The structure of arthropod hemocyanins.

Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. Comparison of the amino acid sequence data for seven different subunits of arthropod hemocyanins from crustaceans and chelicerates shows many highly conserved residues and extensive regions of near identity. This correspondence can be matched closely with the three domain structure established by x-ray crystallography for spiny lobster hemocyanin. The degree of identity is particularly striking in the second domain of the subunit that contains the six histidines which ligate the two oxygen-binding copper atoms. The polypeptide architecture of spiny lobster hemocyanin appears to be the same in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, about 540 to 600 million years ago.

Is outer arm dynein intermediate chain 1 multifunctional?

The outer arm dynein of sea urchin sperm axoneme contains three intermediate chains (IC1, IC2, and IC3; M(r) 128,000, 98,000, and 74,000, respectively). IC2 and IC3 are members of the WD family; the WD motif is responsible for a protein-protein interaction. We describe here the molecular cloning of IC1. IC1 has a unique primary structure, the N-terminal part is homologous to the sequence of thioredoxin, the middle part consists of three repetitive sequences homologous to the sequence of nucleoside diphosphate kinase, and the C-terminal part contains a high proportion of negatively charged glutamic acid residues. Thus, IC1 is a novel dynein intermediate chain distinct from IC2 and IC3 and may be a multifunctional protein. The thioredoxin-related part of IC1 is more closely related to those of two redox-active Chlamydomonas light chains than thioredoxin. Antibodies were prepared against the N-terminal and middle domains of IC1 expressed as His-tagged proteins in bacteria. These antibodies cross-reacted with some dynein polypeptides (potential homologues of IC1) from distantly related species. We propose here that the three intermediate chains are the basic core units of sperm outer arm dynein because of their ubiquitous existence. The recombinant thioredoxin-related part of IC1 and outer arm dyneins from sea urchin and distantly related species were specifically bound to and eluted from a phenylarsine oxide affinity column with 2-mercaptoethanol, indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction.

Natural estrogens induce modulation of microtubules in Chinese hamster V79 cells in culture.

Natural estrogens and their derivatives comprising 30 compounds in total were tested for their ability to induce microtubule disruption in Chinese hamster V79 cells. The cytoplasmic microtubule networks were examined by the indirect immunofluorescence method using anti-beta-tubulin antibody. The effective concentrations of 17 beta-estradiol (E2-17 beta) and 17 alpha-estradiol required for the induction of microtubule distribution in 50% of the cells (EC50) in 1 h were 10 and 9 microM for V79 cells, respectively, and 2-methoxyestradiol showed the strongest activity (EC50 2 microM) among the tested compounds including the catechol estrogens 2-hydroxyestradiol, 4-hydroxyestradiol, 2-hydroxyestrone, and 4-hydroxyestrone. The estrone series of estrogens showed relatively low activity for disruption of microtubule networks compared with E2-17 beta, whereas 3-deoxy-3-methylestradiol showed almost the same EC50 value as E2-17 beta. There was a correlation between the EC50 values of the compounds and their growth-inhibitory activities. The presence of 1 microM taxol in culture protected against 50 microM E2-17 beta-induced microtubule disruption. Cycloheximide and actinomycin D had no preventive action on the effect of E2-17 beta, suggesting that the microtubule-disruptive effect of E2-17 beta is not associated with newly synthesized proteins and mRNAs. The present results indicate that some natural estrogens cause microtubule disruption in a nongenomic manner.

Actin-bundling protein isolated from pollen tubes of lily. Biochemical and immunocytochemical characterization

A 135-kD actin-bundling protein was purified from pollen tubes of lily (Lilium longiflorum) using its affinity to F-actin. From a crude extract of the pollen tubes, this protein was coprecipitated with exogenously added F-actin and then dissociated from F-actin by treating it with high-ionic-strength solution. The protein was further purified sequentially by chromatography on a hydroxylapatite column, a gel-filtration column, and a diethylaminoethyl-cellulose ion-exchange column. In the present study, this protein is tentatively referred to as P-135-ABP (Plant 135-kD Actin-Bundling Protein). By the elution position from a gel-filtration column, we estimated the native molecular mass of purified P-135-ABP to be 260 kD, indicating that it existed in a dimeric form under physiological conditions. This protein bound to and bundled F-actin prepared from chicken breast muscle in a Ca2+-independent manner. The binding of 135-P-ABP to actin was saturated at an approximate stoichiometry of 26 actin monomers to 1 dimer of P-135-ABP. By transmission electron microscopy of thin sections, we observed cross-bridges between F-actins with a longitudinal periodicity of 31 nm. Immunofluorescence microscopy using rhodamine-phalloidin and antibodies against the 135-kD polypeptide showed that P-135-ABP was colocalized with bundles of actin filaments in lily pollen tubes, leading us to conclude that it is the factor responsible for bundling the filaments.

Inhibitory regulation of higher-plant myosin by Ca2+ ions

Myosin isolated from the pollen tubes of lily (Lilium longiflorum) is composed of a 170-kD heavy chain (E. Yokota and T. Shimmen [1994] Protoplasma 177: 153-162). Both the motile activity in vitro and the F-actin-stimulated ATPase activity of this myosin were inhibited by Ca2+ at concentrations higher than 10(-6) M. In the Ca2+ range between 10(-6) and 10(-5) M, inhibition of the motile activity was reversible. In contrast, inhibition by more than 10(-5) M Ca2+ was not reversible upon Ca2+ removal. An 18-kD polypeptide that showed the same mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that of spinach calmodulin (CaM) was present in this myosin fraction. This polypeptide showed a mobility shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a Ca2+-dependent manner. Furthermore, this polypeptide was recognized by antiserum against spinach CaM. By immunoprecipitation using antiserum against the 170-kD heavy chain, the 18-kD polypeptide was coprecipitated with the 170-kD heavy chain, provided that the Ca2+ concentration was low, indicating that this 18-kD polypeptide is bound to the 170-kD myosin heavy chain. However, the 18-kD polypeptide was dissociated from the 170-kD heavy chain at high Ca2+ concentrations, which irreversibly inhibited the motile activity of this myosin. From these results, it is suggested that the 18-kD polypeptide, which is likely to be CaM, is associated with the 170-kD heavy chain as a light chain. It is also suggested that this polypeptide is involved in the regulation of this myosin by Ca2+. This is the first biochemical basis, to our knowledge, for Ca2+ regulation of cytoplasmic streaming in higher plants.

Biochemical and immunocytochemical characterization of two types of myosins in cultured tobacco bright yellow-2 cells

We have isolated a myosin (referred to as 170-kD myosin) from lily pollen tubes, which consists of 170-kD heavy chain and calmodulin (CaM) light chain and is responsible for cytoplasmic streaming. A 170-kD polypeptide that has similar antigenicity to the 170-kD myosin heavy chain of lily pollen tubes was also present in cultured tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells, and possessed the ability to interact with F-actin in an ATP-dependent manner. In addition to this myosin, we identified biochemically another kind of myosin in BY-2 cells. This myosin consisted of a CaM light chain and a 175-kD heavy chain with antigenicity different from the 170-kD myosin heavy chain. In the present study, we referred to this myosin as 175-kD myosin. This myosin was able to translocate rhodamine-phalloidin (RP)-labeled F-actin at an average velocity of about 9 &mgr;m/s in the motility assay in vitro. In contrast, the sliding velocity of RP-labeled F-actin translocated by fractions containing the 170-kD myosin was 3 to 4 &mgr;m/s. The velocity of cytoplasmic streaming in living BY-2 cells ranged from 2 to 9 &mgr;m/s. The motile activity of 175-kD myosin in vitro was inhibited by Ca(2+) at concentrations higher than 10(-6) M. Immunoblot analyses using an antiserum against the heavy chain of 170- or 175-kD myosin revealed that in tobacco plants, the 175-kD myosin was expressed in leaf, stem, and root, but not in germinating pollen, while 170-kD myosin was present in all of these plant parts and in germinating pollen. These results suggest that the two types of myosins, 170 and 175 kD, presumably participate in cytoplasmic streaming in BY-2 cells and other somatic cells of tobacco plants.

Interaction of flagellar inner arm dynein isolated from sea urchin sperm with microtubules in the presence of ATP.

We have isolated C/A dynein, which is considered to be a component of inner arms, from flagellar axonemes of sea urchin sperm (E. Yokota, I. Mabuchi, J. Cell Sci. 107, 345-351 (1994). C/A dynein binds to and bundles the microtubules in the absence of ATP. In contrast to outer arm 21S dynein, C/A dynein is not released from the microtubules in the presence of ATP (E. Yokota, I. Mabuchi, J. Cell Sci. 107, 353-361 (1994)). We further investigated the interaction of C/A dynein with microtubules in the presence of ATP. The turbidity at 350 nm of a mixture of C/A dynein and microtubules increased by the addition of ATP. Both the initial rate and final extent of the turbidity increase were dependent on C/A dynein or ATP concentration and were inhibited by vanadate. ATP hydrolysis by C/A dynein was linear during the time course of the turbidity increase. Negative staining electron microscopy revealed that microtubular bundles which formed in the presence of C/A dynein became thicker and longer after addition of ATP. Furthermore, sliding movements of microtubule(s) in the individual bundles were observed in the presence of ATP. This mode of interaction of C/A dynein with microtubules was distinct from that of flagellar or ciliary dyneins reported so far. These results suggest that C/A dynein, as a component of inner arms, may play a distinct role in the flagellar movement of sea urchin sperm.

Melanoma dynein: evidence that dynein is a general "motor" for microtubule-associated cell motilities.

Platyfish-swordtail hybrid melanoma cells exhibit pigment aggregation in response to adrenergic stimulation or melanophore-concentrating hormone. This translocation of pigment granules is thought to be related to radially arrayed microtubules. Very little is known about the molecular "motor" that powers the translocation. We present evidence that dynein is located on these microtubules and is a candidate for the "motor". Vanadate and erythro-9-[3-(2-hydroxynonyl)]adenine, which are potent inhibitors of dynein ATPase, prevent the transport of melanosome granules in Brij-treated melanoma cells. Direct identification of dynein in melanoma cells and tissues is demonstrated by immunofluorescence microscopy and immunoblotting using anti-fragment A (tryptic fragment of sea urchin sperm dynein) serum. The cytoplasm of melanoma cells is stained with the antiserum and gives rise to a pattern similar to the distribution of microtubules. Western blotting shows that the molecular weight of an immunoreactive polypeptide in melanoma tissues coincides with that of the heavy chain of sea urchin sperm dynein.

The 135-kDa actin-bundling protein from lily pollen tubes arranges F-actin into bundles with uniform polarity.

A plant 135-kDa actin-bundling protein (P-135-ABP) isolated from pollen tubes of Lilium longiflorum (Thunb.) binds stoichiometrically to F-actin filaments and bundles them in vitro (E. Yokota et al., 1998, Plant Physiol. 116: 1421-1429). To further understand the mechanism of actin-filament bundle formation by P-135-ABP, the polarity of each F-actin filament in bundles was examined using myosin subfragment 1 (S-1). Dissociation of F-actin filaments from bundles organized by P-135-ABP was induced by S-1. However, F-actin filaments that remained in a bundle and decorated by S-1 showed uniform polarity. These results indicate that P-135-ABP arranges F-actin filaments into bundles with uniform polarity and consequently plays a key role in the orientation of cytoplasmic streaming in pollen tubes.

Genetic diversities of hantaviruses among rodents in Hokkaido, Japan and Far East Russia.

Seroepizootiologic surveys among wild rodents were carried out in Japan and Far East Russia in 1995 and 1996. Seropositive animals were only identified in Clethrionomys rufocanus (23/134) in Hokkaido, Japan. On the other hand, seropositives were identified in C. rufocanus (1/8), Apodemus agrarius (2/66), Apodemus spp. (2/26) and Microtus fortis (3/22) in Vladivostok, Far East Russia. Total RNA was isolated from lungs of seropositive animals and the S genome segments were amplified by PCR, cloned and sequenced. The S and M genomes of hantavirus, derived from Japanese C. rufocanus (Tobetsu genotype), were most closely related with Puumala viruses (76-79% nucleotide and 95% amino acid identities for S genome, 70-78% nucleotide and 87-92% amino acid identities for M genome). The recombinant nucleocapsid protein of Tobetsu genotype was antigenically quite similar with that of Sotkamo. These suggest that the virus endemic in Japanese C. rufocanus belongs to Puumala virus. Phylogenetic analysis indicates that the genotype forms a distinct lineage within Puumala viruses. Partial S segment (1-1251 nt), derived from seropositive M. fortis in Vladivostok, was sequenced and analyzed. The S genome segment, which was designated Vladivostok genotype, was most closely related with Khabarovsk virus (79% nucleotide and 90% amino acid identities) which was isolated from M. fortis.

The cAMP-dependent protein kinase in sea urchin sperm tails: association of the enzyme with the flagellar axonemes.

When sea urchin spermatozoa were treated with a Triton X-100 solution, cAMP-dependent protein kinase (cA-kinase) activity was extracted. Further extraction with Triton X-100 of axonemes isolated from the Triton-extracted sperm again released a considerable amount of the cA-kinase activity. The activity which remained after extraction three times with Triton X-100 was released by treatment with a low salt solution. These activities found in the various extracts were likely to be due to the same cA-kinase, which was a mammalian type II-like enzyme. The cA-kinase activity that remained in the axonemes after the first Triton X-100 extraction may be involved in the regulation of flagellar movement in the Triton-extracted sperm.

Muscle beta-actinin inhibits elongation of the pointed end of the actin filaments of brush border microvilli.

By using isolated actin bundles of brush border microvilli of chicken intestinal epithelial cells, it was clearly visualized that muscle beta-actinin caps the pointed end of an actin filament, whereas cytochalasin D masks the barbed end. The growth rate at the barbed end in the presence of beta-actinin was markedly slower than in its absence.

Activation of sea urchin sperm flagellar dynein ATPase activity by salt-extracted axonemes.

When 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I.R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsin-treated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KCl. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.

Preparation of tubulin from Caulerpa, a marine green alga, using casein as a protective agent against proteolytic degradation.

Bidirectional organelle movements taking place in the cytoplasm of the rhizomes of Caulerpa, a coenocytic marine green alga, have been indicated to be dependent on microtubules (Kuroda, K. & Manabe, E. (1983) Proc. Jpn. Acad. 59B, 131-134; Manabe, E. & Kuroda, K. (1984) Proc. Jpn. Acad. 60B, 118-121). However, when a crude extract of Caulerpa rhizomes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting with monoclonal anti-tubulin antibody, no reacting band could be detected. This apparent absence of tubulin in the extract was found to be a result of the complete degradation of tubulin by potent intrinsic proteolytic activity. All of the commercially available protease inhibitors so far tested (p-chloromercuriphenylsulfonic acid, phenyl methylsulfonyl fluoride, 1-chloro-4-phenyl-3-tosylamido-2-butanone, 7-amino-1-chloro-3-tosylamido-2-heptanone, p-tosyl-L-arginine methyl ester, soybean trypsin inhibitor, antipain, chymostatin, leupeptin, and pepstatin) failed to inhibit the activity completely. But addition of casein at the concentration of 1% (weight per volume) to the solutions used for preparation was effective in protecting tubulin from proteolytic degradation, thus making it possible to prepare tubulin from the crude extract of Caulerpa. On SDS-PAGE, the Caulerpa alpha-tubulin thus prepared was a little smaller in molecular weight than that of rabbit brain.

Effects of (+)-, (-)- and (+/-)-indenestrols A and B on microtubule distribution and cytotoxicity in Chinese hamster V79 cells.

We have reported that (+)-, (-)- and (+/-)-indenestrols A and B (IA and IB respectively) inhibit the polymerization of microtubule proteins isolated from porcine brain in vitro. In this study, the effects of (+)-, (-)- and (+/-)-IA and IB on the relative plating efficiency, chromosome number and cellular microtubular architecture of Chinese hamster V79 cells, detected with a fluorescent anti-tubulin antibody, were investigated. The results indicated that the effect of (+/-)-IA was similar to that of diethylstilbestrol and that of (+/-)-IB was greater than that of (+/-)-IA. We also determined the effects of the optically active IA and IB isomers and found that the rank order of cytotoxic activity of the IA and IB series was: (-)-IA > (+/-)-IA > (+)-IA and (+/-)-IB > or = (-)-IB > (+)-IB. Furthermore, we studied the intracellular disturbance of microtubule formation induced by these compounds and found that (-)-IA had by far the greatest disruptive effect.

Interpretation of periapical lesions using RadioVisioGraphy.

Density and gray-scale changes in radiographs are important visual features the clinician uses to evaluate changes in bone pattern. With the advent of a new direct digital radiology system, RadioVisioGraphy (RVG), the controlled adjustment of contrast is now possible. The purpose of this study was to compare RVG's diagnostic potential for detecting periapical lesions with that of conventional radiography. Lesions were created in human cadaver specimens and radiographed conventionally and with RVG. Images were evaluated by three endodontists. Results were: (a) when no lesion existed, conventional radiographs were more diagnostic than RVG at a significance level at p < or = 0.05; (b) when lesions were enlarged to involve lamina dura and medullary bone, RVG was superior at p < or = 0.05; and (c) no difference was found between conventional radiography and RVG when the lesion involved cortical bone.

Properties of a broad spectrum beta-lactamase isolated from Flavobacterium meningosepticum GN14059.

A broad substrate-spectrum beta-lactamase was purified from Flavobacterium meningosepticum GN14059. The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated to be about 30,000 and the isoelectric point was 5.1. The enzyme hydrolyzed various beta-lactam antibiotics including oxyiminocephalosporins and aztreonam. Relative rates, with cephaloridine as 100, were cephalothin 200, cefazolin 48, cefuroxime 153, cefotaxime 51, ceftazidime 20, ampicillin 26, carbenicillin 19, and aztreonam 20. The enzyme activity was inhibited by clavulanic acid, sulbactam, imipenem and cephamycins.

[Prosthetic endocarditis caused by Kingella denitrificans in a patient with diabetes mellitus].

Infective endocarditis caused by Kingella denitrificans occurs rarely. A review of the literature reveals only 6 cases of endocarditis caused by the bacillus. K. denitrificans is normally a commensal of the upper respiratory airways, may exceptionally be responsible for endocarditis. A case of possible prosthetic endocarditis caused by K. denitrificans is presented. A 78-year-old male with Type II diabetes was admitted to the hospital complaining of fever, a sore throat and arthralgia. He underwent replacement surgery of a St. Jude medical prosthesis for aortic stenosis at the age of 75. The only physical findings at admission were a temperature of 38.2 degrees C and murmurs of mild mitral regurgitation. The liver and spleen were not palpable, and there were no skin or eye lesions. Laboratory findings were as follows: white blood cell count 9500/microliters with 77% neutrophils, erythrocyte sedimentation rate 71 mm/h (Westergren), blood urea nitrogen 50.2 mg/dl, serum creatinine 1.7 mg/dl and C-reactive protein 22.2 mg/dl. The Gram-negative bacillus isolated from the blood was identified as K. denitrificans by the identification system, namely ID test.FN-20 rapid (Nissui, Japan). Although an echocardiogram detected no vegetation, infective endocarditis was diagnosed because the same bacillus was detected by separate blood cultures and an obvious source of infection was not found other than the prosthetic valve. Initial treatment was flomoxef, which was changed to Ampicillin 2 g/day after K. denitrificans was identified. Ampicillin continued for 6 weeks. The clinical course was good and he did not require further surgery. He has been afebrile for 2 years after completing treatment. This case represents the first report of prosthetic valve endocarditis caused by K. denitrificans in Japan.

[A case of the active intestinal tuberculosis detected during the examination of the right pleural effusion].

A 76-year-old female was admitted to our hospital because of fever and the right pleural effusion. On the analysis of pleural effusion, the total cell count was 6720/microliter with 95% lymphocytes, and ADA was 38.1 U/l. The culture of pleural effusion was negative, and the smear and PCR for Mycobacterium were also negative. For examinations, we performed eterography that showed cicatricial strictures of intestine. X-ray examination of the colonated colonoscopy showed ulcers (circular type), shortening of the colon, Bauhin's value insufficiency and diverticulum-like deformity. Then, she was diagnosed as intestinal tuberculosis. The smear and PCR of biopsy specimens from the lesion were positive, and antituberculotic therapy was effective. Finally, the culture of pleural effusion for Mycobacterium tuberculosis was positive after 8 weeks. We thought intestinal examination may be useful for the diagnosis of tuberculosis, when lymphocyte-rich exudative pleural effusion of unknown etiology is seen.

[Disappearance of Philadelphia chromosomes after remission induction in lymphoid crisis of chronic myelogenous leukemia].

The authors report a rare case of chronic myelogenous leukemia (CML) in which the Ph1 clone disappeared after remission induction of lymphoid crisis. A 58-year-old man was admitted to our hospital because of fever in July 1988. The white cell count was elevated. Bone marrow aspirate showed hypercellularity with myeloid hyperplasia. In the chromosomal analysis, Ph1 chromosomes were detected in 100% of bone marrow cells analysed. Diagnosis of CML was made and treatment was initiated with recombinant interferon-alpha 2a. Hematological remission without cytogenetic improvement was achieved. In March 1990 he developed lymphoid crisis with proliferation of CD10-positive cells. The chromosomal analysis revealed additional abnormalities including, 45, X, -Y, t(9;22) (q34;q11), +1, -8. With vincristine 0.6 mgX4, pirarubicin 15 mgX4, dexamethasone 40 mgX4 therapy complete remission was obtained. In December 1990 the Ph1 positive clone completely disappeared judging from normal karyotypes in the chromosomal analysis and the disappearance of M-bcr gene rearrangement.