The effects of multiple heavy metal stress on the activity of antioxidative enzymes and lipid peroxidation were studied in leaves and roots of two mangrove plants, Kandelia candel and Bruguiera gymnorrhiza, grown under control (10 per thousand NaCl nutrient solution) or five levels of multiple heavy metal stress (10 per thousand NaCl nutrient solution containing different concentration of Pb2+, Cd2+, and Hg2+). Leaves and roots of control and heavy metal-stressed plants were harvested after two months. In leaves of heavy metal-stressed plants superoxide dismutase (SOD) and peroxidase (POD) activities fluctuated in different stress levels compared to the control, while catalase (CAT) activity increased with stress levels in K. candel, but remained unchanged in leaves of B. gymnorrhiza. In comparison with the control, the dynamic tendency of SOD, CAT, and POD activities in roots of heavy metal-stressed plants all ascended, and then declined. The increase in enzyme activities demonstrated that K. candel is more tolerant to heavy metals than B. gymnorrhiza. Lipid peroxidation was enhanced only in leaves of heavy metal-stressed B. gymnorrhiza. These results indicate that in heavy-metal stress antioxidative activities may play an important role in K. candel and B. gymnorrhiza and that cell membrane in leaves and roots of K. candel have greater stability than those of B. gymnorrhiza. For pollution monitoring purposes, POD activity in roots and leaves maybe serve as a biomarker of heavy metal stress in K. candel, while lipid peroxidation maybe serve as biomarker in B. gymnorrhiza.

Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecules (ICAMs) facilitates T cell antigen receptor (TCR)-mediated killing. To dissect TCR and LFA-1 contributions, we evaluated cytolytic activity and granule release by cytotoxic T lymphocytes (CTL) as well as intracellular granule redistribution and morphology of CTL stimulated with natural TCR ligand in the presence or absence of LFA-1 engagement. Although other adhesion mechanisms, e.g., CD2-CD58 interaction, could substitute for LFA-1 to trigger CTL degranulation, productive LFA-1 ligation was indispensable for effective target cell lysis by the released granules. LFA-1-mediated adhesion to glass-supported bilayers containing intercellular adhesion molecule-1 was characterized by a much larger junction area, marked by LFA-1 segregation, and a more compact cell shape compared with those observed for CD2-mediated adhesion to bilayers containing CD58. A larger contact induced by intercellular adhesion molecule 1 determined a unique positioning of granules near the interface. These data provide evidence that LFA-1 delivers a distinct signal essential for directing released cytolytic granules to the surface of antigen-bearing target cells to mediate the effective destruction of these cells by CTL.

Integrin receptors play a central role in cell migration through their roles as adhesive receptors for both other cells and extracellular matrix components. In this study, we demonstrate that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of cell migration. In addition to promoting proliferation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A. Horwitz. 1996. J. Cell Biol. 133:169-184), ectopic expression of the alpha5 integrin in cultures of primary quail myoblasts promotes a striking contact-mediated inhibition of cell migration. Myoblasts ectopically expressing alpha5 integrin (alpha5 myoblasts) move normally when not in contact, but upon contact, they show inhibition of migration and motile activity (i.e., extension and retraction of membrane protrusions). As a consequence, these cells tend to grow in aggregates and do not migrate to close a wound. This phenotype is also seen with ectopic expression of beta1 integrin, paxillin, or activated FAK (CD2 FAK) and therefore appears to result from enhanced integrin-mediated signaling. The contact inhibition observed in the alpha5 myoblasts is mediated by N-cadherin, whose expression is upregulated more than fivefold. Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type and mutant N-cadherins all implicate N-cadherin in the contact inhibition of migration. Ectopic expression of N-cadherin also produces cells that show inhibited migration upon contact; however, they do not show suppressed motile activity, suggesting that integrins and cadherins coordinately regulate motile activity. These observations have potential importance to normal and pathologic processes during embryonic development and tumor metastasis.

Natural killer (NK) cells in rhesus macaques have been variably defined as CD3- CD16+ or CD3- CD8+, although only limited efforts have been made to validate these definitions rigorously. To better understand the role of NK cells in macaque disease models, we undertook a multiparameter analysis of macaque NK cells employing four-colour flow cytometry and a panel of lineage-specific and non-lineage-specific lymphocyte markers. Using this approach, we identified two distinct populations of candidate NK cells: a major CD8bright CD16+ population and a minor CD8bright CD16- population. Further analysis of the major and minor NK cell populations revealed the expression of multiple markers characteristic of NK cells, including CD2, CD7, CD16, CD161, NKG2A and granzyme B. In addition, a CD56+ subset of cells within the minor rhesus NK population was identified which expressed chemokine and lymph node homing receptors similar to those expressed by the CD56bright NK cell population identified in humans. Cytolytic assays confirmed that the phenotypically defined rhesus NK cells lysed NK-susceptible target cells. Our observations support the existence of several distinct subpopulations of rhesus macaque NK cells, which have significant phenotypic and functional similarities to their human counterparts. These improved immunophenotypic definitions of macaque NK cells should facilitate future analysis of innate immune responses in rhesus macaques and the role of NK cells in AIDS pathogenesis in Simian immunodeficiency virus (SIV)-infected macaques.

The GL183 mAb was obtained by immunizing BALB/c mice with the E57 clone (CD7+CD2+CD3-CD16+CD56+) derived from human peripheral blood NK cells. In human peripheral blood, GL183-reactive cells ranged between 2 and 12% (mean 6.5%) in 10 different donors. Double fluorescence and FACS analysis showed that GL183+ cells were consistently included in the CD56+ or CD16+ cell populations. Moreover, since only a fraction of CD56+ or CD16+ cells (approximately 40%) coexpressed GL183 surface antigen, reactivity with GL183 mAb appears to define two subsets within the CD3- lymphocyte population expressing NK cell markers. Although, the majority of GL183+ cells were CD3-, approximately 1% expressed CD3 surface antigens. As shown by clonal analysis, these infrequent CD3+GL183+ cells coexpressed CD56 and CD16 antigens. Cloning of CD3-GL183+ or CD3-GL183- cell populations under limiting dilution conditions yielded clonal progenies that maintained their original surface phenotype. Therefore, expression or lack of expression of GL183 surface antigens represents a stable phenotypic property of a subset of human CD3- NK cells. Immunoprecipitation experiments and two-dimensional PAGE analysis indicated that GL183-reactive molecules were represented in different clones either by a single 58-kD chain or, more frequently, by two chains of approximately 55 and approximately 58 kD, respectively. Analysis of GL183+ or GL183- NK clones for their ability to lyse human (IGROV I) or murine (P815) tumor target cells indicated that GL183- clones were, on average, fivefold more efficient in inducing target cell lysis. GL183+ and GL183- clones produced comparable levels of TNF-alpha in response to PHA plus PMA or anti-CD16 mAb plus PMA. Importantly, production of TNF-alpha was also induced by stimulation of GL183+ clones with GL183 mAb plus PMA. These data indicated that GL183 antigen could mediate cell triggering. This concept was confirmed by the analysis of Ca2+ mobilization, as GL183 mAb induced (in GL183+ clones) increments of [Ca2+]i comparable with those induced by PHA. Moreover, GL183 mAb, or its F(ab')2 fragments, strongly enhanced the cytolytic activity of GL183+ clones against a panel of human tumor target cells, including U937, Raji, IGROV I, M14, and A549. In contrast, GL183 mAb, but not the F(ab')2 fragments, sharply inhibited the cytolytic activity of the same clones against P815, M12, and P3U1 murine target cells. In this case, the effect of GL183 mAb (inhibition) was opposite that of PHA or of stimulatory anti-CD2 or anti-CD16 mAbs, which consistently enhanced the target cell lysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Stable transformants of the Jurkat T-cell line have been obtained that express either of two distinct forms of the type 1 human immunodeficiency virus nef gene: the nef-1-encoded protein (Nef-1) contains alanine, glycine, and valine at positions 15, 29, and 33, respectively; the protein specified by nef-2 (Nef-2) has threonine, arginine, and alanine at the corresponding positions. When Jurkat cells or their Nef-2-expressing transformants are treated with phorbol 12-myristate 13-acetate (PMA) plus either phytohemagglutinin (PHA) or antibodies against CD3 epsilon, T-cell receptor beta chain, or CD2, there is a prompt increase in interleukin 2 (IL-2) mRNA and intracellular calcium and in the IL-2 receptor alpha chain on the cell surface. Although cells expressing Nef-1 also induce calcium mobilization and the production of IL-2 receptor alpha chain, the formation of IL-2 mRNA is blocked in response to these stimuli. Moreover, Nef-1-expressing cells transfected with a plasmid in which the IL-2 promoter is fused to the chloramphenicol acetyltransferase (CAT) gene fail to induce CAT following treatment with PMA and PHA. By contrast, the parental and Nef-2-containing cells induce CAT normally. Nef-1-expressing cells can produce IL-2 mRNA in response to a combination of PMA and ionomycin, although much less efficiently than the parental Jurkat cells or Nef-2-expressing cells. These findings, and others described herein, suggest that the virally encoded Nef protein interferes with a signal emanating from the T-cell receptor complex that induces IL-2 gene transcription.

Previous studies have demonstrated that the HPV-16 E7 gene product encodes the major transforming activity of the virus in rodent cell systems. In this study we have generated a series of point mutations affecting the region of HPV-16 E7, which shows homology to adenovirus E1a conserved domain (CD)1. In conjunction with previously described mutants in the region of E7 with similarity to E1a CD2 and SV40 LT, we have investigated three known activities of the E7 protein; transformation, association with the cellular RB protein and induction of cellular DNA synthesis. The results show that RB binding correlates with the ability of E7 to induce cellular DNA synthesis and mediate cell transformation. In addition an unidentified function of E7, which is necessary for transformation of NIH3T3 cells, but does not affect RB binding or the ability to induce cellular DNA synthesis, has also been demonstrated. This study therefore identifies two separate regions of the E7 gene necessary for transformation of established cells. One of these, in the region of E7 which shows similarity to E1a CD2 and LT, is required for RB binding and DNA synthesis. The other region important for transformation, in the N-terminus of E7, is separable from the RB binding/DNA synthesis function.

OBJECTIVE

Normal human lamina propria lymphocytes manifest increased unstimulated apoptosis compared with peripheral lymphocytes, which are enhanced after stimulation via the CD2 activation pathway. This activation-induced apoptosis down-regulates cell expansion and cytokine production. In previous studies, it was shown that lamina propria T cells from patients with Crohn's disease and ulcerative colitis manifest abnormal proliferation and cytokine production. It was therefore of interest to determine if such cells also showed abnormal patterns of apoptosis.

METHODS

Apoptosis was evaluated by propidium iodide staining of cells followed by flow cytometric analysis. Fas expression and Bcl-2 levels in cells were evaluated by immunofluorescence.

RESULTS

Lamina propria lymphocytes from patients with Crohn's disease and ulcerative colitis as well as from 2 patients with diverticulitis showed defective CD2 pathway-induced apoptosis. Studies of the mechanisms of this defect focusing on cells from patients with Crohn's disease showed that Crohn's disease lamina propria lymphocytes from inflamed tissues express the same amount of cell surface Fas but are less sensitive to Fas-mediated apoptosis than control cells. In addition, lamina propria lymphocytes from inflamed Crohn's disease tissues manifest increased expression of Bcl-2 after CD2 pathway stimulation and elevated Bcl-2 levels in cultures of unstimulated T cells.

CONCLUSIONS

T cells isolated from areas of inflammation in Crohn's disease, ulcerative colitis, and other inflammatory states manifest decreased CD2 pathway-induced apoptosis. Studies of cells from inflamed Crohn's disease tissue indicate that this defect is accompanied by elevated Bcl-2 levels. These changes are probably caused by the chronic inflammation and may aggravate the underlying disease processes that are present.

1. The modulatory effect of internal Ca2+ on the current through the ACh-activated non-selective cation channels (Ins, ACh) was investigated by the whole-cell patch clamp technique in single isolated cells of guinea-pig ileum. 2. Ins, ACh was isolated with caesium aspartate internal solution of low Ca2(+)-buffering capacity (10 microM-EGTA). With preceding depolarizations which evoked voltage-operated Ca2+ currents (ICa), Ins, ACh increased in amplitude and decayed more rapidly. The extent of this 'facilitating' effect depended on the number and duration of the depolarizations. 3. When depolarizing pulses were applied during the sustained phase of Ins, ACh, they were followed by large inward tail currents. These tail currents (tail Ins, ACh) resembled the non-facilitated Ins, ACh recorded without the depolarizing pulse, in regard to voltage-dependent gating and dependence on the extracellular Na+ concentration, thus suggesting that the currents are flowing through the same class of channels. 4. The tail Ins, ACh was apparently composed of two components distinguished by the insensitivity to organic Ca2+ antagonists. The minor component (about 20% of tail Ins, ACh) showed a rapid decay (about 150 ms at -60 mV) which could be attributed to voltage-dependent kinetics. The major component decayed slowly within 5 s and appeared to be related to changes in the intracellular Ca2+ concentration. The latter component was not recorded when Ba2+ or Sr2+ were used as a charge carrier for ICa and was blocked by 10 microM-D600 or nitrendipine, or Cd2+ 0.2-0.5 mM). 5. The tail Ins, ACh increased in proportion to Ca2+ influx when the duration of depolarizing pulses were prolonged from 15 to 200 ms, but this 'facilitating' effect was greatly suppressed when the cell was perfused with 40 mM-EGTA. 6. When the pCa in the pipette was varied using 40 mM-Ca-EGTA, the conductance through Ins, ACh increased in a manner dependent on intracellular Ca2+ concentration. Half-maximal and submaximal activation occurred at about 200 nM and 1 microM, respectively. 7. These results show that the activity of Ins, ACh is very sensitive to the intracellular Ca2+ concentration in the physiological range.

T cells respond to peptide antigen in association with MHC products on antigen-presenting cells (APCs). A number of accessory or costimulatory molecules have been identified that also contribute to T cell activation. Several of the known accessory molecules are expressed by freshly isolated dendritic cells, a distinctive leukocyte that is the most potent APC for the initiation of primary T cell responses. These include ICAM-1 (CD54), LFA-3 (CD58), and class I and II MHC products. Dendritic cells also constitutively express the accessory ligand for <em>CD2</em>8, B7/BB1, which has not been previously identified on circulating leukocytes freshly isolated from peripheral blood. Dendritic cell expression of both B7/BB1 and ICAM-1 (CD54) increases after binding to allogeneic T cells. Individual mAbs against several of the respective accessory T cell receptors, e.g., anti-<em>CD2</em>, anti-CD4, anti-CD11a, and anti-<em>CD2</em>8, inhibit T cell proliferation in the dendritic cell-stimulated allogeneic mixed leukocyte reaction (MLR) by 40-70%. Combinations of these mAbs are synergistic in achieving near total inhibition. Other T cell-reactive mAbs, e.g., anti-CD5 and anti-CD45, are not inhibitory. Lymphokine secretion and blast transformation are similarly reduced when active accessory ligand-receptor interactions are blocked in the dendritic cell-stimulated allogeneic MLR. Dendritic cells are unusual in their comparably higher expression of accessory ligands, among which B7/BB1 can now be included. These are pertinent to the efficiency with which dendritic cells in small numbers elicit strong primary T cell proliferative and effector responses.

The role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca(2+)-activated potassium channels (PK,Ca) and voltage-gated potassium channels (PK,V) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-<em>CD2</em>, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-<em>CD2</em>8, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (IL-2 and interferon gamma) are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,Ca and PK,V, we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for PK,V. These studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca(2+)-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.

1. Calcium channel blockers were tested on excitatory postsynaptic currents (EPSCs) at the synapse formed by the calyx of Held on the principal cells in the medial nucleus of trapezoid body (MNTB) in brainstem slices of 4- to 14-day-old rats. 2. At postnatal day 4-9 (P4-9), EPSCs were irreversibly suppressed by the P/Q-type Ca2+ channel blocker omega-agatoxin-IVA (omega-Aga-IVA, 200 nM) and also by the N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CgTx, 2 microM). A small fraction of EPSCs was resistant to both toxins but abolished by Cd2+ (100 microM). 3. After P7, the omega-CgTx-sensitive EPSC fraction diminished and eventually disappeared after P10. Concomitantly the fraction insensitive to both toxins decreased and became undetectable after P10. 4. In contrast, the omega-Aga-IVA-sensitive EPSC fraction increased with development and became predominant after P10. All through the developmental period examined, the L-type Ca2+ channel blocker nicardipine (10 microM) had no effect. 5. We conclude that presynaptic Ca2+ channel types triggering transmitter release undergo developmental switching during the early postnatal period.

BACKGROUND

Type 1 diabetes results from autoimmune targeting of the pancreatic β cells, likely mediated by effector memory T (Tem) cells. CD2, a T cell surface protein highly expressed on Tem cells, is targeted by the fusion protein alefacept, depleting Tem cells and central memory T (Tcm) cells. We postulated that alefacept would arrest autoimmunity and preserve residual β cells in patients newly diagnosed with type 1 diabetes.

METHODS

The T1DAL study is a phase 2, double-blind, placebo-controlled trial in patients with type 1 diabetes, aged 12-35 years who, within 100 days of diagnosis, were enrolled at 14 US sites. Patients were randomly assigned (2:1) to receive alefacept (two 12-week courses of 15 mg intramuscularly per week, separated by a 12-week pause) or a placebo. Randomisation was stratified by site, and was computer-generated with permuted blocks of three patients per block. All participants and site personnel were masked to treatment assignment. The primary endpoint was the change from baseline in mean 2 h C-peptide area under the curve (AUC) at 12 months. Secondary endpoints at 12 months were the change from baseline in the 4 h C-peptide AUC, insulin use, major hypoglycaemic events, and HbA1c concentrations. This trial is registered with ClinicalTrials.gov, number NCT00965458.

RESULTS

Of 73 patients assessed for eligibility, 33 were randomly assigned to receive alefacept and 16 to receive placebo. The mean 2 h C-peptide AUC at 12 months increased by 0.015 nmol/L (95% CI -0.080 to 0.110) in the alefacept group and decreased by 0.115 nmol/L (-0.278 to 0.047) in the placebo group, and the difference between groups was not significant (p=0.065). However, key secondary endpoints were met: the mean 4 h C-peptide AUC was significantly higher (mean increase of 0.015 nmol/L [95% CI -0.076 to 0.106] vs decrease of -0.156 nmol/L [-0.305 to -0.006]; p=0.019), and daily insulin use (0.48 units per kg per day for placebo vs 0.36 units per kg per day for alefacept; p=0.02) and the rate of hypoglycaemic events (mean of 10.9 events per person per year for alefacept vs 17.3 events for placebo; p<0.0001) was significantly lower at 12 months in the alefacept group than in the placebo group. Mean HbA1c concentrations at week 52 were not different between treatment groups (p=0.75). So far, no serious adverse events were reported and all patients had at least one adverse event. In the alefacept group, 29 (88%) participants had an adverse event related to study drug versus 15 (94%) participants in the placebo group. In the alefacept group, 14 (42%) participants had grade 3 or 4 adverse events compared with nine (56%) participants in the placebo group; no deaths occurred.

CONCLUSIONS

Although the primary outcome was not met, at 12 months, alefacept preserved the 4 h C-peptide AUC, lowered insulin use, and reduced hypoglycaemic events, suggesting efficacy. Safety and tolerability were similar in the alefacept and placebo groups. Alefacept could be useful to preserve β-cell function in patients with new-onset type 1 diabetes.

The mechanism by which low affinity adhesion molecules function to produce stable cell-cell adhesion is unknown. In solution, the interaction of human CD2 with its ligand CD58 is of low affinity (500 mM-1) and the interaction of rat CD2 with its ligand CD48 is of still lower affinity (40 mM-1). At the molecular level, however, the two systems are likely to be topologically identical. Fluorescently labeled glycosylphosphatidylinositol-anchored CD48 and CD58 were prepared and incorporated into supported phospholipid bilayers, in which the ligands were capable of free lateral diffusion. Quantitative fluorescence imaging was used to study the binding of cell surface human and rat CD2 molecules to the fluorescent ligands in contact areas between Jurkat cells and the bilayers. These studies provide two major conclusions. First, CD2/ligand interactions cooperate to align membranes with nanometer precision leading to a physiologically effective two-dimensional affinity. This process does not require the intact cytoplasmic tail of CD2. Second, the degree of membrane alignment that can be achieved by topologically similar receptors deteriorates with decreasing affinity. This suggests an affinity limit for the ability of this mode of cooperativity to achieve stable cell-cell adhesion at approximately 10 mM-1.

No process is more central to T-lymphocyte function than cell-cell adhesion, yet it is only recently that interest in lymphocyte adhesion has burgeoned. Neglect of adhesion is particularly surprising since immunologists are surrounded by a veritable sea of adhesive interactions of lymphocytic cells: transformed lymphocytes grow in aggregates, stimulated lymphocytes aggregate and T cells conjugate with their targets. In retrospect, it is obvious that all lymphocyte adhesion (both antigen-specific and seemingly non-specific adhesive interactions) has to be based on specific receptor-ligand interactions. In this review Malegapuru Makgoba, Martin Sanders and Stephen Shaw focus primarily on the two molecular pathways of lymphocyte adhesion that have been shown to play a critical role in facilitation of antigen-specific recognition, namely CD2 and its ligand, lymphocyte function associated antigen-3 (LFA-3), and LFA-1 and its ligand, intercellular adhesion molecule-1 (ICAM-1). A variety of excellent recent reviews have dealt with this and related aspects of T-cell adhesion. Of particular interest is the review that follows in this issue: it deals with the CD44 molecule which has also been implicated in both adhesion and activation of T cells.

OBJECTIVE

To investigate whether the combination of intravenous immunoglobulin (IVIg) with prednisone improves muscle strength and alters endomysial inflammation in patients with sporadic inclusion body myositis (s-IBM).

BACKGROUND

In a previous controlled trial in s-IBM, IVIg did not significantly improve strength in spite of modest benefits in some muscle groups. The possibility that prednisone may have a synergistic effect with IVIg prompted another controlled trial.

METHODS

Thirty-six patients with biopsy-proven IBM were randomized to receive IVIg or placebo monthly for 3 months. Before infusions, all patients were started on high-dose prednisone for 3 months. Primary outcome measures were differences in the 1) Quantitative Muscle Strength (QMT) testing; and 2) modified Medical Research Council (MRC) scores, between the patients randomized to IVIg + prednisone compared with those randomized to placebo + prednisone. Repeated open muscle biopsies were performed at random in 24 patients to determine changes in the number of autoinvasive T cells and necrotic muscle fibers.

RESULTS

Nineteen patients were randomized to IVIg + prednisone and 17 to placebo + prednisone. No significant change was noted in muscle strength, assessed by QMT and MRC, from baseline to the 2nd, 3rd, or 4th month after treatment between the two groups. The number of necrotic fibers was reduced in the IVIg randomized group (p < 0.01), and the mean number of CD2+ cells was significantly decreased in both groups (p < 0.0001), denoting a steroid effect.

CONCLUSIONS

IVIg combined with prednisone for a 3-month period was not effective in IBM. Endomysial inflammation was significantly reduced after treatment, but the reduction was not of clinical significance.

We report the genomic organization of the human CD2 gene and its expression in transgenic mice. A 28.5 kb segment of DNA consisting of 4.5 kb 5' flanking sequences, 15 kb containing the gene's five exons and 9 kb of 3' flanking sequences can direct the expression of the CD2 gene only on thymocytes, circulating T cells and megakaryocytes of the transgenic mice. The expression of each copy of the human CD2 transgene appears to be as high as the endogenous mouse CD2 gene and as high as the expression on the surface of human T lymphocytes, independent of the site of integration and dependent on the copy number of genes that have integrated.

Repolarization of the action potential in squid axon1 and several types of neurones2-4 involves a voltage-activated potassium (K+) current. Voltage clamp analysis has demonstrated that this current has rapid activation kinetics1,3-5. In several neuronal types, the same technique has also revealed a slowly activated K+ current that is calcium (Ca2+)-sensitive3,5-10. This slow Ca2+-activated K+ current is the major current underlying the late, slower portion of the after-hyperpolarization following an action potential11-14. In several muscle types, fast, transient Ca2+-dependent K+ currents have been described15-17 which may contribute to repolarization of the action potential. Rapidly activating, Ca2+-dependent K+ currents have been observed in sympathetic neurones of the bullfrog and it has been suggested that they contribute to action potential repolarization of those neurones8,9,18. We have studied the membrane currents in bullfrog sympathetic neurones using voltage clamp methods and report here a transient outward current that appears to be composed of two separate currents. One of those currents is a transient, Ca2+-sensitive outward current as indicated by a significant reduction of the current by treatments that reduce or block Ca2+ entry (Mn2+, Cd2+, Co2+, Mg2+ or Ca2+-free Ringer). Such treatments also decreased the rate of action potential repolarization. The results suggest that this current is involved in repolarization of the action potential and consequently may regulate Ca2+ entry into the neurone during spike activity.

In this study, two monoclonal antibodies, IL-A29 and CC15, are described that identify a novel bovine cell surface marker of 215/300 kDa. The antibodies reacted with a discrete population of resting lymphocytes in peripheral blood which, in young animals, constituted about 25% of the mononuclear cells. Thymus, lymph nodes and spleen contained less than 5% positive cells. These cells were negative for surface Ig, a monocyte/granulocyte marker, and the T lymphocyte antigens CD2, CD6, CD4 and CD8. Immunohistological analyses revealed the presence of IL-A29/CC15-positive lymphocytes in the thymic medulla, in the outer cortex of lymph nodes, in the marginal zones of the spleen, in the dermal and epidermal layers of the skin and in the lamina propria of the gut. The IL-A29/CC15+ cells in unfractionated blood mononuclear cells responded in autologous and allogeneic mixed lymphocyte cultures, and when purified they responded to concanavalin A in the presence of recombinant interleukin 2. These observations suggested this population of cells belonged to the T cell lineage. In order to unambiguously define their lineage, cDNA clones encoding bovine T cell receptor (TcR) and CD3 proteins were isolated. Northern blot analyses of IL-A29/CC15+ cell populations and of established cell lines of various lineages demonstrated that they expressed TcR delta and CD3 gamma, delta and epsilon mRNA: TcR alpha was not expressed, whereas only a truncated form of TcR beta mRNA was present. These results indicate that the IL-A29 and CC15 antibodies define a unique population of CD4-CD8-, gamma/delta T cells.

1. The effects of some divalent cations on the A-current (IA) in cultured rat dorsal root ganglion cells (DRGs) were studied using whole-cell patch recording. 2. IA was not affected by omission of calcium from the external medium; however it was significantly depressed by manganese (10 mM) applied by pressure ejection. This depressant effect of manganese resulted from a depolarizing shift of the activation curve by 17 mV, associated with only a slight reduction of the maximum conductance. At 10 mM manganese also caused a depolarizing shift of the steady-state inactivation curve by 34 mV. Divalent cations other than manganese also gave positive shifts of the steady-state activation and inactivation curves for IA but were of different potency; the sequence was: Cd2+ greater than Mn2+ = Co2+ greater than Mg2+. 3. A dose-response curve for the depolarizing shift of the activation and inactivation curves of IA, as a function of manganese concentration, could be fitted by a single binding site model with an apparent dissociation constant of approximately 17 mM. The depolarizing shift of the inactivation curve was on average twice as large as that of the activation curve. 4. In contrast to its effect on IA, manganese (10 mM) did not cause any appreciable change in the voltage dependence of the activation curve for the delayed rectifier K+ current. 5. A low concentration of manganese (1 mM) increased the amplitude of IA recorded at pre-pulse potentials ranging from -50 to -70 mV. This augmentation of IA resulted from a positive shift of the inactivation curve by 6 mV without an appreciable shift of the activation curve; as a result a population of A-channels is released from inactivation over pre-pulse potentials from -50 to -70 mV. 6. These results show that divalent cations can evoke a depolarizing shift of both the activation and inactivation gates controlling IA; this causes either depression or augmentation of IA, depending on the species and concentration of the divalent cation, and also on the pre- pulse potential used to de-inactivate IA. This modulatory effect of divalent cations on the gating of IA appears to reflect binding to a specific, saturable site, either the A-channel protein itself, or phospholipids electrically close to the gating apparatus.

1. Fluorescence measurements of intracellular calcium concentrations ([Ca2+]i) were made on cultured human airway smooth muscle cells using the dye Fura-2. The response to either histamine (100 microM) or bradykinin (1 microM) was biphasic, with a transient increase in [Ca2+]i followed by a sustained [Ca2+]i increase lasting many minutes. The average steady-state (plateau) [Ca2+]i following agonist activation was 267 +/- 5 nM, whereas the average basal [Ca2+]i was 148 +/- 4 nM. 2. The sustained rise in [Ca2+]i required the continued presence of either histamine or bradykinin and was dependent on extracellular Ca2+. The magnitude of the transient rise in [Ca2+]i was not dependent on extracellular Ca2+. Sustained, receptor-activated rises in [Ca2+]i were rapidly abolished by chelation of extracellular Ca2+, or addition of non-permeant polyvalent cations, whereas these agents had minor effects in the absence of agonist. These data indicate that the sustained increase in [Ca2+]i was dependent on receptor-activated Ca2+ influx. 3. Receptor-activated Ca2+ influx was not affected by treatment with organic Ca2+ channel antagonists (nifedipine (10 microM), nisoldipine (10 microM) or diltiazem (10 microM] or agonists (Bay K 8644 (500 nM to 10 microM) or Bay R 5417 (500 nM]. The magnitude of the sustained rise was also not affected by pre-treatment with ouabain (100 microM) indicating little involvement of Na(+)-Ca2+ exchange in the influx mechanism. 4. Receptor-activated Ca2+ influx could be completely inhibited by several polyvalent cations (Co2+, Mn2+, Ni2+, -Cd2+ or La3+). Quantitative estimates of the potency of block were obtained for Ni2+ and La3+. These measurements indicate that the pKi for Ni2+ was 3.6 and for La3+ was 3.5. 5. Both Mn2+ and Co2+ ions caused a time-dependent quench of intracellular Fura-2; however, permeation of neither ion was increased following receptor activation, indicating that the influx pathway is not permeable to these cations. 6. Fura-2 was used to monitor the rate of Ba2+ entry into airway smooth muscle cells by monitoring the Ca(2+)-Fura-2 and Ba(2+)-Fura-2 isosbestic points as well as the 340 and 380 nm signals. Cell activation did not increase the rate of Ba2+ entry indicating that the Ca2+ influx pathway was poorly permeant to Ba2+ ions. Ba2+ (2 mM) was able to inhibit Ca2+ entry as shown by its effects on the Ba(2+)-independent, Ca(2+)-dependent wavelength (371 nm). 7. The voltage dependence of Ca2+ influx was examined before and after agonist-induced activation. The effect of KCl-induced depolarization prior to cell activation was to cause a slight increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

Phytochelatin synthases (PCS) mediate cellular heavy-metal resistance in plants, fungi, and worms. However, phytochelatins (PCs) are generally considered to function as intracellular heavy-metal detoxification mechanisms, and whether long-distance transport of PCs occurs during heavy-metal detoxification remains unknown. Here, wheat TaPCS1 cDNA expression was either targeted to Arabidopsis roots with the Arabidopsis alcohol dehydrogenase (Adh) promoter (Adh::TaPCS1/cad1-3) or ectopically expressed with the cauliflower mosaic virus 35S promoter (35S::TaPCS1/cad1-3) in the PC-deficient mutant cad1-3. Adh::TaPCS1/cad1-3 and 35S::TaPCS1/cad1-3 complemented the cadmium, mercury, and arsenic sensitivities of the cad1-3 mutant. Northern blot, RT-PCR, and Western blot analyses showed Adh promoter-driven TaPCS1 expression only in roots and thus demonstrated lack of long-distance TaPCS1 mRNA and protein transport in plants. Fluorescence HPLC analyses showed that under Cd2+ stress, no PCs were detectable in cad1-3. However, in Adh::TaPCS1/cad1-3 plants, PCs were detected in roots and in rosette leaves and stems. Inductively coupled plasma atomic emission spectrometer analyses showed that either root-specific or ectopic expression of TaPCS1 significantly enhanced long-distance Cd2+ transport into stems and rosette leaves. Unexpectedly, transgenic expression of TaPCS1 reduced Cd2+ accumulation in roots compared with cad1-3. The reduced Cd2+ accumulation in roots and enhanced root-to-shoot Cd2+ transport in transgenic plants were abrogated by l-buthionine sulfoximine. The presented findings show that (i) transgenic expression of TaPCS1 suppresses the heavy-metal sensitivity of cad1-3, (ii) PCs can be transported from roots to shoots, and (iii) transgenic expression of the TaPCS1 gene increases long-distance root-to-shoot Cd2+ transport and reduces Cd2+ accumulation in roots.

Kidney podocytes are highly differentiated epithelial cells that form interdigitating foot processes with bridging slit diaphragms (SDs) that regulate renal ultrafiltration. Podocyte injury results in proteinuric kidney disease, and genetic deletion of SD-associated CD2-associated protein (CD2AP) leads to progressive renal failure in mice and humans. Here, we have shown that CD2AP regulates the TGF-β1-dependent translocation of dendrin from the SD to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-β1. Our study identifies CD2AP as the gatekeeper of the podocyte TGF-β response through its regulation of CatL expression and defines a molecular mechanism underlying proteinuric kidney disease.

Ubiquitously expressed volume-regulated anion channels (VRACs) are chloride channels which are permeable to a variety of small organic anions, including the excitatory amino acids (EAAs) glutamate and aspartate. Broad spectrum anion channel blockers strongly reduce EAA release in cerebral ischaemia and other pathological states associated with prominent astrocytic swelling. However, it is uncertain whether VRAC serves as a major pathway for EAA release from swollen cells. In the present study, we measured swelling-activated release of EAAs as D-[3H]aspartate efflux, and VRAC-mediated Cl- currents by whole-cell patch clamp in cultured rat astrocytes. We compared the pharmacological profiles of the swelling-activated EAA release pathway and Cl- currents. The expression of candidate Cl- channels was confirmed by RT-PCR. The maxi Cl- channel (p-VDAC) blocker Gd3+, the ClC-2 inhibitor Cd2+, and the MDR-1 blocker verapamil did not affect EAA release or VRAC currents. An antagonist of calcium-sensitive Cl- channels (CaCC), niflumic acid, had little effect on EAA release and only partially inhibited swelling-activated Cl- currents. The phorbol ester PDBu, which blocks ClC-3-mediated Cl- currents, had no effect on VRAC currents and up-regulated EAA release. In contrast, DCPIB, which selectively inhibits VRACs, potently suppressed both EAA release and VRAC currents. Two other relatively selective VRAC inhibitors, tamoxifen and phloretin, also blocked the VRAC currents and strongly reduced EAA release. Taken together, our data suggest that (i) astrocytic volume-dependent EAA release is largely mediated by the VRAC, and (ii) the ClC-2, ClC-3, ClC-4, ClC-5, VDAC, CaCC, MDR-1 and CFTR gene products do not contribute to EAA permeability.