9-Cis-retinoic acid exhibits antifibrotic activity via the induction of cyclooxygenase-2 expression and prostaglandin E2 production in scleroderma fibroblasts.

BACKGROUND: The pathogenesis of scleroderma (SSc) is not fully understood, and there is no effective treatment for this chronic disease. Retinoic acid (RA) can modulate connective tissue metabolism, exhibit antifibrotic activity and improve the clinical symptoms of patients with SSc. However, the mechanisms by which RA elicits its antifibrotic actions remain to be determined. OBJECTIVE: To elucidate the underlying mechanisms by which retinoids exert beneficial effects on SSc. METHODS: Cultured skin fibroblasts from patients with SSc were treated with retinoids (9-cis-, 13-cis- and all-trans-retinoic acid) and their effect on the expression of cyclooxygenase (COX)-2, connective tissue growth factor (CTGF) and type I and III collagen and on the production of PGE(2) was examined. COX-2 expression was analysed by western immunoblotting, PGE(2) production by enzyme immunoassay and CTGF expression, and type I and III collagen expression by reverse transcriptase PCR and western immunoblotting. RESULTS: In cultured SSc fibroblasts, 9-cis-RA significantly increased COX-2 protein expression and PGE(2) production and inhibited the expression of CTGF and type I and III collagen. We further found that expression of CTGF and of type I and III collagen mRNA was inhibited by exogenous PGE(2) in SSc fibroblasts. CONCLUSION: In vitro, 9-cis-RA induced COX-2 expression and PGE(2) production in SSc fibroblasts and PGE(2) downregulated CTGF expression, leading to the inhibition of type I and III collagen synthesis. Our results indicate that the clinical effects of 9-cis-RA on SSc are, at least in part, attributable to the induction of PGE(2) and the subsequent suppression of CTGF expression that results in the blockade of collagenogenesis.

Immunoelectron-microscopic detection of globotriaosylceramide accumulated in the skin of patients with Fabry disease.

BACKGROUND: Fabry disease is characterized by the systemic accumulation of glycosphingolipids, particularly in the lysosomes of vascular endothelial cells of most organs due to the deficient activity of alpha-galactosidase A. The major glycolipid accumulated in tissue is globotriaosylceramide (GL-3). To date, no direct detection of GL-3 by immunoelectron microscopy has been reported. OBJECTIVES: To examine whether GL-3 is accumulated exclusively in lysosomes of cutaneous cells using an anti-GL-3 monoclonal antibody (mAb) and immunoelectron microscopy. METHODS: Skin specimens from seven patients with Fabry disease were examined immunohistochemically by light and electron microscopy using an anti-GL-3 mAb. RESULTS: By light microscopy, the cytoplasm of vascular endothelial cells, eccrine gland cells, and perineurium was stained with mouse anti-GL-3 antibody. Electron microscopically, positive signals for GL-3 were limited to dilated lysosomes in the cytoplasm of endothelial cells, pericytes, eccrine gland cells, dermal fibroblasts and perineurium. CONCLUSIONS: Our results demonstrate that the cytoplasmic deposit in Fabry disease was GL-3 and the accumulated GL-3 was localized essentially to lysosomes.

Morphological and biochemical studies of human beta-mannosidosis: identification of a novel beta-mannosidase gene mutation.

BACKGROUND: There are seven well-known lysosomal storage diseases that produce angiokeratoma corporis diffusum clinically. beta-Mannosidosis (MANB1; OMIM248510), first reported in humans in 1986, is a rare hereditary lysosomal storage disease caused by a deficiency of the enzyme beta-mannosidase. Since then, 13 cases of beta-mannosidase deficiency in ten families have been described. A human beta-mannosidase mutation has been reported only by Alkhayat et al. in 1998. OBJECTIVES: To clarify its pathogenesis we did electron microscopic, biochemical and molecular biological investigations of a Japanese patient with beta-mannosidosis. METHODS: Ultrastructural analyses, enzyme assays, cell culture and mRNA and genomic DNA were sequenced to find mutations in the beta-mannosidase gene. RESULTS: Electron microscopy of skin biopsy specimens from the patient showed cytoplasmic vacuolation of lysosomes in blood and lymph vessels, endothelial cells, fibroblasts, secretory portions of eccrine sweat glands, neural cells and basal keratinocytes in the epidermis. This vacuolation was also observed in cultured keratinocytes and fibroblasts. Assays of seven enzyme activities in plasma and cultured skin fibroblasts showed a marked decrease of beta-mannosidase activity. Sequencing the beta-mannosidase cDNA revealed a four-base (ATAA) insertion between exons 7 and 8, resulting in a frameshift at codon 321 and termination at codon 325. Analysis of the patient's genomic DNA revealed a novel homozygous A(+1)-->G splice site mutation in intron 7. CONCLUSIONS: To our knowledge, this is the first case of beta-mannosidosis reported in Japan and the second report in which a gene mutation is identified. The biological importance of beta-mannose moieties in glycoproteins in basal keratinocytes is suggested.

Genetic and physiological analysis of branched-chain alcohols and isoamyl acetate production in Saccharomyces cerevisiae.

Branched-chain alcohols, such as isoamyl alcohol and isobutanol, and isoamyl acetate are important flavor components of yeast-fermented alcoholic beverages. Analysis of a null mutant of the BAT2 gene encoding cytosolic branched-chain amino acid aminotransferase, and a transformant with multi-copy plasmids containing the BAT2 gene showed that the BAT2 gene product plays an important role in the production of branched-chain alcohols and isoamyl acetate. Fermentation tests using the bat2 null mutant transformed with multi-copy plasmids carrying the ATF1 gene, which encodes alcohol acetyltransferase, indicated that modified expression of BAT2 and ATF1 genes could significantly alter the proportion of branched-chain alcohols and isoamyl acetate synthesized. Furthermore, fermentation tests using different ratios of nitrogen source and RNA blot analyses demonstrated that transcription of L-leucine biosynthetic ( LEU) and BAT genes is co-regulated by nitrogen source, that production of isoamyl alcohol depends on this transcription, and that ATF transcription increased with increased concentrations of nitrogen source. Our data suggest that changes in isoamyl alcohol production by nitrogen source are due to transcriptional co-regulation of LEU and BAT genes, and that production of isoamyl acetate is dependent on isoamyl alcohol production and ATF transcription.

A simple method for evaluating abnormal lengthening of the QT interval during the face immersion test.

The slope of the relation between the unadjusted QT interval and heart rate during the face immersion test has been reported to be useful as an index for predicting an abnormal lengthening of the QT interval for children with nonfamilial long QT syndrome. Our goals were to determine whether we can replace the slope of the QT/heart rate relation calculated from all data with that calculated from fewer data and to determine whether we can replace the slope with the corrected QT value by heart rate (QTc value) at the minimum heart rate. We studied 19 children with a prolonged QT interval and 54 control children by using statistical analysis. The slope calculated from the selected data points (at least four) was in agreement with the slope calculated from all data, and the relationship between the slope and the QTc value at the minimum heart rate showed a high correlation. It was determined that we can replace the slope calculated from all data with that calculated from at least four data points and replace the slope with the QTc value at the minimum heart rate as an index for predicting an abnormal lengthening of the QT interval.

Post-pubertal mucinous cystadenoma of the pancreas.

A 14-year-old patient with a mucinous cystadenoma of the pancreas (MCAP) is presented. She presented with a palpable left-sided abdominal mass and underwent a left hemipancreatectomy. MCAP occurs mostly in middle-aged women, and no post-pubertal cases have been reported to date in the English literature.

A new case of alpha-N-acetylgalactosaminidase deficiency with angiokeratoma corporis diffusum, with Ménière's syndrome and without mental retardation.

alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency is a rare hereditary lysosomal storage disease, and only three alpha-NAGA-deficient patients with angiokeratoma corporis diffusum (Kanzaki) have been described. We report a further case in a 47-year-old Japanese woman, the product of a consanguineous marriage. The remarkable findings in this patient were her normal intelligence, Ménière's syndrome, disturbance of peripheral sensory nerves, hearing loss and cardiac hypertrophy. alpha-NAGA enzyme activity in her plasma was 0.77% of the normal value. Other enzyme activities, such as alpha-galactosidase, beta-galactosidase, alpha-L-fucosidase, beta-mannosidase and aspartylglucosaminidase, were within normal limits. A large quantity of amino acid O-glycans was detected in her urine. Gene analysis revealed a novel point mutation (G-->A transition) at nucleotide 11018 (986 in the cDNA) resulting in an Arg-329-Gln substitution. Kanzaki disease has the same enzyme defect as Schindler disease, but the manifestations are quite different.

Risk factors for persistence of pulmonary arterial branch stenosis in neonates and young infants.

BACKGROUND: Pulmonary arterial branch stenosis (PBS) in neonates is considered to be transient. However, PBS has been found not only in neonates, but also in young infants. Among these patients, we encountered several patients whose PBS was still present after the age of 1 year. METHODS: To clarify the natural history of PBS in neonates and young infants, we retrospectively reviewed the records of 103 patients diagnosed with PBS in the neonatal period and early infancy. RESULTS: The PBS findings were improved in all patients. Pulmonary arterial branch stenosis disappeared in 94 patients by the age of 1 year (group A), but persisted after I year of age in nine patients (group B). Group B patients had a significantly lower mean birth weight and greater deltavelocity (the difference in the peak flow velocity between the main pulmonary artery and stenotic branch) at diagnosis than group A patients. Incidences of low birth weight (< 2,500 g) and deltavelocity above 1.2 m/s were significantly higher in group B than group A patients. There was no significant difference in the frequency of premature infants (gestational age < 36 weeks) between the groups, suggesting that intrauterine growth retardation may be related to developmental abnormalities of the pulmonary arterial branch. CONCLUSIONS: All cases of PBS in neonates and young infants were improved. However, PBS persisted in some patients after the age of 1 year. Low birth weight and deltavelocity above 1.2 m/s are risk factors for persistent PBS. Pulmonary arterial branch stenosis was not present after the age of 1 year in 62 of 63 patients without either of these risk factors.

Pyruvate decarboxylase encoded by the PDC1 gene contributes, at least partially, to the decarboxylation of alpha-ketoisocaproate for isoamyl alcohol formation in Saccharomyces cerevisiae.

Isoamyl alcohol is an important flavor component of yeast-fermented alcoholic beverages. To identify the enzyme and gene involved in the decarboxylation of alpha-ketoisocaproate (alpha-KIC) for isoamyl alcohol formation, the enzyme was partially purified and analyzed by mass spectrometry. The pyruvate decarboxylase encoded by the PDC1 gene was considered a likely candidate enzyme. Genetic analysis showed that the activity of alpha-KIC decarboxylase and production of isoamyl alcohol partially decreased in a pdc1 null mutant and increased in a transformant with a multi-copy plasmid carrying the PDC1 gene. These results indicate that pyruvate decarboxylase encoded by the PDC1 gene contributes, at least partially, to the decarboxylation of alpha-KIC for isoamyl alcohol formation.

Cervico-mediastinal bronchogenic cyst occurring in the prenatal period: report of a case.

We experienced a case of cervico-mediastinal bronchogenic cyst in which a cervical cystic mass was detected by prenatal ultrasonography. On prenatal ultrasound, a unilocular, well-defined and hypoechoic mass was detected in the fetal neck. The baby was born by a normal vaginal delivery at 40 weeks of gestation, and had no respiratory distress. Radiological investigations demonstrated a cyst in the cervico-mediastinal region, which displaced the trachea to the left. At the age of 32 days, an elective resection was easily performed through a right inferior collar incision after first aspirating the contents of the cyst. Prenatal sonography showing abnormal findings is effective for identifying cysts in the perinatal period and allows for the timely resection of such cysts before respiratory distress occurs.

Analyses of the transglutaminase 1 gene mutation and ultrastructural characteristics in a Japanese patient with lamellar ichthyosis.

We described a Japanese female with lamellar ichthyosis whose transglutaminase 1 gene (TGM1 gene) was mutated. DNA sequence analysis revealed that the patient had a homozygous mutation, i.e. a point mutation from G to A at nucleotide 1494 resulting in the substitution of glycine for arginine at codon 143. Her mother was heterozygous for this mutation. In situ transglutaminase assay in the patient's skin showed loss of enzyme activity. Ultrastructural examination revealed incomplete formation of cornified cell envelopes and electron-dense materials adjacent to plasma membranes. These results suggest that defective transglutaminase activity caused by homozygous TGM1 gene mutation (G143R) results in disruption of cornified envelope assembly and the clinical phenotype of lamellar ichthyosis.

The expression of cadherins in human neuroblastoma cell lines and clinical tumors.

Cadherins are Ca(2+)-dependent cell-cell adhesion molecules which play crucial roles in the cell-cell interactions during development, tumorigenesis and metastasis. The absence of N (neural)-cadherin is correlated with the onset of neural crest migration and its reappearance is correlated with the cessation of migration and precedes gangliogenesis. We investigated the expression of cadherins including N-cadherin in five cell lines and eleven clinical specimens of human neuroblastomas, which originated from neural crest cells. We found that three of the neuroblastoma cell lines and all the clinical specimens were positive for the expression of the N-cadherin protein. The other two neuroblastoma cell lines were negative for the expression suggesting they originated from migrating neural crest cells. All these cell lines and clinical samples expressed either cadherin-6, cadherin-11 or both, i.e. cadherins expressed on neural crest cells, supporting their neural crest origin.

A malignant rhabdoid tumor of the kidney occurring concurrently with a brain tumor: report of a case.

Malignant rhabdoid tumor of the kidney (MRTK) is one of the most lethal neoplasms to occur in young infants. Cases of MRTK accompanying an embryonal tumor in the central nervous system have occasionally been described. We present herein an interesting case of MRTK that was clinically diagnosed preoperatively. A male infant aged 6 months with both a midline brain tumor and a renal neoplasm was transferred to our institution. Although roentgenographic evaluation suggested that the renal lesion was a Wilms' tumor, midkine (MK), a growth and differentiation factor characteristically present in the urine of patients with Wilms' tumor, was not detected. A preoperative diagnosis of MRTK was established based on the lack of urinary MK in addition to the typical clinical features of the young age and the concurrent brain tumor.

Computed tomographic epidurography: an aid to understanding deformation of the lumbar dural sac by epidural injections.

Local anaesthetics injected into the epidural space may deform the dural sac to a variable degree, thereby contributing to variability in the extent of the block. We investigated deformation of the lumbar dural sac after injection into the lumbar epidural space. The subjects were 26 patients with low-back pain who underwent lumbar epidurography and computed tomographic (CT) epidurography, of whom seven also underwent myelography and computed tomographic myelography. The epidural space was entered via the sacral hiatus in 24 patients and through the L5/S1 interspace in two patients. Ten millilitres of local anaesthetic was then injected into the epidural space followed by 20 mL of contrast medium. Computed tomographic epidurography was undertaken approximately 30-min after the epidural injection at the mid-vertebral and mid-discal levels from the first lumbar through to the first sacral vertebrae. The dural sac usually showed an oval or hexagonal shape on the transverse views at the first and second lumbar vertebral levels, and the shape of an inverted triangle below the level of the third lumbar vertebra. A median line of translucency was also observed on the posteroanterior epidurographic view in 25 of the 26 patients. This line was though to be a manifestation of the dural deformation to the inverted triangle. Dural sac deformation usually shows a specific pattern, although there are individual variations. Dural deformability is an important consideration in any analysis of the spread of epidural block or of the changes of epidural pressure after epidural injection of local anaesthetics.

Aberrations of ammonia metabolism in ornithine carbamoyltransferase-deficient spf-ash mice and their prevention by treatment with urea cycle intermediate amino acids and an ornithine aminotransferase inactivator.

Sparse fur with abnormal skin and hair (spf-ash) mice are deficient in ornithine carbamoyltransferase (OCT) activity, but their OCT protein is kinetically normal. We administered ammonium chloride to spf-ash mice, in order to analyze ammonia metabolism and to find a rationale for the therapy of OCT deficiency. Ammonia concentration in the liver of spf-ash mice increased to a level much higher than in the control. Ammonium chloride injection caused an increase in ornithine (Orn) 5 min after injection and an increase in the sum of Orn, citrulline (Cit) and arginine (Arg) for at least 15 min in the liver of control mice, but no increase in Orn, Cit and Arg in the liver of spf-ash mice. Treatment of spf-ash mice with Arg 5-20 min prior to the injection of ammonium chloride kept the hepatic ammonia concentration at a level comparable to that without the load. A significant reciprocal relationship between ammonia and Orn concentrations in the liver of spf-ash mice 5 min after an ammonium chloride load with or without Arg strongly suggests that ammonia disposal is dependent on the supply of Orn. In spf-ash mice loaded with tryptone as a nitrogen source, Arg supplementation showed a dramatic decrease in urinary orotic acid excretion in a dose-dependent manner. Similar effects were observed with Cit and Orn at the same dose, and a long-lasting effect with an ornithine aminotransferase inactivator, 5-(fluoromethyl)ornithine, at a much lower dose. The rate of urea formation in liver perfused with ammonium chloride was lower in spf-ash mice than in controls, but with the addition of Orn to the medium it increased to a similar level in control and spf-ash mice. These results indicate that OCT is not saturated with Orn in vivo under physiological conditions and that the administration or enrichment of the urea cycle intermediate amino acids enhances the OCT reaction so that the ammonia metabolism of OCT-deficient spf-ash mice is at least partially normalized.

Regulation of metallothionein gene transcription. Identification of upstream regulatory elements and transcription factors responsible for cell-specific expression of the metallothionein genes from Caenorhabditis elegans.

Metallothioneins are small, cysteine-rich proteins that function in metal detoxification and homeostasis. Metallothionein transcription is controlled by cell-specific factors, as well as developmentally modulated and metal-responsive pathways. By using the nematode Caenorhabditis elegans as a model system, the mechanism that controls cell-specific metallothionein transcription in vivo was investigated. The inducible expression of the C. elegans metallothionein genes, mtl-1 and mtl-2, occurs exclusively in intestinal cells. Sequence comparisons of these genes with other C. elegans intestinal cell-specific genes identified multiple repeats of GATA transcription factor-binding sites (i.e. GATA elements). In vivo deletion and site-directed mutation analyses confirm that one GATA element in mtl-1 and two in mtl-2 are required for transcription. Electrophoretic mobility shift assays show that the C. elegans GATA transcription factor ELT-2 specifically binds to these elements. Ectopic expression of ELT-2 in non-intestinal cells of C. elegans activates mtl-2 transcription in these cells. Likewise, mtl-2 is not expressed in nematodes in which elt-2 has been disrupted. These results indicate that cell-specific transcription of the C. elegans metallothionein genes is regulated by the binding of ELT-2 to GATA elements in these promoters. Furthermore, a model is proposed where ELT-2 constitutively activates metallothionein expression; however, a second metal-responsive factor prevents transcription in the absence of metals.

Direct visualization of the elt-2 gut-specific GATA factor binding to a target promoter inside the living Caenorhabditis elegans embryo.

In analyzing the transcriptional networks that regulate development, one ideally would like to determine whether a particular transcription factor binds directly to a candidate target promoter inside the living embryo. Properties of the Caenorhabditis elegans elt-2 gene, which encodes a gut-specific GATA factor, have allowed us to develop such a method. We previously have shown, by means of ectopic expression studies, that elt-2 regulates its own promoter. To test whether this autoregulation is direct, we fused green fluorescent protein (GFP) close to the C terminus of elt-2 in a construct that contains the full elt-2 promoter and the full elt-2 zinc finger DNA binding domain; the construct is expressed correctly (i.e., only in the gut lineage) and is able to rescue the lethality of an elt-2 null mutant. Multicopy transgenic arrays of this rescuing elt-2::GFP construct were integrated into the genome and transgenic embryos were examined when the developing gut has 4-8 cells; the majority of these embryonic gut nuclei show two discrete intense foci of fluorescence. We interpret these fluorescent foci as the result of ELT-2::GFP binding directly to its own promoter within nuclei of the developing gut lineage. Numerous control experiments, both genetic and biochemical, all support this conclusion and support the specificity of the binding. The approach should be applicable to studying other transcription factors binding target promoters, all within the living C. elegans embryo.

[Intraoperative assessment by laser-Doppler skin blood flowmetry of the efficacy of endoscopic thoracic sympathectomy].

We have investigated whether laser-Doppler (L-D) skin blood flowmetry on the finger could be useful for an intraoperative assessment of the efficacy of endoscopic thoracic sympathectomy (ETS) under general anesthesia. Subjects were 5 young adults receiving ETS for palmar hyperhidrosis. ETS was performed with the patients in the semi-sitting position under one lung ventilation. A pair of LDF probes were placed on the palmar side of the both second fingers. Palmar hyperhidrosis disappeared after ETS in all cases, but compensatory hyperhidrosis developed in the back of the body and the thigh. After completion of ETS on one side, the L-D skin blood flow increased to 267.6 +/- 211.1% on the side of ETS, and it increased in 2 other cases and decreased on the contrary in 3 cases on the other side. After ETS on both sides the L-D skin blood flow increased to 265.0 +/- 185.9% on the side of initial ETS and to 211.4 +/- 172.8% on the side of subsequent ETS. The initial EST induced reflex vasoconstriction on the finger of both sides and also on the toe. Spontaneous fluctuation and reflex vasoconstriction of the skin blood flow were still observed, although the periodicity of spontaneous fluctuation between the right and the left finger was lost in some of the cases. An increase in L-D skin blood flow on the side of ongoing ETS is useful for intraoperative assessment of ETS.