Involvement of CCAAT/enhancer-binding protein in regulation of the rat serine:pyruvate/alanine:glyoxylate aminotransferase gene expression.

In the rat liver, transcription of the serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) gene occurs from two sites, +1 and +66, in exon 1, resulting in the formation of two mRNAs, one for a precursor of mitochondrial SPT/AGT and the other for peroxisomal SPT/AGT, respectively. In this study, we attempted to characterize the downstream promoter responsible for generation of peroxisomal SPT/AGT. The minimal downstream promoter was confined to the +21-+90 region. We demonstrated that C/EBPalpha and C/EBPbeta bound around the downstream start site (+66) contribute to the promoter activity. The downstream promoter activity is also regulated positively by a short inverted repeat, located 20-30 bp upstream of the downstream start site, through a protein factor(s) bound to this region. On the other hand, the sequence just downstream of the start site may negatively regulate the promoter activity.

Store depletion by caffeine/ryanodine activates capacitative Ca(2+) entry in nonexcitable A549 cells.

Capacitative Ca(2+) entry is essential for refilling intracellular Ca(2+) stores and is thought to be regulated primarily by inositol 1, 4,5-trisphosphate (IP(3))-sensitive stores in nonexcitable cells. In nonexcitable A549 cells, the application of caffeine or ryanodine induces Ca(2+) release in the absence of extracellular Ca(2+) similar to that induced by thapsigargin (Tg), and Ca(2+) entry occurs upon the readdition of extracellular Ca(2+). The channels thus activated are also permeable to Mn(2+). The channels responsible for this effect appear to be activated by the depletion of caffeine/ryanodine-sensitive stores per se, as evidenced by the activation even in the absence of increased intracellular Ca(2+) concentration. Tg pretreatment abrogates the response to caffeine/ryanodine, whereas Tg application subsequent to caffeine/ryanodine treatment induces further Ca(2+) release. The response to caffeine/ryanodine is also abolished by initial ATP application, whereas ATP added subsequent to caffeine/ryanodine induces additional Ca(2+) release. RT-PCR analyses showed the expression of a type 1 ryanodine receptor, two human homologues of transient receptor potential protein (hTrp1 and hTrp6), as well as all three types of the IP(3) receptor. These results suggest that in A549 cells, (i) capacitative Ca(2+) entry can also be regulated by caffeine/ryanodine-sensitive stores, and (ii) the RyR-gated stores interact functionally with those sensitive to IP(3), probably via Ca(2+)-induced Ca(2+) release.

Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells.

ATP induced a biphasic increase in the intracellular Ca(2+)concentration ([Ca(2+)](i)), an initial spike, and a subsequent plateau in A549 cells. Erythromycin (EM) suppressed the ATP-induced [Ca(2+)](i) spike but only in the presence of extracellular calcium (Ca(2+)(o)). It was ineffective against ATP- and UTP-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation and UTP-induced [Ca(2+)](i) spike, implying that EM perturbs Ca(2+) influx from the extracellular space rather than Ca(2+)release from intracellular Ca(2+) stores via the G protein-phospholipase C-Ins(1,4,5)P(3) pathway. A verapamil-sensitive, KCl-induced increase in [Ca(2+)](i) and the Ca(2+) influx activated by Ca(2+) store depletion were insensitive to EM. 3'-O-(4-benzoylbenzoyl)-ATP evoked an Ca(2+)(o)-dependent [Ca(2+)](i) response even in the presence of verapamil or the absence of extracellular Na(+), and this response was almost completely abolished by EM pretreatment. RT-PCR analyses revealed that P2X(4) as well as P2Y(2), P2Y(4), and P2Y(6) are coexpressed in this cell line. These results suggest that in A549 cells 1) the coexpressed P2X(4) and P2Y(2)/P2Y(4) subtypes contribute to the ATP-induced [Ca(2+)](i) spike and 2) EM selectively inhibits Ca(2+) influx through the P2X channel. This action of EM may underlie its clinical efficacy in the treatment of airway inflammation.

A spectrophotometric method for the determination of glycolate in urine and plasma with glycolate oxidase.

An enzymatic assay was developed for the spectrophotometric determination of glycolate in urine and plasma. Glycolate was first converted to glyoxylate with glycolate oxidase, and the glyoxylate formed was condensed with phenylhydrazine. The glyoxylate phenylhydrazone formed was then oxidized with K(3)Fe(CN)(6) in the presence of excess phenylhydrazine, and A(515) of the resulting 1, 5-diphenylformazan was measured. Since glycolate oxidase also acts on glyoxylate and L-lactate, the incubation of samples with glycolate oxidase was carried out in 120-170 mM Tris-HCl (pH 8.3) to obtain glyoxylate as its adduct with Tris. The pyruvate formed from lactate was removed by subsequent brief incubation with alanine aminotransferase in the presence of L-glutamate, and alpha-ketoglutarate formed was converted back to L-glutamate by glutamate dehydrogenase and an NADPH generating system. Thus the specificity of the assay relies principally on the substrate specificity of glycolate oxidase, and high sensitivity is provided by the high absorbance of 1,5-diphenylformazan at 515-520 nm. Plasma was deproteinized with perchloric acid, and then neutralized with KOH. Plasma and urine samples were then incubated with approximately 5 mM phenylhydrazine, and then treated with stearate-deactivated activated charcoal to remove endogenous keto and aldehyde acids as their phenylhydrazones. The normal plasma glycolate and urinary glycolate/creatinine ratio for adults determined by this method are approximately 8 microM and approximately 0.036, respectively.

Oxalate synthesis in mammals: properties and subcellular distribution of serine:pyruvate/alanine:glyoxylate aminotransferase in the liver.

Primary hyperoxaluria Type 1 (PH1) is caused by a functional deficiency of a liver enzyme, serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), which catalyzes transamination between L-serine or l-alanine as an amino acid substrate and glyoxylate or pyruvate as an alpha-keto acid substrate. A high affinity for glyoxylate is a notable feature of this enzyme, suggesting a role in glyoxylate metabolism in vivo. Another conspicuous feature of SPT/AGT is its species-specific and food habit-dependent subcellular distribution. Thus, the enzyme is located in peroxisomes in herbivores and man, largely in mitochondria in carnivores, and in both the organelles in rodents. The mechanism of the species-specific dual organelle localization of SPT/AGT is either transcription of the gene from two different start sites or loss of the upstream translation initiation ATG codon by mutations. It appears that the mitochondrial versus peroxisomal distribution of SPT/AGT in different animal species is indispensable in meeting the metabolic needs caused by their respective food habits. As for the peroxisomal localization, glycolate is contained in plants much more than in animal tissues, and when ingested, it is converted to glyoxylate, an immediate precursor of oxalate, in liver peroxisomes. Therefore, peroxisomal localization of SPT/AGT may be indispensable for herbivores to convert the glyoxylate formed in peroxisomes into glycine in situ rather than forming oxalate. On the other hand, our recent studies showed that SPT/AGT contributed substantially to serine metabolism in rabbit, human, and dog livers; i.e., irrespective of its mitochondrial or peroxisomal localization. Thus, the mitochondrial localization of SPT/AGT was not a prerequisite for the metabolism of L-serine. Another source of glyoxylate is the metabolism of L-hydroxyproline, and in this case, the enzyme responsible for the glyoxylate formation has been reported to be a mitochondrial matrix enzyme. Collagen accounts for about 30% of total animal proteins and contains about 13% (w/w) hydroxyproline. It is therefore possible that both mitochondrial and peroxisomal SPT/AGT contribute to the metabolism of glyoxylate and serine, but the subcellular site for glyoxylate metabolism is different in herbivores and carnivores.

Primary hyperoxaluria type 1 in Japan.

Glyoxylate is an immediate precursor of oxalate, but in its metabolism the conversion into glycine catalyzed by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) appears to be the main route. When SPT/AGT is missing as in the case of primary hyperoxaluria type 1 (PH1) more glyoxylate is used for the oxalate production, resulting in calcium oxalate urolithiasis and finally systemic oxalosis. SPT/AGT is a unique enzyme of species-specific dual organelle localization; it is located largely in mitochondria in carnivores and entirely in peroxisomes in herbivores and man. For herbivores, the peroxisomal localization of SPT/AGT is indispensable to avoid massive production of oxalate, probably because liver peroxisomes are the main site of glyoxylate production from glycolate, and plants contain glycolate much more than animal tissues. Recently, we took charge of laboratory examination for 8 cases of primary hyperoxaluria in Japan, and felt that symptoms of some of the Japanese PH1 patients are apparently milder than those of Western patients. The reason of this is not clear, but from the above mentioned seemingly indispensable association of grass-eating with the peroxisomal localization of SPT/AGT it may be related, at least in part, to the food habit of Japanese, especially that of old generation, that they prefer boiled greens rather than frying or raw vegetables.

Peroxisomal and mitochondrial targeting of serine:pyruvate/alanine:glyoxylate aminotransferase in rat liver.

Serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide. In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, SPTp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.

A new 30-kDa ubiquitin-related SUMO-1 hydrolase from bovine brain.

SUMO-1 is a ubiquitin-like protein functioning as an important reversible protein modifier. To date there is no report on a SUMO-1 hydrolase/isopeptidase catalyzing the release of SUMO-1 from its precursor or SUMO-1-ligated proteins in mammalian tissues. Here we found multiple activities that cleave the SUMO-1 moiety from two model substrates, (125)I-SUMO-1-alphaNH-HSTVGSMHISPPEPESEEEEEHYC and/or GST-SUMO-1-(35)S-RanGAP1 conjugate, in bovine brain extracts. Of them, a major SUMO-1 C-terminal hydrolase had been partially purified by successive chromatographic operations. The enzyme had the ability to cleave SUMO-1 not only from its precursor but also from a SUMO-1-ligated RanGAP1 but did not exhibit any significant cleavage of the ubiquitin- and NEDD8-precursor. The activity of SUMO-1 hydrolase was almost completely inhibited by N-ethylmaleimide, but not by phenylmethanesulfonyl fluoride, EDTA, and ubiquitin-aldehyde known as a potent inhibitor of deubiquitinylating enzymes. Intriguingly, the apparent molecular mass of the isolated SUMO-1 hydrolase was approximately 30 kDa, which is significantly smaller than the recently identified yeast Smt3/SUMO-1 specific protease Ulp1. These results indicate that there are multiple SUMO-1 hydrolase/isopeptidases in mammalian cells and that the 30-kDa small SUMO-1 hydrolase plays a central role in processing of the SUMO-1-precursor.

Isolation and characterization of cytosolic and membrane-bound deubiquitinylating enzymes from bovine brain.

The deubiquitinylating enzymes (DUBs), that release free ubiquitin (Ub) from its precursors or ubiquitinylated proteins, are known to comprise of a large protein family in eukaryotes, but those in mammalian tissues remain largely unknown. Here we report the existence of unexpectedly large species of DUBs in both soluble and membrane-bound fractions of bovine brain, based on their ability to cleave (125)I-labeled Ub-fused alphaNH-MHISPPEPESEEEEEHYC (designated as Ub-PESTc). Two cytosolic enzymes, tentatively called sDUB-1 and sDUB-2, with molecular masses of about 30 kDa were purified to near homogeneity by Ub-Sepharose affinity chromatography. sDUB-1 and sDUB-2 corresponded to UCH-L3 and UCH-L1/PGP 9.5, respectively. Intriguingly, the particulate fraction of the brain homogenate was found to also contain strong activities against (125)I-Ub-PESTc, which can be solubilized by treatment with 5% n-heptyl-beta-D-thioglucoside and 1% Nonidet P-40, but not by washing with 1 M NaCl. From the solubilized material, two new 30-kDa, membranous DUBs (called mDUB-1 and mDUB-2) were purified to apparent homogeneity by Ub-Sepharose chromatography. Two other Ub-aldehyde sensitive DUBs, designated as mDUB-3 and mDUB-4, were also partially purified by conventional chromatographic operations. These mDUBs differed from each other in substrate specificity and exhibited different characteristics from the sDUBs, revealing that they are a new type of membrane-bound DUB. These results indicate the presence of divergent DUBs in mammalian brain, which may contribute to regulation of numerous pivotal cellular functions mediated by the covalent modification of Ub.

Participation of platelet-activating factor in the lipopolysaccharide-induced liver injury in partially hepatectomized rats.

Platelet-activating factor (PAF) has been shown to be an important mediator in the pathogenesis of lipopolysaccharide (LPS)-induced liver injury in regenerating rat livers. Both LPS and PAF activate nuclear factor-kappa B (NF-kappaB), a key transcription factor for tumor necrosis factor-alpha (TNF-alpha) and cytokine-induced neutrophil chemoattractant (CINC). The aim of this study is to investigate how PAF participates in the LPS-induced and NF-kappaB-mediated regulation of TNF-alpha and CINC in regenerating rat livers. LPS (1.5 mg/kg) was intravenously administered into 70% hepatectomized rats and sham-operated rats 48 hours postoperatively. LPS administration caused a high mortality rate, scattered necrosis in the liver with infiltration of CINC-positive neutrophils, and a continuous CINC messenger RNA up-regulation and activation of NF-kappaB in the liver only in hepatectomized rats. These phenomena were all effectively prevented by pretreatment and posttreatment with a PAF receptor antagonist, TCV-309. Hepatectomized rats showed NF-kappaB staining in hepatocytes, Kupffer cells, and neutrophils around necrosis 4 hours after the LPS injection, representing the activation of this factor in these cells. Based on these results, we propose that PAF contributes to continuous CINC up-regulation and NF-kappaB activation via accumulation and activation of neutrophils, and thereby is involved in LPS-induced liver injury in regenerating rat livers.

Acute portal hypertension increases ileal vulnerability to platelet-activating factor in rats.

BACKGROUND: Patients with portal hypertension can easily develop sepsis of enteric origin after suffering severe trauma and hemorrhagic shock. Platelet-activating factor (PAF) is one of the key mediators of such stress. The aim of this study was to investigate whether portal hypertension increases the vulnerability of the ileum to PAF. MATERIALS AND METHODS: Seven days after surgery, PAF (1.5 microg/kg) was intravenously injected into portal stenosis (PS) rats and sham-operated rats. The levels of tumor necrosis factor-alpha (TNF-alpha), cytokine-induced neutrophil chemoattractant (CINC), and endotoxin in portal plasma were determined. The levels of PAF receptor (PAFR), TNF-alpha, and CINC mRNA in the ileum were also investigated. RESULTS: After PAF administration, PS rats showed (1) significantly higher portal plasma levels of TNF-alpha, CINC, and endotoxin; (2) higher histological damage scores in the ileum; (3) more infiltrating neutrophils in the ileum; and (4) a significantly higher mortality rate than sham-operated rats (P < 0.01). However, PAFR mRNA levels were similar in the two groups. The CINC mRNA level in the ileum of PS rats was increased from 1 to 4 h after PAF administration, while that of the sham-operated rats was transiently increased at 1 h. CONCLUSIONS: Portal hypertension increases the vulnerability of the ileum to PAF. These findings suggest that conditions which causes PAF production may be dangerous in patients with portal hypertension.

Short-chain acyl-CoA-dependent production of oxalate from oxaloacetate by Burkholderia glumae, a plant pathogen which causes grain rot and seedling rot of rice via the oxalate production.

In Burkholderia glumae (formerly named Pseudomonas glumae), isolated as the causal agent of grain rot and seedling rot of rice, oxalate was produced from oxaloacetate in the presence of short-chain acyl-CoA such as acetyl-CoA and propionyl-CoA. Upon purification, the enzyme responsible was separated into two fractions (tentatively named fractions II and III), both of which were required for the acyl-CoA-dependent production of oxalate. In conjugation with the oxalate production from oxaloacetate catalyzed by fractions II and III, acetyl-CoA used as the acyl-CoA substrate was consumed and equivalent amounts of CoASH and acetoacetate were formed. The isotope incorporation pattern indicated that the two carbon atoms of oxalate are both derived from oxaloacetate, and among the four carbon atoms of acetoacetate two are from oxaloacetate and two from acetyl-CoA. When the reaction was carried out with fraction II alone, a decrease in acetyl-CoA and an equivalent level of net utilization of oxaloacetate were observed without appreciable formation of CoASH, acetoacetate or oxalate. It appears that in the oxalate production from oxaloacetate and acetyl-CoA, fraction II catalyzes condensation of the two substrates to form an intermediate which is split into oxalate and acetoacetate by fraction III being accompanied by the release of CoASH.

Flux of the L-serine metabolism in rat liver. The predominant contribution of serine dehydratase.

L-Serine metabolism in rat liver was investigated, focusing on the relative contributions of the three pathways, one initiated by L-serine dehydratase (SDH), another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Because serine hydroxymethyltransferase is responsible for the interconversion between serine and glycine, SDH, SPT/AGT, and GCS were considered to be the metabolic exits of the serine-glycine pool. In vitro, flux through SDH was predominant in both 24-h starved and glucagon-treated rats. Flux through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution under quasi-physiological conditions (1 mM L-serine and 0.25 mM pyruvate) was about (1)/(10) of that through SDH. Flux through GCS accounted for only several percent of the amount of L-serine metabolized. Relative contributions of SDH and SPT/AGT to gluconeogenesis from L-serine were evaluated in vivo based on the principle that 3H at the 3 position of L-serine is mostly removed in the SDH pathway, whereas it is largely retained in the SPT/AGT pathway. The results showed that SPT/AGT contributed only 10-20% even after the enhancement of its activity by glucagon. These results suggested that SDH is the major metabolic exit of L-serine in rat liver.

Flux of the L-serine metabolism in rabbit, human, and dog livers. Substantial contributions of both mitochondrial and peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase.

L-Serine metabolism in rabbit, dog, and human livers was investigated, focusing on the relative contributions of the three pathways, one initiated by serine dehydratase, another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Under quasi-physiological in vitro conditions (1 mM L-serine and 0.25 mM pyruvate), flux through serine dehydratase accounted for only traces, and that through SPT/AGT substantially contributed no matter whether the enzyme was located in peroxisomes (rabbit and human) or largely in mitochondria (dog). As for flux through serine hydroxymethyltransferase and GCS, the conversion of serine to glycine occurred fairly rapidly, followed by GCS-mediated slow decarboxylation of the accumulated glycine. The flux through GCS was relatively high in the dog and low in the rabbit, and only in the dog was it comparable with that through SPT/AGT. An in vivo experiment with L-[3-3H,14C]serine as the substrate indicated that in rabbit liver, gluconeogenesis from L-serine proceeds mainly via hydroxypyruvate. Because an important role in the conversion of glyoxylate to glycine has been assigned to peroxisomal SPT/AGT from the studies on primary hyperoxaluria type 1, these results suggest that SPT/AGT in this organelle plays dual roles in the metabolism of glyoxylate and serine.

Iron dependence of tryptophan hydroxylase activity in RBL2H3 cells and its manipulation by chelators.

Tryptophan hydroxylase requires Fe2+ for in vitro enzyme activity. In this study, the intracellular activity of tryptophan hydroxylase was assessed by applying 3-hydroxybenzylhydrazine (NSD-1015), an inhibitor of aromatic l-amino acid decarboxylase, to monolayer cultures of RBL2H3 cells, a serotonin producing mast cell line. The effect of manipulating intracellular 'free' iron levels on enzyme activity was analyzed by administration of iron chelators. Desferrioxamine (DFO) suppressed the intracellular enzyme activity. Salicylaldehyde isonicotinoyl hydrazone (SIH) also suppressed enzyme activity, but stimulated it when administered in the Fe-bound form. Hemin also stimulated enzyme activity, which progressively increased over several hours to more than sixfold the initial level. DFO and SIH inhibited the hemin stimulatory effect when administered simultaneously with hemin. Both suppression and stimulation with these chelators took place without a significant decrease or increase in the amount of enzyme. These results indicate that there was an inadequate supply of Fe2+ in the cells to support full activity of tryptophan hydroxylase.

IkappaBalpha ubiquitination is catalyzed by an SCF-like complex containing Skp1, cullin-1, and two F-box/WD40-repeat proteins, betaTrCP1 and betaTrCP2.

Destruction of the transcriptional inhibitor IkappaB by the ubiquitin (Ub) system is required for signal-dependent activation of the multifunctional transcriptional factor NF-kappaB, but details of this ubiquitination are largely unknown. We report here that the IkappaBalpha-ubiquitin ligase (IkappaBalpha-E3) is an SCF-like complex containing Skp1, cullin-1, and two homologous F-box/WD40-repeat proteins, betaTrCP1 and betaTrCP2. Intriguingly, all these components are cooperatively recruited to bind to a phosphorylated IkappaBalpha (pIkappaBalpha) produced by tumor necrosis factor-alpha (TNF-alpha) stimulation. IkappaBalpha-E3 bound to pIkappaBalpha catalyzed in vitro ubiquitination of pIkappaBalpha in the presence of ATP, Ub, and E1-activating and E2-conjugating enzymes. Forced expression of betaTrCP1 and betaTrCP2 resulted in dramatic augmentation of the in vitro polyubiquitination activity of IkappaBalpha-E3. These results indicate that the long-sought IkappaBalpha-E3 is an SCF-like complex consisting of multiple proteins which are coordinately assembled during phosphorylation of IkappaBalpha in response to external signals.

Identification and chromosomal assignment of USP1, a novel gene encoding a human ubiquitin-specific protease.

We have cloned a novel gene encoding a human ubiquitin-specific protease (USP1). The product, which consists of 785 amino acids with a deduced molecular mass of 88.2 kDa, possesses His and Cys domains that are highly conserved in all members of the ubiquitin-specific processing (UBP) family of proteases. Recombinant USP1 protein showed genuine UBP activity, correctly cleaving Ub-beta-galactosidase to produce ubiquitin and beta-galactosidase. Chromosomal mapping by fluorescence in situ hybridization and radiation hybrid analyses localized the USP1 gene to the p31.3-p32.1 band of chromosome 1. As losses of heterozygosity or amplifications have been observed in the distal region of the short arm of chromosome 1 in some neuroblastomas, breast cancers, and pancreatic adenocarcinomas, the USP1 gene may be a candidate for either the tumor-suppressive or the oncogenic activities associated with that chromosomal region.

A case of late onset primary hyperoxaluria type I (PH-I) presented with black liver.

A 63-year-old woman who had received hemodialysis therapy since she fell acute on chronic renal failure 4 years ago presented with multiple joint pain. Nephrocalcinosis was not detected by abdominal X-ray when hemodialysis therapy was initiated. Laboratory testing showed azotemia, anemia, hypoproteinemia and mild liver dysfunction but no liver cirrhosis. Biopsied bone tissue demonstrated numerous calcium oxalate crystal depositions. Laparoscopy revealed black liver in macroscopic view. Histological studies showed numerous lipofuscin-like dark brown granules were deposited in hepatocytes. The activity of alanine : glyoxylate aminotransferase (AGT) was less than 0.1 U/g in biopsied patient's liver tissue. Generally, clinical symptoms demonstrated by Japanese primary hyperoxaluria type I (PH-I) patients are milder than those of European patients. Some PH-I patients may successfully avoid urinary tract calcification unless they fall into oliguria by some other causes. The lipofuscin granules are most likely the source of the dark color. Massive deposition of the lipofuscin granules indicated that the duration of the liver metabolic abnormality had lasted for long time. Thus, black liver may be related to a mild form of PH-I.

Morphological analysis of renal cell culture models of calcium phosphate stone formation.

Cell culture models of calcium phosphate renal stone formation were established using the MDCK cell line. Renal microliths were detected within pseudocysts in three-dimensional soft agar cultures, and were also observed in the basal region of cells lining the cell sheet, and immediately beneath domes or blisters in monolayers and collagen gel cultures. Light and scanning electron microscopy indicated that these microliths had a similar lamellated and spherical appearance to those in humans. These microliths were first detected microscopically after 21 days of culture, and were found to be composed of calcium phosphate by X-ray and micro-infrared spectroscopic analyses. These culture models may provide a powerful new tool to study the pathogenesis of renal stone diseases and/or calcium phosphate stone formation in humans and animals.

Induction by peroxisome proliferators and triiodothyronine of serine:pyruvate/alanine:glyoxylate aminotransferase of rat liver.

In rat liver, a single serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) gene is transcribed from two transcription initiation sites. Transcription from the upstream site generates the mRNA encoding the precursor for mitochondrial SPT (pSPTm) and is markedly enhanced by the administration of glucagon or cAMP. In this report we show the increase in the downstream transcript, the peroxisomal SPT (SPTp) mRNA, caused by peroxisome proliferators and triiodothyronine (T3). In the case of T3, the pSPTm mRNA was also increased 72 h after a single administration of the hormone in addition to an earlier increase in SPTp mRNA.