Amino acid sequence comparisons of human topoisomerase I (Topo I) with seven other cellular Topo I enzymes reveal that the enzyme can be divided into four major domains: the unconserved NH2-terminal domain (24 kDa), the conserved core domain (54 kDa), a poorly conserved linker region (5 kDa), and the highly conserved COOH-terminal domain (8 kDa), which contains the active site tyrosine. To investigate this predicted domain organization, recombinant baculoviruses were engineered to express the 91-kDa full-length enzyme, a 70-kDa NH2-terminally truncated enzyme that is missing the first 174 residues, and a 58-kDa NH2- and COOH-terminally truncated core fragment encompassing residues 175-659. The specific activity of the full-length and Topo70 enzymes are indistinguishable from the native human Topo I purified from HeLa cells. Each protein is inhibited by camptothecin, topotecan, and 9-aminocamptothecin, but not by ATP. Activity is stimulated by Mg2+, Ba2+, Ca2+, Mn2+, spermine, and spermidine. The magnitude of the stimulatory effect of Mg2+ is inversely proportional to the salt concentration. Furthermore, at KCl concentrations of 300 mM or greater, the addition of Mg2+ is inhibitory. The effects of Mg2+ and the polycations spermine and spermidine are partially additive, an indication that the stimulatory mechanisms of the two substances are different. Activity was strongly inhibited or abolished by Ni2+, Zn2+, Cu2+, Cd2+, and Co2+. An examination of the hydrodynamic properties of full-length Topo I, Topo70, and Topo58 demonstrates that the core, linker, and COOH-terminal domains fold into a globular structure, while the NH2-terminal domain is highly extended. A comparison of the circular dichroism spectra of full-length Topo I and Topo70 demonstrates that residues 1-174 (approximately 21 kDa) of Topo I are largely if not completely unfolded. This observation is consistent with the fact that the NH2-terminal domain is dispensable for activity.

Recently, T cell subpopulations presumably representing memory T lymphocytes have been described in vitro. Intestinal lamina propria T cells (LP-T) have characteristics resembling those of memory cells. We therefore investigated the expression of surface Ag associated with memory phenotype in vitro on lamina propria lymphocytes (LPL) and PBL and on the T cell subpopulations defined by the bright expression of CD45R0 by flow cytometric analysis of isolated cell populations. LPL had significantly increased percentages of CD45R0 and CD58 positive cells compared with PBL. Whereas PBL showed bimodal expression profiles of CD45R0, CD58, and <em>CD2</em>, the vast majority of LPL was bright for these Ag. Expression of CD45RA was significantly reduced in both frequency and intensity in LPL, and LPL had significantly reduced percentages of CD11a/CD18 and <em>CD2</em>9 positive cells compared with PBL. The CD45R0 bright T cell subpopulations of both PBL and LPL were characterized by a lack of CD45RA. CD45R0 bright T cells from the peripheral blood (PB-T) were predominantly bright for <em>CD2</em>, CD58, <em>CD2</em>9, and CD11a/CD18 whereas CD45R0 dim PB-T had bimodal expression profiles and CD45R0 negative PB-T were dim or even negative for these Ag. CD45R0 bright LP-T were also bright for <em>CD2</em> and CD58 but had significantly reduced surface densities of CD11a/CD18 and <em>CD2</em>9 compared with CD45R0 bright PB-T. The surface density of <em>CD2</em>9 on CD45R0 bright LP-T corresponded to that of CD45R0 negative PB-T, and a significant proportion of CD45R0 bright LP-T was even negative for CD11a/CD18 and <em>CD2</em>9. Additionally, CD45R0 bright LP-T in contrast to PB-T were characterized by a lack of 1-selectin and the expression of CDw49a and the mucosa-specific T cell Ag HML-1 on high percentages of cells. Our results show that the phenotype of CD45R0 bright T cells from the lamina propria clearly deviates from that of memory T cells in vitro and of CD45R0 bright T cells in the peripheral blood. We conclude that memory T cell populations in vivo undergo specific differentiation depending on their tissue localization, leading to unique phenotypic and presumably functional features.

We characterized the response of resting human CD8 T cells to allogeneic endothelial cells (EC). Both resting and IFN-gamma-pretreated EC stimulate similar CD8 T cell proliferative responses (peak, day 5 to 6), whereas only IFN-gamma-pretreated EC stimulate CD4 T cells. The response increases with increasing numbers of CD8 T cells from 25,000 to 400,000/well. The proliferation of CD8 T cells is inhibited by mAbs reactive with CD8 or HLA-A and -B molecules but not with CD4 or HLA-DR. mAb blocking studies show a role for <em>CD2</em>, LFA-3, and CD59, but not for intercellular adhesion molecule-1, intercellular adhesion molecule-2, very late activation Ag-4, vascular cell adhesion molecule-1, <em>CD2</em>8, or <em>CD2</em>8 ligand, as costimulatory molecules. The stimulation of resting CD8 T cells by EC causes secretion of IL-2 and IFN-gamma but not IL-4. Both proliferation and IFN-gamma secretion are inhibited by mAb to the IL-2R alpha subunit (<em>CD2</em>5). Limiting dilution analysis suggests that approximately 1 in 20,000 resting CD8 T cells secrete IL-2 in response to allogeneic EC. EC stimulate greater than 1 in 10,000 CD8/CD45RO+ cells but fewer than 1 in 40,000 CD8/CD45RA+ cells, which indicates that primarily memory CD8 T cells respond to EC. Coculturing CD8 cells with EC stimulates a sufficient level of endothelial class II MHC expression to subsequently support a CD4 T cell proliferative response. The ability of memory CD8 T cells to proliferate against allogeneic EC, a nonclassical APC, and their ability to stimulate EC may contribute to the initiation of vascularized organ graft rejection.

The interaction between a T cell and an antigen-presenting cell (APC) can trigger a signaling response that leads to T cell activation. Prior studies have shown that ligation of the T cell receptor (TCR) triggers a signaling cascade that proceeds through the coalescence of TCR and various signaling molecules (e.g., the kinase Lck and adaptor protein LAT [linker for T cell activation]) into microdomains on the plasma membrane. In this study, we investigated another ligand-receptor interaction (CD58-CD2) that facilities T cell activation using a model system consisting of Jurkat T cells interacting with a planar lipid bilayer that mimics an APC. We show that the binding of CD58 to CD2, in the absence of TCR activation, also induces signaling through the actin-dependent coalescence of signaling molecules (including TCR-zeta chain, Lck, and LAT) into microdomains. When simultaneously activated, TCR and CD2 initially colocalize in small microdomains but then partition into separate zones; this spatial segregation may enable the two receptors to enhance signaling synergistically. Our results show that two structurally distinct receptors both induce a rapid spatial reorganization of molecules in the plasma membrane, suggesting a model for how local increases in the concentration of signaling molecules can trigger T cell signaling.

We have studied the interaction between some heavy metal ions, as compared with earth alkali ions, and calmodulin, a tissue protein which binds Ca2+ and mediates some of its effects. 1. Calmodulin dependent phosphodiesterase was activated with Pb2+, Ca2+, Sr2+, Ba2+, and Cd2+ (EC50 about 0.8 microM). The maximal activation achieved decreases in the order given. Hg2+, Sn2+, Fe2+, Cu2+, Ni2+, Bi3+, and Sb3+ up to 20 microM did not activate. 2. Pb2+ can replace Ca2+ with respect to the calmodulin-dependent phosphorylation of brain membranes. With high Pb2+ concentrations, phosphorylation was inhibited. 3. Calmodulin binding to brain membranes was enhanced with concentrations below 10(-4)M in the following order: Pb2+ greater than or equal to Ca2+ approximately Sr2+ greater than Cd2+ greater than Mn2+ greater than Ba2+. In contrast Mg2+, Hg2+, Sn2+, Fe2+, Ni2+, Co2+, and Cu2+ triggered, if at all, a non-saturable binding of calmodulin. 4. In the flow-dialysis, other ions competed with 45Ca2+ binding to calmodulin in the following order: Pb2+ approximately Ca2+ greater than Mn2+, Ba2+, Cd2+, Sr2+. Thus among the ions investigated Pb2+ is a fully potent substitute for Ca2+ in every calmodulin-dependent reaction investigated. Cd2+ is always much less potent. The earth alkali ions Sr2+ and Ba2+ take an intermediate position. It remains to be shown whether calmodulin is merely a storage site for Pb2+, or whether the resulting functional changes play a role in Pb2+ poisoning.

We report clear evidence that the interaction of the CD38 molecule with the specific mAb A10 on normal human cells and lines modulates the expression of surface activation markers relevant to T, NK, and plasma cell biology and functions. Moreover A10 mAb binding is followed by proliferation effects on all the target cells analyzed, and the phenomenon is accessory cell and IL-2 dependent. The effects of A10 mAb synergizing both CD2 and CD3 activation pathways indicate that CD38 signal transduction mechanism(s) are apparently different from the aforementioned. Nevertheless the decreased A10-driven proliferation after CD3-Ti modulation suggests a possible functional interdependence between these activation pathways. Taken together, the results indicate that the CD38 molecule might play a physiologic role in T, NK, and plasma cell regulation.

Positive selection during thymocyte development is driven by the affinity and avidity of the TCR for MHC-peptide complexes expressed in the thymus. In this study, we show that programmed death-1 (PD-1), a member of the B7/<em>CD2</em>8 family of costimulatory receptors, inhibits TCR-mediated positive selection through PD-1 ligand 1 (PD-L1):PD-1 interactions. Transgenic mice that constitutively overexpress PD-1 on CD4+CD8+ thymocytes display defects in positive selection in vivo. Using an in vitro model system, we find that PD-1 is up-regulated following TCR engagement on CD4+CD8+ murine thymocytes. Coligation of TCR and PD-1 on CD4+CD8+ thymocytes with a novel PD-1 agonistic mAb inhibits the activation of ERK and up-regulation of bcl-2, both of which are downstream mediators essential for positive selection. Inhibitory signals through PD-1 can overcome the ability of positive costimulators, such as <em>CD2</em> and <em>CD2</em>8, to facilitate positive selection. Finally, defects in positive selection that result from PD-1 overexpression in thymocytes resolve upon elimination of PD-L1, but not PD-1 ligand 2, expression. PD-L1-deficient mice have increased numbers of CD4+CD8+ and CD4+ thymocytes, indicating that PD-L1 is involved in normal thymic selection. These data demonstrate that PD-1:PD-L1 interactions are critical to positive selection and play a role in shaping the T cell repertoire.

Podocytes are critical in maintaining the filtration barrier of the glomerulus and are dependent on the slit diaphragm (SD) proteins nephrin, podocin, and CD2-associated protein (CD2AP) to function optimally. The effects of normal human plasma and nephrotic plasma on podocytes were tested, focusing particularly on the SD complex. With the use of a conditionally immortalized human podocyte cell line, it first was shown that exposure to normal and non-nephrotic human plasma leads to a concentration of nephrin, podocin, CD2AP, and actin at the cell surface. Next, the effects of plasma from patients with nephrotic conditions to non-nephrotic conditions were compared. When exposed to all nephrotic plasma samples (and a non-human serum control), nephrin podocin and CD2AP assumed a cytoplasmic distribution; nephrin and synaptopodin were selectively downregulated, and the relocation of nephrin induced by nephrotic plasma could be rescued back to the plasma membrane by co-incubation with non-nephrotic plasma. Furthermore, intracellular calcium signaling was altered by nephrotic plasma, which was mediated by tyrosine kinase phosphorylation. With the use of nephrin mutant human cell lines, it was shown that this signaling and translocation response to normal plasma is nephrin dependent. This work demonstrates that nephrotic plasma seems to be deficient in factors that act via the podocyte SD complex, which are essential in maintaining its physiologic function.

BACKGROUND

Chronic Fatigue Syndrome (CFS) studies from our laboratory and others described decreased natural killer cell cytotoxicity (NKCC) and elevated proportion of lymphocytes expressing the activation marker, dipeptidyl peptidase IV (DPPIV) also known as <em>CD2</em>6. However, neither these assays nor other laboratory tests are widely accepted for the diagnosis or prognosis of CFS. This study sought to determine if NKCC or DPPIV/<em>CD2</em>6 have diagnostic accuracy for CFS.

RESULTS

Subjects included female and male CFS cases and healthy controls. NK cell function was measured with a bioassay, using K562 cells and (51)Cr release. Lymphocyte associated DPPIV/<em>CD2</em>6 was assayed by qualitative and quantitative flow cytometry. Serum DPPIV/<em>CD2</em>6 was measured by ELISA. Analysis by receiver operating characteristic (ROC) curve assessed biomarker potential. Cytotoxic function of NK cells for 176 CFS subjects was significantly lower than in the 230 controls. According to ROC analysis, NKCC was a good predictor of CFS status. There was no significant difference in NK cell counts between cases and controls. Percent <em>CD2</em>+ lymphocytes (T cells and NK cells) positive for DPPIV/C26 was elevated in CFS cases, but there was a decrease in the number of molecules (rMol) of DPPIV/C26 expressed on T cells and NK cells and a decrease in the soluble form of the enzyme in serum. Analyses by ROC curves indicated that all three measurements of DPPIV/<em>CD2</em>6 demonstrated potential as biomarkers for CFS. None of the DPPIV/C26 assays were significantly correlated with NKCC.

CONCLUSIONS

By ROC analysis, NKCC and three methods of measuring DPPIV/C26 examined in this study had potential as biomarkers for CFS. Of these, NKCC, %<em>CD2</em>+<em>CD2</em>6+ lymphocytes and rMol <em>CD2</em>6/<em>CD2</em>+ lymphocyte, required flow cytometry, fresh blood and access to a high complexity laboratory. Soluble DPPIV/C26 in serum is done with a standard ELISA assay, or with other soluble factors in a multiplex type of ELISA. Dipeptidyl peptidase IV on lymphocytes or in serum was not predictive of NKCC suggesting that these should be considered as non-redundant biomarkers. Abnormalities in DPPIV/<em>CD2</em>6 and in NK cell function have particular relevance to the possible role of infection in the initiation and/or the persistence of CFS.

During early fetal development, T cell precursors home from fetal yolk sac and liver to the epithelial thymic rudiment. From cells that initially colonize the thymus arise mature T cells that populate T cell zones of the peripheral lymphoid system. Whereas colonization of the thymus occurs late in the final third of gestation in the mouse, in birds and humans the thymus is colonized by hematopoietic stem cell precursors during the first third of gestation. Using a large series of early human fetal tissues and a panel of monoclonal antibodies that includes markers of early T cells (CD7, CD45), we have studied the immunohistologic location and differentiation capacity of CD45+, CD7+ cells in human fetal tissues. We found that before T cell precursor colonization of the thymus (7-8 wk of gestation), CD7+ cells were present in yolk sac, neck, upper thorax, and fetal liver, and were concentrated in mesenchyme throughout the upper thorax and neck areas. By 9.5 wk of gestation, CD7+ cells were no longer present in upper thorax mesenchyme but rather were localized in the lymphoid thymus and scattered throughout fetal liver. CD7+, CD2-, CD3-, CD8-, CD4-, WT31- cells in thorax and fetal liver, when stimulated for 10-15 d with T cell-conditioned media and rIL-2, expressed CD2, CD3, CD4, CD8, and WT31 markers of the T cell lineage. Moreover, CD7+ cells isolated from fetal liver contained all cells in this tissue capable of forming CFU-T colonies in vitro. These data demonstrate that T cell precursors in early human fetal tissues can be identified using a mAb against the CD7 antigen. Moreover, the localization of CD7+ T cell precursors to fetal upper thorax and neck areas at 7-8.5 wk of fetal gestation provides strong evidence for a developmentally regulated period in man in which T cell precursors migrate to the epithelial thymic rudiment.

High-voltage activated Ca channels in tiger salamander cone photoreceptors were studied with nystatin-permeabilized patch recordings in 3 mM Ca2+ and 10 mM Ba2+. The majority of Ca channel current was dihydropyridine sensitive, suggesting a preponderance of L-type Ca channels. However, voltage-dependent, incomplete block (maximum 60%) by nifedipine (0.1-100 microM) was evident in recordings of cones in tissue slice. In isolated cones, where the block was more potent, nifedipine (0.1-10 microM) or nisoldipine (0.5-5 microM) still failed to eliminate completely the Ca channel current. Nisoldipine was equally effective in blocking Ca channel current elicited in the presence of 10 mM Ba2+ (76% block) or 3 mM Ca2+ (88% block). 15% of the Ba2+ current was reversibly blocked by omega-conotoxin GVIA (1 microM). After enhancement with 1 microM Bay K 8644, omega-conotoxin GVIA blocked a greater proportion (22%) of Ba2+ current than in control. After achieving partial block of the Ba2+ current with nifedipine, concomitant application of omega-conotoxin GVIA produced no further block. The P-type Ca channel blocker, omega-agatoxin IVA (200 nM), had variable and insignificant effects. The current persisting in the presence of these blockers could be eliminated with Cd2+ (100 microM). These results indicate that photoreceptors express an L-type Ca channel having a distinguishing pharmacological profile similar to the alpha 1D Ca channel subtype. The presence of additional Ca channel subtypes, resistant to the widely used L-, N-, and P-type Ca channel blockers, cannot, however, be ruled out.

Sour taste is elicited by acids. How taste cells transduce sour taste is controversial because acids (specifically protons) have diverse effects on cell membranes. Consequently, it is difficult to differentiate between events related to sour taste transduction per se and unrelated effects of protons. We have studied acid taste transduction in mouse taste buds using a lingual slice preparation where it is possible to measure changes in pH and [Ca2+]i simultaneously in taste cells. Focal application of citric acid or HCl to the apical tips of taste buds produced widespread acidification of the entire taste bud. Citric acid was effective at a pH of approximately 4, but HCl only at a pH of approximately 1.5. Despite acidification of the whole taste bud, only a select few taste cells exhibited Ca2+ responses. Acid-evoked Ca2+ responses were dose dependent in a range consistent with them being sour-taste responses. Cells exhibiting acid-evoked Ca2+ responses also responded to KCl depolarization. Acid-evoked Ca2+ responses were blocked by Ba2+ (2 mM) and Cd2+ (500 microM), suggesting that acid responses are generated by Ca2+ influx through depolarization-gated Ca2+ channels. Removing extracellular Ca2+ reduced acid-evoked Ca2+ responses, but depleting intracellular Ca2+ stores with thapsigargin had no effect, suggesting that acid taste responses are generated by an influx of extracellular Ca2+. Neither Cs+ (500 microM) nor amiloride (100 microM) affected acid-evoked Ca2+ responses, suggesting that neither hyperpolarization-activated cyclic nucleotide-gated cation (pacemaker) channels nor epithelial Na+ channels, respectively, transduce sour taste. Collectively, the results indicate that acids, especially weak acids, acidify the taste bud and evoke depolarization-induced Ca2+ entry into a select subset of taste cells. The primary transducer protein(s) for sour taste remain undiscovered.

We studied the clinicopathologic features of 42 cases of nasal-type extranodal natural killer (NK)/T-cell lymphoma in Singapore and compared our findings with other series reported in the Asian and Western populations. A panel of immunohistochemical stains, which included CD2, CD3, CD4, CD8, CD56, T-cell intracellular Antigen-1 and granzyme B, and in situ hybridization for Epstein-Barr virus encoded RNA (EBER) were performed. Polymerase chain reaction for T-cell receptor-gamma gene rearrangement using both gel and capillary electrophoresis were evaluated to determine the proportion of tumors which are of true T-cell lineage. We also studied the functional status of the overexpressed p53 protein in these lymphomas by correlating p53 expression with its downstream target protein, p21. In all, 31 out of 42 cases presented in the upper aerodigestive tract. The other sites of involvement included gastrointestinal tract, skin, soft tissue, testis, liver, spleen, bone marrow and brain. The tumors displayed characteristic morphologic features. In situ hybridization for EBER was detected in 41 out of 42 cases (97.6%). The only significant adverse prognostic factor identified was an International Prognostic Index of two or more. A significantly higher proportion of the tumors (27%), compared to previous studies, demonstrated monoclonal T-cell receptor-gamma gene rearrangement. There was, however, no difference in survival or clinicopathologic features between the true NK-cell tumors and their T-cell counterparts. Overexpression of p53 was present in 40% of the cases, but no significant difference in survival rate was detected in patients with p53 overexpression and there was no association between p53 overexpression with large cell morphology, and advanced stage of disease. These findings suggest that molecular aberrations other than those of the p53 pathway may be operative in the pathogenesis of this malignancy.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed by a clathrin- independent pathway into vesicles named GPI-AP-enriched early endosomal compartments (GEECs). We recently showed that the vacuolating toxin VacA secreted by Helicobacter pylori is endocytosed into the GEECs (Gauthier, N.C., P. Monzo, V. Kaddai, A. Doye, V. Ricci, and P. Boquet. 2005. Mol. Biol. Cell. 16:4852-4866). Unlike GPI-APs that are mostly recycled back to the plasma membrane, VacA reaches early endosomes (EEs) and then late endosomes (LEs), where vacuolation occurs. In this study, we used VacA to study the trafficking pathway between GEECs and LEs. We found that VacA routing from GEECs to LEs required polymerized actin. During this trafficking, VacA was transferred from GEECs to EEs associated with polymerized actin structures. The CD2-associated protein (CD2AP), a docking protein implicated in intracellular trafficking, bridged the filamentous actin (F-actin) structures with EEs containing VacA. CD2AP regulated those F-actin structures and was required to transfer VacA from GEECs to LEs. These results demonstrate that sorting from GEECs to LEs requires dynamic F-actin structures on EEs.

The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules <em>CD2</em>, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, <em>CD2</em>8 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the <em>CD2</em>8 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of <em>CD2</em>8 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were <em>CD2</em>8+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-<em>CD2</em>8-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated <em>CD2</em>8 expression in the CD3- subset as these cells became CD3-<em>CD2</em>8+(bright). Despite the ability of PMA to induce high density <em>CD2</em>8 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-<em>CD2</em>8 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-<em>CD2</em>8 mAb supported proliferation, suggesting that only CD3+ cells could respond to <em>CD2</em>8 signaling. The comitogenic effect of anti-CD3 and anti-<em>CD2</em>8 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of <em>CD2</em>8 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface <em>CD2</em>8 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in <em>CD2</em>8 mRNA levels. Together, these results suggest that <em>CD2</em>8 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the <em>CD2</em>8 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.

The human metallothionein IIA (hMT-IIA) gene contains two enhancer elements whose activity is induced by heavy-metal ions such as Cd2+. To determine the nature of the relationship between the metal-responsive elements and the element(s) responsible for the basal activity of the enhancers, the basal-level enhancer element(s), the hMT-IIA enhancers were subjected to mutational analysis. We show that deletion of the metal-responsive elements had no effect on the basal activity of the enhancer but prevented further induction by Cd2+. On the other hand, replacement of the basal-level enhancer element with linker DNA led to inactivation of the enhancer both before and after treatment with Cd2+. Therefore, the metal-responsive elements seems to act as a positive modulator of enhancer function in the presence of heavy-metal ions. In addition to the two enhancers, the hMT-IIA promoter contained one other element, the GC box, required for its basal expression. Unlike deletion of the basal-level enhancer element, replacement of the GC box with linker DNA had no effect on the ability of the promoter to be induced by Cd2+.

T lymphocyte activation with monoclonal antibodies directed against the CD2 (T,p50) sheep red blood cell receptor antigen and against CD3 (T,p19,29) has been investigated. Co-stimulation of purified T lymphocytes with anti-CD3 (SP34) and anti-CD2 (9-1), which detects a unique epitope on the CD2 molecule, results in T cell activation and cell proliferation. Each antibody alone is unable to mediate this effect. Co-stimulation of purified T cells with two different anti-CD2 antibodies, 9-1 and 9.6, which detect two different epitopes on the CD2 molecule, are also mitogenic. In contrast, the combination of anti-CD3 (SP34) and anti-CD2 (9.6) cannot induce T cell activation. These data suggest that the CD2 epitope defined by the 9-1 antibody is functionally important for T cell activation via the CD3/Ti complex. Furthermore, it is demonstrated that anti-CD3 (SP34) induces epitopic modulation of the CD2 molecule, resulting in enhanced expression of the CD2, 9-1 epitope. This epitope modulation of the CD2 (9-1) epitope by anti-CD3 (SP34) occurs instantaneously at 4 degrees C and in the presence of NaN3. The functional interaction between CD3 and CD2 occurs in spite of any evidence of complex formation between these two molecules. These data suggest that the T cell differentiation antigens CD3 and CD2 are jointly involved in antigen-specific T cell activation. The data are consistent with a model for antigen-specific T cell activation involving both the CD3/Ti complex and subsequent activation of the CD2 complex T cell activation by co-stimulation with anti-CD3 (SP34) and anti-CD2 (9-1) is substantially enhanced by the addition of exogenous, purified interleukin 1 (IL 1). These data would suggest that the CD2 complex, as well as the putative IL 1 receptor, are involved in separate and complementary receptor-ligand interactions, resulting in the amplification of antigen-specific T cell responses.

Rapid progress has recently been made in characterising the structures of leukocyte cell-surface molecules. Detailed analyses of the structure and interactions of CD2 were the first involving a molecule that has not been directly linked to antigen recognition in the manner of antigen receptors or co-receptors. It seems highly likely that the properties of ligand binding by CD2 are relevant to the general mechanisms of cell-cell recognition. As an example of biological recognition, the defining characteristic of cell-cell contact is that it involves the simultaneous interaction of hundreds, if not thousands, of molecules. Affinity and kinetic analyses of ligand binding by CD2 indicated that the protein interactions mediating cell-cell contact, whilst highly specific, are much weaker than initially anticipated, probably due to the requirement that such contacts be easily reversible. Simultaneously, in addressing the mechanism of this mode of recognition, structural and mutational studies focussed on the role of charged residues clustered in the ligand-binding face of CD2, yielding the concept that electrostatic complementarity, rather than surface-shape complementarity, is the dominant feature of specific, low-affinity protein recognition at the cell surface by CD2. The crystallographic analysis of the CD2-binding domain of CD58 strongly supports this concept.

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.

The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.

The backbone dynamics of apo- and (Cd2+)1-calbindin D9k have been characterized by 15N nuclear magnetic resonance spectroscopy. Spin-lattice and spin-spin relaxation rate constants and steady-state [1H]-15N nuclear Overhauser effects were measured at a magnetic field strength of 11.74 T by two-dimensional, proton-detected heteronuclear NMR experiments using 15N-enriched samples. The relaxation parameters were analyzed using a model-free formalism that characterizes the dynamics of the N-H bond vectors in terms of generalized order parameters and effective correlation times. The data for the apo and (Cd2+)1 states were compared to those for the (Ca2+)2 state [Kördel, J., Skelton, N. J., Akke, M., Palmer, A. G., & Chazin, W. J. (1992) Biochemistry 31, 4856-4866] to ascertain the effects on ion ligation on the backbone dynamics of calbindin D9k. The two binding loops respond differently to ligation by metal ions: high-frequency (10(9)-10(12) s-1) fluctuations of the N-terminal ion-binding loop are not affected by ion binding, whereas residues G57, D58, G59, and E60 in the C-terminal ion-binding loop have significantly lower order parameters in the apo state than in the metal-bound states. The dynamical responses of the four helices to binding of ions are much smaller than that for the C-terminal binding loop, with the strongest effect on helix III, which is located between the linker loop and binding site II. Significant fluctuations on slower time scales also were detected in the unoccupied N-terminal ion-binding loop of the apo and (Cd2+)1 states; the apparent rates were greater for the (Cd2+)1 state. These results on the dynamical response to ion binding in calbindin D9k provide insights into the molecular details of the binding process and qualitative evidence for entropic contributions to the cooperative phenomenon of calcium binding for the pathway in which the ion binds first in the C-terminal site.

1. Guinea-pig olfactory cortex neurones in vitro (23-25 degrees C) were voltage clamped by means of a single-micro-electrode sample-and-hold technique. 2. Under current clamp at the resting potential (approximately -80 mV), brief depolarizing stimuli evoked trains of action potentials with little visible after-potential. However, in 90% of recorded cells held at membrane potentials between -70 and -45 mV, depolarizing current pulses evoked a slow after-hyperpolarization (a.h.p.) (approximately 8 mV) lasting several seconds and accompanied by an increase in input conductance. 3. The outward membrane current underlying the a.h.p. was revealed either by switching rapidly to voltage clamp at the end of a spike train ('hybrid' clamp) or by applying brief depolarizing commands from potentials between -60 to -45 mV. The tail current showed a distinct rising phase (time to peak approximately 1 s) and exponential decay (tau approximately 3 s) and was suppressed by removal of external Ca2+, or adding Co2+ (1-2 mM), Cd2+ (200 microM) or Mg2+ (6 mM). The a.h.p. current reversal potential was -96 mV in 3 mM-K+ medium. 4. Low concentrations (1-2 microM) of muscarine, carbachol, oxotremorine or the muscarinic ganglion stimulant, McN-A-343 (1-10 microM) reduced the a.h.p. current and leak conductance and induced a steady inward current, without affecting M-current (IM) relaxations. IM inhibition generally required higher (greater than 10 microM) agonist concentrations, although oxotremorine remained ineffective at up to 50 microM. 5. The a.h.p. current was reduced by noradrenaline and tetraethylammonium (TEA), but not by apamin or tubocurarine. Apart from TEA, these agents had no effect on IM. 6. Addition of tetrodotoxin (TTX, 1 microM) or removing external Na+ depressed the a.h.p. current amplitude recorded under voltage clamp. The residual tail current could be further reduced by adding Cd2+ or muscarinic agonists. 7. Repolarizing tail currents induced following positive voltage commands consisted mainly of IM and slow a.h.p. current with little evidence of a 'fast' Ca2+-activated K+ current (IC). 8. It is concluded that the slow a.h.p. current that underlies the post-burst after-hyperpolarization of olfactory neurones, is a Ca2+-dependent K+ current distinct from IM. It is suggested that the cholinergic modulation of this current (rather than IM) may provide a more subtle control of cell excitability in cortical neurones.

The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase (LpxC) catalyzes the committed step in the biosynthesis of lipid A and is therefore a potential antibiotic target. Inhibition of this enzyme by hydroxamate compounds [Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F. M.; Chen, M. H.; Patchett, A. A.; Stachula, S. S.; Anderson, M. S.; Raetz, C. R. H. (1996) Science 274, 980-982] suggested the presence of a metal ion cofactor. We have investigated the substrate specificity and metal dependence of the deacetylase using spectroscopic and kinetic analyses. Comparison of the steady-state kinetic parameters for the physiological substrate UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc and an alternative substrate, UDP-GlcNAc, demonstrates that the ester-linked R-3-hydroxymyristoyl chain increases kcat/KM (5 x 10(6))-fold. Metal-chelating reagents, such as dipicolinic acid (DPA) and ethylenediaminetetraacetic acid, completely inhibit LpxC activity, implicating an essential metal ion. Plasma emission spectroscopy and colorimetric assays directly demonstrate that purified LpxC contains bound Zn2+. This Zn2+ can be removed by incubation with DPA, causing a decrease in the LpxC activity that can be restored by subsequent addition of Zn2+. However, high concentrations of Zn2+ also inhibit LpxC. Addition of Co2+, Ni2+, or Mn2+ to apo-LpxC also activates the enzyme to varying degrees while no additional activity is observed upon the addition of Cd2+, Ca2+, Mg2+, or Cu2+. This is consistent with the profile of metals that substitute for catalytic zinc ions in metalloproteinases. Co2+ ions stimulate LpxC activity maximally at a stoichiometry of 1:1. These data demonstrate that E. coli LpxC is a metalloenzyme that requires bound Zn2+ for optimal activity.

1. Electrical events and intracellular calcium concentration ([Ca2+]) imaged using fluo-3 and laser scanning confocal microscopy were simultaneously monitored in single smooth muscle cells freshly isolated from guinea-pig vas deferens or urinary bladder. 2. Images obtained every 8 ms, during stepping from -60 to 0 or +10 mV for 50 ms under voltage clamp, showed that a rise in [Ca2+] could be detected within 20 ms of depolarization in five to twenty small (< 2 micrometer diameter) 'hot spots', over 95 % of which were located within 1.5 micrometer of the cell membrane. Depolarization at 30 s intervals activated hot spots at the same places. 3. Cd2+ or verapamil abolished both hot spots and Ca2+-activated K+ current (IK,Ca). Caffeine almost abolished hot spots and markedly reduced IK,Ca. Cyclopiazonic acid, which raised basal global [Ca2+], decreased the rise in hot spot [Ca2+] and IK,Ca amplitude during depolarization. These results suggest that Ca2+ entry caused Ca2+-induced Ca2+ release (CICR). 4. Under voltage clamp, hot spot [Ca2+] closely paralleled the rise in IK,Ca and reached a peak within 20 ms of the start of depolarization, but the rise in global [Ca2+] over the whole cell area was much slower. Step depolarization to potentials positive to -20 mV caused hot spots to grow in size and coalesce, leading to a rise in global [Ca2+] and contraction. Ca2+ hot spots also occurred during the up-stroke of an evoked action potential under current clamp. 5. It is concluded that the entry of Ca2+ in the early stages of an action potential evokes CICR from discrete subplasmalemma Ca2+ storage sites and generates hot spots that spread to initiate a contraction. The activation of Ca2+-dependent K+ channels in the plasmalemma over hot spots initiates IK,Ca and action potential repolarization.