Raloxifene enhances spontaneous microaggregation of platelets through upregulation of p44/p42 MAP kinase: a case report.

UNLABELLED: A 60-year-old postmenopausal woman diagnosed as primary osteoporosis began to take raloxifene. The spontaneous microaggregates of platelets induced by shear stress were accelerated after the treatment, concomitant with the significant upregulation of p44/p42 mitogen-activated protein (MAP) kinase induced by adenosine diphosphate (ADP). After the cessation of raloxifene, the spontaneous microaggregates of platelets and the acceleration of ADP-induced p44/p42 MAP kinase phosphorylation was diminished. We concluded that raloxifene caused platelet hyperaggregability to shear stress and p44/p42 MAP kinase was involved in the pathological state. INTRODUCTION: A 60-year-old postmenopausal woman suffering from severe lumbago was diagnosed as primary osteoporosis with combined vertebral fractures. After the acute phase, she began to take 60 mg daily of oral raloxifene. The spontaneous microaggregates of platelets induced by shear stress were accelerated significantly after 8 weeks from the beginning of raloxifene treatment and observed at 12 weeks. RESULTS: The platelet aggregation induced by ADP was little changed; however, low doses (0.3 and 1 microM) of ADP significantly induced the phosphorylation of p44/p42 MAP kinase in the platelets obtained at 12 weeks. Although there were few subjective complaints except for paroxysmal headache, the medication was stopped with her consent to avoid any adverse effects. The spontaneous microaggregates of platelets gradually decreased after the cessation of medication. At 12 weeks after the cessation, the phosphorylation of p44/p42 MAP kinase induced by low doses of ADP was no more observed. CONCLUSION: These results strongly suggest that raloxifene caused platelet hyperaggregability to shear stress and subclinical thrombus formation in this case and that p44/p42 MAP kinase was involved in the pathological state.

Patch technique for repair of a dural tear in microendoscopic spinal surgery.

A dural tear is a common but troublesome complication of endoscopic spinal surgery. The limitations of space make repair difficult, and it is often necessary to proceed to an open operation to suture the dura in order to prevent leakage of cerebrospinal fluid. We describe a new patch technique in which a small piece of polyglactin 910 is fixed to the injured dura with fibrin glue. Three pieces are generally required to obtain a watertight closure after lavage with saline. We have applied this technique in seven cases. All recovered well with no adverse effects. MRI showed no sign of leakage of cerebrospinal fluid.

Proliferation and apoptosis of tumour cells before and after neoadjuvant therapy for high-grade extremity sarcomas: divergent associations with tumour response and prognosis.

AIMS: To evaluate proliferation and apoptosis in high-grade sarcomas of the extremities before and after preoperative radio-hyperthermo-chemotherapy (RHC) and to determine the relationship between these parameters and treatment outcomes. METHODS AND RESULTS: Pre- and post-RHC specimens of 41 soft tissue and bone tumours were immunohistochemically stained for minichromosome maintenance protein (MCM) 2 and caspase 3 as proliferation and apoptosis markers, respectively, based on a preliminary study comparing them with conventional markers. Indices were calculated as a percentage of positive cells by counting tumour cells in the most frequently labelled areas. MCM2, caspase 3 and MCM2/caspase 3 (growth) indices were 45.3 +/- 21.9%, 4.1 +/- 7.1% and 82.9 +/- 104.5, respectively, in pre-RHC specimens and 35.4 +/- 30.8%, 39.2 +/- 34.6% and 5.3 +/- 11.7, respectively, in post-RHC specimens. Response scores showed positive correlation with pre-RHC MCM2 and post-RHC caspase 3 indices, inverse correlation with post-RHC MCM2 and post-RHC growth indices and no correlation with prognosis. Multivariate analysis revealed high pre-RHC MCM2 and high post-RHC growth indices as significant unfavourable prognostic factors. CONCLUSIONS: High proliferative activity in untreated sarcoma may predict good response to neoadjuvant therapy, but poor prognosis, whereas a high growth index, i.e. high proliferation:apoptosis ratio in a post-neoadjuvant therapy tumour specimen may indicate poor response and poor prognosis.

Surgical treatment of the rheumatoid cervical spine in patients aged 70 years or older.

OBJECTIVE: To investigate whether surgery is appropriate for elderly rheumatoid arthritis patients who are already approaching their statistical life expectancy. PATIENTS AND METHODS: The subjects were 10 patients who underwent cervical spine surgery for rheumatoid arthritis at an age of over 70 yr. The pain grade and neurological deficit class according to Ranawat, peri-operative complications, causes of death and pre-operative cardiopulmonary function were assessed. RESULTS: Good pain relief was achieved. Relief of pain enabled the patients, who could not sit up even in bed because of intolerable neck pain, to ride in a wheel chair without using a neck collar. Only one death was related to surgery. Pre-operative cardiopulmonary function was not significantly different compared with that of elderly patients undergoing other surgical procedures. CONCLUSION: Surgery is a valuable option for the management of elderly patients with rheumatoid cervical spine since it can improve the quality of life.

Synthesis, characterization, and photochemical properties of azobenzene-conjugated Ru(II) and Rh(III) bis(terpyridine) complexes.

We synthesized azobenzene-conjugated bis(terpyridine) Ru(II) and Rh(III) mononuclear and dinuclear complexes and investigated their photochemical properties on excitation of the azo pi-pi band upon 366 nm light irradiation. The Ru mononuclear complex underwent trans-to-cis photoisomerization to reach the photostationary state with only 20% of the cis form, while the Ru dinuclear complex did not isomerize at all photochemically. On the other hand, the mononuclear and dinuclear Rh complexes showed almost complete trans-to-cis photoisomerization behavior. Cis forms of the Rh complexes thermally returned to the trans form at a much slower rate than those of organic azobenzenes, but they did not isomerize photochemically. The reduction potential of the cis forms was 80 mV more negative than that of the trans forms. The photoisomerization quantum yields of the Rh complexes were strongly dependent on the polarity, viscosity, and donor site of the solvents as well as the size of the counterions. We investigated the photoisomerization process of these complexes using femtosecond absorption spectroscopy. For the Rh complexes, we observed S(n) <-- S(2) and S(n) <-- S(1) absorption bands similar to those of organic azobenzenes. For the Ru complexes, we observed very fast bleaching of the MLCT band of the Ru complex, which indicated that the energy transfer pathway to the MLCT was the primary cause of the depressed photoisomerization. The electronic structures, which were estimated from ZINDO molecular orbital calculation, supported the different photochemical reaction behavior between the Ru and Rh complexes.

Unique mutations in mitochondrial DNA of senescence-accelerated mouse (SAM) strains.

Mitochondrial DNA (mtDNA) is exclusively inherited maternally and hence could offer a good method for tracing the lineage of mouse strains. We examined the mtDNA sequence of senescence-accelerated mouse (SAM) strains as well as other laboratory strains of inbred mice to deduce the ancestral strain of SAM. Four unique mutations were identified at bases 2256, 10,847, 11,181, and 13,053 in SAM strains. The mutations were not found in other mouse strains including AKR/J, one of the parental strains of SAM. Comparison of the mtDNA sequences also led to the consensus mtDNA sequence of laboratory strains of inbred mice. The seven laboratory strains of common inbred mice showed polymorphisms at base 9348, thymine repeat from base 9818, and adenine repeat from base 9821, and could be classified into five types by combination of the differences. Although we could not identify mouse strains with the same type of mtDNA as SAM in this study, the polymorphisms would provide a promising clue to ascertain the ancestral strain(s) of SAM. The polymorphism in mtDNA could be used to ascertain the genealogy of other mouse strains as well.

Redox-associated eta(1) to eta(2) conversion of disulfide ligands in dinuclear ruthenium complexes.

Disulfide-bridged dinuclear ruthenium complexes [[Ru(MeCN)(P(OMe)(3))(2)](2)(mu-X)(mu,eta(2)-S(2))][ZnX(3)(MeCN)] (X = Cl (2), Br (4)), [[Ru(MeCN)(P(OMe)(3))(2)](2)(mu-Cl)(2)(mu,eta(1)-S(2))](CF(3)SO(3)) (5), [[Ru(MeCN)(P(OMe)(3))(2)](2)(mu-Cl)(mu,eta(2)-S(2))](BF(4)) (6), and [[Ru(MeCN)(2)(P(OMe)(3))(2)](2)(mu-Cl)(mu,eta(1)-S(2))](CF(3)SO(3))(3) (7) were synthesized, and the crystal structures of 2 and 4 were determined. Crystal data: 2, triclinic, P1, a = 15.921(4) A, b = 17.484(4) A, c = 8.774(2) A, alpha = 103.14(2) degrees, beta = 102.30(2) degrees, gamma = 109.68(2) degrees, V = 2124(1) A(3), Z = 2, R (R(w)) = 0.055 (0.074); 4, triclinic, P1 a = 15.943(4) A, b = 17.703(4) A, c = 8.883(1) A, alpha = 102.96(2) degrees, beta = 102.02(2) degrees, gamma = 109.10(2) degrees, V = 2198.4(9) A(3), Z = 2, R (R(w)) = 0.048 (0.067). Complexes 2 and 4 were obtained by reduction of the disulfide-bridged ruthenium complexes [[RuX(P(OMe)(3))(2)](2)(mu-X)(2)(mu,eta(1)-S(2))] (X = Cl (1), Br (3)) with zinc, respectively. Complex 5 was synthesized by oxidation of 2 with AgCF(3)SO(3). Through these redox steps, the coordination mode of the disulfide ligand was converted from mu,eta(1) in 1 and 3 to mu,eta(2) in 2 and 4 and further reverted to mu,eta(1) in 5. Electrochemical studies of 6 indicated that similar conversion of the coordination mode occurs also in electrochemical redox reactions.

Possible role of xanthobaccins produced by Stenotrophomonas sp. strain SB-K88 in suppression of sugar beet damping-off disease.

Three antifungal compounds, designated xanthobaccins A, B, and C, were isolated from the culture fluid of Stenotrophomonas sp. strain SB-K88, a rhizobacterium of sugar beet that suppresses damping-off disease. Production of xanthobaccin A in culture media was compared with the disease suppression activities of strain SB-K88 and less suppressive strains that were obtained by subculturing. Strain SB-K88 was applied to sugar beet seeds, and production of xanthobaccin A in the rhizosphere of seedlings was confirmed by using a test tube culture system under hydroponic culture conditions; 3 microg of xanthobaccin A was detected in the rhizosphere on a per-plant basis. Direct application of purified xanthobaccin A to seeds suppressed damping-off disease in soil naturally infested by Pythium spp. We suggest that xanthobaccin A produced by strain SB-K88 plays a key role in suppression of sugar beet damping-off disease.

Cysteine-S-conjugate beta-lyase activity and pyridoxal phosphate binding site of onion alliin lyase.

Purification of onion alliin lyase gave two fractions by cation exchange chromatography. Both fractions showed the comparable high catalytic activity of cysteine-S-conjugate beta-lyase with that of alliin lyase using S-(2-chloro-6-nitrophenyl)-L-cysteine and alliin, S-allyl-L-cysteine sulfoxide as substrates. All the active substrates tested with onion alliin lyase were also active to the cysteine-S-conjugate beta-lyase of Mucor javanicus, but the catalytic activity of the Mucor enzyme was lower for all the substrates. The pyridoxal phosphate binding site of the onion alliin lyase was identified as Lys 285 in the amino acid sequence deduced from cDNA which has been reported. This lysine was conserved in all the sequences from the alliin lyase cDNAs, while similarity was not found between the sequences around pyridoxal phosphate binding sites of both the onion alliin lyase and the Mucor cysteine-S-conjugate beta-lyase.

Bilateral pseudo-internuclear ophthalmoplegia in myasthenia gravis.

A 38-year-old male with bilateral pseudo-internuclear ophthalmoplegia (-INO) in myasthenia that could have been misdiagnosed as INO in multiple sclerosis is reported. He experienced fluctuating symptoms including double vision, imbalance, and tinnitus. His eye movements simulated bilateral INO, with a downshoot in abduction. After thymectomy, his eye movements became normal. From our case and a review of the literature, we propose that ptosis, downshoot, and fatigability are likely to be signs of pseudo-INO in myasthenia, whereas an impaired vertical smooth pursuit is unlikely. Dissociated nystagmus and monocular overshoot might be the results of central compensation.

Inhibiting Effects of Lunularic Acid Analogs on the Growth of Liverwort, Watercress, and Timothy Grass.

A number of analogs of lunularic acid varying in the number of methylene carbons between the two benzene rings and in the substituents on their rings were prepared, and their effects on the growth of liverwort gemmaling, watercress, and timothy grass were investigated. Almost all the analogs tested were more inhibitory than lunularic acid, and a correlation between the structure and activity was observed. The differences in the growth-inhibition activity of analogs between higher and lower plants are also discussed.

Plant annexin form homodimer during Ca(2+)-dependent liposome aggregation.

The annexin (p35) was isolated from the fruits of green pepper (Capsicum annum). The partial amino acid sequence of p35 was analyzed. p35 had an endonexin fold as annexin consensus sequences. Purified p35 had other annexin like characters such as strongly bind to phosphatidylserine and phosphatidylinositol, phospholipase A2 inhibition and liposome aggregation. The zero-length crosslinking assay revealed that p35 formed a homodimer during Ca(2+)-dependent liposome aggregation.

Molecular cloning of a cDNA clone encoding a phosphoprotein component related to the Ig receptor-mediated signal transduction.

We have shown previously that a 52-kDa phosphoprotein (p52) co-precipitated with Ig receptor (IgR)-associated MB-1 protein was inducibly phosphorylated by the stimulation with 12-O-tetradecanoyl-phorbol-13-acetate. By immunizing the 52-kDa protein co-precipitated with MB-1, we prepared a mAb (19-14), which can immunoprecipitate the p52 and the associated kinase molecule. Immune complex kinase assay with the 19-14 mAb showed that the p52 is associated with a novel kinase molecule and is involved in IgR-mediated signal transduction. Here we isolated a cDNA clone (alpha 4) from a murine bone marrow cDNA library in lambda gt-11 vector by the 19-14 mAb. The alpha 4 cDNA encodes a novel protein of 340 amino acids with multiple potential phosphorylation sites. The 1.4-kb alpha 4 mRNA is expressed in various cells including cell lines of B lineage, T lineage, monocytic, fibroblast, and normal organs such as liver, spleen, thymus, and brain. To study the molecule encoded by the alpha 4 cDNA, we produced a chimeric protein of the glutathione S transferase (GST)-alpha 4 in pGEX-3X expression vector. The 19-14 mAb binds to the GST-alpha 4 fusion protein. Rabbit anti-alpha 4 Ab prepared by immunizing the GST-alpha 4 fusion protein immunoprecipitated a 52-kDa phosphoprotein from WEHI-231 cells. The 52-kDa phosphoprotein immunoprecipitated by the anti-alpha 4 Ab is tightly associated with kinase activity that is similarly observed with the p52 protein reported previously. Moreover, the 52-kDa phosphoprotein immunoprecipitated with the anti-alpha 4 Ab is identical to the p52 by the protein comparison on non-equilibrium pH gradient gel electrophoresis/SDS-PAGE, isoelectric focusing/SDS-PAGE, and V8 protease mapping. The 52-kDa phosphoprotein is associated with functional components that are inducibly phosphorylated by anti-IgM stimulation. These results suggested that the alpha 4 gene-encoded molecule is functionally involved in the IgR-mediated signal transduction in B cells.

Antimicrobial stress compounds from Hypochoeris radicata.

The exudates of Hypochoeris radicata leaves stressed with cupric chloride afforded two eudesmane- and guaiane-type sesquiterpenes, and two alkenals, (2E,4E)-6-hydroxyhexadienal and (2E,4E)-hexadienedial (mucondialdehyde). Their structures were determined by spectroscopic and synthetic methods.

An enzymatic formation of 13-oxo-trideca-9,11-dienoic acid from 13-hydroperoxylinolenic acid by a homolytic hydroperoxide lyase in elicitor-treated soybean cotyledons.

An activity of homolytic hydroperoxide lyase (HPLS) catalyzing the specific cleavage of 13-hydroperoxylinolenic acid to form a volatile compound and 13-oxo acid was found in the enzyme extract from soybean cotyledons. 2-Penten-1-ol was characterized as a volatile metabolite by gas chromatography-mass spectrometry analysis. The methyl ester derivatives of reaction products were separated and isolated by a normal-phase high-performance liquid chromatography, and identified to be 13-oxo-trideca-9(Z),11(E)-dienoic acid methyl ester and its geometric isomer possessed 9(E),11(E) moiety by the analyses of high resolution mass spectrometry and nuclear magnetic resonance spectrometry. An activity of homolytic HPLS found in soybean cotyledons was evidently enhanced by elicitation. The products of 13-oxo-tridecadienoic acid with alpha, beta, gamma, delta-unsaturation were shown to be antifungal substances by a chromatographic bioassay. The metabolites via lipoxygenase and HPLS pathways may have physiological roles in the resistant action of host plants.

Variations in concentrations of bacterial metabolites, enzyme activities, moisture, pH and bacterial composition between and within individuals in faeces of seven healthy adults.

Variations between and within individuals, and correlations between concentrations of bacterial metabolites, including putrefactive products, ammonia and short chain fatty acids (SCFAs), enzyme activities, moisture and pH, as well as bacterial composition, were studied in faecal samples from seven healthy adults over a period of 7 months. Large variations, both between and within individuals, were observed in concentrations of putrefactive products. Although values for ammonia, SCFAs, enzyme activities, moisture and pH were generally variable, significant person-to-person differences were observed. While ranges of log viable counts of the predominant bacteria such as eubacteria, bifidobacteria and bacteroides in each subject remained between 0.2 and 1.3, those of enterobacteria, streptococci (including enterococci) and lecithinase-negative clostridia varied between 0.4 and 3.0. Levels of bifidobacteria, enterobacteria, streptococci and total aerobic bacteria showed inter-individual variations. Correlations were found among certain of the parameters: moisture correlated negatively with p-cresol (r = -0.707), pH (r = -0.671) and beta-glucosidase activity (GS) (r = -0.608), and positively with acetic acid (r = 0.621), while negative correlations were observed in pH with acetic and butyric acids (r = -0.690 and -0.623, respectively). No significant correlations were found between bacterial compositions, and other faecal factors such as pH, moisture, metabolic enzyme activities and concentrations of putrefactive products.

Prenylated flavonoids in the roots of yellow lupin.

A further investigation of the methanol-soluble compounds in yellow lupin roots has revealed a new diprenylchromone, a new coumaronochromone (lupinalbin H), a new isoflavone 5,7,4'-trihydroxy-8,3'-di-(3,3-dimethylally)isoflavone (isolupalbigenin), and some complex flavanones. The latter compounds have been identified as two known diprenylated flavanones (lonchocarpol A and euchrestaflavanone A), two diasteroisomeric pairs of dihydrofuranoflavanones (lonchocarpols C1 and C2, and lonchocarpols D1 and D2, the structures formerly proposed for lonchocarpols C and D were also reinvestigated), a new furanoflavanone (lupinenol), and three 8-prenylflavanones with an additional (2RS)-hydroxy-3-methyl-3-butenyl side chain. The structures of the latter flavanones were unambiguously identified by spectroscopic (1H NMR) comparison with 6-, 8- and 3'-prenylnaringenins chemically prepared from (2S)-naringenin. The antifungal activity of the prenylated naringenins, and of the various yellow lupin flavanones, was determined by TLC place bioassays using Cladosporium herbarum as the test fungus.

Identification of mucondialdehyde as a novel stress metabolite.

In a survey of antifungal stress compounds induced by cupric chloride we found that leaves of Chenopodium album exuded a highly fungitoxic metabolite mucondialdehyde (trans-2,trans-4-hexadienedial), which was associated with 13-oxo-9,11-tridecadienoic acids (cis-9,trans-11 and trans-9,trans-11 isomers) presumably resulting from beta-scission of 13-hydroperoxy-octadecadi(tri)enoic acid. The biogenesis and role as a general defensive agent in plants are briefly discussed.

Cloning of a DNA Fragment Carrying the 4-Hydroxycinnamate Decarboxylase (pofK) Gene from Klebsiella oxytoca, and Its Constitutive Expression in Escherichia coli JM109 Cells.

The 4-hydroxycinnamate decarboxylase gene (pofK gene) was cloned from Klebsiella oxytoca, an epiphytic bacterium able to decarboxylate hydroxycinnamic acids to styrene derivatives, in Escherichia coli JM109. Colonies of the enzyme activity-positive transformants were screened by a selection assay combined with an antifungal test using Cladosporium herbarum IHU9262 as the bio-indicator. Two positive transformants constitutively producing 4-hydroxycinnamate decarboxylase were obtained. One of the transformants had a recombinant plasmid designated as pTCD100 in which a 9.6 kb HindIII segment carrying pofK gene was contained. As the expression of the pofK gene in K. oxytoca was substrate-inducible, it was most likely that the pofK gene expression in E. coli cells was free from any regulation by a pofK gene repressor postulated in K. oxytoca cells. The decarboxylase synthesized in E. coli cells showed almost the same specific activity as that of K. oxytoca induced by the substrate.