[A patient of MELAS with 3271 mutation with fatal outcome after alcohol intake].

A patient of MELAS is reported. A 28-year-old woman was admitted to Shimada Municipal Hospital because of nausea, vomiting, and right homonymous hemianopsia. She had past history of dizziness and convulsion. A brain magnetic resonance imaging showed an ischemic lesion in the left occipital lobe, which disappeared in the follow-up study. Laboratory examination indicated elevated lactate and pyruvate levels in both blood and cerebrospinal fluid. The muscle biopsy demonstrated ragged-red fibers and strongly SDH-reactive blood vessels. PCR-RFLP analysis of DNA extracted from her muscle and blood as well as her mother's blood revealed a T to C mutation at nucleophile position of 3271 in mitochondrial DNA. She was diagnosed as having MELAS and discharged. One year after the first admission, she re-visited our hospital because of three days' duration of fatigability and generalized muscle pain after alcohol intake. She had severe lactic acidosis, rhabdomyolysis and acute renal failure. Despite a continuous hemodialysis and other intensive efforts, the patient died 20 hours later. Alcohol intake has been reported to induce rhabdomyolysis in myopathy with mitochondrial DNA deletions. The course of this patient suggests that alcohol intake can be an aggravating factor also in MELAS.

Accelerated evolution in the protein-coding region of galectin cDNAs, congerin I and congerin II, from skin mucus of conger eel (Conger myriaster).

Two cDNAs encoding galectins named congerins I and II from the skin mucus of conger eel (Conger myriaster) were isolated and sequenced. Comparison of the nucleotide sequences of congerins I and II showed that the sequence similarities of the 5' and 3' untranslated regions (86 and 88%, respectively) were much higher than those of the protein-coding region (73%). The numbers of nucleotide substitutions per site (KN) for the untranslated regions are smaller than the numbers of nucleotide substitutions per synonymous site (KS) for the protein coding region. Furthermore, nonsynonymous nucleotide substitutions have accelerated more frequently than synonymous nucleotide substitutions in the protein coding region (KA/KS = 2.57). These results suggest that accelerated substitutions have occurred in the protein-coding regions of galectin genes to generate diverse galectins with different molecular properties. Northern blot analysis showed that both congerins were expressed not only in the skin tissues but also in the stomach of conger eel.

Functional and structural characterization of multiple galectins from the skin mucus of conger eel, Conger myriaster.

The complete amino acid sequence of an isogalectin, named congerin II, isolated from the skin mucus of conger eel, was determined by sequencing of the protein and its peptides generated by enzymatic and chemical cleavages. Congerin II consisted of 135 amino acids residues containing an acetylated N-terminus. Congerin II was found to be only 46% homologous in sequence to congerin I which was previously determined (Muramoto K., Kamiya H., Biochem. Biophys. Acta, 1992;1116:129-136), suggesting that the galectins with diverse molecular properties are present in the skin mucus of conger eel. However, it was confirmed by analysis of the secondary structures using circular dichroism that both congerins I and II shared similar folds characterized by beta structures. Congerins I and II showed different molecular properties such as thermostability, pH dependency for hemagglutinating activity and for binding specificity against the pyridylamino derivative of lactose. Congerin I showed more strict recognition specificity for lactose than did congerin II. Furthermore, the effects of chemical modification on congerins I and II were investigated in order to identify the type of amino acids involved in their different lectin activities. Modification of tyrosine and lysine residues did not affect the carbohydrate-binding activities of congerins. However, modification of tryptophan, arginine, histidine, glutamic acid and aspartic acid residues led to considerable loss of their activities, and a different mode of binding activity was observed between modified congerins I and II. These results suggest that multiple galectins from conger eel with the same scaffold have different biological functions and properties.

12-Lipoxygenase in A431 cells: genetic identity, modulation of expression, and intracellular localization.

Human A431 epidermoid carcinoma cells express 12-lipoxygenase enzymatic activity. However, the isoform identity based on cDNA sequence data is not known. Further, the simultaneous characterization of the intracellular distribution of 12-lipoxygenase protein and activity is lacking. Here we report that the cDNA sequence from RT-PCR-amplified 12-lipoxygenase mRNA is identical with the platelet-type 12-lipoxygenase isoform, and the leukocyte-type isoform of 12-lipoxygenase is not expressed in A431 cells. The predominant amount (78%) of 12-lipoxygenase protein resides in the cytosol. In contrast, the predominant (98%) 12-lipoxygenase activity is localized in the membrane fraction. Western blot and immunofluorescence data demonstrate that epidermal growth factor increases total cellular 12-lipoxygenase protein and enhances the association of 12-lipoxygenase protein with perinuclear or nuclear membrane sites. In addition, epidermal growth factor stimulates 12-lipoxygenase activity resulting in generation of 12(S)-hydroxyeicosatetraenoic acid from cellular arachidonate. In contrast, both 12-lipoxygenase protein and activity decrease approximately 80% within 24 h during serum starvation. The recovery of 12-lipoxygenase expression in serum-deprived cells can be induced by readdition of epidermal growth factor or serum. Further, the basal expression of 12-lipoxygenase depends on signal pathways requiring protein tyrosine kinase activity, since genistein, herbimycin A, and tyrphostin 25 reduce the expression of 12-lipoxygenase protein in A431 cells.

[A case of erythroleukemia associated with lung aspergilloma successfully treated with continuous drip infusion of amphotericin B].

In September 1990, a 55-year-old female with erythroleukemia was treated with enocitabine, mitoxantrone, vincristine, and etoposide. Despite prophylaxis of infectious diseases by oral administration of 2,400 mg/day amphotericin B (AMPH), 600 mg/day ofloxacin, and 1,500 mg/day kanamycin, pneumonia with refractory pyrexia appeared and developed cystic lesions with air crescent signs thereafter. Finally, the cystic one formed fungus balls. The pneumonia was diagnosed as aspergillus pneumonia by fungus growth in the tissue in the transbronchial lung biopsy specimens and by an elevation of serum anti-Aspergillus antibody. The patient had continuously been administered with AMPH for 16 days, increasing the drug doses every 2 days. The maximum plasma level of AMPH rose up to 0.78 micrograms/ml, the total amount up to 166 mg. The fungus balls disappeared completely without adverse effects except a transient decrease of plasma potassium level. Pharmacological studies had been reported that tissue AMPH levels elevated more than twice as much as that of the plasma. Although the maximum plasma level was less than that of MIC for Aspergillus, the lung tissue drug level was suspected to have been maintained higher by continuous drip infusion. These findings indicate that continuous drip infusion of AMPH is one of the useful treatment for lung aspergillosis.

Activity and protein distribution of 12-lipoxygenase in HEL cells: induction of membrane-association by phorbol ester TPA, modulation of activity by glutathione and 13-HPODE, and Ca(2+)-dependent translocation to membranes.

The understanding of the intracellular regulation of 12-lipoxygenase requires a knowledge of the distribution of both enzyme protein and its activity. In human erythroleukemia cells, the membrane fraction contains about 90% of the total cellular 12-lipoxygenase activity, whereas only approximately 10% of 12-lipoxygenase activity resides in the cytosol. However, the majority of the cellular 12-lipoxygenase protein is found in the cytosol. Pretreatment of cells for 0-3 days with 160 nM TPA caused a marked, time-dependent increase in membrane-bound 12-lipoxygenase activity and protein, respectively. In contrast, the cytosolic amount of 12-lipoxygenase protein and activity, respectively, were minimally altered by this TPA treatment. Recombining the active membrane fraction with cytosol resulted in no significant inhibition of its 12-lipoxygenase activity, but the addition of GSH to the membrane fraction inhibited 12-lipoxygenase activity in a dose-dependent manner. On the other hand, the cytosolic enzyme can be rendered active in the presence of 1 microM 13-hydroperoxyoctadecadienoic acid. In HEL cell homogenates, a partial translocation of the cytosolic enzyme to the membrane takes place in a Ca(2+)-dependent manner, resulting in an increase in membrane-associated 12-lipoxygenase activity and a concomitant decrease in cytosolic 12-lipoxygenase activity above 0.1 microM Ca2+.

[Anesthetic management of a patient with Bernard-Soulier syndrome].

Bernard-Soulier syndrome (BSS) is a hereditary hemorrhagic disease characterized by prolonged bleeding time due to abnormal platelet aggregation and giant platelets. Transfusion of platelet-rich plasma is the only treatment available for the hemorrhagic episodes in patients with this disease. A 20-year-old female with BBS was scheduled for sagittal osteotomy of the mandibular rami under general anesthesia. Anesthesia was induced with fentanyl and diazepam, and was maintained with nitrous oxide, fentanyl, and 0.5% enflurane. No exacerbation of the bleeding tendency was observed during or after the surgery. We consider that the use of halothane should be avoided in patients with BBS because it may inhibit the aggregation of platelets and prolong the bleeding time.

Evaluation of oral aclarubicin treatment for tumors of the gastrointestinal track.

Aclarubicin (ACR), a drug useful for the treatment not only of tumors of the hematopoietic system but also of those of the gastrointestinal tract, has been administered invariably by the intravenous route. We attempted oral administration of ACR to increase its specificity in the treatment of gastrointestinal tumors. Oral administration resulted in a much higher intratumor concentration but lower peripheral blood cell or bone marrow cell concentration of the drug as compared with intravenous administration. The reduced peripheral cell inhibition and bone marrow suppression permits the administration of the drug in higher doses, and the absence of the drug in the plasma suggests a reduced likelihood of myocardial cell injury and hepatocyte impairment. These favorable findings are considered to warrant early clinical trial of the treatment.

[Idiopathic plasmacytic lymphadenopathy with polyclonal hyperimmunoglobulinemia (IPL) accompanied with severe thrombocytopenia and interstitial pneumonitis].

A case of generalized lymphadenopathy with severe thrombocytopenia and marked polyclonal hypergammaglobulinemia is reported. A 51-year-old man was admitted to the hospital because of nasal bleeding and generalized lymphadenopathy. Laboratory examination revealed a platelet count of 6,000/microliters, a total protein level of 11.6 g/dl, and a gammaglobulin level of 8.7 g/dl including 6,726 mg/dl IgG, 1,119 mg/dl IgA, 265 mg/dl IgM, 265 mg/dl IgD and 226 IU/ml IgE. Neither definite evidence suggesting viral infection nor an autoimmune disease was found except slightly elevated titers of anti-microsome and anti-thyroglobulin antibodies. The chest X-ray revealed diffuse reticular shadowing suggesting interstitial pneumonitis. The cervical lymph node biopsy revealed massive infiltration by IgG-, IgA-, and IgM-positive plasmocytoid cells in the paracortical T cell areas, but no destruction of normal architecture of lymph node. The treatment with prednisolone resulted in a decrease in the severity of the lymphadenopathy, thrombocytopenia and polyclonal hypergammopathy. He is still well; however, interstitial pneumonitis, mild thrombocytopenia, and polyclonal hypergammopathy have persisted for 4 years. The histological findings in this case are similar to those in the case of Castleman lymphoma of a plasma cell type. Thus, considering the clinical data and clinical course, the diagnosis in this case is likely to be idiopathic plasmacytic lymphadenopathy with polyclonal hyperglobulinemia as a subtype of the plasma cell type of Castleman lymphoma. However, the accompanying interstitial pneumonitis and the severe thrombocytopenia, which was caused by excessive platelet destruction or by the suppression of megakaryocytes by proliferated plasma cells, are unique and are not found in any of the previously reported cases.

[Clinical effect and pharmacokinetics of intermediate dose Ara-C therapy in a patient with acute non-lymphocytic leukemia with two CNS recurrences].

A case of two repeated CNS recurrences of acute non-lymphocytic leukemia (M2) was treated with intermediate dose Ara-C therapy and achieved 2 complete remissions. The clinical effect and pharmacokinetics of intermediate dose Ara-C therapy in this patient were discussed. A 55-year-old male with acute non-lymphocytic leukemia (M2) achieved complete remission by combination chemotherapy of Behenoyl-ara-C, Daunorubicin, 6-Mercaptopurine and Prednisolone in July, 1985. He subsequently received consolidation and intensification therapy with periodical intrathecal injection of Methotrexate (MTX), but 13 months later he developed his first CNS recurrence which was resistant to the intrathecal administration of Ara-C and MTX. As he also relapsed systemically, Ara-C was administered in intermediate dose (1 g/m2 every 12 hrs for 5 days) and he achieved complete remission both in the CNS and systemic manifestations. Six months later he was diagnosed as having a second CNS recurrence and another systemic relapse. Intermediate dose Ara-C was administered again, and he achieved complete remission in the CNS and partial remission in systemic manifestations. Pharmacokinetic study revealed high peaks of Ara-C concentration in plasma (6.2 microM immediately after the end of the infusion) and high degree of its penetration into the CNS (5.6 microM at 3 hr after the end of the infusion) suggesting the effective and perhaps a uniform level of Ara-C is achieved throughout the CNS by this therapy. In 3 other patients without CNS involvement 0.88 +/- 0.44 microM of Ara-C, which is enough concentrations for its cytostatic effect, was detected at 3 hr after the end of infusion, suggesting the efficacy of the therapy for CNS prophylaxis. In this case the relapse occurred after repeated administration of antileukemic drugs, including Behenoyl-ara-C, an analog of Ara-C, and was resistant to the intrathecal administration of Ara-C. These findings suggest that intermediate dose Ara-C therapy was effective to overcome a resistance to antileukemic drugs, including Ara-C, and also, in some cases, more effective than intrathecal injection of antileukemic drugs for the treatment of CNS leukemia.

Clinical pharmacology of N4-palmitoyl-1-beta-D-arabinofuranosylcytosine in patients with hematologic malignancies.

The pharmacokinetics of oral N4-palmitoyl-1-beta-D-arabinofuranosylcytosine (PLAC), a lipophilic and deaminase-resistant derivative of 1-beta-D-arabinofuranosylcytosine (ara-C), were determined in patients with hematologic malignancies. The concentration of ara-C and 1-beta-D-arabinofuranosyluracil (ara-U), metabolites of PLAC, were measured by radioimmunoassay and gas chromatography-mass spectrometry-mass fragmentography, respectively. The concentration of PLAC was determined by measuring ara-C, which was derived from PLAC by hydrolyzation. In six patients given an oral bolus of PLAC (300 mg/m2), the plasma-disappearance curve of PLAC corresponded to a one-compartment open model, including first-order absorption. The peak plasma level was 22.9 +/- 6.4 ng/ml, and the predicted time to reach the peak level was 2.5 +/- 1.0 h. The elimination half-life was 3.8 +/- 2.7 h. The plasma ara-C level increased slowly to 6.9 ng/ml during the 1st 2-3 h after administration and remained over 1.0 ng/ml for 12 h. Plasma ara-U was detectable for at least 24 h, with a peak concentration of 376 ng/ml at 6 h. Urinary PLAC excretion was below the limit of detection (5 ng/ml) in all cases. Prolonged urinary ara-C and ara-U excretion was detected, but the total recovery rate was low (6.7% in 24 h) and varied between patients. In spite of the lipophilic nature of the drug, the PLAC concentration in the cerebrospinal fluid, measured at 3 or 6 h, was below the limit of detection in all four patients with no meningeal involvement. This study showed low but persistent levels of PLAC in plasma and tissues, with a continuous release of small amounts of ara-C, which demonstrated antitumor activity in patients with hematologic malignancies.

Reverse effect of guanine on the inhibitory action of mycophenolic acid during nucleic acid synthesis.

Mycophenolic acid (MPA) was demonstrated to inhibit DNA and RNA synthesis in L1210 cells strongly; however these effects were remarkably reduced by guanine. The presence of MPA in the medium decreased the guanine nucleotide contents (GMP, GDP, GTP) of the cells, but the addition of guanine reversed this effect. We have reported previously that MPA had no inhibitory effect on hypoxanthine guanine phosphoribosyltransferase (HGPRTase) activity. Together these findings suggest that the decrease of guanine nucleotides induced by MPA is restored by GMP, which is formed from guanine by HGPRTase in the cells. It is speculated that a suppressor of HGPRTase activity, such as 6-mercaptopurine, may protect the antitumor activity of MPA by preventing the conversion of guanine to GMP.

Pharmacokinetics of aclarubicin and its metabolites in humans and their disposition in blood cells.

The pharmacokinetics of aclarubicin, a new anthracycline antibiotic, was studied in five patients with acute leukemia or in L1210 cell suspension. Aclarubicin disappeared very rapidly from plasma and whole blood after administration at a dose of 20 mg per patient by iv bolus injection. The concentration of active metabolite M1, on the other hand, increased for up to 2 or 4 hrs after administration and exceeded that of aclarubicin, and then remained at much higher concentrations than aclarubicin for up to 24 hrs after administration. In addition, the levels of aclarubicin and its metabolites in whole blood were much higher than the corresponding plasma levels in four of the patients. The drug concentrations in blood cells of 11 patients determined 4 hrs after administration showed a significant positive correlation with leukocyte counts. Moreover, the concentration of aclarubicin and its metabolites was found to be much higher in the leukocyte fraction than in the erythrocyte fraction in vivo and in vitro. These findings indicate that aclarubicin and its metabolites in blood cells were mainly accumulated in leukocytes. In the study of intracellular drug distribution in L1210 cells, the largest amount of aclarubicin was incorporated into the nuclear fraction. This suggests a close relationship between the pronounced drug accumulation in leukocytes and the high affinity of aclarubicin for DNA.