Arpp,a new homolog of carp,is preferentially expressed in type 1 skeletal muscle fibers and is markedly induced by denervation

In this study, we isolated and characterized a murine counterpart of the human Arpp (hArpp) gene. Sequence analysis revealed that the murine Arpp (mArpp) gene is almost identical to the Ankrd2 gene, which has recently been isolated as a mouse gene induced in stretched skeletal muscle. The mArpp gene encodes a protein of 332 amino acids that contains four well-conserved ankyrin-repeat domains in the central portion of the protein. The amino acid sequence of mArpp protein (mArpp) is highly homologous to that of mouse cardiac-restricted ankyrin-repeat protein (Carp), which is proposed to be a putative genetic marker for cardiac hypertrophy. Immunohistochemical analysis revealed that mArpp is preferentially expressed in type 1 skeletal muscle fibers, and that mArpp is localized in both the nucleus and the sarcomeric I-band of muscle fibers, suggesting that Arpp may function as a nuclear and sarcomeric protein. Furthermore, mArpp was also expressed in neurons of the cerebellum and cerebrum, the islets of Langerhans in the pancreas, and the esophageal epithelium, suggesting that mArpp may play a functional physiologic role in brain, pancreas, and esophagus as well as in type 1 muscle fibers. Interestingly, although mArpp was localized in both nucleus and cytoplasm in neurons, its localization was restricted to nucleus in pancreas and esophagus, suggesting that intracellular localization of mArpp is regulated in a tissue-specific manner. Furthermore, we found that mArpp- and Carp-expression in skeletal muscle were markedly up-regulated after denervation. Although the elevated expression level of Carp was kept only for two weeks after denervation, that of Arpp was kept at least for 4 weeks, suggesting that mArpp and Carp may play distinct functional roles in denervated skeletal muscle.     

Further characterization of memory T cells existing in a case of CD8 deficiency

CD8 deficiency is a rare primary immunodeficiency caused by a defect of ZAP-70, which plays a pivotal role in T cell activation. We previously reported the existence of memory phenotype-CD4+ T cells in a case of CD8 deficiency, which demonstrates that activation signals through ZAP-70 are not essential to the phenotypic conversion of T cells from "naive" to "memory." In this study, we further characterized CD45RO+ T cells in a CD8 deficient patient. We showed that the patient's CD45RO+ T cell population had a wide variety of T cell receptor Vbeta-chain gene usage, and contained few clonally expanded T cells, while many clonally expanded T cells were present in the memory T cell population of age-matched healthy children. These results suggest that various kinds of antigens were involved in the differentiation of the patient's T cells, and that the differentiation into memory T cells was not accompanied by profound T cell proliferation. Moreover, our findings confirmed that the patient's CD45RO+CD4+ T cells had acquired effector-cytokine producing ability, indicating that there exists an alternative activation pathway which is independent of ZAP-70 for the acquisition of effector-cytokine producing ability.     

Observation of cavitation in a mechanical heart valve in a total artificial heart

Recently, cavitation on the surface of mechanical heart valves has been studied as a cause of fractures occurring in implanted mechanical heart valves. The cause of cavitation in mechanical heart valves was investigated using the 25 mm Medtronic Hall valve and the 23 mm Omnicarbon valve. Closing of these valves in the mitral position was simulated in an electrohydraulic totally artificial heart. Tests were conducted under physiologic pressures at heart rates from 60 to 100 beats per minute with cardiac outputs from 4.8 to 7.7 L/min. The disk closing motion was measured by a laser displacement sensor. A high-speed video camera was used to observe the cavitation bubbles in the mechanical heart valves. The maximum closing velocity of the Omnicarbon valve was faster than that of the Medtronic Hall valve. In both valves, the closing velocity of the leaflet, used as the cavitation threshold, was approximately 1.3-1.5 m/s. In the case of the Medtronic Hall valve, cavitation bubbles were generated by the squeeze flow and by the effects of the venturi and the water hammer. With the Omnicarbon valve, the cavitation bubbles were generated by the squeeze flow and the water hammer. The mechanism leading to the development of cavitation bubbles depended on the valve closing velocity and the valve stop geometry. Most of the cavitation bubbles were observed around the valve stop and were generated by the squeeze flow.     

Astroglial responses against Abeta initially occur in cerebral primary cortical cultures: species differences between rat and cynomolgus monkey.

In the present study, we investigated how amyloid beta (Abeta) peptides initially affect neuronal cells in primary cerebral cortical cultures from rat and cynomolgus monkey. In these cultures, complicated interactions between glial and neuronal cells occur; moreover, synaptic interactions similar to those observed in vivo also occur between neuronal cells in these cultures. In this study, we applied low concentrations of Abeta to these well-characterized primary cultures to investigate how Abeta initially affects neurons or astroglial cells. In both rat and monkey cortical cultures, treatment with low concentrations of Abeta failed to drastically change or damage of neurons. Abeta treatment, however, significantly activated astrocytes, resulting in increased apolipoprotein E (ApoE) production. Rat astrocytes were more sensitive to Abeta than monkey astrocytes, and responded to Abeta via a different mechanism. In monkey astrocyte cultures, only direct treatment with Abeta increased ApoE production. In rat astrocyte cultures, however, treatment with conditioned media from cortical cultures grown with Abeta increased ApoE production, indicating that some sort of neuron-derived soluble factor(s) was also involved in activating rat astrocytes. These species differences suggest that monkey cortical cultures would be more useful as an in vitro model system to understand the details of how Abeta accumulates in the human brain, since monkeys are phylogenetically more similar to humans.     

Identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies

Summary Thirteen monoclonal antibodies (MAbs) to the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana were prepared and examined for their effects on various biological activities of VSV, including in vitro infection, hemagglutination, adsorption to cells, and mediation of cell fusion. Competitive binding assays with these MAbs revealed the presence of at least seven distinct antigenic determinants (epitopes) on the G protein. In some cases, overlappings among epitopes to various degrees were observed as partial inhibition or binding enhancement. The MAbs to all the epitopes but one (epitopes 1–6) reacted with the denatured G protein in a Western immunoblot analysis. Four of the epitopes (epitopes 2, 4, 5, and 7) were involved in neutralization and two (epitopes 1 and 2) in hemagglutination inhibition. None of the MAbs inhibited the adsorption of radiolabeled VSV to BHK-21 cells; the MAbs to epitope 2 slightly enhanced the virus adsorption. All neutralization epitopes except epitope 2 (epitopes 4, 5, and 7) were associated with inhibition of VSV-mediated cell fusion. These results show a direct spatial relationship between the epitopes recognized by the MAbs and functional sites on G protein and further insights into the structure and function of G protein.     

Expression and localization of chromogranin A gene and protein in human submandibular gland

Human saliva chromogranin A (CgA) is clinically promising as a psychological stress marker. However, expression of CgA is poorly understood in humans, although salivary gland localization of CgA in other mammals, such as rodents and horses, has been demonstrated. In the present study, we investigated the expression and localization of CgA in the human submandibular gland (HSG) using various methods. CgA was consistently localized in serous and ductal cells in HSG, as detected by immunohistochemistry and in situhybridization. Reactivity was stronger in serous cells than in ductal cells. In addition, strong immunoreactivity for CgA was observed in the saliva matrix of ductal cavities. Western blotting gave one significant immunoreactive band of 68 kDa in the adrenal gland, HSG and saliva. Finally, CgA was detected in secretory granules of serous and ductal cells by immunoelectron microscopy. In conclusion, CgA in humans is produced by HSG and secreted into saliva.     

Usefulness of immunoplatelet measurement using the flow cytometric method for accurate platelet counting in hematological patients with severe thrombocytopenia

We measured platelet counts in 95 patients with hematological disorders accompanied by thrombocytopenia (platelet counts < 5.0 x 10(4)/microliter) including 35 patients with severe thrombocytopenia(platelet counts < 2.0 x 10(4)/microliter). We used four methods based on different principles and compared the results, i.e., the flow cytometric method (BEADS method) utilizing platelet-specific monoclonal antibody (SZ2, antiGPIb) in conjunction with fluorescent reference beads (Flow-Count Fluorospheres), manual hemocytometry, and two automated blood cell counters, the NE-8000 (impedance method) and the Technicon H-2 (optical method). The BEADS method was superior to the other methods in linearity of serial dilutions, and the coefficient variations of the BEADS method(2.5-5.2%) were superior to the other methods. The platelet counts measured by the automated blood cell counters were higher(0.6-0.9 x 10(4)/microliter) than those by the BEADS method and manual hemocytometry. Furthermore, the BEADS method was able to measure accurate platelet counts in samples containing red blood cell fragments. The BEADS method may be an accurate and useful method for measuring samples with severe thrombocytopenia, and, especially, samples containing red blood cell fragments.     

Identification of neural genes using Xenopus DNA microarrays.

To isolate novel genes regulating neural induction, we used a DNA microarray approach. As neural induction is thought to occur by means of the inhibition of bone morphogenetic protein (BMP) signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips representing over 17,000 unigenes. We have identified 77 genes that are induced in animal caps after inhibition of BMP signaling, and all of these genes were subjected to whole-mount in situ hybridization analysis. Thirty-two genes showed specific expression in neural tissues. Of the 32, 14 genes have never been linked to neural induction. Two genes that are highly induced by BMP inhibition are inhibitors of Wnt signaling, suggesting that a key step in neural induction is to produce Wnt antagonists to promote anterior neural plate development. Our current analysis also proves that a microarray approach is useful in identifying novel candidate factors involved in neural induction and patterning.     

Effect of interferon treatment on serum 2',5'-oligoadenylate synthetase levels in hepatitis C-infected patients

Interferon (IFN) is widely used for patients with hepatitis C. Less than half of treated patients respond to IFN therapy, however, and increased resistance to IFN is particularly observed in genotype 1b patients. Recently, genotype 1b patients with the wild type sequence in the NS5A gene were shown to be resistant to therapy, suggesting that the NS5A protein may be involved to IFN resistance. Thus, we investigated the serum 2',5'-oligoadenylate synthetase (2',5'-OAS) levels before and during IFN treatment. In addition, other biochemical markers and NS5A mutations were also examined in 30 HCV genotype 1b-positive patients. Before IFN treatment, 2',5'-OAS activity in sera was significantly lower in wild type patients than in mutant type patients. All patients were subsequently enrolled in IFN therapy, and 2',5'-OAS activity was elevated both in wild and mutant type patients, irrespective of the number of mutations in NS5A. Logistic regression analysis revealed that clearance of serum HCV RNA was independently related to the pretreatment viral load and NS5A mutations, but not to serum 2',5'-OAS activity. We concluded that the NS5A protein, that is associated with the outcome of IFN therapy, affects the kinetics of IFN-induced molecules, such as 2', 5'-OAS. 2',5'-OAS activity does not, however, seem to be related to long-term virological response to IFN therapy.