Direct ex vivo analysis of human CD4(+) memory T cell activation requirements at the single clonotype level

CD4(+) memory T cells continuously integrate signals transmitted through the TCR and costimulatory molecules, only responding when the intensity of such signals exceeds an intrinsic activation threshold. Recent data suggest that these activation thresholds can be regulated independently of TCR specificity, and that threshold tuning may constitute a major mechanism for controlling T cell effector activity. In this work we take advantage of the profound clonotypic hierarchies of the large human CD4(+) T cell response to CMV to study activation thresholds of fresh (unexpanded) memory T cells at the clonotypic level. We identified dominant responses to CMV matrix determinants mediated by single TCRB sequences within particular TCR-Vbeta families. The specific response characteristics of these single, Ag-specific, TCRB-defined clonotypes could be unequivocally determined in fresh PBMC preparations by cytokine flow cytometry with gating on the appropriate Vbeta family. These analyses revealed 1) optimal peptides capable of eliciting specific responses by themselves at doses as low as 2 pg/ml, with each log increase in dose eliciting ever-increasing frequencies of responding cells over a 4- to 5-log range; 2) significant augmentation of response frequencies at all submaximal peptide doses by CD28- and CD49d-mediated costimulation; 3) differential dose response and costimulatory characteristics for IFN-gamma and IL-2 responses; and 4) no association of activation requirements with the CD27-defined CD4(+) T cell memory differentiation pathway. Taken together these data confirm that triggering heterogeneity exists within individual CD4(+) memory T cell clonotypes in vivo and demonstrate that such single clonotypes can manifest qualitatively different functional responses depending on epitope dose and relative levels of costimulation.     

Functional state of the axonemal dyneins during flagellar bend propagation

When mouse spermatozoa swim in media of high viscosity, additional waves of bending are superimposed on the primary traveling wave. The additional (secondary) waves are relatively small in scale and high in frequency. They originate in the proximal part of the interbend regions. The initiation of secondary bending happens only in distal parts of the flagellum. The secondary waves propagate along the interbends and then tend to die out as they encounter the next-most-distal bend of the primary wave, if that bend exceeds a certain angle. The principal bends of the primary wave, being of greater angle than the reverse bends, strongly resist invasion by the secondary waves; when a principal bend of the primary wave propagates off the flagellar tip, the secondary wave behind it suddenly increases in amplitude. We claim that the functional state of the dynein motors in relation to the primary wave can be deduced from their availability for recruitment into secondary wave activity. Therefore, only the dyneins in bends are committed functionally to the maintenance and propagation of the flagellar wave; dyneins in interbend regions are not functionally committed in this way. We equate functional commitment with tension-generating activity, although we argue that the regions of dynein thus engaged nevertheless permit sliding displacements between the doublets.     

Purification and folding of recombinant bovine oxoglutarate/malate carrier by immobilized metal-ion affinity chromatography

A major obstacle to investigating the structure of membrane proteins is the difficulty in obtaining sufficient amounts of functional protein. The oxoglutarate carrier, an intrinsic membrane-transport protein of the inner membranes of bovine-heart mitochondria, has been cloned as a fusion protein containing a C-terminal hexa-histidine tag. This fusion protein has been expressed at an abundant level in a mutant strain of Escherichia coli BL21 (DE3) called C41 (DE3). The protein accumulated as inclusion bodies and none was detected in the bacterial inner membrane. The denatured protein was refolded to reconstitute functional properties similar to the native protein. Solubilized inclusion body protein was immobilized using nickel-chelating affinity chromatography, and purified and refolded in a single step. The protein eluted as a monomer which was stable in mild detergent, at a yield equivalent to 15 mg active protein/liter bacterial culture. The reconstituted fusion protein displayed the same transport characteristics as the wild-type, demonstrating that the tag does not perturb the structure of the protein. The oxoglutarate carrier is one member of an extensive family of mitochondrial transport proteins. These proteins transport many different metabolites across the inner mitochondrial membrane and share a common mechanism of action. Therefore, it is likely that this folding protocol can be applied successfully to other mitochondrial transport proteins, thus providing sufficient protein for extensive crystallization trials with a wide range of family members.     

Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation of rat adipocytes

Adipocyte lipolysis was compared with hormone-sensitive lipase (HSL)/perilipin subcellular distribution and perilipin phosphorylation using Western blot analysis. Under basal conditions, HSL resided predominantly in the cytosol and unphosphorylated perilipin upon the lipid droplet. Upon lipolytic stimulation of adipocytes isolated from young rats with the beta-adrenergic agonist, isoproterenol, HSL translocated from the cytosol to the lipid droplet, but there was no movement of perilipin from the droplet to the cytosol; however, perilipin phosphorylation was observed. By contrast, upon lipolytic stimulation and perilipin phosphorylation in cells from more mature rats, there was no HSL translocation but a significant movement of perilipin away from the lipid droplet. Adipocytes from younger rats had markedly greater rates of lipolysis than those from the older rats. Thus high rates of lipolysis require translocation of HSL to the lipid droplet and translocation of HSL and perilipin can occur independently of each other. A loss of the ability to translocate HSL to the lipid droplet probably contributes to the diminished lipolytic response to catecholamines with age.