Two mouse cofilin isoforms, muscle-type (MCF) and non-muscle type (NMCF), interact with F-actin with different efficiencies

Two cofilin isoforms, a muscle-type (MCF) and a non-muscle-type (NMCF), are co-expressed in developing mammalian skeletal and cardiac muscles. To clarify how they are involved in the actin filament dynamics during myofibrillogenesis, we examined their localization in muscle tissues and cultured muscle cells using immunocytochemical methods, and their interaction with F-actin in vitro. NMCF was mostly detected in a diffuse pattern in the cytoplasm but MCF was partly localized to the striated structures in myofibrils. The location of chicken cofilin, a homologue of MCF, in the I-bands of myofibrils was determined by an immunocytochemical method. It is suggested that MCF could be associated with actin filaments in muscle cells more efficiently than NMCF. Using purified recombinant MCF and NMCF, their interaction with F-actin was examined in vitro by a cosedimentation assay method. We observed that MCF was precipitated with F-actin more effectively than NMCF. When MCF and NMCF were simultaneously incubated with F-actin, MCF was preferentially associated with F-actin. MCF and NMCF inhibited the interaction of F-actin with tropomyosin, but the former suppressed the actin-tropomyosin interaction more strongly than the latter. These results suggest that MCF interacts with F-actin with higher affinity than NMCF, and although both of them are involved in the regulation of actin assembly in developing myotubes, the two proteins may play somewhat different roles.     

Unusual heme binding in the bacterial iron response regulator protein: spectral characterization of heme binding to the heme regulatory motif

We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single "heme-regulatory motif", HRM, and plays a key role in the iron homeostasis of a nitrogen-fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where (29)Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside of the HRM. The Raman line for the Fe-S stretching mode observed at 333 cm(-1) unambiguously confirmed heme binding to Cys. The lower frequency of the Fe-S stretching mode corresponds to the weaker Fe-S bond, and the broad Raman line of the Fe-S bond suggests multiple configurations of heme binding. These structural characteristics are definitely different from those of typical hemoproteins. The unusual heme binding in Irr was also evident in the EPR spectra. The characteristic g-values of the 5-coordinate Cys-ligated heme and 6-coordinate His/His-ligated heme were observed, while the multiple configurations of heme binding were also confirmed. Such multiple heme configurations are not encountered for typical hemoproteins where the heme functions as the active center. Therefore, we conclude that heme binding to HRM in the heme-regulated protein, Irr, is quite different from that in conventional hemoproteins but characteristic of heme-regulated proteins using heme as the signaling molecule.     

Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes

Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.     

Modulation of cellular rhythm and photoavoidance by oscillatory irradiation in the Physarum plasmodium

We studied responses of cellular rhythm and light-induced movement to periodic irradiation in a unicellular amoeboid organism, the Physarum plasmodium. The intrinsic frequency of the contraction rhythm, which is based on biochemical oscillations, became synchronized with the frequency of periodic irradiation with light when both frequencies were close enough. In order to study the role of the synchronization in light-induced movement, periodic irradiation was applied to only part of the plasmodium. The rate of avoidance of light was modulated in the frequency band in which the synchronization occurred. The synchronization property of the contraction oscillation underlies the regulation of tactic movement in plasmodium.     

Histochemical and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog submandibular glands.

p-Nitrophenyl phosphatase (p-NPPase) activity of (Na+-K+)-activated adenosine triphosphatase ((Na+-K+)-ATPase) on the acinar cells of dog submandibular gland was demonstrated by using light microscopy. The reaction products of p-NPPase of fresh frozen sections were seen to be localized on the basal parts of acini, and disappeared when the sections were incubated in medium containing 10(-3) Mouabain or in a K-free medium. Under the electron microscope, the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells. On the microvilli of the luminal plasma membrane of the acinar cell, a small quantity of the reaction products was also present. This localization of ATPase reaction products on the serous and mucous cells seemed to coincide well with that of p-NPPase activity observed on the acini under light microscopy. Possible explanations are given regarding distribution of the above mentioned enzymes in relation to the cation transport of the plasma membrane. Structural and functional asymmetrical properties of acinar cells of the dog submandibular gland are also discussed.     

The 99 and 170 loop-modified factor VIIa mutants show enhanced catalytic activity without tissue factor

To elucidate the functions of the surface loops of VIIa, we prepared two mutants, VII-30 and VII-39. The VII-30 mutant had all of the residues in the 99 loop replaced with those of trypsin. In the VII-39 mutant, both the 99 and 170 loops were replaced with those of trypsin. The k(cat)/K(m) value for hydrolysis of the chromogenic peptidyl substrate S-2288 by VIIa-30 (103 mm(-)1s(-)1) was 3-fold higher than that of wild-type VIIa (30.3 mm(-)1 s(-)1) in the presence of soluble tissue factor (sTF). This enhancement was due to a decrease in the K(m) value but not to an increase in the k(cat) value. On the other hand, the k(cat)/K(m) value for S-2288 hydrolysis by VIIa-39 (17.9 mm(-)1 s(-)1) was 18-fold higher than that of wild-type (1.0 mm(-)1 s(-)1) in the absence of sTF, and the value was almost the same as that of wild-type measured in the presence of sTF. This enhancement was due to not only a decrease in the K(m) value but also to an increase in the k(cat) value. These results were in good agreement with their susceptibilities to a subsite 1-directed serine protease inhibitor. In our previous paper (Soejima, K., Mizuguchi, J., Yuguchi, M., Nakagaki, T., Higashi, S., and Iwanaga, S. (2001) J. Biol. Chem. 276, 17229-17235), the replacement of the 170 loop of VIIa with that of trypsin induced a 10-fold enhancement of the k(cat) value for S-2288 hydrolysis as compared with that of wild-type VIIa in the absence of sTF. These results suggested that the 99 and the 170 loop structures of VIIa independently affect the K(m) and k(cat) values, respectively. Furthermore, we studied the effect of mutations on proteolytic activity toward S-alkylated lysozyme as a macromolecular substrate and the activation of natural macromolecular substrate factor X.     

Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells

Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrP^Sc) in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrP^Sc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrP^Sc degradation were evaluated. The accumulation of PrP^Sc in cells persistently infected with the prion strain Fukuoka-1 (FK), derived from a patient with Gerstmann–Sträussler–Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA) but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrP^Sc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrP^Sc from either the 22L or the Chandler strain, indicating that the degradation of PrP^Sc in host cells might be strain-dependent.     

DNA of a new parvo-like virus isolated from the silkworm,Bombyx mori

Parvo-like virus, which was designated as “Ina-flacherie virus (Ina-FV),” was isolated from the silkworm, Bombyx mori, and the properties of its DNA were characterized. Purified Ina-FV had a diameter of 22 ± 0.5 nm and a sedimentation coefficient of 102 S. On density gradient separation in CsCl, particles were found at densities of 1.40 and 1.45 g/ml. The DNA content of Ina-FV was 28 ± 2%. The DNA in low-salt buffer possessed properties typical of a single-stranded (ss) molecule. Double-stranded (ds) DNA was extracted under conditions of appropriate high salt and elevated temperature. Electron microscopical examination revealed that the ds DNA was composed of linear molecules with an average length of 1.7 μm and other less well-defined structures. The linear ds molecule had a molecular weight of about 3.4 × 10⁶ determined by electron microscopy (EM) and agarose gel electrophoresis. When the ds DNA was alkali-denatured and examined in an EM, linear ss molecules with approximate length of 1.7 μm were observed, indicating that the linear ds molecule was formed from the annealing of the linear ss molecules of unit length. These data suggest that Ina-FV is closely related to members of the densovirus subgroup.     

Relationship between survival rates and numbers of natural teeth in an elderly Japanese population

Objectives: The objective of this study was to assess whether elderly people with 20 or more natural teeth were more likely to live longer than a cohort with less than 20 teeth. Materials and methods: Groups of elderly people over 80 years of age (24 males and 35 females) with 20 or more teeth (≥20 group) were compared with elderly people (24 males and 35 females) with less than 20 teeth (<20 group). Follow‐up studies were conducted at regular intervals for 10 years from July 1992 to July 2002. The cumulative survival rate of the ≥20 group (average ± SE tooth number of teeth – males, 23.9 ± 0.6; females, 23.8 ± 0.4) was compared with the <20 group (average number of teeth – males, 3.8 ± 1.1; females, 2.6 ± 0.8). The multivariate Cox proportional hazard models with the number of teeth in a group (≥20 group or <20 group). Smoking status and alcohol intake as covariates were used to adjust the cumulative survival rate. Results: The male participants in the ≥20 group had a significantly higher cumulative survival rates (p < 0.05) than the <20 group at 18 and 21 months from baseline. There were no significant differences in survival rates between the female groups. Adjusted cumulative survival rate was significantly different at 72, 75 and 78 months between the ≥20 group and <20 group for males but not for females. Conclusion: Having 20 or more natural teeth was associated with increased survival rate in elderly males, but not among the elderly females.     

Bone marrow stroma cells are susceptible to prion infection

Abnormal protease-resistant prion protein (PrP-res) is the only surrogate biochemical marker for prion diseases, and a sensitive technique to detect PrP-res in blood or tissues is urgently needed. Primary cultured bone marrow stromal cells (MSCs) expressed PrP and were capable of supporting stable human prion infection. Using a mouse-adapted BSE strain, we demonstrated that PrP-res can be detected in expanded MSCs. We then analyzed the bone marrow cells collected at autopsy from two individuals with sporadic Creutzfeldt-Jakob disease (CJD), and, in both cases, cultured MSCs were positive for PrP-res. These data would suggest that ex vivo MSC expansion accompanied by PrP-res analysis could be a helpful tool in the definitive diagnosis of prion disease at an earlier stage in the disease process than is currently possible, and with considerably less distress to the patient.