Endothelial leukocyte adhesion molecule-1 in endotoxin-induced uveitis.

Expression of endothelial leukocyte adhesion molecule-1 (ELAM-1) on endothelial cells leads to the attachment of polymorphonuclear leukocytes. The sequential expression of ELAM-1 and major histocompatibility complex (MHC) class II antigen was examined in the eyes of 59 Lewis rats with endotoxin-induced uveitis (EIU) after the injection of Salmonella typhimurium endotoxin. The eyes were enucleated at 2-hr intervals. Hematoxylin and eosin-stained paraffin-embedded sections and immunohistochemically stained cryostat sections were graded by two masked observers. The MHC class II antigen was expressed on cells in the iris and ciliary body 4 hr after injection of endotoxin and on the corneal endothelium, 8 hr postinjection. It was found that ELAM-1 was expressed first on cells of the ciliary body and iris 10 hr after the injection of endotoxin and on the corneal endothelium, 22 hr postinjection. Clinical and histopathologic disease developed 16 hr postinjection. Adherence of polymorphonuclear cells to the corneal endothelium was observed at the time of ELAM-1 expression. In conclusion, expression of ELAM-1 on ocular tissue occurred in EIU and appeared to promote polymorphonuclear cell accumulation in the anterior segment of the eye.


In vitro mutagenesis of HLA-B27: single and multiple amino acid substitutions at consensus B27 sites identify distinct monoclonal antibody-defined epitopes.

The consensus HLA-B27 sequence includes a unique constellation of amino acid residues along the peptide-binding cleft. To investigate the potential role of this region in the antigenic structure of HLA-B27, a panel of transfected cell lines was produced expressing 24 mutant B27 molecules with single or multiple substitutions within this constellation of residues. The cells were analyzed by flow cytometry with a panel of four anti-B27 mAb: ME1, GSP5.3, GS145.2, and B27M2. Previous studies have suggested that position 67 exerts a conformational effect on the ME1, GSP5.3, and GS145.2 epitopes. This was further supported in these studies by the observation that additional substitutions at the flanking residues 63 and 70 could reverse the disruption of these mAb epitopes by large residues at 67. Substitutions at positions 69-71 disrupted the binding of ME1 and GSP5.3, apparently by a direct effect. Individual substitutions at either of the two positions bearing residues unique to B27, 70 and 97, had no significant influence on the binding of any of the four mAb. The region of amino acid positions 63-71 in HLA-B27 thus appears to participate in the formation of at least three distinct epitopes shared by B27 and B7, identified by ME1, GSP5.3, and GS145.2.


Reversion of radiosensitivity in azacytidine-treated XRS5 cells does not result in full radioprotection by WR-1065.

A series of cell lines were previously generated from the radiation sensitive Chinese hamster ovary line xrs5 after treatment with azacytidine. Six of these lines have been examined for their resistance to killing by 0 to 20 Gray of 60Co gamma rays and the amount of radioprotection afforded by treatment with the drug 2-[(aminopropyl)amino]ethanethiol (WR-1065). As xrs5 cells have lost the ability to be protected by WR-1065, studies were performed to determine whether reversion to radio-resistance correlated with recovery of aminothiol radioprotection. Treatment of azacytidine-treated, radiation sensitive and resistant cells with four millimolar WR-1065 30 minutes prior to irradiation enhanced survival after exposure to gamma radiation, although the enhancement in survival was less than for wild type Chinese hamster ovary K1 cells. The data suggest that there is not an absolute linkage between recovery of gamma ray radiation resistance and protection by WR-1065 and other factors, such as chromatin organization, must play a role.


Self-psychological approach to posttraumatic stress disorder: neurobiological aspects of transmuting internalization.

This essay presents the case of a young man with severe posttraumatic stress disorder (PTSD) in order to demonstrate how a self-psychological orientation and psychotherapeutic approach enables acute symptoms to be relieved, while concomitantly addressing vulnerability in character structure. The self-psychological conceptualization of the psychodynamics of PTSD is discussed, along with a recently proposed neuropsychological theory of PTSD. After viewing the pertinent neurobiological research, an integration of the self-psychological concepts of transmuting internalization (a purported curative factor in psychotherapy) with neurobiological data and behavioral observations is presented.


Effects of recombinant canine granulocyte colony-stimulating factor on white blood cell production in clinically normal and neutropenic dogs.

Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered to clinically normal dogs, cyclic-hematopoietic dogs, and dogs undergoing autologous bone marrow transplantation, to determine whether rcG-CSF could be used to stimulate WBC production and function in normal and neutropenic dogs. To the normal dogs, rcG-CSF was administered by SC injection at rates of 1 microgram/kg of body weight, q 12 h; 2 micrograms/kg, q 12 h; or 5 micrograms/kg, q 12 h. A significant dose-dependent increase in the WBC count resulted from the stimulation of bone marrow progenitor cells. The increased WBC count was characterized by mature neutrophilia and monocytosis. Neutrophil myeloperoxidase and phagocytic activity were normal in rcG-CSF-treated normal dogs, demonstrating the production of normal functional neutrophils in response to rcG-CSF treatment. Recombinant canine G-CSF prevented neutropenia and associated clinical signs but did not completely eliminate the cycling of neutrophils in cyclic-hematopoietic dogs when it was administered at rates of 1 microgram/kg, q 12 h, and 2.5 micrograms/kg, q 12 h. The time to bone marrow reconstitution was not decreased in dogs treated with rcG-CSF at a rate of 2.5 micrograms/kg, q 12 h, for 13 days following autologous bone marrow transplantation. On the basis of our findings, we suggest that treatment with rcG-CSF is an effective way to stimulate myelopoiesis in dogs, but that the dose of rcG-CSF required to stimulate WBC production will vary depending on the cause of neutropenia. Recombinant canine G-CSF should be useful in stimulating production and maintaining function of WBC for treatment of clinical diseases seen commonly in veterinary practice.


Purification of insoluble nitric oxide synthase from rat cerebellum.

Nitric oxide synthase [EC 1.14.23] from the particulate fraction of rat cerebella was purified and characterized. The homogenate of rat cerebella was centrifuged to obtain a pellet, which was washed and incubated with Triton X-100 containing buffer. The enzyme activity appeared in the 100,000 x g supernatant after incubation with the detergent. The solubilized enzyme was then purified by sequential affinity chromatography using adenosine 2',5'-diphosphate agarose and calmodulin Sepharose 4B, which gave a product that migrated as a single protein band on SDS/PAGE with a molecular mass of about 150 kDa. The purified enzyme exhibited an absolute requirement for FAD, in addition to NADPH and Ca2+/calmodulin. Thus, there is an insoluble nitric oxide synthase in rat cerebellum that has similar characteristics to the soluble type.


Mapping of a linear autoantigenic epitope within the human thyroid peroxidase using recombinant DNA techniques.

Autoantibodies directed against the thyroid peroxidase (TPO), the thyroid microsomal antigen, are widely used to diagnose human autoimmune thyroid disease. A cloned 3.088 kb cDNA coding for the entire mature human TPO was isolated from a cDNA library derived from a pathological thyroid gland of a Graves' disease patient and used further to generate a so-called TPO epitope cDNA library in order to map linear autoantigenic epitopes involving a recombinant molecular biology approach. The TPO epitope cDNA library consisting of randomly fragmented cDNA sequences inserted in the expression vector pGEX-2T was expressed in Escherichia coli and screened with characterized anti-TPO autoantisera from Hashimoto's disease patients. All the sera were positively tested with a purified thyroid microsomal antigen fraction (TMA/TPO). Only about 1% of examined autoantisera were able to recognize bacterial expressed recombinant TPO representing sequential antigenic determinants. A corresponding autoantigenic epitope with 61 amino acids in length was located at the C-terminus of human TPO.


Expression of mRNAs for alpha-fetoprotein (AFP) and albumin and incorporation of AFP and docosahexaenoic acid in baboon fetuses.

alpha-Fetoprotein and albumin, two members of a multigene family, reversibly bind fatty acids with high affinity. The origin of alpha-fetoprotein (AFP) and albumin present in fetal tissues other than the liver and yolk sac is a subject of controversy. In this work, we have searched for the presence of the albumin and AFP mRNA molecules in different fetal organs of the baboon (Papio cinocephalus), using a highly sensitive gel-blot hybridization assay with human albumin and AFP cDNA probes. Large amounts of albumin and AFP mRNA molecules were found in the fetal liver; significant quantities were also present in the gastrointestinal tract and in the kidney. No detectable levels were found in the other tissues examined (brain, skin, spleen, pancreas, muscle, heart, thymus, placenta, and amnion). After injection of radiolabeled AFP into pregnant baboons, all fetal tissues took up the protein. White adipose tissue, kidney, intestine, lung, liver, and cerebral cortex showed a great uptake of exogenous AFP. [14C]Docosahexaenoic acid (22:6, n-3), injected at the same time, was actively transferred from the maternal compartment across the placenta and incorporated into cellular lipids by all fetal tissues and particularly by liver (around 70% of total incorporation). The levels of [14C]docosahexaenoic acid per gram of tissue increased in the order: maternal blood less than placenta less than fetal liver, indicating a selective accumulation of this fatty acid by the fetus. These results indicate that intracellular AFP in non-hepatic tissues of the developing baboon is, for the most part, of plasma origin.


Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase.

Endothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO), is derived from a guanidino nitrogen of L-arginine via its metabolism by nitric oxide synthase (NOS). Herein, we report the molecular cloning of a cDNA encoding the constitutive calcium-calmodulin (Ca2+/CaM)-regulated nitric oxide synthase (ECNOS). A full-length ECNOS clone was isolated by screening a bovine aortic endothelial cell cDNA library using a fragment of rat brain NOS (bNOS) cDNA. This cDNA has an open reading frame of 3615 nucleotides encoding a 1205-amino acid protein. Membranes prepared from COS cells transfected with the ECNOS cDNA demonstrated NADPH- and Ca2+/CaM- dependent conversion of L-, but not D-, arginine to NO and citrulline that was inhibited by NG-nitro-L-arginine methyl ester. Comparison of the deduced amino acid sequence of ECNOS to the bNOS and macrophage NOS (Mac-NOS) sequences revealed 57 and 50% identity, respectively. In addition, ECNOS contains a unique N-myristylation consensus sequence (not shared by bNOS or Mac-NOS) that may explain its membrane localization.


Metabolic turnover of methotrexate polyglutamates in lysosomes derived from S180 cells. Definition of a two-step process limited by mediated lysosomal permeation of polyglutamates and activating reduced sulfhydryl compounds.

Transport and metabolic turnover of methotrexate (MTX) polyglutamates were examined in lysosomes derived from S180 cells. These studies extend prior work from this laboratory (Barrueco, J. R., and Sirotnak, F. M. (1991) J. Biol. Chem 266, 11732-11737) which described basic properties of a facilitative transport system in lysosomes capable of mediating intralysosomal accumulation of MTX polyglutamates. In the present report, we show that the rate of turnover of MTX polyglutamates in lysosomes, which releases MTX in the extralysosomal space, is limited by the extent of mediated intralysosomal accumulation of the polyglutamate and reduced sulfhydryls that activate the enzyme folylpolyglutamate hydrolase. Evidence is presented that cysteine functions as the naturally occurring reduced sulfhydryl compound in lysosomes being equipotent to 2-mercaptoethanol as an activator of folylpolyglutamate hydrolase. Folylpolyglutamate hydrolase in permeabilized lysosomes from S180 cells exhibited a low pH optimum characteristic of a lysosomal enzyme, was activated at concentrations of reduced sulfhydryl at 0.1 mM and above, and exhibited Km values in the range of 0.2-3 microM that decreased with increase in polyglutamate chain length. Values for Km for MTX polyglutamates of folylpolyglutamate hydrolase activity were 100-200-fold lower than values for Km or Ki for facilitated intralysosomal transport, whereas capacities for both processes were similar. This relationship between the kinetic properties of each process ensures efficient hydrolysis of MTX polyglutamates within the lysosome.


Characterization of a gene family encoding abscisic acid- and environmental stress-inducible proteins of alfalfa.

The phytohormone abscisic acid (ABA) has been proposed as a common mediator controlling adaptive plant responses to a variety of environmental stresses, including water deficit, salinity, wounding, and low temperature. We have recently isolated three cDNAs, pUM90-1, pUM90-2, and pUM91-4, from a cDNA library of ABA-induced mRNAs of alfalfa. These cDNA clones exhibit a very high degree of sequence homology with one another and sequence similarities with certain regions of several stress- and ABA-inducible genes. The polypeptides encoded by these cDNAs are very rich in glycine (35-40%), histidine (7-15%), asparagine (8-14%), and tyrosine (5-10%) and have no tryptophan and proline. All of the encoded polypeptides contain characteristic tandem repeats comprising glycine residues intercepted with histidine and/or tyrosine. The RNAs corresponding to a representative cDNA, pUM90-1, were induced after treatment of seedlings with low temperature, drought, salt, and wounding stress, but not by heat; the induction was maximal under low temperature treatment. ABA and ABA analog rapidly induced the expression of these genes, whereas gibberellic acid treatment exhibited no induction whatsoever. These genes appear to be specifically induced in the shoot tissues. Analysis of ABA induction of genes corresponding to pUM90-1 in alfalfa seedlings of different age groups demonstrated that these genes were inducible in seedlings/plants of all age groups examined. Taken together these results suggest that these cDNA clones encode a group of proteins that are inducible by ABA and multiple environmental stresses and correspond to a new family of genes of plants, designated as ABA- and environmental stress-inducible genes.


Poly(A)+ RNA from rabbit intestinal mucosa induces b0,+ and y+ amino acid transport activities in Xenopus laevis oocytes.

Injection of poly(A)+ RNA (mRNA) isolated from rabbit intestinal mucosa into Xenopus laevis oocytes results in an increase in sodium-independent uptake of L-[3H]leucine, L-[35S]cystine, and L-[3H]arginine. This uptake activity is related to an mRNA species corresponding to the recently isolated rabbit kidney cortex cDNA clone rBAT (related to b0,+ amino acid transporter; Bertran, J., Werner, A., Stange, G., Markovich, D., Moore, M. L., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 281, 717-723) and to a protein involved in amino acid transport via system y+. This conclusion is based on the following observations: 1) mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, induces sodium-independent uptake of L-leucine, L-cystine, and L-arginine. 2) In Northern blot analysis, mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, hybridizes to an rBAT cDNA probe, with signals of 2.2-2.3 kilobases and 3.7-3.9 kilobases. 3) mRNA isolated from mucosa of jejunum induces sodium-independent uptake of L-leucine and L-cysteine which shows an inhibition pattern corresponding to system b0,+; the inhibition pattern of mRNA-induced uptake of L-arginine is compatible with the contribution of system b0,+ and y+. 4) Hybrid depletion with an rBAT antisense oligonucleotide greatly prevents the mRNA-dependent induction of uptake of L-cystine (greater than 90%) and of L-leucine (approximately 75%); it reduces to about 50% the induction of L-arginine uptake. 5) After separation of mRNA on a sucrose density gradient, the fractions resulting in expression of b0,+ transport activity were also those hybridizing with rBAT cDNA; induction of transport activity from these fractions was also sensitive to hybrid depletion. 6) The mRNA-induced component of L-arginine uptake which is resistant to rBAT hybrid depletion is inhibited by L-homoserine, only in the presence of sodium; thus, it is related to a system y(+)-like activity.


A lineage-specific Ca(2+)-activated K+ conductance in HL-60 cells.

Cells of the human promyelocytic cell line HL-60 can be controllably induced to terminally differentiate into either granulocytes or monocyte/macrophages. HL-60 promyelocytes and terminally differentiated macrophages express a K(+)-selective ion channel which is activated by intracellular free Ca2+ concentrations above 10(-7) M. Because of its voltage independence, this channel can be distinguished from the voltage- and Ca(2+)-activated family of outward-rectifying channels. The channel is selective for K+ against Na+ and is blocked by Ba2+, thus it may be similar to the Ca(2+)-activated K+ channel previously described in human macrophages. In its sensitivity to block by charybdotoxin, this channel also resembles a Ca(2+)-activated K+ channel of lymphocytes, which plays a role in activation-dependent hyperpolarization. In contrast to promyelocytes and macrophages, functional expression of the Ca(2+)-activated K+ channel is suppressed to nearly undetectable levels in granulocytes derived from HL-60 cells by retinoic acid-induced differentiation. These data suggest that signals which produce elevation of intracellular Ca2+ will hyperpolarize promyelocytes and differentiated macrophages by activating this conductance; however, signals which elevate free Ca2+ in granulocytes must act on other effectors, which may produce a different final influence on membrane potential.


Platelet-derived growth factor BB-dimer suppresses the expression of macrophage colony-stimulating factor in human vascular smooth muscle cells.

Vascular smooth muscle cell is a major cell component involved in the process of atherosclerosis. In the present study, we investigated the effects of platelet-derived growth factor (PDGF)-BB dimer on the expression of macrophage-colony stimulating factor (M-CSF) in vascular smooth muscle cells isolated from human umbilical artery. On Northern blot analysis of total RNAs isolated from smooth muscle cells, with human cDNA for M-CSF, a marked dose-dependent reduction of mRNA level was found in PDGF-BB-treated smooth muscle cells. Cellular production of M-CSF was estimated by immunoblot analysis of cell lysate with specific polyclonal antibody against recombinant human M-CSF. A concentration of 10 ng/ml PDGF-BB significantly reduced M-CSF mass in smooth muscle cells compared with that in the absence of PDGF-BB. These results suggest that PDGF-BB plays an important role in the cellular metabolism of vascular wall by regulating the rate of M-CSF production in vascular smooth muscle cells.


Expression of the c-Harvey ras oncogene alters peptide synthesis in the neurosecretory cell line AtT20.

Ras proteins are enriched in neurosecretory cells suggesting that ras may play an important role in regulating the differentiated properties of such cells. We introduced the human H-ras oncogene, EJ-ras, into the model secretory cell line AtT20 to determine the effects of ras oncogene expression on neuropeptide synthesis and release. We report here that both of these processes are changed in ras-transfected AtT20 cells. Stimulated release of the pituitary hormone corticotropin is reduced, and transcription of the gene encoding its precursor, proopiomelanocortin, is down-regulated. At the same time, expression of other genes, both housekeeping and neural-specific, remain relatively unchanged. The alteration of proopiomelanocortin expression in AtT20 cells following ras oncogene transformation supports the hypothesis that ras may play a role in the determination of the differentiated phenotype of neurosecretory cells.


The RNA polymerase II elongation complex. Factor-dependent transcription elongation involves nascent RNA cleavage.

Regulation of transcription elongation is an important mechanism in controlling eukaryotic gene expression. SII is an RNA polymerase II-binding protein that stimulates transcription elongation and also activates nascent transcript cleavage by RNA polymerase II in elongation complexes in vitro (Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). Here we show that SII-dependent in vitro transcription through an arrest site in a human gene is preceded by nascent transcript cleavage. RNA cleavage appeared to be an obligatory step in the SII activation process. Recombinant SII activated cleavage while a truncated derivative lacking polymerase binding activity did not. Cleavage was not restricted to an elongation complex arrested at this particular site, showing that nascent RNA hydrolysis is a general property of RNA polymerase II elongation complexes. These data support a model whereby SII stimulates elongation via a ribonuclease activity of the elongation complex.


Recognition of a tetranucleotide loop of signal recognition particle RNA by protein SRP19.

The interaction of protein SRP19 with the RNA component of human signal recognition particle (SRP) was studied by site-directed mutagenesis of the SRP RNA. The effects of nucleotide changes in the tetranucleotide loop (tetraloop) of helix 6 showed that SRP19 recognizes a tetraloop in a sequence-specific manner. Adenosine 149 at the third position of the tetraloop was essential for binding. In contrast, changes of the base at the second position had no effect. Mutations that disrupt or compensate individual SRP RNA helices were generated to investigate the importance of base pairing and to identify other binding sites. Considerable base pairing was essential in helix 6. Another SRP19-binding site was located in the distal part of helix 8. The primary sequences of the tetraloop-binding protein SR19 and of bacterial ribosomal protein S15 are shown to be similar.


Three genes for the human high affinity Fc receptor for IgG (Fc gamma RI) encode four distinct transcription products.

Three distinct but closely related classes of receptors that bind the Fc portion of immunoglobulin G (Fc gamma RI, -II, and -III) have been identified in humans. Only Fc gamma RI has high affinity for ligand and has a unique third extracellular domain (EC3). We have characterized three genes for human Fc gamma RI (A, B, and C). Each gene consists of six exons, spans 9.4 kilobase pairs, and localizes to chromosome 1. Although they are remarkably similar, genes B and C are notably different from A; in-frame stop codons are present in the EC3 domain of genes B and C, and deletions occur in a splice donor sequence of gene B. Four distinct Fc gamma RI transcripts were analyzed. One transcript, from gene A, would encode a transmembrane receptor with three external domains. A second transcript, an alternatively spliced product of gene B, would encode a two-external domain transmembrane receptor. Two transcripts, from genes B and C, have stop codons in EC3 and would be predicted to generate secreted receptors.


Cyclic AMP-dependent protein kinase type I mediates the inhibitory effects of 3',5'-cyclic adenosine monophosphate on cell replication in human T lymphocytes.

Human T lymphocytes were used as a model system to study the expression and roles of cAMP-dependent protein kinase isozymes (cAKI and cAKII) in cAMP-induced inhibition of cell replication. Human peripheral blood T lymphocytes expressed mRNA for the alpha-subforms (RI alpha and RII alpha) of the regulatory subunits of cAKI and cAKII and for the alpha- and beta-subforms (C alpha and C beta) of the catalytic subunits of cAK. At the protein level, RI alpha represented approximately 75% of the total R subunit activity, whereas RII alpha (phospho and dephospho forms) accounted for the remaining 25%. RII beta was not detected at either the mRNA or the protein level. The RI alpha protein was mainly (greater than 75%) cytosolic, whereas RII alpha was almost exclusively (greater than 90%) particulate associated. Treatment of proliferating T lymphocytes (activated through the CD3 cell surface marker) with 10 different cAMP analogs demonstrated that all inhibited cell replication in a concentration-dependent manner. The potency (as measured by the concentration giving 50% inhibition, IC50) of the cAMP analogs ranged from 30 microM for 8-chlorophenylthio-cAMP to 1100 microM for 8-piperidino-cAMP. A cAMP analog pair directed to activate cAKI (8-aminohexylamino-cAMP and 8-piperidino-cAMP) synergized in the inhibition of T lymphocyte proliferation, whereas a cAKII-directed cAMP analog pair (8-chlorophenylthio-cAMP and N6-benzoyl-cAMP) did not. We conclude that activation of cAKI is sufficient to inhibit T lymphocyte proliferation. The membrane-bound cAKII may mediate cAMP actions not related to cell replication.


Molecular cloning and expression of rat liver N-heparan sulfate sulfotransferase.

N-Heparan sulfate sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the nitrogen of glucosamine in heparan sulfate. The enzyme has been previously purified to apparent homogeneity from rat liver (Brandan, E., and Hirschberg, C. B. (1988) J. Biol. Chem. 263, 2417-2422). We have now cloned the rat liver enzyme using the following strategy: (a) the amino acid sequence was obtained from tryptic peptides of the purified protein, (b) mixed oligonucleotides were generated based on the sequence of the tryptic peptides, (c) a polymerase chain reaction fragment was obtained using mixed oligonucleotide interprimer amplification of cDNA, and (d) this fragment was used to screen rat liver lambda gt 10 and lambda ZAP libraries. Three clones were obtained, one of which seems to contain the complete coding sequence of the N-heparan sulfate sulfotransferase (N-HSST). Evidence that the cDNA clone corresponds to the previously purified and characterized N-HSST was the following: (a) the predicted sequence of the N-HSST contains all of the 11 tryptic peptides obtained from the purified protein, (b) when a cDNA containing the sequence coding for the N-HSST was introduced in a eukaryotic expression vector and transfected in COS-1 cells, the enzyme activity was expressed 9-fold over controls, and (c) the characteristic of the predicted protein fits with the purified protein in terms of molecular weight, membrane localization, and its being an N-linked glycoprotein. The size of the longest cDNA isolated is 4.1 kilobases, which is in close agreement with the 4.2-kilobase size of one of the mRNA observed in Northern analyses. In addition, messages of 7.0 and 8.5 kilobases were also observed, suggesting that a large portion is untranslated. The latter messages were the major mRNA species detected.


Characterization of myelin basic protein thyroid hormone response element and its function in the context of native and heterologous promoter.

In this report we have characterized further the myelin basic protein (MBP) gene thyroid hormone response element (TRE) by functional and binding analysis. Mutation and deletion experiments revealed that this TRE, confined to the sequences -184 to -167 of the MBP promoter, is able to function as a classical regulatory element in the context of the native and a heterologous promoter. It is comprised of two regions, containing a motif that is highly conserved among other TREs: AGGACA, arranged as an inverted palindrome. Any mutation within the footprinted region impaired receptor binding and function. Moreover, the deletion of sequences outside of the receptor footprinted region (MBP-TRE-18) resulted in a higher triiodothyronine responsiveness and a concomitant increase in receptor-dependent, hormone-independent repression. Results of transfection assays showed that both receptors alpha and beta elicit indistinguishable triiodothyronine responses when the MBP-TRE functions as a regulator of a heterologous promoter activity. However, a preferential beta receptor transactivation was observed when the MBP-TRE was placed in the context of its native promoter.


Biochemical studies on the reverse transcriptase and RNase H activities from human immunodeficiency virus strains resistant to 3'-azido-3'-deoxythymidine.

A series of biochemical investigations to compare the DNA polymerase and RNase H functions of the reverse transcriptases (RTs) corresponding to azidothymidine (AZT)-sensitive and -resistant human immunodeficiency virus (HIV) strains are described. Steady-state kinetic studies with purified recombinant enzymes utilizing several templates and three inhibitors, 3' azido-3' deoxythymidine triphosphate (AZTTP), 3-amino-thymidine 5'-triphosphate, and 2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate, found consistent 2-4-fold differences between the enzymes from the two strains over a wide pH range. A strong pH dependence for all three inhibitors was found at pH values below 7.4 and suggested an ionizable group on the enzyme with a pK of about 7. The sensitivities of the RNase H activities of the two enzymes to AZTTP and AZTMP were also compared and found to be similar. The nucleotide incorporation fidelities of recombinant RTs corresponding to AZT-sensitive and -resistant clinical isolates were compared and the error specificities determined. No significant differences were found. Both enzymes were equally able to incorporate AZTTP into an elongating M13 DNA strand with concomitant chain termination. Purified wild-type and mutant virions from cell-culture supernatants were compared in "endogenous" DNA synthesis reactions, and the sensitivities of this activity to AZTTP were found to be similar. The contrast between the small differences found in this study and the high level of viral resistance in tissue culture presumably reflects an incomplete understanding of AZT inhibition of HIV in the cell.


cDNA cloning of a novel human ubiquitin carrier protein. An antigenic domain specifically recognized by endemic pemphigus foliaceus autoantibodies is encoded in a secondary reading frame of this human epidermal transcript.

Autoantibodies from a patient suffering from endemic pemphigus foliaceus (EPF), a blistering skin disease, were used to screen a lambda gt11 human keratinocyte cDNA library. One immunoreactive cDNA clone (lambda EPF5) containing a 900-base pair insert was isolated and subjected to further analysis. Eight of 25 EPF sera were shown to react with the EPF5 fusion protein on immunoblots. The EPF5 cDNA insert hybridized with a 1.2-kilobase epidermal RNA transcript on a Northern blot. Sequence analysis revealed that lambda EPF5 contained the complete coding sequence for a 24-kDa polypeptide exhibiting significant sequence homology with a family of enzymes known as ubiquitin carrier proteins, or E2s, which are an essential component of the ubiquitin-protein conjugation system. The homology was particularly high in the core region containing the active site cysteine. The keratinocyte ubiquitin carrier protein expressed in bacteria, and isolated either intact or as a glutathione S-transferase fusion protein, exhibited the ability to form a thiol ester linkage with ubiquitin in a ubiquitin activating enzyme (E1)-dependent manner, a characteristic property of ubiquitin carrier proteins. The E2 enzyme encoded by clone EPF5 is the first member of this protein family to be cloned from an epidermal source. Interestingly, the EPF autoantibody-reactive epitope and the ubiquitin carrier protein were shown to be encoded in two different translational reading frames. The relevance of the cloned EPF epitope in the pathogenesis of this autoimmune disorder remains to be determined.


Myosin light chain-2 luciferase transgenic mice reveal distinct regulatory programs for cardiac and skeletal muscle-specific expression of a single contractile protein gene.

To examine the relationship between the cardiac and skeletal muscle gene programs, the current study employs the regulatory (phosphorylatable) myosin light chain (MLC-2) as a model system. Northern blotting, primer extension, and RNase protection studies documented the high level expression of the cardiac MLC-2 mRNA in both mouse cardiac and slow skeletal muscle (soleus). Transgenic mouse lines harboring a 2100- or a 250-base pair rat cardiac MLC-2 promoter/luciferase fusion gene were generated, demonstrating high levels of luciferase activity in cardiac muscle, and only background luminescence in slow skeletal muscle and non-muscle tissues. As assessed by in situ hybridization, immunofluorescence, and luminescence assays of luciferase reporter activity in various regions of the heart, both the endogenous MLC-2 gene and the MLC-2 luciferase fusion gene were expressed exclusively in the ventricular compartment, with expression in the atrium at background levels. Point mutations within the conserved regulatory sites HF-1a and HF-1b significantly cripple ventricular muscle specificity, while mutation of the single E-box site was without effect, suggesting that ventricular muscle-specific expression occurs through an E-box-independent pathway. This study provides direct evidence that the cis regulatory sequences in the cardiac/slow twitch MLC-2 gene which confer cardiac and skeletal muscle-specific expression can be clearly segregated, suggesting that distinct regulatory programs may have evolved to control the tissue-specific expression of this single contractile protein gene in cardiac and skeletal muscle.


Inhibition by dexamethasone of beta 3-adrenergic receptor responsiveness in 3T3-F442A adipocytes. Evidence for a transcriptional mechanism.

Modulation of beta 3-adrenergic receptor (beta 3AR) expression by dexamethasone was investigated in the murine 3T3-F442A adipocytic cell line. In untreated cells, a major population of binding sites (62,000-114,000 sites/cell) of low affinity for (-)-[3H] CGP12177 and (-)-[125I]iodocyanopindolol (corresponding to the beta 3AR subtype) was present along with a minor population (6,500-8,000 sites/cell) of sites of high affinity for the radioligands (corresponding to a mixture of the beta 1 and beta 2AR subtypes). Long-term exposure of the cells to 250 nM dexamethasone led to a sharp decrease in beta 3AR density (less than 5,000 sites/cell) which paralleled a diminished potency of the beta 3AR-selective agonists BRL37344 and CGP12177 to stimulate the production of intracellular cAMP. Analysis of RNA by polymerase chain reaction and nuclear run-on assays indicated that dexamethasone inhibited the synthesis of beta 3AR mRNA, resulting in 4-8-fold decrease in the steady-state levels of this mRNA. The down-regulation of beta 3AR protein and cellular mRNA appeared to be mediated by the receptor for glucocorticoids as assessed by the antagonistic action of the anti-glucocorticoid RU38486.


Interaction of tRNA(Lys-3) with multiple forms of human immunodeficiency virus reverse transcriptase.

The interaction of several forms (p51, p66, and p66/p51) of recombinant human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with a synthetic derivative of its cognate replication primer, tRNA(Lys-3), has been determined by gel-mobility shift analysis. While p66/p51 RT is proficient in tRNA binding, preparations of p66 and p51 display only weak binding at elevated protein:tRNA ratios, despite the former containing both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activity. Gel permeation analysis of purified p66 RT indicate this to be predominantly monomeric, suggesting that dimerization may be a prerequisite for efficient tRNA binding. Prolonged incubation of a mixture of the 66- and 51-kDa polypeptides results in heterodimer reconstitution, restoration of tRNA binding, and recovery of appreciable levels of RNA-dependent DNA polymerase activity. Under the same conditions, both the tRNA binding and RNA-dependent DNA polymerase activities of the 66- and 51-kDa polypeptides are unaffected, suggesting that they remain in the monomeric conformation.


Dimerization and activation of the kit receptor by monovalent and bivalent binding of the stem cell factor.

The protooncogene c-kit encodes a tyrosine kinase receptor for the stem cell factor (SCF). Mutants of c-kit were shown to confer a pleiotropic defective phenotype and often display negative dominance in heterozygous mice. To explore the involvement of receptor dimerization in this genetic phenomenon, we employed both a human ligand, which does not recognize the murine receptor, and a rodent SCF, which binds to the human receptor with 100-fold reduced affinity as compared with human SCF. SCF binding to living cells was found to induce rapid and complete receptor dimerization that involved activation of the catalytic tyrosine kinase function. Although receptor dimerization can be attributed to the dimeric nature of the ligand, no dissociation of Kit dimers occurred at high excess of SCF, suggesting that receptor-receptor interactions are also involved in dimer stabilization. This was supported by in vitro formation of heterodimers between the human and murine Kit proteins through monovalent binding of species-specific human SCF. By coexpression of human and mouse Kit in murine fibroblasts, we found that receptor heterodimerization in living cells involved an increase in the affinity of human Kit for rat SCF and also an accelerated rate of receptor down-regulation. When a human Kit mutant lacking the kinase insert domain was coexpressed with the murine wild-type receptor, we observed a significant decrease in both the activation of the intact tyrosine kinase and its coupling to an effector protein, namely phosphatidylinositol 3'-kinase. Our results favor a receptor activation model that assumes an initial step of monovalent ligand binding, followed by an intermediate receptor dimer bound by one arm of the ligand molecule. This model predicts the existence of an intrinsic receptor dimerization site and provides a structural basis for genetic dominance of mutant SCF receptors.


Abnormal localization of cystic fibrosis transmembrane conductance regulator in primary cultures of cystic fibrosis airway epithelia.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane glycoprotein that forms Cl- channels. Previous work has shown that when some CF-associated mutants of CFTR are expressed in heterologous cells, their glycosylation is incomplete. That observation led to the hypothesis that such mutants are not delivered to the plasma membrane where they can mediate Cl- transport. Testing this hypothesis requires localization of CFTR in nonrecombinant cells and a specific determination of whether CFTR is in the apical membrane of normal and CF epithelia. To test the hypothesis, we used primary cultures of airway epithelia grown on permeable supports because they polarize and express the CF defect in apical Cl- permeability. Moreover, their dysfunction contributes to disease. We developed a semiquantitative assay, using nonpermeabilized epithelia, an antibody directed against an extracellular epitope of CFTR, and large (1 microns) fluorescent beads which bound to secondary antibodies. We observed specific binding to airway epithelia from non-CF subjects, indicating that CFTR is located in the apical membrane. In contrast, there was no specific binding to the apical membrane of CF airway epithelia. These data were supported by qualitative studies using confocal microscopy: the most prominent immunostaining was in the apical region of non-CF cells and in cytoplasmic regions of CF cells. The results indicate that CFTR is either missing from the apical membrane of these CF cells or it is present at a much reduced level. The data support the proposed defective delivery of some CF-associated mutants to the plasma membrane and explain the lack of apical Cl- permeability in most CF airway epithelia.


Repression of myogenic differentiation by aFGF, bFGF, and K-FGF is dependent on cellular heparan sulfate.

We have proposed a model in which fibroblast growth factor (FGF) signalling requires the interaction of FGF with at least two FGF receptors, a heparan sulfate proteoglycan (HSPG) and a tyrosine kinase. Since FGF may be a key mediator of skeletal muscle differentiation, we examined the synthesis of glycosaminoglycans in MM14 skeletal muscle myoblasts and their participation in FGF signalling. Proliferating and differentiated MM14 cells exhibit similar levels of HSPG, while differentiated cells exhibit reduced levels of chondroitin sulfate proteoglycans and heparan sulfate chains. HSPGs, including syndecan, present in proliferating cells bind bFGF, while the majority of chondroitin sulfate and heparan sulfate chains do not. Treatment of skeletal muscle cells with chlorate, a reversible inhibitor of glycosaminoglycan sulfation, was used to examine the requirement of sulfated proteoglycans for FGF signalling. Chlorate treatment reduced glycosaminoglycan sulfation by 90% and binding of FGF to high affinity sites by 80%. Chlorate treatment of MM14 myoblasts abrogated the biological activity of acidic, basic, and Kaposi's sarcoma FGFs resulting in terminal differentiation. Chlorate inhibition of FGF signalling was reversed by the simultaneous addition of sodium sulfate or heparin. Further support for a direct role of heparan sulfate proteoglycans in fibroblast growth factor signal transduction was demonstrated by the ability of heparitinase to inhibit basic FGF binding and biological activity. These results suggest that activation of FGF receptors by acidic, basic or Kaposi's sarcoma FGF requires simultaneous binding to a HSPG and the tyrosine kinase receptor. Skeletal muscle differentiation in vivo may be dependent on FGFs, FGF tyrosine kinase receptors, and HSPGs. The regulation of these molecules may then be expected to have important implications for skeletal muscle development and regeneration.


Direct activation of second messenger pathways mimics cell adhesion molecule-dependent neurite outgrowth.

We present evidence that direct activation of neuronal second messenger pathways in PC12 cells by opening voltage-dependent calcium channels mimics cell adhesion molecule (CAM)-induced differentiation of these cells. PC12 cells were cultured on monolayers of control 3T3 cells or 3T3 cells expressing transfected N-cadherin in the presence of KCl or a calcium channel agonist Bay K 8644. Both potassium depolarization and agonist-induced activation of calcium channels promoted substantial neurite outgrowth from PC12 cells cultured on control 3T3 monolayers and increased neurite outgrowth from those cultured on N-cadherin-expressing 3T3 monolayers. The potassium-induced response could be inhibited by L- and N-type calcium channel antagonists and by kinase inhibitor K-252b but was unaffected by pertussis toxin. In contrast activators of protein kinase C did not stimulate neurite outgrowth, and the neurite outgrowth response induced by activation of protein kinase A was not inhibited by calcium channel antagonists or pertussis toxin. These studies support the postulate that CAM-induced neuronal differentiation involves a specific transmembrane signaling pathway and suggest that activation of this pathway after CAM binding may be more important for the neurite outgrowth response than CAM-dependent adhesion per se.


Disulfides modulate RGD-inhibitable cell adhesive activity of thrombospondin.

Thrombospondin (TSP) contains the Arg-Gly-Asp (RGD) sequence that is thought to be important for cell adhesion mediated by several cell-surface integrin receptors. The RGD sequence is located in the type 3 repeat region of TSP that has multiple Ca2+ binding sites and is subject to a complex intramolecular thiol-disulfide isomerization. TSP that we isolated from thrombin-activated human platelets using buffers containing 0.1 mM Ca2+, in which Cys974 is the major labeled cysteine, did not have RGD-inhibitable adhesive activity. However, one of our preparations of TSP and TSP purified following alternative procedures using greater than or equal to 0.3 mM Ca2+ did have RGD-inhibitable adhesive activity. Reduction of TSP with DTT, either before or after adsorption to surfaces, enhanced its adhesive activity. Reduced TSP supported robust cell spreading when coated at concentrations as low as 1 micrograms/ml, whereas "adhesive" TSP not treated with DTT was active at coating concentration of greater than 20 micrograms/ml and supported only modest cell spreading. Lower DTT concentrations were required for enhancement of the adhesive activity of TSP if Ca2+ was chelated with EDTA. Cellular adhesion to DTT-treated TSP was inhibited by RGD-containing peptide and by mAb to a functional site of the alpha v beta 3 integrin. Cell blots of reduced proteolytic fragments of TSP localized the adhesive activity to the RGD-containing type 3 repeat region. These results suggest a novel mechanism for regulation of integrin-ligand interactions in which the ligand can isomerize between inactive and active forms.


Calcium alginate beads as a slow-release system for delivering angiogenic molecules in vivo and in vitro.

A method previously used in this laboratory for entrapment of tumor cells in alginate beads has been extended to provide a slow release delivery system for growth factors with known in vivo angiogenic activity. Protein growth factors were entrapped in alginate beads in amounts sufficient to cause incorporation of 3H-thymidine by COMMA-D cells in vitro, and in vivo neovascularization when injected subcutaneously into Balb/c mice. Entrapment of 125I-labelled growth factors showed that the amount of molecule entrapped in alginate beads may vary with the charge of the molecule. In vitro cell proliferation studies showed that entrapment in alginate beads may provide a slow-release system or a stabilizing environment for the protein. In some cases biological activity of the growth factor in solution was increased by the presence of control alginate beads. When alginate-entrapped growth factors were injected into Balb/c mice, induction of new blood vessels could be monitored qualitatively by macroscopic photography and assessed quantitatively by measuring the pooling of radiolabelled red blood cells at the experimental site. Subcutaneous injection of purified angiogenic factors not entrapped in alginate beads did not cause neovascularization. Diffusion of 125I-labelled growth factors from alginate beads in the animal showed that release in vivo may depend on the charge of the protein molecule. These results indicate that injection of purified molecules entrapped in alginate beads provides an effective localized and slow-release delivery of biologically active molecules. This delivery system may extend the time of effectiveness of biologically active molecules in vivo compared to direct injection without alginate entrapment. The method of entrapment and injection has potential for identifying active factors in tumor-induced angiogenesis and testing new compounds as modulators of neovascularization.


[Synergic effect of Dextran 70 at 32% and an antibiotic in the prevention of peritoneal adhesions. Experimental study in rats].

In an experimental study we evaluate the anti-adherential effect of four different solutions: physiological serum, Chloramphenicol, Dextran 70 (32%) and the association of these last two solutions. Synergic effect is shown when using an antibiotic (Chloramphenicol) with a non-bactericid solution (Dextran). Comparing our results with current literature, we are confident on the efficacity of similar association concerning other solutions than Dextran 70 (32%).


Evaluation of the interaction of protein alpha-amino groups with M(II) by immobilized metal ion affinity chromatography.

The adsorption properties of various peptides and proteins, lacking histidyl groups, on immobilized Cu(II), Ni(II), Zn(II) and Co(II) ions are described; at pH 6 and below they were little retarded. At higher pH the retention became pronounced for iminodiacetate (IDA)-Cu(II) gel. This effect seems to be related to the presence of a terminal alpha-amino group; in the absence of this group the retention of the protein was largely eliminated. At pH 8.5 a terminal alpha-amino group is adsorbed as strongly as a histidyl group. IDA-Ni(II), IDA-Zn(II) and IDA-Co(II) gels display little or no attraction for the terminal alpha-amino group of a protein.


Purification, characterization and crystallization of recombinant HIV-1 reverse transcriptase.

The pol I gene from HIV-1 encoding the protease, reverse transcriptase (RT) and endonuclease has been expressed in Escherichia coli. By modifying the fermentation conditions and developing a new purification scheme, the yield of purified RT has been increased substantially compared with that obtained in an earlier procedure. The expressed RT was purified to homogeneity by ammonium sulphate fractionation followed by chromatography on DEAE Sepharose, Heparin Sepharose, S Sepharose and Poly(A)-Sepharose. The purified HIV-RT is a heterodimer (p66/p51) with an isoelectric point close to 8 and with a tendency to aggregate. The proteolytic product (p51), corresponding to the N-terminal end of the RT molecule, was isolated and identified, as were also some bacterial polypeptides that co-elute with HIV-RT during the early stages of the purification. The heterodimer was crystallized in several morphological forms using the vapour-diffusion hanging drop technique. To concentrate the protein and to change the buffer for crystallization, reverse-salt-gradient chromatography and micropreparative columns were used. The best crystals diffracted to 9 A resolution. The best crystals of native RT diffracted to 9 A resolution and in complex with nucleic acids to 4.5 A resolution (using a rotating anode X-ray source).


Purification of complex protein mixtures by ion-exchange displacement chromatography using spacer displacers.

The anion-exchange separation of complex protein mixtures by displacement chromatography using spacer displacers driven by high-affinity final displacers is demonstrated. Guinea pig serum was separated on a medium-resolution adsorbent using a single heterogeneous mixture of carboxymethyldextran displacers to space the protein components. Mouse liver cytosol was separated on a low-resolution adsorbent using six carboxymethyldextran spacer displacers of increasing column affinity. The demonstration of the purification of alkaline phosphatase from E. coli periplasm by displacement chromatography on a high-performance liquid chromatography column is reviewed. The benefits of spacer displacers for separating minor components from complex biological protein mixtures is discussed. A simplified method for preparing carboxymethyldextran displacers is presented.


Lectin affinity electrophoresis of alpha-fetoprotein. Increased specificity and sensitivity as a marker of hepatocellular carcinoma.

alpha-Fetoprotein (AFP) is widely used as a marker of hepatocellular carcinoma (HCC) for assisting diagnosis and also for screening purposes, even though its sensitivity has been decreased slightly as a result of the earlier detection of HCC by ultrasonography. Using lectin-dependent fractionation of AFP, the diagnostic sensitivity as well as the specificity of AFP can be increased compared with measurement of total AFP. Furthermore, lectin-reactive forms of AFP, AFP-L3 and AFP-P4, have been shown to serve as preclinical markers of HCC. Accordingly, AFP is still the most reliable marker of HCC in screening and monitoring high-risk patients.


Expression of the insulin-like growth factor-II/mannose-6-phosphate receptor in multiple human tissues during fetal life and early infancy.

The insulin like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor has been detected in many cells and tissues. In the rat, there is a dramatic developmental regulation of IGF-II/M6P receptor expression, the receptor being high in fetal and neonatal tissues and declining thereafter. We have systematically studied the expression of the human IGF-II/M6P receptor protein in tissues from 10 human fetuses and infants (age 23 weeks gestation to 24 months postnatal). We have asked 1) whether there is differential expression among different organs, and 2) whether or not the human IGF-II/M6P receptor is developmentally regulated from 23 weeks gestation to 24 months postnatal. Protein was extracted from human tissues using a buffer containing 2% sodium dodecyl sulfate and 2% Triton X-100. Aliquots of the protein extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using an anti-IGF-II/M6P receptor antiserum (no. 66416) and 125I-protein A or an immunoperoxidase stain. IGF-II/M6P receptor immunoreactivity was detected in all tissues studied with the highest amount of receptor being expressed in heart, thymus, and kidney and the lowest receptor content being measured in brain and muscle. The receptor content in ovary, testis, lung, and spleen was intermediate. The apparent molecular weight of the IGF-II/M6P receptor (220,000 kilos without reduction of disulfide bonds) varied among the different tissues: in brain the receptor was of lower molecular weight than in other organs. Immunoquantitation experiments employing 125I-protein A and protein extracts from human kidney at different ages revealed a small, albeit not significant, difference of the receptor content between fetal and postnatal tissues: as in other species, larger amounts of receptor seemed to be present in fetal than in postnatal organs. In addition, no significant difference of the receptor content between human fetal liver and early postnatal liver was measured employing 125I-protein A-immunoquantitation in three fetal and five postnatal liver tissue samples. The distribution of IGF-binding protein (IGEBP) species, another abundant and major class of IGF binding principles, was also measured in human fetal and early postnatal lung, liver, kidney, muscle, and brain using Western ligand blotting with 125I-IGF-II: as with IGF-II/M6P receptor immunoreactivity there was differential expression of the different classes of IGFBPs in the various organs.(ABSTRACT TRUNCATED AT 400 WORDS)


Role of protein kinase-C in regulation of insulin-like growth factor-binding protein-1 production by HepG2 cells.

Insulin-like growth factor binding protein-1 (IGFBP-1) is a liver-derived protein that modulates the mitogenic actions of the insulin-like growth factors (IGFs). IGFBP-1 production is potently inhibited by insulin both in vivo and in HepG2 human hepatoma cells. To further define the pathways of IGFBP-1 regulation, we studied the effects of modulators of protein kinase-C (PKC) on HepG2 cell IGFBP-1 production. Phorbol 12-myristate 13-acetate (PMA) stimulated IGFBP-1 production in a time- and dose-dependent manner, with maximal stimulation occurring at 10-100 nmol/L. The degree of stimulation was dependent on cell density, ranging from about 2-fold in confluent to more than 10-fold in sparse cultures. Preincubation with PMA abolished the inhibitory effect of insulin, while preincubation with insulin did not inhibit PMA stimulation. The transient PKC activator diC8 had no effect, while studies with the PKC inhibitors sphinganine and H-7 were limited by solvent vehicle cytotoxicity. Staurosporine (STS), a potent PKC inhibitor, stimulated IGFBP-1 production 2- to 4-fold and augmented the stimulatory effect of PMA. Concanavalin-A, an inhibitor of PMA-stimulated PKC translocation and down-regulation, inhibited the effects of PMA and STS. Our findings indicate that PKC is involved in the regulation of hepatic IGFBP-1 production. The effects of PMA, which causes rapid activation, followed by membrane translocation and down-regulation of PKC, are similar to those of STS and are countered by Concanavalin-A. These data suggest that PKC activity may mediate tonic inhibition of IGFBP-1 production, while PKC downregulation stimulates the production of this regulatory protein.


Growth hormone (GH)-releasing hormone-(1-29) twice daily reverses the decreased GH and insulin-like growth factor-I levels in old men.

Aging is associated with decreased GH and insulin-like growth factor-I (IGF-I) levels and lean body mass, and increased body fat. Recombinant human GH treatment of old men partially reverses body composition changes. Administration of GH-releasing hormone (GHRH) to GH-deficient children and young adults increases GH and IGF-I levels while preserving physiological GH release. We investigated whether GHRH injections restore GH and IGF-I levels in old men to the levels in young men. Healthy young (n = 9; 26.2 +/- 4.1 yr; mean +/- SD) and old (n = 10; 68.0 +/- 6.2 yr) nonobese men underwent baseline blood sampling for measurements of IGF-I and 24-h profiles of GH release, followed by iv bolus GHRH stimulation tests. Old men then took, randomly, both low (0.5 mg) and high (1 mg) dose GHRH-(1-29) sc injections twice daily for 14 days, with an intervening 14-day nontreatment period. The study protocol was repeated on day 14 of each treatment. At baseline, the mean peak duration of spontaneous GH release (P less than 0.005) and IGF-I levels (P less than 0.0001) were lower in the old men. GHRH treatment evoked dose-related increases in all parameters, with significant differences (vs. old basal values) in mean 24-h GH (P less than 0.001), area under peaks (P less than 0.001), peak amplitude (P less than 0.05), and IGF-I (P less than 0.005) only at the high dose. After high dose treatment, there were no significant differences in these parameters between age groups. Peak and integrated responses to iv GHRH stimulation tests did not differ between young and old men either before or during GHRH treatment. Baseline serum levels of both testosterone (P less than 0.01) and phosphate (P less than 0.05) were lower in the older men. Phosphate levels increased (P less than 0.05) during GHRH treatment. GHRH treatment did not affect fasting glucose, urinary C-peptide, blood pressure, or chemistry and hematology profiles. Thus, short term sc administration of GHRH to healthy old men reverses age-related decreases in GH and IGF-I, suggesting that prolonged treatment could improve age-related alterations in body composition.


Insulin-like growth factor-binding protein-3 proteolysis is induced after elective surgery.

The insulin-like growth factors (IGFs) are bound to several binding proteins (IGFBPs) that appear to regulate IGF transport, receptor binding, and action. The concentrations of these peptides are altered by catabolic conditions. To determine if IGF-I and IGFBP levels change after surgery, sera were obtained from 16 patients before and after cholecystectomy. Immunoreactive IGF-I measured in plasma samples from which IGFBPs had been extracted did not change postoperatively. In contrast, IGF-I determined in unextracted samples increased roughly 3-fold postoperatively, presumably due to changes in IGFBPs. Two days postoperatively, IGFBP-3 levels, determined by ligand blot, averaged 36% of preoperative values, whereas levels of IGFBP-2 and a 24,000 mol wt IGFBP did not change significantly. Similarly, by immunoblot, intact IGFBP-3 was decreased 84.2 +/- 20.2%, and a 31,000 mol wt IGFBP-3 fragment increased 57.5 +/- 47.4% postoperatively. Coincubation of postoperative, but not preoperative, sera with control sera resulted in a significant decrease in IGFBP-3 and production of proteolytic fragments. IGFBP-3 proteolytic activity in postoperative sera was markedly inhibited by antipain, Na-p-tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonylfluoride, aprotinin, o-phenanthroline, and EDTA, but not by leupeptin or N-tosyl-L-phenylalanine chloromethyl ketone. This pattern of inhibition is consistent with a metal-dependent trypsin-like serine protease. We speculate that proteolysis of IGFBP-3 may alter tissue uptake of IGF-I and thereby help to counteract the catabolic state caused by surgery.


Effects of recombinant insulin-like growth factor-I (IGF-I) and growth hormone on serum IGF-binding proteins in calorically restricted adults.

To determine the effects of exogenous insulin-like growth factor-I (IGF-I) and GH on IGF-binding proteins (IGFBP)-1, -2, and -3, six healthy nonobese adult volunteers underwent two 2-week periods of diet restriction (20 Cal/kg.day), and during the last 6 days of the first period received either IGF-I (12 micrograms/kg.h by iv infusion over 16 h) or GH (0.05 mg/kg.day by sc injection). During the second 2-week study period, the alternate hormone was given. IGFBP-1 and -2 concentrations were determined by specific RIA, and changes in IGFBP-3 were assessed by ligand blotting. Free IGF-I concentrations were measured by size-exclusion high pressure liquid chromatography, followed by RIA. Diet restriction alone did not affect either IGFBP-1 or -2 significantly. IGF-I treatment increased IGFBP-1 from 78 +/- 46 ng/mL (mean pretreatment) to 137 +/- 64 ng/mL (P less than 0.001; mean for the last 4 days of IGF-I). IGF-I also caused an increase in IGFBP-2 from 315 +/- 136 to 675 +/- 304 ng/mL (P less than 0.001). GH injections caused a modest decline in IGFBP-1 concentrations but had no effect on IGFBP-2 concentrations. By ligand blotting, both IGF-I and GH caused a modest increase in IGFBP-3 band intensity. In three subjects diet restriction alone caused a small decrease in IGFBP-3 hand intensity, and this was reversed by hormone treatment. Free IGF-I concentrations in serum were increased from 1.6% to 4.4% of the total IGF-I during IGF-I infusions. GH injections caused a smaller increase in free IGF-I concentrations. The results show significant increases in IGFBP-1 and -2 during IGF-I infusion. The change in IGFBP-3, while significant, is quantitatively less than that in experimental animals that have been given IGF-I while undergoing dietary restriction. The net effect of the changes in these three forms of IGFBPs is not sufficient to maintain a normal IGF-I-binding capacity in serum, because free IGF-I levels were increased disproportionately during the IGF-I infusions. Because hypoglycemia was noted in these subjects despite insulin suppression, these alterations in IGFBPs might have changed the tissue bioavailability of IGF-I and facilitated its hypoglycemic effects.


Dihydrotestosterone accumulation in genital skin fibroblasts derived from elderly men with prostatic hyperplasia.

The conversion of testosterone to dihydrotestosterone (DHT) by 5 alpha-reductase and the interconversion between DHT and 5 alpha-Androstane-3 alpha,17 beta-diol (3 alpha-diol) by 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) were studied in fibroblasts derived from the genital skin of 15 prepubertal boys (2-10 yr), 17 young men (20-40 yr), 13 elderly men (60-78 yr) without clinically evident prostatic pathology, and 17 elderly men (61-88 yr) with benign prostatic hyperplasia (BPH). Respective DHT formations from testosterone (5 alpha-reduction) and 3 alpha-diol (3 alpha-HSOR oxidation) were not different among genital skin fibroblasts of the 4 groups. However, DHT degradation to 3 alpha-diol (3 alpha-HSOR reduction) was significantly lower in fibroblasts from elderly men with BPH than in those from the prepubertal boys (P less than 0.01), the young men (P less than 0.01), and the elderly men without BPH (P less than 0.05). 3 beta-HSOR reduction in fibroblasts of the BPH group was significantly lower (P less than 0.05) than in those of the elderly men without BPH; however, it did not differ from values for the prepubertal boys and the young men. (3 alpha + 3 beta)-HSOR reduction was also significantly lower (P less than 0.05) in the BPH group than respective values of the three other groups. These results indicate that DHT accumulation may occur in genital skin fibroblasts from elderly men with BPH, resulting from a shift in the overall balance of androgen metabolism, which favors the net formation of DHT.


Heterogeneity of serum peptides with immunoactivity detected by a radioimmunoassay for proinsulin-like growth factor-II E domain: description of a free E domain peptide in serum.

We have reported that normal human sera contain immunoactivity (IA) detected by a RIA directed against the first 21 amino acids of the E domain of proinsulin-like growth factor-II (pro-IGF-II). Marked elevations of E-21 IA were found in the serum of patients with nonislet cell hypoglycemia (NICTH) and patients with renal failure receiving chronic hemodialysis. In this paper we describe some of the properties of the E-21 IA of normal and abnormal sera. The E-21 IA eluted from a calibrated acid Sephadex G-50 column as two major peaks. In normal serum the first major peak had a mol wt (Mr) between 14,000-15,000, and the second peak had a Mr between 5,000-6,000. When E-21 IA from serum of a patient with NICTH was similarly studied, most of the IA was present as a Mr 11,000 peak and only a small amount was present as a 5,000-6,000 Mr peak. In contrast, most of the E-21 IA present in the sera of patients on hemodialysis was present in the smaller molecular form, which eluted from a reverse phase column as a single component. This small Mr peak lacked determinants for the IGF-II monoclonal antibody (Amano), for pooled serum IGF-binding proteins, and for the IGF-I receptors on human placental membranes. We suggest that the 15- and 11-kilodalton peaks represent the glycated and unglycated forms of pro-IGF-II (E-21) reported by others. The glycated form appears to predominate in normal serum, whereas the nonglycated form predominates in the serum of patients with NICTH and renal failure. The 5,000-6,000 Mr E-21 IA probably represents a fragment of the free E domain of pro-IGF-II. Its size is consistent with cleavage of the free E domain between Arg46 and Arg47. The accumulation of this E-21 IA in renal failure is evidence that the kidney has a major role in the clearance of this fragment, which is not accomplished by the membranes used in hemodialysis.


Localization of the growth hormone receptor, identified by immunocytochemistry, in second trimester human fetal tissues and in placenta throughout gestation.

Pituitary GH secretion appears largely unnecessary for the attainment of normal birth size in many species, including man. This is believed to be due to an immaturity and/or an absence of GH receptors in many fetal tissues. However, in vitro studies using late first trimester human fetal tissues have demonstrated mitogenic actions of GH on liver and stimulation of insulin biosynthesis in pancreas. To resolve this discrepancy, we have employed immunocytochemistry to identify the presence and distribution of GH receptors in various human fetal tissues. Fetuses of 14-16 weeks gestation were obtained after therapeutic abortion, tissues were fixed, and immunocytochemistry was performed using monoclonal antibodies against purified rat or rabbit GH receptor. The specificity of staining was confirmed by preabsorption of the antibodies with 1) adult rat liver membranes or 2) human fetal liver membranes, both of which possess specific GH-binding sites, or 3) human fetal skeletal muscle membranes, which do not specifically bind GH. Positive staining was seen in a subpopulation of liver parenchymal cells, in the ductal and endocrine tissue of pancreas, in the germinal layer of the epidermis and the deeper dermal layers of skin, and in the tubular epithelium of kidney. No immunopositive staining was seen in skeletal or cardiac muscle, epiphyseal growth plate, lung, intestine, or adrenal. Positive staining was present in the neuronal cell bodies of the cerebral cortex. GH receptor was also detectable as early as 8 weeks gestation in syncytial layers of the placenta and was maintained until term. Results demonstrate the presence of immunoreactive GH receptor/binding protein in some human fetal tissues early in development. In particular, these results would support a role for GH in the growth and function of liver and pancreas.


The expression of human chorionic gonadotropin/luteinizing hormone receptors in human endometrial and myometrial blood vessels.

hCG/human LH (hLH) receptors have recently been found in human endometrial and myometrial cells and uterine vasculature. The present study was undertaken to further corroborate the immunocytochemical evidence for the presence of vascular receptors. Northern blot and in situ hybridization analyses have revealed that human uterus contains a major 4.3-kilobase and a minor 2.6-kilobase hCG/hLH receptor mRNA transcript and that these transcripts are present in part in endometrial and myometrial vascular smooth muscle cells and vascular endothelial cells. Immunoblot and immunocytochemical analyses have revealed that human uterus also contains a single immunoreactive receptor protein, and that this receptor protein in part is present in endometrial and myometrial vascular smooth muscle and vascular endothelium. The expression of receptor mRNA and/or immunoreactive receptor protein was higher in myometrial than in endometrial blood vessels, and higher in vessels of both uterine compartments from the secretory compared to proliferative phase, postmenopause, or pregnancy. The blood vessels in omentum, broad ligament, and parametrium did not immunostain for hCG/hLH receptors. A blood vessel seen traversing through parametrium immunostained for the receptor protein only after it entered the myometrium. The blood vessels in nontarget tissues did not immunostain, whereas those in some target tissues, but not all of them, immunostained for the receptor protein. In summary, the present study demonstrates for the first time that human endometrial and myometrial vascular smooth muscle and endothelium express hCG/hLH receptor mRNA and immunoreactive receptor protein. These findings suggest that hCG/hLH may directly regulate blood flow in human uterus and other target tissues. The reproductive state dependency of uterine vascular receptors suggests that these receptors are probably regulated by other reproductive hormones.


Intrauterine progestin induces continuous insulin-like growth factor-binding protein-1 production in the human endometrium.

The effect of local intrauterine progestin on endometrial insulin-like growth factor-binding protein-1 (IGFBP-1) production was studied in 60 women using a levonorgestrel-releasing intrauterine device (IUD). Intrauterine progestin was a potent stimulator of stromal cell IGFBP-1 production, with 97% of endometrial specimens showing positive staining by immunohistological methods. After 5 yr or more of intrauterine progestin exposure, 100% (n = 20) of the tissues remained strongly positive for IGFBP-1. The IGFBP-1 content in endometrial tissue homogenates reached values as high as 10 micrograms/mg protein when measured by immunoradiometric assay. In contrast to the continuous endometrial IGFBP-1 production induced by local progestin, no such effect could be found in endometria from subjects with sc progestin-releasing implants or copper IUDs. Although the levonorgestrel-releasing IUD had a striking effect on local endometrial IGFBP-1 production, it had no effect on serum IGFBP-1 levels. By Western ligand blot analysis, the domainating IGF-binding species in endometria exposed to intrauterine progestin was of 28K mol wt, corresponding to IGFBP-1, whereas no IGFBP species of 31-43K, corresponding to IGFBP-2 or IGFBP-3, were detected.


Changes induced in serum protein profiles by ovarian stimulation during in-vitro fertilization--embryo transfer treatment: a comparison between conception and non-conception cycles.

The profiles of plasma protein concentrations during the follicular phase in unstimulated women and in women undergoing ovarian stimulation for in-vitro fertilization--embryo transfer (IVF-ET) treatment are described. Plasma protein concentrations are correlated with those of total oestradiol (protein-bound and free) and total progesterone. In addition, 10 conception cycles and 18 non-conception cycles are compared in an attempt to identify predictors of successful treatment. Ovarian stimulation caused a significant increase in follicular phase in serum concentrations of sex hormone binding globulin (SHBG), cortisol binding protein (CBP) and insulin-like growth factor binding protein 1 (IGFBP1). In contrast no increase was observed in unstimulated cycles. Serum levels of endometrial protein PP14 decreased significantly during the follicular phase in both stimulated and unstimulated cycles. Levels of pregnancy zone protein (PZP) were more than doubled at the time of oocyte aspiration compared to the unstimulated cycles. Albumin concentrations were unchanged by the stimulation. Throughout the follicular phase, levels of SHBG were significantly higher, and total oestradiol significantly lower in women who became pregnant, than in those who did not. Therefore, a low concentration of free, biologically active oestradiol seemed to favour pregnancy, as the concentration of albumin is similar in the two groups. The endometrial protein PP14 was significantly lower during the follicular phase in conception than in non-conceptional cycles. On day 2 of the treatment cycle, the PP14 concentration showed a 75% correct prediction of conception and non-conception cycles. These results suggest that levels of PP14 may predict successful IVF cycles even before hormonal treatment is commenced.


The acrosome reaction-inducing activity of individual human follicular fluid samples is highly variable and is related to the steroid content.

In this study, we have evaluated the relationship between the acrosome reaction-inducing activity of individual human follicular fluid samples and their steroid content. Eighteen samples of follicular fluid were obtained during egg retrieval in six patients undergoing assisted fertilization. Motile spermatozoa were incubated in modified Tyrode's medium (26 mg/ml bovine serum albumin) for 20 h at 1 x 10(7) cells/ml. In a single experiment, aliquots of a semen specimen were simultaneously treated with an aliquot of each follicular fluid sample. The percentage of acrosome reacted spermatozoa was determined using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) lectin. The fluids were also analysed by radioimmunoassay to determine the levels of progesterone, 17 alpha-hydroxy-progesterone, testosterone and oestradiol. The results showed that there was a positive, highly significant correlation between the acrosome reaction-inducing activity and the progesterone level of each follicular fluid sample (r = 0.72, P less than 0.005). Additionally, treatment of the follicular fluid samples with charcoal-dextran caused both a decrease in progesterone concentration and the total loss of the acrosome reaction-inducing activity. The addition of progesterone restored the acrosome reaction-inducing ability in 88% of samples. These data support the idea that progesterone in follicular fluid is the molecule responsible for inducing the acrosome reaction in human spermatozoa.


The expression of retinoic acid receptors in cultured human endometrial stromal cells and effects of retinoic acid.

Patterns of expression of retinoic acid receptors (RAR) in cultures of human endometrial stromal cells are described. Transcripts for all three classes of RAR were expressed in these cells but RAR-beta was expressed at a low level by comparison with RAR-alpha and RAR-gamma. The abundance of RAR-beta transcripts was elevated by treating the cells with retinoic acid, but there was no effect on the level of expression of RAR-alpha and RAR-gamma. The induction of RAR-beta by retinoic acid was detectable within 4 h and at low concentrations of retinoic acid (10(-10) M). Adenosine 3':5'-cyclic monophosphate (cAMP) analogues and forskolin, an adenylate cyclase activator, had no effect on the retinoic acid-mediated induction of RAR-beta, contrary to recent observations on embryonal carcinoma cells. However, the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), forskolin and 8-bromo-cAMP depressed basal levels of RAR-beta expression. These data suggest that endometrial stromal cells may be a target tissue of retinoic acid in vivo, and imply a role for retinoic acid in the cyclical differentiation of human endometrium.


The value of serum levels of oestradiol, progesterone and beta-human chorionic gonadotrophin in the prediction of early pregnancy loss.

Serial serum levels of oestradiol, progesterone and the beta-subunit of human chorionic gonadotrophin (beta-HCG) had been performed in 674 cycles in women conceiving a singleton pregnancy, either spontaneously or as a result of assisted conception. To determine the value of these estimations in the prediction of early pregnancy loss, frequency distribution curves and receiver operating characteristic curves were derived for the respective hormones measured at weeks 4-7 of gestation and expressed as multiples of the median (MoM) values in pregnancies occurring both with and without ovarian stimulation. A cut-off level of beta-HCG less than 0.5 MoM gave a sensitivity of 68% with an odds ratio of 4.0 at 7 weeks in unstimulated cycles in the prediction of pregnancy failure. A cut-off of 0.8 MoM for progesterone gave a sensitivity of 59% and an odds ratio of 2.8. Prospective hormonal monitoring during the early weeks of gestation may be useful in the prediction of early pregnancy loss and should help to avoid the emergency presentation of some of the complications of early pregnancy, in particular ectopic pregnancy. The limitations imposed by multiple pregnancies and uncertain gestation due to menstrual data may restrict the use of this strategy to specialist fertility centres.


Improved sensitivity and specificity of a single measurement of serum progesterone over serial quantitative beta-human chorionic gonadotrophin in screening for ectopic pregnancy.

The sensitivity and specificity of a single serum progesterone measurement was compared against two beta-human chorionic gonadotrophin (HCG) measurements 48 h apart in screening for abnormal pregnancy, i.e. ectopic pregnancy, completed or incomplete abortion. Of 1120 patients in the first trimester presenting with a positive urinary pregnancy test, 116/1120 (10.4%) had an ectopic pregnancy, 755/1120 (67.4%) had ultrasonographically confirmed intra-uterine pregnancies, and 249/1120 (22.2%) had abnormal intra-uterine pregnancies documented as complete, incomplete or missed abortions. Of the ectopic pregnancies, 113/116 (97.4%) had a serum progesterone level less than 25 ng/ml while 516/755 (68.3%) viable intra-uterine pregnancies had a serum progesterone level greater than or equal to ng/ml. Of the 1120 patients screened, 402 (35.9%) had both a serum progesterone and two HCG measurements and were eligible for inclusion in this study. Setting a cut-off of 25 ng/ml, the sensitivity and specificity of a single serum progesterone measurement was then compared against two serial HCG measurements, utilizing receiver operating characteristic curves. This analysis demonstrated that a single serum progesterone measurement was significantly more sensitive (P less than 0.05) than two HCG measurements in screening for an abnormal pregnancy. In some patients, a single serum progesterone makes possible the diagnosis of ectopic pregnancy 2 days earlier than two HCG determinations because a second blood sample was not required. We conclude that a single serum progesterone measurement should be added to serial HCG determinations as a standard diagnostic screening test for ectopic pregnancy.


Computer-aided analysis of DNA curves on transverse gradient gels.

Transverse pore gradient polyacrylamide gel electrophoresis of DNA restriction fragments was used to generate gel patterns describing migration distance as a function of gel concentration (Ferguson curves). These Ferguson curves were digitized, traced and analyzed with the aid of a personal computer. The traced curves were plotted semi-logarithmically and the plots were subjected to least-squares linear regression analysis to yield values of the slope (KR) and the intercept at %T = 0 (YO). These values are highly precise since they are based on approx. 100 measurements per curve. The computerized method reduces the errors due to manual measurements of migration distances and is time and labor saving. The method is still limited to intra-experimental comparison of Ferguson curves, since it does not as yet comprise a determination of gel concentration. At present, curve tracing remains semi-automated, requiring manual intervention when Ferguson curves cross or approach one another. Potentially, the importance of the computerized analysis of transverse pore gradient gels lies in the rapid quantitative interpretation of Ferguson curves for detection of anomalously migrating DNA species. Potentially, that application provides a more sensitive and informative mode of detection than either the mere visual observation of crossing Ferguson curves or of a shift in mobility at a single gel concentration.


Staining of argininosuccinate lyase activity in polyacrylamide gel.

A procedure for the direct staining of argininosuccinate lyase activity in polyacrylamide gel is described. The method was based on coupling one of the enzymatic products fumarate with fumarase and malic enzyme catalyzed reactions. Fumarate was first converted to L-malate by fumarase. Malic enzyme then catalyzed the oxidative decarboxylation of L-malate to give CO2 and pyruvate with concomitant reduction of NADP+ to NADPH. Finally the reducing power of NADPH was coupled to phenazine methosulfate and in turn to nitroblue tetrazolium yielding a deeply colored insoluble formazan which may be quantitized or semiquantitized by densitometer.


[Orbital myositis, recurrence of Whipple's disease].

Whipple's disease, a chronic systemic and multi-organ disease can be associated with ophthalmological manifestations with or without CNS involvement. Myositis rarely occurs during Whipple's disease. Antibiotic treatment prevents CNS recurrence. We report here the association of an orbital pseudotumor with Whipple's disease. This myositis was discovered on a follow-up CT examination. It appeared only one year after the end of a long course of antibiotic treatment, but it resolved within a few weeks when treatment was reintroduced. Relations between these two diseases are discussed.


Effect of the organophosphorous insecticide, chlorpyrifos (Dursban), on growth, fecundity and mortality of Biomphalaria alexandrina and on the production of Schistosoma mansoni cercariae in the snail.

Exposure of Biomphalaria alexandrina to sublethal concentrations (0.125, 0.25 and 0.05 ppm) of the organophosphorous insecticide, chlorpyrifos (Dursban), induced a reduction in egg production and egg hatchability. Exposure of Schistosoma mansoni miracidia to the insecticide (60 min, 0.50 ppm) prior to infection of B. alexandrina did not affect the subsequent production of cercariae. However, exposure of S. mansoni-infected snails to the insecticide until day 55, from day 20 to day 62 and from day 35 to 62 following infection resulted in blockage of cercarial shedding. Cercarial shedding commenced in some snails when the treatment stopped. Exposure to the insecticide in concentrations of 0.125 and 0.25 ppm during the first 20 days following infection did not affect the subsequent production of cercariae, but exposure to 0.5 ppm during the first 20 days affected markedly the production of cercariae due to a high snail mortality. The findings indicate that the cercaria is the target stage for the activity of chlorpyrifos on the intramolluscan larval development. It is suggested that S. mansoni cercarial production in B. alexandrina may be a useful system for monitoring the effect of low concentrations of pesticides on the aquatic environment, and that the ability by chemical means to interrupt the cercarial production might be a useful tool in further analyses of important aspects of the snail/parasite relationship.


Pseudomonas cepacia pulmonary infection in adults with cystic fibrosis: is nosocomial acquisition occurring?

Ribotyping of 25 isolates of Pseudomonas cepacia taken from the sputum of 21 adults with cystic fibrosis (CF) who were registered at the Royal Brompton Hospital between 1987 and 1991, revealed that seven patients (33.3%) shared strains of a similar ribotype pattern with others, while 14 patients (63.7%) harboured strains unique to each individual. Constancy of sputum strain carriage was seen in two of three patients sampled twice over a 3-month period. Although no evidence for patient-to-patient transmission of P. cepacia within this group of patients was found, the fact that one third of CF patients shared strains of the same ribotype with others, suggests that nosocomial acquisition of this organism may have occurred.


Generation of monoclonal antibodies to human lymphocyte cell surface antigens using insect cells expressing recombinant proteins.

We have expressed human CD40 and human B7 in insect cells using the baculovirus expression system and have used these insect cells to immunize mice for the generation of monoclonal antibodies. We demonstrate here that specific monoclonal antibodies to human CD40 and human B7 were obtained using this approach. One significant advantage of this method is that immunizing mice with insect cells did not evoke an immune response to human cells and, therefore, EBV-transformed human B cells could be used to screen for specific antibody production by the hybridoma clones.


Agonistic properties of anti-B cell antibodies purified on staphylococcal protein A may be due to contaminating protein A.

Some antibodies directed to cell surface receptors may mimic the physiological ligands by inducing the transmission of activation or growth signals. Such agonistic antibodies have proven very useful when studying functional properties of various receptor molecules on, e.g., lymphoid cells. However, while investigating the agonistic effects on tonsillar B cells of the anti-CD43 monoclonal antibody (mAb) D4B11 and the anti-CD40 mAb S2C6, we made some observations which emphasize the need for caution when using antibodies purified by protein A affinity chromatography. Both antibody preparations were found to elicit changes in the intracellular free calcium concentration ([Ca2+]i) as well as promoting proliferation of phorbol ester activated cells. However, a closer analysis showed that the increase in [Ca2+]i could be attributed to soluble staphylococcal protein A (SpA) desorbed during antibody purification. By using pure soluble SpA, we were able to show that nanogram amounts were sufficient to increase [Ca2+]i by a mechanism that involved both a mobilization from intracellular stores and an influx across the B cell membrane. A similar effect on cytosolic Ca2+ in B cells was also noted for streptococcal protein G (protein G), another bacterial component used for antibody purification. However, in contrast to SpA, protein G had little effect on cell proliferation. These observations suggest that the presence of trace amounts of SpA or protein G in antibodies purified on these bacterial components may lead to incorrect interpretations of the agonistic properties of such antibodies. When the above findings were taken into account, it was found that the CD43 mAb D4B11, like the CD40 mAb S2C6, stimulated growth of B cells without causing any measurable increase in [Ca2+]i. Both CD40 and CD43 may thus be coupled to signalling pathways which do not involve breakdown of membrane phosphoinositides.


Determination of monoclonal antibody specificity by immunoadsorption and western blotting.

A rapid and simple method has been developed for identifying the specificity of monoclonal antibodies at an early stage in the production of hybridomas. The technique is a micro-method utilizing biotinylated crude antigen and the surface of microtiter plates as an immunoaffinity matrix. The monoclonal antibodies to be tested are adsorbed to the microtiter wells and incubated with the labeled antigen preparation. Non-specific binding can be reduced by blocking and repeated washing steps. The specific antigen is then eluted by SDS-containing buffers, subjected to SDS-PAGE, blotted onto nitrocellulose and detected by enzyme-labeled avidin in a Western blot assay. The amount of bound and removed antigen can be quantitated by developing eluted and non-eluted control wells by ELISA techniques. Since this ELISA can be performed rapidly, only samples which contain sufficient specific material can be selected for electrophoresis and blotting. The major advantages of the technique are (i) the use of a non-radioactive label resulting in an easy and time-saving procedure, (ii) the possibility of quantitating the amount of captured and detached antigen by ELISA, (iii) the need for only a minimal amount of antigen, (iv) the use of unpurified antibodies of all isotypes, (v) a high signal-to-noise ratio, and (vi) as with all immunoprecipitation techniques, the possibility of detecting SDS-sensitive epitopes and of using crude antigen preparations. Using this method we were able to characterize monoclonal antibodies against both soluble proteins (mouse and human C1q) and membrane determinants (human pan T cell CD5 and CD7).


Production of monoclonal antibodies against the human anaphylatoxin C5a des Arg and their application in the neoepitope-specific sandwich-ELISA for the quantification of C5a des Arg in plasma.

A panel of ten murine monoclonal antibodies (MAbs) was raised against the human anaphylatoxin C5a des Arg. The MAbs were shown to abrogate or significantly inhibit the chemotactic activity in zymosan activated serum. MAb 4A2E10E2 and MAb 3G3C4 were used as capture and detecting antibody, respectively, in a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of C5a des Arg. This ELISA was shown to be very sensitive (detection limit 20 pg/ml) and could be applied directly to plasma/serum samples. The lack of interference by plasma components, in particular C5, suggested specificity for an epitope on C5a (des Arg) which is concealed in native C5 and exposed on the activation fragment only, i.e., a 'neoepitope'. The mean C5a des Arg level in EDTA-plasma from 25 healthy individuals assessed at a 1/20 dilution was 11.2 ng/ml (SD 3.4; range 6.4-16.8 ng/ml). The applicability of the assay was investigated in patients treated with haemodialysis using different membranes. Markedly elevated plasma levels of C5a des Arg were found in blood returning from the dialyzer following contact with cuprophane membranes.


Increased ELISA sensitivity using a modified method for conjugating horseradish peroxidase to monoclonal antibodies.

We investigated the importance of monoclonal antibody (MCA) purity and the input molar ratio of horseradish peroxidase (HRPO)/IgG used for MCA conjugation on various immunoenzymometric assay (IEMA) parameters. The sensitivity of IEMAs for human follicle stimulating hormone (hFSH), human chorionic gonadotropin (hCG) and the free alpha subunit of hCG (hCG alpha) could be increased up to 6-fold, whereas non-specific binding remained within tolerable limits (E less than 0.1), when MCAs purified by high performance liquid chromatography (HPLC) using a hydroxylapatite column (HPHT) were conjugated with an input molar HRPO/IgG ratio of four instead of the usual ratio of two.


Role of endogenous interferon in the high sensitivity of C3H/10T1/2 cells to the antiviral activity of interferon.

Addition of anti-mouse interferon (IFN) serum to untransformed 10T1/2 cells at the time of virus challenge led to a reduction of the IFN titers as determined by inhibition of CPE. The titers were reduced to values comparable to those usually observed for transformed 10T1/2 cells. In contrast, no change in IFN titers was observed when anti-IFN serum was added to transformed 10T1/2 cells. There was no significant difference between the two types of cells in the level of IFN induced by the challenge virus, VSV. The IFN level was low and did not increase after pretreatment of the cells with IFN. The distinct effect exerted by anti-IFN serum on untransformed and x-ray transformed 10T1/2 cells was not unique and comparable results were obtained in several other normal and transformed cells.


The interferon system in patients with malignant disease.

Since the interferon (IFN) system involves both IFN producing and IFN responding cells, it is possible to study separately these phenomena, relating them to disease entities as well as to response to therapy. Numerous studies in animals and man suggest effectiveness of IFN and IFN inducer therapy in cancer. However, the competency of the various components of the endogenous IFN system in malignancy has received little attention. These studies show that in malignancy there may be (1) a high incidence of elevated blood levels of IFN; (2) a deficient response of peripheral blood mononuclear cells to endogenous and exogenous IFN; and (3) increased uninduced in vitro IFN production by these cells. These findings indicate that cancer patients are equipped with the ability to produce IFN and suggest that it may be the deficient response of their cells to IFN that plays a role in the development and progression of the disease. Furthermore, the finding of increased spontaneous "uninduced" production of IFN by cells from cancer patients suggests the possibility of an intracellular inducer such as found in persistently virus-infected cells.


Suppression of macrophage Ia antigen expression by endogenous interferon-alpha/beta.

Exogenous interferon-alpha (IFN-alpha) and interferon-beta (IFN-beta) (type I IFNs) are known to suppress the IFN-gamma-dependent expression of class II MHC (Ia) antigens on macrophages (M phi). We report here that the endogenous type I IFNs produced by M phi in response to IFN inducers regulate Ia expression of the M phi themselves. Coculture of M phi with IFN-gamma and polyinosinic-polycytidylic acid [poly(I):poly(C)] resulted in the reduction of Ia expression in comparison with those cultured without poly(I):poly(C). Pretreatment of M phi with poly(I):poly(C) or a bacterial lipopolysaccharide (LPS), which is also a potent IFN inducer, in vitro or in vivo, before being exposed to IFN-gamma was also effective in suppressing the Ia expression. Such suppression was abolished by the addition of anti-IFN-alpha/beta antibodies to the M phi culture along with IFN-gamma. M phi cultured with L-cell conditioned medium (LCM) containing M-CSF were less capable of expressing Ia antigens than those cultured without LCM. The Ia-expressing ability of LCM-treated M phi was also restored by the addition of anti-IFN-alpha/beta antibodies. M phi in the early stage of sterile inflammation were less responsive to IFN-gamma than those in the late stage. These results suggest that endogenous type I IFNs, which are produced in response to natural or synthetic IFN-inducers, regulate M phi Ia expression in an autocrinal manner.


Decreased interferon response by lymphocytes from children with chronic hepatitis.

Since a decreased interferon response has been linked with certain chronic viral infections, a preliminary study was undertaken to determine whether an altered interferon response may be involved in the pathogenesis of chronic hepatitis, a complication of type B hepatitis. The production of interferon by lymphocytes from patients with chronic hepatitis was compared with the production by lymphocytes from normal subjects. The amount of interferon produced by paramyxovirus-stimulated lymphocytes from 16 patients was substantially smaller than that produced by lymphocytes from 12 healthy donors. The results indicate that decreased production of interferon may be responsible for the chronicity of the disease. However, further studies are necessary to establish causality.


Interferon production in severe hemophiliacs with and without HIV antibodies.

The interferon (IFN) system, both serum IFN levels and the in vitro IFN production, was investigated in 38 clinically asymptomatic multitransfused hemophiliacs, half positive and half negative for HIV antibodies. In most patients, no circulating IFN was detected; similar levels of IFN-alpha were obtained after peripheral blood mononuclear cell (PBMC) stimulation with Sendai virus both in hemophiliacs and controls, while production of IFN-gamma following stimulation with phytohemagglutin (PHA) was diminished in a large number of patients irrespective of their HIV serology. These data indicate that the deficiency in IFN-gamma generation is not only related to HIV contamination but may be a direct consequence of the chronic antigenic stimulation through Factor VIII concentrates.


Cell cycle regulation (G1) by autocrine interferon and dissociation between autocrine interferon and 2',5'-oligoadenylate synthetase expression.

In tertiary MEF undergoing cell cycle progression, autocrine interferon (IFN) is released and constitutive levels of 2',5'-oligoadenylate (2-5A) synthetase activity, low through the cell cycle, surge into a peak within S phase. Treatment of MEF with the autocrine IFN they produce elicits a 2-5A synthetase response from cells positioned in G0 but not from cells in G1 and from cells in S phase. Neutralization of the autocrine IFN by antibody shortens the length of G1 leaving unaltered the kinetics of progression through S and G2 and has no effect on the S phase-linked expression of 2-5A synthetase activity. The growth controlling effect of the autocrine IFN has been mapped to the second part of G1.