Mutational analysis of third cytoplasmic loop domains in G-protein coupling of the HM1 muscarinic receptor. We measured dose-response curves for carbachol stimulation of phosphatidyl inositol (PI) turnover with mutants of the Hm1 muscarinic cholinergic receptor having various deletions from amino acids 219 to 358 of the large third intracellular (i3) loop (208 to 366). These deletions had only small or no effects on the ability of Hm1 transfected into HEK 293 cells to stimulate PI turnover. This result indicates that only small regions of 9 to 11 amino acids adjacent to trans-membrane domains (TMDs) 5 and 6 can be directly involved in G protein coupling. Point mutations were constructed to test the role of charged amino acids in these junctions. A triple point mutation of Hm1 (E214 A/ E216K/ E221 K), which mimics the charge distribution in Hm2 (negatively coupled to cAMP) over the first 14 amino acids of i3, and a double point mutation in the N terminal junction, K359A/K361A, both failed to affect carbachol stimulated PI turnover. Therefore, charge distribution in the loop junctions appears to play a minor role in G protein coupling of Hm1 in HEK 293 cells.


Human trifunctional protein deficiency: a new disorder of mitochondrial fatty acid beta-oxidation. In this paper we report the identification of a new disorder of mitochondrial fatty acid beta-oxidation in a patient which presented with clear manifestations of a mitochondrial beta-oxidation disorder. Subsequent studies in fibroblasts revealed an impairment in palmitate beta-oxidation and in addition, a combined deficiency of long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA-dehydrogenase and long-chain 3-oxoacyl-CoA thiolase. The recent identification of a multifunctional, membrane-bound beta-oxidation enzyme protein catalyzing all these three enzyme activities (Carpenter et al. (1992) Biochem. Biophys. Res. Commun. 183, 443-448; Uchida et al. (1992) J. Biol. Chem. 267, 1034-1041) suggested an underlying basis for this peculiar combination of three enzyme deficiencies. We show by means of size-exclusion chromatography that there is, indeed, a deficiency of the multifunctional beta-oxidation enzyme protein in this patient.


Multiple cis-acting elements of the proximal promoter region are required for basal level transcription of the H1(0) histone gene. Basal level transcription of the mouse histone H1(0) gene is mediated by 531 base pairs of the promoter region. Deletion of the most distal upstream 80 bp of this fragment reduces transcription to very low values. By in vitro footprinting we demonstrate now that multiple factors bind to the DNA fragment localized between the 80 bp and the cap nucleotide. In addition to the presence of motifs for the binding of SP1, H1-box, H4TF-2 and TATA-box-factors, other not yet described protein-binding elements were identified. Internal deletions in the wild type promoter enclosing these motifs strongly restrict transcription. Furthermore, when one of these motifs was modified by site-directed mutagenesis a strong impairment of transcription followed. Thus for basal level transcription, in addition to the 80 bp distal fragment, cis-acting elements localized in the 450 bp proximal promoter region are required.


Intracellular [Ca2+] and K+ and Cl- efflux responses in submandibular cells of neonatal and adult rats. The effects of graded doses (5 x 10(-8) to 10(-5)) acetylcholine on intracellular Ca2+ and on 86Rb and 36Cl efflux were compared in submandibular cell clusters of 1 and 7 day-old and adult rats. Initial Ca2+ peaks were similar at agonists concentrations lower than 10(-7) M but the release of Rb+ and Cl- were smaller in cells of young animals. At higher agonist concentrations, Ca2+ peaks were higher in immature cells; however, initial Cl- (but not Rb+) efflux was similar to that of mature cells. Plateau Ca2+ levels were independent of age and agonist concentrations but the content of Cl- and Rb+ varied greatly and differences between age groups were less evident. These data confirm a dissociation between intracellular Ca2+ levels and Ca(2+)-mediated ion transport in immature salivary cells.


In vitro induction of cecropin genes--an immune response in a Drosophila blood cell line. The Drosophila melanogaster cell line mbn-2 was explored as a model system to study insect immune responses in vitro. This cell line is of blood cell origin, derived from larval hemocytes of the mutant lethal (2) malignant blood neoplasm (1(2)mbn). The mbn-2 cells respond to microbial substances by the activation of cecropin genes, coding for bactericidal peptides. The response is stronger than that previously described for SL2 cells, and four other tested Drosophila cell lines were totally unresponsive. Bacterial lipopolysaccharide, algal laminarin (a beta-1,3-glucan), and bacterial flagellin were strong inducers, bacterial peptidoglycan fragments gave a weaker response, whereas a formyl-methionine-containing peptide had no effect. Experiments with different drugs indicate that the response may be mediated by a G protein, but not by protein kinase C or eicosanoids, and that it requires a protein factor with a high rate of turnover.


PI-specific phospholipase C "alpha" from sheep seminal vesicles is a proteolytic fragment of PI-PLC delta. Phosphatidylinositide-specific phospholipase C enzymes (PLCs) catalyze the conversion of the phosphoinositides to biologically important signal transducing molecules. These enzymes may be grouped into "families" which share similar structures and modes of regulation. The existence of a structurally distinct family of PLC termed "alpha" has been recently called into question. In the current paper we show by immunoblotting experiments that PLC "alpha" from sheep seminal vesicles is recognized by monoclonal antibodies raised against the delta 1 isoform of bovine brain PLC, and appears to be derived from a higher molecular weight band at 85 kDa. We also show that antibodies raised against PLC alpha efficiently immunoprecipitate the 85-kDa PLC delta 1 isoform from bovine brain and Chinese hamster lung fibroblasts. These data provide strong evidence that the PLC alpha from sheep seminal vesicles is a proteolytic fragment of PLC delta 1. Thus, there is still no conclusive evidence for a separate "alpha" class of PLC.


Platelet derived growth factor induces c-fos and c-myc mRNA in rat aortic smooth muscle cells in primary culture without elevation of intracellular Ca2+ concentration. Platelet derived growth factor (PDGF) has been shown to induce c-fos and c-myc proto-oncogenes. In the present study, we investigated the effects of genistein, a tyrosine kinase inhibitor, and NiCl2, a Ca2+ influx blocker, on PDGF-induced Ca2+ transient and on expression of c-fos and c-myc mRNA. In the presence of extracellular Ca2+, PDGF induced elevation of intracellular Ca2+ concentration ([Ca2+]i) and increases in c-fos and c-myc mRNA, as detected by reverse transcription polymerase chain reaction (RT-PCR) and Northern hybridization. PDGF-induced [Ca2+]i elevation was composed of an initial transient increase (first component) followed by steady state elevation (second component). Genistein (10 microM) blocked the 1st, but not the 2nd, component of [Ca2+]i elevation induced by PDGF. NiCl2 (1 mM) and removal of extracellular Ca2+ inhibited the 2nd, but not the 1st, component. In the presence of 10 microM genistein and 1 mM NiCl2, PDGF induced c-fos and c-myc mRNA, although the [Ca2+]i elevation could be completely blocked by these two agents. These results indicate that elevation of [Ca2+]i is not a prerequisite condition for PDGF to induce c-fos and/or c-myc mRNA in rat aortic smooth muscle cells in primary culture.


Specific blockade of basic fibroblast growth factor gene expression in endothelial cells by antisense oligonucleotide. The migration and proliferation of endothelial cells play a pivotal role in various vascular diseases. To elucidate the role of endogenous basic fibroblast growth factor (bFGF) produced within endothelial cells on cell growth, we introduced the antisense oligonucleotide complementary to bFGF mRNA into cultured bovine aortic endothelial cells by cationic liposome to block the production of autocrine bFGF. The treatment of the endothelial cells with the specific antisense oligomer efficiently inhibited the synthesis of bFGF with the concomitant suppression of endothelial proliferation, indicating the significant role of bFGF as an endothelial growth promotor. The neutralizing antibody against bFGF had no inhibition on basal DNA synthesis of the endothelial cells, in contrast to marked suppressive action of bFGF antisense oligomer. The results provide the new analytic and therapeutic implications in the use of the antisense methodology for the study of vascular biology.


Molecular cloning and sequencing of an alternatively spliced form of the human thyrotropin receptor transcript. In the present study, we report the molecular cloning and sequencing of an alternatively spliced form of the human thyrotropin receptor (hTSHR) mRNA transcript, which has previously been detected on Northern blot analysis of human thyroid cells. The smaller hTSHR cDNA, designated hTSHR cDNA-I, is approximately 1 kb in size and encodes a protein of 253 amino acids. Comparison of the nucleotide sequence of hTSHR cDNA-I with available hTSHR genomic sequence data reveals that the cDNA-I contains exons 1-8 and unidentified DNA tract, presumably an intron. Thus, the hTSHR cDNA-I encodes for the N-terminal half of the extracellular domain of the hTSHR (approximately 60%). The truncated TSHR-I may be secreted and function as a TSH binding protein.


Unusual pressure dependence of the lateral motion of pyrene-labeled phosphatidylcholine in bipolar lipid vesicles. The lateral mobility of a pyrene-labeled phosphatidylcholine probe in liposomes containing archaebacterial bipolar lipids has been studied isothermally as a function of pressure. The pressure-dependence of the probe mobility, R, is found to be slightly positive or zero in the temperature range of 17 - 48 degrees C. At temperatures > 48 degrees C, R becomes negative and decreases with temperature. The data indicate that lateral mobility only becomes appreciable at high temperatures. In addition, the R values obtained with other lipid membranes are much lower than that obtained with bipolar liposomes, implying that the membranes of archaebacterial liposomes are laterally immobile, as compared to other lipid membranes.


Models of stratum corneum intercellular membranes: the sphingolipid headgroup is a determinant of phase behavior in mixed lipid dispersions. During formation of the intercellular membranes of mammalian stratum corneum, sphingomyelin and glucosylceramide are converted enzymatically to ceramide. To model in isolation the possible effect of such a lipid modification on the phase behavior of the ensemble, we used proton and deuterium nuclear magnetic resonance to compare an equimolar dispersion of bovine brain sphingomyelin, cholesterol, and perdeuterated palmitic acid (at pH 6.2), with an equivalent dispersion in which bovine brain ceramide was substituted for sphingomyelin. While the sphingomyelin dispersions remain in a homogeneous fluid lamellar phase from 20-75 degrees C under these conditions, those containing ceramide display complex polymorphism.


Alternative splicing of the mouse amelogenin primary RNA transcript contributes to amelogenin heterogeneity. A heterogeneous population of amelogenin proteins is derived from a single copy of the mouse amelogenin gene. To investigate the one gene--multiple protein enigma, we designed a study to distinguish between alternative splicing and proteolytic cleavage models. A pulse of [35S]methionine labeling demonstrated that multiple amelogenins are synthesized concurrently, a result consistent with an alternative splicing mechanism. Using reverse transcription and polymerase chain reaction we cloned a segment from the 5' end of a mouse amelogenin mRNA and connected it to a previously isolated abbreviated cDNA clone. Four additional cDNAs derived from alternatively spliced amelogenin mRNAs have been cloned and characterized. The five transcripts encode amelogenins 180, 156, 141, 74, and 59 amino acids in length.


Studies of the relationship between cell proliferation and cell death. II. Early gene expression during concanavalin A-induced proliferation or dexamethasone-induced apoptosis of rat thymocytes. Several data in the literature suggest that an intriguing relationship exists between cell proliferation and cell death. Accordingly, we studied the early expression of different genes in the same cells, i.e. rat thymocytes, undergoing cell proliferation upon stimulation with Concanavalin A or cell death following dexamethasone treatment. We showed that an early accumulation of c-fos, c-jun and c-myc mRNA occurred in both phenomena but with different kinetics. It can be speculated that the early induction of nuclear oncogenes is necessary to allow the later induction of other genes probably regulated at the transcriptional level by the AP-1 complex and/or by Myc protein. The accumulation of the transcript for another gene, i.e. poly(ADP-ribose)polymerase, an enzyme responsible for posttranslational modifications of several nuclear proteins, could instead be related to chromatin modifications occurring in both processes.


Enhanced production of platelet-activating factor in stimulated rat leukocytes pretreated with triacsin C, A novel acyl-coA synthetase inhibitor. Triacsin C, a product of Streptmyces sp. SK-1894, was previously reported as an inhibitor of long chain acyl-CoA synthetase. Pretreatment with triacsin C (500 nM) for 1h enhanced production of platelet-activating factor in rat neutrophils, followed by stimulation with A23187 or fMLP. Amount of lyso-PAF was also augumented. Triacsin C alone did not increase PAF content and did not modulate enzymatic activities of acytransferase, cholinephosphotransferase, acetylhydrolase, acetyltransferase or phospholipase A2. These results suggest that triacsin C might enhance supply of substrate for PAF synthesis, i.e. accumulation of lyso-PAF by interfering reacylation pathway.


Characterization of high mobility group protein binding to cisplatin-damaged DNA. cis-Diamminedichloroplatinum (II) (cisplatin, CDDP) is a widely used chemotherapeutic agent. While many tumors are highly responsive to CDDP, certain tumors are resistant to this drug, limiting its efficacy. The anti-tumor activity of CDDP is believed to result from its coordination bonding to chromosomal DNA. Alterations in tumor cell sensitivity to CDDP may result from the presence or absence of protein(s) which specifically recognize CDDP-damaged DNA. We have developed a damaged-DNA affinity precipitation assay that allows the direct identification of cellular proteins that bind to CDDP-damaged DNA. Using this procedure, we have identified several proteins which specifically bind to CDDP-damaged DNA. Two of these proteins have been identified as high mobility group proteins (HMG) 1 and 2 in the current report, we have characterized the binding of these proteins to CDDP-DNA. The calculated Kd of binding to CDDP-damaged DNA was 3.27 x 10(-10) for HMG1 and 1.87 x 10(-10) for HMG2. Using highly specific chemical modifying reagents, we have determined that Cys residues play an important role in protein binding. We also observed that HMG2 will bind to DNA modified with carboplatin and iproplatin although to a lesser extent than to DNA damaged with CDDP. Thus, our results indicate that HMG 2 binds with high affinity to DNA modified with therapeutically active platinum compounds. In addition, our findings suggest that thiol groups play an essential role in the binding of HMG1 and HMG2 to CDDP-DNA.


Phospholipase A2 activity is not involved in the tumor necrosis factor-triggered apoptotic DNA fragmentation in bovine aortic endothelial cells. Using the arachidonic acid release as a probe of phospholipase A2 activity, we tested the involvement of this enzyme in the TNF-triggered apoptotic cell death in the bovine aortic endothelial cells. We observed that TNF induced a liberation of arachidonic acid from these cells which was comparable to that obtained from the L929 cells. An augmentation of the amount of released arachidonic acid or the reduction of the TNF-stimulated phospholipase A2 activity did not modify the TNF-induced DNA fragmentation in the endothelial cells. We suggest that these events are not required for TNF apoptotic cytotoxicity in the endothelial cells.


Human neutrophil proteinase 3: mapping of the substrate binding site using peptidyl thiobenzyl esters. A series of peptidyl thiobenzyl esters was used to map the active site of human leukocyte proteinase 3. The steady-state kinetics parameters reveal the following features regarding the substrate specificity of proteinase 3 and its putative active site: (a) the preferred P1 residue is a small hydrophobic amino acid such as aminobutyric acid, norvaline, valine or alanine (in decreasing order of preference); (b) the enzyme has an extended active site; and (c) its active site is similar to that of the related serine proteinases leukocyte elastase and leukocyte cathepsin G.


Up-regulation of MSH receptors by MSH in Cloudman melanoma cells. MSH can up-regulate MSH binding capacity of cultured Cloudman melanoma cells in a dose- and time-dependent fashion. Binding is mediated through proteins exhibiting an apparent molecular weight of 50-53kDa, consistent with previous studies implicating them as the principal MSH receptors on Cloudman cells. Pre-incubation of cells with MSH stimulates expression of the receptor proteins both on the plasma membrane surface as well as in internal sites associated with coated vesicles. The effects of MSH are additive with those of UV light, suggesting that UV and MSH might stimulate receptor expression through separate mechanisms.


Evidence that a ryanodine receptor triggers signal transduction in the osteoclast. We have investigated the effect of the alkaloid ryanodine on the release of intracellularly stored Ca2+ in response to activation of the osteoclast Ca2+ receptor by the surrogate agonist, Ni2+, Ni2+ (6 mM) in the presence of ethylene-glycol bis-(aminoethyl ether) tetraacetic acid (EGTA) (1.2 mM) and valinomycin (5 microM) induced a transient elevation of cytosolic [Ca2+] in fura 2-loaded osteoclasts. This transient was superimposed upon a small steady elevation of cytosolic [Ca2+] induced by the initial application of valinomycin alone. Ryanodine (10 microM) completely abolished such responsiveness. However, cytosolic [Ca2+] transients were restored when osteoclasts were depolarized by the extracellular inclusion of 100 mM-[K+] in the same solution. Thus, we demonstrate a sensitivity of the osteoclast signal transduction system to ryanodine for the first time to our knowledge.


Molecular cloning and characterization of rabbit pancreatic triglyceride lipase. Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) has been cloned from a gt11 cDNA library made from poly A+ RNA of adult rabbit pancreas. Pancreatic lipase (PL) assists the absorption of dietary triglycerides by hydrolyzing them at 1 and 3 positions to free fatty acids and 2-monoacylglycerol in the presence of bile acids and colipase in the intestinal lumen. Since rabbits are classifically used for the study of the diet induced changes in the lipid metabolism, as a prelude to studying the diet and age dependent changes in the expression of this enzyme, a full length PL cDNA clone was obtained from its pancreas. The coding region of rabbit pancreatic lipase cDNA consists of 1407 base pairs contained in an open reading frame encoding 469 amino acids including the 16 that constitute the signal peptide. Northern blot analysis revealed a band around 1.5 kb. When rabbit enzyme is compared to other species, an over all homology of 70-80% was observed at the nucleotide level. High homology in the amino acid sequence and composition is also apparent between rabbit and other species like dog (65%), pig (76%) and rat (63%). Highest homology is found to be around active-site serine. The regions of homology with other species may help to define sites of interaction of lipase with co-lipase.


Identification of jun-B as third member in human antioxidant response element-nuclear proteins complex. Deletion mutagenesis in human NAD(P)H:Quinone Oxidoreductase (NQO1) gene and transfection studies into mammalian cells identified a segment of DNA designated as human Antioxidant Response Element (hARE) responsible for high basal expression in tumor cells and its induction by beta-naphthoflavone (beta-NF). The twenty four base pairs of the hARE contains an essential cis-element AP1 binding site and has been shown to bind to jun-D and c-fos proteins from mouse hepatoma (Hepa-1) nuclear extract. In the present report, we have identified jun-B as the third major protein in the hARE-Hepa-1 proteins complex observed in the band shift assays.


The role of protein kinase C in the induction of nitric oxide synthesis by murine macrophages. The role of protein kinase C (PKC) in the induction of nitric oxide synthesis by interferon-gamma (IFN-gamma) was investigated using two murine macrophage cell lines, J774 and RAW 264.7. Nitric oxide (NO) production was markedly reduced by a PKC inhibitor, Ro31-8220 in a dose-dependent manner. Incubation of cells with IFN-gamma resulted in translocation of PKC to the cell membrane. Prolonged incubation of cells with a high concentration of phorbol ester, which down-regulated PKC activity, also reduced nitric oxide production. These findings provide evidence that PKC is involved in the induction of nitric oxide synthesis by IFN-gamma.


Isolation of a novel nuclear glycoprotein from pig kidney. A nuclear glycoprotein with an apparent Mr of 66,000 Da has been isolated from pig kidney chromatin after extraction with urea, guanidine-HCl and 2 M NaCl, and some of its structural features have been characterized. It belongs to the group of N-glycosylated proteins, which in the nucleus has so far received little attention. From its monosaccharide composition and recognition by lectins its oligosaccharides appear to be of high mannose and/or hybrid types. Some properties of its protein moiety suggest that it has a role in the packing of the DNA loops in the condensed chromatin.


Calcium uptake by liver mitochondria from hypothyroid rats is inhibited in vitro by triiodothyronine. The rate of Ca2+ uptake by liver mitochondria from hypothyroid and euthyroid rats was determined as a function of the mitochondrial membrane potential (delta psi). At large values of delta psi Ca2+ uptake was a linear function of delta psi and its dependence on delta psi was the same in mitochondria from euthyroid and hypothyroid rats. However, at small values of delta psi the rates of Ca2+ uptake for mitochondria from euthyroid or hypothyroid rats deviated slightly from each other. Triiodothyronine (T3), at a concentration of 10 nM, inhibited the rate of Ca2+ uptake in vitro, suggesting that T3 in vivo may regulate mitochondrial Ca2+ transport and thus alter the matrix free Ca2+ concentration, which has control over respiration.


Purification and properties of urease from the cyanobacterium Anabaena cylindrica. A purification procedure has been developed by which urease activity in extracts from the cyanobacterium Anabaena cylindrica was enriched 500-fold. The procedure involves MgSO4 precipitation at 55 degrees C and chromatography on hydroxylapatite and diethylaminoethyl sephadex. Its molecular weight was measured by sedimentation equilibrium in an airfuge to be 197,000 +/- 2000 with an estimated subunit molecular weight of 32,000 as determined by polyacrylamide gel electrophoresis. The pH- and temperature-dependence of the enzyme were determined and the activity found to be optimal at pH 8 and 30 degrees C, respectively. The concentration-dependence of the activation of the enzyme by Mg++ was measured, as were the effects on activity of a range of other metal ions.


Effect of endotoxin on rat liver regeneration after partial hepatectomy. Lipopolysaccharide administered intraperitoneally to rats just after partial (70%) hepatectomy at the dose from 0.04 to 4 mg/kg body weight inhibited the induction of thymidylate synthase and thymidine kinase which are rate-determining enzymes of DNA synthesis, resulting in the impairment of DNA synthesis in the regenerating liver. This effect may be due to the hepato-toxic nature of endotoxin.


Molecular analysis of genetic mutation in electrophoretic variant of human lactate dehydrogenase-A(M) subunit. An electrophoretic variant of lactate dehydrogenase-A (M) subunit was discovered in a patient with multiple myeloma. DNA analysis of the variant allele revealed a nucleotide substitution (transition) of C to T at codon 314 (CGT-TGT), and this mutation resulted in the replacement of an arginine by a cysteine (R314C). This amino acid replacement affects the net charge of the subunit and makes the LDH-A variant have a faster electrophoretic mobility. The responsible missense mutation created a new restriction site, AGGCCT, which can be simply detected by endonuclease AatI digestion. In addition, four synonymous substitutions with no amino-acid replacements were found at codons 51, 119, 163 and 175 in the LDH-A gene from the patient.


Age-specific development of malate-aspartate shuttle in the liver and kidney of mice. The activities of malate-aspartate shuttle enzymes were measured in the liver and kidney of 15-, 30-, and 60-day old mice. The results indicate that the activities (U/mg protein) of both isoenzymes (cytosolic and mitochondrial) of both malate dehydrogenase and aspartate aminotransferase are significantly higher in the liver of 15-day old mice than in the liver of 30- and 60-day old animals. However, the shuttle enzymes showed a peak value in the kidney of 30-day old mice. In vitro reconstitution of malate-aspartate shuttle showed a similar pattern of activity in the tissues studied. These findings suggest that the activity of malate-aspartate shuttle is expressed differentially in these tissues of mice at different postnatal ages.


Changes in ADP-ribosylation of rho-related protein by exoenzyme C3 from Clostridium botulinum with maturation in rat testis. A rho-related protein, which was ADP-ribosylated by exoenzyme C3 from Clostridium botulinum, was found in both cytosol and membrane fractions of the testes of adult rats. This protein was observed in the cytosol fraction of the testes already in the newborn, did not significantly change in level up to about 7 weeks, and thereafter became clearly reduced. In the membrane fraction, in contrast, the protein was not found in the newborn rats. These observations suggest that there are some relationships between the maturation of the testes and changes in the subcellular distribution of this rho-related protein.


Six-base deletion occurring in messages of human cytochrome P-450 in the CYP2C subfamily results in reduction of tolbutamide hydroxylase activity. We isolated and expressed a clone, hPA6, possibly corresponding to the CYP2C9 cDNA. Compared with the other CYP2C9 cDNA clones, hPA6 showed a 6-nucleotide deletion near its middle. From the same cDNA library, we could also isolate another cDNA clone, named hPA22, which retained the 6 bases. For clarification of the effect of the 2-amino acid deletion resulting from the 6-base deletion on enzymatic activities, both clones were expressed in yeast. The expressed enzymes showed tolbutamide hydroxylase activities, and these activities were inhibited by antibodies against P-450-HM2, a probable CYP2C9. The activity of the enzyme encoded by hPA6 was lower than that encoded by hPA22; thus the 2-amino acid deletion in the CYP2C9 reduced the enzymatic activity.


Cell proliferation-related production of matrix metalloproteinases 1 (tissue collagenase) and 3 (stromelysin) by cultured human rheumatoid synovial fibroblasts. We investigated the effects of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin on the cell proliferation of and the production of matrix metalloproteinases (MMPs) by rheumatoid synovial fibroblasts in order to determine the role of these agents in rheumatoid arthritis. PDGF stimulated rheumatoid synovial fibroblasts to increase DNA synthesis and the production of precursor forms of MMP-1 of M(r) = 53,000 and -3 of M(r) = 57,000. EGF and insulin also increased DNA synthesis and the production of these enzymes, but the amount of DNA or MMPs was smaller than that induced by PDGF. Since the production of matrix macromolecules and their degradation is essential for the remodelling of synovial tissue in rheumatoid arthritis, these data suggest that the production of MMP-1 and-3 by rheumatoid synovial fibroblasts in relation to cell proliferation plays an important role in the pathological process of rheumatoid arthritis.


Decrease in immunoreactivity and vasoactivity of endothelin-1 after exposure to oxygen. Endothelin-1 (ET-1) caused a dose-dependent contraction of porcine coronary arterial rings. Preincubation of ET-1 in oxygenated Krebs solution at 37 degrees C resulted in a progressive loss of the contractile activity and immunoreactivity of ET-1. The half-life of ET-1 contractile activity in oxygenated and non-oxygenated buffer was 16.1 and 86.6 min., respectively. The molecular weight of ET-1 exposed to oxygen was identical to that of ET-1. These results indicate that during the contraction of smooth muscle preparations the levels of ET-1 in the tissue bath are steadily decreasing due to exposure to high pO2 levels.


Purification and partial characterization of a trypsin inhibitor from chick skeletal muscle. A protein capable of inhibiting trypsin and a number of other serine proteases was purified from chicken skeletal muscle. It has an apparent molecular weight of 64,000 as determined by gel filtration. The inhibitor molecule binds trypsin at a molar ratio of 1:1 to form a stable complex, in which trypsin can be completely inhibited. In this complex, the inhibitor is extensively digested by trypsin but retains its inhibitory activity and tertiary structure by intramolecular disulfide bonds. In addition, its activity was found to markedly increase during development of embryonic muscle. The physiological role of this inhibitor, however, remains unknown.


Cu(2+)-induced lipid oxidation in plasma: questionable relation between cholesterol oxidation and LDL modification. Oxidatively modified low-density lipoproteins (LDL) may be involved in the process of cholesterol deposition in arteries. Because of their cytotoxicity, oxysterols resulting from cholesterol oxidation could be a contributing factor in this process. Studies in this area have generally been performed on purified LDL, but in our research whole plasma was exposed to the oxidizing action of copper. Oxidation of the ring structure, which is at the origin of oxysterols, apparently occurs once most polyunsaturated fatty acids have disappeared. Hydrated LDL density reaches a value identical to that obtained during oxidation of LDL by endothelial cells, whereas the ring structure remains unmodified.


Allosteric activation by nucleotides of the inactive by phosphatase ornithine decarboxylase of Escherichia coli. ODC was purified to homogeneity from E. coli K12 MG1655 strain transformed with a pBR322 plasmid carrying the ODC gene. This preparation was homogeneous as it was analyzed by SDS-polyacrylamide gel electrophoresis. From this preparation the amino-terminal sequence analysis was obtained. The native ODC of E. coli is activated by ATP, GTP, CTP and UTP at 10(-3) M concentration to around 170-300%. Our results indicate that the recombinant ODC is activated only by GTP and UTP at 10(-3) M 370% and 300%, respectively. When the recombinant ODC was incubated with calf intestine alkaline phosphatase, this inactive ODC can be reversibly activated allosterically only by GTP or UTP at a concentration of 10(-6) or 10(-5) M. That GTP or UTP can allosterically convert the inactive form of ODC to an active form suggests that these analogues may be the in vivo physiological regulators of ODC.


Characterization of bovine heparin-binding neurotrophic factor (HBNF): assignment of disulfide bonds. The topology of the disulfides in native heparin-binding neurotrophic factor (HBNF) isolated from bovine brain was studied by proteolytic digestion using trypsin, Asp-N endoproteinase and chymotrypsin and peptide mapping. Disulfide-linked peptides were identified by automated Edman degradation. It has been shown that there are disulfide bonds between Cys15-Cys44, Cys23-Cys53, Cys30-Cys57, Cys67-Cys99 and Cys77-Cys109.


Studies on variations of glycoproteins and lysosomal hydrolases in human uterine cervical carcinoma. Variations of glycoprotein components such as hexose, hexosamine, and sialic acid and lysosomal glycosidases such as beta-D-glucosidase, beta-D-galactosidase, and N-acetyl-beta-D-glucosaminidase were studied in the tumor tissue from various stages of cervical carcinoma of the uterus. Carbohydrate components of glycoprotein were found to be markedly reduced, and the reduction was very much significant in hexosamine and sialic acid in the advanced stages. Lysosomal glycosidases exhibited a significant increase, and among them N-acetyl-beta-D-glucosaminidase exhibited a prominent increase, in the advanced stages of cervical carcinoma of the uterus.


Occurrence of free D-aspartic acid in marine macroalgae. The biologically rare D-aspartic acid was found in extracts of marine macroalgae. DL-aspartic acid was isolated from the Phaeophyta, Hizikia fusiformis, by ion-exchange chromatography, and crystallized from aqueous ethanol. It was characterized by optical-rotatory-dispersion analysis and reversed-phase HPLC analysis. The crystal consisted of equal amounts of D and L isomers. The presence of free D-aspartic acid was verified in 15 species from among 20 species of marine macroalgae by the same method (HPLC). Generally, Phaeophyta contained a high concentration of D-aspartic acid in contrast with the low concentration in Phycophyta and Rhodophyta. Particularly in Costaria costata, Hizikia fusiformis and Sargassum yezoensis, belonging to Phaeophyta, D-aspartic acid was found in concentrations proportional to those of L-aspartic acid.


Anticoagulant action of rare earth metals. Some of the lanthanides, the rare earth metals, lanthanum (La), cerium (Ce), neodymium (Nd), samarium (Sm), terbium (Tb), dysprosium (Dy), erbium (Er) and ytterbium (Yb) prolonged the clotting time of normal human plasma in a dose-dependent manner when clotting was induced either by thromboplastin or by kaolin in the presence of cephalin and Ca2+. They also prolonged the activated factor X induced clotting time of platelet-rich plasma. The amidolytic activities of activated factor X and thrombin progressively decreased with increasing amount of rare earth metals. These results suggested that the rare earth metals appear to show their anticoagulant effect with mechanisms in part the inhibition of the enzymatic activities of both activated factor X and thrombin.


Action of a nuclease from rat nuclei on UV-irradiated DNA. An Mg(2+)-dependent nuclease was highly purified from rat-liver nuclei. The nuclease activity was enhanced in ultraviolet light (UV)-irradiated dsDNA, but not in unirradiated dsDNA. Irrespective of UV irradiation, ssDNA was readily cleaved by this enzyme. UV-irradiated plasmid DNA was incubated with this enzyme and subjected to the template primer activity assay with a Klenow polymerase. With increasing incubation time, the activity was enhanced in the circular-relaxed and linear forms, but not in the superhelical form. These results implied that this enzyme excises UV-damaged sites in dsDNA to form single strand gaps for repair synthesis.


Insulin and IGF1 receptors in a model renal epithelium: receptor localization and characterization. Insulin and IGF1 stimulate transepithelial Na+ transport in the urinary bladder of the toad Bufo marinus, a model renal epithelium. The signal transduction mechanisms for the natriferic action are unknown. Ultrastructural techniques were used to localize both receptors and ligands in the epithelium. Electron microscopy using gold-labelled anti-insulin or anti-IGF1 receptor antibodies demonstrated the majority of the insulin receptors were associated with the basolateral membrane while IGF1 receptors were found on basolateral and apical membranes. Both insulin and IGF1 receptors were found in endosomes and on the membranes surrounding subapical granules. In intact tissues incubated with iodinated IGF1 or insulin, both ligands were associated with the basolateral membrane. IGF1 was internalized to a greater extent than insulin and only IGF1 accumulated in cell nuclei.


Polymeric ovotransferrin and its ability to bind and deliver iron to chick-embryo red blood cells. Polymeric forms of hen ovotransferrin have been obtained by storage at 4 degrees C for 5 years or at 57 degrees C for 14 days and fractionated as dimers and tetramers by gel filtration on Sephadex. The ability of tyrosine to undergo nitration was reduced in the tetrameric protein, so that one could hypothesize that dityrosine formation is somewhat responsible of such polymerization process. Experimental data on the biological functionality showed that: i) dimeric ovotransferrin was able neither to bind nor to deliver iron; ii) tetrameric ovotransferrin was able to bind but not to deliver iron.


Cholera toxin diminishes tyrosine kinase activity of the insulin receptor. We have examined the effect of cholera toxin (CT) on the insulin receptor tyrosine kinase. Incubation of intact rat hepatoma cells FaO with CT (1 microgram/ml/2h) inhibited insulin-induced receptor autophosphorylation by 30% in vivo. This effect persisted after receptor purification in vitro. CT did not alter hormone binding of the insulin receptor, indicating that the toxin affects signal transduction of insulin at the level of the receptor kinase. Experiments using chinese hamster ovary (CHO) cells transfected either with the human insulin receptor (HIR) or a mutant lacking the last 43 amino acids of the receptor beta-subunit (HIR delta CT) showed, that the carboxy-terminal tail of the insulin receptor does not play a role in the suppressive effect of the toxin on the insulin receptor kinase.


Relationship between Ca2+ and L-asparagine in the induction of ornithine decarboxylase in H-35 rat hepatoma cells. The activity of ornithine decarboxylase (ODC) in H-35 hepatoma cells depleted of Ca2+ by washing with 2 mM EGTA increased 35-fold after incubating for 4 h in a simple salt-glucose solution containing 10 mM L-asparagine and only if Ca2+ was replenished. Actinomycin D (5 micrograms/ml) and cycloheximide (20 microM) reduced the stimulatory effect by 84 and 100% respectively. Increase of active enzyme protein was also demonstrated by a 3-fold increase in alpha-difluoromethylornithine binding. Asparagine prolonged the half-life of induced ODC by 20% whereas Ca2+ reduced it by 32%. The observed inductive effects are not accounted for entirely by a direct influence of Ca2+ and asparagine on the turnover of ODC protein. These factors are likely to be parts of a signalling pathway leading to amplification of cellular ODC.


Detection and characterization of mRNA and proteins encoded by human rab2 low molecular weight GTP-binding protein gene. Three RNA species of 3.5, 2.4 and 1.4 kb were detected in all human hematopoietic, fibroblastic and tumor cell lines examined, as the mRNA of human rab2 gene presumably involved in intracellular transport. The mRNA differed in the length of the 3'-non-translated regions due to termination and/or processing at the alternative polyadenylation sites. The 1.4-kb rab2 RNA was predominant in Molt-4 and U-937 of mononuclear cell origin, while the 2.4-kb RNA was dominant in the other cells examined. The degradation of all rab2 RNA was similar and as slow as beta-actin RNA. The rab2 proteins were ubiquitously detected in the human cells as two phosphorylated peptides of major pp24rab2 and minor pp25rab2. pp25rab2 was slightly more phosphorylated than pp24rab2. Both rab2 proteins were abundantly detected in neural PC12 and NB3 cells, and mostly pp24rab2 was produced in the other human and rodent cells.


Urea synthesis in rats fed diet containing kidney beans. When rats were fed a diet containing kidney bean (Phaesolus vulgaris) urea excretion was increased 3-5 fold. Isolated liver mitochondria from rats fed the kidney bean diet produced 40% more citrulline in the presence of arginine than mitochondria isolated from control rats. Mitochondrial activities of urea cycle enzymes and N-acetylglutamate synthetase were similar in animals fed diets containing kidney bean or lactalbumin. The possible mechanisms causing acute urea production in rats fed with kidney bean are discussed.


Interactions between the dopamine D2 receptor and GTP-binding proteins. The dopamine D2 receptor in bovine brain striatum exists in a high- and a low-affinity state for dopamine. The high-affinity state is believed to arise from coupling with a guanine nucleotide-binding protein, to be disrupted by the addition of GTP. We found that antibodies specific to the C-terminal region of the alpha-subunit of Go and Gi cause a shift from high- towards low-affinity, demonstrating that the C-terminus of G alpha is involved in the receptor G protein contact. Purification of the D2 receptor by affinity chromatography via immobilized agonists or antagonists results in the copurification of both Go and Gi proteins. However, this copurification is of a nonspecific nature and prevents the detection of putative specific receptor.G protein complexes.


Changes in membrane fluidity during human liver development. The physico-chemical properties of the hepatic plasma membrane during prenatal period of development and in adult human liver were studied. Fluorescence polarization studies using the lipid probe pyrene, clearly demonstrated a significant reduction in the membrane fluidity with liver maturation. Lipid analysis showed an age dependent reduction in lipid/protein ratio while there was an increase in membrane cholesterol throughout the prenatal period and in adult human liver which decreased the membrane fluidity. Hepatic plasma membrane from prenatal liver also showed a decrease in phosphatidyl choline/phosphatidyl ethanolamine ratio and an increase in sphingomyelin/phosphatidyl choline ratio. These results suggest that there is a gradual decrease in lipid content and membrane fluidity during the prenatal period of development which might regulate the differentiation of membrane associated function in human liver.


Relationship between serum lipid peroxidation products in hypercholesterolemic subjects and vitamin E status. The relationship of serum lipid peroxidation products in hypercholesterolemic subjects to their vitamin E intake was examined in 15 such subjects with no other associated significant disease process in a 3 month trial with vitamin E supplementation. These patients with elevated serum cholesterol levels also have elevated thiobarbituric acid reactive substances (TBARS) and lipid oxidation products (LOPS). Vitamin E supplementation of 800 IU daily normalized the lipid peroxidation products but did not significantly change serum lipids.


Purification and characterization of N-acetyl-alpha-D-galactosaminidase from Gallus domesticus. Exoglycosidases modify carbohydrate epitopes on glycoproteins and glycolipids. The N-acetyl-alpha-D-galactosaminidase from the domestic chicken, Gallus domesticus, is an important exoglycosidase which degrades the human blood group A epitope. This enzyme has never been demonstrably purified or thoroughly characterized. We have developed a technique to purify this enzyme to homogeneity. The isolated enzyme has a molecular weight of 49.1 kDa by SDS PAGE and 145.0 kDa by gel filtration. The enzyme is highly selective for PNP-N-acetyl-alpha-D-galactosaminide and is inactive against other low molecular weight substrates. The enzyme hydrolyzes the terminal N-acetyl-alpha-D-galactosaminide residues from blood group A2 erythrocytes. Protease activity is below detectable limits. The enzyme has a pH optima of 3.7, a pI of 8.15, is relatively unaffected by ionic strength, and is stable at 4 degrees C.


Purification of a membraneous actin binding protein from bovine adrenal medulla. An actin binding protein having a molecular mass of 39,000 was purified from the Triton extract of bovine adrenal medulla membrane fraction using DNase I affinity column and following chromatographies. Specific antibody was produced against the protein and immunoblotting analysis of tissue extract showed the purified protein was not a breakdown product of a larger protein and also showed the presence of this protein in bovine adrenal medullary chromaffin cells and rat pheochromocytoma cells (PC12). An immunoblotting analysis of membrane fractions treated with nucleotides and their analogues showed that this protein was specifically solubilized in the presence of GTP gamma-S.


Purification and properties of 2,3-dihydroxy-p-cumate-3,4-dioxygenase from Bacillus species. 2,3-Dihydroxy-p-cumate-3,4-dioxygenase, an enzyme involved in the catabolism of p-cymene, was purified to homogeneity from Bacillus species by affinity chromatography. Purification of the dioxygenase allowed the observation of the immediate ring cleavage product of 2,3-dihydroxy-p-cumate. The enzyme was optimally active at pH 8.2 and at 35 degrees C. The Km value for 2,3-dihydroxy-p-cumate was 32 microM. The enzyme had a broad substrate specificity for 3-substituted catechols. The activity of the enzyme was inhibited by heavy metals, sulphydryl inhibitors, iron-chelating agents, and substrate analogues. Fe2+ was suggested as a cofactor.


Halophilic malate dehydrogenase--a case history of biophysical investigations: ultracentrifugation, light-, X-ray- and neutron scattering. Halophilic malate dehydrogenase (hMDH) from Haloarcula marismortui has been isolated, purified and characterized by biochemical and biophysical solution studies. A stabilization mechanism at extremely high concentrations of salt, based on the formation of co-operative hydrate bonds between the protein and hydrated salt ions, was suggested from thermodynamic analysis of native enzyme solutions. Recently the gene coding for hMDH was isolated and sequenced and an active enzyme cloned (F. Cendrin, J. Chroboczek, G. Zaccai, H. Eisenberg and M. Mevarech, unpublished work). A study of the crystal structure of hMDH in a high-salt physiological medium is in progress (O. Butbul-Dym & J. Sussman, personal communication). Here we discuss in depth implications of these recent developments on our earlier results.


Biotechnological potential of halobacteria. The extremely halophilic archaebacteria (halobacteria) became an early focus of scientific interest owing to their role in salted food deterioration. In more recent times their peculiar physiology involving extreme adaptation to the salt environment and other unique features have allowed the development of other applied interests. Their similarities to eukaryotic cells at the level of cell division justifies their use in the prescreening for anti-cancer drugs, and some of their antigens could be used for cancer diagnosis. Their unique retinal proteins can be used as light-biosensors and the use of the purple membrane (pm) as reversible holographic medium has already been developed. Halobacterial enzymes are an extremely tough raw material for enzyme technology, particularly for applications in which the reaction mixture has very low water activity. Thanks to their peculiar lipids and to the production of polysaccharides by some halobacteria, their cultures could be used for enhanced oil recovery. Some halobacteria are excellent producers of industrially interesting biopolymers. The use of halobacteria as producers of polyhydroxyalkanoates, biological polyesters such as poly-3-hydroxybutyrate, with the properties of biodegradable thermoplastics, is being considered.


Enzymes from thermophilic archaebacteria: current and future applications in biotechnology. The one guaranteed property of enzymes isolated from extremely thermophilic micro-organisms is their thermostability. Most significantly, almost any such enzyme will be more thermostable than the functionally similar enzyme from a lower temperature source. Thermostability is not an isolated property: resistance to heat denaturation imparts stability to a number of other denaturing influences (detergents, organic solvents, etc). These characteristics of hyperthermophilic enzymes are the most likely basis for the development of new biotechnological applications. A limited number of hyperthermophilic enzymes have found application in specialist biotechnological applications; others have visible potential in growing areas of biotechnology. Existing and potential applications are discussed using DNA manipulation enzymes, dehydrogenases, and esterases as examples.


Biotechnological potential of methanogens. Methane produced microbiologically is currently used as an energy source, especially by cities and industries, albeit at a level far below its potential; the incentive is currently to save money on disposal costs for waste problems. Anaerobic digestion can be helpful in degrading several halogenated hydrocarbon wastes, and methanogens are partly responsible. Ethane instead of methane may be a future product of interest. Some pure cultures of methanogens may be suitable for production of B-12, or perhaps the speciality biochemical F420, a 5-deazaflavin of interest to both methanogen and streptomyces researchers. Methanogens can cause a variety of problems, including biocorrosion, increased atmospheric methane, and ruminant nutrition loss. Studies of tropical wetlands, including rice paddies and swamps, and the study of a variety of ruminants in the tropics are particularly interesting and appropriate at this time, with respect to methane produced in these ecosystems. In some cases, it may be possible to control methane production by the use of inhibitors or ecological control mechanisms.


Archaebacterial lipids: structure, biosynthesis and function. The foregoing review of membrane lipids in archaebacteria has revealed a remarkable variety of polar lipids classes, including phospholipids, glycolipids, phosphoglycolipids and sulpholipids, all derived from the one basic core structure, diphytanylglycerol (1) and an equally remarkable set of novel pathways for their biosynthesis. Even with the relatively limited knowledge that we have of the physical properties of these lipids, it is clear that they are well-adapted as membrane components to the particular environmental conditions of the three groups of archaebacteria, extreme halophiles, methanogens, and thermoacidophiles. However, much remains to be learned concerning the precise asymmetric arrangement of the lipids in the membrane bilayers or monolayers, the interaction of the lipids with the membrane proteins, and the function of this membrane lipid asymmetry with respect to ion transport, permeability to nutrients, proton transport and conductance, and energy transduction. Perhaps then these unusual lipids will not appear so strange and our knowledge of them will help us to understand the function of the more familiar lipids in the eubacteria and eukaryotes.


The enzymology of archaebacterial pathways of central metabolism. From a comparison of the pathways of central metabolism in the archaebacteria, eubacteria and eukaryotes, it is clear that the basic pathways were established before the divergence of the three kingdoms, but that the notable differences may provide important clues to their evolution. From these comparisons, enzymes found in all evolutionary groups have been chosen for detailed structural studies; given the range of extreme phenotypes found within the archaebacteria, these studies will be crucial to our understanding of the structural basis for protein stability and how such features may be engineered into a protein of choice.


Alternative functional relationships between ELF field exposure and possible health effects: report on an expert workshop. If exposure to 60 Hz fields poses risks to public health, the relationship between exposure and risk may involve something other than the product of field strength and time. Such alternative possible relations, or "effects functions," are of great interest to epidemiologists, engineers, risk analysts, and regulators. A structured survey and workshop were used to explore whether leading researchers in bioelectromagnetics share similar views about alternative possible effects functions. Substantial agreement was found about several effects functions in a few specific contexts such as calcium-ion efflux and cell signalling, and biosynthesis pathways. No significant agreement emerged in many other contexts. No effects function possibilities were ruled out. Further effort of this sort was judged unlikely to yield greater consensus until more complete scientific understanding becomes available. However, a series of structured workshops on research planning and priority setting were judged to hold great potential for useful results.


Rats are not aversive when exposed to 60-Hz magnetic fields at 3.03 mT. Thirty-two male rats were tested in two replicates of an experiment to determine whether body currents induced by 60-Hz magnetic fields might lead to avoidance behavior comparable to that which results from exposure to strong 60-Hz electric fields. The test apparatus was a two-compartment Plexiglas shuttlebox enclosed in a sound-attenuating plywood chamber, which in turn was encompassed by two copper bus bars that, when energized, served as a source of 60-Hz magnetic fields. Location of the rat, and traverse activity in the shuttlebox were monitored by nine infra-red photo detectors equally spaced along the length of the apparatus. Rats were divided into 2 groups: 1 group of rats (n = 8 per group per replicate) was sham exposed while rats in the other group (n = 8 per group per replicate) were exposed to a 3.03 mT (30.3 G), 60-Hz magnetic field whenever they traversed to or were located on the side (L or R) predetermined as the exposed side. To control artifact incident to side preference, the side exposed (L or R) was alternated over the exposed rats. Each rat was tested individually in a 1-h session. A 2-factor ANOVA (exposed vs. control, replicate 1 vs. replicate 2) failed to reveal any significant effects due to either factor or to an interaction between factors. These data demonstrate that rats do not avoid exposure to 60-Hz magnetic fields at a flux density of 3.03 mT and further imply that the avoidance by rats of high level 60-Hz electric fields is mediated by something other than the internal body currents induced by the exposure.


Relative-risk-estimate bias and loss of power in the Mantel test for trend resulting from the use of magnetic-field point-in-time ("spot") measurements in epidemiological studies based on an ordinal exposure scale. We assessed the merits of various point-in-time ("spot") measurement protocols in case-control studies based on an ordinal exposure scale. After classifying a number of houses on the basis of prolonged monitoring of the ambient, extremely low frequency (ELF) magnetic field, we determined the probability of misclassification for each "spot" measurement protocol. We calculated the effect of this misclassification on the relative risk estimates and on the Mantel test for trend. We found that classification based on a small group of point-in-time measurements allows an adequate estimate of the relative risk, although the statistical significance of the dose-response gradient may be seriously underestimated. However, the use of automated ambient-field monitors, which results in loss of information on spatial variability, can lead to similar consequences. Therefore, manually collected point-in-time measurements remain a viable option for exposure assessment.


Increased sensitivity of the non-human primate eye to microwave radiation following ophthalmic drug pretreatment. Previous studies in our laboratory have established that pulsed microwaves at 2.45 GHz and 10 mW/cm2 are associated with production of corneal endothelial lesions and with disruption of the blood-aqueous barrier in the non-human primate eye. In the study reported here we examined ocular damage in monkeys (M. mulatta and M. fascicularis) following topical treatment with one of two ophthalmic drugs (timolol maleate and pilocarpine) that preceded exposure to pulsed microwaves. Anesthetized monkeys were sham exposed or exposed to pulsed, 2.45 GHz microwaves (10 microseconds, 100 pps) at average power densities of 0.2, 1, 5, 10, or 15 mW/cm2 4 h a day for 3 consecutive days (respective SARs were 0.052, 0.26, 1.3, 2.6, and 3.9 W/kg). Immediately before microwave exposure, one or both eyes were treated topically with one drop of 0.5% timolol maleate or of 2% pilocarpine. Following administration of a drug, we observed a significant reduction in the power-density threshold (from 10 to 1 mW/cm2) for induction of corneal endothelial lesions and for increased vascular permeability of the iris. Diagnostic procedures (in vivo specular microscopy and fluorescein iris angiography) were performed following each exposure protocol. In addition, increased vascular permeability was confirmed with horseradish peroxidase tracer techniques. Although we did not measure intraocular temperatures in experimental animals, the results suggest that a mechanism other than significant heating of the eye is involved. Our data indicate that pulsed microwaves at an average SAR of 0.26 W/kg, if administered after pretreatment with ophthalmic drugs, can produce significant ocular effects in the anesthetized primate.


Effects of a 30 kV/m, 60 Hz electric field on the social behavior of baboons: a crossover experiment. Using a crossover experimental design, we evaluated our earlier findings that exposure to a 30 kV/m, 60 Hz electric field for 12 hours per day, 7 days per week for 6 weeks produced significant changes in the performance rates of social behaviors among young adult male baboons. In the crossover experiment, the former control group was exposed to a 30 kV/m, 60 Hz electric field for 3 weeks. Only an extremely small, incidental magnetic field was generated by the exposure apparatus. We found that electric-field exposure again produced increases in the performance rates that index Passive Affinity, Tension, and Stereotypy. These findings, combined with results from our other electric-field experiments, indicate that exposure to strong electric fields, in the absence of associated magnetic fields, consistently produces effects that are expressed as increases in rates of performance of social behaviors in young adult male baboons.


Uniform magnetic fields and double-wrapped coil systems: improved techniques for the design of bioelectromagnetic experiments. A common mistake in biomagnetic experimentation is the assumption that Helmholtz coils provide uniform magnetic fields; this is true only for a limited volume at their center. Substantial improvements on this design have been made during the past 140 years with systems of three, four, and five coils. Numerical comparisons of the field uniformity generated by these designs are made here, along with a table of construction details and recommendations for their use in experiments in which large volumes of uniform intensity magnetic exposures are needed. Double-wrapping, or systems of bifilar windings, can also help control for the non-magnetic effects of the electric coils used in many experiments. In this design, each coil is wrapped in parallel with two separate, adjacent strands of copper wire, rather than the single strand used normally. If currents are flowing in antiparallel directions, the magnetic fields generated by each strand will cancel and yield virtually no external magnetic field, whereas parallel currents will yield an external field. Both cases will produce similar non-magnetic effects of ohmic heating, and simple measures can reduce the small vibration and electric field differences. Control experiments can then be designed such that the only major difference between treated and untreated groups is the presence or absence of the magnetic field. Double-wrapped coils also facilitate the use of truly double-blind protocol, as the same apparatus can be used either for experimental or control groups.


Small integrating meter for assessing long-term exposure to magnetic fields. A small, lightweight meter has been developed for magnetic-field measurements, particularly those needed for exposure-assessment purposes. This meter, known as the AMEX-3D, continuously measures all three axes of magnetic-flux density and electronically combines the data into a single estimate of cumulative exposure to the root-mean-square (rms) resultant flux density. The AMEX-3D weighs about 120 g, measures 2.7 cm x 5.1 cm x 10.2 cm, and is battery powered. Two panel-mounted jacks are provided for measuring battery voltage and for reading cumulative exposure data from the unit. The instrument has, within 3 dB, a flat response to magnetic flux densities at all frequencies in its 30-1,000 Hz bandwidth. A detailed analysis of error sources in the AMEX-3D leads to an estimate of +/- 20% as the accuracy of the instrument over its dynamic range, which extends from 0.02 to 15 microT. The AMEX-3D was tested in the field by asking electric-utility distribution linemen to wear AMEX-3D and EMDEX meters simultaneously while working. Agreement between the two measures of exposure was excellent.


Statistical review of the henhouse experiments: the effects of a pulsed magnetic field on chick embryos. This paper analyzes data from a study conducted by the United States Office of Naval Research on the effects of pulsed magnetic fields on chick embryos. The experiment involved incubation of eggs under carefully controlled conditions in six different laboratories. The original analysis included inappropriate statistical methodology for analyzing the experimental results. Since the conclusions from this study rest so heavily on the results of statistical analyses, choosing the proper methodology is imperative. The major aim of this paper then is to introduce more appropriate analytic tools and illustrate their use in the present context. Qualitatively our results agree with those of the original analysis; our findings about interactions between effects, however, makes interpretation of these effects more subtle. We apply linear logistic modeling to counts of damaged embryos, using as covariates factors corresponding to exposure, laboratory, incubator, run, and measurements of background radiation. This facilitates estimation of the size of the effects. The effects of laboratory, incubator, and run are explored both as fixed and random effects. We find statistically significant exposure and laboratory effects, in accordance with the original study. However, we also find that the inter-laboratory variation in exposure effect is at least as large as the exposure effect itself. The presence of such effects fundamentally alters the interpretation of the fitted model, as is graphically presented.


Whole-body microwave dosimetry based on a single, gradient-layer calorimeter. A simple, cost-effective, and accurate technique to measure the whole-body-averaged specific absorption rate (SAR) in Sprague-Dawley rat carcasses by a single-gradient-layer calorimeter is described. The results of SAR determinations showed a highly linear relation between the average power density of the incident field (1.25 GHz) and the normalized heat loading of the carcasses.


[Site-directed chemical restriction of a single-stranded DNA fragment by an alkylating derivative of the tetranucleotide d(pApGpCpA) in the presence of tetranucleotide effectors]. Alkylation of a single-stranded DNA 302-mer by a 5'-O-phosphoryl-[4-(N-2-chloroethyl-N-methylamino)benzyl]amide derivative of the tetradeoxyribonucleotide d(pApGpCpA) in the presence of 3',5'-di-N-(2-hydroxyethyl) phenazinium derivatives of tetranucleotides as effectors led to specific chemical cleavage of the target at the guanosine residues of the sites ... pTpGppT. The reagent can be selectively addressed to one of three alkylation sites with the aid of a pair of tetranucleotide effectors flanking the chemically reactive tetranucleotide in the complex with the target DNA. The yield of the cleavage depends on the concentration of both the reagent and effectors, and can be enhanced, if a chain of two or more effectors from each side of the reagent is used. In this case, 3',5'-di-Phn-tetranucleotide effectors are to immediately flank the reagent.


[Study of the spatial structure of the duplex (Phn-NH(CH2)NH)pd(CCAAACA).pd(TGTTTGGC) with covalently bound 10-(2-hydroxyethyl)phenazine by in aqueous solution by 2D-(1)H-NMR and by limited molecular mechanics]. Detailed investigation of the spatial structure of duplex (Phn-NH(CH2)2NH) x pd(CCAAACA).pd(TGTTTGGC) having a covalently linked N-(2-hydroxyethyl)-phenazine in aqueous solution was continued by means of one- and two-dimensional 1H-NMR spectroscopy. Distances between the protons of the oligonucleotides as well as distances between the phenazinium and the nearest nucleotide groups protons were determined from the series of one-dimensional NOE experiments. The effective correlation time tau c determined for some proton pairs shows the phenazinium fragment to have greater internal motion than the heterocyclic bases. The deoxyribose protons coupling constants show the sugars to be in 2'-endo-conformation. The restrained molecular mechanics have yielded a possible structure of duplex in the aqueous solution fitting the experimental set of interproton distances.


[Directed mutagenesis of genes of some plant proteins]. Mutagenesis of two previously cloned plant genes, maize storage protein cZ22B1 gene and barley Photosystem II protein D1 gene (psbA), was carried out. To improve the nutritional quality of zein, the DNA region corresponding to the protein sixth alpha-helix rod was substituted by a synthetic segment bearing three codon changes for Lys. Additional stabilization of this helix was achieved by three more codon changes for Glu. By means of oligonucleotide directed mutagenesis five different copies of psbA gene were obtained, bearing single codon change of Ser264 (wild type) for Gly, Ala, Cys, Asn, and Thr, respectively. These constructs can be used for studying functional topography of protein D1 and core region.


[Physico-chemical and immunochemical characteristics of molecular forms of carcinoembryonic antigen (CEA) not bound with concanavalin A]. A molecular form of CEA non-binding Con A (CEAnC) was isolated from colon adenocarcinoma metastases in liver as a fraction of CEA having no affinity to Con-A-Sepharose. CEAnC was shown to be immunochemically identical to CEA, but to differ substantially with regard to the amino acid and sugar composition, and structure of the sugar moiety, possibly containing non only N-, but also O-glycosyl carbohydrate chains. The antigens studied were also found to possess different spatial structures. The differences between CEA and CEAnC suggest CEAnC to be a new molecular form of CEA.


[Protection of naturally susceptible animals against foot-and-mouth disease with a peptide, synthesized on a lysine matrix]. A peptide VP1-(142-158)-MAP (Multiple antigen peptide system) consisting of two parts: a lysine matrix made up of three levels of lysine residues coupled with each other and amino acid sequence 142-158 of VP1 of FMD virus strain A(22)550--has been synthesized. Guinea-pigs inoculated with 20 mkg of the peptide incorporated with Freund's complete adjuvant were protected against challenge with 500 ID50 of homologous FMD virus. Sheep were immunized with a single inoculation of the peptide in a dose of 1.0 mg. Cattle inoculated twice with 1.5 mg of the peptide with incomplete adjuvant on the basis of synthetic oil developed high virus-specific antibody titres both after the first (5.3-7.6 log2 ND50/0.1 ml) and the second inoculation (10.2-11.0 log2 ND50/0.1 ml). The peptide-immunized animals were resistant to challenge with homologous virulent virus in a dose of 10(4) ID50. The immunogenic and protective capacities of the peptide VP1-(142-158)-MAP were shown to be greater as compared with those of its linear analogue-peptide VP1-(141-160).


[Transformation products of vitamin E and its analog--chroman C1 in an oxidizing ethyl linoleate medium]. Spirodimers, dimers, quinones of alpha-tocopherol and chroman C1 have been synthesized by the alpha-tocopherol and chroman Ci oxidation with K3[Fe(CN)6] and FeCl3. Products of the oxidative transformation of alpha-tocopherol and chroman Ci, viz. dimers, quinones and trimers, in the media of ethyllinoleate and nonane have been identified by HPLC, TLC, UV, IR and 1H NMR spectroscopy. The mechanism of the transformation and modifying activity of chromans under various oxidation conditions are discussed with regards to the experimental data.


[Molecular and crystalline structure of 3'-methylamino-2',3'-dideoxyribosylthymine--a potential inhibitor of HIV]. The structure of 3'-methylamino-2',3'-dideoxyribosylthymine [ddT(3'NHMe)] was determined by X-ray analysis. The space group is P2(1)2(1)2(1). Cell dimensions are: a 5.132(1), b 13.718(1), c 16.947(2) A, V 1193.2 A3, Z 4. The structure was solved by directed methods and refined by the full-matrix least square method to R 4.8%. The molecule of ddT(3'NHMe) has anti-conformation with respect to the glycosidic bond (chi (O4'-C1'-N1-C2) = -106.7 degrees), C3'-endo-C4'-exo puckering of the sugar moiety (P -28.8 degrees, psi m -31.5 degrees) and gauche-gauche conformation about exocyclic C4'-C5' bond (psi(C3'-C4'-C5'-O5') 45.8 degrees). The structure of ddT(3'NHMe) was compared with those of 3'-amino-3'-deoxythymidine, 3'-azido-3'-deoxythymidine and natural thymidine.


[Achievements and prospects of the directed search for antiviral agents in the nucleoside series and their derivatives]. Recent advances of antiviral drug design among nucleosides and their derivatives have been summarized. The first chapter deals with the history of nucleic acids components and further developments in this area. Next part discusses the mechanism of action of biologically active nucleosides: 2',3'-dideoxynucleosides, acyclic analogues, phosphonate derivatives and nucleoside antibiotics. The third chapter describes planning of complicated synthesis of nucleoside analogues from branched-chain sugars and stereo-specific formation of glycosidic bond upon synthesis of ribonucleoside and 2'-deoxyribonucleoside. The last part outlines further perspectives, i. e. preparation of antiviral compounds and use of nucleoside analogues in oligonucleotide synthesis.


[The mechanism of anticholinesterase action of acetylene organophosphorus inhibitors]. Introduction of the triple bond in the leaving group of the organophosphorus inhibitor molecule gives a sharp raise of the inhibitor activity but does not change principal characteristics of the cholinesterase inhibition mechanism. The reactivation experiments suggest that inactivation of cholinesterases by these compounds occurs due to phosphorylating of the serine hydroxyl by the corresponding phosphoric acid. A close similarity was shown between acetylenic and saturated organophosphorus inhibitors in altering ka upon change of pH and tetraalkylammonium ions action. It is demonstrated that S-alkynyl esters of thioacetic acid are slowly hydrolyzed by acetylcholinesterase and cholinesterase without irreversible inhibition of the enzymes.


[Pepsin in the enzymatic synthesis of esters and n-nitroanilide peptides]. Pepsin was shown to catalyze synthesis of esters or p-nitroanilides tri-, tetra-, penta- and hexapeptides of general formula Z-X-Y-B, where X = Ala-Phe, Phe-Met, Ala-Ala-Glu, Ala-Ala-Phe, Ala-Ala-Leu, Ala-Ala-Trp, Ala-Ala-Met. Y = Ala, Leu, Val, Phe, Arg, Ala-Ala, Gly-Gly, Leu-Ala-Ala, Phe-Ala-Ala. B = OMe, pNA. The reactions were carried out in dimethylformamide-water solutions at pH 4.6 by equimolar ratio of amino- and carboxyl components (with the exception of Arg-pNA taken in 2-fold excess). The amount of pepsin in the reaction approached 1:1700 enzyme: substrate molar ratio although it might be improved--up to 1:3.10(5) for relatively long peptides.


[Synthesis and expression in Escherichia coli of the gene for the albumin-binding domain of Streptococcus protein G]. The chemical-enzymatic synthesis of a gene coding for A2B2 repeats of the albumin-binding domain of streptococcal protein G has been accomplished. The codon usage of the natural gene has been modified to adapt an artificial sequence for the efficient translation in E. coli. The gene (238 b.p.) was cloned in the polylinker plasmid pUCL1 and then fused in frame to the 3'-terminus of the gene for the IgG-binding domain of staphylococcal protein A, which was earlier cloned in the expression plasmid pUCL2. A fused polypeptide composed of the E and B domains of protein A and A2B2 repeats of protein G was produced in E. coli cells under the lac promoter control. The resulted product was isolated by affinity chromatography on IgG-sepharose and (or) albumin-sepharose.


[Preparation of 5-(125)iodine-2'-deoxyuridine triphosphate and incorporation of the labeled nucleotide into DNA by polymerase chain reaction]. Radioiodine was substituted into deoxyuridine triphosphate by radioiodination of 5-mercuri-2'-deoxyuridine triphosphate. The purified 125I-nucleotide was incorporated into DNA in reactions by the protocol of the polymerase chain reaction. The incorporation of the radioactive nucleotide required added thymidine triphosphate for the synthesis of full length molecules during the one minute elongation period. The iodine substituent in the DNA remained covalently bound during both heat and alkaline denaturation.


[Change in the cleavage site of synthetic substrates of SsoII restriction endonucleases upon introduction of non-nucleotide inserts in the recognition segment]. The cleavage of synthetic DNA duplexes containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy]methylguanine (glG) residues instead of one of dG residues or one of the nucleosides of the central base pair of the recognition site by SsoII restriction endonuclease (decreases CCNGG) has been studied. It is found that the non-nucleotide insertions (except for glG) result in a change of the SsoII cleavage site and an increase of the efficiency of the cleavage. The novel noncanonical cleavage occurs at the phosphodiester bond adjoining the non-nucleotide insert from the 5'-end.


Informed consent and the prescription of nonsteroidal antiinflammatory drugs. To examine disclosure of side effects of nonsteroidal antiinflammatory drugs (NSAIDs) and to identify patient- and physician-specific factors associated with greater disclosure. Forty-six encounters between rheumatologists and new adult outpatients who were prescribed an NSAID they had not been taking prior to the visit were audiotaped. Reviewers coded the NSAID prescribed, specific side effects mentioned, demographic features of patients and physicians, and patient clinical characteristics. Neither patients nor physicians were aware that side effect disclosure was being studied. A mean of 1.7 side effects was mentioned per encounter. Epigastric discomfort was mentioned in 72% of encounters, while other side effects, including hepatic, renal, hematologic, or central nervous system effects, were mentioned in < or = 15% of encounters. Three factors were identified as independent predictors of less disclosure of side effects: senior clinician (versus less experienced), patient not taking another NSAID immediately prior to the visit, and patient age < 40. Increased disclosure by less experienced clinicians occurred exclusively with patients who were taking another NSAID prior to the visit. Disclosure of side effects other than epigastric discomfort to patients who are prescribed a new NSAID is limited. Patients not taking NSAIDs previously, who presumably have the most to gain from such discussions, are told the least. These results have implications with regard to doctor-patient decision-making and malpractice litigation in the outpatient setting.


Psychological well-being among people with recently diagnosed rheumatoid arthritis. Do self-perceptions of abilities make a difference? Satisfaction with abilities and perceived importance of abilities are 2 factors involved in the process of self-evaluation. We examined the role that these factors play in adjustment to rheumatoid arthritis (RA). Data were collected, via telephone interview and mailed questionnaire, from 234 individuals with recently diagnosed RA. Disease severity information was obtained from participants' physicians. Consistent with study hypotheses, satisfaction was associated with psychological well-being only among those who viewed as very important the abilities being evaluated. These findings increase understanding of the conditions under which low levels of satisfaction are likely to be associated with psychological distress.


Coping with arthritis. Current status and critique. Our understanding of the complex role of coping in the psychological adjustment to arthritis has improved over the past decade. Studies have consistently demonstrated a relationship between certain coping strategies and psychological outcomes. However, unresolved problems in the methodology and theory concerning these studies cloud the picture, limiting the conclusions that can be drawn. There are general problems with the ways in which coping has been conceptualized and measured by researchers evaluating stress and coping, and there are problems more specific to the ways coping concepts and measures have been used to study patients with arthritis. A complete understanding of the relationship between coping and mental health in this population would have important theoretical and clinical implications. Such studies would serve as a model for investigations of stress and coping in patients with other illnesses, and would point to effective psychosocial interventions for improving the quality of life for this population.


Defective hypothalamic response to immune and inflammatory stimuli in patients with rheumatoid arthritis. To determine the integrity of the hypothalamic-pituitary-adrenal (HPA) axis responses to immune/inflammatory stimuli in patients with rheumatoid arthritis (RA). Diurnal secretion of cortisol and the cytokine and cortisol responses to surgery were studied in subjects with active RA, in subjects with chronic osteomyelitis (OM), and in subjects with noninflammatory arthritis, who served as controls. Patients with RA had a defective HPA response, as evidenced by a diurnal cortisol rhythm of secretion which was at the lower limit of normal in contrast to those with OM, and a failure to increase cortisol secretion following surgery, despite high levels of interleukin-1 beta (IL-1 beta) and IL-6. The corticotropin-releasing hormone stimulation test in the RA patients showed normal results, thus suggesting a hypothalamic defect, but normal pituitary and adrenal function. These findings suggest that RA patients have an abnormality of the HPA axis response to immune/inflammatory stimuli which may reside in the hypothalamus. This hypothalamic abnormality may be an additional, and hitherto unrecognized, factor in the pathogenesis of RA.


Regulation of synovial cell growth. Coexpression of transforming growth factor beta and basic fibroblast growth factor by cultured synovial cells. To demonstrate expression of transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) by cultured rheumatoid arthritis (RA) synovial cells and to investigate their role as synovial cell mitogens. Polypeptide growth factors were detected and identified by immunocytochemical staining and Western blot analysis. Messenger RNA (mRNA) transcripts encoding TGF beta and bFGF were identified by polymerase chain reaction analysis. The influence of neutralizing growth factor monoclonal antibodies (MAb) on RA synovial cell growth was investigated. TGF beta bioactivity was determined by Mv1Lu assay. Lysates of RA, as compared with normal, synovial cells contained greater amounts of TGF beta and bFGF. Western blot analysis identified a single TGF beta band (MW approximately 25 kd) in each of the cell lysates examined. Western blot analysis using MAb DE6 identified a doublet of bFGF bands (MW approximately 18.0 kd) in normal synovial cell lysates and 4 bFGF bands (MW approximately 18.0, 22.0, 22.6, and 25.2 kd) in RA synovial cell lysates. RA and normal synovial cells expressed mRNA transcripts encoding TGF beta 1 but not TGF beta 2, and FGF-2 (basic FGF). Additional mRNA transcripts encoding FGF-5 and FGF-7 were expressed by RA, but not normal, synovial cells in culture. In contrast to MAb 1D11.16, which caused a dose-dependent decrease in RA synovial cell growth, MAb DG2 (up to 100 micrograms/ml) had no effect on cell growth. RA and normal synovial cells cultured in serum-free medium express TGF beta 1 and native bFGF. However, only RA synovial cells in culture express higher molecular weight isoforms of bFGF. TGF beta 1 appears to regulate synovial cell growth in vitro through an external autocrine loop. Despite expression of high-affinity bFGF receptors on cultured synovial cells, the mechanisms by which bFGF modulates synovial cell growth are unknown.


Quantitation of chondroitin 4-sulfate and chondroitin 6-sulfate in pathologic joint fluid. To investigate the correlation between joint disease and the composition of chondroitin sulfate in the joint fluid, unsaturated disaccharide isomers of chondroitin 4-sulfate (delta di-4S) and chondroitin 6-sulfate (delta di-6S) were measured in joint fluids obtained from patients with osteoarthritis (OA), rheumatoid arthritis (RA), or traumatic arthritis (TA). These pathologic joint fluids were digested with chondroitinase ABC, and the delta di-4S and delta di-6S produced were determined by high performance liquid chromatography combined with fluorometry. Total content of delta di-4S plus delta di-6S was 71.8 +/- 30.0 nmoles/ml (mean +/- SD) in OA, 55.4 +/- 29.3 nmoles/ml in RA, and 211 +/- 149 nmoles/ml in TA joint fluids. The ratio of delta di-6S to delta di-4S was 3.81 +/- 0.992 in OA, 1.13 +/- 0.527 in RA, and 5.75 +/- 2.46 in TA joint fluids. Differences between groups were statistically significant. These results strongly suggest that the levels of chondroitin sulfate isomers and the delta di-6S: delta di-4S ratio in joint fluid reflect the proteoglycan metabolism of joint tissues, particularly of articular cartilage; hence, they could be used to diagnose joint diseases and to predict articular cartilage destruction from such joint diseases.


Calcium-dependent cysteine proteinase (calpain) in human arthritic synovial joints. To study the roles of calpains in the synovial joint in rheumatoid arthritis (RA) and osteoarthritis (OA) and to verify the hypothesis that calpains present in the synovial fluid come from the synovium. We performed immunohistochemical, biochemical, and immunoblotting analyses for calpains in synovial tissues, synovial cell cultures, and synovial fluids. Immunohistochemical staining of RA synovium demonstrated specific cytoplasmic staining of cells in the synovial lining layer, storomal fibroblasts, and endothelial cells. OA synovium showed almost the same intensity and distribution of calpain staining. DEAE-cellulose chromatography of RA and OA synovial extracts and synovial fluids showed a peak of caseinolytic activity attributable to calpain, as well as an inhibitory peak of calpastatin, a specific inhibitor protein of calpains. Immunoblotting using the anticalpain antibody from the calpain peak of RA and OA synovium and synovial fluid showed identity with the heavy subunit of calpain (80 kd). Similarly, calpain existed in the same form (80 kd) in conditioned media (supernatant) obtained from synovial cell cultures, as well as in the synoviocytes. The total specific activity of the 2 calpains in the synovial fluid of RA patients was higher than that of calpastatin. The findings suggest that the extracellular appearance of calpains could be due to the secretion of these proteins from the synovial cells and that calpains may play a role in cartilage damage of RA and OA that occurs in synovial joints.


Efficacy and safety of 10-deazaaminopterin in the treatment of rheumatoid arthritis. A one-year continuation, double-blind study. To determine the long-term safety and efficacy of 10-deazaaminopterin (10-DAM) in the treatment of rheumatoid arthritis (RA). A 1-year continuation of an initial 15-week randomized, double-blind clinical trial of 10-DAM and methotrexate (MTX). 10-DAM (n = 10) and MTX (n = 8) had comparable safety and efficacy profiles. One 10-DAM-treated and 2 MTX-treated patients experienced transient side effects; 1 MTX-treated patient experienced recurrent nausea and discontinued MTX. 10-DAM appears to be as beneficial and as safe as MTX for the treatment of RA.


The treatment of Wegener's granulomatosis with glucocorticoids and methotrexate. To identify alternatives to daily low-dose cyclophosphamide (CYC) in the treatment of Wegener's granulomatosis (WG). An open-label pilot study of weekly low-dose methotrexate (MTX) plus glucocorticoids (GC) for treatment of patients with WG was performed. Twenty-nine patients who did not have immediately life-threatening disease were included. Outcome was determined by clinical characteristics, pathologic findings, course of illness, laboratory and radiographic findings, and successful withdrawal of GC therapy. Weekly administration of MTX (at a mean stable dosage of 20 mg) and GC resulted in marked improvement in 76% of the 29 patients. Remission was achieved in 69% of the patients, 7% improved but had intermittent smoldering disease that precluded total withdrawal of GC, and 17% had progressive disease within 2-6 months of starting the study treatment. Two patients who initially achieved remission later had relapses after GC was discontinued. Of those who remain in remission (mean followup time 14.5 months), 72% have not required GC for a mean period of 10 months. Although standard therapy for WG (daily CYC and GC) has dramatically improved outcome in this often-fatal disease, treatment morbidity has led to attempts to identify effective interventions that have less toxicity. Weekly low-dose MTX was shown in this study to be a feasible alternative to CYC in patients whose illness was not immediately life-threatening or in whom prior CYC treatment was ineffective or produced serious toxicity. Although these results are preliminary, they are encouraging and justify further studies in which MTX, CYC, and other alternative therapeutic approaches are compared concurrently.


Quantitative electroencephalography. A new approach to the diagnosis of cerebral dysfunction in systemic lupus erythematosus. Neuropsychiatric manifestations are common in patients with systemic lupus erythematosus (SLE), but accurate diagnosis is often difficult. We conducted a prospective study to determine the utility of neurometric quantitative electroencephalography (QEEG) as an indicator of cerebral dysfunction in SLE patients. Fifty-two SLE patients were divided into 4 groups based on the results of neuropsychiatric evaluations. These included patients with objective evidence of neuropsychiatric SLE (NPSLE), patients with neuropsychiatric symptoms, patients with no evidence of NPSLE, and patients with a prior history of NPSLE: All QEEG findings were compared with data in an age-regressed normative database and with findings in an independent sample of normal subjects. QEEG sensitivity was 87%, and specificity was 75%. QEEG results were abnormal in 74% of the SLE patients with neuropsychiatric symptoms and in 28% of the patients with no evidence of active NPSLE: QEEG profiles varied as a function of the severity and type of neuropsychiatric manifestation present. Within this patient population, QEEG was more sensitive than magnetic resonance imaging, computed tomography scanning, or conventional EEG. Neurometric QEEG may be a sensitive indicator of cerebral dysfunction in patients with NPSLE and can differentiate patients with diverse neuropsychiatric manifestations. When combined with a careful clinical history and evaluation, QEEG provides information that may be useful for the early detection of NPSLE and for serial evaluation of disease activity and treatment efficacy.


The spectrum of pericardial tamponade in systemic lupus erythematosus. Report of ten patients. To describe the range of clinical manifestations and the outcome of pericardial tamponade in patients with systemic lupus erythematosus (SLE). Patients with pericarditis and with pericardial tamponade were identified from our computerized database of 395 SLE patients. Medical records were reviewed to establish activity of SLE at the time of tamponade, as well as clinical and laboratory features, treatment, and outcome of the tamponade. Pericarditis occurred in 75 patients (19%), with 11 episodes of tamponade in 10 of them (13%; 2.5% of entire series). Tamponade was the initial manifestation of SLE in 4 patients. Seven episodes occurred during active lupus, with nephritis present in 6. Signs of venous congestion, including ascites and facial or peripheral edema, were the most common manifestation of tamponade. Pericardial fluid was exudative, and pericardial tissue demonstrated a range of findings including fibrinous and fibrotic changes, acute and chronic inflammatory infiltrates, and vascular proliferation. Tamponade was fatal in 1 patient, and 2 patients each had recurrent effusions and pericardial thickening. Pericardial tamponade may occur at any point in the course of SLE, and should be considered in patients with unexplained signs of venous congestion. The differential diagnosis includes active SLE, uremia, and infection. Treatment with high-dose steroids and either pericardiocentesis or placement of a pericardial window is indicated, but recurrent effusions or pericardial thickening may develop.


The effect of cyclosporin A on early and late stages of experimental lupus. To investigate the effect of cyclosporin A (CSA) on the development of lupus in an experimental model. Lupus was induced in naive mice following injection of a human anti-double-stranded DNA (anti-dsDNA) monoclonal antibody carrying the 16/6 idiotype (Id). CSA was injected into the mice at an early stage of the disease (2 months after immunization) and at a late stage (4 months after immunization). CSA was found to have a suppressive effect on autoantibody production, as well as on the appearance of other disease manifestations, in the mice with lupus. The effects of the drug were more prominent when the mice were treated at an early stage. This was reflected by a dramatic decrease, to normal levels, in autoantibodies to dsDNA, histones, cardiolipin, Sm, RNP, SS-A/Ro, SS-B/La, and anti-DNA 16/6 Id. Similar effects on the erythrocyte sedimentation rate, white blood cell count, and urinary protein levels were noted. These data were supported by electron microscopy analysis showing a lack of immunoglobulin deposition in the kidneys of mice in which treatment was started early. This study demonstrates that, similar to findings in other autoimmune conditions (e.g., insulin-dependent diabetes mellitus), administration of CSA at an early stage in systemic lupus erythematosus may be more beneficial than if the drug is given at a later stage.


Cardiac involvement in systemic sclerosis. To assess the incidence and extent of cardiac involvement in systemic sclerosis (SSc) patients with no apparent cardiac symptoms. Surface electrocardiography, ambulatory electrocardiography, radionuclide ventriculography, myocardial scintigraphy, and echocardiography were performed in 18 patients. These studies demonstrated ventricular tachycardia in 1 patient, nonsustained ventricular tachycardia in 5, supraventricular tachycardia in 6, decreased left ventricular ejection fraction in 2, decreased right ventricular ejection fraction in 8, and stress-induced reversible myocardial perfusion abnormalities in 6. These observations demonstrate a high rate of cardiac abnormalities in SSc patients without cardiac symptoms.


Restricted junctional usage of T cell receptor V beta 2 and V beta 13 genes, which are overrepresented on infiltrating T cells in the lips of patients with Sjögren's syndrome. To analyze the clonality of T cell receptor (TCR) V beta 2- and V beta 13-positive T cells, which are predominantly expressed in the lips of patients with Sjögren's syndrome (SS). The junctional sequences of complementary DNA clones encoding TCR V beta 2 and V beta 13 genes were determined by the polymerase chain reaction. Forty-one V beta 2 and 45 V beta 13 clones established from the lips of 3 SS patients were sequenced. The V beta 2/J beta 2.3 pair was enriched in 2 of the 3 patients (44% and 46% of the clones, respectively), and the V beta 13/J beta 2.1 sequence was dominant in 2 of the 3 (23% and 45%). These pairs were not used preferentially in peripheral blood lymphocytes from the same patients. Infiltrating V beta 2- and V beta 13-positive T cells from the lips of all 3 patients with SS were polyclonal, but the junctional usage of cells from 2 lip samples was restricted, compared with cells from peripheral blood. This suggests that not all expanded cells from the lips of SS patients are stimulated by superantigens.


Serum anti-SS-B/La and IgA rheumatoid factor are markers of salivary gland disease activity in primary Sjögren's syndrome. To identify serologic markers of salivary gland disease activity in 43 patients with primary Sjögren's syndrome. Comparison of salivary gland biopsies (focus scores) and flow rates with serum concentrations of IgA and IgM rheumatoid factor (RF), total serum IgG, serum anti-SS-B/La antibodies, and the erythrocyte sedimentation rate. Serum anti-SS-B/La antibody levels correlated with focus scores (rs = 0.477, P < 0.0025). Serum IgA-RF concentrations correlated inversely with stimulated parotid gland salivary flow rates (rs = -0.394, P < 0.01). Measuring serum levels of anti-SS-B/La and IgA-RF would be useful when monitoring salivary responses in therapeutic trials, especially in patients with minimal salivary function.


Antineutrophil cytoplasmic autoantibody-associated vasculitis presenting as Sjögren's syndrome. A 63-year-old woman, in whom a diagnosis of Sjögren's syndrome was initially made, proved to have systemic vasculitis with salivary gland involvement and necrotizing and crescentic glomerulonephritis. Antineutrophil cytoplasmic autoantibodies (ANCA) against myeloperoxidase were positive. ANCA-associated vasculitis should be considered in the differential diagnosis of Sjögren's syndrome. A positive finding on immunoassay for ANCA against myeloperoxidase or proteinase 3 may help establish the diagnosis.


Heavy and light chain composition of serum IgA and IgA rheumatoid factor in Henoch-Schönlein purpura. To characterize the heavy and light chain composition of serum IgA and IgA rheumatoid factor (RF) in 34 children with Henoch-Schönlein purpura (HSP). Serum IgA and IgA subclass concentrations were measured by radial immunodiffusion. IgA-RF and the light chain composition of serum IgA and IgA-RF were determined by enzyme immunoassay. Serum IgA and IgA1 concentrations were higher in HSP patients than controls (P = 0.0001), but there was no difference in IgA2 concentrations between the two groups. IgA-RF was present in 19 of 34 HSP patients; it was composed predominantly of IgA1. The kappa:lambda ratio of serum IgA was virtually identical in patients and controls, but the kappa:lambda ratio of IgA-RF was significantly higher than that of the serum IgA from the 19 IgA-RF seropositive patients (P = 0.0003). Serum IgA1 is preferentially increased in children with HSP, but the light chain composition of IgA is not different from that in controls. IgA-RF is common in HSP; it is composed predominantly of IgA1 and it is enriched in kappa light chains.


Suppression of renal disease and arthritis, and prolongation of survival in MRL-lpr mice treated with an extract of Tripterygium wilfordii Hook f. Treatment of MRL-lpr/lpr mice with Tripterygium wilfordii Hook f (TWHf) to evaluate its effects on mortality, renal disease, and arthritis. Mice were fed water (group A, control), TWHf (group B), or first water and then TWHf (group C) from age 7 weeks until age 21 weeks. Arthritis and glomerulonephritis were decreased in groups B and C mice, and survival was increased in group B mice. TWHf decreases the mortality rate, and severity of glomerulonephritis and arthritis in MRL-lpr/lpr mice.


Premature death with bladder outlet obstruction and hyperprolactinemia in New Zealand black X New Zealand white mice treated with ethinyl estradiol and 17 beta-estradiol. To determine causes of death, estrogen toxicity, and hyperprolactinemia in a murine model of systemic lupus erythematosus (SLE). Female New Zealand Black x New Zealand White (NZB x NZW) mice were implanted with ethinyl estradiol, 17 beta-estradiol, or empty capsules (controls). Estrogen-treated mice developed striking hyperprolactinemia and died prematurely with genitourinary complications. Implanted estrogens, including 17 beta-estradiol in a dose reported previously to stimulate SLE, contribute to premature death in NZB x NZW mice, through toxic effects. Estrogen therapy increases the level of prolactin, an immunostimulatory hormone.


In vitro studies on the influence of L-ascorbic acid 2-[3,4-dihydro- 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6yl-hy drogen phosphate] potassium salt on lipid peroxidation and phospholipase A2 activity. The effects of L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2- (4,8,12-trimethyltridecyl)-2H-1-benzopyran-6yl-hydrogen phosphate] potassium salt (EPC-K1, CAS 127061-56-7), a new compound for ischemia-reperfusion injuries, on lipid peroxidation and phospholipase A2 activity were studied in vitro using rat brain homogenates and human plasma. EPC-K1 inhibited phospholipase A2 activity in human plasma in a concentration-dependent manner (IC50 = 7.3 x 10(-4) mol/l), whereas a mixture of alpha-tocopherol and ascorbic acid did not exhibit this effect. In rat brain homogenates, EPC-K1 also inhibited lipid peroxidation in a concentration-dependent manner (IC50 = 2.3 x 10(-6) mol/l). alpha-Tocopherol was less active than EPC-K1. These properties of EPC-K1 suggest that EPC-K1 may prove useful in the treatment of ischemia-reperfusion injuries.