Changes in fibre-type composition and myosin heavy-chain IId isoform in rat soleus muscle during recovery period after hindlimb suspension

We examined the changes in myosin heavy-chain (HC) isoforms and fibre-type composition in rat soleus muscle using both myosin adenosine triphosphatase staining and sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) analyses during the recovery period after 4 weeks of hindlimb suspension. Although there was no change in type IIc fibres after the suspension, an increase in this type of fibres was observed during the 1- to 4-week recovery period. The increase in type Ilc fibres was considered to be due to a shift from type Ila to IIc fibres. The SDS-PAGE analysis revealed the presence of the HC IId isoform, which was not observed in the control muscle, after a 4-week hindlimb suspension. The HC IId isoform gradually decreased over 3 weeks of recovery and disappeared in the 4th week of recovery after the suspension. These results suggest that the hypogravity conditions induced by hindlimb suspension stimulated the synthesis of the HC IId isoform, whereas an increase in mechanical load to the muscle accelerated the degradation of the HC IId isoform and the synthesis of type Ilc fibres during the recovery period after hindlimb suspension.     

Split tolerance between spleen and lymph node cells in severe combined immunodeficiency mice grafted with AKR fetal liver cells

Severe combined Immunodeficient (SCID) mice defective in stemcells for T and B cells appear to be an ideal host for constructionof chimeric mice. When bone marrow cells are used as a sourceof stem cells, however, host SCID mice do not always show sufficientreconstitutlon. In this study, fetal liver cells from AKR embryoswere transplanted into SCID mice without prior irradiation.This treatment induced full reconstltution of lymphopoiesisas evaluated by flow cytometry analysis and serum Ig production2 months after transplantation. Thus, fetal liver cells seemto be a better source for reconstitutlon of SCID mice than bonemarrow cells. Lymph node (LN) cells of these mice (FLT mice)had no proliferatlve or cytotoxlc activities against eitherhost-type (C.B-17) or donor-type (AKR) spleen cells. However,spleen cells from FLT mice exhibited marked proliferatlve andcytotoxlc activities against C.B-17 cells, with no activitiesagainst AKR cells. Spirt tolerance against C.B-17 cells In spleenand LN cells was not a transient phenomenon, since similar resultswere obtained from a cytotoxic T lymphocyte assay 4 months later.In spite of the strong host reactivity in vitro, aberrationof clonal deletion or development of a graft-versus-host diseasewas not seen in FLT mice. As IL-2 induced the host reactivityof LN cells in a mixed lymphocyte reaction, potentially host-reactiveT cells were present in LN but were rendered anerglc. Tolerancein FLT mice seems to be regulated by a peripheral mechanism.We supposed that the split tolerance in FLT mice was inducedby the different antigenicity between the spleen and LN.     

Immunocytochemical demonstration of heat shock protein 25 in the rat temporomandibular joint

The expression of heat shock protein 25 (Hsp 25) was investigated in the rat temporomandibular joint by immunocytochemistry combined with confocal and electron microscopy. Immunostaining with an antibody to Hsp25 was able to demonstrate various cellular elements in the synovial membrane of the joint. Intense immunoreaction for Hsp25 was recognized in certain cells comprising the synovial lining layer. Confocal microscopic observation revealed two characteristic profiles of the Hsp25-positive cells with cytoplasmic processes: one extended thick and long processes towards the articular cavity, and the other prejected horizontally slender processes which covered the synovial membrane. Under the electron microscope, the immunoreactive synovial lining cells were characterized by a well-developed rough endoplasmic reticulum and secretory granules, suggesting that they can be categorized as fibroblastic type B cells. The covering by the cytoplasmic extensions was confirmed by immuno-electron microscopic observations. This cytoplasmic covering presumably performs a barrier function and expedites the effective secretion/resorption of synovial fluids. Since it has been proposed that Hsp 25 is associated with an estrogen receptor, the immunopositive synovial lining cells were considered estrogen-target cells. Immunoreactivity for Hsp25 was also observed in the chondrocytes of the maturative and hypertrophic cell layers as well as in the cells of the articular disk. A suggestion was made that Hsp25 might be involved in the inhibition of apoptosis of those cells.     

Pathogenic capacity of proteases from Serratia marcescens and Pseudomonas aeruginosa and their suppression by chicken egg white ovomacroglobulin.

The pathogenicities of three proteases from Serratia marcescens, two proteases from Pseudomonas aeruginosa, and one thermolysin from Bacillus stearothermophilus were examined. All proteases tested caused acute liquefactive necrosis of the cornea and descemetocele formation in guinea pig eyes after intrastromal injection, with the exception of the 60-kilodalton protease from S. marcescens, which produced only an opaque lesion. When injected into guinea pig skin, the protease also enhanced vascular permeability, which was followed by edema formation. The permeability-enhancing activity of the proteases increased in parallel with the concentration of the enzymes. When tested in vitro for its effect on these bacterial proteases, chicken egg white ovomacroglobulin (ovoM) inhibited the enzymatic activity of all the proteases after a short incubation period at an enzyme/inhibitor ratio (molar) of 1:1 to 1:4 or at a lower concentration after a longer incubation period. Such treatment of the proteases with chicken egg white ovoM before injection intrastromally into the eyes or intradermally into the clipped flanks of guinea pigs protected the cornea from destruction or completely prevented the permeability reaction and edema formation. No inhibitory effects of plasma protease inhibitors against these bacterial proteases were noted. Since the proteases are critical in the pathogenic processes caused by the bacteria, these results suggest a beneficial effect of ovoM against bacterial infections.     

Detection of antibodies to hepatitis C virus (HCV) structural proteins in anti-HCV-positive sera by an enzyme-linked immunosorbent assay using synthetic peptides as antigens.

We have defined 10 linear immunogenic regions encoded by the putative hepatitis C virus (HCV) structural proteins (core and envelope) by employing an enzyme-linked immunosorbent assay (ELISA) and by using 17 sequential synthetic peptides covering the N-terminal 330 amino acids of the structural polyproteins as antigens. These peptides correspond to amino acids 1 to 24, 21 to 44, 42 to 68, 64 to 91, and 100 to 120 of the putative core protein and amino acids 192 to 212, 223 to 238, 236 to 258, 250 to 266, and 307 to 330 of the putative envelope protein. In particular, the peptide covering amino acids 21 to 44 of the core protein was reactive with all but one (40 of 41) of the serum samples giving a positive signal in the passive hemagglutination assay (PHA) using the core and nonstructural proteins (NS 3/4) of the virus as antigens. We detected the HCV genome in 25 (61%) of 41 PHA-positive serum samples by the polymerase chain reaction (PCR) test. Of 25 PCR-positive serum samples, 17 serum samples had reactivity to the peptides derived from the envelope protein. On the other hand, only 1 of the 16 PCR-negative serum samples had reactivity to the peptides derived from the envelope protein. Interestingly, we often observed high serum alanine aminotransferase levels in PCR-positive individuals bearing antibodies to the envelope protein.     

Linear antigenic regions of the structural proteins of human T-cell lymphotropic virus type I detected by enzyme-linked immunosorbent assays using synthetic peptides as antigens.

We synthesized 46 sequential peptides 21 to 39 amino acids long over the structural protein of human T-cell leukemia virus type I (HTLV-I; the p19 and p24 gag protein and the gp46 and p20E env proteins) and tested their reactivities against antibodies in sera from HTLV-I healthy carriers and patients diagnosed as having human T-cell leukemia-lymphoma (ATLL) and myelopathy (HAM) by using an enzyme-linked immunosorbent assay. Of the 46 synthetic peptides, 18 peptides (2 corresponding to the p19 gag protein, 2 corresponding to the p24 gag protein, 8 corresponding to the gp46 env protein, and 6 corresponding to the p20E env protein) reacted with antibodies in the sera from HTLV-I healthy carriers. In particular, the peptides comprising amino acids 100 to 119 and 119 to 130 of the gag and 175 to 199, 213 to 236, 253 to 282, and 288 to 317 of the env proteins reacted with antibodies in sera from more than 30% of HTLV-I healthy carriers. These peptides also showed high reactivities to the antibodies in the sera from patients with ATLL and HAM. The results indicate that the predominant antigenic regions of the structural protein of HTLV-I were located at the C-terminal end of the p19 gag protein and the C-terminal half of the gp46 env protein, and the corresponding peptides proved to be useful antigens in detecting antibodies in the sera from individuals infected with HTLV-I.     

A NEW METHOD FOR VITAL STAINING OF CENTRAL NERVOUS TISSUES WITH NEUTRAL RED

A new vital staining method with neutral red has been established where by cerebral ganglion cells can be stained in vivo .     

Mechanical characteristics of skinned and intact muscle fibres from the giant barnacle,Balanus nubilus

Intact muscle fibres fromBalanus nubilus develop tensions of up to 600 kN sd m⁻² during electrical stimulation. The rise of tension occurs with a half-time (177 ms at 12° C) about fivefold longer than that of tetanised frog muscle at the same temperature. The response of myofibrillar bundles to a rapid stretch resembles that of frog muscle but has a y_o value (i.e. the size of an instantaneous release necessary to just discharge tension) which is ca. 2.5 times smaller, and phase 2 of the tension transient (the “quick phase”) occurs at a rate comparable to that of frog muscle. In contrast, the ATPase activity (0.018 mmoles · kg wet weight⁻¹ · s⁻¹) of this preparation and its maximum shortening velocity (0.15–0.16 muscle lengths · s⁻¹) are both at least fivefold slower than frog muscle. These findings can be accounted for by a cross-bridge cycle in barnacle muscle in which events prior and subsequent to the tension generating step(s) occur at a rate at least fivefold slower than comparable steps in frog muscle, but the step(s) associated with tension development occur at similar rates in the two preparations. Since the rate of mechanical relaxation in barnacle muscle is modified in the presence of intracellular calcium buffers and by depolarisation-induced elevation of the free calcium during the relaxation phase, it is proposed that the time course of relaxation is not determined exclusively by the kinetics of the cross-bridge cycle, but is also dependent on the free calcium concentration during relaxation.     

Local production and localization of transforming growth factor-beta in tuberculous pleurisy.

Transforming growth factor-beta (TGF-beta) is one of the cytokines which play an immunosuppressive role in an inflammatory process. To investigate the local production of TGF-beta, we evaluated the levels of TGF-beta in tuberculous pleural effusions (TBPE) and non-tuberculous benign pleural effusions (non-TBPE) by the growth inhibition assay with Mv1Lu mink lung epithelial cells. The mean level of TGF-beta in TBPE (46.1 +/- 31.5 pM; mean +/- s.d.) was higher than in non-TBPE (21.7 +/- 12.3 pM) (P < 0.05). Although the level of interferon-gamma (IFN-gamma) in TBPE measured by ELISA was significantly higher than in non-TBPE, there was no significant difference in the levels of tumour necrosis factor-alpha (TNF-alpha) measured by ELISA between these two groups. Moreover, to elucidate localization of TGF-beta in tuberculous pleurisy, immunohistochemical studies of pleura, using the rabbit polyclonal antibody Ab39 against latent TGF-beta 1 binding protein (LTBP) were performed. Results revealed that LTBP was localized in immature fibrotic areas where infiltrations of T lymphocytes and macrophages were absent. Importantly, the major sources of LTBP in these areas were thought to be mesothelial cells and fibroblasts. LTBP was not found in granulomas and mature fibrotic areas. Our data suggest that TGF-beta in tuberculous pleurisy may play important roles for regression of granulomatous inflammation and pleural fibrosis for tissue repair.     

Effect of serum amyloid P component level on transthyretin-derived amyloid deposition in a transgenic mouse model of familial amyloidotic polyneuropathy.

To elucidate the pathogenesis of amyloid deposition associated with familial amyloidotic polyneuropathy (FAP), we developed several transgenic mouse lines carrying the human mutant transthyretin (TTR) gene. We found that human TTR and mouse serum amyloid P component (SAP) are deposited as amyloid in tissues of these mouse lines. Because SAP is a major acute-phase reactant in mice, we asked whether repeated injections of Escherichia coli lipopolysaccharide (LPS) would enhance the amyloid deposition in one of these transgenic mouse lines. During the course of repeated LPS injections, serum levels of SAP in the transgenic mice remained between severalfold to about 50-fold higher than seen in the absence of stimulation. As no significant difference was detected in the onset, progression, and tissue distribution of TTR-derived amyloid (ATTR) deposition between the LPS-stimulated and unstimulated transgenic mice, the induction of SAP synthesis by acute inflammation probably does not affect the onset and extent of ATTR deposition.