Validity of NIR spectroscopy for quantitatively measuring muscle oxidative metabolic rate in exercise.

The purpose of this study was to examine the validity of the quantitative measurement of muscle oxidative metabolism in exercise by near-infrared continuous-wave spectroscopy (NIRcws). Twelve male subjects performed two bouts of dynamic handgrip exercise, once for the NIRcws measurement and once for the (31)P-magnetic resonance spectroscopy (MRS) measurement as a standard measure. The resting muscle metabolic rate (RMRmus) was independently measured by (31)P-MRS during 15 min of arterial occlusion at rest. During the first exercise bout, the quantitative value of muscle oxidative metabolic rate at 30 s postexercise was evaluated from the ratio of the rate of oxyhemoglobin/myoglobin decline measured by NIRcws during arterial occlusion 30 s after exercise and the rate at rest. Therefore, the absolute values of muscle oxidative metabolic rate at 30 s after exercise [VO(2NIR(30))] was calculated from this ratio multiplied by RMRmus. During the second exercise bout, creatine phosphate (PCr) resynthesis rate was measured by (31)P-MRS at 30 s postexercise [Q((30))] under the same conditions but without arterial occlusion postexercise. To determine the validity of NIRcws, VO(2NIR(30)) was compared with Q((30)). There was a significant correlation between VO(2NIR(30)), which ranged between 0.018 and 0. 187 mM ATP/s, and Q((30)), which ranged between 0.041 and 0.209 mM ATP/s (r = 0.965, P < 0.001). This result supports the application of NIRcws to quantitatively evaluate muscle oxidative metabolic rate in exercise.

Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts.

While CD28 functions as the major T cell costimulatory receptor, a number of other T cell molecules have also been described to induce T cell costimulation. Here, we investigated the mechanisms by which costimulatory molecules other than CD28 contribute to T cell activation. Non-CD28 costimulatory molecules such as CD5, CD9, CD2, and CD44 were present in the detergent-insoluble glycolipid-enriched (DIG) fraction/raft of the T cell surface, which is rich in TCR signaling molecules and generates a TCR signal upon recruitment of the TCR complex. Compared with CD3 ligation, coligation of CD3 and CD5 as an example of DIG-resident costimulatory molecules led to an enhanced association of CD3 and DIG. Such a DIG redistribution markedly up-regulated TCR signaling as observed by ZAP-70/LAT activation and Ca2+ influx. Disruption of DIG structure using an agent capable of altering cholesterol organization potently diminished Ca2+ mobilization induced by the coligation of CD3 and CD5. This was associated with the inhibition of the redistribution of DIG although the association of CD3 and CD5 was not affected. Thus, the DIG-resident costimulatory molecules exert their costimulatory effects by contributing to an enhanced association of TCR/CD3 and DIG.

cDNA cloning and characterization of a novel receptor-type protein tyrosine phosphatase expressed predominantly in the brain.

Protein tyrosine phosphatase has the potential to control various cellular events by negatively regulating the extent of tyrosine phosphorylation. Here, we report the isolation of a murine receptor protein tyrosine phosphatase, PTPBR7, which is expressed almost exclusively in the brain. Though the cytoplasmic portion of PTPBR7 reveals high similarity to HePTP/LC-PTP and STEP, these are, unlike PTPBR7, non-receptor protein tyrosine phosphatases. Unlike most receptor protein tyrosine phosphatases, PTPBR7 has only one cytoplasmic phosphatase domain, and its extracellular domain reveals no obvious structural similarity to known molecules. Thus, PTPBR7 defines a new subfamily of receptor-type protein tyrosine phosphatases. The putative extracellular domain of PTPBR7 was expressed in COS-7 cells as a chimeric fusion protein with an immunoglobulin Fc portion (PTPBR7-Fc). PTPBR7-Fc was secreted in the culture supernatant, confirming the capability of the extracellular domain of PTPBR7 to translocate across the cytoplasmic membrane. The cytoplasmic portion of PTPBR7 was expressed as a fusion protein in bacteria and was demonstrated to have catalytic activity. The expression of PTPBR7 was detectable in brain and especially in cerebellum but undetectable in liver, lung, heart, kidney, thymus, bone marrow, and spleen. In situ hybridization analysis revealed the most prominent signal in Purkinje cells. The predominant expression of PTPBR7 in the brain suggests that PTPBR7 may have role(s) in neuronal cells.

cDNA cloning of a novel protein tyrosine phosphatase with homology to cytoskeletal protein 4.1 and its expression in T-lineage cells.

Reversible tyrosine phosphorylation plays important regulatory roles in various cellular events including the differentiation and function of lymphocytes. Here we report the cDNA cloning of a non-receptor type protein tyrosine phosphatase, PTP36, which is expressed in murine thymus. PTP36 was a new member of a tyrosine phosphatase subfamily defined by MEG-01 and PTPH1, which had a C-terminal phosphatase domain as well as an N-terminal domain with homology to cytoskeletal-associated proteins like band 4.1, ezrin, and talin. In addition, we found a putative SH3-binding motif in PTP36 but not in MEG-01 or PTPH1. PTP36 was expressed in cells of both hematopoietic and non-hematopoietic origins. In thymocytes subpopulations, PTP36 was preferentially expressed in double positive stage cells. The change of PTP36 expression level along with T cell maturation suggests its involvement in the regulation of T cell development.

Enhanced production of interleukin-6 in mice with type II collagen-induced arthritis.

We established an interleukin-6 (IL-6)-dependent cell line from murine plasmacytoma MOPC-104E cells. This cell line (designated PIL-6) was found to respond to murine and to human IL-6, but not to any other cytokines. We used this cell line to investigate the involvement of IL-6 production in type II collagen-induced arthritis in DBA/1 mice. Only marginal IL-6 activity was detected in sera from DBA/1 mice inoculated with Freund's complete adjuvant (FCA) alone, with an unrelated protein (bovine serum albumin) plus FCA, or with type II collagen plus Freund's incomplete adjuvant. However, enhanced IL-6 activity was observed in DBA/1 mice that had been injected with type II collagen plus FCA to induce arthritis. The elevated level of serum IL-6 activity was associated with high levels of IL-6 produced when lymph node cells from arthritic mice were stimulated in vitro with type II collagen. We also found that the L3T4+ T cell subset is responsible for the enhanced production of IL-6 in arthritic mice. The results are discussed in the context of potential roles of IL-6 in the induction and/or expression of chronic, progressive arthritis.

Polyclonal B cell activation by a B cell differentiation factor, B151-TRF2. III. B151-TRF2 as a B cell differentiation factor closely associated with autoimmune disease.

We demonstrated previously that B151K12 T cell hybridoma produces two distinct B cell differentiation factors, B151-TRF1 and B151-TRF2, capable of inducing differentiation of antigen-activated and unstimulated B cells into antibody-forming cells, respectively. In the present study we investigated the pathophysiologic relation of these factors with factors obtained from MRL/MP-lpr/lpr(MRL/lpr) mice and (C57BL/6 X DBA/2)F1 (BDF1) mice undergoing chronic graft-vs-host reaction (GVHR), representing a murine model of systemic lupus erythematosus with polyclonal B cell activation associated with the T cell hyperfunction. The functional and biochemical analyses revealed that B151-TRF2-like, but not B151-TRF1-like, activity was found in culture fluid supernatant (CFS) of lymphoid cells from MRL/lpr mice with lymphoproliferative syndrome. On the other hand, both B151-TRF1- and B151-TRF2-like activities were detected in CFS prepared from spleen cells of BDF1 mice undergoing chronic GVHR by the inoculation of parental DBA/2 spleen cells. Interestingly, spleen cells of BDF1 mice transferred with DBA/2 thymocytes preferentially elaborated B151-TRF1-like factor. Because BDF1 mice transferred with DBA/2 spleen cells but not with DBA/2 thymocytes developed a SLE-like syndrome exemplified by the appearance of Coombs' antibody and proteinuria, it seemed likely that production of B151-TRF2-like factor was closely associated with the onset of autoimmune disease. In fact, B151-CFS containing B151-TRF2 but not B151-TRF1 activity could induce a striking autoantibody production both in vivo and in vitro as detected by PFC responses of normal mice to bromelain-treated mouse red blood cells (BrMRBC). Moreover, it was demonstrated that in vitro anti-BrMRBC PFC responses induced by semipurified B151-TRF2 was markedly inhibited by addition of relevant anti-Ia antibody to the culture. Thus, the present study demonstrates that B151-TRF2 represents one of the B cell differentiation factors responsible for polyclonal B cell activation leading to autoantibody production.

Cholecystokinin-8-like immunoreactive neuron pathway from the supramammillary region to the ventral tegmental nucleus of Gudden of the rat.

The existence of a pathway from the supramammillary region (SM) to the ventral tegmental nucleus of Gudden (NTV) containing cholecystokinin-8 (CCK)-like immunoreactive (CCKI) structures was demonstrated by experimental manipulations in the rat. Destruction of the SM resulted in the disappearance of CCKI fibers in the NTV.

The mechanism of tolerance induction in thymus-derived lymphocytes; I. intracellular inactivation of hapten-reactive helper T lymphocytes by hapten-nonimmunogenic copolymer of D-amino acids.

Treatment of a p-azobenzoate (PAB) derivative of a copolymer of D-glutamic acid and D-lysine (D-GL) induced a profound state of unresponsiveness to PAB-reactive helper T lymphocytes generated in PAB-mouse gamma globulin (MGG)-primed mice. This unresponsiveness in T lymphocytes was specific for PAB-reactive cells, since the bacterial alpha-amylase-, keyhole limpet hemocyanin-, or ovalbumin-primed helper T lymphocytes were not suppressed by PAB-D-GL treatment. Taking advantage of the relative ease with which PAB-D-GL can induce specific unresponsiveness to helper T lymphocytes in an animal previously primed with PAB-MGG, it was possible to approach certain questions concerning the mechanisms of tolerance-induction and the fate of tolerant helper T lymphocytes in the PAB-D-GL model by utilizing a classical adoptive cell transfer systemmelimination of the possibility of carry-over of the tolerogen with cells or of the generation of suppressor cells as the result of PAB-D-GL treatment as an explanation of the suppression of helper T-cell activity strongly inplicates the existence of a central intracellular mechanism of specific tolerance on the helper T-cell level. The possibility that suppression of the activity of PAB-reactive helper T lymphocytes by PAB-D-GL reflects simple blocking of surface receptor molecules on T lymphocytes was ruled out as it was found that the helper activity of PAB-reactive cells was minimally suppressed even when PAB-D-GL was directly exposed in vitro to helper T lymphocytesmmoreover, the most conclusive evidence on te the tolerant state induced by in vivo exposure of primed T cells to PAB-D-GL. It appears, therefore, that specific tolerance induced by PAB-D-GL' TO PAB-reactive helper T lymphocytes is an example of irreversible inhibition of T-cell reactivity to antigen, reflecting yet to be determined events at the intra- and subcellular levels.