Association of ghrelin receptor gene polymorphism with bulimia nervosa in a Japanese population.

Eating disorders (EDs) have a highly heterogeneous etiology and multiple genetic factors might contribute to their pathogenesis. Ghrelin, a novel growth hormone-releasing peptide, enhances appetite and increases food intake, and human ghrelin plasma levels are inversely correlated with body mass index. In the present study, we examined the 171T/C polymorphism of the ghrelin receptor (growth hormone secretagogue receptor, GHSR) gene in patients diagnosed with EDs, because the subjects having ghrelin gene polymorphism (Leu72Met) was not detected in a Japanese population, previously. In addition, beta3 adrenergic receptor gene polymorphism (Try64Arg) and cholecystokinin (CCK)-A receptor (R) gene polymorphism (-81A/G, -128G/T), which are both associated with obesity, were investigated. The subjects consisted of 228 Japanese patients with EDs [96 anorexia nervosa (AN), 116 bulimia nervosa (BN) and 16 not otherwise specified (NOS)]. The age- and gender-matched control group consisted of 284 unrelated Japanese subjects. The frequency of the CC type of the GHSR gene was significantly higher in BN subjects than in control subjects (chi(2) = 4.47, p = 0.035, odds ratio = 2.05, Bonferroni correction: p = 0.070), while the frequency in AN subjects was not different from that in controls. The distribution of neither beta3 adrenergic receptor gene nor CCK-AR polymorphism differed between EDs and control subjects. Therefore, the CC type of GHSR gene polymorphism (171T/C) is a risk factor for BN, but not for AN.

Flow cytometric studies of the host-regulated cell cycle in algae symbiotic with green paramecium.

Paramecium bursaria (green paramecium) possesses endosymbiotically growing chlorella-like green algae. An aposymbiotic cell line of P. bursaria (MBw-1) was prepared from the green MB-1 strain with the herbicide paraquat. The SA-2 clone of symbiotic algae was employed to reinfect MBw-1 cells and thus a regreened cell line (MBr-1) was obtained. The regreened paramecia were used to study the impact of the host's growth status on the life cycle of the symbiotic algae. Firstly, the relationship between the timing of algal propagation and the host cell division was investigated by counting the algal cells in single host cells during and after the host cell division and also in the stationary phase. Secondly, the changes in the endogenous chlorophyll level, DNA content, and cell size in the symbiotic algae were monitored by flow cytometry and fluorescence microscopy. The number of algae was shown to be doubled prior to or during the host cell division and the algal population in the two daughter cells is maintained at constant level until the host cell cycle reenters the cytokinesis, suggesting that algal propagation and cell cycle are dependent on the host's cell cycle. During the host's stationary growth, unicellular algal vegetatives with low chlorophyll content were dominant. In contrast, complexes of algal cells called sporangia (containing 1-4 autospores) were present in the logarithmically growing hosts, indicating that algal cell division leading to the formation of sporangia with multiple autospores is active in the dividing paramecia.

Oxidative burst by acellular haemoglobin and neurotransmitters.

Acellular haemoglobin (Hb) has intrinsic toxicity to the tissues since harmful reactive oxygen species are readily produced during auto-oxidation of Hb. On the other hand, Hb is known to have peroxidase-like activity monovalently oxidizing various peroxidase substrates. Thus, monovalently oxidized organic free radical species may be produced. This may relay the radical reactions leading to the production of reactive oxygen species such as superoxide. Such substrates possibly generating superoxide, include aromatic monoamines such as neurotransmitters and their precursors rich in neural a tissues. Based on our knowledge on the reactivity of haemoproteins against phenolics and aromatic monoamines, we proposed a hindered danger in use of Hb as a reperfusion agent. Clinical use of recently developing Hb-based blood substitutes must be reconsidered.

Differential localization of non-muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC-5 fibroblasts.

We investigated the localization of non-muscle myosin II isoforms and mono- (at serine 19) and diphosphorylated (at serine 19 and threonine 18) regulatory light chains (RLCs) in motile and non-motile MRC-5 fibroblasts. In migrating cells, myosin IIA localized to the lamella and throughout the posterior region. Myosin IIB colocalized with myosin IIA to the posterior region except at the very end. Diphosphorylated RLCs were detected in the restricted region where myosin IIA was enriched. In non-motile cells, myosin IIA was enriched in peripheral stress fibers with diphosphorylated RLCs, but myosin IIB was not. Our results suggest that myosin IIA may be highly activated by diphosphorylation of RLCs and primarily involved in cell migration.

Cytotoxic activity of benzothiepins against human oral tumor cell lines.

A total of 11 newly synthesized benzothiepins and structurally-related compounds were investigated for cytotoxic activity against both normal and tumor cells. All these compounds showed higher cytotoxic activity against three human oral tumor cell lines (HSC-2, HSC-3, HSG) than against normal human gingival fibroblast (HGF), suggesting tumor-specific cytotoxic action. In general, 3,4-dihydro-1-benzothiepin-5(2H)-ones [1-6] showed higher cytotoxic activity than 2,3-dihydro-1-benzothiepins [7-11]. Compounds 4 (4-bromo-3,4-dihydro-2-(2-oxo-2-phenylethyl)-1-benzothiepin-5(2H)-one), 5 (4-bromo-3,4-dihydro-2-(2-oxopropyl)-1-benzothiepin-5(2H)-one) and 6 (4-bromo-3,4-dihydro-2-[1-(methoxycarbonyl)-1-methylethyl]-1-benzothiepin-5(2H)-one), showed higher cytotoxic activity than compounds 1, 2 and 3, respectively, which had Cl instead of Br at C-4 position. Agarose gel electrophoresis demonstrated that these compounds induced large DNA fragments in oral tumor cells, whereas they produced smear pattern of smaller DNA fragments in human promyelocytic leukemia cells HL-60. These data suggest the medicinal efficacy of benzothiepins.

Fungal auxin antagonist hypaphorine competitively inhibits indole-3-acetic acid-dependent superoxide generation by horseradish peroxidase.

Plant peroxidases (EC 1.11.1.7) including horseradish peroxidase (HRP-C), but not the nonplant peroxidases, are known to be highly specific indole-3-acetic acid (IAA) oxygenases which oxidize IAA in the absence of H2O2, and superoxide anion radicals (O2*-) are produced as by-products. Hypaphorine, a putative auxin antagonist isolated from ectomycorrhizal fungi, inhibited the IAA-dependent generation of O2*- by HRP-C, which occurs in the absence of H2O2. Hypaphorine has no effect on the nonspecific heme-catalyzed O2*- generation induced by high concentration of ethanol. It is probable that the inhibitory effect of hypaphorine on O2*- generation is highly specific to the IAA-dependent reaction. The mode of inhibition of the IAA-dependent O2*--generating reaction by hypaphorine was analyzed with a double-reciprocal plot and determined to be competitive inhibition, indicating that hypaphorine competes with IAA by binding to the putative IAA binding site on HRP-C. This implies the importance of structural similarity between hypaphorine and IAA. This work presented the first evidence for antagonism between IAA and a structurally related fungal alkaloid on binding to a purified protein which shares some structural similarity with auxin-binding proteins.

Identification of myosin II kinase from sea urchin eggs as protein kinase CK2.

Here we purified and identified a myosin II kinase from sea urchin eggs. The activity of this myosin II kinase in the egg extract was not significantly affected by Ca(2+)/calmodulin (CaM). Using sequential column chromatographies, we purified the myosin II kinase from the egg extract as a complex composed of 36- (p36) and 28-kDa (p28) proteins. Partial amino acid sequences of these two components were highly coincident with those of the alpha and beta subunits of protein kinase CK2 (formerly casein kinase II) in sea urchin eggs, respectively. To confirm that the purified myosin II kinase was CK2, we obtained a cDNA which encodes p36 from a cDNA library of sea urchin eggs. The amino acid sequence derived from the obtained cDNA showed over 70% homology to CK2 from various eukaryotes. Furthermore, recombinant p36, as well as the purified myosin II kinase, phosphorylated MRLC. One dimensional phosphopeptide mapping revealed that the phosphorylation site(s) of MRLC by both recombinant p36 and the purified myosin II kinase was identical. These clearly showed that the Ca(2+)/CaM-independent myosin II kinase activity in sea urchin eggs was identical to CK2.

Lewis acid-promoted cycloaddition reaction of cyclopropanes with allylsilanes.

The treatment of cyclopropanes having donor and acceptor substituents at the vicinal positions on the cyclopropane ring with a Lewis acid readily generates a 1,3-zwitterion, which reacted with allylsilanes to produce cycloadducts and allylic products. It was found that the yield of the cycloadduct depends on the steric demand of the alkyl substituents on the silicon atom.

Efficient generation of the cartesian coordinates of truncated icosahedron and related polyhedra.

Efficient algorithms for deriving the analytical expressions of the rectangular coordinates of the vertices of regular polyhedra and truncated icosahedron inscribed in a cube is described and the results are exposed. Various characteristic quantities of the geometrical structure of truncated icosahedron are obtained. Kaleidoscopes for projecting the truncated icosahedron are discussed.

Model for regulation of non-muscle myosins.

Diverse morphological changes of non-muscle eukaryotic cell are usually accompanied by dynamic remodeling in its cytoskeleton. The conformation/functional state of myosins is presumed to be markedly altered during cytoskeletal reorganization. Although myosins play critical roles in various forms of cellular movement and shape changes, the molecular basis for regulation of their activity is complicated and far from understood. A numerous experimental data show the phosphorylatable light chain (20 kDa), known as a regulatory light chain (RLC), as one of the key modulators of regulatory signals in the structure of myosin-II molecule. However, the clue that coordinates its phosphorylation state remains to be enigmatic. In the present work, we propose the most rationale mechanism which might regulate the state of RLC phosphorylation, and therefore, might control organization and activity of non-muscle myosins.

Graph-theoretical analysis of tunneling electron transfer in large polycyclic aromatic hydrocarbon networks.

Effect of a single nitrogen atom substitution to a number of large polycyclic aromatic hydrocarbon (PAH) molecules was calculated systematically, and it was found that especially in parallelogram-type PAH abnormal electron transfer (called tunneling electron transfer, TET) was observed. That is, fairly large amount of pi-electron is withdrawn to an electronegative nitrogen atom from almost the farthest end of a conjugated aromatic hydrocarbon molecule, leaving almost no change in the interior of the molecule. This change can be simulated by the Kekulé structure counting for subgraphs of the parent molecule.

Growth kinetics of algal populations exsymbiotic from Paramecium bursaria by flow cytometry measurements.

BACKGROUND: The ciliate Paramecium bursaria normally exists as a green paramecium system because each animal cell carries several hundred, unicellular, green, algal cells in its cytoplasm. One of the remarkable and poorly understood pecularities of this system is the steady state in the number of algae per protozoan cell. A major point in the study of mechanisms governing the persistence of symbiont numbers is adequate understanding of the algal life cycle. METHODS: Asynchronously growing cell populations of several algal strains (SA-1, SA-3, and SA-9) exsymbiotic from P. bursaria were characterized by flow cytometry. Algal endogenous chlorophyll and DNA contents were monitored to analyze cell growth kinetics at logarithmic and stationary culture phases. Cell sorting visualized the morphology of algae corresponding to the hyperhaploid (2C and 4C) DNA peaks. RESULTS: Cell-division cycle-dependent changes in chlorophyll and DNA content distributions were most dramatic in logarithmically growing algal populations (an increase in the number of S-phase cells and cells with more chlorophyll), which are thought to be associated with accelerated DNA and chlorophyll metabolism in log-phase algal cultures. Upon reaching the stationary phase of growth, algal populations distinctly showed, in addition to one haploid (1C) DNA peak, two hyperhaploid peaks (2C and 4C) corresponding mainly to cells with two and four nuclei, respectively. CONCLUSIONS: Growth characteristics of algae exsymbiotic from P. bursaria monitored by flow cytometry provide valuable information for the analysis of the algal life cycle, which is important for understanding the regulation mechanisms of symbiont numbers.

Diphosphorylated MRLC is required for organization of stress fibers in interphase cells and the contractile ring in dividing cells.

Activity of nonmuscle myosin II is regulated by phosphorylation of its regulatory light chain (MRLC). Phosphoryration of MRLC at both Thr18 and Ser19 (diphosphorylation) results in higher MgATPase activity and in promotion of the assembly of myosin II filaments than does that of MRLC at Ser19 (monophosphorylation) in vitro. To determine the roles of the diphosphorylated MRLC in vivo, we transfected three kinds of MRLC mutants, unphosphorylated, monophosphorylated and diphosphorylated forms (MRLC2(T18AS19A), substitution of both Ser19 and Thr18 by Ala; MRLC2(T18AS19D), Ser19 by Asp and Thr18 by Ala; and MRLC2(T18DS19D), both Ser19 and Thr18 by Asp, respectively), into HeLa cells. Cells overexpressing the mutant MRLC2(T18DS19D) contained a larger number of actin filament bundles than did those overexpressing the mutant MRLC2(T18AS19D). Moreover, cells overexpressing the nonphosphorylatable mutant MRLC2(T18AS19A) showed a decrease in the number of actin filament bundles. Taken together, our data suggest that diphosphorylation of MRLC plays an important role in regulating actin filament assembly and reorganization in nonmuscle cells.

HeLa ZIP kinase induces diphosphorylation of myosin II regulatory light chain and reorganization of actin filaments in nonmuscle cells.

Dlk/ZIP kinase is a serine/threonine kinase highly homologous to DAP kinase. We have reported that HeLa ZIP kinase (hZIPK) phosphorylated the regulatory light chain of myosin II (MRLC) at both Ser19 and Thr18 in vitro. In this study, we demonstrate that hZIPK also induces the diphosphorylation of MRLC in nonmuscle cells. Peptide mapping revealed that transient transfection of hZIPK into HeLa cells caused diphosphorylation of MRLC. In contrast, transfection of the kinase inactive mutant of hZIPK did not induce any phosphorylation of MRLC. Using antibodies specific for mono- or diphosphorylated MRLC, we showed that diphosphorylated MRLC induced by the overexpression of hZIPK was concentrated in striking aggregates or bundles of actin filaments in HeLa cells, while monophosphorylated MRLC showed no prominent localization to these aggregates. Overexpression of hZIPK also induced dramatic changes in cell shape and disruption of nuclear morphology reminiscent of changes during apoptosis. These effects of hZIPK were suppressed by the coexpression of a mutant MRLC where both phosphorylation sites were replaced with alanine, indicating that the changes in actin organization were a consequence of MRLC diphosphorylation. These results suggested that hZIPK plays a role in regulating actin organization and cell morphology in non-muscles and at least part of its effects are mediated through the diphosphorylation of MRLC.

Myosin II regulatory light chain as a novel substrate for AIM-1, an aurora/Ipl1p-related kinase from rat.

Previous studies demonstrated that the phosphorylated myosin II regulatory light chain (MRLC) is localized at the cleavage furrow of dividing cells, suggesting that phosphorylation of MRLC plays an important role in cytokinesis. However, it remains unclear which kinase(s) phosphorylate MRLC during cytokinesis. AIM-1, an Aurora/Ipl1p-related kinase from rat, is known as a serine/threonine kinase that is required for cytokinesis. Here we examined the possibility that AIM-1 is a candidate for a kinase that phosphorylates MRLC during cytokinesis. As a result, we showed that AIM-1 monophosphorylated MRLC at Ser19 using two-dimensional phosphopeptide mapping analysis and several MRLC mutants. Furthermore, AIM-1 was colocalized with monophosphorylated MRLC at the cleavage furrow of dividing cells. We propose here that AIM-1 may participate in monophosphorylation of MRLC during cytokinesis.

Effects of anticancer drugs, metals and antioxidants on cytotoxic activity of benzothiepins/benzoxepins.

Among 11 benzothiepins/benzoxepins, 4-chloro-3,4-dihydro-2-(2-oxo-2-phenylethyl)-1-benzothiepin-5-(2H)-one [1] showed the highest cytotoxicity against human oral squamous cell carcinoma HSC-2 cells, followed by 2,3-dihydro-2-(2-oxopropyl)-2-phenyl-1-benzoxepin [2]. Popular antioxidants, such as N-acetyl-L-cysteine and sodium ascorbate significantly reduced the cytotoxic activity of [1] but not that of [2]. Compound [1] induced internucleosomal DNA fragmentation in human promyelocytic leukemic HL-60 cell line, but produced large DNA fragmentation in human oral tumor cell lines (HSC-2, HSG). Compounds [1] and doxorubicin additively reduced the viable cell number of HSC-2 cells. These data, taken together with their tumor specific action, demonstrate for the first time, the medicinal efficacy of benzothiepins/benzoxepins.

Urinary excretions of albumin and type IV collagen in normotensive and hypertensive subjects.

Plasma albumin leaks into urine as a result of glomerular hypertension and basement membrane injury, while urinary type IV collagen derives from mesangial matrix and glomerular basement membrane. The purpose of this study was to elucidate the pathophysiological significance of these urinary microproteins as an indicator of cardiovascular organ injuries in hypertension. In health-checkup participants without diabetes, proteinuria, or microhematuria, and who were not being treated for hypertension or any other disease at the time of enrollment, urinary albumin and type IV collagen were measured and their relations to organ injuries and cardiovascular risk factors were evaluated. Of 1,079 subjects (40- to 65-year-old; 256 men and 823 women) enrolled in the study, 120 (11.1%) had untreated hypertension exceeding 140/90 mmHg. Urinary albumin was positively correlated with both age (r=0.16, p<0.001) and systolic blood pressure (r=0.27, p<0.001). Urinary type IV collagen was not only positively correlated with age (r=0.12, p<0.001) and diastolic blood pressure (r=0.14, p<0.001) but also negatively correlated with blood hemoglobin (r=-0.12, p<0.001). Urinary albumin, but not type IV collagen, had a significant relation to electrocardiographic signs of left ventricular hypertrophy (p=0.012) and retinal arteriosclerosis on fundoscopy (p <0.001). Thus both albumin and type IV collagen would seem to have increased in association with age and hypertension in this cohort. It is suggested that urinary albumin is an indicator not only of renal injury, but also possibly of development of cardiac hypertrophy and arteriosclerotic changes. Urinary type IV collagen, on the other hand, may be associated with renal tissue injuries that affect erythrokinetics.

Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria.

BACKGROUND: The stable symbiotic association between Paramecium bursaria and algae is of interest to study such mechanisms in biology as recognition, specificity, infection, and regulation. The combination of algae-free strains of P. bursaria, which have been recently established by treating their stocks of green paramecia with herbicide paraquat (Hosoya et al.: Zool Sci 12: 807-810, 1995), with the cloned symbiotic algae isolated from P. bursaria (Nishihara et al.: Protoplasma 203: 91-99, 1998), provides an excellent clue to gain fundamental understanding of these phenomena. METHODS: Flow cytometry and light microscopy have been employed to characterize the algal cells after they have been released from the paramecia by ultrasonic treatment. Algal optical properties such as light scattering and endogenous chlorophyll fluorescence intensity have been monitored for symbiotic and free-living strains, and strains at stages of interaction with a host. RESULTS: Neither algal morphology nor chlorophyll content has been found to be altered by sonication of green paramecia. This fact allows to interpret in adequate degree changes in the optical properties of symbiont that just has been released from the association with a host (decreased forward light scatter and chlorophyll fluorescence signals). Optical characterization of both symbiotic and free-living algal strains with respect to their ability to establish symbioses with P. bursaria showed that chlorophyll content per cell volume seems to be a valuable factor for predicting a favorable symbiotic relationship between P. bursaria and algae. CONCLUSIONS: Flow cytometry combined with algae-free paramecia and cloned symbiotic algae identifies algal populations that may be recognized by host cells for the establishment of symbioses.