Mechanism of erythrocyte accumulation of methylation inhibitor S-adenosylhomocysteine in uremia

We have recently demonstrated that methyl esterification of erythrocyte membrane proteins, a reaction involved in recognition and repair of specifically damaged proteins, is impaired in uremia. This is accompanied by a significant increase in intracellular S-adenosylhomocysteine (AdoHcy), a potent inhibitor of methyltransferases. AdoHcy accumulation is normally prevented by its enzymatic hydrolysis to homocysteine (Hcy) and adenosine, a reversible reaction catalyzed by AdoHcy hydrolase. To assess the contribution that Hcy offers in the elevation of AdoHcy, we measured plasma and red blood cell Hcy, AdoHcy, adenosine, and S-adenosylmethionine (AdoMet) intracellular concentrations, as well as RBC AdoHcy hydrolase specific activity, in standard hemodialysis patients and normal subjects. Plasma and red blood cell Hcy levels are significantly higher in the dialysis group, and are positively correlated to AdoHcy levels. Adenosine and AdoMet levels, and AdoHcy hydrolase specific activity are not significantly different between the two groups. The enzymatic formation of labeled AdoHcy from Hcy and tracer adenosine appears to be significantly increased, in vitro, in erythrocytes from both control and uremic patients, when 50 microM Hcy (concentration comparable to plasma levels actually found in vivo in uremic patients) is added to the incubation medium. When erythrocytes from uremic patients are incubated in vitro in absence of Hcy, a significant reduction of intracellular AdoHcy is observed with time compared to identical samples incubated in presence of 50 microM Hcy, with a T1/2 of approximately 270 minutes. The results allow us to conclude that plasma and red cell Hcy levels actually found in uremia can be effectively responsible for the intracellular accumulation of the toxic compound AdoHcy.     

Effects of 4'-modified analogs of aristeromycin on the metabolism of S-adenosyl-L-homocysteine in murine L929 cells

(1'R,2'S,3')-9-(2',3'-Dihydroxycyclopentan-1'-yl)adenine (DHCaA), (1'R,2'S,3'R)-9-(2',3'-dihydroxycyclopentan-1'-yl)-3-deazaadenine (3-deaza-DHCaA), (4'R)-4'-methyl-DHCaA, and (4'R)-4'-vinyl-DHCaA, which are analogs of the carbocyclic nucleoside aristeromycin, were synthesized earlier by our laboratory and were shown to be potent inhibitors of purified bovine liver S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1). In the present study, these analogs were shown to produce rapid (within 15 min) and concentration-dependent (0.03-10 microM) inhibition of AdoHcy hydrolase in cultured murine L929 cells [relative order of inhibitory activity, DHCaA = 3-deaza-DHCaA > (4'R)-4'-vinyl-DHCaA = (4'R)-4'-methyl-DHCaA]. The relative potencies of these inhibitors on the L929 AdoHcy hydrolase were consistent with their inhibitory effects on the recombinant forms of rat liver and human placental enzymes. This inhibition of L929 cellular AdoHcy hydrolase persisted for up to 48 hr. The inhibition of the L929 AdoHcy hydrolase resulted in a significant increase in the cellular concentrations of AdoHcy, whereas the cellular S-adenosylmethionine (AdoMet) levels remained relatively constant, thereby elevating the AdoHcy/AdoMet ratios. Maximum increases in AdoHcy levels and AdoHcy/AdoMet ratios occurred within 6 hr of exposure to the inhibitors and persisted for at least 24 hr. At a concentration of 1 microM, DHCaA and 3-deaza-DHCaA increased AdoHcy/AdoMet ratios to approximately 0.8 (after 24 hr of exposure to the inhibitors), whereas (4'R)-4'-vinyl-DHCaA and (4'R)-4'-methyl-DHCaA elevated AdoHcy/AdoMet ratios to approximately 0.15, compared with control levels of 0.05. Treatment of L929 cells with concentrations of DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA up to 10 microM did not result in changes in cellular levels of endogenous nucleotides (e.g., CTP, UTP, ATP, and GTP). In contrast, cells treated with 10 microM aristeromycin for 6 hr contained reduced cellular levels of CTP, ATP, and GTP and significant levels of aristeromycin triphosphate and a GTP metabolite of this carbocyclic nucleoside. These data clearly show that the 4'-modified analogs [DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA] retain inhibitory activity toward cellular AdoHcy hydrolase, causing elevated levels of AdoHcy and elevated AdoHcy/AdoMet ratios. However, these analogs are devoid of substrate or inhibitory activity toward cellular adenosine kinase. In addition, aristeromycin is rapidly metabolized in murine L929 cell lysates, i.e., > 60% of the aristeromycin had been metabolized in 6 hr. In contrast, neither DHCaA nor 3-deaza-DHCaA showed any decrease in concentration after incubation with cell lysates for up to 6 hr.     

Increased expression of 3-hydroxyanthranilate 3,4-dioxygenase gene in brain of epilepsy-prone El mice

The El mouse is an established animal model for human epilepsy. We previously reported that the level of quinolinic acid (QUIN), an excitotoxin, was high in the brain of epilepsy-prone El mice and that the increased production of QUIN was secondary to an increased activity of 3-hydroxyanthranilate 3,4-dioxygenase (3-HAO, EC 1.13.11. 6) in the brains of these mice. In this study, we cloned and sequenced the cDNA for 3-HAO and showed that its expression in the brain of El mice was higher than that of control ddY mice. These results suggest that a genetic defect leading to derepression of the 3-HAO gene expression in the brain may be involved in the pathogenesis for the epileptic diseases of El mice.     

Carbocyclic adenosine analogues as S-adenosylhomocysteine hydrolase inhibitors and antiviral agents: recent advances

Various carbocyclic analogues of adenosine, including aristeromycin (carbocyclic adenosine), carbocyclic 3-deazaadenosine, neplanocin A, 3-deazaneplanocin A, the 5'-nor derivatives of aristeromycin, carbocylic 3-deazaadenosine, neplanocin A and 3-deazaneplanocin A, and the 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A have been recognized as potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase. This enzyme plays a key role in methylation reactions depending on S-adenosylmethionine (AdoMet) as methyl donor. AdoHcy hydrolase inhibitors have been shown to exert broad-spectrum antiviral activity against pox-, paramyxo-, rhabdo-, filo-, bunya-, arena-, and reoviruses. They also interfere with the replication of human immunodeficiency virus through inhibition of the Tat transactivation process.     

Crystal structure of apo-glycine N-methyltransferase (GNMT)

The crystal structure of the recombinant apo-form of glycine N-methyltransferase (GNMT) has been determined at 2.5 A resolution. GNMT is a tetrameric enzyme (monomer Mr = 32,423Da, 292 amino acids) that catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to glycine with the formation of S-adenosylhomocysteine (AdoHcy) and sarcosine (N-methylglycine). GNMT is a regulatory enzyme, which is inhibited by 5-methyltetrahydrofolate pentaglutamate and believed to control the ratio of AdoMet to AdoHcy in tissues. The crystals belong to the orthorhombic space group P2(1)2(1)2 (a = 85.39, b = 174.21, c = 44.71 A) and contain one dimer per asymmetric unit. The AdoMet-GNMT structure served as the starting model. The structure was refined to an R-factor of 21.9%. Each monomer is a three-domain structure with a large cavity enclosed by the three domains. The tetramer resembles a square with a central channel about which N-terminal domains are intertwined. Only localized changes of the residues involved in the binding pocket are observed for the apo-GNMT structure when compared to that determined in the presence of substrate and substrate analog.     

Striatal dopamine depletion, tremors, and hypokinesia following the intracranial injection of S-adenosylmethionine: a possible role of hypermethylation in parkinsonism

The major symptoms of Parkinson disease (PD) are tremors, hypokinesia, rigidity, and abnormal posture, caused by the degeneration of dopamine (DA) neurons in the substantia nigra (SN) and deficiency of DA in the neostriatal DA terminals. Norepinephrine (NE) and serotonin (5-HT) levels in the neostriatum and tyrosine hydroxylase and melanin pigments in the substantia nigra are also decreased, and brain cholinergic activity is increased. The cause of PD is unknown, but PD is an age-related disorder, suggesting that changes that occur during the aging process may help to precipitate PD. Methylation increases in aging animals. Increased methylation can deplete DA, NE, and 5-HT; increase acetylcholine; and cause hypokinesia and tremors. These effects are similar to changes seen in PD, and interestingly also, they are similar to some of the changes that are associated with the aging process. It is suggested, therefore, that increased methylation may be an inducing factor in parkinsonism. Accordingly, the effects of an increase in methylation in the brain of rats were studied. S-adenosylmethionine (AdoMet), the limiting factor in the methylation process, was injected into the lateral ventricle of rats. Specific behavioral changes that resemble changes seen in PD were investigated. The results showed that AdoMet caused tremors, rigidity, hypokinesia, and depleted DA. The hypokinetic effects of a single dose of AdoMet lasted for about 90 min. AdoMet has a dose-dependent hypokinetic effect. A dose of 9.4 nmol reduced movement time (MT) by 68.9% and increased rest time (RT) by 20.7%, and a dose of 400 nmol reduced MT by 92.4% and increased RT by 27.6%. The normethyl analog of AdoMet, S-adenosylhomocysteine, did not cause hypokinesia or tremors, but it blocked the AdoMet-induced motor effects. L-dopa, the precursor of DA, also blocked the AdoMet-induced motor effects. These data suggest that the methyl group of AdoMet as well as DA depletion are involved in the AdoMet-induced motor effects. A dose of 0.65 mumol of AdoMet depleted DA in the ipsilateral caudate nucleus (CN) or neostriatum by 50.1%, and DA in the contralateral CN was reduced by 9.3%. Double the dose of AdoMet did not increase the depletion of DA on the ipsilateral CN, but DA in the contralateral CN was decreased by 26.3%. Taken together, the results suggest that increased methylation may contribute to the symptoms of PD.     

Function of Pro-185 in the ProCys of conserved motif IV in the EcoRII [cytosine-C5]-DNA methyltransferase

ProCys in the conserved sequence motif IV of [cytosine-C5]-DNA methyltransferases is known to be part of the catalytic site. The Cys residue is directly involved in forming a covalent bond with the C6 of the target cytosine. We have found that substitution of Pro-185 with either Ala or Ser resulted in a reduced rate of methyl group transfer by the EcoRII DNA methyltransferase. In addition, we observed an increase in the Km for substrate S-adenosyl-L-methionine (AdoMet), but a decrease in the Km for substrate DNA. This is reflected in minor changes in kcat/Km for DNA, but in 10- to 100-fold reductions in kcat/Km for AdoMet. This suggests that Pro-185 is important to properly orient the activated cytosine and AdoMet for methyl group transfer by direct interaction with AdoMet and indirectly via the Cys interaction with cytosine.     

Stereospecificity of 6'-C-neplanocin A analogues as inhibitors of S-adenosylhomocysteine hydrolase activity and human immunodeficiency virus replication

The R- and S-isomers of 6'-C-neplanocin A analogues, which are all known as inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, were studied for their inhibitory effects on Human Immunodeficiency Virus type 1 (HIV-1) replication and HIV-1 Tat-mediated transactivation. The R-isomers showed much greater activity against AdoHcy hydrolase than the S-isomers. The same differential activity was observed against the HIV-1 replication and the Tat transactivation.     

The structure of the C-terminal domain of methionine synthase: presenting S-adenosylmethionine for reductive methylation of B12

BACKGROUND: In both mammalian and microbial species, B12-dependent methionine synthase catalyzes methyl transfer from methyltetrahydrofolate (CH3-H4folate) to homocysteine. The B12 (cobalamin) cofactor plays an essential role in this reaction, accepting the methyl group from CH3-H4folate to form methylcob(III)alamin and in turn donating the methyl group to homocysteine to generate methionine and cob(I)alamin. Occasionally the highly reactive cob(I)alamin intermediate is oxidized to the catalytically inactive cob(II)alamin form. Reactivation to sustain enzyme activity is achieved by a reductive methylation, requiring S-adenosylmethionine (AdoMet) as the methyl donor and, in Esherichia coli, flavodoxin as an electron donor. The intact system is controlled and organized so that AdoMet, rather than methyltetrahydrofolate, is the methyl donor in the reactivation reaction. AdoMet is not wasted as a methyl donor in the catalytic cycle in which methionine is synthesized from homocysteine. The structures of the AdoMet binding site and the cobalamin-binding domains (previously determined) provide a starting point for understanding the methyl transfer reactions of methionine synthase. RESULTS: We report the crystal structure of the 38 kDa C-terminal fragment of E.coli methionine synthase that comprises the AdoMet-binding site and is essential for reactivation. The structure, which includes residues 901-1227 of methionine synthase, is a C-shaped single domain whose central feature is a bent antiparallel betasheet. Database searches indicate that the observed polypeptide has no close relatives. AdoMet binds near the center of the inner surface of the domain and is held in place by both side chain and backbone interactions. CONCLUSIONS: The conformation of bound AdoMet, and the interactions that determine its binding, differ from those found in other AdoMet-dependent enzymes. The sequence Arg-x-x-x-Gly-Tyr is critical for the binding of AdoMet to methionine synthase. The position of bound AdoMet suggests that large areas of the C-terminal and cobalamin-binding fragments must come in contact in order to transfer the methyl group of AdoMet to cobalamin. The catalytic and activation cycles may be turned off and on by alternating physical separation and approach of the reactants.     

A hydrolase enzyme inactivating endogenous ligands for cannabinoid receptors

Cannabinoids are psychoactive components of marijuana, and bind to specific G protein-coupled receptors in the brain and other mammalian tissues. Anandamide (arachidonoylethanolamide) was discovered as an endogenous agonist for the cannabinoid receptors. Hydrolysis of anandamide to arachidonic acid and ethanolamine results in the loss of its biological activities. The enzyme responsible for this hydrolysis was solubilized, partially purified from the microsomes of porcine brain, and referred to as anandamide amidohydrolase. In addition to the anandamide hydrolysis, the enzyme preparation catalyzed anandamide synthesis by the condensation of arachidonic acid with ethanolamine. Several lines of enzymological evidence suggested that a single enzyme catalyzes both the hydrolysis and synthesis of anandamide. This reversibility was confirmed by the use of a recombinant enzyme of rat liver overexpressed in COS-7 cells. However, in consideration of the high Km value for ethanolamine as a substrate for the anandamide synthesis, the enzyme was presumed to act as a hydrolase rather than a synthase under physiological conditions. The recombinant enzyme acted not only as an amidase hydrolyzing anandamide and other fatty acid amides but also as an esterase hydrolyzing methyl ester of arachidonic acid. 2-Arachidonoylglycerol, which was found recently to be another endogenous ligand, was also efficiently hydrolyzed by the esterase activity of the same enzyme. The anandamide hydrolase and synthase activities were detected in a variety of rat organs, and liver showed by far the highest activities. A high anandamide hydrolase activity was also detected in small intestine but only after the homogenate was precipitated with acetone to remove endogenous lipids inhibiting the enzyme activity. The distribution of mRNA of the enzyme was in agreement with that of the enzyme activity.     

Biopharmaceutics of didanosine in humans and in a model for acid-labile drugs, the pentagastrin-pretreated dog

1. A. proteinaceous inhibitor for S-adenosyl-L-methionine (AdoMet)-dependent transmethylation reactions has been purified to apparent homogeneity from rat liver cytosolic fraction. 2. The peptide was made up of 29 amino acid residues with a molecular weight of 2,584. Glycine accounted for 52% of the total amino acids. 3. Employing AdoMet: protein-carboxyl O-methyltransferase (Protein methylase II) and bovine serum gamma-globulin as in vitro substrate, the mode of inhibition was found to be non-competitive with Ki value of 1.9 x 10(-8) M. 4. When the inhibitor was present in the reaction mixture together with S-adenosyl-L-homocysteine (AdoHcy), which is a competitive inhibitor for AdoMet, the extent of inhibition exceeded that exerted by each individual inhibitor alone, suggesting that the sites of the inhibitors on the enzyme molecule are different. 5. Almost a stoichiometric relationship exists between the enzyme and the inhibitor molecule, the ratio being approx one.     

Unusual cloacal exstrophy in a male neonate with a normal penis and urethra

El mouse is a mutant which has epileptic convulsions after tossing-up stimulations and has a hippocampal dysfunction. Platelet-derived growth factor B-chain homodimer has been reported to be a trophic factor of hippocampal neurons. We found that a recombinant PDGF-BB suppressed the convulsions of El mice in a dose-dependent manner. Furthermore, thrombin-treated mice manifested no convulsions, but thrombin receptor activating peptide-treated ones had convulsions. These findings suggest that an abnormality in PDGF-BB release may make El mice susceptible to tonic-clonic convulsions.     

Calcium-contraction relationship in rat mesenteric arterial smooth muscle. Effects of exogenous and neurogenic noradrenaline

First, we determined the cerebral localization of reduced glutathione (GSH) in normal mice by means of autoradiography using 99mTc-meso-hexamethyl propylene oxime. A highly specific localization of GSH in the cerebellum and hippocampus was observed. Secondly, we measured the elevation of GSH level in the brain after low-dose gamma-irradiation. The cerebral GSH levels increased soon after irradiation with 50 cGy of gamma-rays, reaching a maximum at 3 h post-treatment, then remaining significantly higher than that of the non-irradiated control until 12 h and returning to the control level by 24 h. Thirdly, we examined the induction of the activities and the mRNAs of proteins involved in the synthesis and regeneration of GSH in the brain of mice subjected to low-dose gamma-ray irradiation. The level of mRNA for gamma-glutamylcysteine synthetase was significantly increased at 0.5 h, and remained high until 2 h post-irradiation (50 cGy). The level was transiently lowered to the non-irradiated control level at 3 h and slightly increased again after 6 h post-irradiation. gamma-Glutamylcysteine synthetase activity was significantly increased 3 h after irradiation, and remained high up to 24 h post-irradiation. As for glutathione reductase, the mRNA level was increased at 0.5 h, and peaked strongly at 2 h, while the enzyme activity was significantly increased at 6 h after irradiation, and continued to increase up to 24 h. The level of mRNA for thioredoxin, which contributes to GSH biosynthesis by supplying cysteine to the de novo pathway, peaked between 0.5 h and 2 h post-irradiation, and rapidly declined thereafter. The content of thioredoxin showed a transient decrease immediately after irradiation, but was then remarkably elevated, reaching a maximum at 3 h, and thereafter declining sharply. These results indicate that the increase in endogenous GSH in mouse brain soon after low-dose gamma-ray irradiation is a consequence of the induction of GSH synthesis-related proteins and occurs via both the de novo synthesis and the regeneration pathways.     

Aristeromycin-5'-carboxaldehyde: a potent inhibitor of S-adenosyl-L-homocysteine hydrolase

In an earlier study, Liu et al. (Bioorg. Med. Chem. Lett. 1992, 2, 1741-1744) showed that both the E and Z isomers of 4',5'-didehydro-5'-fluoroaristeromycin were very potent irreversible inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase. However, it was unclear from a mechanistic standpoint whether these vinyl fluorides were themselves type-I mechanism-based inhibitors causing reduction of enzyme-bound NAD+ or whether they were prodrug for aristeromycin-5'-carboxaldehyde, which was the ultimate type-I inhibitor. To elucidate this mechanism of enzyme inhibition, (4'S)- and (4'R)-aristeromycin-5'-carboxaldehydes (1a,b) were synthesized in this study and shown to be potent type-I mechanism-based inhibitors of AdoHcy hydrolase with k2/Ki values of 4.4 x 10(6) and 8.2 x 10(4)M-1min-1, respectively. However, Using 19F NMR and HPLC, it was shown that (4'S)-4,5'-dedehydro-5'-fluoraristeromycin in the presence of AdoHcy hydrolase did not release fluoride ion or generate aristeromycin-5'-carboxaldehyde (1a,b). These results suggest that the E and Z isomers of 4',5'-didehydro-5'-fluoroaristeromycin are inactivating AdoHcy hydrolase by directly reducing NAD+ to NADH and not using the hydrolytic activity of the enzyme to generate aristeromycin-5'-carboxaldehyde.     

Tissue plasminogen activator mRNA expression in granule neurons coincides with their migration in the developing cerebellum

Tissue plasminogen activator activity in the developing cerebellum, as quantified by zymography of cerebellar homogenates from embryonic day (E) 17 to adult mice, shows a peak of activity at postnatal day (P) 7, followed by a steady 75% decrease into adulthood. Northern blot analysis reveals a similar pattern for tissue plasminogen activator mRNA levels, which are low at E17 but increase dramatically, reaching their highest levels of specific mRNA/micrograms RNA in P1-P7 mice and declining about threefold in the adult mouse. In situ hybridization of whole mouse brain sections with a tissue plasminogen activator antisense cRNA probe shows pronounce reactivity in the cerebellum. Although some binding is associated with the cerebellar meninges, the external granule layer is devoid of tissue plasminogen activator mRNA at all ages. However, highly labeled elongated cells, which also bind antibody to neuronal nuclear antigen and are adjacent to Bergmann glial fibers (i.e., migrating granule neurons), are readily visible throughout the molecular and Purkinje layers at P7 and P14. In the adult mouse cerebellum, tissue plasminogen activator mRNA labeling is restricted to cells in the Purkinje/internal granule layers. Thus, tissue plasminogen activator gene expression is induced as granule neurons leave the external granule layer and begin their inward migration.     

Cyclin E2: a novel CDK2 partner in the late G1 and S phases of the mammalian cell cycle

We report here the cloning and characterization of human and mouse cyclin E2, which define a new subfamily within the vertebrate E-type cyclins, while all previously identified family-members belong to the cyclin El subfamily. Cyclin E2/CKD2 and cyclin E/CDK2 complexes phosphorylate histone H1 in vitro with similar specific activities and both are inhibited by p27Kip1. Cyclin E2 mRNA levels in human cells oscillate throughout the cell cycle and peak at the G1/S boundary, in parallel with the cyclin E mRNA. In cells, cyclin E2 is complexed with CDK2, p27 and p21. Like cyclin E, cyclin E2 is an unstable protein in vivo and is stabilized by proteasome inhibitors. Cyclin E2-associated kinase activity rises in late G1 and peaks very close to cyclin E activity. In two malignantly transformed cell lines, cyclin E2 activity is sustained throughout S phase, while cyclin E activity has already declined and cyclin A activity is only beginning to rise. We speculate that cyclin E2 is not simply redundant with cyclin E, but may regulate distinct rate-limiting pathway(s) in G1-S control.     

Selective effects of the endogenous cannabinoid arachidonylethanolamide (anandamide) on regional cerebral blood flow in the rat

Chronic focal epilepsy is associated with synaptic plasticity and growth of new connections. Brain-derived neurotrophic factor (BDNF) is associated with each of these processes in normal brain and shows acute up-regulation in models of generalized epilepsy. Here, using an experimental model of focal epilepsy, we show persistent up-regulation of BDNF mRNA, independent of that of other growth factors, in association with the development and persistence of chronic seizures. In situ hybridization histochemistry revealed that rats perfused within 2-3 days after seizure onset had widespread increases in BDNF mRNA levels in the neocortex. Rats perfused at later times, however, showed focal up-regulation of BDNF mRNA at the injection site and down-regulation in a surrounding cortical zone. Nerve growth factor and neurotrophin-3 mRNAs were not significantly altered. These reciprocal changes in BDNF gene expression in the epileptic focus and the cortical surround may contribute to plastic changes in epileptic neuronal circuits that accompany the transition from acute to chronic epilepsy. BDNF down-regulation in the surround is likely to be associated with the inhibitory surround that hampers seizure spread, but facilitates the persistence of a chronic epileptic focus.     

Up-regulation of rho A and rho-kinase mRNAs in the rat myometrium during pregnancy

We have recently demonstrated that some tenascin (TN) gene-knockout mice display abnormal behaviors, and that these abnormal behaviors stem from a low level of dopamine transmission in the brain. In the present study, we elucidated that tyrosine hydroxylase (TH) activity in the frontal cortex, striatum, and hippocampus of TN-knockout mice which showed abnormal behavior was significantly decreased. Also, the TH mRNA level of the midbrain was decreased by 43% in these animals compared with values for wild-type mice. These results suggest that the low dopamine turnover rate in some areas of the brain of TN-knockout mice accompanied by motor defects is due, at least in part, to the reduction in TH activity caused by diminished TH mRNA expression, and that TN-knockout mice exhibit abnormal behaviors in the presence of low levels of TH-gene expression.     

Enhanced production of glomerular extracellular matrix in a new mouse strain of high serum IgA ddY mice

To investigate the relationship between high serum levels of IgA and glomerular lesions, selective mating was performed in high serum IgA ddY mice, a murine model of spontaneously developing mesangioproliferative glomerulonephritis mimicking human IgA nephropathy. The selection and mating of high IgA ddY mice were accomplished when the mice were three to four months old. In the 12th generation of high IgA ddY (HIGA) mice, significantly higher levels of serum IgA from 10 age weeks to 60 weeks (P < 0.0002 to 0.0001) were observed in comparison with BALB/c mice. Relatively high proteinuria was observed at 40 weeks of age, although hematuria was consistently negative. Microscopic observations of renal tissue disclosed a marked glomerular mesangial matrix increase and a reduction of cell proliferation with age by both semiquantitative and morphometric analyses with moderate tubulointerstitial damage. These mesangial matrices were stained markedly by antisera for collagen type IV and by fibronectin, but not by collagen type I. Localization of TGF-beta protein was also detected in the mesangium of the HIGA mice. The positive mesangial IgA deposition was maintained consistently by this mating procedure and became more marked with age. Size analysis of IgA from ten pooled HIGA mice aged 50 to 60 weeks revealed dominant polymeric IgA in sera and dimeric IgA in glomerular eluates. Clonal analysis of serum IgA disclosed heterogeneous spectrotypes in a wide pH range (4.5 to 6.5), in contrast to very limited spectrotypes in the acidic pH range (4.5 to 5.2) of IgA in the glomerular eluates from these mice. The analyses of retroviral gp70 antigen involvement in the HIGA mice disclosed a significant increase of serum levels of gp70 anti-gp70 immune complexes with age, with no relationship to the severity of glomerular gp70 deposition. Northern blot analysis of renal tissue revealed markedly high mRNA expression of collagen type I, IV, fibronectin and TGF-beta even in 10-week-old HIGA mice in comparison with BALB/c mice. The expression became more significant in 60-week-old animals. The genetic background required to induce the expansion of IgA-producing B-cell clones is suggested to be closely related to the increased gene expression of TGF-beta, which induces enhanced glomerular extracellular matrix (especially fibronectin) accumulation in HIGA mice, being possibly mediated by the mesangial deposition of dimeric and highly acidic IgA. This newly established strain may provide a model for investigating the relationship between progressive glomerular sclerotic lesions and the induction of pathogenic IgA in human IgA nephropathy.