CD80(B7-1) and CD86(B7-2) co-stimulatory molecules have been reported to activate Th1/Th2 development pathways differentially. It is well known that Langerhans cells (LC), potent antigen-presenting dendritic cells in the epidermis, express several co-stimulatory molecules and that this expression is modulated by several cytokines. Based on the recently reported effect of interferon (IFN)-gamma and interleukin (IL-)-10 on the expression of CD80 and CD86 by LC, we examined the effects of these cytokines on the expression of CD54 (intercellular adhesion molecule-1) and CD40 in addition to CD80 and CD86 on LC, and correlated the expression of each co-stimulatory molecule with antigen presentation for a Th1 clone by cultured LC (cLC) treated with these cytokines. LC cultured for 72 h significantly up-regulated MHC class II antigen expression and all the co-stimulatory molecules were examined. As previously reported, IL-10 or IFN-gamma inhibited the up-regulation of CD80 expression. Granulocyte/macrophage-colony-stimulating factor (GM-CSF) partially restored the suppression of CD80 expression induced by IFN-gamma on cultured LC, while it had virtually no effect on the inhibition induced by IL-10. Antigen presentation for the myoglobin-specific syngeneic Th1 clone by cLC, which were pre-incubated with these cytokines, correlated well with their CD80 expression. In addition, among the antibodies for CD80, CD86, CD28 or CD40, the suppression of the Th1 clone stimulation by LC was found to occur only with anti-CD80 and anti-CD28 antibodies. Finally, we studied the effects of IFN-gamma and IL-10 on GM-CSF production by epidermal keratinocytes (KC). We could show that only IFN-gamma, but not IL-10, suppressed GM-CSF production by KC. These findings suggest that both IFN-gamma and IL-10 suppress antigen presentation by LC for Th1 cells by suppressing their CD80 expression. The inhibitory effect of IFN-gamma on CD80 expression on LC appears to be partially mediated through the suppression of GM-CSF production by KC.

The function of the central nervous system depends on the correct regulation of ion channels by interacting proteins. Here, we identified cereblon as a new interactor of the voltage-gated chloride channel ClC-2. A distal region of the ClC-2 C-terminus interacts with the Lon domain of cereblon. Cereblon is expressed in several brain regions including the retina. There, we detected cereblon in nuclear and synaptic layers and localized the protein in the same subcellular region of bipolar cell bodies previously reported to express ClC-2. Our data suggest that cereblon might be associated with voltage-gated chloride channels in the central nervous system.

The ClC channel family consists of chloride channels important for various physiological functions. Two members in this family, ClC-0 and ClC-1, share approximately 50-60% amino acid identity and show similar gating behaviors. Although they both contain two subunits, the number of pores present in the homodimeric channel is controversial. The double-barrel model proposed for ClC-0 was recently challenged by a one-pore model partly based on experiments with ClC-1 exploiting cysteine mutagenesis followed by modification with methanethiosulfonate (MTS) reagents. To investigate the pore stoichiometry of ClC-0 more rigorously, we applied a similar strategy of MTS modification in an inactivation-suppressed mutant (C212S) of ClC-0. Mutation of lysine 165 to cysteine (K165C) rendered the channel nonfunctional, but modification of the introduced cysteine by 2-aminoethyl MTS (MTSEA) recovered functional channels with altered properties of gating-permeation coupling. The fast gate of the MTSEA-modified K165C homodimer responded to external Cl(-) less effectively, so the P(o)-V curve was shifted to a more depolarized potential by approximately 45 mV. The K165C-K165 heterodimer showed double-barrel-like channel activity after MTSEA modification, with the fast-gating behaviors mimicking a combination of those of the mutant and the wild-type pore, as expected for the two-pore model. Without MTSEA modification, the heterodimer showed only one pore, and was easier to inactivate than the two-pore channel. These results showed that K165 is important for both the fast and slow gating of ClC-0. Therefore, the effects of MTS reagents on channel gating need to be carefully considered when interpreting the apparent modification rate.

Members of the ClC family of voltage-gated chloride channels are found from bacteria to mammals with a considerable degree of conservation in the membrane-inserted, pore-forming region. The crystal structures of the ClC channels of Escherichia coli and Salmonella typhimurium provide a structural framework for the entire family. The ClC channels are homodimeric proteins with an overall rhombus-like shape. Each ClC dimer has two pores each contained within a single subunit. The ClC subunit consists of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a chloride selectivity filter within the 15-A neck of a hourglass-shaped pore. Three Cl(-) binding sites within the selectivity filter stabilize ions by interactions with alpha-helix dipoles and by chemical interactions with nitrogen atoms and hydroxyl groups of residues in the protein. The Cl(-) binding site nearest the extracellular solution can be occupied either by a Cl(-) ion or by a glutamate carboxyl group. Mutations of this glutamate residue in Torpedo ray ClC channels alter gating in electrophysiological assays. These findings reveal a form of gating in which the glutamate carboxyl group closes the pore by mimicking a Cl(-) ion.

Chiral nematic liquid crystals--otherwise referred to as cholesteric liquid crystals (CLCs)--are self-organized helical superstructures that find practical application in, for example, thermography, reflective displays, tuneable colour filters and mirrorless lasing. Dynamic, remote and three-dimensional control over the helical axis of CLCs is desirable, but challenging. For example, the orientation of the helical axis relative to the substrate can be changed from perpendicular to parallel by applying an alternating-current electric field, by changing the anchoring conditions of the substrate, or by altering the topography of the substrate's surface; separately, in-plane rotation of the helical axis parallel to the substrate can be driven by a direct-current field. Here we report three-dimensional manipulation of the helical axis of a CLC, together with inversion of its handedness, achieved solely with a light stimulus. We use this technique to carry out light-activated, wide-area, reversible two-dimensional beam steering--previously accomplished using complex integrated systems and optical phased arrays. During the three-dimensional manipulation by light, the helical axis undergoes, in sequence, a reversible transition from perpendicular to parallel, followed by in-plane rotation on the substrate surface. Such reversible manipulation depends on experimental parameters such as cell thickness, surface anchoring condition, and pitch length. Because there is no thermal relaxation, the system can be driven either forwards or backwards from any light-activated intermediate state. We also describe reversible photocontrol between a two-dimensional diffraction state, a one-dimensional diffraction state and a diffraction 'off' state in a bilayer cell.

The ClC chloride channels and transporters constitute a large family of membrane proteins that is involved in a variety of physiological processes. All members share a conserved molecular architecture that consists of a complex transmembrane transport domain followed by a cytoplasmic domain. Despite the strong conservation, the family shows an unusually broad variety of functional behaviors as some members work as gated chloride channels and others as secondary active chloride transporters. The conservation in the structure and the functional resemblance of gating and coupled transport suggests a strong mechanistic relationship between these seemingly contradictory transport modes. The cytoplasmic domains constitute putative regulatory components that are ubiquitous in eukaryotic ClC family members and that in certain cases interact with nucleotides thus linking ion transport to nucleotide sensing by yet unknown mechanisms.

The suprachiasmatic nuclei (SCN) of the anterior hypothalamus comprise a self-sustained biological clock generating an endogenous ∼24-h circadian rhythm, driving many overt daily rhythms in the body. An important remaining question is how the SCN neurons communicate with their efferent targets to control the daily oscillations in behavior and physiology. In this chapter, we summarize several signaling factors that may serve as such SCN output factors. Whereas vasopressin may be involved in the regulation of circadian hormone rhythms, SCN-derived prokineticin 2 (PK2), TGF-α, and cardiotrophin-like cytokine (CLC) may serve as output factors for other circadian rhythms, including locomotor activity, body temperature, and energy metabolism. The circadian rhythm in firing activity of SCN neurons is also likely to be a critical output signaling mechanism. The likely involvement of these output factors in the generation of the circadian rhythm in SCN neuronal firing activity is also discussed.

Renal stone disease (nephrolithiasis) affects 5% of adults and is often associated with hypercalciuria. Hypercalciuric nephrolithiasis is a familial disorder in more than 35% of patients, and may occur as a monogenic disorder, or as a polygenic trait involving 3 to 5 susceptibility loci in man and rat, respectively. Studies of monogenic forms of hypercalciuric nephrolithiasis in man, for example, Bartter syndrome, Dent's disease, autosomal dominant hypocalcemic hypercalciuria (ADHH), hypercalciuric nephrolithiasis with hypophosphatemia, and familial hypomagnesemia with hypercalciuria have helped to identify a number of transporters, channels, and receptors that are involved in regulating the renal tubular reabsorption of calcium. Thus, Bartter syndrome, an autosomal recessive disease, is caused by mutations of the bumetanide-sensitive Na-K-Cl (NKCC2) cotransporter, the renal outer-medullary potassium channel (ROMK), the voltage-gated chloride channel, CLC-Kb, or in its beta subunit, Barttin. Dent's disease, an X-linked disorder characterized by low molecular weight proteinuria, hypercalciuria, and nephrolithiasis, is due to mutations of the chloride/proton antiporter, CLC-5; ADHH is associated with activating mutations of the calcium-sensing receptor, which is a G protein-coupled receptor; hypophosphatemic hypercalciuric nephrolithiasis associated with rickets is due to mutations in the type 2c sodium-phosphate cotransporter (NPT2c); and familial hypomagnesemia with hypercalciuria is due to mutations of paracellin-1, which is a member of the claudin family of membrane proteins that form the intercellular tight junction barrier in a variety of epithelia. These studies have provided valuable insights into the renal tubular pathways that regulate calcium reabsorption and predispose to kidney stones and bone disease.

The clc genomic island is a 105 kb integrative and conjugative element (ICE) in Pseudomonas sp. strain B13, which encodes metabolism of 3-chlorocatechol. The clc island is integrated in a tRNAGly gene, but can excise and form a circular intermediate in which both ends are connected. The integrase gene (intB13) of the clc genomic island is located at the right end, 202 bp from the junction site facing inwards. Fragments upstream of intB13 in the circular form and in the integrated form were fused to a promoterless gfp gene for Green Fluorescent Protein and introduced in monocopy onto the chromosome of strain B13. Quantitative GFP fluorescence measurements in individual cells of the different B13-derivatives revealed that the circular form fragment contained a strong constitutive promoter (Pcirc) driving intB13 expression in all cells. By using primer extension Pcirc could be mapped near the left end of the clc element and Pcirc can therefore only control intB13 expression when left and right ends are connected as in the circular form. Expression from intB13 upstream fragments from the integrated clc element was weaker than that from Pcirc and only occurred in maximally 15% of individual cells in a culture. A promoter (Pint) could be roughly mapped in this region by using reverse-transcription PCR and by successively shortening the fragment from the 5' end. Transposon mutants in cloned left end sequences of the clc element were selected which had lost the activation potential on the Pint promoter and those which resulted in overexpression of GFP from Pint. The DNA sequence of the region of the transposon insertions pointed to a relatively well conserved area among various other genomic islands. The activator mutants mapped in an open reading frame (ORF) encoding a 175 amino acid protein without any significant similarity to functionally characterized proteins in the databases.

Voltage-gated chloride channels (ClC) are highly conserved during evolution and appear to participate in a variety of physiological functions. Recently, ClC-2 was proposed to play a role in stabilizing the chloride equilibrium potential near or below the resting membrane potential in neurons expressing ligand-gated chloride channels. Because rod bipolar cells in mammalian retina express three forms of inhibitory ligand-gated chloride channels, we decided to study ClC-2 localization and function in the rat retina. RNA encoding ClC-1, -2, -3, -4, and -5 was detected by reverse transcription-PCR in the rat retina. ClC-2-specific antibodies identified protein on cell bodies and in synaptic layers. Double-immunofluorescence staining revealed that intense ClC-2 immunoreactivity colocalized with PKC-stained rod bipolar cells. Patch-clamp experiments performed with individual rod bipolar cells demonstrated the presence of a time-dependent, inwardly rectified current activated at hyperpolarizing membrane potentials. This current demonstrated selectivity for different anions (Cl(-)>> I(-)>> gluconate), was inhibited by Cd(2+), and was minimally reduced by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid. These features are consistent with currents generated by ClC-2 channels. Our data indicate that functional ClC-2 channels are present in retinal rod bipolar cells and support a role for ClC-2 in maintaining Cl(-) homeostasis in neurons with ligand-gated chloride channels.

OBJECTIVE

Previously, we have found that the ClC-3 chloride channel is involved in endothelin-1 (ET-1)-induced rat aortic smooth muscle cell proliferation. The present study was to investigate the role of ClC-3 in cell cycle progression/distribution and the underlying mechanisms of proliferation.

METHODS

Small interference RNA (siRNA) is used to silence ClC-3 expression. Cell proliferation, cell cycle distribution and protein expression were measured or detected with cell counting, bromodeoxyuridine (BrdU) incorporation, Western blot and flow cytometric assays respectively.

RESULTS

ET-1-induced rat basilar vascular smooth muscle cell (BASMC) proliferation was parallel to a significant increase in endogenous expression of ClC-3 protein. Silence of ClC-3 by siRNA inhibited expression of ClC-3 protein, prevented an increase in BrdU incorporation and cell number induced by ET-1. Silence of ClC-3 also caused cell cycle arrest in G(0)/G(1) phase and prevented the cells' progression from G(1) to S phase. Knockdown of ClC-3 potently inhibited cyclin D1 and cyclin E expression and increased cyclin-dependent kinase inhibitors (CDKIs) p27(KIP) and p21(CIP) expression. Furthermore, ClC-3 knockdown significantly attenuated phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta) induced by ET-1.

CONCLUSIONS

Silence of ClC-3 protein effectively suppressed phosphorylation of the Akt/GSK-3beta signal pathway, resulting in down-regulation of cyclin D1 and cyclin E, and up-regulation of p27(KIP) and p21(CIP). In these BASMCs, integrated effects lead to cell cycle G(1)/S arrest and inhibition of cell proliferation.

CLC-5 is a member of the CLC family of voltage-gated chloride channels. Mutations disrupting CLC-5 lead to Dent's disease, an X-linked renal tubular disorder, characterised by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, and renal stones. Sequence analysis of CLC-5 reveals a 746 amino acid protein with an intracellular amino-terminus, transmembrane spanning domains, and two CBS domains within its intracellular carboxy-terminus. CBS domains have been implicated in intracellular targetting and trafficking as well as protein-protein interactions. We investigate subcellular localisation of three naturally occurring CLC-5 mutants which all lead to a truncated protein, disrupting the second CBS domain. These mutants are unable to traffic normally to acidic endosomes but are retained in perinuclear compartments, colocalising with the Golgi complex. This is the first identification of the cellular pathogenesis of CBS domain mutations of CLC-5.

We introduce an approach based on the recently introduced functional mode analysis to identify collective modes of internal dynamics that maximally correlate to an external order parameter of functional interest. Input structural data can be either experimentally determined structure ensembles or simulated ensembles, such as molecular dynamics trajectories. Partial least-squares regression is shown to yield a robust solution to the multidimensional optimization problem, with a minimal and controllable risk of overfitting, as shown by extensive cross-validation. Several examples illustrate that the partial least-squares-based functional mode analysis successfully reveals the collective dynamics underlying the fluctuations in selected functional order parameters. Applications to T4 lysozyme, the Trp-cage, the aquaporin channels Aqy1 and hAQP1, and the CLC-ec1 chloride antiporter are presented in which the active site geometry, the hydrophobic solvent-accessible surface, channel gating dynamics, water permeability (p(f)), and a dihedral angle are defined as functional order parameters. The Aqy1 case reveals a gating mechanism that connects the inner channel gating residues with the protein surface, thereby providing an explanation of how the membrane may affect the channel. hAQP1 shows how the p(f) correlates with structural changes around the aromatic/arginine region of the pore. The CLC-ec1 application shows how local motions of the gating Glu(148) couple to a collective motion that affects ion affinity in the pore.

An HEK-293 cell line stably expressing the human recombinant ClC-2 Cl(-) channel was used in patch-clamp studies to study its regulation. The relative permeability P(x)/P(Cl) calculated from reversal potentials was I(-)>> Cl(-) = NO(3)(-) = SCN(->>/=Br(-). The absolute permeability calculated from conductance ratios was Cl(-) = Br(-) = NO(3)(-)>>/= SCN(-)>> I(-). The channel was activated by cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleic acid (C:18 cisDelta9), elaidic acid (C:18 transDelta9), arachidonic acid (AA; C:20 cisDelta5,8,11,14), and by inhibitors of AA metabolism, 5,8,11,14-eicosatetraynoic acid (ETYA; C:20 transDelta5,8,11,14), alpha-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and 2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2 Cl(-) channels were activated by a combination of forskolin plus IBMX and were inhibited by the cell-permeant myristoylated PKA inhibitor (mPKI). Channel activation by reduction of bath pH was increased by PKA and prevented by mPKI. AA activation of the ClC-2 Cl(-) channel was not inhibited by mPKI or staurosporine and was therefore independent of PKA or protein kinase C activation.

Ion gradients across intracellular membranes contribute to the physicochemical environment inside compartments. CLC anion transport proteins that localise to intracellular organelles are anion-proton exchangers involved in anion sequestration or vesicular acidification. By homology, the only CLC protein of Saccharomyces cerevisiae, Gef1, belongs to this family of intracellular exchangers. Gef1 localises to the late Golgi and prevacuole and is essential in conditions of iron limitation. In the absence of Gef1, a multicopper oxidase involved in iron uptake, Fet3, fails to acquire copper ion cofactors. The precise role of the exchanger in this physiological context is unknown. Here, we show that the Gef1-containing compartment is adjusted to a more alkaline pH under iron limitation. This depends on the antiport function of Gef1, because an uncoupled mutant of Gef1 (E230A) results in the acidification of the lumen and fails to support Fet3 maturation. Furthermore, we found that Gef1 antiport activity correlates with marked effects on cellular glutathione homeostasis, raising the possibility that the effect of Gef1 on Fet3 copper loading is related to the control of compartmental glutathione concentration or redox status. Mutational inactivation of a conserved ATP-binding site in the cytosolic cystathione beta-synthetase domain of Gef1 (D732A) suggests that Gef1 activity is regulated by energy metabolism.

OBJECTIVE

Given the crucial role of the skeletal muscle chloride conductance (gCl), supported by the voltage-gated chloride channel CLC-1, in controlling muscle excitability, the availability of ligands modulating CLC-1 are of potential medical as well as toxicological importance. Here, we focused our attention on niflumic acid (NFA), a molecule belonging to the fenamates group of non-steroidal anti-inflammatory drugs (NSAID).

METHODS

Rat muscle Cl(-) conductance (gCl) and heterologously expressed CLC-1 currents were evaluated by means of current-clamp (using two-microelectrodes) and patch-clamp techniques, respectively. Fura-2 fluorescence was used to determine intracellular calcium concentration, [Ca(2+)](i), in native muscle fibres.

RESULTS

NFA inhibited native gCl with an IC(50) of 42 muM and blocked CLC-1 by interacting with an intracellular binding site. Additionally, NFA increased basal [Ca(2+)](i) in myofibres by promoting a mitochondrial calcium efflux that was not dependent on cyclooxygenase or CLC-1. A structure-activity study revealed that the molecular conditions that mediate the two effects are different. Pretreatment with the Ca-dependent protein kinase C (PKC) inhibitor chelerythrine partially inhibited the NFA effect. Therefore, in addition to direct channel block, NFA also inhibits gCl indirectly by promoting PKC activation.

CONCLUSIONS

These cellular effects of NFA on skeletal muscle demonstrate that it is possible to modify CLC-1 and consequently gCl directly by interacting with channel proteins and indirectly by interfering with the calcium-dependent regulation of the channel. The effect of NFA on mitochondrial calcium stores suggests that NSAIDs, widely used drugs, could have potentially dangerous side-effects.

In rats treated with high-dose corticosteroids, skeletal muscle that is denervated in vivo (steroid-denervated) develops electrical inexcitability similar to that seen in patients with acute quadriplegic myopathy. To determine whether changes in muscle gene transcription might underlie inexcitability of steroid-denervated muscle we performed RNase protection assays to quantitate adult (SkM1) and embryonic (SkM2) sodium channel isoforms and chloride channel (CLC-1) mRNA levels in control, denervated, steroid-innervated, and steroid-denervated skeletal muscle. While SkM1 mRNA levels were relatively unaffected by denervation or steroid treatment, SkM2 mRNA levels were increased by both. These effects were synergistic and high levels of SkM2 mRNA were expressed in denervated muscle exposed to corticosteroids. Skeletal muscle CLC-1 mRNA levels were decreased by denervation. To better understand the marked upregulation of SkM2 in steroid-denervated muscle we examined changes in myogenin and glucocorticoid receptor mRNA levels. However, changes in these mRNA levels cannot account for the upregulation of SkM2 in steroid-denervated muscle.

RNA repair has been proposed as a novel gene-based therapeutic strategy. Modified Tetrahymena group I intron ribozymes have been used to mediate trans-splicing of therapeutically relevant RNA transcripts, but the efficiency of the ribozyme-mediated RNA repair process has not been determined precisely and subsequent restoration of protein function has been demonstrated only by indirect means. We engineered a ribozyme that targets the mRNA of a mutant canine skeletal muscle chloride channel (cClC-1) (mutation T268M in ClC-1 causing myotonia congenita) and replaces the mutant-containing 3' portion by trans-splicing the corresponding 4-kb wild-type sequence. Repair efficiency assessed by quantitative RT-PCR was 1.2% +/- 0.1% in a population of treated cells. However, when chloride channel function was examined in single cells, a wide range of electrophysiological activity was observed, with 18% of cells exhibiting significant functional restoration and some cells exhibiting complete rescue of the biophysical phenotype. These results indicate that RNA repair can restore wild-type protein activity and reveal considerable cell-to-cell variability in ribozyme-mediated trans-splicing reaction efficiency.

Ecosystems are often indirectly connected through consumers with complex life cycles (CLC), in which different life stages inhabit different ecosystems. Using a structured consumer resource model that accounts for the independent effects of two resources on consumer growth and reproductive rates, we show that such indirect connections between ecosystems can result in alternative stable states characterized by adult-dominated and juvenile-dominated consumer populations. As a consequence, gradual changes in ecosystem productivity or mortality rates of the consumer can lead to dramatic and abrupt regime shifts across different ecosystems, hysteresis and counterintuitive changes in the consumer abundances. Whether these counter intuitive or abrupt responses occur depend on the relative productivity of both habitats and which consumer life-stage inhabits the manipulated ecosystem. These results demonstrate the strong yet complex interactions between ecosystems coupled through consumers with CLC and the need to think across ecosystems to reliably predict the consequences of natural or anthropogenic changes.

OBJECTIVE

Calu-3 cells are derived from serous cells of human lung submucosal glands, a prime target for therapy in cystic fibrosis (CF). Calu-3 cells can be cultured to form epithelia capable of transepithelial transport of chloride. A CF Calu-3 cell is not available.

METHODS

A retroviral vector was used to cause persistent down regulation of CFTR using siRNA methodology, in Calu-3 cells. A Calu-3 cell line with CFTR content less than 5% of the original line has been established. Epithelia grown using the modified cells have been used in comparative studies of transporting capability.

RESULTS

All aspects of cAMP activated chloride secretion were attenuated in the epithelia with reduced CFTR content. However transporting capability was reduced less than the CFTR content. From studies with the CFTR channel inhibitor, GlyH-101, it was concluded that wild type Calu-3 cells have a reserve of CFTR channels not located in the membrane, but available for replacement, while in the modified Calu-3 cell line there was little or no reserve. Lubiprostone, a putative ClC-2 activator, increased transepithelial chloride secretion in both modified and wild type Calu-3 epithelia. Modified Calu-3 epithelia with the residual CFTR currents blocked with GlyH-101 responded equally well to lubiprostone as those without the blocking agent.

CONCLUSIONS

It appears that lubiprostone is capable of stimulating a non-CFTR dependent transepithelial chloride secretion in Calu-3 monolayers, with obvious implications for CF therapy. Cell lines, however, do not always reflect the behaviour of the native tissue with integrity.

The ortho-cleavage pathways of catechol and 3-chlorocatechol are central catabolic pathways of Pseudomonas putida that convert aromatic and chloroaromatic compounds to tricarboxylic acid (TCA) cycle intermediates. They are encoded by the evolutionarily related catBCA and clcABD operons, respectively. Expression of the cat and clc operons requires the LysR-type transcriptional activators CatR and ClcR, respectively, and the inducer molecules cis,cis-muconate and 2-chloro-cis,cis-muconate, respectively. The regulation of the cat and clc promoters has been well studied, but the extent to which these operons are repressed by growth in TCA cycle intermediates has not been explored. We demonstrate by transcriptional fusion studies that the expression from the clc promoter is repressed when the cells are grown on succinate, citrate, or fumarate and that this repression is ClcR dependent and occurs at the transcriptional level. The presence of these organic acids did not affect the expression from the cat promoter. In vitro transcription assays demonstrate that the TCA cycle intermediate fumarate directly and specifically inhibits the formation of the clcA transcript. No such inhibition was observed when CatR was used as the activator on either the cat or clc template. Titration studies of fumarate and 2-chloromuconate show that the fumarate effect is concentration dependent and reversible, indicating that fumarate and 2-chloromuconate most probably compete for the same binding site on ClcR. This is an interesting example of the transcriptional regulation of a biodegradative pathway by the intracellular sensing of the state of the TCA cycle.

HL-60 promyelocytic leukemia cells differentiated to eosinophils and eosinophilic precursors when cultured under mildly alkaline conditions (pH 7.6-7.8) for 7 d without refeeding. New cytoplasmic granules appeared blue in the least mature cells and red in the most mature cells when stained with Wright-Giemsa. The granules also stained with Luxol-fast-blue, a characteristic of eosinophil granules. Furthermore, most cells contained the eosinophil major basic protein (MBP); the Charcot-Leyden Crystal (CLC) protein (lysophospholipase), eosinophil peroxidase, acid phosphatase, and arylsulfatase were also detected in a portion of these cells. The eosinophil major basic protein was found in a high proportion of undifferentiated cells, and thus may be constituitively produced. By examining finely banded chromosomes, translocation break points were demonstrated at q22 on one chromosome 16 and at q23 on the other homologue; abnormalities in this region of the long arm of 16 are a characteristic finding in the recently described syndrome of acute myelomonocytic leukemia (AMMoL) with abnormal bone marrow eosinophils. In common with the bone marrow eosinophils in these patients, the HL-60 eosinophil granules contained chloroacetate esterase and periodic-acid Schiff (PAS) reactive material; crystalloid inclusions were rare. Therefore, the HL-60 cell line appears to be an in vitro model for eosinophilopoiesis and may be specially suited for the study of the abnormal eosinophils seen in certain malignant conditions.

The role(s) of the eosinophil Charcot-Leyden crystal (CLC) protein in eosinophil or basophil function or associated inflammatory processes is yet to be established. Although the CLC protein has been reported to exhibit weak lysophospholipase activity, it shows virtually no sequence homology to any known member of this family of enzymes. The X-ray crystal structure of the CLC protein is very similar to the structure of the galectins, members of a beta-galactoside-specific animal lectin family, including a partially conserved galectin carbohydrate recognition domain (CRD). In the absence of any known natural carbohydrate ligand for this protein, the functional role of the CLC protein (galectin-10) has remained speculative. Here we describe structural studies on the carbohydrate binding properties of the CLC protein and report the first structure of a carbohydrate in complex with the protein. Interestingly, the CLC protein demonstrates no affinity for beta-galactosides and binds mannose in a manner very different from those of other related galectins that have been shown to bind lactosamine. The partial conservation of residues involved in carbohydrate binding led to significant changes in the topology and chemical nature of the CRD, and has implications for carbohydrate recognition by the CLC protein in vivo and its functional role in the biology of inflammation.