Aggressive natural killer-cell leukemia (ANKL) is a rare form of large granular lymphocyte leukemia, which is characterized by a systemic proliferation of NK cells. The clinical features of 22 ANKL cases were analyzed. Hepatomegaly (64%), splenomegaly (55%) and lymphadenopathy (41%) were also frequently observed. Leukemic cells were identified as CD1-, <em>CD2</em>+, surface CD3-, CD4-, CD5-, CD7+, CD8+/-, CD10-, CD11b+/-, CD13-, CD16+, CD19-, <em>CD2</em>0-, <em>CD2</em>5-, CD33(-), CD34-, CD38+, CD56+, CD122+, HLA-DR+ and TCR-. Two of the 16 cases examined for CD57 were positive and three of the seven cases examined for cytoplasmic CD3. Epstein-Barr virus was detected in the tumor cells of 11 of the 13 cases examined. No common cytogenetic abnormalities were identified and 6q anomaly was detected in only one. Three of 13 patients treated with chemotherapy containing anthracycline/anthraquinone attained complete remission, in contrast to none of the eight who were treated with regimens without anthracycline. Although the overall prognosis was poor with a median survival of 58 days, those who attained remission showed better prognosis (P=0.005). These findings suggest that ANKL is an entity of mature cytotoxic NK-cell neoplasms with distinct phenotype and disease presentations. Intensive treatment for ANKL may result in a better prognosis.

The czcR gene, one of the two control genes responsible for induction of resistance to Co2+, Zn2+, and Cd2+ (czc system) in the Alcaligenes eutrophus plasmid pMOL30, was cloned and characterized. The 1,376-bp sequence upstream of the czcCBAD structural genes encodes a 41.4-kDa protein, the czcR gene product, transcribed in the opposite direction of that of the czcCBAD genes. The putative CzcR polypeptide (355 amino acid residues) contains 11 cysteine and 14 histidine residues which might form metal cation-binding sites. A czcC::lacZ reporter gene translational fusion was constructed, inserted into plasmid pMOL30 in A. eutrophus, and expressed under the control of CzcR. Zn2+, Co2+, and Cd2+, as well as Ni2+, Cu2+, Hg2+, and Mn2+ and even Al3+, served as inducers of beta-galactosidase activity. Besides the CzcR protein, the membrane-bound CzcD protein was essential for induction of czc. The CzcR and CzcD proteins display no sequence similarity to two-component regulatory systems of a sensor and a response activator type; however, CzcD has 34% identity with the ZRC-1 protein, which mediates zinc resistance in Saccharomyces cerevisiae (A. Kamizomo, M. Nishizawa, Y. Teranishi, K. Murata, and A. Kimura, Mol. Gen. Genet. 219:161-167, 1989).

To gain insight into the secondary structure of the ion conduction pathway of a voltage-gated K+ channel, we used sulfhydryl-specific reagents of different diameters to probe amino acid side-chain accessibilities in the pore of the channel after cysteine-substitution mutagenesis. We identified five positions at which modified amino acid side chains are accessible from the aqueous lumen of the external channel vestibule. Covalent coupling of the 2-trimethylammonium-thioethyl group to cysteine thiols leads to position-dependent current reduction, suggesting a gradual narrowing of the pore. The fact that the modified side chains of two adjacent amino acids are reactive is not compatible with the ion conduction pathway forming a regular beta-pleated sheet at these positions. The smaller thiol reagent Cd2+ reacts with modified side chains that are also accessible to the larger (2-trimethylammoniumethyl)methanethiosulfate (MTSET) [corrected]. Our results imply that the outer vestibule of a potassium-selective ion channel narrows over a short distance of three amino acids near a position where a regular beta-structure is unlikely.

Metallothioneins (MTs) are low molecular weight, heavy metal binding proteins unique in their high cysteine content and high affinity for Zn2+, Cd2+, Hg2+, Ag2+ and Cu2+ (refs 1--3). The synthesis of MTs is induced by zinc or cadmium in the liver and kidney and in cultured cells. More recently MT induction by the steroid hormone dexamethasone (Dex) has been demonstrated in HeLa cells and adrenalectomized rats. Because glucocorticoid hormones lead to an intracellular accumulation of zinc, the question arises of whether the induction of MT gene expression by steroids is a 'primary induction response' (ref. 18), or due to elevated intracellular Zn2+. The glucocorticoid-induced transport of Zn2+ is dependent on concurrent protein synthesis. We now show that, in contrast to glucocorticoid-stimulated Zn2+ transport, the Zn2+ and Dex induction of translatable MT-mRNA is independent of concomitant protein synthesis but not RNA synthesis; that is, MT induction by either agent is a primary induction response.

Monoclonal antibodies against the CD3 antigen and certain lectins can induce interleukin 2 dependent antigen-specific T cell clones to mediate non-antigen specific cytotoxicity. On the basis of this observation, we predicted that it may be possible to identify cytotoxic T lymphocytes (CTL) in peripheral blood without knowing the antigen specificity of these in vivo primed CTL. By using this strategy, peripheral blood lymphocytes were separated into low and high-density fractions on Percoll gradients and were tested for cytotoxic activity in the presence or absence of concanavalin A (Con A) or anti-Leu-4 antibody. Lectin-dependent cellular cytotoxicity (LDCC) and anti-CD3 induced cytotoxicity against both natural killer (NK)-insensitive and NK-sensitive targets were exclusively mediated by low-density CD3+ T lymphocytes. Additional studies indicated that low-density CD3+ T lymphocytes co-expressing Leu-7 antigen preferentially mediated this activity, although in some individuals, significant activity was also observed in the low-density T cells lacking Leu-7. In contrast, high-density CD3+ T lymphocytes and CD16+ (Leu-11+) NK cells (both Leu-7 and Leu-7+) did not mediate nonantigen-specific cytotoxicity under these conditions. The finding that NK cell-mediated cytotoxicity was unaffected by these lectins refutes the hypothesis that lectin-dependent cellular cytotoxicity is simply a result of effector and target agglutination. T cell-mediated cytotoxicity was both lectin and antibody specific. Phytohemagglutinin, Con A, and pokeweed mitogen induced cytolytic activity in the Leu-7+ T cells, whereas wheat germ agglutinin did not. Of the antibodies against T cell-associated differentiation antigens (anti-Leu-2,3,4, and 5), only anti-Leu-4 induced cytotoxicity. This anti-CD3-induced cytotoxicity was essentially completely inhibited by the presence of anti-LFA-1 or anti-CD2 monoclonal antibodies, implicating these molecules in the triggering process. A proportion of the CD3+, Leu-7+ CTL expressed HLA-DR antigens, indicating possible in vivo activation. Because previous clinical studies have indicated that lymphocytes with this phenotype may be elevated in clinical situations associated with immunosuppression and chronic viral infection, this unique subset of CD3+ T lymphocytes may represent a population of in vivo primed CTL possibly against viral antigens.

Human intraepithelial lymphocytes (IEL) are CD8+ T cells located between intestinal epithelial cells, capable of only minimal proliferation to mitogens but brisk proliferation to mitogens combined with sheep red blood cells. This study examines this differential response of IEL. Both IEL and CD8+ T lymphocytes from the peripheral blood are predominantly CD2+, CD3+, CD4-, CD5+, CD8+, and express the alpha beta subunits of the T-cell receptor. Human IEL express the same densities of the CD2, CD3, and CD8 antigens but a lower density of the CD5 antigen than do peripheral blood CD8+ T cells. The proliferation of IEL is significantly less than that of peripheral blood CD8+ T lymphocytes in response to phytohemagglutinin, to concanavalin A, or to anti-CD3 antibody bound to Sepharose (p less than 0.05). Supplementing IEL with interleukin-1, interleukin-2, or autologous peripheral blood macrophages does not completely reconstitute the proliferative response of IEL to these stimuli. Rather, the low proliferation of IEL to these stimuli is due to incomplete activation, as demonstrated by the low percentage of CD2CD2CD2 receptor such as the combination of anti-T11(2) and anti-T11(3) antibodies or mitogen and sheep red blood cells. The sheep red blood cells enhance the mitogen-induced response of IEL by augmenting events of activation, both interleukin-2 production and interleukin-2 receptor expression. Thus, IEL represent an unusual compartment of CD2+, CD3+ T lymphocytes that are activated more completely by stimuli of the CD2 receptor than by stimuli of the CD3 receptor or by T-cell mitogens.

Other investigators have previously reported that TNF has been induced from macrophages by bacteria and, more recently, from NK cells by certain tumor cells. Sendai virus has also been reported to induce TNF from macrophages. We report here that an opportunistic fungi, Candida albicans, can also induce TNF, not only from human monocytes, but also from Percoll-fractionated large granular lymphocytes (LGL) which mediate NK function. Incubation of monocytes of LGL with C. albicans for 8 h was sufficient for detection of TNF release and peak induction was observed at 24 h. Induction of TNF from LGL did not require the participation of monocytes or T cells because treatment of the LGL with CD14 or CD15 to eliminate contaminating monocytes and CD3, CD4, or CD8 to eliminate contaminating T cells did not decrease the level of TNF produced from the treated LGL. Small T cells recovered from the denser fractions of the Percoll gradient had no ability to produce TNF, even when 10% monocytes were added to the T cells to provide accessory function. The phenotype of the TNF-producing LGL was CD2+, CD11+, CD16+, NKH1+, LEU7-. The TNF produced by both monocytes and LGL was neutralized by specific monoclonal and polyclonal anti-TNF but not by monoclonal antilymphotoxin. These results indicate that TNF production is a normal response of monocytes and LGL to stimulation by fungi such as C. albicans and that the release of TNF may be related to its ability to activate effector function to control Candida growth, which we have shown earlier for neutrophils with TNF.

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca2+/calmodulin-dependent cyclic nucleotide (AMP) phosphodiesterase activity in rat liver cytosol was investigated. The addition of Ca2+ (50 microM) and calmodulin 160 U/ml in the enzyme reaction mixture caused a significant increase in cyclic AMP phosphodiesterase activity. This increase was inhibited by the presence of regucalcin (0.5-3.0 microM); the inhibitory effect was complete at 1.0 microM. Regucalcin (1.0 microM) did not have an appreciable effect on basal activity without Ca2+ and calmodulin. The inhibitory effect of regucalcin was still evident even at several fold higher concentrations of calmodulin (160-480 U/ml). However, regucalcin (1.0 microM) did not inhibit Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in the presence of 100 and 200 microM Ca2+ added. Meanwhile, Cd2+ (25-100 microM)-induced decrease in Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity was not reversed by the presence of regucalcin (1.0 microM). The present results suggest that regucalcin can regulate Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity due to binding Ca2+ in liver cells.

Bacillus stearothermophilus T-6 produces an extracellular xylanase that was shown to optimally bleach pulp at pH 9 and 65 degrees C. The enzyme was purified and concentrated in a single adsorption step onto a cation exchanger and is made of a single polypeptide with an apparent M(r) of 43,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Xylanase T-6 is an endoxylanase that completely degrades xylan to xylose and xylobiose. The pIs of the purified protein were 9 and 7 under native and denaturing conditions, respectively. The optimum activity was at pH 6.5; however, 60% of the activity was still retained at pH 10. At 65 degrees C and pH 7, the enzyme was stable for more than 10 h; at 65 degrees C and pH 9, the half-life of the enzyme was approximately 6 h. Kinetic experiments at 55 degrees C gave Vmax and Km values of 288 U/mg and 1.63 mg/ml, respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by Zn2+, Cd2+, and Hg2+. Xylan completely protected the protein from inactivation by N-bromosuccinimide. The N-terminal sequence of the first 45 amino acids of the enzyme showed high homology with the N-terminal region of xylanase A from the alkalophilic Bacillus sp. strain C-125.

This study was designed to compare cytokine release in lamina propria lymphocytes (LPLs) and PBLs activated by Abs against CD3, CD2, and CD2CD2 ligation than PBL, as determined by release of IFN-gamma, IL-2, IL-4, and TNF-alpha. Moreover, CD2CD2>> CD3 dominance in cytokine induction. PHA-activated PBLs expressed more CD2 receptors than freshly isolated LPLs, but were less responsive to activation through CD2, indicating that postreceptor pathways in LPL may be adapted specifically to facilitate CD2-mediated cytokine secretion. Antiphosphotyrosine (APT) immunoblotting revealed inducible substrate phosphorylation during CD2, but not CD3, ligation in whole LPLs, as well as LPL-derived T cell lines. PBLs cocultured with an irradiated B cell line, Daudi, and IL-2 for 5 days attained a CD2-dominant cytokine-secretion pattern with identical tyrosine phosphorylation profiles as freshly isolated LPL or LPL T cell lines. PHA-activated PBLs did not produce these tyrosine phosphorylation profiles. This suggests that B lymphocytes in the lamina propria may contribute to a T cell differentiation process in which CD2, possibly by potentiation of its postreceptor pathway, becomes a prominent receptor for induction of cytokine secretion.

To study the in vivo role of IL-4-expressing cells, we developed a strategy to tag these cells, by generating mice in which one IL-4 allele was replaced with a cDNA encoding the human CD2 (huCD2) cell-surface molecule. Expression of the huCD2 reporter was, like IL-4, restricted to the appropriately polarized T helper 2 cells. However, most of the cells expressed only the IL-4 or the targeted allele. Analysis of the frequency of monoallelic versus biallelic expression suggests that the activation of each individual allele is regulated by a stochastic process whose probability can be augmented by increasing the strength of signal delivered through the TCR. Allele-specific activation may be a general feature of cytokine regulation that contributes to the functional diversity within T helper cell subpopulations.

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.

71 leukaemic patients having HLA-matched bone-marrow transplants (BMT) were randomised to receive whole marrow (group A) or marrow depleted of T cells by treatment with monoclonal antibodies (anti CD4-CD5-CD8, group B; anti CD2-CD5-CD7, group C) plus complement. All patients received cyclophosphamide and total body irradiation before transplantation and cyclosporin after BMT. Marrow treatment removed 97% of T cells (median) in group B and 99% in group C. Although both serious and mild graft-versus-host disease (GVHD) were reduced in T-cell depleted patients, graft failure and relapse were increased. Graft failure was caused by GVHD and transplant complications in the controls and by rejection and relapse in the T-cell depleted groups; relapse-free survival did not differ between the groups. Without better control of host immunity and of the residual leukaemia T-cell depletion of the marrow, BMT should not be pursued in standard-risk patients.

The frequency of circulating alloreactive human memory T cells correlates with allograft rejection. Memory T cells may be divided into effector memory (T(EM)) and central memory (T(CM)) cell subsets, but their specific roles in allograft rejection are unknown. We report that CD4+ T(EM) (CD45RO+ CCR7- CD62L-) can be adoptively transferred readily into C.B-17 SCID/bg mice and mediate the destruction of human endothelial cells (EC) in vascularized human skin grafts allogeneic to the T cell donor. In contrast, CD4+ T(CM) (CD45RO+ CCR7+ CD62L+) are inefficiently transferred and do not mediate EC injury. In vitro, CD4+ T(EM) secrete more IFN-gamma within 48 h in response to allogeneic ECs than do T(CM). In contrast, T(EM) and T(CM) secrete comparable amounts of IFN-gamma in response to allogeneic monocytes (Mo). In the same cultures, both T(EM) and T(CM) produce IL-2 and proliferate in response to IFN-gamma-treated allogeneic human EC or Mo, but T(CM) respond more vigorously in both assays. Blockade of LFA-3 strongly inhibits both IL-2 and IFN-gamma secretion by CD4+ T(EM) cultured with allogeneic EC but only minimally inhibits responses to allogeneic Mo. Blockade of CD80 and CD86 strongly inhibits IL-2 but not IFN-gamma production by in response to allogeneic EC or Mo. Transduction of EC to express B7-2 enhances allogeneic T(EM) production of IL-2 but not IFN-gamma. We conclude that human CD4+ T(EM) directly recognize and respond to allogeneic EC in vitro by secreting IFN-gamma and that this response depends on <em>CD2</em> but not <em>CD2</em>8. Consistent with EC activation of effector functions, human CD4+ T(EM) can mediate allogeneic EC injury in vivo.

Transforming growth factor (TGF)-beta added to cultures of highly purified human splenic B cells induced high levels of IgA synthesis in the presence of PWM and activated cloned CD4+ T cells. TGF-beta had no effect on IgM or IgG production. The induction of IgA synthesis by TGF-beta reflected IgA switching, because a strong induction of IgA production was also observed, when sIgA- B cells were cocultured with cloned activated CD4+ T cells in the presence of pokeweed mitogen. Resting CD4+ T cell clones or activated CD8+, TCR-gamma delta + CD4-,CD8- T cell clones failed to provide the co-stimulatory signal that in addition to TGF-beta and pokeweed mitogen was required for induction of IgA switching and IgA synthesis. mAb against CD4 or class II MHC molecules inhibited TGF-beta induced IgA synthesis, indicating that CD4-class II MHC interactions are required for productive T-B cell contacts resulting in IgA production. In contrast, anti-LFA-1, anti-CD2, and anti-class I MHC mAb were ineffective. TGF-beta failed to induce IgA synthesis by sIgA+ B cells under these culture conditions. Interestingly, induction of IgA production by sIgA- B cells required neutralization of TGF-beta activity by addition of the anti-TGF-beta mAb 1D11.1G 24 h after onset of the cultures. IgA production was prevented when the anti-TGF-beta mAb was added at the start of the cultures, indicating the specificity of the reaction. IgA synthesis was completely suppressed when TGF-beta was present during the total culture period of 11 days. These findings indicate that TGF-beta can act as a specific switch factor for IgA, provided it is only present at early stages of the cultures.

BACKGROUND

To obtain a better understanding of the mechanism underlying different modalities of immunotherapy, we investigated the types of tumor-infiltrating cells present at the tumor site, with special attention to the presence of macrophages.

METHODS

Frozen sections of carcinomas of the kidney, colon, breast, lung, ovary, and thyroid gland, as well as malignant melanoma were investigated with a panel of monoclonal antibodies against macrophage, T cell and NK cell associated antigens. Both type and pattern of the tumor-infiltrating cells were analyzed.

RESULTS

All tumor-infiltrating cells accumulated preferentially in the stromal bands between tumor cells. In all types of tumor, CD11c+, CD14+, CD68+ and alpha-naphthyl-acetate-esterase positive monocytes/macrophages accounted for most tumor-infiltrating cells. Next in frequency were T lymphocytes (<em>CD2</em>+, CD3+, TCR alpha beta +). Only a few B lymphocytes (<em>CD2</em>2+), and T cells expressing the T cell receptor gamma delta (TCR gamma delta) were found. Hardly any lymphoid cells with an NK phenotype (CD3-, CD56+) were present in the tumors studied. Large numbers of CD16+ cells were found, which could be identified as macrophages on the basis of their morphology, positive staining with a panel of monocyte/macrophage markers, and the results of double staining with CD11c.

CONCLUSIONS

We have demonstrated the presence of a large number of macrophages in the cellular infiltrates of several types of tumors. The largest numbers of CD16+ macrophages were found in renal cancer, melanoma, and colonic-carcinoma. These are the tumors that are most susceptible to immunotherapy with lymphokine activated killer cells, suggesting that these CD16+ macrophages may be involved in antitumor cytotoxicity. Furthermore, these findings suggest that new strategies of immunotherapy aimed at the use of macrophages present in many tumors could be developed.

The activation requirements for antigen-dependent proliferation of CD4+ T cells are well documented, while the events leading to the inactivation phase are poorly understood. Here, we tested the hypothesis that the lymphocyte-activation gene 3 (LAG-3), a second major histocompatibility complex (MHC) class II ligand, plays a regulatory role in CD4+ T lymphocyte activation. CD4+ class II-restricted T cell clones were stimulated by their relevant antigen (hemagglutinin peptide or diphteria toxoid) and antigen-presenting cells with or without anti-LAG-3 monoclonal antibody (mAb). Kinetic studies were performed to monitor different activation parameters, including the measurement of thymidine incorporation, expression of activation antigens and cytokine secretion. Results showed that the time course from the initial time points up to the peak time point was not modified in the presence of anti-LAG-3 mAb. However, addition of these antibodies, either as whole IgG or as Fab fragments, led to increased thymidine incorporation values for late time points and, hence, to a shift in the decreasing proliferation curve. We also showed that expression of activation antigens, such as CD2CD2 mAb, immobilized anti-CD3 or anti-T cell receptor mAb were not altered by anti-LAG-3 mAb. The allogeneic proliferative response of a CD8+ T cell clone was also not affected. Overall, the present analysis reveals a modulating effect of anti-LAG-3 mAb, mediated specifically on antigen-dependent, MHC class II-restricted responses of CD4+ T cell lines. These results support the view that LAG-3/MHC class II interaction down-regulates antigen-dependent stimulation of CD4+ T lymphocytes.

Action potential firing rates are generally limited by the refractory period, which depends on the recovery from inactivation of voltage-gated Na channels. In cerebellar Purkinje neurons, the kinetics of Na channels appear specialized for rapid firing. Upon depolarization, an endogenous open-channel blocker rapidly terminates current flow but prevents binding of the "fast" inactivation gate. Upon repolarization, unbinding of the blocker produces "resurgent" Na current while allowing channels to recover rapidly. Because other cerebellar neurons, including granule cells, unipolar brush cells, and neurons of the cerebellar nuclei, also fire rapidly, we tested whether these cells might also express Na channels with resurgent kinetics. Neurons were acutely isolated from mice and rats, and TTX-sensitive Na currents were recorded under voltage clamp. Unlike Purkinje cells, the other cerebellar neurons produced only tiny resurgent currents in solutions optimized for voltage-clamping Na currents (50 mM Na+; Co2+ substitution for Ca2+). Under more physiological ionic conditions (155 mM Na+; 2 mM Ca2+ with 0.03 mM Cd2+), however, granule cells, unipolar brush cells, and cerebellar nuclear cells all produced robust resurgent currents. The increase in resurgent current, which was greater than predicted by the Goldman-Hodgkin-Katz equation, appeared to result from a combination of knock-off of open-channel blockers by permeating ions as well as relief of divalent block at negative potentials. These results indicate that resurgent current is typical of many cerebellar neurons and suggest that rapid open-channel block and unblock may be a widespread mechanism for restoration of Na channel availability in rapidly firing neurons.

1. Ganglion cells were dissociated from the enzyme-treated rat retina, identified with specific fluorescent labels, and maintained in vitro. Electrophysiological properties of solitary retinal ganglion cells were investigated with both conventional intracellular and patch-clamp recordings. Although comparable results were obtained for most measurements some important differences were noted. 2. The input resistance of solitary retinal ganglion cells was considerably higher when measured with 'giga-seal' suction pipettes than with conventional intracellular electrodes. Under current-clamp conditions with both intracellular and patch pipettes, these central mammalian neurones maintained resting potentials of about -60 mV and displayed action potentials followed by an after-hyperpolarization in response to small depolarizations. The membrane currents during this activity, analysed under voltage clamp with patch pipettes, consisted of five components: Na+ current (INa), Ca2+ current (ICa), and currents with properties similar to the delayed outward, the transient (A-type), and the Ca2+-activated K+ currents (IK, IA and IK(Ca), respectively). 3. Ionic substitution, pharmacological agents, and voltage-clamp experiments revealed that the regenerative currents were carried by both Na+ and Ca2+. 100 nM-1 microM-tetradotoxin (TTX) reversibly blocked the fast spikes carried by the presumptive INa, which under voltage-clamp analysis had classical Hodgkin-Huxley-type activation and inactivation. 4. Single-channel recordings of the Na+ current (iNa) permitted comparison of these 'microscopic' events with the 'macroscopic' whole-cell current (INa). The inactivation time constant (tau h) fitted to the averaged single-channel recordings of iNa in outside-out patches was slower than the tau h obtained during whole-cell recordings of INa. 5. In the presence of 1-40 microM-TTX and 20 mM-TEA, slow action potentials appeared in intracellular recordings and were probably mediated by Ca2+. The potentials were abrogated by 3 mM-Co2+ or 200 microM-Cd2+; conversely, increasing the extracellular Ca2+ concentration from 2.5 to 10-25 mM or substitution of 1 mM-Ba2+ for 2.5 mM-Ca2+ enhanced their amplitude. ICa was measured directly in whole-cell recordings with patch pipettes after blocking INa with extracellular 1 microM-TTX and K+ currents with intracellular 120-mM Cs+ and 20 mM-TEA. 6. During whole-cell recordings with patch electrodes, extracellular 20 mM-TEA suppressed IK and, to a lesser extent, IA. Extracellular 5 mM-4-AP or a pre-pulse of the membrane potential to -40 mV prior to stronger depolarization completely blocked IA.(ABSTRACT TRUNCATED AT 400 WORDS)

The mechanism of block of voltage-dependent Na+ channels by extracellular divalent cations was investigated in a quantitative comparison of two distinct Na+ channel subtypes incorporated into planar bilayers in the presence of batrachotoxin. External Ca2+ and other divalent cations induced a fast voltage-dependent block observed as a reduction in unitary current for tetrodotoxin-sensitive Na+ channels of rat skeletal muscle and tetrodotoxin-insensitive Na+ channels of canine heart ventricular muscle. Using a simple model of voltage-dependent binding to a single site, these two distinct Na+ channel subtypes exhibited virtually the same affinity and voltage dependence for fast block by Ca2+ and a number of other divalent cations. This group of divalent cations exhibited an affinity sequence of Co congruent to Ni greater than Mn greater than Ca greater than Mg greater than Sr greater than Ba, following an inverse correlation between binding affinity and ionic radius. The voltage dependence of fast Ca2+ block was essentially independent of CaCl2 concentration; however, at constant voltage the Ca2+ concentration dependence of fast block deviated from a Langmuir isotherm in the manner expected for an effect of negative surface charge. Titration curves for fast Ca2+ block were fit to a simplified model based on a single Ca2+ binding site and the Gouy-Chapman theory of surface charge. This model gave similar estimates of negative surface charge density in the vicinity of the Ca2+ blocking site for muscle and heart Na+ channels. In contrast to other divalent cations listed above, Cd2+ and Zn2+ are more potent blockers of heart Na+ channels than muscle Na+ channels. Cd2+ induced a fast, voltage-dependent block in both Na+ channel subtypes with a 46-fold higher affinity at 0 mV for heart (KB = 0.37 mM) vs. muscle (KB = 17 mM). Zn2+ induced a fast, voltage-dependent block of muscle Na+ channels with low affinity (KB = 7.5 mM at 0 mV). In contrast, micromolar Zn2+ induced brief closures of heart Na+ channels that were resolved as discrete substate events at the single-channel level with an apparent blocking affinity of KB = 0.067 mM at 0 mV, or 110-fold higher affinity for Zn2+ compared with the muscle channel. High-affinity block of the heart channel by Cd2+ and Zn2+ exhibited approximately the same voltage dependence (e-fold per 60 mV) as low affinity block of the muscle subtype (e-fold per 54 mV), suggesting that the block occurs at structurally analogous sites in the two Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

The CD44 inhibitor Lutheran [In(Lu)]-related p80 molecule has recently been shown to be identical to the Hermes-1 lymphocyte homing receptor and to the human Pgp-1 molecule. We have determined the effect of addition of CD44 antibodies to in vitro activation assays of PBMC. CD44 antibodies did not induce PBMC proliferation alone, but markedly enhanced PBMC proliferation induced by a mitogenic CD2 antibody pair or by CD3 antibody. CD44 antibody addition had no effect upon PBMC activation induced by PHA or tetanus toxoid. CD44 antibody enhancement of CD2 antibody-induced T cell activation was specific for mature T cells as thymocytes could not be activated in the presence of combinations of CD2 and CD44 antibodies. CD44 antibody enhancement of CD2-mediated T cell triggering occurred if CD44 antibody was placed either on monocytes or on T cells. In experiments with purified monocyte and T cell suspensions, CD44 antibodies A3D8 and A1G3 augmented CD2-mediated T cell activation by three mechanisms. First, CD44 antibody binding to monocytes induced monocyte IL-1 release, second, CD44 antibodies enhanced the adhesion of T cells and monocytes in CD2 antibody-stimulated cultures, and third, CD44 antibodies augmented T cell IL-2 production in response to CD2 antibodies. Thus, ligand binding to CD44 molecules on T cells and monocytes may regulate numerous events on both cell types that are important for T cell activation. Given that recent data suggest that the CD44 molecule may bind to specific ligands on endothelial cells (vascular addressin) and within the extracellular matrix (collagen, fibronectin), these data raise the possibility that binding of T cells to endothelial cells or extracellular matrix proteins may induce or up-regulate T cell activation in inflammatory sites.

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define 'S-phase promoting factor' (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

Cells of Escherichia coli strain B develop large intracellular vacuoles and exhibit an abnormally long lag phase when inoculated into a defined medium to glucose and salts containing 3 times 10-6 M Cd2+. Early in this lag, about 95% of the cells fail to form colonies when plated on nutrient broth-NaCl-agar. Prior to the initiation of proliferation, the morphology of these cells becomes normal. They regain viability in the absence of deoxyribonucleic acid replication. The rate and extent of growth are normal once proliferation begins. This reversible phenomenon of accommodation to a growth-inhibiting concentration of Cd2+ does not appear to result from a selection of mutant cells. Cells which are proliferating in the presence of Cd2+ accumulate the ion to a very high concentration. In membranes and 31% in the cytoplasm. In unaccommodated cells, the figures are 2%, 75%, and 23%, respectively. The activity of alkaline phosphatase, a zinc-metalloenzyme which is inhibited by cadmum and is located between cell wall and membrane, is not abnormally low in accommodated cells, suggesting that the cadmim is compartmentalized in these cells. Molecular sieve chromatography of cell extracts shows that the Cd2+ is associated with two classes of macromolecules. It appears that accommodation of E. coli to the presence of Cd2+ involves exclusion of the ion from the cell and reversal of damage caused by prior exposure to the ion.

Manganese transport regulator (MntR) is a member of the diphtheria toxin repressor (DtxR) family of transcription factors that is responsible for manganese homeostasis in Bacillus subtilis. Prior biophysical studies have focused on the metal-mediated DNA binding of MntR [Lieser, S. A., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2003) Biochemistry 42, 12634-12642], as well as metal stabilization of the MntR structure [Golynskiy, M. V., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2005) Biochemistry 44, 3380-3389], but only limited data on the metal-binding affinities for MntR are available. Herein, the metal-binding affinities of MntR were determined by using electron paramagnetic resonance (EPR) spectroscopy, as well as competition experiments with the fluorimetric dyes Fura-2 and Mag-fura-2. MntR was not capable of competing with Fura-2 for the binding of transition metal ions. Therefore, the metal-binding affinities and stoichiometries of Mag-fura-2 for Mn2+, Co2+, Ni2+, Zn2+, and Cd2+ were determined and utilized in MntR/Mag-fura-2 competition experiments. The measured Kd values for MntR metal binding are comparable to those reported for DtxR metal binding [Kd from 10(-)7 to 10(-4) M; D'Aquino, J. A., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 18408-18413], AntR [a homologue from Bacillus anthracis; Sen, K. I. et al. (2006) Biochemistry 45, 4295-4303], and generally follow the Irving-Williams series. Direct detection of the dinuclear Mn2+ site in MntR with EPR spectroscopy is presented, and the exchange interaction was determined, J = -0.2 cm-1. This value is lower in magnitude than most known dinuclear Mn2+ sites in proteins and synthetic complexes and is consistent with a dinuclear Mn2+ site with a longer Mn...Mn distance (4.4 A) observed in some of the available crystal structures. MntR is found to have a surprisingly low binding affinity (approximately 160 microM) for its cognate metal ion Mn2+. Moreover, the results of DNA binding studies in the presence of limiting metal ion concentrations were found to be consistent with the measured metal-binding constants. The metal-binding affinities of MntR reported here help to elucidate the regulatory mechanism of this metal-dependent transcription factor.