycerol (DAG) and/or stimulation of a CAPK by ceramide. Ceramide and DAG levels of A431 cells are determined by thin layer chromatography (TLC) after treatment with either TGF beta 1 or with EGF. 100 pM TGF beta 1 treatment for 1 hr increases the cellular contents of DAG 2-fold. 20 nM EGF treatment for 15 min decreases it 0.5-fold. Ceramide levels are reduced 2-fold by TGF beta 1 and almost unaffected by EGF. To evaluate the involvement of other components of signal transduction, the effects of TGF beta 1 and EGF on PKC activity are studied. 20 nM EGF decreases membrane PKC activity to 0.5-fold of controls, whereas 100 pM TGF beta 1 treatment of A431 cells increases this activity 4-fold. Modulation of PKC activity is paralled by translocation of the enzyme between the cytosol and the membrane as determined by Western immunoblot analysis. These studies suggest that TGF beta 1 and EGF may have regulatory effects on both sphingolipid and phospholipid metabolisms which could transmodulate both the CAPK and the PKC mediated signal tranduction pathways.
 
Naphthalene metabolism and toxicity have been extensively investigated and these data indicate that the lung and eye toxicity are mediated by reactive intermediates. The elegant studies conducted in Buckpitt’s laboratory implicate a 1,2-epoxide as one reactive intermediate which is extensively conjugated with glutathione (Buckpitt et al., 1987). Data from this laboratory also suggest that the liver may form a precursor metabolite or reactive metabolite which is transported to the lung (Buckpitt and Warren, 1983). Studies of the ocular effects of naphthalene suggest that the well known liver metabolite, naphthalene-1,2-ihydrodiol (DIOL), may be transported to the eye where it is oxidized to the catechol metabolite, 1,2-dihydroxynaphthalene (VanHeyningen and Pirie, 1967). The catechol is easily oxidized non-enzymatically presumably to potentially reactive quinones and semiquinones. A more recent report also suggests the involvement of quinones in the ocular toxicity of naphthalene (Wells et al., 1989) and an in vitro study indicates that multiple quinone-derived metabolites are formed from D1OL when its oxidation is catalyzed by hepatic dihydrodiol dehydrogenase (Smithgall et al., 1988). Naphthalene is extensively metabolized and at least two epoxides as well as the the catechol/quinone metabolites are potentially reactive, toxic metabolites (Horning et al., 1980).
 
Enhanced labelling of phosphatidylinositol occurs in different type of cells during stiinulation (1,2). This effect includes phosphatidylinositol breakdown and resynthesis (see cycle of reaction in Hawthorne et al., this Synposium) and has recently been related in various tissues to α -adrenergic and muscarinic cholinergic agonists (2). According to the available information it seems most probably that the site of control of this effect is confined to phosphatidylinositol breakdown to 1,2-diacylglycerol. Pancreas, parotid and longitudinal ileum smooth muscle show a fall in the concentration of phosphatidylinositol during stimulation (3,4,5). It is then possible that studies of phosphatidylinositol labelling are only a reflection of the primary event which is phosphatidylinositol breakdown. A specific phospholipase-C type of activity against phosphatidylinositol has been found in particulate fractions of brain and vas deferens smooth muscle (6-8). This activity produces breakdown of phosphatidylinositol with production of 1,2- diacylglycerol, myoinositol 1,2-cyclic phosphate and myoinositol 1-phosphate (8,9)
 
PAP, a very polar substance, is highly metabolized in mice and excreted principally in urine in the form of the 2-hydroxy-3-phenylaminopropanoic acid of each enantiomer. Thus, the major route of PAP elimination in these strains is alkyl chain oxidation. In particular, S-PAP is eliminated principally in the form of that metabolite, whereas R-PAP enantiomer showed further oxidized species at the aromatic ring and alkyl chain, yielding potential decarboxylated compounds and iminoquinones. All these metabolites may have toxicologic implications. On the other hand, OOPAP intestinal hydrolysis in favour of one PAP enantiomer might be expected since lipases show chiral hydrolysis (unpublished data, manuscript in preparation). In this respect, enantiomeric distribution and metabolic differences should be taken into account in the toxicokinetics of these compounds and their potential association with Toxic Oil Syndrome symptoms.
 
In clinical use, the therapeutic safety margin with any iron chelator is likely to be narrow compared with commonly used pharmaceuticals. This is because there is inevitably a fine balance between excess iron and iron depletion within cells. This safety margin may be increased in the presence of iron overload but as not all cells will be equally iron overloaded, some will be more susceptible to iron deprivation than others. It is likely that minor modifications to chelator structure will make important differences to the therapeutic safety margin in clinical practice. The approach of our group has therefore been to identify the principles determining the efficacy and/or toxicity of iron chelators so that compounds which can be effective at lower doses or have less toxicity, than for example L1, can be identified. The hydroxypyridin-4-ones (HPOs) (Hider et al, 1982) are an appropriate group of compounds for such studies as their relatively simple structure allows a systematic investigation of structure-function relationships. The problems in identifying models which will predict the behaviour of chelators in iron overloaded humans has not helped in the development of iron chelators; indeed the preclinical and clinical experience with the HPOs has highlighted these shortcomings.
 
Benzoate 1,2-dioxygenase which has so far been referred to as benzoate hydroxylase catalyzes the double hydroxylation of benzoate, namely the incorporation of two hydroxyl groups into benzoate, resulting in the formation of l,2-dihydro-l,2-dihydroxybenzoate (DHB). Both oxygen atoms of the two hydroxyl groups introduced into the substrate are derived from the molecular oxygen (1,2).
 
Vinyl chloride is recognized as a human carcinogen. 2-Chlorooxirane, its biological reactive intermediate formed by the P450 enzyme system in the liver, and the rearrangement product chloroacetaldehyde give rise to the etheno (ε) DNA adducts. This type of modified DNA bases appears to be the molecular basis for the genotoxic effects of the parent compound, and site specific mutagenesis studies (Basu et al., 1993) show its mutagenicity. There is evidence that ε adducts might not only be formed by vinyl chloride metabolites but also be present at a certain level due to endogeneous sources (Misra et al., 1994).
 
1,2-Dibromoethane (DBE) is widely used as an insecticide, fungicide and gasoline additive.1 A number of adverse effects of this compound have been reported, notably its mutagenicity towards bacteria2 and carcinogenicity towards rats and mice.3 Apart from its direct alkylating ability, two mechanisms for these toxic actions can be envisaged, both of which involve biotransformation. The first one consists of oxidation, followed by loss of hydrogen bromide, leading to bromoacetaldehyde, a highly reactive substance which can bind covalently to macromolecules.4, 5 However, bromoacetaldehyde has been reported to be non-mutagenic.6 The second possible mechanism involves conjugation to glutathione (GSH). As has been shown for 1,2-dichloroethane7 and cis-1,2-dichlorocyclohexane,8 the 2-halogenothioether resulting from substitution of one of the halogen atoms by glutathione is responsible for enhanced mutagenic activity of these compounds in the presence of GSH and GSH-S-transferases (see Fig. 1).
 
Glutathione (GSH) is a ubiquitous tripeptide involved in cellular defense mechanisms and the metabolism of xenobiotic compounds. Detoxification of free radicals and activated oxygen species by direct reaction, as well as that mediated by the enzymic activity of glutathione peroxidase, is a well known and described biochemical function.Equally well known and described is the role of GSH in conjugation. Conjugation with GSH occurs nonenzymatically and through the action of GSH S-transferases. Leukotrienes, active autacoids thought to be involved in inflammatory processes, are formed by the conjugation of GSH with fatty acids derived from arachidonic acid. One important function of GSH S-transferases is the conjugation of GSH with certain xenobiotic compounds, thus enhancing excretion of the xenobiotic. Furthermore, activated metabolites produced from xenobiotic compounds by the action of mixed function oxygenase may also be conjugated with GSH. This occurs both directly and enzymatically. The net result of conjugation with active metabolites is detoxification of these reactive chemical species. In certain cases, the GSH conjugate may ultimately result in toxic reactions. Stepwise, enzymatic degradation of certain GSH conjugates may result in reactive intermediates that result in tissue injury. Some glutathione conjugates, for example, may be enzymatically toxified by the sequential actions of gamma-glutamyl transferase and cysteine conjugate beta-lyase (Dohn and Anders, 1982). Other GSH conjugates may undergo intramolecular rearrangement to unstable intermediates that also result in toxic reactions. For example, the conjugation of vicinaldihaloalkanes may result in an intramolecular rearrangement to a reactive, transitory episulfonium ion (van Bladeren et al., 1980; Schasteen and Reed, 1981; Livesay and Anders, 1982).
 
1,2,3-Trichloropropane (TCP) is used as an industrial solvent and as an intermediate in pesticide and polysulfide rubber manufacturing. It is structurally similar to other halogenated alkanes with known carcinogenic potential (1,2- dibromoethane and 1,2-dibromo-3-chloropropane). Preliminary evidence from a chronic bioassay study conducted by the National Toxicology Program indicates that TCP administration results in an increased incidence of tumors in both male and female F-344 rats and B6C3F1 mice (Mahmood et al.,1988).
 
Isoquinolines derived from DA and excreted in the urine of Parkinsonian patients under L-dopa therapy are called “Mammalian TIQ”.3,4 Pictet-Spengler synthesis5 provides an easy route for the preparation of optically inactive TIQ. Many TIQ have been prepared from DA and carbonyl compounds (phenylpyruvic acids, aldehydes, ketones) in this way and under so-called “physiological” conditions, suggesting that this reaction might have biological significance.6 If so, then similar reactions could take place with the two catecholamines (±)-NE or (±)-E or their optical isomers, resulting in the formation of diastereomeric 4,6,7-trihydroxy TIQ or their optical isomers. The preparation of TIQ alcohols 17 and 28 (Figure 1) belonging to this class of compounds, and prepared from (±)-NE and formaldehyde and (-)-E and acetaldehyde respectively, has been reported.
 
Exogenous 1,25-(OH)2-D3, administered to vitamin D-replete animals on a high calcium diet, induces biosynthesis of the duodenal, cytosolic calcium-binding protein (CaBP) in less than 2 h. This process can be blocked by simultaneously administered cycloheximide, but not by actinomycin D. In vitamin D-replete animals on a low Ca diet, on the other hand, 1,25-(OH)2-D3 administration leads to new CaBP synthesis only after about 7 h; this process can be blocked by actinomycin D. In vitamin D-deficient animals on a high calcium diet who have no CaBP, treatment with 1,25-(OH)2-D3 induces CaBP formation in congruent to 8 h; this process is known to be blocked by actinomycin D. Thus in D-replete animals on a low calcium diet and in D-deficient animals, CaBP biosynthesis proceeds by a transcriptional route, whereas in D-replete animals on a high calcium diet the rapid response appears to be posttranscriptional. This finding points to the possibility of a more rapid regulatory action of vitamin D than previously reported and how vitamin D might function in the D-replete state.
 
Previous studies from our laboratory have demonstrated that 1,25-dihydroxyvitamin D3 (1,25D) stimulates separate calcium (Ca) and inorganic phosphate (P) absorptive mechanisms in rat intestine (1,2). The proportion of Ca to P “pumps” varies in different regions of the intestine. Thus, there is a preponderance of Ca pumps in the duodenum, a preponderance of P pumps in the jejunum and an equal distribution of Ca and P pumps in the ileum (3). Interestingly, the colon has only 1,25D-responsive Ca pumps (2). The active Ca absorptive mechanisms in duodenum, ileum and colon appear to share certain characteristics, all exhibiting a Km of approximately 1 mM (2,3). The expression of this 1,25D-induced Ca absorption in colon does not appear to require the presence of extracellular sodium ions (4). In the present study we examined the sodium (Na) requirement of 1,25D-stimulated P transport processes in rat jejunum.
 
Patients with recurrent calcium oxalate/apatite nephrolithiasis in association with hypercalciuria (idiopathic hypercalciuria) may exhibit low serum inorganic phosphate concentrations (1,2,3,4) in association with normal rates of urinary phosphate excretion implying the existence of impaired renal tubular phosphate reabsorption. In fact, such calcium stone formers exhibit impaired renal phosphate conservation in response to ingestion of a diet low in phosphate (5). They also exhibit higher than normal salivary phosphate concentrations (6) implying the presence of a more generalized abnormality of phosphate transport. Dietary phosphate deprivation in animals is accompanied by increased synthesis of the calcium mobilizing steroid 1,25-(OH)2-vitamin D3 by the kidney (7) and elevated serum levels of this hormone (8). Similarly renal synthesis of 1,25-(OH)2-D is increased in hypercalciuric kidney stone formers (9) and high serum 1,25-(OH)2-D concentrations among these patients may be inversely related to the degree of hypophosphatemia (4).
 
Parathyroid hormone (PTH) has been shown to stimulate the formation of 1,25-dihydroxycholecalciferol (1,25(OH)2D3), the most biologically active form of vitamin D3, by the kidney (1, 2). It has been postulated that 1,25(OH)2D3 or other vitamin D3 metabolites might directly influence the secretion of PTH. Receptors for 1,25(OH)2D3 have been identified in chick (3) and pig (4) parathyroid glands and in human parathyroid adenoma (5). Accumulation of 1,25(OH)2D3 by the parathyroid gland has been reported in the chick (6), and 1,25(OH)2D3 when administered to-gether with 24,25-dihydroxycholecalciferol (24,25(OH)2D3), caused the size of the parathyroid glands in vitamin D-deficient chicks to regress (7). The effect of vitamin D metabolites on PTH secretion has been examined in several species. Studies several years ago by Oldham, et al, showed that the administration of pharmacological amounts of 25-hydroxycholecalciferol (250H D3) to vitamin D-deficient dogs appeared to decrease the circulating concentrations of immunoreactive parathyroid hormone (IPTH) before significant increases in serum calcium concentration occurred (8). Chertow, et al, reported a decrease in IPTH in rats administered 1,25(OH)2D3 and an inhibition of IPTH released from slices of bovine parathyroid tissue in vitro when 1,25(OH)2D3 was added to the culture medium (9).
 
Vitamin D-dependent rickets, type II, is a rare, hereditary syndrome characterized by early onset rickets, hypocalcemia, secondary hyperparathyroidism, normal vitamin D intake, and normal or elevated circulating levels of 1,25-dihydroxyvitamin D (1,25(OH)2D). It is thought to result from target tissue resistance to the action of 1,25(OH)2D in a manner analogous to the resistance to androgens and glucocorticoids seen in patients with decreased hormone action in the face of elevated blood levels of those hormones.1,2 We will refer to this form of rickets as hereditary, hypocalcemic 1,25(OH)2D3-resistant rickets (HHDR).
 
Evidence has accumulated from investigations performed in this laboratory and in others that the renal tubular effect of 1,25-dihydroxy vitamin D3 (1,25 D3), the active form of the vitamin, is to increase phosphate reabsorption by the kidney (1–3). We recently reported that the infusion of 2.5 ng of 1,25 D3 per hour along with 0.2 U of PTH/hr. for six hours resulted in an antiphosphaturia in thyroparathyroidectomized (TPTX), vitamin D-deficient (−D) rats (3). This dose of the metabolite has been considered to be in the physiological range, and was not effective unless given with the non-phosphaturic “permissive” dose of PTH. The development of the brush border vesicle (BBV) transport technique (4) provided us with an opportunity to study the locus of this antiphosphaturic action of the metabolite. Furthermore, glucose metabolism has been reported to be linked to phosphate (Pi) transport events; Rasmussen and his coworkers have reported (5) that parathyroid hormone (PTH), a substance which diminishes phosphate uptake by brush border vesicles (4), increases gluconeogenesis (GNG). Accordingly, we examined the effect of the antiphosphaturic agent 1,25 D3, on BBV Pi uptake and on GNG.
 
Harrison and Harrison1 first proposed that vitamin D acts as an important regulator of phosphate homeostasis. Since then, it has has been demonstrated that the active form of the vitamin, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3], enhances, by an unknown mechanism, the intestinal absorption of phosphate2,3. The effect of 1,25-(OH)2D3 on the reabsorption of phosphate in the kidney is less clear. In some studies, infusion of the vitamin has been found to decrease phosphate excretion4,5, whereas in other investigations 1,25-(OH)2D3 has been reported to be Phosphaturie6 or without effect on urinary phosphate7. Some of the difficulties in resolving the apparent discrepancies stem, in part, from the complex interactions in the in vivo animal of the vitamin D metabolic and phosphate transport systems with various regulatory factors, including parathyroid hormone and other endocrines, dietary status, and calcium8,9. This prompted us to initiate studies on a simpler model system, namely, the isolated renal cell. In this report we will examine the effects of 1,25-(OH)2D3 on the uptake of phosphate by these isolated cells; first, after the vitamin is administered to a D-deficient chick in vivo, and second, when 1,25-(OH)2D3 is incubated with the cells in vitro.
 
Calcium and phosphorus homeostasis is exquisitely regulated by the three hormones, calcitonin, parathyroid hormone, and cholecalciferol (vitamin D3). The most notable advance in our understanding of the mechanism of action of vitamin D has been the elucidation of the complex metabolic pathway which has evolved to produce the biologically active form, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Coupled with these developments concerning our understanding of the metabolic pathway of conversion of vitamin D into its active form, has been the realization that the mechanism of action of the fat soluble vitamin D is in reality similar to that of many of the classical steroid hormones, e.g. aldosterone, testosterone, estrogen, hydrocortisone, and ecdysterone. It should be noted that chemically vitamin D is in reality a steroid, in particular a seco steroid. Seco steroids are those in which one of the rings has undergone fission; in the instance of calciferol, this is ring B.
 
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Peter Vaupel
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Atilla Engin
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