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.

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.

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.

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.

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.

[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.

Expression of vascular endothelial growth factor in sera and lymph nodes of the plasma cell type of Castleman's disease.

To evaluate the possible involvement of vascular endothelial growth factor (VEGF) in the pathogenesis of Castleman's disease, we studied VEGF levels in sera and supernatants of cultured lymph nodes from two patients with the plasma cell type of Castleman's disease, and analysed the expression of VEGF immunohistochemically in the lymph nodes. Clinically, one patient was classified as the localized type and the other as the multicentric type. Histologically, mature plasma cells and hyalinized vessels were prominent in the interfollicular region. The VEGF levels of the sera and the supernatants of cultured lymph nodes of both patients were higher than those of normal controls. VEGF was strongly expressed in plasma cells in the interfollicular region of the lymph nodes of both patients, but rarely in normal lymph nodes. Our results suggest that VEGF may be involved in the marked vascular proliferation in the interfollicular region of the lymph nodes of the plasma cell type of Castleman's disease.

Reprogramming of early embryonic blastomeres into endodermal progenitors by a Caenorhabditis elegans GATA factor.

The END-1 GATA factor has been implicated in specifying endoderm in Caenorhabditis elegans and is the earliest known zygotic protein expressed in the lineage of E, the clonal endoderm progenitor. We report that ubiquitous end-1 expression during a critical period in embryogenesis causes all non-endodermal lineages to produce endoderm instead of ectoderm and/or mesoderm. END-1 expression bypasses the requirement for maternal SKN-1 and the maternal Wnt signaling pathway in endoderm formation. This suggests that a primary function of these maternal factors is to regulate zygotic end-1 expression, which is then sufficient to initiate the entire program for endoderm development.

Appendiceal abscess mimicking infected urachal cyst in a child with intestinal malrotation.

An appendiceal abscess with intestinal malrotation can occur anywhere in the abdomen, not only in the right lower quadrant. We report a case presenting a midline mass of the lower abdomen whose computed tomography (CT) and ultrasonography (US) findings mimicked a urachal abscess. A retrospective review of CT findings led to the correct diagnosis by showing malposition of the ascending colon.

pha-4 is Ce-fkh-1, a fork head/HNF-3alpha,beta,gamma homolog that functions in organogenesis of the C. elegans pharynx.

The C. elegans Ce-fkh-1 gene has been cloned on the basis of its sequence similarity to the winged-helix DNA binding domain of the Drosophila fork head and mammalian HNF-3alpha,beta,gamma genes, and mutations in the zygotically active pha-4 gene have been shown to block formation of the pharynx (and rectum) at an early stage in embryogenesis. In the present paper, we show that Ce-fkh-1 and pha-4 are the same gene. We show that PHA-4 protein is present in nuclei of essentially all pharyngeal cells, of all five cell types. PHA-4 protein first appears close to the point at which a cell lineage will produce only pharyngeal cells, independently of cell type. We show that PHA-4 binds directly to a 'pan-pharyngeal enhancer element' previously identified in the promoter of the pharyngeal myosin myo-2 gene; in transgenic embryos, ectopic PHA-4 activates ectopic myo-2 expression. We also show that ectopic PHA-4 can activate ectopic expression of the ceh-22 gene, a pharyngeal-specific NK-2-type homeodomain protein previously shown to bind a muscle-specific enhancer near the PHA-4 binding site in the myo-2 promoter. We propose that it is the combination of pha-4 and regulatory molecules such as ceh-22 that produces the specific gene expression patterns during pharynx development. Overall, pha-4 can be described as an 'organ identity factor', completely necessary for organ formation, present in all cells of the organ from the earliest stages, capable of integrating upstream developmental pathways (in this case, the two distinct pathways that produce the anterior and posterior pharynx) and participating directly in the transcriptional regulation of organ specific genes. Finally, we note that the distribution of PHA-4 protein in C. elegans embryos is remarkably similar to the distribution of the fork head protein in Drosophila embryos: high levels in the foregut/pharynx and hindgut/rectum; low levels in the gut proper. Moreover, we show that pha-4 expression in the C. elegans gut is regulated by elt-2, a C. elegans gut-specific GATA-factor and possible homolog of the Drosophila gene serpent, which influences fork head expression in the fly gut. Overall, our results provide evidence for a highly conserved pathway regulating formation of the digestive tract in all (triploblastic) metazoa.

Extrarenal Wilms' tumour.

Extrarenal Wilms' tumour is rare and its imaging has received scant mention in the literature. We describe a 2-year-old boy with a firm mass in the right flank. CT, MRI and ultrasonography showed an inhomogeneous solid mass located in the retroperitoneum, which was separate from the right kidney. Angiography showed an enlarged right gonadal artery and irregularly tortuous vessels in the tumour similar to intrarenal Wilms' tumour ("spider leg" or "creeping vine" appearance). Histopathological examination confirmed an extrarenal Wilms' tumour.

Undifferentiated (embryonal) sarcoma of the liver: report of three cases.

Undifferentiated (embryonal) sarcoma is a rare malignant tumor of the liver. It typically presents in late childhood and its prognosis is poor. We experienced three cases of such a tumor during the period of 1976-1989; two of these patients are still alive without disease. Each case was independently treated with a combination of surgery and pre- and/or postoperative chemotherapy, which was found to be effective. In one surviving patient, cyclophosphamide and vincristine were found to be effective, while in the other a combination of cisplatin, Adriamycin (ADM), vincristine and cyclophosphamide was observed to induce a rapid reduction in the size of the recurrent tumor. Thus, an adequate combination of surgery and chemotherapy may improve the prognosis of this tumor.

Establishment and characterization of a cell line of congenital primitive neuroectodermal tumor of soft tissue.

A new human cell line, termed Muraoka, has been established from the recurrent tumor of a case of congenital primitive neuroectodermal tumor (PNET) arising at the temporofacial region of a male infant. The microscopic findings of this cell line were epithelioid, and the xenografted tumor in a nude mouse consisted of the malignant epithelioid cells. Immunohistochemically, the cells were positive for neuron-specific enolase, S-100 protein, carcinoembryonic antigen, cytokeratin, epithelial membrane antigen, and glial fibrillary acidic protein. These findings were quite similar to those of the epithelioid cells in the original tumor and of the xenografted tumor cells. Neither chromosomal abnormalities nor N-myc amplification were observed. Morphological differentiation after treatment with N6-2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (Bt2-cAMP), all-trans-retinoic acid (RA), prostaglandin E1 (PGE1), and 5-bromo-2'-deoxyuridine (BrdU) showed two different results. Bt2-cAMP and PGE1 induced neuronal differentiation with the extension of neurites, whereas RA and BrdU predominantly induced Schwannian differentiation (flat cells). In these respects, the cell line Muraoka seems to be useful for studying characteristics of PNET as well as for developing the new treatments against such tumors.

Different effects of vitamin E on the growth and morphological differentiation of 4 murine neuroblastoma cell lines in vitro.

The effects of vitamin E (vit.E, d-alpha-tocopherol acid succinate) on the growth and the morphological differentiation of 4 representative murine neuroblastoma cell lines: C1300 (wild type), NS-20 (cholinergic type), N-18 (inactive type), N1E-115 (adrenergic type), and the combined effects of vit.E and cis-diamminedichloroplatinum(II)(cisplatin, CDDP) were studied in culture. Vitamin E inhibited the growth of all clonal cells in a dose-dependent manner, especially that of NS-20 cells, and it caused significant morphological differentiation only in NS-20 cells. In addition, vit.E enhanced the growth inhibition of C1300 cells by cisplatin in an additive manner, but not that of NS-20 cells. These suggested that both phenomena induced by vit.E may not always occur in parallel in each clonal neuroblastoma, and both mechanisms might be different.

Combined effects of vitamin E (alpha-tocopherol) and cisplatin on the growth of murine neuroblastoma in vivo.

Combined effects of vitamin E (alpha-tocopherol) and cisplatin on the growth of two murine neuroblastomas (C1300, NS-20) was investigated in vivo. Five groups of mice were prepared; group 1 were fed the control diet, group 2 were fed a vitamin E-deficient diet, group 3 were fed a vitamin E-supplemented diet, group 4 were fed the control diet and plus vitamin E solution given intraperitoneally during the treatment (solvent i.p. group), and group 5 were given vitamin E in the same manner (20 mg/kg/day; vitamin E i.p. group). Cisplatin (6 mg/kg) was injected intraperitoneally into the mice of each group during the treatment. In case of the C1300 neuroblastoma, the antitumor activity of cisplatin was most enhanced in the mice receiving vitamin E i.p., and the intra-tumor vitamin E and platinum levels were significantly higher in this group than in the other groups (P less than 0.01, and P less than 0.05 respectively). In contrast, in animals transplanted with the NS-20 murine neuroblastoma, which proved to be a cisplatin-tolerant tumor in separate experiments, no combined effect of those drugs was observed, although the intra-tumor level of platinum was elevated. The possibility was that vitamin E increases the influx of cisplatin into the tumor cells and acts after incorporation of cisplatin through the plasma membrane. Vitamin E did not accentuate the cisplatin-induced renal impairment in vitamin E-loaded groups. Those results suggested that vitamin E should be considered as a co-agent of cisplatin for the treatment of neuroblastoma.