Differences emerge in visceral adipose tissue accumulation after selection for innate cardiovascular fitness.

Cardiorespiratory fitness (CRF) has been reported to be inversely associated with visceral adipose tissue (VAT) accumulation, independent of body weight. However, the confounding effect of physical activity on the association between CRF and VAT remains inadequately addressed. On the basis of maximal oxygen uptake (VO(2 max)), 143 sedentary, overweight women were dichotomized into high-fit (HF) and low-fit (LF) groups. Body composition and VAT were measured using dual-energy X-ray absorptiometry and computed tomography, respectively, and activity-related energy expenditure (AEE) was calculated using the doubly labeled water technique. No differences were observed between HF and LF for body mass index (HF 28.2 ± 1.3; LF 28.3 ± 1.31 kg m(-2)), total body weight (HF 77.5 ± 6.8; LF 77.9 ± 7.3 kg), total fat mass (HF 33.5 ± 5.1; LF 33.9 ± 4.4 kg) or AEE (HF 439.9 ± 375.4; LF 517.9 ± 298.7 kcal day(-1)). Significant differences in visceral adiposity (HF 68.5 ± 30.4; LF 91.2 ± 31.8 cm(2); P<0.001) and insulin sensitivity (HF 5.1 ± 1.8; LF 3.1 ± 2.4 S(I) × 10(-4) min(-1) µIU(-1) ml(-1); P<0.01) were observed between the HF and LF groups, independent of age, race and AEE. This study affirms previous findings that CRF is an important determinant of the accumulation of VAT, and this relationship is independent of physical activity.

Functional characterization of a signal transducing motif present in the T cell antigen receptor zeta chain.

A conserved sequence motif has been identified in a number of signaling subunits associated with hematopoietic cell antigen receptors. Here, we characterize signaling by a 17 amino acid motif that is triplicated in the T cell antigen receptor zeta chain. Analysis of zeta truncations and constructs containing the isolated motif demonstrates that this motif is sufficient for the induction of both proximal and distal events associated with T cell activation. Stimulation of truncations that contain either one, two, or three copies of the motif results in induction of an identical pattern of tyrosine phosphoproteins. Moreover, triplication of the NH2-terminal zeta motif results in enhanced signaling, suggesting a redundant role in signal amplification for the three motifs in zeta. Finally, we demonstrate the association of a recently identified protein tyrosine kinase ZAP-70 with this motif, and provide evidence for its involvement in zeta function.

Thymocyte development in the absence of pre-T cell receptor extracellular immunoglobulin domains.

Immature thymocytes express a pre-T cell receptor (pre-TCR) composed of the TCRbeta chain paired with pre-Talpha. Signals from this receptor are essential for passage of thymocytes through a key developmental checkpoint in the thymus. These signals were efficiently delivered in vivo by a truncated form of the murine pre-TCR that lacked all of its extracellular immunoglobulin domains. De novo expression of the truncated pre-TCR or an intact alphabetaTCR was sufficient to activate characteristic TCR signaling pathways in a T cell line. These findings support the view that recognition of an extracellular ligand is not required for pre-TCR function.

Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases.

The T cell antigen receptor (TCR) initiates signals by interacting with cytoplasmic protein tyrosine kinases (PTKs) through a 17-residue sequence motif [called the antigen recognition activation motif (ARAM)] that is contained in the TCR zeta and CD3 chains. TCR stimulation induces the tyrosine phosphorylation of several cellular substrates, including the ARAMs. Lck kinase activity is required for phosphorylation of two conserved tyrosine residues in an ARAM. This phosphorylation leads to the recruitment of a second cytoplasmic PTK, ZAP-70, through both of the ZAP-70 Src homology 2 domains and its phosphorylation. Thus, TCR signal transduction is initiated by the sequential interaction of two PTKs with TCR ARAMs.

Regulation of interleukin-2 gene enhancer activity by the T cell accessory molecule CD28.

The mechanism by which cell surface molecules regulate T cell production of lymphokines is poorly understood. Production of interleukin-2 (IL-2) can be regulated by signal transduction pathways distinct from those induced by the T cell antigen receptor. Stimulation of CD28, a molecule expressed on most human T cells, induced the formation of a protein complex that bound to a site on the IL-2 gene distinct from previously described binding sites and increased IL-2 enhancer activity fivefold. The CD28-responsive complex bound to the IL-2 gene between -164 and -154 base pairs from the transcription start site. The sequence of this element is similar to regions conserved in the 5' flanking regions of several other lymphokine genes.

Evaluation of multi-frequency bioelectrical impedance analysis for the assessment of body composition in individuals with obesity.

OBJECTIVE: The purpose of this study was to compare body composition measurements estimated by multi-frequency bioelectrical impedance analysis (MF-BIA) with air displacement plethysmography (ADP) in individuals with obesity. METHODS: Bariatric patients were recruited from Geisinger's Center for Nutrition and Weight Management Clinic in Danville, Pennsylvania. Sixty-two participants (age = 52.4 ± 9.3 years; body mass index = 38.9 ± 8.0 kg m-2) reported for a same-day testing visit. Body composition was measured using a common MF-BIA analyzer (InBody 720, Biospace Co., Beverly Hills, CA) and ADP. RESULTS: Strong relationships were observed between MF-BIA and ADP methods (r = 0.88-0.96, P < 0.001). There were no differences between MF-BIA and ADP measures of per cent body fat, fat mass or fat-free mass for the total sample or when examined by gender. CONCLUSIONS: The InBody 720 MF-BIA analyzer produced body composition measurements that were similar to ADP supporting the use of this technology in the obese population.

New insights into T-cell antigen receptor structure and signal transduction.

Recent studies have provided insights into how the complex structure of the T-cell antigen receptor relates to its signal transduction function. Both the CD3 and zeta subunits contain functioning signaling modules that regulate the activation of tyrosine kinases and phosphorylation of cellular substrates.

Signaling checkpoints during the development of T lymphocytes.

Two major lineage decisions face immature T cells as they develop in the thymus. At an early stage in their development, they must first commit to either the gamma delta or alpha beta lineages. If they opt for the alpha beta lineage, then at a later stage they must also choose between a CD4+ or CD8+ fate before they can pass through the thymic medulla and exit to the periphery. Thymocyte survival at key developmental checkpoints is determined by signaling from cytokine receptors and the T-cell receptor. Recent advances have been made in contemporary understanding of the signals that regulate thymocyte survival, proliferation and lineage decisions.

Electronically captured, patient-reported physical function: an important vital sign in obesity medicine.

OBJECTIVES: Impaired physical function (i.e., inability to walk 200 feet, climb a flight of stairs or perform activities of daily living) predicts poor clinical outcomes and adversely impacts medical and surgical weight management. However, routine assessment physical function is seldom performed clinically. The PROMIS Physical Function Short Form 20a (SF-20a) is a validated questionnaire for assessing patient reported physical function, which includes published T-score percentiles adjusted for gender, age and education. However, the effect that increasing levels of obesity has on these percentiles is unclear. We hypothesized that physical function would decline with increasing level of obesity independent of gender, age, education and comorbidity. MATERIALS AND METHODS: This study included 1,627 consecutive weight management patients [(mean ± SEM), 44.7 ± 0.3 years and 45.1 ± 0.2 kg/m2] that completed the PROMIS SF-20a during their initial consultation. We evaluated the association between obesity level and PROMIS T-score percentiles using multiple linear regression adjusting for gender, age, education and Charlson Comorbidity Index (CCI). RESULTS: Multiple linear regression T-score percentiles were lower in obesity class 2 (-12.4%tile, p < 0.0001), class 3 (-17.0%tile, p < 0.0001) and super obesity (-25.1%tile, p < 0.0001) compared to class 1 obesity. CONCLUSION: In patients referred for weight management, patient reported physical function was progressively lower in a dose-dependent fashion with increasing levels of obesity, independent of gender, age, education and CCI.

Production and characterization of monoclonal antibodies specific for the murine T cell receptor zeta chain.

The T cell receptor (TCR) comprises an antigen-specific alpha beta heterodimer non-covalently associated with the CD3 gamma delta epsilon and TCR zeta subunits. Both the CD3 and TCR zeta subunits are proposed to be responsible for the intracellular signal-transduction events. We report here the production of eight monoclonal antibodies (mAbs) that bind in an ELISA assay to a 113 amino acid synthetic peptide corresponding to the cytoplasmic domain of TCR zeta. Western blot analysis of anti-CD8 precipitates of lysates of transfectants expressing chimeric CD8/zeta constructs encoding increasing COOH-terminal truncations of TCR zeta indicates that four of these mAbs recognized the region of TCR zeta chain comprising the last 29 COOH-terminal residues. Thus, this region of TCR theta may encode an immunodominant epitope. Furthermore, one of these mAbs, G3, is capable of precipitating both non-phosphorylated and tyrosine phosphorylated TCR zeta. The G3 mAb should be useful for elucidating the structural and signalling characteristics of the TCR zeta chain.

Surface chimeric receptors as tools in study of lymphocyte activation.

In this chapter we have described a powerful technology that has allowed the functional dissection of individual subunits from oligomeric receptors. We have focused primarily on chimeras derived from antigen receptors or their downstream signaling components to illustrate the wide utility of the approach; however, the technology has been applied to numerous multimeric receptors of the immune system including cytokine receptors, Fc receptors, and natural killer (NK) cell inhibitory receptors. Although the significance of the structural complexity of oligomeric receptors is by no means understood, it is certain that valuable benefits must be derived from the integrated function of their subunits. In the case of antigen receptors, the multiplicity of ITAMs likely allows the cell to distinguish subtle variations in ligand affinities with exquisite sensitivity. Clearly, an isolated subunit that is ligated with antibodies cannot confer such complex function. For instance, it cannot reveal the subtle changes in signal transduction that likely occur on stimulation with altered antigenic peptide ligands or during a complex cell-cell interaction. However, before the intricacies of integrated receptor function can be appreciated, the potential or unique functional properties contributed by each individual receptor component must first be understood. Providing a tool to acquire this kind of understanding has been the greatest asset of this technology. Acknowledging its limitations, the use of surface chimeric receptors remains an invaluable approach toward our understanding the complex function of oligomeric receptors.

Reliability of the VmaxST portable metabolic measurement system.

The purpose of this study was to evaluate the reliability of the VmaxST portable metabolic measurement system. Forty-five healthy adults (age = 25.7 +/- 5.9 yr; height = 171.8 +/- 9.1 cm; weight = 69.6 +/- 12.8 kg; VO2peak) = 40.7 ml/kg/min; percent fat = 21.7 +/- 11.0) performed two separate and identical exercise routines on different days consisting of treadmill walking at 2.0 mph (53.6 m/min), 3.0 mph (80.5 m/min), and 4.0 mph (107.3 m/min) and running at 6.0 mph (160.9 m/min). VE and gas exchange were measured continuously breath-to-breath. A random effects model on log-transformed data yielded coefficients of variation (CV) and intraclass correlation coefficients (ICC) for VO2 and VE of 5.2 - 7.6 %, and 0.77 - 0.92, respectively, for all walking and running trials. For VCO2, CVs were higher (10 - 12 %) and ICCs lower (0.70 - 0.81). Ordinary least squares regression between the individual difference scores and the individual mean scores for VE, VO2 and VCO2, respectively, indicated no systematic bias (all p > 0.05). Bland-Altman analysis also illustrated no systematic bias between repeated measurements. The VmaxST provides reliable measurements of VO2 and VE during walking and running eliciting VE and VO2 at least up to approximately 56 and 2.2 l/min, respectively. The system appears to be less reliable for measuring VCO2.

The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways.

The function of the T cell antigen receptor (TCR) invariant chains, CD3 gamma, delta, epsilon, and zeta, is poorly understood. Evidence suggests that CD3 couples receptor ligand binding to intracellular signaling events. To examine the role of the CD3 zeta chain in TCR-mediated signal transduction, a chimeric protein linking the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of the zeta chain was constructed. The CD8/zeta chimera is expressed independently of the TCR and is capable of transducing signals that, by criteria of early and late activation, are indistinguishable from those generated by the intact TCR. These data indicate that CD8/zeta can activate the appropriate signal transduction pathways in the absence of CD3 gamma, delta, and epsilon, and suggest that the role of CD3 zeta is to couple the TCR to intracellular signal transduction mechanisms.

The zeta chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein.

Stimulation of the T-cell antigen receptor (TCR) leads to tyrosine phosphorylation of a number of cellular proteins, including phospholipase C (PLC) gamma 1 and the TCR zeta chain. We describe here a 70-kDa tyrosine phosphoprotein (ZAP-70) that associates with zeta within 15 sec following TCR stimulation. The phosphorylation of ZAP-70 and its association with zeta is independent of the other TCR chains since stimulation of a functional CD8/zeta chimeric receptor in a TCR-negative T cell leads to coprecipitation of ZAP-70 with the chimeric protein. In a Jurkat cell expressing the TCR and the CD8/zeta chimeric protein, tyrosine phosphorylation and association of ZAP-70 occurs exclusively with the stimulated receptor complex. In addition, a tyrosine kinase that does not appear to be fyn associates with the cytoplasmic domain of zeta and phosphorylates zeta and ZAP-70 in vitro.

Signal transduction by the T cell antigen receptor.

The T cell antigen receptor (TCR) must recognize antigen, and translate this recognition event into intracellular signal transduction events. Two signal transduction events are regulated by the TCR: the activation of a protein tyrosine kinase (PTK) and phospholipase C (PLC). Recent studies suggest that the TCR-activated PTK regulates PLC activation by the phosphorylation of tyrosine residues of PLC gamma 1. The complex structure of the TCR is now being related to its signal transduction function. Studies with chimeric receptors reveal that the antigen binding Ti heterodimer communicates with the subunits involved with signal transduction, the CD3 chains and zeta dimers, through the carboxy-terminal regions of the Ti chains that surround and include the transmembrane domains. Other chimeras have helped demonstrate that the zeta chain family of dimers function to couple the TCR to intracellular signal transduction mechanisms. The signal transduction function of the TCR can be regulated in a number of ways and by other T cell surface molecules. The plasma membrane tyrosine phosphatase CD45, plays a critical role to specifically regulate TCR-mediated activation of PTK's and PLC. Thus, an understanding of the complex structure of the TCR and the intricacies of its signal transduction function is rapidly emerging.

Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs.

p56lck, a lymphocyte-specific member of the src family of cytoplasmic protein-tyrosine kinases, is associated noncovalently with the cell surface glycoproteins CD4 and CD8, which are expressed on functionally distinct subpopulations of T cells. Using transient coexpression of p56lck with CD4 or CD8 alpha in COS-7 cells, we show that the unique N-terminal region of p56lck binds to the membrane-proximal 10 and 28 cytoplasmic residues of CD8 alpha and CD4, respectively. Two cysteine residues in each of the critical sequences in CD4, CD8 alpha, and p56lck are required for association. Our results suggest a novel role for cysteine-mediated interactions between unrelated proteins and provide a model for the association of other src-like cytoplasmic kinases with transmembrane proteins.