Clarification of muscle synergy structure during standing-up motion of healthy young, elderly and post-stroke patients.

Standing-up motion is an important daily activity. It has been known that elderly and post-stroke patients have difficulty in performing standing-up motion. The standing-up motion is retrained by therapists to maximize independence of the elderly and post-stroke patients, but it is not clear how the elderly and post-stroke patients control their redundant muscles to achieve standing-up motion. This study employed the concept of muscle synergy to analyze how healthy young adults, healthy elderly people and post-stroke patients control their muscles. Experimental result verified that four muscle synergies can represent human standing-up motion. In addition, it indicated that the post-stroke patients shift the weights of muscle synergies to finish standing-up motion comparing to healthy subjects. Moreover, different muscle synergy structures were associated with the CoM and joint kinematics.

HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination.

An ATM-dependent cellular signal, a DNA-damage response, has been shown to be involved during infection of human immunodeficiency virus type-1 (HIV-1), and a high incidence of malignant tumor development has been observed in HIV-1-positive patients. Vpr, an accessory gene product of HIV-1, delays the progression of the cell cycle at the G2/M phase, and ATR-Chk1-Wee-1, another DNA-damage signal, is a proposed cellular pathway responsible for the Vpr-induced cell cycle arrest. In this study, we present evidence that Vpr also activates ATM, and induces expression of gamma-H2AX and phosphorylation of Chk2. Strikingly, Vpr was found to stimulate the focus formation of Rad51 and BRCA1, which are involved in repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), and biochemical analysis revealed that Vpr dissociates the interaction of p53 and Rad51 in the chromatin fraction, as observed under irradiation-induced DSBs. Vpr was consistently found to increase the rate of HR in the locus of I-SceI, a rare cutting-enzyme site that had been introduced into the genome. An increase of the HR rate enhanced by Vpr was attenuated by an ATM inhibitor, KU55933, suggesting that Vpr-induced DSBs activate ATM-dependent cellular signal that enhances the intracellular recombination potential. In context with a recent report that KU55933 attenuated the integration of HIV-1 into host genomes, we discuss the possible role of Vpr-induced DSBs in viral integration and also in HIV-1 associated malignancy.

Low or no antibody responses to human immunodeficiency virus type 1 Nef in infected carriers with subtype E, in contrast to subtype B that showed antibodies preferentially recognizing subtype-specific Nef epitopes.

The viral accessory gene product Nef has been shown to play an important role in human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis. Only little information is available regarding the differences in the host immune responses against Nef protein and its function in vivo among different subtypes of HIV-1. In the present study, we showed marked differences in the immune responses to Nef protein between subtypes B and E. The amino acid sequence in subtype E Nef showed 72% homology with that in subtype B. Most murine monoclonal antibodies obtained by immunization with subtype B or E Nef protein showed cross-reactivity with both Nef proteins (80 and 67%, respectively). Next, we focused on the immune responses among infected Japanese and Thai individuals. Subtyping of the individuals into B and E was carried out by enzyme-linked immunosorbent assay (ELISA) using synthetic peptides corresponding to the V3 loop representing the principal neutralizing domain. Most of the sera from these individuals reacted strongly with Gag p24 proteins derived from subtypes B and E at similar levels. However, the immune responses among these individuals to Nef protein were markedly different. Some subtype B-infected Japanese and Thai individuals (40 and 35%, respectively) showed higher levels of anti-Nef antibodies, although these antibodies preferentially recognized epitopes specific to subtype B. On the other hand, most of the subtype E-infected Japanese and Thai individuals showed low or no antibody responses to Nef proteins. Thus, immune responses to Nef were markedly different between subtypes B- and E-infected carriers, suggesting different function(s) for Nef in AIDS pathogenesis. Further, vaccine design must take into account the different subtypes of HIV-1.

Differential susceptibility of resting CD4(+) T lymphocytes to a T-tropic and a macrophage (M)-tropic human immunodeficiency virus type 1 is associated with their surface expression of CD38 molecules.

Recent evidence has accumulated which definitively shows that chemokine receptors CCR5 and CXCR4 play an essential role as coreceptors for human immunodeficiency virus type 1 (HIV-1) infection. Flow cytometric analysis permitted us to detect CD38, a surface marker of early differentiation, as well as activation of T cells, on about half of healthy donor-derived CD4(+) T cells. In this study, we focused on the susceptibility of CD38(+) and CD38(-) subsets of CD4(+) T cells to HIV-1 infection with different coreceptor tropisms. About 20% of peripheral blood mononuclear cell-derived resting CD4(+) T cells were recovered into the CD38(+) subset fraction by panning with a monoclonal antibody to CD38. Most of the cells in this CD38(high) fraction also expressed CD45RA and CD62L at higher intensities compared with those of CD38(low) fraction. CCR5(+) T cells predominated in the CD38(-) subset, although cell surface expression of CD4 and CXCR4 was almost similar between both subsets. This difference was consistent with a significantly higher susceptibility of the CD38(-) subset to a macrophage (M)-tropic HIV-1 strain. In contrast, it was shown that a T-tropic strain of HIV-1 could replicate more efficiently in the CD38(+) subset, although viral adsorption rates were similar between both subsets. Thus, the differential susceptibility of CD4(+) T cells to M(-) and T-tropic HIV-1 was associated with their surface expression of CD38.

Ribosomal protein L7 included in tuberculin purified protein derivative (PPD) is a major heat-resistant protein inducing strong delayed-type hypersensitivity.

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis. It consists of more than 100 denatured proteins in a culture filtrate of a heated culture of Mycobacterium tuberculosis. In two-dimensional electrophoretic analysis of PPDs from M. tuberculosis and M. bovis BCG, most proteins were diffusely separated and could not be seen as spots because of denaturation, whereas a few proteins showed relatively clear spots, indicating heat resistance. Two such proteins corresponded to ribosomal proteins L7 and L12. The mixture of these proteins L7/L2 induced a strong delayed-type hypersensitivity reaction. Another protein showing a clear spot was a GroES analogue, but this did not induce delayed-type hypersensitivity. There were a few other unidentified proteins. It is well known that L7 and L12 are encoded by the same gene and that they differ from each other only by an acetylic post-translational modification that occurs at the N-terminus of L12 converting it to L7 in Escherichia coli. L12, but not L7, was found in two-dimensional electrophoresis of BCG ribosomes, although we found two proteins corresponding to L7 and L12 in PPDs and a native culture filtrate of BCG. We compared the delayed-type hypersensitivity reaction elicited by L7/L12 derived from a culture filtrate of BCG and L12 derived from BCG ribosomes. L7/L12 from the culture filtrate could induce delayed-type hypersensitivity, but L12 from ribosomes could not, indicating that L7 was attributable to the induction of delayed-type hypersensitivity. The activity of L7/L12 was heat resistant. Neither glycosylation nor phosphorylation of L7/L12 from a culture filtrate could be detected. The acetylation at N-terminal of L12 was essential for the delayed-type hypersensitivity activity.

[Development of slide-method to distinguish alive and dead mycobacteria by fluorescent staining--a trial for solving the biohazard problem in TB laboratories].

An acid-fast staining can detect mycobacteria in clinical specimens rapidly and specifically. It equally stains living and dead bacteria. It would be of more clinical use if the viability of mycobacteria in a sample was determined by the staining. In the present paper, the problems of FDA/EB staining, which detects live or dead bacteria, were solved by establishing a new technique, a slide-method. An air-dried smear of Mycobacterium bovis BCG (Tokyo 172) on a glass slide was covered by a filter paper fully soaked in the staining solution (500 micrograms FDA and 40 micrograms EB per ml PBS). This was kept in an incubator at 37 degrees C for 20 min. The filter paper was removed after incubation and the slide was examined using a fluorescent microscope with a blue filter. Live bacteria were stained greenish yellow taking the FDA stain in while dead bacteria were stained red with EB. This new slide technique eliminated the problems associated with FDA/EB staining. Moreover, stained smears appeared to be more stable compared with the conventional tube method. To overcome the biohazard problems in smear examination of tubercle bacilli, heating of the slides on a heat block at 100 degrees C for 20 min or passing air dried smears in a flame 5 to 30 times was tried to kill the bacteria. The heat-treated slides were stained with FDA/EB and the number of green and red bacteria were counted. Samples of the smeared bacteria were taken after heating and cultured on a solid medium to determine the presence of any colony-forming unit. It was found that no CFU was observed after heating and the morphology of the stained sample was the same to that before heating. These facts suggest that the above mentioned method is a simple, safe yet inexpensive diagnostic tool for mycobacterial clinical specimens.

Host immune responses to ribosome, ribosomal proteins, and RNA from Mycobacterium bovis bacille de Calmette-Gúerin.

The ribosomes from BCG strongly induced delayed type hypersensitivity (DTH) skin reactions in guinea pigs immunized with live BCG or heat killed Mycobacterium tuberculosis H37Rv, and also induced lymphocyte proliferative response in mice immunized with ribosomes. In contrast, neither ribosomal proteins nor RNA alone induced both DTH skin reactions and lymphocyte proliferative responses. Particle form consisted of ribosomal proteins and RNAs might be absolutely required for the activation of immune responses.

[The role of some cellular components of bacterial parasites in determining the incidence of tuberculosis: studies on mycobacterial antigens, with special reference to mycobacterial immunoreactive ribosomal and secreted proteins].

Tuberculosis remains as major disease, affecting more than 20 million people. The elimination of the disease with vaccination, rapid diagnosis, and and efficient therapy is an important objective of our study. To realize the objective, the characterization of antigens is essential. We have chosen two kinds of antigens for our study, the ribosomal antigens and and an antigenic proteins secreted by mycobacteria. The biochemical and immunological characterization of ribosomal fraction was carried out. Ribosomal proteins were purified and assessed for DTH reaction. The N-terminal amino acids sequences were determined. Total structures of S19, S7 and S12 in 30S and L7/L12 in 50S subunits were elucidated. L7/L12 had 66% homology with analogue from S. griseus which showed GTPase activity in protein synthesis. This protein was secreted in culture medium and induced strong DTH. Secreted antigenic proteins are of great interest for us. Secreted antigens may be recognized rapidly by immune system and therefore may induce rapid and high level immune response. It is also expected that it may contain protective antigens, since live BCG protect disease more efficiently than heat killed BCG. We have determined and published the total structure of four proteins (MPB64, MPB70, MPB57 and alpha antigen). We attempted to utilize this antigen for the diagnosis and the design of vaccine. The structures of alpha antigens from M. avium, M. intracellulare, M. scrofulaceum, M. kansasii and BCG were determined and its potential for application to diagnosis was presented. Using the operon of M. kansasii, alpha antigen and V3 region of HIV-1 were expressed by recombinant BCG which induced CTL in mice.

Immunological properties of ribosomal proteins from Mycobacterium bovis BCG.

Two proteins with molecular mass 65 kDa, a heat shock protein, and an S1-like protein were found in a 30S ribosomal subunit from Mycobacterium bovis BCG. The 17-kDa protein in the 30S subunit was homologous to alpha-crystallin heat shock protein, and the 16-kDa protein in the 50S subunit was homologous to the L7/L12 protein. The latter provoked a strong delayed-type hypersensitivity reaction in the sensitized guinea pigs. The GroES-like protein (12 kDa) loosely associated with ribosomes.

[Consistency in viabilities of mycobacteria detected by FDA/EB staining and colony forming units on the medium].

We have previously reported that the staining of mycobacteria with fluorescein diacetate (FDA) and ethidium bromide (EB) detects viable bacteria and distinguish them from heat-killed bacteria. Whether this method can be applied to clinical specimens has been an important question. In the present experiment, we treated mycobacteria with either UV-irradiation, 70% ethanol, 0.2% benzalconium chloride, 5% saponated cresol solution, or 5% phenol for 24 hours, and stained with FDA/EB to evaluate the effect of sterilization. We also stained samples of sputum from a tuberculosis patient with FDA/EB after treatment with 4% NaOH for 30 min. and neutralized with 1N H2SO4. All the samples were determined for the colony forming units on 1% Ogawa egg media. A good correlation was observed between the results of FDA/EB staining and colony formation. We believe that FDA/EB staining is useful method to detect viable mycobacteria in clinical samples.

Effect of a synthetic adjuvant for inducing anti-tumour immunity.

An acylated derivative of muramyl dipeptide (MDP), 6-O-(2-tetradecyl-hexadecanoyl)-muramyl-dipeptide (B30-MDP) is a strong adjuvant effective in inducing cell-mediated immunity. We used B30-MDP as an adjuvant for induction of anti-tumour immunity. Guinea-pigs which were injected repeatedly with a mixture of X-ray-treated leukaemic cells and B30-MDP dissolved in phosphate buffered saline resisted a challenge of leukaemia cells and showed no sign of leukocytosis. The immunity induced was tumour-specific and retained for more than 100 days. These results suggest that B30-MDP is useful as a simple but potent immunotherapeutic tool.

Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis.

Five actively secreted proteins (MPT32, MPT45, MPT51, MPT53, and MPT63) and the MPT46 protein were purified to homogeneity from Mycobacterium tuberculosis culture fluid and compared with proteins previously purified by ourselves and other investigators. Antisera were obtained by immunization of rabbits with all of the newly isolated proteins identified to be immunogenic. Two-dimensional electrophoresis of culture fluids obtained each week for 2 to 10 weeks of culturing of M. tuberculosis revealed characteristic changes, permitting identification of two distinct groups of proteins being actively secreted from the mycobacterial cells or appearing later in the culture fluids as a result of the release of soluble proteins from the cytosol after lysis of bacteria. The N-terminal amino acid sequences of five MPTs were shown to be identical to those of proteins previously isolated by other investigators and given different designations, and five new sequences are given. These sequences and the use of the antisera may serve to identify these proteins with mycobacterial constituents isolated by other investigators. The previously identified but not isolated MPT45 protein was shown to correspond to the C component of the antigen 85 complex. The 27-kDa MPT51 protein was demonstrated to cross-react with the three components of the antigen 85 complex, and the N-terminal amino acid sequences of MPT51 and MPT59 showed 60% homology. This finding and the extensive cross-reactivity between the components of the antigen 85 complex may indicate that there is a family of closely related secreted proteins in mycobacteria.

[Effect of atypical mycobacterial infection on the cell-mediated immunity induced by Mycobacterium bovis BCG in mice. I. Suppression of delayed-type hypersensitivity].

Mycobacterium avium Mino grows well in the lung, spleen or liver of susceptible mice, such as C57BL/6, C57BL/10, and B10 congenics. Infection with Mino inhibited the induction of delayed-type hypersensitivity (DTH) to BCG in the mouse receiving subcutaneous injection of BCG 2 weeks later. Post-infection with Mino did not affect DTH to BCG already established. Preceding infection with avirulent strains of atypical mycobacteria weakly suppressed the induction of DTH by BCG. On the other hand, Mino infection did not affect DTH to SRBC. DTH to Mino and DTH to BCG detected by PPD-I and PPDs hardly showed cross-reactivity. These results suggest that tuberculin skin reaction after BCG vaccination could be suppressed by the infection with atypical mycobacteria in humans, especially where these bacterial infections are prevailing.

[Detection of viable or non-viable mycobacteria by hydrolysis of fluorogenic substrate].

Acid-fast staining is broadly used for the detection of mycobacteria in smears or pathological preparations, but it does not distinguish viable and non-viable bacteria. Enzymatic hydrolysis of fluorescein diacetate (FDA) was reported as a tool to detect viable mammalian cells or protozoa. We have applied this method to mycobacteria samples to detect viable or non-viable bacteria in the smear. Mycobacterium bovis BCG (Tokyo), 20 mg/ml suspension of standard vaccine, was used as the sample of viable bacteria. A part of the same suspension was heated at 100 degrees C for 30 min and used as the sample of non-viable bacteria. Three samples, viable, non-viable, and 1:1 mixture of the two, were stained with acid-fast staining and FDA-EB (ethidium bromide) mixture, respectively. For FDA/EB staining, stock solution of FDA (5 mg/ml acetone) was diluted 1:50 in PBS and 25 microliters of FDA and 25 microliters of EB (20 micrograms/ml PBS) were mixed with 50 microliters of bacterial suspension. Preparations were observed with either light or fluorescein microscope. Living bacteria were stained in yellow green in FDA/EB staining while non-viable bacteria were red. Mixed sample of live and dead bacilli showed differential staining with green or red in FDA/EB staining, but no difference was shown in acid-fast staining between viable and non-viable bacteria.

Comparative studies with various substrains of Mycobacterium bovis BCG on the production of an antigenic protein, MPB70.

A protein, isolated and purified from the unheated culture filtrate of Mycobacterium bovis BCG (substrain Tokyo 172) and designated MPB70, elicited a delayed skin reaction in guinea pigs sensitized with viable cells of BCG but not in those sensitized with heat-killed cells. The skin reaction reached the maximum 4 to 8 weeks after the inoculation of the BCG and then decreased gradually, resulting in conversion to negative after 20 weeks, whereas the skin reaction to purified protein derivative (PPD) continued to be positive. Guinea pigs immunized with viable cells of various substrains of BCG were skin tested with MPB70 and PPD. Guinea pigs immunized with the BCG substrain Tokyo 172 and the substrain Moreau (Brazil) showed strong delayed skin reactions to both MPB70 and PPD. On the other hand, guinea pigs immunized with the Pasteur substrain 1173P2, the Glaxo substrain 1077, the Copenhagen substrain 1331, the Tice substrain, or the Beijing substrain 64-42 showed negative skin reactions to MPB70, whereas they were strongly positive to PPD. In a two-dimensional acrylamide gel electrophoretic analysis of proteins from the culture filtrates of the BCG substrains, the culture filtrates of the Tokyo and Moreau substrains showed the spot of MPB70 on the gel slabs, whereas those of the other BCG substrains did not.