The effect of increasing [K+]0 on 3H-glycogen levels was examined in mouse cerebral cortical slices. K+ stimulates in a time- and concentration-dependent manner the hydrolysis of 3H-glycogen. Over 70% of the maximal effect is reached within 30 sec and the EC50 for the glycogenolytic action of K+ is 11 mM. Significant 3H-glycogen hydrolysis occurs at 5-12 mM [K+]0, concentrations reached by the ion in the extracellular space during neuronal activity. The K+-evoked glycogenolysis is Ca2+-dependent, and is inhibited by Ca2+-channel blockers such as Ni2+ and Mn2+, but not by Cd2+, nifedipine, and omega-conotoxin. Furthermore, the effect of K+ is not enhanced by the Ca2+-channel agonist Bay K 8644. This type of pharmacological profile suggests that the activation of voltage-sensitive Ca2+ channels of the T subtype mediates the glycogenolytic action of K+. This set of observations suggests that K+ released in the extracellular space by active neurons may promote the mobilization of energy substrates and therefore play a role in the coupling between neuronal activity and energy metabolism.

Human T lymphocytes can be activated through either the antigen/MHC receptor complex T3-Ti (CD3-Ti) or the T11 (CD2) molecule to proliferate via an IL-2 dependent mechanism. To investigate the relationship of these pathways to one another, we generated and characterized Jurkat mutants which selectively express either surface CD3-Ti or CD2. Here we show that CD3-Ti- mutants fail to be stimulated by either pathway to increase phosphoinositide turnover, mobilize calcium or induce the IL-2 gene. The activation capacity of these mutants via CD2 as well as CD3-Ti can be restored following reconstitution of surface CD3-Ti expression upon appropriate DNA transfer (e.g. Ti beta subunit cDNA into Ti beta- Jurkat variants). Collectively, these results demonstrate that CD3-Ti and CD2 pathways are interdependent and that phosphoinositide turnover is linked to the CD3-Ti complex.

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber-Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu2+ ions either alone or in the presence of ascorbate (Cu2+/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu2+ alone displayed Cu2+-binding, which was significantly increased in the presence of Zn2+, Hg2+, Cd2+, Ag+1 and As3+. Treatment of microsomes with Cu2+ in the microM range decreased the microsomal thiol content; in the presence of ascorbate, Cu2+ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu2+ concentration to>> or =50 microM led to non-detectable lipoperoxidation levels. On the other hand, microM Cu2+ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.

The synthesis of phytochelatins (PCs) is essential for the detoxification of nonessential metals and metalloids such as cadmium and arsenic in plants and a variety of other organisms. To our knowledge, no direct evidence for a role of PCs in essential metal homeostasis has been reported to date. Prompted by observations in Schizosaccharomyces pombe and Saccharomyces cerevisiae indicating a contribution of PC synthase expression to Zn2+ sequestration, we investigated a known PC-deficient Arabidopsis (Arabidopsis thaliana) mutant, cad1-3, and a newly isolated second strong allele, cad1-6, with respect to zinc (Zn) homeostasis. We found that in a medium with low cation content PC-deficient mutants show pronounced Zn2+ hypersensitivity. This phenotype is of comparable strength to the well-documented Cd2+ hypersensitivity of cad1 mutants. PC deficiency also results in significant reduction in root Zn accumulation. To be able to sensitively measure PC accumulation, we established an assay using capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry of derivatized extracts. Plants grown under control conditions consistently showed PC2 accumulation. Analysis of plants treated with same-effect concentrations revealed that Zn2+-elicited PC2 accumulation in roots reached about 30% of the level of Cd2+-elicited PC2 accumulation. We conclude from these data that PC formation is essential for Zn2+ tolerance and provides driving force for the accumulation of Zn. This function might also help explain the mysterious occurrence of PC synthase genes throughout the plant kingdom and in a wide range of other organisms.

Cell populations of human pregnancy decidua, obtained by enzymic digestion from first trimester samples, were analysed by flow cytometry after labelling with monoclonal antibodies. The majority of these decidual cells (75%) were of bone marrow origin. The most abundant cell type expressed antigens characteristic of large granular lymphocytes (LGL), although macrophages and small numbers of classical T cells were also present. Three subsets of decidual LGL can be defined by single-and double-antibody labelling. Most decidual LGL are positive for NKH1, a marker of peripheral blood LGL, but negative for CD16, the Fc receptor of NK cells, and for the T-cell markers CD3 and CD5. About half the NKH1-positive cells also express CD2, associated with the E-rosette receptor, and are identical to the CD3-negative/CD2-positive cells reported previously in early pregnancy decidua. The NKH1-positive cells apparently correspond to a minor subset of peripheral blood LGL. The remaining decidual LGL are positive for CD16 and negative or only dimly positive for NKH1, and are similar to the major type of peripheral blood LGL. After purification by flow cytometry, the NKH1-positive cells were demonstrated to be of similar size to, but slightly higher granularity than, lymphocytes, whereas the CD16-positive cells were larger and more granular. The possible role of decidual LGL in modulating placental development is discussed.

We have recently developed a mAb, anti-1F7, which defines a family of structures found to include the molecule recognized by anti-Ta1 (CD2CD2. The proliferative response induced via the CD3-1F7 or CD2-1F7 pathways is associated with the IL-2 autocrine pathway, including IL-2 production. IL-2R expression and anti-IL-2R (Tac) inhibition. Furthermore, solid-phase immobilization of anti-1F7 but not anti-Ta1 acts in conjunction with submitogenic doses of PMA to mediate a comitogenic effect in the absence of anti-CD3 or anti-CD2, leading to CD4+ T cell proliferation. PMA treatment, in the meantime, leads to enhanced expression of 1F7 on the T cell surface. Despite its functional association with both pathways of activation, however, the 1F7 structure is not comodulated with the CD3/TCR complex nor the CD2 molecule. These findings thus suggest that the CD2CD2-induced human CD4+ T cell activation.

NK cells, lymphocytes of the innate immune system, are important for defense against infectious pathogens and cancer. Classically, the CD56dim NK cell subset is thought to mediate antitumor responses, whereas the CD56bright subset is involved in immunomodulation. Here, we challenge this paradigm by demonstrating that brief priming with IL-15 markedly enhanced the antitumor response of CD56bright NK cells. Priming improved multiple CD56bright cell functions: degranulation, cytotoxicity, and cytokine production. Primed CD56bright cells from leukemia patients demonstrated enhanced responses to autologous blasts in vitro, and primed CD56bright cells controlled leukemia cells in vivo in a murine xenograft model. Primed CD56bright cells from multiple myeloma (MM) patients displayed superior responses to autologous myeloma targets, and furthermore, CD56bright NK cells from MM patients primed with the IL-15 receptor agonist ALT-803 in vivo displayed enhanced ex vivo functional responses to MM targets. Effector mechanisms contributing to IL-15-based priming included improved cytotoxic protein expression, target cell conjugation, and LFA-1-, CD2-, and NKG2D-dependent activation of NK cells. Finally, IL-15 robustly stimulated the PI3K/Akt/mTOR and MEK/ERK pathways in CD56bright compared with CD56dim NK cells, and blockade of these pathways attenuated antitumor responses. These findings identify CD56bright NK cells as potent antitumor effectors that warrant further investigation as a cancer immunotherapy.

Costimulatory molecules on the APC regulate T cell growth by providing signals that regulate responses to TCR occupancy. One such molecule is B7/BB-1, which triggers a T cell activation pathway by binding the <em>CD2</em>8 and/or CTLA-4 cell-surface molecules. Expression and signaling activity of <em>CD2</em>8 have been shown to increase after T cell activation by various polyclonal activators. Here we show that <em>CD2</em>8 expression and signaling activity in activated T cells decrease after ligand binding to <em>CD2</em>8. Stimulation of <em>CD2</em>8 on PHA- or PMA-activated T cells by cross-linked mAb 9.3 or by co-culture with B7+ Chinese hamster ovary (CHO) cells caused a marked reduction of <em>CD2</em>8 mRNA levels within 4 h. The decrease in <em>CD2</em>8 mRNA was transient, and by 24 h of <em>CD2</em>8 stimulation, <em>CD2</em>8 mRNA was found at approximately initial levels. In contrast, CTLA-4 mRNA levels were usually up-regulated by <em>CD2</em>8 triggering. Cell-surface expression of <em>CD2</em>8, but not <em>CD2</em> or CD3, decreased by 12 to 24 h after addition of B7+ CHO cells, but returned to initial levels or higher by 48 h. The ability of <em>CD2</em>8 cross-linking on PMA-activated CD4+ cells to trigger calcium mobilization was also reduced by treatment with B7+ CHO cells, and remained reduced even after cell-surface expression of <em>CD2</em>8 returned to normal levels. Thus, engagement of the <em>CD2</em>8 receptor by its natural ligand B7/BB-1 leads to a transient down-regulation of <em>CD2</em>8 synthesis and a prolonged unresponsiveness to <em>CD2</em>8 signaling. This represents a novel mechanism for regulation of costimulatory signals delivered by interactions of <em>CD2</em>8 with the B7/BB-1 counter receptor.

Neurons in the nucleus laminaris detect the coincidence of binaural signals, and are the first neurons to calculate the interaural time difference for the sound source localization in birds. In this paper, we have studied contributions of synaptic depression to the coincidence detection in the nucleus laminaris in a slice preparation of the chick embryo (E16-18), using the whole-cell patch recording technique. Under voltage clamp, EPSCs decreased progressively in their amplitude during the course of tetanic stimuli. This synaptic depression was primarily ascribed to the reduction of transmitter release from the presynaptic terminal, because the depression was decreased by reducing transmitter release with 2.5 microm Cd2+ but was not affected by reducing desensitization of postsynaptic AMPA receptors with 20 microm cyclothiazide. Under current clamp, trains of 10 stimuli of 100 Hz were applied bilaterally with changing the time intervals systematically between both sides. Response window, defined as the time interval corresponding to the half-maximum firing probability, was narrowed during the course of the stimulus train, and this occurred in parallel with a decrease in the EPSP amplitude. In addition, the reduction of the EPSP amplitude due to 2.5 microm Cd2+ or 2 microm CNQX improved the accuracy of coincidence detection. These results indicate that the synaptic depression may improve the coincidence detection in the chick laminaris neurons.

The T cell antigen receptor (TCR) and its ligand peptide-major histocompatibility complex (MHC) are small (approximately 7 nm) compared with other abundant cell surface molecules such as integrins, CD43, and CD45 (23-50 nm). We have proposed that molecules at the T cell/antigen-presenting cell (APC) interface segregate according to size, with small "accessory" molecules (e.g., CD2, CD4, CD8, CD2CD2 ligand, CD48, and examining how this affects T cell antigen recognition. Although the interaction of CD2 on T cells with wild-type or shortened forms of CD48 on APCs enhances T cell antigen recognition, the interaction of CD2 with elongated forms of CD48 is strongly inhibitory. Further experiments indicated that elongation of the CD2/CD48 complex inhibited TCR engagement of peptide-MHC, presumably by preventing the formation of sufficiently intimate contacts at the T cell/APC interface. These findings demonstrate the importance of small size in CD2/CD48 function, and support the hypothesis that T cell antigen recognition requires segregation of cell surface molecules according to size.

A native structure of the cytochrome b(6)f complex with improved resolution was obtained from crystals of the complex grown in the presence of divalent cadmium. Two Cd(2+) binding sites with different occupancy were determined: (i) a higher affinity site, Cd1, which bridges His143 of cytochrome f and the acidic residue, Glu75, of cyt b(6); in addition, Cd1 is coordinated by 1-2 H(2)O or 1-2 Cl(-); (ii) a second site, Cd2, of lower affinity for which three identified ligands are Asp58 (subunit IV), Glu3 (PetG subunit) and Glu4 (PetM subunit). Binding sites of quinone analogue inhibitors were sought to map the pathway of transfer of the lipophilic quinone across the b(6)f complex and to define the function of the novel heme c(n). Two sites were found for the chromone ring of the tridecyl-stigmatellin (TDS) quinone analogue inhibitor, one near the p-side [2Fe-2S] cluster. A second TDS site was found on the n-side of the complex facing the quinone exchange cavity as an axial ligand of heme c(n). A similar binding site proximal to heme c(n) was found for the n-side inhibitor, NQNO. Binding of these inhibitors required their addition to the complex before lipid used to facilitate crystallization. The similar binding of NQNO and TDS as axial ligands to heme c(n) implies that this heme utilizes plastoquinone as a natural ligand, thus defining an electron transfer complex consisting of hemes b(n), c(n), and PQ, and the pathway of n-side reduction of the PQ pool. The NQNO binding site explains several effects associated with its inhibitory action: the negative shift in heme c(n) midpoint potential, the increased amplitude of light-induced heme b(n) reduction, and an altered EPR spectrum attributed to interaction between hemes c(n) and b(n). A decreased extent of heme c(n) reduction by reduced ferredoxin in the presence of NQNO allows observation of the heme c(n) Soret band in a chemical difference spectrum.

The pig is a useful model for the heterogeneity of the mammalian immune system and has also recently received attention as a possible source of organs for human transplantation. Here we report a detailed analysis of porcine lymphocyte phenotypes. Peripheral blood alpha beta T cells consisted of four subsets (CD4+8-, CD4+8lo, CD4-8lo and CD4-8hi) and gamma delta T cells of three (CD2-4-8-, CD2+4-8lo and CD2+4-8-). There were in addition, a large proportion of non-T-non-B lymphocytes with CD2+3-4-8lo surface immunoglobulin-negative phenotype containing natural killer (NK) activity. A striking observation was the relatively low frequency of alpha beta T cells in the blood of young pigs. Similar phenotypes were also identified in the cells from peripheral lymphoid tissues, though the proportions of the gamma delta T cells and the non-T-non-B lymphocytes in the lymph nodes and tonsil were much lower and the majority of the gamma delta T cells in the lymphoid tissues bore CD2 and or CD8. In thymus, the small thymocytes were predominantly CD3-4+8+ while the mature large thymocytes displayed phenotypes similar to those of peripheral T cells. Thus this work has directly defined porcine alpha beta and gamma delta T cells demonstrated the T-cell nature of the unique CD4+8+ subset of peripheral lymphocytes, revealed the high heterogeneity of the CD8+ cells, and established the phenotype of NK cells. The functional properties of these defined porcine lymphocyte subsets can now be experimentally determined in health and disease.

Plant growth is severely affected by toxic concentrations of heavy metals. On characterizing the heavy metal-induced signalling pathways, the effects of cadmium (CdCl2) and copper (CuCl2) on MBP (myelin basic protein) kinase activities in Oryza sativa L. cv. TNG67 were analysed and it was found that Cd2+-induced 42 kDa MBP kinase has the characteristics of a mitogen-activated protein (MAP) kinase. This study confirmed that the 42 kDa kinase-active band contains, at least, the activities of OsMPK3 and OsMPK6. Then, the heavy metal signal transduction pathways leading to MAP kinase activation in rice roots were examined. Pretreatment with sodium benzoate, a hydroxyl radical scavenger, attenuated Cd2+- or Cu2+-induced MAP kinase activation. The Cd2+-, but not Cu2+-, induced MAP kinase activities were suppressed by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and Cd2+ induced NADPH oxidase-like activities, suggesting that NADPH oxidases may be involved in Cd2+-induced MAP kinase activation. Using a Ca2+ indicator, it was demonstrated that Cd2+ and Cu2+ induce Ca2+ accumulation in rice roots. The Cd2+- and Cu2+-induced MAP kinase activation required the involvement of Ca2+-dependent protein kinase (CDPK) and phosphatidylinositol 3-kinase (PI3 kinase) as shown by the inhibitory effect of a CDPK antagonist, W7, and a PI3 kinase inhibitor, wortmannin, respectively. Furthermore, bongkrekic acid (BK), a mitochondrial permeability transition pore opening blocker, suppressed Cd2+-, but not Cu2+-, induced MAP kinase activation, indicating that Cd2+-induced MAP kinase activities are dependent on the functional state of mitochondria. Collectively, these findings imply that Cd2+ and Cu2+ may induce MAP kinase activation through distinct signalling pathways. Moreover, it was found that the 42 kDa MAP kinase activities are higher in Cd-tolerant cultivars than in Cd-sensitive cultivars. Therefore, the Cd-induced 42 kDa MAP kinase activation may confer Cd tolerance in rice plants.

The action of ethosuximide (ETX) on Na+, K+, and Ca2+ currents and on tonic and burst-firing patterns was investigated in rat and cat thalamic neurons in vitro by using patch and sharp microelectrode recordings. In thalamocortical (TC) neurons of the rat dorsal lateral geniculate nucleus (LGN), ETX (0.75-1 mM) decreased the noninactivating Na+ current, INaP, by 60% but had no effect on the transient Na+ current. In TC neurons of the rat and cat LGN, the whole-cell transient outward current was not affected by ETX (up to 1 mM), but the sustained outward current was decreased by 39% at 20 mV in the presence of ETX (0.25-0.5 mM): this reduction was not observed in a low Ca2+ (0.5 mM) and high Mg2+ (8 mM) medium or in the presence of Ni2+ (1 mM) and Cd2+ (100 microM). In addition, ETX (up to 1 mM) had no effect on the low-threshold Ca2+ current, IT, of TC neurons of the rat ventrobasal (VB) thalamus and LGN and in neurons of the rat nucleus reticularis thalami nor on the high-threshold Ca2+ current in TC neurons of the rat LGN. Sharp microelectrode recordings in TC neurons of the rat and cat LGN and VB showed that ETX did not change the resting membrane potential but increased the apparent input resistance at potentials greater than -60 mV, resulting in an increase in tonic firing. In contrast, ETX decreased the number of action potentials in the burst evoked by a low-threshold Ca2+ potential. The frequency of the remaining action potentials in a burst also was decreased, whereas the latency of the first action potential was increased. Similar effects were observed on the burst firing evoked during intrinsic delta oscillations. These results indicate an action of ETX on INaP and on the Ca2+-activated K+ current, which explains the decrease in burst firing and the increase in tonic firing, and, together with the lack of action on low- and high-threshold Ca2+ currents, the results cast doubts on the hypothesis that a reduction of IT in thalamic neurons underlies the therapeutic action of this anti-absence medicine.

Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 45Ca2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45Ca2+ uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd2+, Ni2+, and Mg2+. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.

Patients with the X-linked lymphoproliferative disorder (XLPD) are unable to control Epstein-Barr virus (EBV)-induced infections and lymphoproliferation. This disease is caused by a deficit of SAP, an adapter protein involved in the signal transduction of several cell surface receptors of the CD2 superfamily. One of these receptors, called 2B4, is expressed on NK cells, cytotoxic T cells and myeloid cells and activates NK cell cytotoxicity. Here we show that XLPD patients have a defect of 2B4 receptor-mediated NK cell cytotoxicity. This defect may contribute to the pathogenesis of XLPD by reducing NK cell lysis of EBV-infected B cells.

In contrast to cyclin D1 nulls (cD1-/-), mice without cyclin D2 (cD2-/-) lack cerebellar stellate interneurons; the reason for this is unknown. In the present study in cortex, we found a disproportionate loss of parvalbumin (PV) interneurons in cD2-/- mice. This selective reduction in PV subtypes was associated with reduced frequency of GABA-mediated inhibitory postsynaptic currents in pyramidal neurons, as measured by voltage-clamp recordings, and increased cortical sharp activity in the EEGs of awake-behaving cD2-/- mice. Cell cycle regulation was examined in the medial ganglionic eminence (MGE), the major source of PV interneurons in mouse brain, and differences between cD2-/- and cD1-/- suggested that cD2 promotes subventricular zone (SVZ) divisions, exerting a stronger inhibitory influence on the p27 Cdk-inhibitor (Cdkn1b) to delay cell cycle exit of progenitors. We propose that cD2 promotes transit-amplifying divisions in the SVZ and that these ensure proper output of at least a subset of PV interneurons.

Signaling via the alpha-beta T cell Ag receptor (Ti)-CD3 complex is a complicated event that implicates several protein kinases, most notably protein kinase C (PKC). We have recently identified a serine kinase in T lymphocytes with the following characteristics: molecular mass 43 kDa, in vitro substrate affinity for microtubule associated protein 2 (MAP-2) with a preference for Mn2+ during the catalytic reaction, and elution from DEAE resin over a salt range 100 to 200 mM NaCl. This kinase is activated in a rapidly reversible fashion during ligation of CD3/Ti by a process which involves prior phosphorylation; in vitro exposure of activated 43-kDa MAP-2 kinase (MAP-K) to an immobilized phosphatase abrogated its kinase activity. We now show that a MAP-2K response could also be obtained during treatment with mAb to Ti and the specific PKC agonist, PMA. Although the kinetics of the former response was rapidly reversible, PMA elicited a more prolonged response. The dose responsiveness for PMA was similar to the requirements for PKC activation in intact lymphocytes. Moreover, as with PKC, we found that the CD3-induced MAP-2K response could be further enhanced by using a second layer cross-linking antibody. The specificity of CD3/Ti in the Jurkat cell response is demonstrated by the fact that OKT-11(CD2) and anti-CD4 mAb did not stimulate a MAP-2K response. It was also not possible to elicit a response in a Jurkat cell mutant that lacks surface expression of CD3 and Ti. The specificity of PKC in these events was further explored with the cell permeant diacylglycerol, 1-oleoyl-2-acetylglycerol, and the nonagonist phorbol ester, 4 alpha-phorbol 12,13-didecanoate: whereas the former was an effective inducer of the MAP-2K response, the latter failed to yield any stimulation. Prior exposure of Jurkat cells to 100 mM PMA for 24 h eliminated greater than 60% of the MAP-2K response during anti-CD3 treatment. This response could also be inhibited in dose-dependent fashion by prior treatment of Jurkat cells with the potent PKC inhibitor 1-(5-isoquinolinesulfonyl) 2-methylpiperazine dihydrochloride. Although a Ca2(+)-ionophore failed to synergize with PMA at inducing a MAP-2K response, depletion of extracellular Ca2+ by EGTA abrogated anti-CD3 responsiveness. The events culminating in MAP-2K activation were slightly inhibited in the presence of cholera toxin but not pertussis toxin.(ABSTRACT TRUNCATED AT 400 WORDS)

Precursor tRNA (ptRNA) substrates carrying a single Rp or Sp-phosphorothioate modification at the RNase P cleavage site were used as tools to study the cleavage mechanism of RNase P RNA from Bacillus subtilis. Both the Sp and the Rp-diastereomer reduced the rate of processing at least 10(4)-fold under conditions where the chemical step is essentially rate-limiting. Neither the Rp nor the Sp-phosphorothioate modification affected ptRNA ground state binding to B. subtilis RNase P RNA. Processing of the Rp-diastereomeric ptRNA could be restored in the presence of Mn2+or Cd2+, demonstrating direct metal ion coordination to the pro -Rp oxygen during catalysis. With Cd2+, processing required the presence of another metal ion, such as Ca2+or Mg2+, to mediate substrate binding. This is in contrast to Escherichia coli RNase P RNA, which promotes cleavage of Rp-diastereomeric ptRNA in the presence of Cd2+as the sole divalent metal ion. Analysis of [Cd2+]-dependent processing of the Rp-diastereomeric substrate by B. subtilis RNase P RNA was consistent with the involvement of at least two metal ions in catalysis. The presence of two catalytic metal ion binding sites is also supported by the inhibition mode of Ca2+on cleavage of unmodified ptRNA. In the presence of an Sp-phosphorothioate modification at the scissile bond, neither Mn2+nor Cd2+were able to restore significant cleavage at this location. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Unaffected ground state binding of the Sp-diastereomeric ptRNA but a>>/=10(4)-fold reduced hydrolysis rate may indicate a crucial role of the pro -Sp oxygen in transition state stabilization or may be attributed to steric exclusion of catalytic metal ions. Based on our comparative analyses of B. subtilis and E. coli RNase P RNA, each representing the main structural subtypes of bacterial RNase P RNA, common features in terms of active site constraints and role of catalytic metal ions can now be formulated for bacterial RNase P RNAs. On the other hand, substantial and unexpected differences with respect to the overall metal ion requirements and tRNA binding modes have been observed for the two catalytic RNAs.

BACKGROUND

The invasion and destruction of nonnecrotic muscle fibers by CD8+ cytotoxic T cells is considered a hallmark of polymyositis. In the cases of polymyositis reported so far, the autoinvasive CD8+ T cells expressed the common form of T-cell receptor for the recognition of antigen, the so-called alpha/beta T-cell receptor. We describe a 69-year-old man with polymyositis mediated by CD4-, CD8- T cells expressing the recently discovered, uncommon gamma/delta T-cell receptor.

METHODS

We used immunofluorescence or immunoperoxidase techniques to study frozen sections of muscle from our patient, who had mild weakness of cervical and proximal limb muscles, and from control patients with polymyositis, inclusion-body myositis, dermatomyositis, or granulomatous myopathy with monoclonal antibodies against T-cell-related antigens (<em>CD2</em>, CD3, CD4, CD8, and gamma/delta T-cell receptor), B cells (<em>CD2</em>2), major histocompatibility complex (MHC) and MHC-related antigens (MHC Class I, CD1a, CD1b, and CD1c), and the 65-kd heat-shock protein. The membrane contacts between the autoinvasive cells and the sarcolemma were investigated by electron microscopy.

RESULTS

In the patient described here, but not in 28 others with inflammatory myopathies, myriad gamma/delta T cells surrounded and invaded nonnecrotic muscle fibers. All muscle fibers were highly reactive for MHC Class I antigen and the 65-kd heat-shock protein. Treatment with prednisone improved the clinical and histologic findings.

CONCLUSIONS

Polymyositis can be mediated by gamma/delta T cells. This new form of polymyositis appears to be highly responsive to steroids.

The purpose of this study was to characterize excitation-contraction (e-c) coupling in myotubes for comparison with e-c coupling of adult skeletal muscle. The whole cell configuration of the patch clamp technique was used in conjunction with the calcium indicator dye Fluo-3 to study the calcium transients and slow calcium currents elicited by voltage clamp pulses in cultured myotubes obtained from neonatal mice. Cells were held at -80 mV and stimulated with 15-20 ms test depolarizations preceded and followed by voltage steps designed to isolate the slow calcium current. The slow calcium current had a threshold for activation of about 0 mV; the peak amplitude of the current reached a maximum at 30 to 40 mV a and then declined for still stronger depolarizations. The calcium transient had a threshold of about -10 mV, and its amplitude increased as a sigmoidal function of test potential and did not decrease again even for test depolarizations sufficiently strong >> or = 50 mV) that the amplitude of the slow calcium current became very small. Thus, the slow calcium current in myotubes appears to have a negligible role in the process of depolarization-induced release of intracellular calcium and this process in myotubes is essentially like that in adult skeletal muscle. After repolarization, however, the decay of the calcium transient in myotubes was very slow (hundreds of ms) compared to adult muscle, particularly after strong depolarizations that triggered larger calcium transients. Moreover, when cells were repolarized after strong depolarizations, the transient typically continued to increase slowly for up to several tens of ms before the onset of decay. This continued increase after repolarization was abolished by the addition of 5 mM BAPTA to the patch pipette although the rapid depolarization-induced release was not, suggesting that the slow increase might be a regenerative response triggered by the depolarization-induced release of calcium. The addition of either 0.5 mM Cd2+ + 0.1 mM La3+ or the dihydropyridine (+)-PN 200-110 (1 microM) reduced the amplitude of the calcium transient by mechanisms that appeared to be unrelated to the block of current that these agents produce. In the majority of cells, the decay of the transient was accelerated by the addition of the heavy metals or the dihydropyridine, consistent with the idea that the removal system becomes saturated for large calcium releases and becomes more efficient when the size of the release is reduced.

We initiated a clinical trial of nonmyeloablative haploidentical stem-cell transplantation (SCT) using MEDI-507, an immunoglobulin-G1 monoclonal anti-CD2 antibody. The trial was based on a preclinical major histocompatibility complex-mismatched bone marrow transplant model in which graft-versus-host disease (GVHD) was prevented and mixed chimerism as a platform for adoptive cellular immunotherapy was reliably induced. Twelve patients (three cohorts of four patients each) received cyclophosphamide, MEDI-507, and haploidentical unmanipulated bone marrow (n=8) or ex vivo T-cell-depleted peripheral blood stem cells (n=4) for chemorefractory hematologic malignancy. A two-dose regimen and schedule modifications of MEDI-507 were undertaken because of graft loss in the first cohort of four patients and GVHD in the second cohort. With ex vivo T-cell-depleted peripheral blood SCT, mixed chimerism occurred in all four patients without GVHD. Two patients, however, subsequently lost their grafts. Nonmyeloablative preparative therapy with MEDI-507 and haploidentical SCT have led to the reliable induction of at least transient mixed chimerism as a potential platform for adoptive cellular immunotherapy.

Preemptive lamivudine in lymphoma patients undergoing intensive chemotherapy can effectively prevent chemotherapy-related HBV reactivation. Nevertheless, the safety profile after withdrawal of lamivudine and the impact of rituximab-containing chemotherapy on HBV reactivation has not been defined. To illustrate the necessity of prolonged surveillance after cessation of preemptive lamivudine in lymphoma patients treated with rituximab and chemotherapy, four patients with B-cell NHL carrying HBV received rituximab plus CHOP. Preemptive lamivudine therapy was administered 1 week before chemotherapy until 4 weeks after completion of chemotherapy. Serial serum alanine aminotransferase (ALT), total bilirubin, and HBV-DNA levels were prospectively monitored in three patients. The fourth patient was closely monitored for ALT. The HBV DNA was checked after development of clinical overt hepatitis. The peripheral blood CD2CD2+ lymphocytes were totally depleted when HBV DNA started to increase. Delayed HBV reactivation can occur in lymphoma patients receiving R+CHOP after withdrawal of preemptive lamivudine. More protracted lamivudine therapy may be an alternative to close monitoring following chemotherapy, and further studies are needed to define optimal duration of lamivudine therapy.

We have demonstrated that endothelial cells (EC) augment IL-2 production by PHA-stimulated PBMC or purified CD4+ T cells and that the increase is apparent both in the amount of soluble IL-2 secreted and in the level of specific mRNA detectable by Northern blot hybridization. The ability of EC to affect levels of IL-2 cannot be reproduced by soluble factors, including the cytokines IL-1, IL-6, IFN-gamma, or TNF, conditioned medium from resting EC or IL-1, IFN-gamma- or TNF-treated EC, or from resting PBMC + EC cultures. Separation of the EC and PBMC by a Transwell membrane demonstrated that cell contact was required for augmentation of IL-2 synthesis and that this effect was unlikely to be mediated by a short-lived soluble signal. The cell-cell interaction required the ligand pair CD2/LFA-3, since augmentation could be inhibited by antibodies to these structures. Antibodies to ICAM-1, LFA-1, CD4, and MHC class II were without effect. A contact-dependent pathway involving CD2/LFA-3 interactions also may be used by EC to augment IL-2 production from T cells stimulated more specifically through the TCR/CD3 complex with antibody OKT3. This pathway provides a proliferative advantage to T cells stimulated with OKT3 in the presence of EC and may also be involved in the proliferative response of resting T cells to allogeneic class II MHC-expressing EC. We propose that EC augmentation of T cell IL-2 synthesis may be critical in the ability of EC to elicit primary T cell antigen responses and may have consequences for the development of localized cell-mediated immune reactions.