Histone deacetylase 6 (HDAC6) is a critical target for drug design because of its role in oncogenic transformation and cancer metastasis, and is unique among all histone deacetylases in that it contains tandem catalytic domains designated CD1 and CD2. We now report the crystal structures of CD2 from Homo sapiens HDAC6 and of CD1 and CD2 from Danio rerio HDAC6. We correlated these structures with activity measurements using 13 different substrates. The catalytic activity of CD2 from both species exhibited broad substrate specificity, whereas that of CD1 was highly specific for substrates bearing C-terminal acetyllysine residues. Crystal structures of substrate complexes yielded unprecedented snapshots of the catalytic mechanism. Additionally, crystal structures of complexes with eight different inhibitors, including belinostat and panobinostat (currently used in cancer chemotherapy), the macrocyclic tetrapeptide HC toxin, and the HDAC6-specific inhibitor N-hydroxy-4-(2-((2-hydroxyethyl)(phenyl)amino)-2-oxoethyl)benzamide, revealed surprising new insight regarding changes in Zn(2+) coordination and isozyme-specific inhibition.

In this study, we conducted synchrotron radiation Fourier transform infrared (IR) spectroscopy, potentiometric titration, and metal sorption experiments to characterize metal-cyanobacteria sorption reactions. Infrared spectra were collected with samples in solution for intact cyanobacterial filaments and separated exopolymeric sheath material to examine the deprotonation reactions of cell surface functional groups. The infrared spectra of intact cells sequentially titrated from pH 3.2 to 6.5 display an increase in peak intensity and area at 1400 cm(-1) corresponding to vibrational COO- frequencies from the formation of deprotonated carboxyl surface sites. Similarly, bulk acid-base titration of cyanobacterial filaments and sheath material indicates that the concentration of proton-active surface sites is higher on the cell wall compared to the overlying sheath. A three-site model provides an excellent fit to the titration curves of both intact cells and sheath material with corresponding pKa values of 4.7 +/- 0.4, 6.6 +/- 0.2, 9.2 +/- 0.3 and 4.8 +/- 0.3, 6.5 +/- 0.1, 8.7 +/- 0.2, respectively. Finally, Cu2+, Cd2+, and Pb2+ sorption experiments were conducted as a function of pH, and a site-specific surface complexation model was used to describe the metal sorption data. The modeling indicates that metal ions are partitioned between the exopolymer sheath and cell wall and that the carboxyl groups on the cyanobacterial cell wall are the dominant sink for metals at near neutral pH. These results demonstrate that the cyanobacterial surfaces are complex structures which contain distinct surface layers, each with unique molecular functional groups and metal binding properties.

The interaction between transition metals (Ag+, Cd2+ and Hg2+) and selenium (Se) in the bloodstream was studied in vitro by means of the HPLC--inductively coupled argon plasma-mass spectrometry (ICP MS) method. Transition metal ions and selenide (produced in vitro from selenite in the presence of glutathione) or sulfide (Na2S) formed a (metal-Se/S) complex, which then bound to a plasma protein, selenoprotein P (Sel P), to form a ternary complex, (metal-Se/S)-Sel P. The molar ratios of metals to Se were 1:1 for Hg/Se and Cd/Se, but either 1:1 or 2:1 for Ag/Se, depending on the ratio of their doses. The results indicate that the interaction between transition metals and Se occurs through the general mechanism, i.e., transition metal ions and selenide form the unit complex (metal-Se)n, and then the complex binds to selenoprotein P to form the ternary complex ¿(metal-Se)n¿m--seleno-protein P in the bloodstream.

BACKGROUND

Proteinuria is a major cause of progression in renal disease. The glomerular ultrafiltration barrier, containing highly differentiated podocytes, normally restricts protein access to the urine. Patients with urinary protein in the nephrotic range (>3.5 g daily) often have effaced podocyte foot-processes. Slit diaphragms span the gaps between foot processes as a barrier to macromolecules. Nephrin and podocin are slit-diaphragm proteins identified in families with congenital nephrotic syndromes. CD2-associated protein (CD2AP) is an adapter protein originally identified as a novel ligand interacting with the T-cell-adhesion protein CD2. CD2AP knockout (-/-) mice develop a congenital nephrotic syndrome with podocyte foot-process effacements and die at 6 weeks of age from renal failure. CD2AP localises to the slit diaphragm and links nephrin and podocin to phosphoinositide 3-OH kinase; this complex has cell-signalling properties.

BACKGROUND

The CD2AP +/- heterozygous mice developed by Jeong Kim and colleagues (Science 2003; 300: 1298-300) are haploinsufficient and develop glomerular changes at 9 months of age with a histological pattern similar to that in human focal segmental glomerulosclerosis. These researchers found that 2 of 30 African-American patients with idiopathic focal segmental glomerulosclerosis had a CD2AP mutation that ablated expression of one allele. WHERE NEXT? Further studies should address the normal distribution of the CD2AP heterozygous mutation in different ethnic populations, because the association with human idiopathic focal segmental glomerulosclerosis could be accidental. Decreased expression of CD2AP in podocytes of individuals with the CD2AP heterozygous mutation would help to understand how the haploinsufficiency translates into increased susceptibility to renal disease. Transfection of podocytes with mutated CD2AP or study of cultured podocytes from CD2AP +/- mice would provide further insight into whether the nephrin-podocin-CD2AP signal-transduction pathway is altered and leads to increased apoptosis of podocytes. Assuming that a decrease in CD2AP attenuates clearance of glomerular immune complexes, patients with other types of idiopathic glomerulonephritis should also have a CD2AP mutation. However, first studies looking at the most common form of glomerulonephritis, IgA nephropathy, have failed to show decreased renal CD2AP expression.

The mechanism of Cd2+ block of Ca2+ currents (ICa) was explored in squid neurons using whole-cell patch clamp. Control currents activated sigmoidally, more rapidly at more positive potentials, and did not inactivate significantly. External Cd2+ up to 250 microM reduced ICa reversibly. For small depolarizations, the current for a step of 10 ms increased to a maintained value, resembling the control; but for Vm greater than 0 mV, the increase was followed by a decrease, as Cd2+ block became greater. Final block was greater for larger depolarizations. At 0 mV the half-blocking concentration was 125 microM. Tail currents, measured as channels close, had an initial "hook" when recorded in Cd2+: currents increased transiently, then decreased. This suggests that Cd2+ escapes from some channels, which then conduct briefly before closing. Analysis of tail currents shows that Cd2+ does not slow channel closing. The data can be explained if Cd2+ is a permeant blocker of Ca2+ channels and if channels can close when occupied by Cd2+. Cd2+ permeates the channels, but binds transiently to a site in the pore, obstructing the passage of other ions (e.g., Ca2+). Dwell time depends on the transmembrane potential, becoming shorter for more negative internal potentials. A five-state model was used to simulate the steady-state and kinetic features. It combines a Hodgkin-Huxley type m2 gating scheme and a one-site Woodhull ionic blockage model for a permeant blocker and includes a closed blocked state. To fit the data, the binding site for Cd2+ had to be near the outer end of the pore, with a well depth of -12.2 RT, and with a barrier at each end of the pore. The model predicts that the Cd2+ entry rate is nearly voltage independent, but the exit rate is steeply voltage dependent (e-fold/17 mV). Analysis further suggests that the channel closes at a normal rate with Cd2+ in the pore.

In order to study a possible mechanism for rhythmic firing of dopaminergic (DA) neurons, intracellular recordings were obtained from 56 rhythmically firing DA neurons in the rat substantia nigra compacta (SNc), using in vitro slice preparations. In the presence of TTX, spontaneous oscillation of the membrane potential was induced in SNc DA neurons when the membrane potential was depolarized more positive from -60 to -40 mV. Each oscillation wave was characterized by a pacemaker-like slow depolarization (PLSD) followed by a relatively prompt repolarization. As the DC depolarization was increased from -60 to -40 mV, the oscillation frequency increased from 0.5 to 5 Hz, but the amplitude of the wave decreased. Of 17 neurons tested in the presence of TTX, the maximum amplitudes of the oscillation varied from 10-15 mV in 8 neurons and were less than 5 mV in 9 neurons. In those 9 neurons, an application of TEA greatly enhanced (up to 15 mV) the amplitude of oscillation. The oscillation ceased when the membrane was hyperpolarized more negative than -60 mV. At the membrane potential more negative than -60 mV in the presence of TTX an injection of a depolarizing current pulse could evoke PLSD which was an all-or-nothing regenerative spike potential. The rate of rise of the PLSD changed depending on the intensity of injected current pulses but their amplitude remained constant. Its time-to-peak was slow (up to 1400 ms), while the decay time was relatively brief (< 500 ms). The threshold membrane potential for evoking PLSD was -53.7 +/- 3.2 mV (n = 10). This was higher than the previously reputed threshold for low threshold Ca2+ spike (LTS) (< -60 mV) and lower than that for high threshold Ca2+ spike (HTS) >> -35 mV) in SNc DA neurons. Even at a holding potential of -45 mV, a depolarizing current pulse could trigger PLSD while LTS was completely inactivated. Cd2+ (0.4 mM) abolished the oscillation and PLSD without marked effects on the LTS (n = 6). A low Ca2+ and high Mg2+ Ringer's solution also abolished the oscillation and PLSD (n = 4). An intracellular injection of EGTA markedly prolonged the decay time course of PLSD characterized by a slow and a relatively fast falling phase (n = 5). This would suggest an involvement of Ca(2+)-dependent K+ conductance and/or Ca2+ dependent inactivation of Ca2+ conductance during repolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

It is demonstrated that acetylcholine released from cholinergic interneurons modulates the excitability of neostriatal projection neurons. Physostigmine and neostigmine increase input resistance (RN) and enhance evoked discharge of spiny projection neurons in a manner similar to muscarine. Muscarinic RN increase occurs in the whole subthreshold voltage range (-100 to -45 mV), remains in the presence of TTX and Cd2+, and can be blocked by the relatively selective M1,4 muscarinic receptor antagonist pirenzepine but not by M2 or M3 selective antagonists. Cs+ occludes muscarinic effects at potentials more negative than -80 mV. A Na+ reduction in the bath occludes muscarinic effects at potentials more positive than -70 mV. Thus, muscarinic effects involve different ionic conductances: inward rectifying and cationic. The relatively selective M2 receptor antagonist AF-DX 116 does not block muscarinic effects on the projection neuron but, surprisingly, has the ability to mimic agonistic actions increasing RN and firing. Both effects are blocked by pirenzepine. HPLC measurements of acetylcholine demonstrate that AF-DX 116 but not pirenzepine greatly increases endogenous acetylcholine release in brain slices. Therefore, the effects of the M2 antagonist on the projection neurons were attributable to autoreceptor block on cholinergic interneurons. These experiments show distinct opposite functions of muscarinic M1- and M2-type receptors in neostriatal output, i.e., the firing of projection neurons. The results suggest that the use of more selective antimuscarinics may be more profitable for the treatment of motor deficits.

Bradyrhizobium japonicum hypB encodes a protein containing an extremely histidine-rich region (24 histidine residues within a 39-amino-acid stretch) and guanine nucleotide-binding domains. The product of the hypB gene was overexpressed in Escherichia coli and purified by Ni(2+)-charged metal chelate affinity chromatography (MCAC) in a single step. In SDS/PAGE, HypB migrated at 38 kDa--slightly larger than the calculated molecular mass (32.8 kDa). Purified HypB has GTPase activity with a kcat of 0.18 min-1 and a Km for GTP of 7 microM, and it has dGTPase activity as well. HypB exists as a dimer of molecular mass 78 kDa in native solution as determined by fast protein liquid chromatography on Superose 12. It binds 9.0 +/- 0.14 divalent nickel ions per monomer (18 Ni2+ per dimer) with a Kd of 2.3 microM; it also binds Zn2+, Cu2+, Co2+, Cd2+, and Mn2+. In-frame deletion of the histidine-rich region (deletion of 38 amino acids including 23 histidine residues) resulted in a truncated HypB that did not bind to the MCAC column, whereas in-frame deletion of 14 amino acids including 8 histidine residues within HypB resulted in a truncated HypB that still bound to the column. The results indicate that the histidine residues within the histidine-rich region of HypB are involved in metal binding.

In this study we have used the expression of perforin to characterize subsets of porcine cytotoxic lymphocytes. Perforin positive lymphocytes expressed both CD2 and CD8alpha, most were small dense lymphocytes (SDL) and up to 90% were CD3 negative. However, the numbers of perforin positive T-cells increased with the age of the animal and their populations increased after specific antigen stimulation in vitro. The remaining perforin positive lymphocytes were large and granular and contained more CD3+CD5+CD6+ T-cells (-40%) of which a substantial proportion also co-expressed CD4. Perforin was expressed in subpopulations of both CD8alphaalpha and CD8alphabeta lymphocytes, but was not expressed in gammadelta T-cells or monocyte/macrophages. The perforin positive CD3- subset was phenotypically homogeneous and defined as CD5-CD6-CD8beta-CD16+CD11b+. This population had NK activity and expressed mRNA for the NK receptor NKG2D, and adaptors DAP10 and DAP12. Perforin positive T-cells (CD3+) could be divided into at least three subsets. The first subset was CD4-CD5+CD6+CD11b-CD16- most were small dense lymphocytes with cytotoxic T-cell activity but not all expressed CD8beta. The second subset was mainly observed in the large granular lymphocytes. Their phenotype was CD4+CD5+CD6+CD8beta+CD16-CD11b- and also showed functional CTL activity. Thus not all of double positive T-cells are memory helper T-cells. The third subset did not express the T-cell co-receptor CD6, but up to half of them expressed another T-cell co-receptor CD5. The majority of this subset expressed CD11b and CD16, thus the third perforin positive T-cell subset was CD3+CD4-CD5+CD6-CD8beta+/-CD11b+CD16+, and possessed MHC-unrestricted cytotoxicity and LAK activity.

Induction and maintenance of a state of T cell unresponsiveness to specific alloantigen would have significant implications for human organ transplantation. Using human histocompatibility leukocyte antigen DR7-specific helper T cell clones, we demonstrate that blockade of the B7 family of costimulatory molecules is sufficient to induce alloantigen-specific T cell clonal anergy. Anergized cells do not respond to alloantigen and a variety of costimulatory molecules, including B7-1, B7-2, intercellular adhesion molecule-1 (ICAM-1), and lymphocyte function-associated molecule (LFA)-3. However, after culture in exogenous interleukin (IL)-2 for at least 7 d, anergized cells can respond to alloantigen in the presence of LFA-3. LFA-3 costimulation subsequently restores responsiveness to alloantigen in the presence of previously insufficient costimulatory signals. Expression of CD2R epitope is downregulated on anergic cells and is restored after 7 d of IL-2 culture. The loss of the CD2R is temporally associated with the inability of anergized cells to respond to LFA-3. These results suggest that in addition to blockade of B7 family members, inhibition of CD2 and, potentially, other costimulatory pathways that might reverse anergy will be necessary to maintain prolonged alloantigen-specific tolerance.

To define the role of the CD2-lymphocyte function-associated antigen 3 (LFA-3) interaction in T-cell activation, we have expressed a cDNA encoding the human CD2 molecule in a murine antigen-specific T-cell hybridoma. Expression of the CD2 molecule greatly enhances T-cell responsiveness to antigen; this enhancement is inhibited by anti-CD2 and anti-LFA-3 monoclonal antibodies (mAbs). CD2+ hybridomas produce interleukin 2 in response to combinations of anti-CD2 mAbs 9.6 and 9-1 and, in the presence of mAb 9-1, to sheep erythrocytes or to the LFA-3 antigen. Furthermore, hybridomas expressing a mutant CD2 molecule that has lost mAb 9.6 binding do not exhibit the enhanced response to antigen or the ability to respond to LFA-3 plus mAb 9-1, but these hybridomas retain the ability to respond to combinations of anti-CD2 mAbs. The role of the CD2-LFA-3 interaction in T-cell activation and the potential for other physiologic ligands for CD2 are discussed.

There are two major subsets of gammadelta T cell in humans. Vgamma2Vdelta2 T cells predominate in the circulation and significantly expand in vivo during a variety of infectious diseases. Ags identified for the Vdelta2 T cells are nonpeptide phosphate, amine, and aminobisphosphonate compounds. In contrast, Vdelta1-encoded TCRs account for the vast majority of gammadelta T cells in tissues such as intestine and spleen. Some of these T cells recognize CD1c and MHC class I-related chain (MICA/B) molecules [correction]. These T cells are cytotoxic and use both perforin- and Fas-mediated cytotoxicity. A fundamental question is how these gammadelta T cells are activated during microbial exposure to carry out effector functions. In this study, we provide evidence for a mechanism by which Vdelta1 gammadelta T cells are activated by inflammatory cytokines in the context of the Vdelta1 TCR. Dendritic cells are necessary as accessory cells for microbial Ag-mediated Vdelta1 gammadelta T cell activation. Cytokine (IL-12), adhesion (LFA3/CD2, LFA1/ICAM1) and costimulatory (MHC class I-related chain (MICA/B) molecules/NK-activating receptor G2D) molecules play a significant role along with Vdelta1 TCR in this activation.

Detoxification of xenobiotic compounds and heavy metals is a pivotal capacity of organisms, in which glutathione (GSH) plays an important role. In plants, electrophilic herbicides are conjugated to the thiol group of GSH, and heavy metal ions form complexes as thiolates with GSH-derived phytochelatins (PCs). In both detoxification processes of plants, phytochelatin synthase (PCS) emerges as a key player. The enzyme is activated by heavy metal ions and catalyzes PC formation from GSH by transferring glutamylcysteinyl residues (gamma-EC) onto GSH. In this study with Arabidopsis, we show that PCS plays a role in the plant-specific catabolism of glutathione conjugates (GS-conjugates). In contrast to animals, breakdown of GS-conjugates in plants can be initiated by cleavage of the carboxyterminal glycine residue that leads to the generation of the corresponding gamma-EC-conjugate. We used the xenobiotic bimane in order to follow GS-conjugate turnover. Functional knockout of the two PCS of Arabidopsis, AtPCS1 and AtPCS2, revealed that AtPCS1 provides a major activity responsible for conversion of the fluorescent bimane-GS-conjugate (GS-bimane) into gamma-EC-bimane. AtPCS1 deficiency resulted in a gamma-EC-bimane deficiency. Transfection of PCS-deficient cells with AtPCS1 recovered gamma-EC-bimane levels. The level of the gamma-EC-bimane conjugate was enhanced several-fold in the presence of Cd2+ ions in the wild type, but not in the PCS-deficient double mutant, consistent with a PCS-catalyzed GS-conjugate turnover. Thus AtPCS1 has two cellular functions: mediating both heavy metal tolerance and GS-conjugate degradation.

Regulation of the cadA cadmium and zinc resistance determinant of Staphylococcus aureus plasmid pI258 was demonstrated by using gene fusions and direct measurements of transcription. In growth experiments, cells harboring the intact cadA operon were induced with different cations and challenged by an inhibitory concentration of ZnCl2, a substrate of the CadA resistance system. Uninduced cells did not grow for 8 h after Zn2+ addition, whereas induced cells grew in the presence Zn2+. Cd2+ was a strong inducer, and Bi3+ and Pb2+ also induced well; Co2+ and Zn2+ were weak inducers. A translational beta-lactamase fusion to the cadA gene showed the same induction specificity as that seen with growth experiments with the intact cadA operon. A short beta-lactamase transcriptional fusion to the cadC gene also showed the same pattern of induction, establishing that the cadC gene was not involved in regulation. In Northern (RNA) blot hybridization experiments, a cadmium-inducible, 2.6-kb, operon-length transcript was detected. Primer extension experiments determined that Cd(2+)-inducible transcription of the cadA operon begins at nucleotides 676 and 677 of the published sequence (G. Nucifora, L. Chu, T. K. Misra, and S. Silver, Proc. Natl. Acad. Sci. USA 86: 3544-3548, 1989).

A method has been developed that combines electrospray ionization mass spectrometry with pH control to provide analysis of metals in native or reconstituted metallothioneins. These metalloproteins cooperatively bind seven divalent metal ions, most commonly Zn2+ and Cd2+. Since the protein is denatured and metal ions are lost below pH3, the pH of the electrospray solution is critical to successful results. The metal-free apoprotein was detected with its most abundant ions in a charge state of 6+, while the folded metallothionein-metal complexes were observed with lower charge states. The retention of seven metals in the molecular ions detected is consistent with the hypothesis that metallothionein retains its conformation in the gas phase. This mass spectrometric technique can be used to determine rapidly and accurately how many and what cations are incorporated per molecule of protein. Information about molar distributions and estimates of relative abundances of various complexes in the sample can be acquired in a single measurement.

Using in situ hybridisation histochemistry in combination with patch-clamp recordings and specific pharmacological tools, the molecular nature of the channels underlying Ca2+-dependent K+ currents was determined in dorsal vagal neurones (DVNs) of rat brainstem slices. In situ hybridisation analysis at cellular resolution revealed the presence of 'big'-conductance Ca2+- and voltage-activated K+ (BK) channel alpha-subunit mRNA, and of only one 'small'-conductance Ca2+-activated K+ (SK) channel subunit transcript, SK3, at very high levels in DVNs. By contrast, SK1 and SK2 mRNAs were below the threshold limit of detection. The SK channel-mediated after-hyperpolarising current (IAHP) was blocked by apamin with a half-maximal inhibitory concentration of approximately 2.2 nM. This is consistent with homomultimeric SK3 channels mediating IAHP in DVNs. IAHP was also blocked by scyllatoxin (20-30 nM) and curare (100-200 microM). Application of apamin (100 nM) or scyllatoxin (20 nM) invariably caused a substantial increase to 146.1 +/- 10.4 and 181.8 +/- 12.9 % of control, respectively, in the spontaneous firing rate of DVNs. Action potential duration was not affected by these SK channel blockers. The selective BK channel blocker iberiotoxin (50 nM) increased action potential duration by 22.5 +/- 7.3 %, as did low concentrations of tetraethylammonium (0.5 mM; 99.3 +/- 16.4 %) and the Ca2+ channel blocker Cd2+ (100 microM; 49.5 +/- 20.9 %). BK channel blockade did not significantly affect the firing rate of DVNs. These results allow us to establish a tight correlation between the properties of cloned and native BK and SK channels, and to achieve an understanding, at the molecular level, of their role in regulating the spontaneous firing frequency and in shaping single action potentials of central neurones.

Cytokinesis requires membrane trafficking coupled to actin remodeling and involves a number of trafficking molecules. CD2-associated protein (CD2AP) has been implicated in dynamic actin remodeling and membrane trafficking that occurs during endocytosis leading to the degradative pathway. In this study, we present several arguments for its implication in cytokinesis. First, endogenous CD2AP was found concentrated in the narrow region of the midzone microtubules during anaphase and in the midbody during late telophase. Moreover, we found that CD2AP is a membrane- and not a microtubule-associated protein. Second, the overexpression of the first two Src homology 3 domains of CD2AP, which are responsible for this localization, led to a significant increase in the rate of cell multinucleation. Third, the CD2AP small interfering RNA interfered with the cell separation, indicating that CD2AP is required for HeLa cells cytokinesis. Fourth, using the yeast two-hybrid system, we found that CD2AP interacted with anillin, a specific cleavage furrow component, and the two proteins colocalized at the midbody. Both CD2AP and anillin were found phosphorylated early in mitosis and also CD2AP phosphorylation was coupled to its delocalization from membrane to cytosol. All these observations led us to propose CD2AP as a new player in cytokinesis.

The association of various surface molecules with the cytoskeleton in resting peripheral blood T cells was examined by assaying the capacity of detergent to solubilize them. Cytoskeletal association was assessed by staining T cells with a fluorescein-conjugated mAb, resuspending the cells in buffer with or without the nonionic detergent, NP-40, and determining the capacity of the detergent to remove the mAb from the cell surface by using flow microfluorimetry. MAb to CD3, the TCR, and CD45 were completely removed from the cell surface by detergent. In contrast, 7 to 50% of mAb to CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules were resistant to detergent solubilization, demonstrating that a fraction of these molecules was constitutively associated with the cytoskeleton. The effect of cross-linking these molecules with a mAb and a secondary goat anti-mouse Ig was also examined. Cross-linking CD3 or the TCR induced cytoskeletal association of these molecules. In addition, cross-linking increased the fraction of CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules that was associated with the cytoskeleton. In contrast, cross-linking CD45 did not induce an association with the cytoskeleton. The effect of T cell activation on the cytoskeletal association of these molecules was also examined. Stimulation of T cells with ionomycin and PMA greatly increased the expression of CD2 and CD44 without increasing the number of molecules associated with the cytoskeleton. Stimulation with PMA alone had no effect on the expression of CD2 or CD44, but was found to decrease the percentage of these molecules associated with the cytoskeleton. Stimulation with ionomycin and PMA increased both the expression of class I MHC molecules and the number of molecules associated with the cytoskeleton proportionally. Finally, stimulation with ionomycin and PMA decreased CD3 expression, but increased the number of CD3 molecules associated with the cytoskeleton. The data establish a pattern of cytoskeletal association of T cell-surface molecules that is a characteristic of each individual molecule and can be altered by cross-linking. Moreover, the results indicate that the association of various T cell surface molecules with the cytoskeleton is a dynamic process that varies with the state of activation and or differentiation of the cells.

The czc determinant, which mediates resistance to Co2+, Zn2+ and Cd2+ in Alcaligenes eutrophus CH34 by cation efflux, is regulated by a two-component regulatory system composed of the sensor histidine kinase CzcS and the response activator CzcR (in addition to other components previously described). Regulatory genes are arranged in an upstream regulatory region (URR) and a downstream regulatory region (DRR). Transcription of czcCBA and of the URR was regulated by heavy-metal cations. DNA sequencing of the region downstream of czcD revealed the presence of the czcR and czcS genes which together with czcD form the DRR. Regulation of the DRR was studied with a czcD::lacZ translational fusion and a czcS::lux transcriptional fusion. Expression of both genes is also regulated by heavy metals. The genes of the URR yielded three mRNAs of approx. 1200, 500 and 200 nucleotides, respectively. The genes czcCBA for the cation/proton antiporter CzcCBA were transcribed by one operon as a transcript of 6200 nucleotides.

Differentiation of immature double positive (DP) CD4+ CD8+ thymocytes into single positive (SP) CD4+ and CD8+ T cells is referred to as positive selection and requires physical contact with thymic cortical epithelium. We now have identified "coinducer" molecules on DP thymocytes that, together with TCR, signal DP thymocytes to differentiate into SP T cells in vitro in the absence of thymic epithelium. A remarkable number of different molecules on DP thymocytes possessed "coinducing" activity, including <em>CD2</em>, CD5, <em>CD2</em>4, <em>CD2</em>8, CD49d, CD81, and TSA-1. Interestingly, in vitro differentiation occurred in the absence of lineage-specific signals, yet resulted in the selective generation of CD4+CD8- T cells. Thus, the present study has identified surface molecules that can signal DP thymocytes to differentiate into SP T cells in the absence of thymic epithelium and has characterized a default pathway for CD4+ T cell differentiation.

OBJECTIVE

Primitive human hematopoietic cells contain higher levels of aldehyde dehydrogenase (ALDH) activity than their terminally differentiating progeny but the particular stages when ALDH levels change have not been well defined. The objective of this study was to compare ALDH levels among the earliest stages of hematopoietic cell differentiation and to determine whether these could be exploited to obtain improved purity of human cord blood cells with long-term lympho-myeloid repopulating activity in vivo.

METHODS

ALDEFLUOR-stained human cord blood cells displaying different levels of ALDH activity were first analyzed for co-expression of various surface markers. Subsets of these cells were then isolated by multi-parameter flow cytometry and assessed for short-and long-term repopulating activity in sublethally irradiated immunodeficient mice.

RESULTS

Most short-term myeloid repopulating cells (STRC-M) and all long-term lympho-myeloid repopulating cells (LTRC-ML) stained selectively as ALDH+. Limiting dilution analysis of the frequencies of both STRC-M and LTRC-ML showed that they were similarly and most highly enriched in the 10% top ALDH+ cells. Removal of cells expressing <em>CD2</em>, CD3, CD7, CD14, CD16, <em>CD2</em>4, CD36, CD38, CD56, CD66b, or glycophorin A from the ALDH+ low-density fraction of human cord blood cells with low light side-scattering properties yielded a population containing LTRC-ML at a frequency of 1/360.

CONCLUSIONS

Elevated ALDH activity is a broadly inclusive property of primitive human cord blood cells that, in combination with other markers, allows easy isolation of the stem cell fraction at unprecedented purities.

1. A three electrode voltage-clamp was used to investigate membrane currents in the skeletal muscle fibres of the stick insect, Carausius morosus. Contraction was blocked by hypertonic solutions. 2. Membrane currents elicited by step depolarizations consisted of an inward current, an early outward current and a delayed outward current. 3. The reversal potential of the delayed outward current did not change when SO4(2-) was substituted for Cl-, but shifted by 14.1 mV when [K]0 was increased from 20 mM to 40 mM in SO4(2-) solution, suggesting that the delayed current is carried by K+. Both early and delayed outward currents were substantially reduced by 120 mM-tetraethylammonium (TEA) ions. 4. The small size of the shift in the reversal potential of the delayed outward current with increased pulse duration suggests that the delayed current measured flows mainly across the surface membrane. 5. Increasing [Ca]o made the apparent reversal potential for the inward current (120 mM-TEA Ringer) more positive and increased the size of the maximum inward current. However, Ca-currents showed saturation with increasing [Ca]o, indicating that there is a site to which Ca ions bind during their passage through the membrane. The dissociation constant of this site was 7.3 mM at 0 mV and was voltage-dependent. 6. Inward currents were blocked by 1 mM-La3+ or Cd2+, or by substitution of Co2+ or Ni2+ for Mg2+. Strontium and barium were able to permeate the channel but Na+ and Mg2+ appear impermeant. 7. As expected from the low intracellular Ca concentration, the instantaneous current-voltage relation of the Ca current rectified strongly in the inward direction. 8. Both constant field theory and the simplest, single site, Eyring rate theory model predict the rectification of the instantaneous current-voltage relation. The rate theory model also predicts saturation of the Ca current with [Ca]o.

The P segments of the voltage-dependent Na+ channel line the outer mouth and selectivity filter of the pore. The residues that form the cytoplasmic mouth of the pore of the channel have not been identified. To study the structure of the inner pore mouth, the presumed selectivity filter residues (D400, E755, K1237, and A1529), and three amino acids just amino-terminal to each of these residues in the rat skeletal muscle Na+ channel, were mutated to cysteine and expressed in tsA 201 cells. These amino acids are predicted (by analogy to K+ channels) to be on the cytoplasmic side of the putative selectivity filter residues. Inward and outward Na+ currents were measured with the whole-cell configuration of the patch-clamp technique. Cysteinyl side-chain accessibility was gauged by sensitivity to Cd2+ block and by reactivity with methanethiosulfonate (MTS) reagents applied to both the inside and the outside of the cell. Outward currents through the wild-type and all of the mutant channels were unaffected by internal Cd2+ (100 microM). Similarly, 1 mM methanethiosulfonate ethylammonium (MTSEA) applied to the inside of the membrane did not affect wild-type or mutant outward currents. However, two mutants amino-terminal to the selectivity position in domain III (F1236C and T1235C) and one in domain IV (S1528C) were blocked with high affinity by external Cd2+. The Na+ current through F1236C and S1528C channels was inhibited by MTSEA applied to the outside of the cell. The accessibility of these mutants to externally applied cysteinyl ligands indicates that the side chains of the mutated residues face outward rather than inward. The K+ channel model of the P segments as protein loops that span the selectivity region is not applicable to the Na+ channel.

The sidedness of isolated rabbit cardiac sarcolemmal vesicles was studied by observing the effects of several permeability-increasing agents on measurements of the amount of sialic acid released by neuraminidase, specific ouabain binding, and K+-phosphatase and (Na+ + K+)-ATPase activities. The results suggest that the vesicles are sealed and are about 80% right-side-out. Na+-Ca2+ exchange exhibited by these vesicles could be attributed to the sarcolemma rather than to some contaminating organelle. Ca2+ uptake was stimulated by preloading the vesicles with NaCl (and not KCl). Increasing the external [Na+] induced a rapid Ca2+ loss, which could not be mimicked by K+, Li+, Rb+ or choline+. The Nai+-dependent Ca2+ uptake was inhibited by certain cations: Cd2+>> La3+>> Y3+>> Mn2+>> Co2+>> Mg2+. The Nai+-dependent Ca2+ influx was enhanced by an inside positive membrane potential and inhibited by an inside negative membrane potential. Potentials were induced by a K+-valinomycin system.