Hsp27 suppresses the formation of inclusion bodies induced by expression of R120G alpha B-crystallin, a cause of desmin-related myopathy.

The R120G mutation in the small heat shock protein (sHSP) alpha B-crystallin has been identified in a family suffering from desmin-related myopathy. In this study, we characterized the features of transiently expressed R120G alpha B-crystallin in mammalian cells. In addition, we examined interactions of this mutant alpha B-crystallin with Hsp27, another representative sHSP. In HeLa cells, transiently expressed R120G alpha B-crystallin was mainly fractionated in the insoluble fraction, although wild-type alpha B-crystallin was predominantly found in the soluble fraction. In immunofluorescence studies, we found 15-25% of R120G alpha B-crystallin-expressing cells to contain multiple cytosolic inclusion bodies, in which Hsp27 was also localized. When R120G alpha B-crystallin and Hsp27 were transiently co-expressed in HeLa cells, the amount of R120G alpha B-crystallin in the soluble fraction was greater than with expression of R120G alpha B-crystallin alone. Moreover, co-expression resulted in reduced formation of inclusion bodies, suggesting that Hsp27 acts as a molecular chaperone for R120G alpha B-crystallin.

A solitary bronchial papilloma with malignant changes.

We describe a case of solitary papilloma of the bronchus and provide a review of 38 similar cases reported in Japan. A 70-year-old man complained of cough and sputum. Chest X-rays and CT scans revealed atelectasis of the right middle lobe. On bronchoscopy, a polypoid tumor was found at the orifice of the bronchus of the right middle lobe. The tumor was histologically diagnosed as a squamous papilloma with moderate atypia. Because of elevated tumor markers and the reported high incidence of malignant changes in papillomas, the tumor was endoscopically resected by electrosurgical snare. While this procedure resulted in improvement of atelectasis, the chest CT taken subsequently revealed a mass adjacent to the resected polypoid tumor in the middle lobe bronchus. Percutaneous needle biopsy followed by histopathological examination confirmed the tumor to be a squamous cell carcinoma. Only three cases of malignant changes in papillomas have been previously reported in Japan. Electrosurgical snare, which allows the identification of tissue at the tumor base, should be the treatment of choice rather than YAG laser surgery.

Evaluation of the electroosmotic medium pump system for preparative disk gel electrophoresis.

This paper describes an improved electroosmotic elution system for preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on the epochal idea of H. V. Tan et al. (Nucleic Acids Res. 1988, 16, 1921-1930). In this elution system, a semipermeable membrane, mounted under the gel terminal end, works as the elution pump as well as the partition of the elution chamber. We refer to this system as the "electroosmotic medium pump system." Operation of the constructed apparatus (3.6 cm i.d. disk gel column) and resolution of the protein bands were examined by separation of the model protein mixture (bovine serum albumin (BSA), ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor) and purification of the membrane protein, dipeptidyl peptidase IV (DPP IV). The Spectra/Por 7 dialysis membrane provided a better flow profile for the elution buffer. The four model proteins of the protein mixture were able to be completely separated from each other and recovered without dilution. The maximum protein concentration of eluate achieved was 93 mg/ml, when applying a single component, BSA fraction V, as a sample. Furthermore, the multifunctional ectoenzyme, DPP IV, was purified in a single step.

Primary intestinal T-cell lymphoma resembling lymphomatous polyposis: report of a case.

We report an interesting case of primary intestinal T-cell lymphoma (ITL) resembling lymphomatous polyposis (LP) in a 24-year-old man. The neoplasm macroscopically showed numerous small polyps throughout the colon and microscopically showed diffuse proliferation of small-sized tumor cells with occasionally cleaved or irregularly shaped nuclei. The tumor cells were immunohistochemically positive for CD3, CD8, TIA-1, and CD56, and a polymerase chain reaction study showed a single band, indicating monoclonal rearrangement of the T-cell receptor beta gene. The phenotypic features in the current case are consistent with those of ITL derived from cytotoxic CD56+ CD8+ intraepithelial lymphocytes. This is the second documented case of primary ITL with a morphologic pattern of LP.

[Diagnosis of neurodegenerative disease by mass spectrometry].

We analyzed wild-type and variant transthyretins (TTRs) by mass spectrometry and reported that all TTR preparations demonstrated free TTR, TTR conjugated with thiol compounds and several minor components. We previously described a component with a molecular mass 80 Da larger than free TTR, which was proven to be TTR conjugated with bisulfite. The amyloid fibril formation of purified TTR was monitored by the turbidity at 330 nm, and by a Congo red binding assay as a function of pH. The S-sulfonated TTR showed clear elevation of the turbidity and Congo red binding under acidic conditions. In contrast, TTR reduced by dithiothreitol, which was free of the S-sulfonated component, did not show evidence of amyloid fibril formation. We analyzed rabbit serum TTR obtained from a rabbit fed a diet containing sulfite and from a rabbit on a sulfite-free diet. Compared to that in the rabbit fed a sulfite-containing diet, sulfonated TTR was decreased on the 7th day of a sulfite-free diet. These results suggested that the S-sulfonated wild-type TTR is highly amyloidogenic, and that prolonged ingestion of antimicrobial and antioxidant agents containing sulfite/bisulfite, may cause senile systemic amyloidosis.

Detection by mass spectrometry of highly increased amount of S-sulfonated transthyretin in serum from a patient with molybdenum cofactor deficiency.

Serum transthyretin has several isoforms, most of which are caused by disulfide linkage with cysteine residue at position 10. We found an ion peak 80 D larger than unmodified transthyretin by electrospray ionization mass spectrometry and assigned it to S-sulfonated transthyretin. The peak height was <2% of total transthyretin in control sera from more than 200 individuals including infants. Transthyretin from a patient with molybdenum cofactor deficiency was analyzed, and the peak was prominent, higher than 85% of total transthyretin. In patients with this disease, the presence of elevated levels of sulfite leads to the formation of S-sulfonated cysteine. The peak can be used as a diagnostic marker for molybdenum cofactor deficiency, although more sera from patients with this disease should be tested.

A new amyloidogenic transthyretin variant, [D38A], detected by electrospray ionization/mass spectrometry.

A new variant of transthyretin (TTR) was detected by mass spectrometry (MS) in a 63-year-old Japanese female patient suffering from amyloidosis. TTR was analyzed by 2-dimensional liquid chromatography coupled with electrospray ionization MS. Variant TTR showed extra peaks in addition to normal TTR peaks. The extra peaks were about 44 Da smaller than normal TTR peaks, and the abundance of variant peaks showed about 80% of the corresponding normal free and adduct peaks. Direct genomic DNA sequencing of TTR exon 2 showed both adenine and cytosine in the position corresponding to the second base of codon 38. This codes for a variant alanine (GCT) as well as the normal aspartic acid (GAT), indicating that the case is heterozygous for the substitution, [D38A].

Enhanced amyloidogenicity of sulfonated transthyretin in vitro, a hypothetical etiology of senile amyloidosis.

Genetic variants of transthyretin (TTR) cause systemic amyloidosis and wild-type TTR may also in some situations produce amyloid fibrils. We have analyzed wild-type and variant TTRs by mass spectrometry and found that TTR preparations from all individuals demonstrated free TTR, TTR conjugated with thiol compounds and several minor components. We previously described a component which had a molecular mass 80 Da larger than free TTR and was proved to be TTR conjugated with sulfite. Here, the amyloid fibril formation of the TTR isoforms was monitored by the turbidity at 330 nm, and by a Congo red-binding assay as a function of pH, according to the method of Lai et al. The S-sulfonated TTR showed the highest level of amyloid fibril formation. In contrast, TTR reduced by dithiothreitol, which was free of the S-sulfonated component, showed neither elevation of the turbidity nor the Congo red binding. Commercially purchased TTR without further treatment containing free, S-sulfonated and other species of TTR molecules showed an intermediate elevation. These results suggested that the S-sulfonated wild-type TTR is highly amyloidogenic. Although further experiments are needed to apply the observation to in vivo phenomenon, exogenous sulfite may be a cause of senile systemic amyloidosis.

Expression of the Ets-1 proto-oncogene correlates with malignant potential in human astrocytic tumors.

The protein encoded by the Ets-1 proto-oncogene is a transcription factor that regulates expression of matrix proteases. It has been associated with tumor invasion and angiogenesis. Glioma progression is characterized by increased invasiveness and neovascularization, so we hypothesized that expression of Ets-1 proto-oncogene might play a role in the progression of these tumors. Therefore, we examined the expression of Ets-1 protein by immunohistochemical means and in situ hybridization in tissues obtained from 81 primary and 20 recurrent astrocytic tumors. Twenty-eight (65%) of 43 glioblastomas (Grade IV astrocytomas) stained for Ets-1. The percentage of positive cells in glioblastomas varied from 10 to 90%. Of the 16 anaplastic astrocytomas (Grade III), 4 (25%) were moderately positive (<50% of cells) for Ets-1. None of 22 cases of low-grade astrocytomas (Grade II) expressed endogenous Ets-1. The staining score was significantly associated with tumor grade (P < .0001). Normal brain tissues did not express Ets-1 protein, whereas recurrent astrocytoma cases expressed significantly more positivity for Ets-1 than did primary tumors (P = .03). The Ets-1 protein was observed mainly in the nucleus and corresponded to the cytoplasmic Ets-1 mRNA localization by in situ hybridization. Western and Northern blot analyses confirmed overexpression of Ets-1 protein and mRNA in high-grade tumors. We conclude that Ets-1 protein expression correlates with the malignant potential of tumors of astroglial origin.

Ets-1 transcription factor-mediated urokinase-type plasminogen activator expression and invasion in glioma cells stimulated by serum and basic fibroblast growth factors.

Overexpression of urokinase plasminogen activator (uPA) has been proposed to be one of the mechanisms by which malignant glioma cells cause pericellular proteolysis of stromal structures during brain-tissue invasion. However, knowledge about the mechanisms that regulate uPA in glioma cells is still rather scant. Growth factors, particularly epidermal growth factor and basic fibroblast growth factor (bFGF), regulate uPA synthesis in various nonglial cells. Because these factors have been associated with glioma invasion, we thought that perhaps similar events may occur in glioma cells. In this study, we demonstrate that growth factors regulate uPA gene transcription in glioma cells by induction of Ets-1 transcription factor. Serum and bFGF treatment concomitantly stimulated baseline levels of Ets-1 and uPA mRNA and protein, and this was associated with a marked increase in the migration and invasion potentials of glioma cells in vitro. Treatment of cells with antisense Ets-1 but not the control sense oligonucleotides concurrently inhibited the expression of Ets-1 and uPA, and this blocked glioma cell migration and invasion by more than 50%. In addition, medium conditioned by antisense Ets-1 oligonucleotide-treated cells showed markedly reduced uPA activity, as determined by casein zymography. These results strongly suggest that serum and bFGF control glioma invasion by inducing synthesis of Ets-1 transcription factor, which directly up-regulates expression of uPA in glioma cells. Antisense inhibition of Ets-1 expression may therefore provide a potential and exciting therapeutic target for preventing invasion by glioma cells.

A simple and reliable method of detecting variant transthyretins by multidimensional liquid chromatography coupled to electrospray ionization mass spectrometry.

Transthyretin (TTR) variants cause amyloidosis. A method, originally reported by us, of detecting the variants by high performance liquid chromatography/electrospray ionization mass spectrometry (ESIMS) using materials precipitated with anti-TTR antiserum, has been successfully applied by several institutions. The method is simple and reliable, but some variants may not precipitate with the antiserum or may precipitate in different yields compared to normal TTR. Moreover, unidentified minor peaks were observed, which may have been derived from cross reactive materials. We have now devised a new procedure to overcome these problems. An anion exchange and reversed phase liquid chromatography system and ESI mass spectrometer were connected in a tandem fashion using a 6 port valve and a protein trap cartridge. The profile of ion peaks by the method was the same as that by MS with immunoprecipitates. The minor peaks were proved not to be derived from cross reactive materials, and the molecular species of these peaks were characterized. This method is faster than immunoprecipitation method and using no antibody is a great benefit. The method can be applied widely to the study various proteins, when antibodies are not available.

Skin tumor risk among atomic-bomb survivors in Japan.

OBJECTIVES: Elevated risks of skin cancer following high doses of ionizing radiation have long been known. Recent reports on atomic-bomb survivors indicate that nonmelanoma skin cancer can be induced at low to medium doses. We studied atomic-bomb survivors to determine the effects of radiation on specific histologic types of skin cancer and to describe the dose-response relationship. METHODS: Cases of melanoma, nonmelanoma skin cancers, and Bowen's disease were ascertained between 1958 and 1987 for the 80,000 cohort members through the population-based Hiroshima and Nagasaki (Japan) tumor registries augmented by searches of other records. RESULTS: An excess of basal cell carcinoma (n = 80), with some suggestion of a non-linear dose-response, was observed. The excess risk decreased markedly as age at exposure increased, and there was no evidence for an interaction between ionizing and ultraviolet radiation. No dose-response was found for squamous cell carcinoma (n = 69). The excess relative risk point-estimates were large, but statistically nonsignificant for both melanoma (n = 10) and Bowen's disease (n = 26). CONCLUSIONS: The basal layer of the epidermis appears to be quite sensitive to radiation carcinogenesis, particularly at a young age. The suprabasal layer seems to be more resistant, as shown by the lack of an association for squamous cell carcinomas.

Complex gangliosides are essential in spermatogenesis of mice: possible roles in the transport of testosterone.

Mice, homozygous for disrupted ganglioside GM2/GD2 synthase (EC 2.4. 1.94) gene and lacking all complex gangliosides, do not display any major neurologic abnormalities. Further examination of these mutant mice, however, revealed that the males were sterile and aspermatogenic. In the seminiferous tubules of the mutant mice, a number of multinuclear giant cells and vacuolated Sertoli cells were observed. The levels of testosterone in the serum of these mice were very low, although testosterone production equaled that produced in wild-type mice. Testosterone was found to be accumulated in interstitial Leydig cells, and intratesticularly injected testosterone was poorly drained in seminiferous fluid in the mutant mice. These results suggested that complex gangliosides are essential in the transport of testosterone to the seminiferous tubules and bloodstream from Leydig cells. Our results provide insights into roles of gangliosides in vivo.

[Langerhans cell histiocytosis in the hypothalamus: a case report].

A case of Langerhans cell histiocytosis (LCH) in the hypothalamus is presented. A 52 year old man with eosinophilic granuloma of the lung was referred to our hospital. He complained of headache, easy fatigability, high fever, and voiding large quantities of urine, but was otherwise asymptomatic. T1-weighted images demonstrated a small mass in the hypothalamus. After about six months, the mass had grown to 2 cm in diameter. Surgery was performed by a pterional approach and a biopsy specimen was collected. Light microscopic examination of sections of the biopsy specimen showed polymorphous cellular infiltration by histiocytic cells, eosinophils, neutrophils, and lymphocytes. Electron microscopy showed Langerhans cells and Birbeck granules in the cytoplasm. The histological diagnosis was LCH, multifocal type. After low-dose radiation therapy, the mass gradually decreased in size. The clinicopathological features, neuroradiological findings, and treatment are briefly discussed.

Malignant peripheral nerve sheath tumors developing multifocally in the central nervous system in a patient with neurofibromatosis type 2.

We describe autopsy findings of multifocal malignant peripheral nerve sheath tumors (MPNSTs) appearing in the central nervous system in a 45-year-old Japanese female with neurofibromatosis type 2. Multiple MPNSTs were detected in both III and VIII, left IV, and V cranial nerves, and a number of nerve roots of the spinal cord. Neurofibromata were on the other hand evident on some nerve roots of the spinal cord and femoral and sciatic nerves. Our results suggest that a mutation of p53 gene may have played a role in the malignant transformation of nerve tumors in this patient since p53 protein was immunohistochemically detected in MPNST cells but not in tumor cells of the neurofibromata.

Hb Peterborough [beta 111(G13)Val-->Phe] in Japan; rapid identification by ESI/MS using proteolytic digests of oxidized globin.

A variant hemoglobin was found in a Japanese female whose hemoglobin was studied to clarify the cause of a low Hb A1c value, found during a routine medical examination. The detection and identification of the variant was performed by electrospray ionization mass spectrometry. Its structure was revealed to be the same as Hb Peterborough [beta 111(G13)Val-->Phe]. For sequence determination, oxidized globin as well as non-derivatized globin were cleaved by trypsin and lysyl endopeptidase. An abnormal peptide was found in digests of oxidized globin, as shown by electrospray ionization mass spectrometry. Cysteic acid in oxidized peptides enhanced the abundance of fragment ions in tandem mass spectrometry, which helped to quickly and accurately determine the substitution in beta T-12, a peptide in the core region. Electrospray ionization mass spectrometry analysis of the hemolysate also showed a low level of glycated hemoglobin. The patient's hemolysate showed decreased stability in the isopropanol test. An abnormal band was detected on isoelectrofocusing on the cathodic side of normal Hb A. This is the second report of Hb Peterborough and the first of its occurrence in Japan.

Meningeal melanocytoma in the left frontal region.

We report a rare case of meningeal melanocytoma in the left frontal region. A 45-year-old man complained of a headache. Magnetic resonance (MR) scanning showed characteristic patterns: a slightly high signal intensity mass in the left frontal region on the T1-weighted image and a low signal intensity on the T2-weighted image. The patient underwent gross total removal of the tumor. The postoperative course was uneventful. After two years, there was a small local recurrence. The histological finding of the tumor showed meningeal melanocytoma. To our knowledge, this is the second published report of a meningeal melanocytoma in the supratentorial region.

A new nonamyloid transthyretin variant, G101S, detected by electrospray ionization/mass spectrometry. Mutations in brief no. 201. Online.

Familial amyloidotic polyneuropathy is caused by transthyretin (TTR) variants. The identification of new variants with and without amyloidosis may help to clarify the mechanism of amyloid fibril formation. We detected several variant TTRs from patients with and without symptoms of amyloidosis using mass spectrometry (MS). TTR was isolated by mixing test serum with anti-transthyretin antiserum, and the generated immunoprecipitate was analyzed by high performanced liquid chromatography/electrospray ionization (HPLC/ESI) MS. Variant TTRs showed extra peaks in addition to normal TTR peaks. A variant found in nonamyloid group was sequenced by HPLC/ESI tandem MS using peptides obtained by protelytic digestion of TTR and by DNA analysis. The structure was new, [G101S], and was found in a 74 years old Japanese male. This mutation results from substitution in a CpG hot spot. The substitution in the surface loop, 98-102, between F and G b-strands may not cause amyloid formation.

Immunohistochemical analysis of progesterone receptor and Ki-67 labeling index in astrocytic tumors.

BACKGROUND: Intracranial tumors such as meningiomas express steroid hormone receptors but little is known regarding progesterone receptor (PR) in astrocytic tumors. The authors evaluated expression of PR in 86 astrocytic tumors in relation to tumor proliferative potential. METHODS: Paraffin embedded tumor sections were stained with polyclonal antiprogesterone antibody by the peroxidase-antiperoxidase method and with monoclonal MIB-1-Ki-67 antibody by avidin-biotin complex immunohistochemistry. RESULTS: Sixty-three of the 86 astrocytic tumors (73%) showed positive PR immunoreactivity. PR expression was observed in 4 of 9 pilocytic astrocytomas, 13 of 24 Grade 2 astrocytomas, 15 of 20 anaplastic astrocytomas, and 31 of 33 glioblastomas. In addition to the tumor cells, cells of microvascular endothelial proliferation and the smooth muscle of tumor vessel walls were frequently PR positive. Glioblastomas had a significantly higher percentage of PR positive cells compared with anaplastic (P < 0.0008) and low grade (P < 0.0001) astrocytomas. Patients with PR positive astrocytomas were of an older age than patients with PR negative astrocytomas (48.71 +/- 21.95 years vs. 37.09 +/- 24.69 years; P < 0.04). The mean Ki-67 labeling index (LI) was significantly higher in the high grade (3-4) astrocytomas compared with low grade (1-2) astrocytomas (P < 0.0001). PR positive astrocytic tumors had higher Ki-67 LI than PR negative tumors. PR expression was not correlated with tumor recurrence and patient survival. CONCLUSIONS: The current study suggests that PR in the astrocytic tumors correlates with histologic grade and PR may participate in the growth of these tumors and tumor angiogenesis. The measurement of PR in these tumors may indirectly represent tumor growth potential.

A histopathologic and immunohistochemical study of small nodules of renal angiomyolipoma: a comparison of small nodules with angiomyolipoma.

Small mesenchymal nodules (SNs) are observed in some cases of renal angiomyolipoma (AML), with or without tuberous sclerosis. They are composed of blood vessels and/or nonvascular smooth muscle cells (SMCs) and/or fat cells. We examined 20 cases of AML, performed detailed histopathologic and immunohistochemical studies of SNs, verified the histologic relationship between SNs and AMLs, and compared the SNs of the tuberous/nontuberous sclerosis groups. Seventy-seven SNs were observed in five cases of AML. The SNs were 0.11 mm to 20.0 mm in diameter. The location of small-sized SNs in the kidney was variable; almost all of the SNs larger than 3.25 mm were in the renal capsule. The small-sized SNs contained mainly epithelioid-type nonvascular SMCs. Blood vessels and fat cells were not observed in the small-sized SNs but appeared gradually in the large-sized SNs. Almost all of the SNs were rounded lesions, and no fusion was observed between the SNs. Nonvascular SMCs of all of the SNs and AMLs were positive for vimentin, alpha-SM actin, and S-100 protein. The SNs less than 1.13 mm in diameter were negative for HMB-45; the nonvascular SMCs of AMLs and of SNs greater than 1.13 mm in diameter were positive for HMB-45. Nonvascular SMCs of SNs and AMLs showed a neurogenic phenotype. The SNs of the nontuberous sclerosis group contained only SMC components, whereas the same-size SNs of the tuberous sclerosis group contained SMCs, fat cells, and blood vessels. The SNs of the nontuberous sclerosis group may not increase in size or may grow slowly. Some of the SNs of patients with tuberous sclerosis grow to become AML. Although the SNs in patients with nontuberous sclerosis do not contain any blood vessels or fat cells, their SMCs show the histologic and immunohistochemical characteristics of AMLs; this indicates that SNs are the "buds" of AML.