Impact of screening with thyroid ultrasonography in myasthenia gravis patients.

OBJECTIVES: This study was conducted to screen thyroid abnormality evaluated with ultrasonography (US) in patients with myasthenia gravis (MG) and investigate further when malignancy is suspected. METHODS: Thyroid screening using US was conducted in 162 patients with MG. In cases where malignancy was suspected, further investigations were performed. RESULTS: Abnormal US findings were detected in 125 of 162 patients with MG (72 patients with nodules, 74 patients with cysts, 27 patients with diffuse findings such as enlargement, atrophy, a hypoechoic pattern or a heterogenous echoic pattern, and 28 patients with calcification). From among these 125 subjects, 30 patients underwent further examinations such as needle aspiration cytology. As a result, six patients (3.7% of 162 cases) were positive for papillary carcinoma. The size of the carcinoma in three patients was <10 mm, yet the stage of thyroid carcinomas was high (stage III or IVa) in all six cases. CONCLUSIONS: Our data suggest that the prevalence of thyroid carcinoma in cases of MG may be higher than that of the general population. Furthermore, in patients with MG, there is a possibility that the stage of the carcinoma is higher even when the carcinoma is of a very small size. Patients with MG, when diagnosed, should be advised to undergo US screening of the thyroid because most cases of thyroid carcinoma are highly curable.

Reversal of resistance to vincristine in P388 leukemia by various polycyclic clinical drugs, with a special emphasis on quinacrine.

We investigated several lipophilic drugs with a polycyclic structure for their effect on the net uptake of vincristine in vincristine-resistant P388 leukemia cells. Fourteen of 23 agents promoted vincristine uptake in the resistant cells. The net increase in vincristine uptake was caused by prevention of its outward transport rather than by stimulation of inward transport. Some of these drugs, e.g., quinacrine, dilazep, syrosingopine, simetride, etc., remarkably potentiated the cytotoxicity of vincristine against the resistant cells in vitro. Quinacrine, an antimalarial drug which had the greatest effect on vincristine uptake and relatively low host toxicity, exhibited potent therapeutic synergism in combination with vincristine in resistant leukemia-bearing mice.

SHP-1 is involved in neuronal differentiation of P19 embryonic carcinoma cells.

Accumulating evidence suggests that tyrosine phosphorylation plays an important role in the development of the central nervous system and in the differentiation of neuronal cells. To identify protein tyrosine phosphatases (PTPs) that might regulate signaling events leading to neuronal cell differentiation, we cloned PTP genes from the murine P19 embryonic carcinoma cell line and examined the change of their expression during differentiation. P19 cells are known to be pluripotent and the aggregate formation and subsequent replating in the presence of retinoic acid (RA) induce growth arrest and neuronal differentiation. The results demonstrated that among several PTP genes expressed in P19 cells, a cytosolic Src homology region 2 domain-containing PTP, SHP-1, is expressed highly in undifferentiated P19 cells, but is reduced to an undetectable level at day 3 after replating in the presence of RA. Further, SHP-1 was tyrosine-phosphorylated and activated at day 1 after replating. When ectopic SHP-1 was constitutively expressed, P19 cells continued to proliferate and failed to differentiate upon stimulation with RA. Collectively, these results suggest that the regulated expression and activity of SHP-1 may be involved in the neuronal differentiation of P19 cells.

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., by shoot tip encapsulation in calcium-alginate and storage at 12-25°C.

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., at above-freezing temperatures following alginate-bead encapsulation was attempted. Shoot tips excised from in vitro plantlets were encapsulated in calcium-alginate beads and stored on different substrates at 12, 20, and 25 °C. Percent viability when encapsulated shoot tips were stored on substrate containing only water solidified with 1% (wt/vol) agar was 80% after 12 months at 12°C forC. odorata, 90% after 12 months at 25°C forG. crinita, and 70% after 6 months at 20°C forJ. mimosaefolia.

[Retroperitoneal leiomyosarcoma found 5 cm in size: a case report].

A 76-year-old woman presented with dull pain in left flank. Excretory urogram showed no function of left kidney. Retrograde pyelography, ultrasonography, computed tomography and magnetic resonance imaging demonstrated hydronephrosis of the left kidney and a mass lesion surrounding the ureter. The tumor was removed together with the upper region of the left ureter and kidney. The tumor was 5 x 3 x 2.5 cm in diameter and the pathological diagnosis was a leiomyosarcoma. Although recurrent tumor arose in the pelvis at 14 months after the operation, the patient has been in good general condition without showing any other evidence of recurrent lesion. This case may indicate the importance of early resection of retroperitoneal leiomyosarcoma.

[A resected case of large cell neuroendocrine carcinoma].

Large cell neuroendocrine carcinoma (LCNEC) is a rare lung cancer and it has a poor prognosis. We describe our experience with a patient in whom LCNEC was diagnosed. 38-year-old woman who complained of a cough and low grade fever up was admitted to our hospital. Chest X-ray and CT shoued 6.5 x 5.0 mass in rt-S1 and S2. It was suspected as LCNEC of the right lung because of broncopscopic cytology. The upper lobectomy of the right lung and desection of mediastinal lymph nodes were performed. Pathological diagnosis was III B (T2N2M0) LCNEC. Four weeks after the operation, one cycle of chemotherapy (CDDP + VP - 16 + VDS) and one cycle of chemoradiotherapy (thoracic radiation of 40 Gy, CDDP + 5 - FU) were performed. There are no findings of tumor recurrence 7 months after operation.

Biochemical characterization of mouse brain necdin.

Necdin is a protein encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells (P19). Necdin of mouse brain was characterized by Western blotting and silver-staining analysis by using affinity purified antibodies to 17 synthetic peptides of deduced C-terminal amino acids. Necdin exhibits a molecular mass of 51 kDa on SDS/PAGE, and is localized in the S1 and S2 nucleosomal fractions. Sonicated necdin is found in all fractions of Sephacryl S-300 gel filtration chromatography, with a peak at 700 kDa. Necdin is released on microsomal nuclease digestion, which is essential for electrophoretic migration on acetic acid/urea/Triton gels, suggesting that it could be a DNA-binding protein. Nucleosomal necdin shows two peaks at approx. 10 S and approx. 20 S on sucrose gradient centrifugation in the presence of 0.6 M NaCl, and a single peak in the presence of 2.0 M NaCl. Necdin forms a huge complex through chemical cross-linking with glutaraldehyde or dimethyl sulphate. The silver-staining intensity of the 51 kDa band corresponds to the decrease in the immuno-staining in a reagent concentration-dependent manner. Necdin binds tightly to a double-stranded DNA affinity chromatography column, and can be eluted from it with 2.0 M NaCl after washing with 0.6 M NaCl (approx. 100 ng per ml of gel). This purified necdin exhibits of pI of 9.1 on isoelectric focusing. The nucleosomal necdin complex (>200 kDa) was adsorbed on an organomercurial agarose affinity chromatography column and was eluted with 10 mM DTT, revealing that necdin is possibly involved in the transactive nucleosomal complex. These data show that necdin is a nuclear basic DNA-binding protein that associates with other molecules to regulate transcriptionally active genes and nuclear function.

Characterization of over-expressed alkaline phosphodiesterase I in tumour-derived fibroblasts from patients with neurofibromatosis.

Alkaline phosphodiesterase I from cultured fibroblasts from patients with neurofibromatosis was partially purified and characterized following extraction with Triton X-100, and fractionation with high-performance liquid chromatography. Some properties were compared with the enzyme extracted from normal-appearing fibroblasts. The isoelectric points of both the tumour and normal-appearing cell enzymes were 6.0. The enzyme required Zn2+ for its activity, was heat labile, and nicked superhelical covalently closed circular phi X174 DNA. The activity was inhibited by GTP, DTT and EDTA. The native molecular weight of alkaline phosphodiesterase I was determined to be 430,000. No differences were found in properties of the tumour-derived and normal cell enzymes. On purification it was observed that the peak pattern of enzyme activity corresponded to that of 125 kDa protein, which was more abundant upon SDS-PAGE analysis in tumour cells than in normal cells. The most active fraction of isoelectric focusing, which was performed using disulfide cross-linked polyacrylamide gel, was used to produce an antibody. The bands of 125, 60 and 40 kDa were immuno-stained in tumour cell preparation. These results indicate that alkaline phosphodiesterase I, of which the molecular weight is probably 125 kDa, is over-expressed in tumour-derived fibroblasts from neurofibromatosis patients.

Deficiency of a 42-kilodalton protein in tumor-derived fibroblastic cells in neurofibromatosis.

Cell proteins obtained from cultured normal appearing skin and neurofibromas of neurofibromatosis patients, and normal skin of normal donors were compared by SDS-PAGE and isoelectric focusing analysis. Essentially, identical protein patterns were obtained for the pellet fractions of all the strains. The lysate fraction binding patterns were also similar to each other, but a deficiency of a 42-kilodalton protein with pI 4.3 was observed in the four tumor-derived cell strains examined. These results raise the possibility that tumor-derived fibroblastic cells are of the same cell origin as skin fibroblasts, and that the deficiency of a 42-kilodalton protein could be related to the tumorigenicity in neurofibromatosis.

Purification and characterization of neutral and acid sphingomyelinases from rat brain.

Neutral and acid sphingomyelinases were copurified from a rat brain P2 fraction by extraction with 1% Triton X-100, followed by (NH4)2SO4 fractionation, acetone powdering, extraction with 1% Triton X-100, (NH4)2SO4 fractionation, Sepharose CL-6B chromatography, and chromatofocusing. The neutral sphingomyelinase was eluted with buffer containing 0.4 M NaCl after the acid sphingomyelinase had been eluted with Polybuffer at pH 5.3. The neutral sphingomyelinase exhibited specific activity of 48,300 nmol/h/mg of protein, with 254-fold purification; the corresponding value for acid sphingomyelinase was 25,300 nmol/h/mg protein, with 668-fold purification from the P2 fraction. The purified neutral sphingomyelinase had no acid sphingomyelinase activity, and vice versa. The properties of the two enzymes were examined. A single band corresponding to a molecular weight of 67,000 was obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for both enzymes. The pI was estimated to be 5.5 for both on isoelectric focusing. The native molecular weights of the neutral and acid sphingomyelinases were found to be 434,000 and 284,000, respectively, on gel filtration with Sepharose CL-6B. The single band obtained for each enzyme on SDS-PAGE was identified as an antigen with antibody raised against the purified neutral sphingomyelinase. Their amino acid compositions were very similar. The neutral and acid sphingomyelinases probably consist of common polypeptides and are immunologically cross-reactive.

Purification and amino acid microsequencing of alkaline phosphodiesterase I from calf kidney.

Alkaline phosphodiesterase I was purified from calf kidney following extraction with 0.5% Triton X-100 and then fractionation with a Rotfor cell and high-pressure liquid chromatography. The isoelectric point of alkaline phosphodiesterase I was 5.9. Both the native and the subunit molecular weight of alkaline phosphodiesterase I was determined to be 125 kDa. The N-terminus of its amino acid sequence appeared to be blocked. The immobilized protein, separated by gel electrophoresis and transblotted to a polyvinylidene difluoride membrane, was digested on the membrane by proteases and separated by reverse-phase high-performance liquid chromatography. Eighty one amino acids were sequenced. One of the internal sequences was identical with that of an active-site-containing fragment from calf intestinal enzyme, as reported by Culp et al. (1985).

Nicotinamide-induced activity of alkaline phosphodiesterase I toward tumor-derived cultured cells from neurofibromatosis patients.

Membrane-bound alkaline phosphodiesterase I was investigated in control fibroblasts and tumor-derived fibroblasts from patients with neurofibromatosis. Alkaline phosphodiesterase I activity of tumor-derived cells increased in a dose response to nicotinamide (0-9 mM) in culture; 5'-thymidine p-nitrophenyl phosphate was used as substrate. The enzyme activity increased 1.7-fold after 30 h of incubation with 9 mM nicotinamide, and after 3 weeks increased 5-fold. The nicotinamide-dependent enhancement of alkaline phosphodiesterase I activity was inhibited by actinomycin D, which specifically blocks RNA synthesis, but not by cycloheximide. These results suggest that the increase in the enzyme activity caused by nicotinamide was due to induction of alkaline phosphodiesterase I at the transcriptional level in tumor-derived cells. The metabolic effect of nicotinamide on alkaline phosphodiesterase I may be related to tumorigenicity in neurofibromatosis.

The effect of tacrine (THA) on cycloheximide- and basal forebrain lesion-induced memory deficit in rats.

The effects of 9-amino-1,2,3,4-tetrahydroacridine (tacrine), an active acetylcholinesterase inhibitor, on cycloheximide- and basal forebrain (BF) lesion-induced memory deficit in the water maze and passive avoidance task were investigated. While cycloheximide (1.5 mg/kg, s.c.) produced amnesia in the passive avoidance task, chronic administration of tacrine (1, 3 and 10 mg/kg, once a day for 1 week) improved the amnesia. BF lesion produced amnesia in both the water maze and passive avoidance tasks. Chronic tacrine (0.1-3 mg/kg, passive avoidance task, or 0.3 mg/kg, water maze task, once a day for 1 week) improved BF lesion-induced amnesia in the passive avoidance and water maze tasks. These results suggest that tacrine may be useful for senile dementia.

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Cryptomeria japonica D. Don.

This report describes the successful plant regeneration via somatic embryogenesis from immature zygotic embryos of Cryptomeria japonica D. Don. For the induction of embryogenic tissue, we determined that the optimal medium contained N6-benzyladenine and 2,4-dichlorophenoxyacetic acid. Immature zygotic embryos that were collected at the end of June yielded embryogenic tissue at the highest frequency. Embryogenic tissues that had proliferated in liquid medium included small and loosely packed cells and elongating or elongated cells. We used ten cell lines to determine the optimal medium for the development of somatic embryos. Induced somatic embryos germinated with synchronous sprouting of cotyledons, hypocotyls and roots. Gibberellin A3 in the germination medium had a positive effect on both the elongation of hypocotyls and the survival of seedlings. The frequencies of induction and germination of somatic embryos differed among the cell lines examined. Most of the seedlings grew normally. This system of somatic embryogenesis required 4-5 months for the regeneration of C. japonica plantlets from immature zygotic embryos.