Large-scale purification of human BACE expressed in mammalian cells and removal of the prosegment with HIV-1 protease to improve crystal diffraction.

BACE, or beta-secretase, is an attractive target in the treatment of Alzheimer's Disease because of its involvement in the generation of amyloid beta peptides. BACE is a type I transmembrane aspartyl protease composed of pre-, pro-, catalytic, transmembrane and cytoplasmic domains. For the present study, the coding sequence was truncated just before the transmembrane domain and the resulting construct was extended with the C-terminal addition of a (His)(6) and expressed in several mammalian host cells. The enzyme expressed in CHO cells had the best crystallographic behavior and was purified in large quantities in a three step procedure. The purified BACE was comprised of two forms, namely the full length proBACE construct beginning with Thr(1), and a derivative missing the first 24 amino acids beginning with E(25). These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 A resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F(39)-V(40) bond, leaving the V(40)EM...ES(432) (His)(6) derivative that was purified yielding an enzyme that was no more active than untreated BACE but co-crystallized with inhibitors producing well shaped, bipyramidal co-crystals diffracting to 2.6 A resolution.

Cloning, sequencing and phylogenetic analysis of a human 5-hydroxytryptamine 1D receptor pseudogene.

A third member of the human 5HT1D gene family has been identified using a combination of homology cloning and DNA sequence analysis. This human gene is most related to the 5HT1D alpha subtype (77% shared identity) and is a pseudogene, based on the lack of an open reading frame (ORF) caused by multiple in-frame stop codons and nucleotide (nt) deletions relative to the functional 5HT1D alpha gene (encoding the 5-hydroxytryptamine 1D alpha receptor). The 5HT1D pseudogene also contained an insertion that shares 87% identity to the Alu consensus sequence. Phylogenetic analysis of the three human genes in this family reveals that although the two functional genes, 5HT1D alpha and 5HT1D beta, are detected in all mammalian species examined, the 5HT1D pseudogene is only detected in a subset of primates (catarrhines) that evolved approximately 35-45 million years (Myr) ago. Alternatively, based on the 23% divergence between the functional 5HT1D alpha gene and the 5HT1D pseudogene, we estimate that these two genes began to diverge approximately 50 Myr ago.

Nucleotide sequence analysis of the human KCNJ1 potassium channel locus.

Detailed analyses of transcripts encoding various isoforms of the human potassium (K+, inward rectifying) channel ROM-K (also referred to as K(ir)1.1) revealed the existence of at least five distinct transcripts [Shuck et al., J. Biol. Chem. 269 (1994) 24261-24270]. These five hROM-K transcripts appear to be the result of alternative splicing of five exons. The nucleotide sequence of the genomic DNA including and spanning these exons (the KCNJ1 locus) was obtained directly from lambda and P1 clones (a total of 40 kb). The organization of the hKCNJ1 gene was determined by combining this sequence information with data obtained from primer extension and RT-PCR experiments. It appears that the hKCNJ1 gene utilizes multiple promoters, with promoter-like elements found 5' of exons 1, 4, or 5. The promoter 5' of exon 5 was unexpected; thus, it appears that the hKCNJ1 gene is capable of producing six distinct hROM-K transcripts via the use of three promoters and alternative splicing of five exons. Comparisons of the rat and human ROM-K cDNA sequences find human homologs (orthologs) for two of the three distinct rROM-K transcripts. A search of the complete human KCNJ1 sequence with the exon sequence that defines the other rROM-K transcript located a region of shared nucleotides, a putative sixth exon, in the hKCNJ1 gene. This finding suggests that the rKCNJ1 gene may contain an exon that is no longer or infrequently used in transcripts derived from the hKCNJ1 gene.

Pitfalls in the quantitative estimation of the secretion of granule proteins by eosinophils.

The secretion of preformed granule proteins by eosinophils is an important correlate of eosinophil activation. However, a review of the literature reveals large disparities in the amounts of these substances which were reportedly secreted when eosinophils were activated. In the present study we report that our attempts to quantitate the secretion of eosinophil peroxidase and eosinophil-derived neurotoxin from activated eosinophils by measuring these substances in the incubation supernatants were uniformly unsuccessful. We found that, once they were secreted, both eosinophil peroxidase and eosinophil-derived neurotoxin were promptly lost to assay and presumably destroyed. Thus the measurement of the difference in the concentration of these substances in eosinophils prior to and after activation, revealed that as much as 65% of the eosinophil-derived neurotoxin and 62% of the peroxidase in the eosinophils were lost to assay during activation of the cells whereas the largest amount of these substances which could be measured in the incubation supernatants never exceeded 2%. Evidence is presented that the destruction of eosinophil-derived neurotoxin must occur prior to the release of this substance into the medium. Attempts to inhibit the destruction of eosinophil peroxidase and of eosinophil-derived neurotoxin by incorporating various inhibitors into the incubations were unsuccessful. These results emphasize the need to monitor the overall recoveries of secreted products from activated eosinophils and suggest that meaningful estimates of the secretion of these granule proteins from activated eosinophils can only be obtained by measuring the residual content of these substances in eosinophils after they have been activated and comparing these values to the contents of eosinophils prior to activation.

Structural characterization of the oligosaccharides formed by depolymerization of heparin with nitrous acid.

Heparin was cleaved with nitrous acid at pH 1.5 and the products were reduced with Na+ boro[3H]hydride to generate a mixture of di- and tetrasaccharides having anhydro-D-[3H]mannitol (AManR) residues on their reducing terminals. The products were purified to homogeneity by gel filtration and high-performance liquid chromatography. For each oligosaccharide, the proportions of D-glucuronic acid (GlcUA), L-iduronic acid (IdoUA), N-acetyl-D-glucosamine (GlcNAc), and AManR and the monosaccharide sequence were determined by quantification of the products of acid hydrolysis. The tetrasaccharide sequences were determined by comparison of the disaccharide units formed by hydrazinolysis and deamination with previously characterized disaccharides. The following new oligosaccharides were identified: GlcUA(2-SO4)-AManR, GlcUA(2-SO4)-AManR(6-SO4), GlcUA-AManR(3,6-diSO4), GlcUA-GlcNAc-GlcUA-AManR, IdoUA-GlcNAc-GlcUA-AManR, GlcUA-GlcNAc(6-SO4)-GlcUA-AManR, IdoUA(2-SO4)-GlcNAc-GlcUA-AManR, IdoUA-GlcNAc(6-SO4)-GlcUA-AManR, IdoUA(2-SO4)-GlcNAc-GlcUA-AManR(6-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(6-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(3-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(3,6-diSO4), and IdoUA(2-SO4)-GlcNAc(6-SO4)-GlcUA-AManR(6-SO4). Then the disaccharides and the tetrasaccharides were readily resolved by high-performance anion-exchange liquid chromatography and were quantified on the basis of the amount of 3H counts/min in each. The structures are discussed in terms of their implications regarding heparin biosynthesis and anticoagulant activity.

Kinetics of proteoheparan sulfate synthesis, secretion, endocytosis, and catabolism by a hepatocyte cell line.

The metabolism of heparan sulfate proteoglycan was studied in monolayer cultures of a rat hepatocyte cell line. Late log cells were labeled with 35SO4(2-) or [3H] glucosamine, and labeled heparan sulfate, measured as nitrous acid-susceptible product, was assayed in the culture medium, the pericellular matrix, and the intracellular pools. Heparan sulfate in the culture medium and the intracellular pools increased linearly with time, while that in the matrix reached a steady-state level after a 10-h labeling period. When pulse-labeled cells were incubated in unlabeled medium, a small fraction of the intracellular pool was released rapidly into the culture medium while the matrix heparan sulfate was taken up by the cells, and the resulting intracellular pool was rapidly catabolized. The structures of the heparan sulfate chains in the three pools were very similar. Both the culture medium pool and the cell-associated fraction of heparan sulfate contained proteoheparan sulfate plus a polydisperse mixture of heparan chains which were attached to little, if any, protein. Pulse-chase data suggested that the free heparan sulfate chains were formed as a result of catabolism of the proteoglycan. When NH4Cl, added to inhibit lysosomal function, was present during either a labeling period or a chase period, the total catabolism of the heparan sulfate chains to monosaccharides plus free SO2-4 was blocked, but the conversion of the proteoglycan to free heparan sulfate chains continued at a reduced rate.

Nonenzymatic reduction of prostaglandin H by lipoic acid.

Lipoic acid has recently been found to stimulate prostaglandin biosynthesis by sheep vesicular gland microsomes (Marnett, L. J., and Wilcox, C. L. (1977). Biochim. Biophys. Acta 487, 222). The increase in oxygenated products is predominantly in the formation of prostaglandin F and its structure has been verified by gas chromatography-mass spectrometry. Endoperoxide trapping experiments employing reduced glutathione show that the conversion of prostaglandin H to prostaglandin F is slow in lipoate containing incubation mixtures. Therefore, the net effect of the addition of lipoic acid to vesicular gland microsomes is the stimulation of prostaglandin endoperoxide biosynthesis. Further experiments reveal that the reduction of prostaglandin H to prostaglandin F by lipoate is nonenzymatic and occurs after the termination of biosynthesis in the work-up mixture. The reduction takes place preferentially in the organic phase of a Folch extract (chloroform-methanol-2% formic acid 8:4:3). Authentic prostaglandin H2 is reduced by lipoic acid to prostaglandin F 2alpha in high yield under these conditions.

Cloning and pharmacological characterization of a novel human 5-hydroxytryptamine1D receptor subtype.

The canine RDC4 gene was used to isolate two distinct human serotonin receptor genes. The receptor encoded by clone RH-6 was the species homolog of RDC4 and was identical to a human serotonin 5-hydroxytryptamine1D (5-HT1D) receptor that was recently reported [Mol. Pharmacol. 40:143-148 (1991)]. The receptor encoded by RH-2 was a novel 5-HT receptor that was 61% identical to RH-6 and showed the greatest homology with the rat 5-HT1B receptor sequence (94%). The RH-2 gene contained an intronless, 1170-base pair, open reading frame that encoded a 390-amino acid protein that contained all of the hallmarks of a guanine nucleotide-binding protein-linked receptor. Heterologous expression of the RH-2 gene in Chinese hamster ovary cells led to the appearance of high affinity binding sites for 5-HT (Kd = 2.6 nM, Bmax = 2.9 pmol/mg of membrane protein), and the receptor expressed in Chinese hamster ovary cells was coupled to inhibition of adenylyl cyclase. Competition binding experiments using compounds that are selective for various 5-HT receptor subtypes showed the highest correlation with a 5-HT1D-like receptor (r = 0.89) and a low correlation with 5-HT1B-like receptors. Examples of the 5-HT1D-like pharmacology displayed by RH-2 include high affinity for the 5-HT1D-selective compound sumatriptan (Ki = 9.4 nM) and for the alpha 2-adrenergic receptor antagonist rauwolscine (Ki = 47 nM). Therefore, despite the close genetic relationship between RH-2 and the rat 5-HT1B receptor, our results indicate that the receptor encoded by RH-2 2 is best classified as a human 5-HT1D receptor subtype and defines a second member of the human 5-HT1D receptor family.

Regulation of prostaglandin H synthase mRNA levels and prostaglandin biosynthesis by platelet-derived growth factor.

Stimulation of serum-starved NIH-3T3 cells with 20 ng/ml recombinant platelet-derived growth factor BB (rPDGF) results in the synthesis of prostaglandin E2 (PGE2) that is detectable within 10 min and which peaks after 2 h. Inhibition of translation with 36 microM cycloheximide inhibits rPDGF-stimulated PGE2 synthesis (greater than 90%), suggesting that de novo synthesis of prostaglandin H synthase (PGHS) is required for growth factor stimulation of PGE2 synthesis. Paradoxically, the addition of 10 microM exogenous arachidonate to the serum-starved cells resulted in 2-fold more PGE2 synthesis, and maximal synthesis occurred at 30 min compared to 2 h following rPDGF treatment. These data suggest that the serum-starved cells have ample biosynthetic capacity to support maximal rPDGF-stimulated PGE2 synthesis without de novo enzyme synthesis. Kinetic analysis of PGHS mRNA levels showed that although rPDGF did elevate the steady-state level of PGHS mRNA, the increase occurred after maximal PGE2 synthesis was achieved. Quantification of PGHS levels by immunoblot showed that there was no significant increase in enzyme levels following rPDGF stimulation even under conditions where PGHS mRNA levels were elevated. Irreversible inactivation of PGHS by treatment with aspirin and subsequent stimulation with rPDGF synchronized the maximal elevation of PGHS mRNA levels and PGE2 synthesis; both peaked at 3 h. Interestingly, aspirin pretreatment increased both the rate and amplitude of rPDGF-stimulated PGHS mRNA induction. The enhanced induction was not simply the result of a loss in PGE2 production since treatment with exogenous PGE2 also induced PGHS mRNA levels. Since rPDGF-stimulated PGE2 synthesis is blocked by both transcription and translation inhibitors but arachidonate-stimulated PGE2 synthesis is unaffected, the requirement for protein synthesis does not appear to involve de novo PGHS synthesis. The synthesis of a protein required for coupling of the rPDGF receptor to phospholipase activation and arachidonic acid mobilization is a more likely interpretation. In summary, our data suggest that rPDGF can induce PGHS mRNA levels, but that rPDGF-stimulated PGE2 synthesis is not dependent upon de novo PGHS synthesis.

Controlled tryptic digestion of prostaglandin H synthase. Characterization of protein fragments and enhanced rate of proteolysis of oxidatively inactivated enzyme.

Treatment of prostaglandin H (PGH) synthase (70 kDa) with trypsin generates fragments of 33 and 38 kDa. Each of the fragments was purified by reverse-phase high performance liquid chromatography (HPLC) using acetonitrile/water/trifluoroacetic acid gradients. Amino acid sequence analysis indicates that the 33-kDa protein contains the NH2 terminus of PGH synthase. Neither the 33- nor 38-kDa fragment isolated by HPLC exhibits any PGH synthase activity; however, cleavage of intact enzyme to 33- and 38-kDa fragments to the extent of 90% only reduces cyclooxygenase activity by 40%. This implies that the cleaved proteins or a complex formed between them retains the conformation necessary for enzyme activity. Extensive attempts to resolve active fragments from each other or from intact enzyme were unsuccessful; intact enzyme and digestion fragments cochromatograph under all conditions employed. Treatment of PGH synthase with [3H]acetylsalicylic acid followed by trypsin digestion introduces [3H]acetyl moieties into the intact protein and the 38-kDa fragment (0.8-0.9 acetyl group/subunit). Nearly complete conversion of PGH synthase to 33- and 38-kDa fragments by exposure to high concentrations of trypsin prior to [3H]acetylsalicylic acid treatment results in labeling of the 38-kDa fragment, but not the 33-kDa fragment. The present findings are consistent with the presence of a membrane-binding domain (33 kDa) and an active site domain (38 kDa) in the 70-kDa subunit of PGH synthase. They also suggest that, following cleavage, the 38-kDa fragment retains the structural features responsible for the cyclooxygenase activity and selective aspirin labeling of PGH synthase. PGH synthase undergoes self-catalyzed inactivation by oxidants generated during its catalytic turnover. When PGH synthase, inactivated by treatment with arachidonic acid or hydrogen peroxide, was treated with trypsin it was cleaved two to three times faster than unoxidized enzyme. Addition of heme to oxidized PGH synthase did not reconstitute cyclooxygenase activity or resistance to trypsin cleavage. Spectrophotometric studies demonstrated that oxidatively inactivated enzyme did not bind heme. This implies that oxidation of protein residues as well as the heme prosthetic group is an important determinant of proteolytic sensitivity. Oxidative modification may mark PGH synthase for proteolytic cleavage and turnover.

Inhibition of thromboxane A synthesis in U937 cells by glucocorticoids. Lack of evidence for lipocortin 1 as the second messenger.

The mechanism of inhibition of eicosanoid synthesis by glucocorticoids has been investigated using differentiated U937 cells as a model. These cells synthesize thromboxane A2 (TXA2) in response to a variety of agonists, and synthesis of TXA2 initiated by certain stimuli was inhibited by pretreatment of the cells with glucocorticoids. The inhibitory response was specific for glucocorticoid steroids and required receptor occupancy based on both the rate of onset of the inhibitory activity and the correlation between potency and receptor affinity of various analogs. The inhibitory response was also specific for the agonist used to initiate TXA2 synthesis. Both lipopolysaccharide- and zymosan-induced TXA2 synthesis were inhibited by increasing concentrations of dexamethasone (greater than 80%, IC50 10 nM), while synthesis initiated by addition of either exogenous arachidonic acid or the Ca2+ ionophore A23187 was unaffected over the same concentration range. The latter result indicates that the dexamethasone block is upstream from release of esterified arachidonic acid. Attempts to localize the block more accurately showed that although dexamethasone was not acting as a generalized inhibitor of transcription or translation, its ability to inhibit TXA2 synthesis was mimicked by the activity of actinomycin D and cycloheximide. The role of the purported phospholipase inhibitor protein lipocortin 1 in mediating the dexamethasone inhibition of TXA2 synthesis was studied by examining the effect of dexamethasone on lipocortin 1 metabolism. Under conditions which gave maximal inhibition of lipopolysaccharide- or zymosan-stimulated TXA2 synthesis, dexamethasone had no effect on the steady state level of lipocortin 1 mRNA or protein, indicating that lipocortin 1 induction by dexamethasone is not responsible for the observed inhibition. Furthermore, lipocortin 1 was not secreted from the cells under any conditions examined, and the intracellular form had a relatively long half-life (greater than 21 h). The lack of induction of lipocortin 1 by dexamethasone and the fact that it is not released from the cells are both inconsistent with the properties previously described for lipocortin-like activities and indicate that lipocortin 1 is not a glucocorticoid second messenger in this experimental model. Although the data are consistent with a mechanism involving inhibition of a factor that activates TXA2 synthesis, we cannot rule out a mechanism involving glucocorticoid induction of a phospholipase inhibitor protein distinct from lipocortin 1.

Elevated prostaglandin H synthase gene expression in ras-transformed cells.

1. rPDGF stimulates PGE2 release in wild type, but not ras transformed NIH-3T3 cells. 2. Ras transformation blocks PGE2 release by inhibiting phospholipase C activation, IP3 synthesis, and Ca2+ mobilization. 3. rPDGF stimulation of wild type NIH-3T3 cells increases both prostaglandin H synthase (PGHS) mRNA levels and PGHS enzyme levels as measured by immunoblot. However, PGHS gene transcription is not required for PDGF-stimulated PGE2 release. 4. Ras transformed NIH-3T3 cells display elevated basal PGE2 synthesis, and very high levels of both PGHS mRNA and enzyme. rPDGF does not further stimulate PGHS gene transcription. 5. Exogenous PGE2 attenuates rPDGF-stimulated cell proliferation in both wild type and ras transformed cells. 6. These data suggest that increased PGHS gene expression and enhanced basal PGE2 synthesis may be in response to the unregulated growth of ras transformed cells.

BACE2 functions as an alternative alpha-secretase in cells.

BACE1 and BACE2 define a new subfamily of membrane-anchored aspartyl proteases. Both endoproteases share similar structural organization including a prodomain, a catalytic domain formed via DTG and DSG active site motifs, a single transmembrane domain, and a short C-terminal tail. BACE1 has been identified as the Alzheimer's beta-secretase, whereas BACE2 was mapped to the Down's critical region of human chromosome 21. Herein we show that purified BACE2 can be autoactivated in vitro. Purified BACE2 cleaves human amyloid precursor protein (APP) sequences at the beta-secretase site, and near the alpha-secretase site, mainly at A beta-Phe(20)--Ala(21) and also at A beta-Phe(19)--Phe(20). Alternatively, in cells BACE2 has a limited effect on the beta-secretase site but efficiently cleaves the sequences near the alpha-secretase site. The in vitro specificity of APP processing by BACE2 is distinct from that observed in cells. BACE2 localizes in the endoplasmic reticulum, Golgi, trans-Golgi network, endosomes, and plasma membrane, and its cellular localization patterns depend on the presence of its transmembrane domain. BACE2 chimeras that increase localization of BACE2 in the trans-Golgi network do not change its APP processing patterns. Thus, BACE2 can be distinguished from BACE1 on the basis of autoprocessing of the prosegment, APP processing specificity, and subcellular localization patterns.

Cloning, heterologous expression and characterization of murine interleukin 1 receptor antagonist protein.

A cDNA coding for the human interleukin 1 receptor antagonist protein (IL 1Ra) was used to clone the corresponding murine cDNA. The nucleotide sequence of the open reading frame coding for the processed form of mIL 1Ra predicted a 152-residue protein that was 77% identical to human IL 1Ra. The cellular and tissue distribution of murine IL 1Ra (mIL 1Ra) transcripts showed high levels in macrophages and skin while lower levels were detected in tissues that contain significant numbers of resident macrophages. The portion of the mIL 1Ra cDNA that codes for the mature form of the protein was placed under the control of a Trp promoter and expressed in E. coli at a level of 37% of total cell protein. The expressed protein was secreted into the periplasm and was purified to homogeneity in a single step by cation-exchange chromatography. Recombinant mIL 1Ra competitively inhibited 125I-labeled IL 1 alpha binding to murine type I IL 1R present on EL4 6.1 cells (Ki value of 0.21 nM) and antagonized IL 1-stimulated co-mitogenesis in murine thymocytes (0.7 x 10(6)-1.1 x 10(6) units/mg).

Peptide alignment of the porcine 21-hydroxylase cytochrome P-450 using a cDNA sequence of the corresponding bovine enzyme.

The amino acid sequence deduced from a cDNA clone of the bovine adrenal steroid 21-hydroxylase cytochrome P-450 has been utilized to align peptide sequences derived from the corresponding porcine enzyme. Homology analysis revealed that only fifty percent of the amino acid sequence predicted by the cDNA clone overlapped with peptide sequences from the porcine enzyme. The homology in the remaining portions of the bovine sequence was restored by considering amino acid sequences predicted by the two additional DNA reading frames of the cDNA sequence. Forty eight percent of the bovine sequences predicted by the two alternate reading frames showed strong homology with the porcine peptide sequences. A minimum of 4 nucleotide sequencing errors account for the observed reading frame alterations and the approximate position of each error in the bovine cDNA sequence has been established.

Sedimentation equilibrium analysis of five lipocortin-related phospholipase A2 inhibitors from human placenta. Evidence against a mechanistically relevant association between enzyme and inhibitor.

Five proteins from human placenta capable of inhibiting pancreatic phospholipase A2 were purified. Two of these proteins were identified as lipocortins I and II. The other three proteins were immunologically distinct from lipocortins I and II and had apparent subunit molecular masses of 32, 33, and 73 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequence analysis of peptides produced by cyanogen bromide digestion indicated sequence homology of these proteins with lipocortin I and the heavy chain subunit of lipocortin II. Two of these proteins were identified as endonexin II and 67-kDa calelectrin. The third protein appears to be the human form of bovine endonexin I, also characterized as porcine protein II. Sedimentation equilibrium analysis of lipocortin I, endonexin I and II, and the 67-kDa calelectrin suggested monomer-dimer equilibria with dissociation constants in the range of 0.33-1.3 X 10(-3) M and monomer molecular masses of 38,050, 36,400, 36,850, and 73,610 Da, respectively. Self-association of lipocortin II was described by dimerization of a protomer (K12 = 5.3 x 10(-7) M), followed by an indefinite self-association of the dimer (isodesmic dissociation constant, Kiso = 3.6 x 10(-6) M). The protomer molecular mass was 48,800 Da, consistent with a heterodimeric structure composed of one heavy (38,600 Da) and one light (10,944 Da) chain as previously characterized for lipocortin II. Sedimentation equilibrium analysis of mixtures of individual protein inhibitors and purified pancreatic phospholipase A2 indicated weak association between enzyme and inhibitor (Kd greater than or equal to 3 x 10(-5) M), insufficient to account for the observed inhibition of enzyme activity.

Suppression of monocyte function and differential regulation of IL-1 and IL-1ra by IL-4 contribute to resolution of experimental arthritis.

IL-4 has diverse effects on hematopoietic cells, including the ability to suppress certain mononuclear cell functions. To evaluate the effect of IL-4 on the evolution of acute and chronic arthritis, murine recombinant IL-4 was administered systemically to animals receiving an arthropathic dose of group A streptococcal cell wall fragments. Daily treatment with IL-4 had a minimal effect on the acute phase, but significantly suppressed the chronic, destructive phase. By 4 wk after initiation of disease, the articular index of IL-4-treated animals was reduced > 60% (articular index = 4 +/- 0.9) compared with the untreated rats (11.5 +/- 0.48, p < 0.001). A substantial decrease in the influx of inflammatory cells and virtual elimination of pannus and erosions occurred after IL-4 therapy. Associated with the reduced accumulation of mononuclear leukocytes was a decrease in their proinflammatory functions including cytokine production and reactive oxygen intermediate metabolism. These observations are consistent with the selective effects of IL-4 on phagocytic cell function demonstrated in vitro. Furthermore, IL-4 induced gene expression for IL-1ra, a protein that antagonizes the action of IL-1 by binding to the IL-1 receptor without agonist activity. Through an expanding spectrum of effects on monocyte-macrophage phenotypic and functional parameters, IL-4 is emerging as an important inhibitor of cell-mediated immune responses and pathogenic processes.

Cloning and characterization of two K+ inward rectifier (Kir) 1.1 potassium channel homologs from human kidney (Kir1.2 and Kir1.3).

The DNA sequence encoding the rat brain inward rectifier-10 K+ channel was amplified from rat brain RNA using reverse transcription-polymerase chain reaction and used to clone the human homolog. Low stringency screening of a human kidney cDNA library and subsequent DNA sequence analysis identified two related K+ inward rectifier cDNAs, referred to as Kir1.2 and Kir1.3, which were derived from transcription of distinct human genes. Kir1.2 represents the human homolog of the rat BIRK-10 sequence, whereas Kir1.3 was unique compared with all available sequence data bases. The genes that encode Kir1.2 and Kir1.3 were mapped to human chromosomes 1 and 21, respectively. Both genes showed tissue-specific expression when analyzed by Northern blots. Kir1.2 was only detected in brain >> kidney and was detected at high levels in all brain regions examined. Kir1.3 was most readily detected in kidney and was also expressed in pancreas > lung. Comparative analysis of the predicted amino acid sequences for Kir1.2 and Kir1.3 revealed they were 62% identical. The most remarkable difference between the two polypeptides is that the Walker Type A consensus binding motif present in both Kir1.1 and Kir1.2 was not conserved in the Kir1.3 sequence. Expression of the Kir1.2 polypeptide in Xenopus oocytes resulted in the synthesis of a K+-selective channel that exhibited an inwardly rectifying current-voltage relationship and was inhibited by external Ba2+ and Cs+. Kir1.2 current amplitude was reduced by >85% when the pH was decreased from pH 7.4 to 5.9 using the membrane-permeant buffer acetate but was relatively unaffected when pH was similarly lowered using membrane-impermeant biphthalate. The inhibition by intracellular protons was voltage-independent with an IC50 of pH 6.2 and a Hill coefficient of 1.9, suggesting the cooperative binding of 2 protons to the intracellular face of the channel. In contrast, Kir1.3 expression in Xenopus oocytes was not detectable despite the fact that the cRNA efficiently directed the synthesis of a polypeptide of the expected Mr in an in vitro translation system. Co-expression of Kir1.3 with either Kir1.1 or Kir1.2 reduced currents resulting from expression of these inward-rectifier subunits alone, consistent with a dominant negative influence on Kir1.1 and Kir1.2 expression.