Molecular weight-dependent lymphatic transfer of exogenous macromolecules from large intestine of renal insufficiency rats.

To study the lymphatic delivery of exogenous macromolecules via the enteral route in renal insufficiency, we determined the transfer selectivity to the systemic blood and lymph of fluorescein isothiocyanate-labeled dextrans of different average molecular weight (10, 18, 39, and 69kD). The compounds were administered into the large intestinal lumen of rats with occluded renal circulation, with the aid of lipid-surfactant mixed micelles as an absorption prometer. Whereas concentrations of the smaller dextrans (molecular weight under 18 kD) in the lymph of the thoracic duct and in the peripheral plasma were similar, levels of dextrans over 39 kD were significantly higher in the lymph than in the plasma. Further, dextran plasma concentrations decreased in inverse proportion to increasing molecular weight. The molecular weight threshold for a high lymph-to-blood level ratio (18-39 kD) was higher than that previously found in rats with normal renal function (10-18 kD). This difference was accounted for by reduced renal clearance of the low molecular weight dextrans in renal failure. These results are useful in the design of lymphotropic drug delivery in disease states.


Folding in vitro of bovine pancreatic trypsin inhibitor in the presence of proteins of the endoplasmic reticulum.

The rates of folding and disulfide bond formation in reduced BPTI were measured in vitro in the presence and absence of total protein from the endoplasmic reticulum. The rates were increased substantially by the endoplasmic reticulum proteins, but only to the extent expected from the known content and activity of protein-disulfide-isomerase. No effects of added ATP or Ca2+ were observed, even though protein-disulfide-isomerase binds Ca2+ tightly.


Empirical solvation models in the context of conformational energy searches: application to bovine pancreatic trypsin inhibitor.

Continuum solvation models that estimate free energies of solvation as a function of solvent accessible surface area are computationally simple enough to be useful for predicting protein conformation. The behavior of three such solvation models has been examined by applying them to the minimization of the conformational energy of bovine pancreatic trypsin inhibitor. The models differ only with regard to how the constants of proportionality between free energy and surface area were derived. Each model was derived by fitting to experimentally measured equilibrium solution properties. For two models, the solution property was free energy of hydration. For the third, the property was NMR coupling constants. The purpose of this study is to determine the effect of applying these solvation models to the nonequilibrium conformations of a protein arising in the course of global searches for conformational energy minima. Two approaches were used: (1) local energy minimization of an ensemble of conformations similar to the equilibrium conformation and (2) global search trajectories using Monte Carlo plus minimization starting from a single conformation similar to the equilibrium conformation. For the two models derived from free energy measurements, it was found that both the global searches and local minimizations yielded conformations more similar to the X-ray crystallographic structures than did searches or local minimizations carried out in the absence of a solvation component of the conformational energy. The model derived from NMR coupling constants behaved similarly to the other models in the context of a global search trajectory. For one of the models derived from measured free energies of hydration, it was found that minimization of an ensemble of near-equilibrium conformations yielded a new ensemble in which the conformation most similar to the X-ray determined structure PTI4 had the lowest total free energy. Despite the simplicity of the continuum solvation models, the final conformation generated in the trajectories for each of the models exhibited some of the characteristics that have been reported for conformations obtained from molecular dynamics simulations in the presence of a bath of explicit water molecules. They have smaller root mean square (rms) deviations from the experimentally determined conformation, fewer incorrect hydrogen bonds, and slightly larger radii of gyration than do conformations derived from search trajectories carried out in the absence of solvent.


Variability of conformations at crystal contacts in BPTI represent true low-energy structures: correspondence among lattice packing and molecular dynamics structures.

The structures of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X-ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the molecule, although the regions that display most variability are associated with the loop structures (residues 14-17 and 24-29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higher B-factors, although it is not apparent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X-ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H-bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance; the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96-psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X-ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low-energy conformers that have been preferentially selected by crystallization factors.


Structural effects induced by mutagenesis affected by crystal packing factors: the structure of a 30-51 disulfide mutant of basic pancreatic trypsin inhibitor.

The X-ray structure of the C30V/C51A disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) has been analyzed at 1.6 A resolution. The mutant crystallizes in a cell having two molecules in the asymmetric unit. The packing environments of these two molecules are quite different, allowing for an assessment of which among the observed structural changes result from the mutation and which are produced by lattice packing considerations. The removal of the 30-51 disulfide bridge has little apparent affect on the B-factors of segments of adjacent polypeptide chain, although there are distinct differences in the structure compared to wild-type BPTI crystal structures. Both of the two C30V/C51A molecules show differences at the mutation site when compared to another 30-51 disulfide mutant, C30A/C51A, presumably due to the larger steric bulk of a valine versus an alanine at residue 30. A comparison of the two independent C30V/C51A molecules indicates that there are significant differences between them even at the site of mutation. The description of the specific structural differences of each molecule differs in detail and suggests different conclusions about the nature of structural perturbation near 30-51. In addition, when these two molecules are compared to two different wild-type structures, which had been determined from different space groups, a somewhat different pattern of changes is observed. These findings indicate that crystal packing can influence the observed perturbations in mutant structures.


In vitro selection of an RNA epitope immunologically cross-reactive with a peptide.

An antiserum raised against a peptide was used to select a unique RNA species from a degenerate pool of RNAs designed to resemble an autoantibody recognition site in U1 RNA. The peptide and the selected RNA epitope could compete for antibody binding, suggesting that both RNA and peptide epitopes occupy the same or overlapping antigen-combining sites. Thus, the RNA epitope functioned as a specific inhibitor of the antibody-antigen interaction. We demonstrate that the RNA epitope can be used to tag unrelated RNA molecules and also to detect the presence of the antibody. We propose that sequence-specific recognition of RNA by antibodies may involve protein-RNA contacts similar to those occurring in other nucleic acid-binding proteins. In addition, these findings are compatible with the suggestion that nucleic acid-binding autoantibodies may arise through immunological cross-reactivity between proteins and nucleic acids.


Major histocompatibility complex determinants select T-cell receptor alpha chain variable region dominance in a peptide-specific response.

Dominant expression of T-cell receptor (TCR) alpha or beta chain variable region (V alpha or V beta) gene families has been observed in the T-cell response to some conventional peptide antigens. Current models for the interaction of TCR V region elements with different determinants of a major histocompatibility complex (MHC)-peptide complex, the normal TCR ligand, suggest that the TCR V-J junctional region (CDR3, where J is joining) is the primary contact with a peptide epitope and that other TCR V region segments may interact directly with neighboring MHC determinants. This suggests that V alpha or V beta dominance in a specific response can be MHC-selected. In this case, if related peptides bind to an MHC molecule in a similar orientation, they could select for identical V alpha or V beta dominance even if they are noncrossreactive at the level of T-cell activation. We have screened for this possibility by introducing minimal conservative substitutions in a synthetic peptide, YYEELLKYYEELLK, that is presented to T cells in association with an uncommon A beta E alpha d mixed Ia isotype. We report here that the peptide variant FFEELLKFFEELLK is noncrossreactive with YYEELLKYYEELLK but appears to preserve the same MHC binding motif since T-cell responses are restricted to the same mixed A beta E alpha isotype. Although the two peptides are noncrossreactive in either direction, the same members of the V alpha 4 gene family are dominantly expressed in T cells specific for either peptide. We conclude that the similar topography of the two MHC-peptide complexes gives functional significance to a unique A beta E alpha determinant that selects for V alpha 4 dominance.


GreA protein: a transcription elongation factor from Escherichia coli.

A protein identified as the 158-amino acid product of the greA gene was isolated from Escherichia coli. When added to a halted ternary transcription complex, the GreA protein induced cleavage and removal of the 3' proximal dinucleotide from the nascent RNA. The new 3' terminus generated by the cleavage could be extended into longer transcripts. GreA-mediated cleavage of a transcript appears to permit a ternary complex to resume transcription from a state of indefinite elongation arrest induced by a specific DNA site. The GreA protein tended to interact with RNA polymerase during purification and recycled between RNA polymerase molecules in the course of the in vitro cleavage reaction. Similar biochemical activities have been reported in eukaryotic RNA polymerases, indicating that transcript cleavage and restart of elongation may be a general transcriptional mechanism.


Spatial pattern of receptor expression in the olfactory epithelium.

A PCR-based strategy for amplifying putative receptors involved in murine olfaction was employed to isolate a member (OR3) of the seven-transmembrane-domain receptor superfamily. During development, the first cells that express OR3 appear adjacent to the wall of the telencephalic vesicle at embryonic day 10. The OR3 receptor is uniquely expressed in a subset of olfactory cells that have a characteristic bilateral symmetry in the adult olfactory epithelium. This receptor and its specific pattern of expression may serve a functional role in odor coding or, alternatively, may play a role in the development of the olfactory system.


Cloning of ASH, a ubiquitous protein composed of one Src homology region (SH) 2 and two SH3 domains, from human and rat cDNA libraries.

The protein ASH (for abundant Src homology), composed of one Src homology region (SH) 2 and two SH3 domains, was cloned by screening human and rat cDNA libraries with an oligonucleotide probe directed to a consensus sequence of the SH2 domains. The rat-derived ASH peptide was comprised of 217 amino acids with a molecular mass of 25-28 kDa and was found to be ubiquitous in rat tissues. A human cDNA clone was also found to code for part of the same protein, suggesting that ASH is common to human and rat. The amino acid sequence of ASH was strikingly similar to Sem-5, the product of a nematode cell-signaling gene, and ASH is most probably a mammalian homologue of Sem-5. ASH bound in vitro to phosphotyrosine-containing proteins, including activated epidermal growth factor receptor, the ASH SH2 domain being responsible for the binding. Induced expression of an antisense ASH cDNA led to a reduction in cell growth. Considering these observations and the structural homology to Sem-5, ASH is likely to function as a ubiquitous signal transducer, possibly resembling Sem-5, which communicates between a receptor protein tyrosine kinase and a Ras protein.


Cis-acting, orientation-dependent, positive control system activates pheromone-inducible conjugation functions at distances greater than 10 kilobases upstream from its target in Enterococcus faecalis.

The prgB gene encodes the surface protein, Asc10, which mediates cell aggregation, resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline-resistance plasmid pCF10 in Enterococcus faecalis. Messenger RNA analysis by Northern blot hybridization and primer extension indicates that prgB transcription is pheromone-inducible and monocistronic. Previous transposon mutagenesis and sequencing analysis of a 12-kilobase (kb) region of pCF10 indicated that several genes including prgR and prgS are required to activate expression of prgB. The distance (3-4 kb) between these regulatory genes and prgB suggested that the activation might function in trans. To test this, a promoterless lacZ gene fusion to prgB was constructed and cloned without some or all of the regulatory genes. Several restriction fragments of the regulatory region were cloned in a higher copy-number plasmid, and numerous complementation studies were carried out in E. faecalis. Complementation in trans was not observed in any of these experiments. However, when the regulatory region and target genes were cloned in different sites of the same plasmid, separated by as much as 12 kb, activation of prgB was observed. Interestingly, this activation occurred only when the regions were cloned in the same relative orientation in which they exist on wild-type pCF10. These results suggest that one or more regulatory molecules may bind to an upstream cis-acting site and track along the DNA to reach a target site to activate prgB transcription.


Integrins as a primary signal transduction molecule regulating monocyte immediate-early gene induction.

Integrins are cell surface receptors found on monocytes that facilitate adhesion to both cellular and extracellular substrates. These integrins are thought to be involved in the selective gene induction observed after monocyte adhesion to various extracellular matrices. To investigate this hypothesis, we stimulated monocytes with monoclonal antibodies to different integrin receptors to specifically mimic the integrin receptor-ligand interactions. Engagement of the common beta chain of the beta 1 subfamily of integrins resulted in expression of the inflammatory mediator genes, interleukin 1 beta, interleukin 1 receptor antagonist, and monocyte adherence-derived inflammatory gene 6 (MAD-6), whereas engagement of the common beta chain of the beta 2 family did not. Furthermore, to characterize integrin-mediated gene induction, we examined the ability of antibodies to the alpha chain of integrin receptors to regulate gene expression. Engagement of the very late antigen 4 (VLA-4) receptor resulted in induction of all the mediator genes. Receptor crosslinking was required because individual Fab fragments were unable to stimulate gene induction whereas the divalent F(ab')2 fragment and the whole IgG molecule could. Interleukin 1 beta secretion was dependent on the anti-integrin antibody used. Some antibodies required a second signal and, for others, direct engagement was sufficient for protein production. In conclusion, engagement of integrin receptors regulated the production of both inflammatory mediator mRNA and protein. These results suggest that integrin-dependent recognition and adherence may provide the key signals for initiation of the inflammatory response during monocyte diapedesis.


Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.

Normal human breast epithelial cells show a high degree of phenotypic plasticity in monolayer culture and express many traits that otherwise characterize tumor cells in vivo. Paradoxically, primary human breast carcinoma cells are difficult to establish in culture: most outgrowths arise from the normal tissue surrounding the tumor. These characteristics have posed major obstacles to the establishment of simple reliable criteria for mammary epithelial transformation in culture. In the present study, we show that a reconstituted basement membrane (BM) can be used to culture all normal human breast epithelial cells and a subset of human breast carcinoma cells. The two cell types can be readily distinguished by virtue of the ability of normal cells to reexpress a structurally and functionally differentiated phenotype within BM. Twelve specimens of normal breast tissue and 2 normal breast epithelial cell lines (total 14 samples) embedded in BM as single cells were able to form multicellular spherical colonies with a final size close to that of true acini in situ. Sections of mature spheres revealed a central lumen surrounded by polarized luminal epithelial cells expressing keratins 18 and 19 and sialomucin at the apical membrane. Significantly, two-thirds of normal spheres deposited a visible endogenous type IV collagen-containing BM even though they were in contact with exogenously provided BM. Growth was arrested completely within the same time period. In contrast, none of 6 carcinoma cell lines or 2 cultures of carcinoma from fresh samples (total 8 samples) responded to BM by growth regulation, lumen formation, correct polarity, or deposition of endogenous BM. These findings may provide the basis of a rapid assay for discriminating normal human breast epithelial cells from their malignant counterparts. Furthermore, we propose that the ability to sense BM appropriately and to form three-dimensional organotypic structures may be the function of a class of "suppressor" genes that are lost as cells become malignant.


Activation of phosphatidylinositol 3-kinase by epidermal growth factor, basic fibroblast growth factor, and nerve growth factor in PC12 pheochromocytoma cells.

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF), which stimulate the phosphorylation of proteins on tyrosine in PC12 cells, initiate these modifications through ligand-specific cell surface receptors that contain the causative tyrosine kinases. One apparent substrate for these enzymes is phosphatidylinositol 3-kinase (PI 3-kinase), an enzyme that phosphorylates the D-3 position of the inositol ring and associates with several protein tyrosine kinases, as indicated by the fact that it is immunoprecipitated from EGF-, bFGF-, and NGF-stimulated PC12 cells by an anti-phosphotyrosine antibody. All three growth factors increase immunoprecipitable PI 3-kinase activity after 2 min of addition at concentrations able to stimulate either mitogenic or neurotrophic responses in PC12 cells. The level of stimulation of PI 3-kinase activity by EGF, bFGF, and NGF is 15- to 20-fold, 2- to 3-fold, and 8- to 10-fold, respectively. Moreover, tyrosine phosphorylation of PI 3-kinase was detected in EGF-, bFGF-, and NGF-stimulated PC12 cells, and the amount of the phosphorylation correlated with the level of stimulation of enzyme activity. In contrast, phosphatidylinositol 4-kinase, which produces the inositol phospholipids cleaved by phospholipase C-gamma to yield diacylglycerol and inositol-1,4,5-trisphosphate, is not affected by these growth factors. The pattern of stimulation of PI 3-kinase does not correlate with the induction of neurite outgrowth but rather with the mitotic responses, suggesting that PI 3-kinase and its products may be more important for signaling in cell division than in trophic processes. However, the levels of phosphatidylinositol 3-phosphate do not coincide with the stimulation of [3H]thymidine incorporation by these growth factors, rendering its role in mitotic functions, at least in PC12 cells, also uncertain.


D1-dopamine receptors activate c-fos expression in the fetal suprachiasmatic nuclei.

The existence of an activatable dopamine system within the hypothalamic suprachiasmatic nuclei (SCN), the site of a biological clock, was investigated in rats during fetal life. In situ hybridization studies revealed that D1-dopamine receptor mRNA was highly expressed in the fetal SCN and not expressed in other hypothalamic regions. Cocaine injected into pregnant rats or directly into rat fetuses on day 20 of gestation selectively activated c-fos gene expression in the fetal SCN; cocaine did not induce c-fos expression elsewhere in the fetal brain or in the maternal SCN. This cocaine-induced activation of c-fos expression in fetal SCN was mediated in part through D1-dopamine receptors, as the cocaine-induced activation was partially blocked by the D1-dopamine receptor antagonist SCH 23390. In addition, the selective D1-dopamine receptor agonist SKF 38393 induced high levels of c-fos expression in the fetal SCN. The presence of an activatable dopamine system within the fetal SCN provides a mechanism through which maternal signals could entrain the fetal biological clock and through which maternally administered psychotropic drugs could alter normal development of the circadian timing system.


Higher plant Ca(2+)-ATPase: primary structure and regulation of mRNA abundance by salt.

Calcium-dependent regulatory mechanisms participate in diverse developmentally, hormonally, and environmentally regulated processes, with the precise control of cytosolic Ca2+ concentration being critical to such mechanisms. In plant cells, P-type Ca(2+)-ATPases localized in the plasma membrane and the endoplasmic reticulum are thought to play a central role in regulating cytoplasmic Ca2+ concentrations. Ca(2+)-ATPase activity has been identified in isolated plant cell membranes, but the protein has not been characterized at the molecular level. We have isolated a partial-length cDNA (LCA1) and a complete genomic clone (gLCA13) encoding a putative endoplasmic reticulum-localized Ca(2+)-ATPase in tomato. The deduced amino acid sequence specifies a protein (Lycopersicon Ca(2+)-ATPase) of 1048 amino acids with a molecular mass of 116 kDa, eight probable transmembrane domains, and all of the highly conserved functional domains common to P-type cation-translocating ATPases. In addition, the protein shares approximately 50% amino acid sequence identify with animal sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases but less than 30% identity with other P-type ATPases. Genomic DNA blot hybridization analysis indicates that the Lycopersicon Ca(2+)-ATPase is encoded by a single gene. RNA blot hybridization analysis indicates the presence of three transcript sizes in root tissue and a single, much less abundant, transcript in leaves. Lycopersicon Ca(2+)-ATPase mRNA levels increase dramatically upon a 1-day exposure to 50 mM NaCl. Thus this report describes the primary structure of a higher-plant Ca(2+)-ATPase and the regulation of its mRNA abundance by salt stress.


Identification and prevalence of a genetic defect that causes leukocyte adhesion deficiency in Holstein cattle.

Two point mutations were identified within the gene encoding bovine CD18 in a Holstein calf afflicted with leukocyte adhesion deficiency (LAD). One mutation causes an aspartic acid to glycine substitution at amino acid 128 (D128G) in the highly conserved extracellular region of this adhesion glycoprotein, a region where several mutations have been found to cause human LAD. The other mutation is silent. Twenty calves with clinical symptoms of LAD were tested, and all were homozygous for the D128G allele. In addition, two calves homozygous for the D128G allele were identified during widespread DNA testing, and both were subsequently found to exhibit symptoms of LAD. The carrier frequency for the D128G allele among Holstein cattle in the United States is approximately 15% among bulls and 6% among cows. This mutation is also prevalent among Holstein cattle throughout the world, placing this disorder among the most common genetic diseases known in animal agriculture. All cattle with the mutant allele are related to one bull, who through the use of artificial insemination sired many calves in the 1950s and 1960s. The organization of the dairy industry and the diagnostic test described herein will enable nearly complete eradication of bovine LAD within 1 year. These results also demonstrate that bovine LAD is genetically homologous and phenotypically similar to human LAD, thus providing a useful animal model for studies of LAD and beta 2 integrin function.


Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity.

A cDNA that expresses a receptor for very low density lipoprotein (VLDL) was isolated from a rabbit heart cDNA library and characterized. The deduced amino acid sequence of the cDNA revealed that the cDNA encodes a protein with striking homology to the low density lipoprotein (LDL) receptor. Like the LDL receptor, the mature protein consists of the following five domains spanning 846 amino acids: 328 N-terminal amino acids including an 8-fold repeat of 40 amino acids homologous to the ligand binding repeat of the LDL receptor; 396 amino acid residues homologous to the epidermal growth factor precursor including three cysteine-rich repeats; a region immediately outside of the plasma membrane rich in serines and threonines; 22 amino acids traversing the plasma membrane; and 54 amino acids including the NPVY sequence that is required for clustering of the LDL receptor in coated pits and that projects into the cytoplasm. LDL-receptor-deficient Chinese hamster ovary cells transfected with the cDNA bound and internalized VLDL, beta-migrating VLDL, and intermediate density lipoprotein but did not bind LDL with high affinity. The 3.6- and 9.5-kilobase mRNAs for the VLDL receptor are highly abundant in heart, muscle, and adipose tissue. Barely detectable amounts of the mRNAs were present in liver. Based on the structural features, ligand specificity, and tissue expression of the mRNAs, we suggest that this VLDL receptor may mediate uptake of apolipoprotein E-containing lipoproteins enriched with triglyceride in nonhepatic tissues that are active in fatty acid metabolism.


mu-crystallin is a mammalian homologue of Agrobacterium ornithine cyclodeaminase and is expressed in human retina.

mu-Crystallin is the major component of the eye lens in several Australian marsupials. The complete sequence of kangaroo mu-crystallin has now been obtained by cDNA cloning. The predicted amino acid sequence shows similarity with ornithine cyclodeaminases encoded by the tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens. Until now, neither ornithine cyclodeaminase nor any structurally related enzymes have been observed in eukaryotes. RNA analysis of kangaroo tissues shows that mu-crystallin is expressed at high abundance in lens, but outside the lens mu-crystallin is preferentially expressed in neural tissues, retina, and brain. An almost full-length cDNA for mu-crystallin was cloned from human retina. In human tissues, mu-crystallin mRNA is present in neural tissue, muscle, and kidney. This pattern of expression and relationship to an enzyme involved in unusual amino acid metabolism suggests the interesting possibility that mammalian mu-crystallins could be enzymes participating in processes such as osmoregulation or the metabolism of excitatory amino acids.


CD5 acts as a tyrosine kinase substrate within a receptor complex comprising T-cell receptor zeta chain/CD3 and protein-tyrosine kinases p56lck and p59fyn.

T-cell antigens including CD2, CD4, CD6, CD8, and CD28 serve as coreceptors with the T-cell receptor (TCR)/CD3 complex in control of T-cell growth. The molecular basis by which these antigens fulfill this role has remained a major issue. An initial clue to this question came with our finding that the sensitivity of in vitro kinase labeling (specifically using protein-tyrosine kinase p56lck) allowed detection of a physical association between CD4-p56lck and the TCR/CD3 complexes. Another T-cell antigen, CD5, is structurally related to the macrophage scavenger receptor family and, as such, can directly stimulate and/or potentiate T-cell proliferation. In this study, we reveal that in Brij 96-based cell lysates, anti-CD5 antibodies coprecipitated TCR zeta chain (TCR zeta)/CD3 subunits as well as the protein-tyrosine kinases p56lck and p59fyn. Conversely, anti-CD3 antibody coprecipitated CD5, p56lck, and p59fyn. Indeed, anti-CD5 and anti-CD3 gel patterns were virtually identical, except for a difference in relative intensity of polypeptides. Anti-CD4 coprecipitated p56lck, p32, and CD3/TCR zeta subunits but precipitated less CD5, suggesting the existence of CD4-TCR zeta/CD3 complexes distinct from the CD5-TCR zeta/CD3 complexes. Consistent with the formation of a multimeric CD5-TCR zeta/CD3 complex, anti-CD5 crosslinking induced tyrosine phosphorylation of numerous T-cell substrates, similar to those phosphorylated by TCR zeta/CD3 ligation. Significantly, as for TCR zeta, CD5 was found to act as a tyrosine kinase substrate induced by TCR/CD3 ligation. The kinetics of phosphorylation of CD5 (t1/2 = 20 sec) was among the earliest of activation events, more rapid than seen for TCR zeta (t1/2 = 1 min). CD5 represents a likely TCR/CD3-associated substrate for protein-tyrosine kinases (p56lck or p59fyn) and an alternative signaling pathway within a multimeric TCR complex.


Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro.

A panel of 20 recombinant Fab fragments reactive with the surface glycoprotein gp120 of human type 1 immunodeficiency virus (HIV-1) were examined for their ability to neutralize MN and IIIB strains of the virus. Neutralization was determined as the ability of the Fab fragments to inhibit infection as measured in both a p24 ELISA and a syncytium-formation assay. One group of closely sequence-related Fab fragments was found to neutralize virus in both assays with a 50% neutralization titer at approximately 1 micrograms/ml. Another Fab neutralized in the p24 ELISA but not in the syncytium assay. The other Fab fragments showed weak or no neutralizing ability. The results imply that virion aggregation or crosslinking of gp120 molecules on the virion surface is not an absolute requirement for HIV-1 neutralization. Further, all of the Fab fragments were shown to be competitive with soluble CD4 for binding to gp120 and yet few neutralized the virus effectively, implying that the mechanism of neutralization in this case may not involve receptor blocking. The observation of a preponderance of high-affinity Fab fragments with poor or no neutralizing ability could have implications for vaccine strategies.


Expression patterns of gamma-aminobutyric acid type A receptor subunit mRNAs in primary cultures of granule neurons and astrocytes from neonatal rat cerebella.

Using a competitive polymerase chain reaction assay, we have quantitated the absolute amounts of mRNA encoding 14 distinct subunits of the gamma-aminobutyric acid type A (GABAA) receptor in primary cultures of rat cerebellar granule neurons and cerebellar astrocytes. We found that the total amount of GABAA receptor subunit mRNA in astrocytes was 2 orders of magnitude lower than in neuronal cells. Furthermore, granule cell cultures expressed all 14 different GABAA subunit mRNAs, while the astroglial cultures contained detectable amounts of all the subunits expressed by granule cells except the alpha 6 and the gamma 2L subunits. Of the alpha subunit family members, the alpha 1, alpha 5, and alpha 6 mRNAs were prominent in granule cells, while the alpha 1 and alpha 2 mRNAs were abundant in astrocytes. Of the beta receptor subunit mRNAs, the beta 1 and beta 3 mRNAs were abundantly expressed in both cultures. The gamma 2S and gamma 2L mRNAs constituted the great majority of gamma subunit mRNAs in neurons, while the gamma 1 subunit mRNA was the most abundant gamma subunit mRNA in astrocytes. When various allosteric modulators of GABAA receptors were tested electrophysiologically, methyl 6,7-dimethoxy-4-ethyl-beta-carboline- 3-carboxylate (DMCM) was the only one to modulate chloride currents elicited by GABA in a significantly different manner in granule cells (negative modulation) compared with astrocytes (positive modulation). The latter effect was previously observed in transiently expressed recombinant GABAA receptors containing a gamma 1 instead of a gamma 2 subunit. Our quantitative mRNA results suggest that an important molecular determinant responsible for the DMCM-positive modulatory effect on astroglial native GABAA receptors is the presence of the gamma 1 subunit in the receptor assembly.


A bifunctional enzyme (delta 1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants.

Many plants synthesize and accumulate proline in response to osmotic stress. Despite the importance of this pathway, however, the exact metabolic route and enzymes involved in the synthesis of proline in plants have not been unequivocally identified. We report here the isolation of a mothbean (Vigna aconitifolia) cDNA clone encoding a bifunctional enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), with both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities that catalyzes the first two steps in proline biosynthesis. The two enzymatic domains of P5CS correspond to the ProB and ProA proteins of Escherichia coli and contain a leucine zipper in each domain, which may facilitate inter- or intramolecular interaction of this protein. The Vigna P5CS enzyme activity is feedback regulated by proline but is less sensitive to end-product inhibition than is the E. coli gamma-glutamyl kinase. The P5CS gene is expressed at high levels in Vigna leaves and is inducible in roots subjected to salt stress, suggesting that P5CS plays a key role in proline biosynthesis, leading to osmoregulation in plants.


A phylogenetic analysis of the myxobacteria: basis for their classification.

The primary sequence and secondary structural features of the 16S rRNA were compared for 12 different myxobacteria representing all the known cultivated genera. Analysis of these data show the myxobacteria to form a monophyletic grouping consisting of three distinct families, which lies within the delta subdivision of the purple bacterial phylum. The composition of the families is consistent with differences in cell and spore morphology, cell behavior, and pigment and secondary metabolite production but is not correlated with the morphological complexity of the fruiting bodies. The Nannocystis exedens lineage has evolved at an unusually rapid pace and its rRNA shows numerous primary and secondary structural idiosyncrasies.


Radioprotection of mice by recombinant rat stem cell factor.

Treatment with recombinant rat stem cell factor (rSCF) protects mice from the lethal effects of irradiation. Mice treated with a single dose of rSCF prior to irradiation of up to 1150 rads [given as a split dose (1 rad = 0.01 Gy)] resulted in > 80% long-term survival, whereas a single injection given after the last dose of irradiation was not radioprotective. The combination of pre- and posttreatment (-20 h, -2 h, and +4 h) with rSCF resulted in 100% survival of otherwise lethally irradiated mice. Using this optimum schedule of rSCF administration, a radioprotective factor of 1.3-1.35 was achieved. The major cause of death in the control animals was massive bacteremia consisting of enteric organisms. The rSCF-treated animals had a much lower frequency of septicemia, due primarily to a rapid hematopoietic recovery of bone marrow function not evident in control animals.


High-resolution mapping of mammalian genes by in situ hybridization to free chromatin.

Fluorescence in situ hybridization to metaphase chromosomes or chromatin fibers in interphase nuclei is a powerful technique in mapping genes and DNA segments to specific chromosome region. We have been able to release the chromatin fibers from cells arrested at G1 and G2 phases using different drugs and a simple alkaline lysis procedure. We have also demonstrated specific hybridization of fluorescence-labeled probes to single-copy genomic DNA sequences on the free chromatins. Fluorescence in situ hybridization signals have been detected for sequences separated as close as 21 kilobase pairs and as far as 350 kilobase pairs, with excellent correspondence between the observed and expected distances. The resolution of this technique should approach 10 kilobase pairs and its coverage should span millions of base pairs. Therefore, free chromatin mapping can be generally used to study the structure and organization of mammalian genomes.


Association of protein-tyrosine kinase with phospholipase C-gamma 1 in bone marrow-derived mouse mast cells.

Bone marrow-derived mouse mast cells contain phospholipase C-gamma 1 (PLC-gamma 1), which is phosphorylated at tyrosine residues upon cross-linking of cell-bound IgE antibodies with multivalent antigen. It was found that immune complexes formed from digitonin lysates of the mast cells by monoclonal anti-PLC-gamma 1 antibodies contained protein-tyrosine kinase (PTK), which phosphorylated PLC-gamma 1 in vitro. The tyrosine kinase activity coprecipitated with PLC-gamma 1-anti-PLC-gamma 1 complexes markedly increased when the cell lysates were obtained immediately after antigen challenge. The results indicate that PTK is associated with PLC-gamma 1 in the mast cells and that the kinase is activated upon cross-linking of Fc epsilon RI. Neither beta nor gamma subunit of Fc epsilon RI nor src family PTK was coprecipitated with the PLC-gamma 1-anti-PLC-gamma 1 complexes. In situ denaturation/renaturation experiments, which detect autophosphorylated kinases, indicated that the PTK associated with PLC-gamma 1 was a 44-kDa protein.


Direct analysis of the binding of Src-homology 2 domains of phospholipase C to the activated epidermal growth factor receptor.

A number of proteins involved in intracellular signaling contain regions of homology to the product of the src oncogene that are termed Src-homology (SH) 2 domains. SH2 domains are believed to mediate the association of these proteins with various tyrosine-phosphorylated receptors in a growth factor-dependent manner. We have examined the kinetic characteristics of one of these interactions, the binding of the SH2 domains of phospholipase C gamma 1 with the receptor for epidermal growth factor (EGF). Bacterial fusion proteins were prepared containing the two SH2 domains of PLC gamma 1 and labeled metabolically with [35S]methionine/cysteine. A fusion protein containing both SH2 domains bound to the purified EGF receptor from EGF-treated cells, whereas no binding to receptors from control cells was detected. Binding was rapid, reaching apparent equilibrium by 10 min. Dissociation of the complex occurred only in the presence of excess unlabeled SH2 protein and exhibited two kinetic components. Similarly, analysis of apparent equilibrium binding revealed a nonlinear Scatchard plot, further indicating complex binding kinetics that may reflect cooperative behavior. The binding of the fusion protein containing both SH2 domains was inhibited by a fusion protein containing only the amino-terminal SH2 domain, although at concentrations an order of magnitude higher than that observed with the complete fusion protein. Fusion proteins containing SH2 domains from the GTPase-activating protein, the p85 regulatory subunit of phosphatidylinositol 3'-kinase, or the Abl oncoprotein competed less effectively. Binding of the PLC gamma 1 SH2 fusion protein to a mutant EGF receptor lacking the two carboxyl-terminal tyrosine phosphorylation sites exhibited a significantly lower affinity than that observed with the wild type, suggesting that this region of the receptor may play an important role. This binding assay represents a means with which to evaluate the pleiotropic nature of growth factor action.


A fraction of the mRNA 5' cap-binding protein, eukaryotic initiation factor 4E, localizes to the nucleus.

The 5' cap structure m7GpppN (where N is any nucleotide) is a ubiquitous feature of cellular eukaryotic mRNAs. The cap is multifunctional as it is involved in translation, nucleocytoplasmic transport, splicing, and stabilization of mRNA against 5' exonucleolytic degradation. The cap binding protein, eukaryotic initiation factor 4E (eIF-4E), is a translation initiation factor that binds to the cap structure and is part of a complex (eIF-4F) that promotes mRNA binding to ribosomes. Overexpression of eIF-4E in fibroblasts results in cell transformation. To test the hypothesis that some of the biological effects of eIF-4E might be effected by a nuclear function, we determined the cellular distribution of eIF-4E. By means of indirect immunofluorescence experiments using polyclonal and monoclonal antibodies against eIF-4E as well as transfected epitope-tagged eIF-4E, we demonstrate that a fraction of eIF-4E localizes to the nucleus. These results suggest that eIF-4E is also involved in a nuclear function.


Reverse transcriptase of human immunodeficiency virus can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in vitro primer-utilization assay.

Although the reverse transcriptase (RT) of human immunodeficiency virus (HIV) uses human tRNA(3Lys) as a primer of viral genome DNA synthesis in vivo, HIV RT binds Escherichia coli glutamine tRNA and in vitro-made human lysine tRNA with nearly equivalent affinities. We show that HIV RT can use either tRNA(3Lys) or tRNA(2Gln) as a primer for DNA synthesis in vitro without the addition of any other host or viral proteins. E. coli tRNA(2Gln) can serve as a primer for HIV RT if a primer-binding site sequence complementary to the 3' end of tRNA(2Gln) is at the 3' end of the template. With this reduced template, the specificity of binding the proper tRNA is due to base-pairing between a bound tRNA to the primer-binding site of the viral RNA template rather than sequence-specific recognition of tRNA(3Lys) by RT. If an 8-nucleotide viral sequence 3' to the primer-binding site is included in the template, then addition of Zn2+ or Co2+ is required for tRNA(3Lys)-primed synthesis, and tRNA(2Gln) now fails to prime synthesis. The latter result implies that a template sequence adjacent to the primer-binding site and containing 6 nucleotides complementary to the anticodon loop of human tRNA(3Lys) plays an active role in tRNA discrimination.


Regulation of the reverse transcriptase of human immunodeficiency virus type 1 by dNTPs.

Reverse transcriptase (RNA-directed DNA polymerase, EC 2.7.7.49) of human immunodeficiency virus type 1 has been examined with respect to the steady-state kinetics of polymerization of dNTPs into product DNA. With dNTPs as variable substrate, the kinetics of polymerization deviated from standard Michaelis-Menten kinetics. Substrate inhibition was observed at high substrate concentrations and negative cooperativity was seen at lower substrate concentrations. Examination of incorporation of substrate dNMPs in the presence of nucleotides not complementing the template demonstrated that dNTPs may act as noncompetitive inhibitors, as well as substrate. The Ki of the enzyme for dNTPs was 104 microM. A working model is presented that accounts for the substrate inhibition. In this model, the reverse transcriptase is a multisubunit holoenzyme, where noncompetitive inhibition is mediated by one subunit binding nucleotide and down-regulating the enzymatically active 64-kDa subunit. With additional assumptions, this model can accommodate the negative cooperativity observed.


Oncogenic ras triggers the activation of 42-kDa mitogen-activated protein kinase in extracts of quiescent Xenopus oocytes.

Quiescent, full-grown Xenopus oocytes, which are arrested at the G2/M border of meiosis, contain an inactive 42-kDa mitogen-activated protein kinase (p42MAPK) that is activated when oocytes are stimulated to resume the meiotic cell cycle. We have made extracts from these oocytes that respond to four cell cycle activators: oncogenic [Val12]Ras protein, clam cyclins A delta 60 and B delta 97, and the phosphatase inhibitor okadaic acid. All four induce the tyrosine phosphorylation and activation of p42MAPK. Both cyclins and okadaic acid, but not [Val12]Ras, also lead to activation of the endogenous cyclin B/cdc2 kinase complexes in extracts of quiescent oocytes. Using extracts prepared from cycloheximide-arrested interphase cells, we show that although p42MAPK activation can occur in response to cyclin-activated cdc2, the Ras-induced activation of p42MAPK occurs without intervening cdc2 activation. Neither the nononcogenic [Gly12]Ras nor [Val12,Arg186]Ras, a mutant that lacks the C-terminal consensus sequence directing prenylation and subsequent membrane association, is an effective activator of p42MAPK in vitro.


Antirepression function in Escherichia coli for the cAMP-cAMP receptor protein transcriptional activator.

The cAMP receptor protein (CRP) complex (cAMP-CRP) is a global regulator of gene expression. It influences transcription from a number of promoters in Escherichia coli, including two divergently oriented promoters in the pap pili-adhesin gene system. To further define the role of cAMP-CRP in pap regulation we monitored protein-DNA interactions in vitro and levels of pap transcription in vivo in wild-type and mutant pap-containing clones. The results showed that activation was mediated by a single cAMP-CRP-binding site centered at nucleotide positions -215.5 and -115.5 relative to the transcriptional start points. A target for the pap-specific regulatory protein PapB was localized adjacent to the cAMP-CRP-binding site. The long-range effects exerted from the protein-binding sites were consistent with the idea that cAMP-CRP caused a change in the local DNA conformation and that a nucleoprotein complex (involving cAMP-CRP and PapB) was formed in the region between the pap promoters. Moreover, transcription became independent of activation of cAMP-CRP and the PapB protein in a mutant lacking the nucleoid-associated protein H-NS. Our findings suggest that the cAMP-CRP complex mediates its positive regulatory function by alleviating transcriptional silencing and, as such, plays a role as antirepressor.


Kinetic role of nonnative species in the folding of bovine pancreatic trypsin inhibitor.

We have shown previously that during the oxidative folding of bovine pancreatic trypsin inhibitor only intermediates with native disulfide bonds are well populated. Nevertheless, these studies also confirmed the earlier conclusion [Creighton, T. E. (1977) J. Mol. Biol. 113, 275-293] that the rate-limiting transition in the kinetically preferred route for folding involves intramolecular disulfide bond rearrangements. Consequently, intermediates with nonnative disulfide bonds must form transiently during folding. Two specific nonnative species, denoted [30-51; 5-14] and [30-51; 5-38], in which numbers indicate residues participating in a disulfide bond, can be detected at low levels in kinetic folding experiments with bovine pancreatic trypsin inhibitor. By working with purified reversibly trapped intermediates, the role of these two nonnative species has been examined directly. These species are found to be in relatively rapid exchange with each other and with an initially formed native two-disulfide intermediate [30-51; 14-38]. Thus, the low abundance of the two nonnative species detected in kinetic folding experiments reflects primarily their low thermodynamic stability as compared to this native intermediate. To a small extent, these nonnative species form the productive native intermediate [30-51; 5-55], which is the immediate precursor to the native protein. However, an equal amount of [5-55; 14-38], a nonproductive dead-end intermediate, is also produced. Thus, the nonnative species detected during the folding of bovine pancreatic trypsin inhibitor are not committed to forming the productive native intermediate, nor do they serve to direct folding specifically toward a productive route.


Roles of mechano-sensitive ion channels, cytoskeleton, and contractile activity in stretch-induced immediate-early gene expression and hypertrophy of cardiac myocytes.

Mechanical loading of cardiac and skeletal muscles in vivo and in vitro causes rapid activation of a number of immediate-early (IE) genes and hypertrophy of muscle cells. However, little is known as to how muscle cells sense mechanical load and transduce it into intracellular signals of gene regulation. We examined roles of putative cellular mechanotransducers, mechanosensitive ion channels, the cytoskeleton, and contractile activity in stretch-induced hypertrophy of cardiac myocytes grown on a deformable silicone sheet. Using the patch-clamp technique, we found a single class of stretch-activated cation channel that was completely blocked by gadolinium (Gd3+). Inhibition of this channel by Gd3+ did not affect either the stretch-induced expression of IE genes or the increase in protein synthesis. Neither disruption of microtubules with colchicine nor that of actin microfilaments by cytochalasin D prevented the stretch-induced IE gene expression and increase in protein synthesis. Arresting contractile activity of myocytes by high K+, tetrodotoxin, or Ba2+ did not affect the stretch-induced IE gene expression. Tetrodotoxin-arrested myocytes could increase protein synthesis in response to stretch. These results suggest that Gd(3+)-sensitive ion channels, microtubules, microfilaments, and contractile activity may not be necessary for transduction of mechanical stretch into the IE gene expression and hypertrophy. The stimulus of membrane stretch may be transmitted to the cell nucleus through some mechanisms other than electrical or direct mechanical transduction in cardiac myocytes.


Mouse mammary epithelium produces a soluble heat-sensitive macromolecule that inhibits differentiation of 3T3-L1 preadipocytes.

Coculture of normal mouse mammary gland (NMMG) epithelial cells with 3T3-L1 preadipocytes resulted in inhibition of triglyceride accumulation. This inhibition was also observed when the NMMG cells were grown in inserts and placed within a 100-mm dish containing confluent 3T3-L1 cells. As the number of NMMG-containing inserts was increased, there was a progressive decline in triglyceride content of the 3T3-L1 cells. Conditioned medium from NMMG cells also resulted in a dose-dependent inhibition of adipocyte formation, and when concentrated 10-fold by passage through a filter with a cutoff of 30 kDa, all of the inhibitory activity was recovered. Heating the concentrated conditioned medium at 98 degrees C for 30 min resulted in complete loss of activity. Of several peptides tested, transforming growth factor-beta, platelet-derived growth factor, tumor necrosis factor, interleukin 6, and basic fibroblast growth factor showed inhibitory activity, whereas epidermal growth factor, insulin-like growth factor I, and transforming growth factor-alpha did not.


Orally administered interferons exert their white blood cell suppressive effects via a novel mechanism.

Interferons (IFN) have been approved for a number of clinical uses. The accepted routes of administration are intramuscular, subcutaneous, and intravenous. Recently, interferons administered by the oral route have been shown to exert a systemic effect. Oral administrations of IFN-alpha, IFN-beta, and IFN-gamma have been shown to cause a suppression of the peripheral white blood cell (WBC) count in mice. This study investigates the mechanism by which this suppression occurs. The results show that, in contrast to their intraperitoneal administration, oral administration of rHuIFN-alpha A/D or rMuIFN-gamma does not result in the presence of detectable levels of interferons in the blood. In addition, although the presence of circulating specific antibody to interferon blocks the peripheral WBC suppressive effects of intraperitoneally administered MuIFN-beta or rMuIFN-gamma, the presence of those antibodies does not block the peripheral WBC suppressive effects of the orally administered interferons. The peripheral WBC suppressive effect of orally administered rHuIFN-alpha A/D and rMuIFN-gamma can be transferred by injection of blood from oral interferon-treated donor mice to recipient mice. Recipient mice receiving plasma from donor mice showed no peripheral WBC suppression. Recipient mice receiving blood cells from donor mice showed significant peripheral WBC suppression. No effect of orally administered rHuIFN-alpha A/D on the relative percentages of lymphocytes, neutrophils, and monocytes was noted. These results indicate that the mechanism by which orally administered interferons exert their WBC suppressive effect differs from that of intraperitoneally administered interferons. WBC suppression resulting from orally administered interferons may involve cell to cell transfer of the interferons' effects, rather than the systemic distribution of the interferons in the blood. These studies further suggest that there may be a role for oral administration as a new route of interferon administration and provide a glimpse into the mechanism by which the orally administered interferons exert their systemic effects.


Potential antitumor agents XIX [1]. Synthesis and antitumor activity of tricyclic compounds related to lotifazole.

The synthesis of phenothiazine and anthraquinone derivatives, bearing at least one fragment present in lotifazole, is reported. Some of the new compounds showed antitumor activity in vitro (P388 leukemia cells) and in vivo (Ehrlich ascites tumor cells in mice).


Altered systemic and tissue prostacyclin in cerulein induced acute pancreatitis in rats.

Prostacyclin metabolism in rat acute pancreatitis was evaluated by measuring the tissue levels of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and the urinary excretion of 2, 3-dinor 6-keto-PGF1 alpha. Acute pancreatitis was induced by i.v. cerulein perfusion and was confirmed by the pancreas enzyme changes and the histological findings. Significantly enhanced tissue and urinary prostacyclin levels were found in acute pancreatitis rats, when compared to the controls. Concomitantly, an enhanced tissue phospholipase A2 (PLA2) activity was also found. These data show the importance of 2, 3-dinor PGF1 alpha as an inflammatory marker in cerulein-induced pancreatitis.


Evaluation of a prostacyclin analog in prevention of pulmonary oxygen toxicity.

Prolonged exposure to high concentrations of oxygen can result in significant lung injury, although newborn animals are tolerant relative to adults. We previously reported that relative O2 tolerance in the rabbit is lost by 10 days of age, and is coincident with a decline in lung prostacyclin. In the current study we administered iloprost, a stable prostacyclin analog, by continuous infusion to maturing rabbits exposed to greater than 95% oxygen. Compared to vehicle-treated controls, iloprost-treated rabbits had significantly lower protein in bronchoalveolar lavage fluid at 84 h, a smaller percentage of neutrophils at 65 and 84 h, and lower mortality at 96 h. The partial protection against pulmonary oxygen toxicity afforded by iloprost is likely due to its membrane stabilizing effect, and its inhibitory actions on neutrophil migration, activation, production of oxygen radicals and proteolytic enzymes.


Behavioral and neurochemical changes in response to acute stressors: influence of previous chronic exposure to immobilization.

The effect of daily (2 h) exposure to immobilization (IMO) for 15 days on the behavioral and neurochemical responses of adult male rats to acute stress caused by 2-h IMO or 2-h tail-shock was studied. The brain areas studied were frontal cortex, hippocampus, hypothalamus, midbrain, and pons plus medulla. Chronic exposure to IMO did not alter noradrenaline (NA), 3-methoxy,4-hydroxyphenyletileneglycol-SO4 (MHPG-SO4), serotonin, or 5-hydroxindoleacetic acid (5-HIAA) concentrations in any brain area as measured approximately 20 h after the last exposure to IMO. Exposure to behavioral tests did not modify neurochemical variables except NA levels in the hypothalamus of nonchronically stressed (control) rats. Both exposure to 2-h IMO or 2-h shock significantly decreased NA levels in hypothalamus and midbrain of nonchronically stressed rats. These decreases in response to the two acute stressors were not observed in chronically stressed rats. However, MHPG-SO4 levels increased to the same extent in control and chronically stressed rats after exposure to the acute stressors. Likewise, increased 5-HIAA concentrations observed in response to acute stressors were similar in control and chronically stressed rats. The inhibition of activity (areas crossed and rearing) in the holeboard caused by acute IMO was less marked in rats previously exposed to the same stressor than in control rats, but the response to shock was similar. In the forced swim test, acute IMO decreased struggling in control rats but tended to increase it in chronically stressed rats. The response to shock followed the same pattern as that to IMO, although it was slight.(ABSTRACT TRUNCATED AT 250 WORDS)


Dopaminergic and serotonergic function following isolation rearing in rats: study of behavioural responses and postmortem and in vivo neurochemistry.

This series of experiments compared isolation-reared and socially reared rats for their locomotor activity, behavioural stereotypy, and monoamine function both postmortem and in vivo using intracerebral dialysis. In Experiment 1, isolates showed an altered time course of locomotor activity following d-amphetamine sulphate (AMPH) administration (0.5, 2.0, 3.0, or 5.0 mg/kg, SC). Isolation-reared rats also showed increased sensitivity to the sedative effects of a low dose of apomorphine hydrochloride (0.1 mg/kg) but did not differ from social controls following higher doses of the drug (0.5, 1.5, or 3.0 mg/kg, SC). Isolates showed a decrease in the intensity of apomorphine-induced stereotyped behaviours but no change in stereotypy induced by AMPH. In Experiment 2, isolates had higher postmortem dopamine (DA) concentrations and an altered asymmetry in DA function in the medial prefrontal cortex (PFC) but not in the nucleus accumbens (NAC) or caudate putamen (CPu). Isolated rats also had a lower 5-hydroxyindoleacetic acid (5-HIAA)/5-hydroxytryptamine (5-HT) ratio in the NAC (but not in the PFC or CPu) compared to controls. Experiment 3 used intracerebral dialysis to examine monoamine function in vivo following isolation rearing. Isolates showed greater increases in extracellular DA and greater decreases in DOPAC in response to 2 mg/kg AMPH SC in both the NAC and CPu. There were no apparent differences in the perfusate concentrations of either dopamine (DA), dihydroxyphenylacetic acid (DOPAC), or homovanillic acid (HVA) prior to drug administration. However, consistent with the results of Experiment 2, isolates had a reduced basal perfusate concentration of 5-HIAA from the NAC but not from the CPu. Experiment 4 measured postsynaptic DA function in CPu tissue slices following isolation. Isolation rearing did not affect cAMP accumulation in response to stimulation of D1 DA receptors by DA (0, 2.7, 9, or 30 microM). In addition, isolation rearing did not affect the coupling between D1 and D2 receptors, as measured by the increase in cAMP accumulation with 1 microM 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1 H-3-benzazepin (SK&F 38393) and its reduction by 10 microM quinperole hydrochloride (LY 171555). These results are discussed in terms of the possible relationship between these neurochemical findings and the behavioural disturbances following isolation rearing of rats.


Astringent-tasting compounds alter ion transport across isolated canine lingual epithelia.

The effects of acid and astringent compounds on ion transport across isolated canine lingual epithelia were measured in an Ussing chamber. Lowering the pH from 7.4 to 3.2 decreases ion transport, as measured by the short-circuit current (Isc), when the dorsal surface of the tongue is bathed in 0.5 M NaCl and increases Isc when it is bathed in 0.05 M NaCl, tannic acid (0.1 M) inhibits Isc at both pH 3.2 and 7.4. At 0.05 M NaCl, pH 7.4 tannic acid also inhibits Isc. Thus, inhibition of Isc by tannic acid does not depend upon the pH, meaning that the reduction in transport arises from tannic acid. In the presence of NaCl (at both 0.05 and 0.5 M NaCl), 0.1 M AlK (SO4)2 or 0.1 M AlNH4(SO4)2 also inhibit Isc. For these salts, the decrease in Isc arises from the aluminum ion and not from K+, NH4+, or SO(4-)-. Other less astringent compounds (gallic and tartaric acids) had only slight effects on Isc. The main findings of this study are that both tannic acid and the aluminum salts inhibited ion transport, likely Na+ influx, via amiloride-inhibitable channels in isolated lingual epithelia. Inhibition of such Na+ channels may contribute to astringent taste.


Ethacrynic acid and sulphasalazine inhibit the generation of leukotriene C4 in rat stomachs: a possible gastric anti-ulcer mechanism in cold-restraint-stressed rats.

The role of gastric glandular mucosal leukotriene C4 in gastric ulceration, produced by restraint at 4 degrees C (stress) for 2 h in rats, was studied in relation to the ulcer-preventing effects of ethacrynic acid, sulphasalazine and its constituents (sulphapyridine and 5-aminosalicylic acid), AA-861 and ONO-1078. Stress itself significantly raised mucosal leukotriene C4 levels; pretreatment with ethacrynic acid, sulphasalazine, sulphapyridine or AA-861 antagonised these changes and reduced the severity of gastric ulceration. Mucosal mast cell degranulation was prevented by ethacrynic acid, sulphasalazine, 5-aminosalicylic acid, AA-861 or ONO-1078; the mucus-depleting effect of stress was also reversed by all these drugs, except for 5-aminosalicylic acid. The anti-ulcer effect of ethacrynic acid and sulphasalazine appears to be related to their influence on glandular mucosal leukotriene C4 levels.


Central and peripheral motor effects of galanin on the duodenojejunum and colon in fed rats.

The effects of intraperitoneal, intrathecal and intracerebroventricular injections of the peptide galanin (GAL) on duodenojejunal and colonic motility were studied in conscious fed rats. At 0.3-3.0 nmol/rat, intraperitoneal GAL restored the 'fasted pattern' of duodenojejunal activity, i.e. the migrating myo-electric complex (MMC) was restored for a short period of time, and the MMC frequency was not significantly different from that observed before feeding. In addition, the activity of the proximal but not of the distal colon was significantly increased by GAL administration. The intracerebroventricular administration of GAL (0.03-0.3 nmol/rat) induced an MMC fasted pattern on the duodenojejunum after a latency period of about 1 h. In these experiments proximal colonic motor activity was significantly increased for 120-180 min. GAL given intrathecally (0.03-0.3 nmol/rat) induced a long-lasting fasted pattern of the intestinal activity within 10-20 min which was not dose dependent in duration, while the motility index of the proximal colon was significantly increased. Pretreatment with naloxone prevented the specific effects of GAL, given intracerebroventricularly or intrathecally, on the duodenojejunum and colon and the colonic response, but not the restoration of the MMC pattern on the duodenojejunum, induced by GAL given intraperitoneally. Ketoprofen pretreatment was completely ineffective. These observations indicate a plurality of sites of action of GAL on digestive tract motility including local duodenal receptors and suggest the importance of a spinal component in the control of motility by GAL when given intrathecally. Moreover the present results indicate the involvement of opioid receptors in the fasted pattern induced by GAL given intracerebroventricularly or intrathecally and in the colonic effects regardless of the route of administration.


Risperidone in the treatment of disorders with a combined psychotic and depressive syndrome--a functional approach.

In vitro receptor-binding profiles and in vivo pharmacological studies have shown risperidone to be a potent mixed serotonin-S2 dopamine-D2-like receptor antagonist. While anti-D2 activity may relate to the antipsychotic potency of neuroleptic drugs, an antidepressive efficacy of substances with anti-S2 activity has been suggested. In an open pilot-study, ten patients with schizodepressive disorders or a DSM-III-R diagnosis of psychotic major depressive episodes were treated with risperidone (2-10 mg/d) for six weeks. Weekly psychopathological evaluation was performed, including BPRS, SANS, SAPS, VAS scales, and AIMS and UKU for the assessment of side-effects. Generally, the psychotic syndrome (BPRS, SANS and SAPS) decreased markedly in all patients; seven patients also showed a clinically significant improvement of depressive symptoms (BPRS). Except for two patients who needed biperiden because of extrapyramidal side-effects, the tolerance of risperidone was good. The antipsychotic and antidepressive properties of risperidone shown in our pilot study are promising enough to merit full double-blind controlled trials for further evaluation of its therapeutic value in this broad spectrum of psychiatric disorders.


Influence of early undernutrition in rats on their body fat and RNA content in VMH neurons.

The RNA content of the ventromedial hypothalamus (VMH) and lateral hypothalamic area (LHA) neurons, and the epididymal, retroperitoneal, and liver fat content of 150-day-old male rats submitted to neonatal undernutrition, were investigated. The neonatal undernutrition was carried out by two different ways. First, by reducing the litters to two pups per nest from birth to weaning. The pups from normal litters (eight pups/nest) served as controls. Secondly, by separating half of the pups (four pups) from normal litters for 8 h daily during the first 5 postnatal days. The remaining nonseparated pups served as their controls. The data show that both groups of early undernourished pups had a significantly increased RNA content in the VMH neurons (RNA content in the LHA neurons was unchanged) and a significantly decreased epididymal, retroperitoneal, and liver fat content in comparison with their controls. The results indicate that early undernutrition leads to a permanent increase in the functional activity of VMH and alteration in fat metabolism in rats.


Prostate cancer screening.

No screening test has been proven to reduce prostate cancer mortality. DRE has been the traditional method of screening, and it is often used to detect other diseases in addition to prostate cancer. Newer modalities, such as TRUS and PSA, can identify patients with nonpalpable prostate cancer, but the use of these tests will also result in many false-positives. In addition, it is not known whether the use of these tests will reduce prostate cancer mortality, or instead cause harm to those patients screened. Given the potential for harm, and the extraordinary expense, routine screening of asymptomatic men with newer modalities should be considered experimental.


Conditioned tolerance in human opiate addicts.

Repeated administration of opioid drugs results in tolerance, a lessening of the drug's effect. There is pre-clinical evidence suggesting a conditioning component to drug tolerance. In the present study, six former opiate dependent subjects received i.v. opiate either by un-signalled infusion or by signalled self-injection and the effects were compared with those of saline under double-blind conditions. The subjects' pre-injection rituals constitute a signal which reliably predict the appearance of the opiate. These rituals produced drug-opposite physiological responses which resulted in an attenuation of the effects of the drug. Thus, tolerance was observed when the subjects injected the opiate, but not when the same dose was received by un-signaled intravenous infusion. These results are consistent with a conditioning explanation for the observed drug tolerance.


Effect of ACTH, adrenalectomy and the combination treatment on the density of 5-HT2 receptor binding sites in neocortex of rat forebrain and 5-HT2 receptor-mediated wet-dog shake behaviors.

The effect of ACTH and/or adrenalectomy on serotonin (5-HT)2 receptor binding sites was evaluated in the neocortex of rat forebrain. One day after the adrenalectomy or sham operation, ACTH (50 micrograms/day) was injected subcutaneously into adult male SD rats for 10 consecutive days. Saturation analysis showed that subchronic ACTH treatment significantly increased the Bmax values for 3H-ketanserin binding without any change in the Kd values. Moreover, this ACTH-induced increase in the Bmax values was prevented by adrenalectomy. The concentrations of 5-HT and 5-hydroxyindole acetic acid (5-HIAA) measured by HPLC-ECD were not altered by these manipulations. Ten-day administration of corticosterone (20 and 50 mg/kg) also increased 5-HT2 receptor density in the neocortex of rat forebrain. 5-HT2 (and 5-HT1C) receptor agonist, (+/-)DOI-induced wet-dog shakes in ACTH and/or adrenalectomy-treated rats were also examined. Ten-day administration of ACTH enhanced (+/-)DOI-induced wet-dog shakes and this increase was prevented by adrenalectomy. These results indicate that subchronic adrenocorticotropin-adrenal axis activation of rats increases both the number of 5-HT2 receptors in neocortex of forebrain and the wet-dog shake responses induced by (+/-)DOI.


Transfection of the Escherichia coli nth gene into radiosensitive Chinese hamster cells: effects on sensitivity to radiation, hydrogen peroxide, and bleomycin sulfate.

The Escherichia coli nth gene encodes endonuclease III, which catalyses the glycolytic removal of various oxidized thymine residues from DNA. A truncated version of nth, with the prokaryotic regulatory sequences removed, was ligated into the retrovirus-based vector pZipneoSV(X)1 and transfected into the radiosensitive Chinese hamster ovary cell line, xrs7. Following selection with G418, two clones (x7nth1 and x7nth6) were shown by Southern analysis to contain the nth gene. No substantial difference in gamma-ray sensitivity was detected between xrs7, clones x7nth1 and x7nth6, and the parent vector transfected clone (x7neo1). However, clones containing the nth gene were more resistant to hydrogen peroxide cytotoxicity [D0's for x7nth1 and x7nth6 were 0.072 microgram/ml (4 microM) and 0.046 microgram/ml, respectively, compared with D0's of 0.034 and 0.027 microgram/ml for xrs7 and x7neo1, respectively] but markedly more sensitive to bleomycin sulfate cytotoxicity than xrs7 and x7neo1 (e.g., 1D0's for x7nth6 and xrs7 were 0.05 and 0.12 microgram/ml, while 2D0's for x7nth1 and xrs7 were 0.35 and 0.48 microgram/ml, respectively). Alterations in sensitivity to hydrogen peroxide and bleomycin sulfate could not be explained by differences in the distribution of the cell-cycle phases and growth rate of nth-containing clones and control cell lines. These results are consistent with the hypothesis that modified thymine lesions are potentially cytotoxic. Hence, when cells incur a high level of endonuclease III-repairable damage relative to strand breakage, such as after treatment with hydrogen peroxide, increased repair capacity increases survival. Gamma radiation produces a lower level of endonuclease III-repairable damage relative to all the other types of lesions produced; hence increased repair capacity has no measurable effect on cell survival. The increased sensitivity of x7nth1 and x7nth6 to bleomycin sulfate toxicity may indicate that, when thymine damage and single-strand breaks are in close proximity on opposite strands of the DNA, endonuclease III, which incises DNA at the site of damaged residues, can increase the number of double-strand breaks and hence decrease the level of cell survival.


Transrectal US in evaluation of patients after radical prostatectomy. Part II. Transrectal US and biopsy findings in the presence of residual and early recurrent prostatic cancer.

The anatomic appearance of the prostatic fossa on transrectal ultrasound (TRUS) scans obtained after radical retropubic prostatectomy (RRP) for carcinoma was studied in 16 patients in whom local recurrence was suspected on the basis of rising serum prostate-specific antigen (PSA) levels above 0.4 ng/mL, negative pelvic computed tomographic scans, and negative bone scans. Findings in samples obtained with ultrasound (US)-guided biopsy were compared with those in samples obtained with digitally guided biopsy (DGB); each patient was his own control. When the postoperative anatomic appearance on TRUS scans was compared with that in patients without suspected recurrence of cancer, no significant difference was seen. Needle biopsy was positive for carcinoma in eight patients (50%): US-guided biopsy, in seven patients; DGB, in five patients; and both US-guided biopsy and DGB, in four patients. US-guided biopsy has limited usefulness over DGB in patients with rising PSA levels after RRP, but use of both DGB and US-guided biopsy may maximize sensitivity. The main value of TRUS may be in accurate positioning of the biopsy needle about the vesicourethral anastomosis.


Release of endogenous neurochemicals may increase vascular permeability, induce edema and influence cell changes in trauma to the spinal cord.

Trauma to the spinal cord induces a series of electrophysiological, immunological and biochemical events, but it is still unclear how such reactions are initiated and maintained. Most likely release of neurochemicals, breakdown of microvascular permeability and the formation of vasogenic edema play important roles in the pathophysiology of spinal cord trauma. In an animal model we have focused the attention to the possible involvement of endogenous serotonin, prostaglandins and opioid peptides in the formation of edema and associated disturbances of vascular permeability. The trauma was produced in anesthetized rats by making a focal lesion in the right dorsal horn at the T10-11 segments. This injury resulted in a profound increase in the microvascular permeability to 131I-sodium and an elevation of water content in the rostral T9 and caudal T12 segments as measured 5 h after the onset of the injury. Light microscopy of the perifocal changes in the T9-T12 segments using Nissl stain and immunohistochemistry to glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) showed profound cellular changes which were most severe in the ipsilateral ventral horn. Many nerve cell bodies were shrunken and the tissue had a spongy edematous appearance. There was a marked increase of GFAP immunoreactivity as well as a significant diminution of MBP staining. Pre-treatment with p-chlorophenylalanine (p-CPA, an endogenous serotonin depletor and synthesis inhibitor) or indomethacin (an endogenous prostaglandin synthesis inhibitor) or naloxone (an opioid receptor antagonist) significantly reduced the permeability changes and the edema formation.(ABSTRACT TRUNCATED AT 250 WORDS)


Non-vasopressinergic, non-oxytocinergic neuropeptides in the rat hypothalamo-neurohypophyseal tract: experimental immunohistochemical studies.

The hypothalamo-neurohypophyseal tract is known to contain the classical neurohypophyseal hormones vasopressin and oxytocin. Additionally, dynorphin, methionine- and leucine-enkephalin, cholecystokinin (CCK), corticotropin-releasing factor (CRF), and galanin are co-stored with vasopressin and/or oxytocin. Recent immunohistochemical studies have revealed the existence of a low to moderate number of substance P-, vasoactive intestinal peptide (VIP)-, neuropeptide Y (NPY)- and somatostatin-immunoreactive nerve fibers within the rat neurohypophysis. VIP-, substance P- and NPY-immunoreactive fibers were distributed throughout the organ, whereas somatostatin-immunoreactive fibers were present in the proximal part of the organ. The positive nerve endings were either large in size resembling classical nerve terminals related to perivascular spaces, or smaller similar to peptidergic fibers as described in the CNS. These results indicate that these neuropeptides may be either co-stored with the classical neurohypophyseal hormones or contained in another system of afferents to the organ. The probably distinct functional roles of these neuropeptides in the physiology of the neurohypophysis are discussed.


Role of the OVLT in the febrile response to circulating pyrogens.

The available data suggest that circulating endogenous pyrogens (EPs) probably do not penetrate the brain, but interact with sensory elements in the organum vasculosum laminae terminalis (OVLT) which may involve 5HT and SP as neurotransmitters. It is proposed that substance P (SP) may affect thermo-regulatory neurons in the preoptic area (POA) directly or induce the local synthesis of cytokines that secondarily act on these neurons. Recent evidence indicates that endothelial cells in the OVLT bind circulating cytokines to receptors on their luminal surface. This may result in the release of putative neuroregulators which then process the original signals inwardly to the POA, where they then affect neuronal functions leading to fever production. Thus, trans-BBB passage of cytokines is prevented, but the brain site mediating their pyrogenic effect is informed and the appropriate responses are activated. It is emphasized, however, that this suggested mechanism is still speculative.


The role of radiotherapy in the treatment of primary mediastinal seminoma.

Nine patients with primary mediastinal seminoma were treated with radiotherapy. All patients achieved complete response on chest radiography. None of the three patients treated with whole mediastinal irradiation relapsed. Four of the six patients with involved-field irradiation had marginal relapses, suggesting the efficacy of the whole mediastinal irradiation.


Diurnal variation of amylase secretion is coupled with alterations of beta-adrenoceptors in the rat parotid gland.

Diurnal changes in the neurotransmitter receptors are important for studying the receptor function in neurophysiology. The purpose of this study is to gain an insight into the regulatory mechanisms of the diurnal variation of amylase secretion. Rat salivary amylase levels showed a diurnal variation with two peaks, a marked peak at 13 h and a lesser peak at 21 h. This increase in salivary amylase levels was completely inhibited by pretreatment of rats with the beta-adrenergic antagonist propranolol, but not by the alpha-adrenergic antagonist phentolamine. Amylase level in parotid tissue homogenate also showed a diurnal change, but there was only one peak, at 13 h. The number of maximal binding sites (Bmax) for [3H]dihydroalprenolol (DHA) in parotid membranes showed a diurnal variation with two marked peaks at 13 and 21 h, but the affinity of parotid beta-adrenoceptors for agonists or antagonists did not show any diurnal changes. Phosphorylation of nuclear non-histone proteins in the rat parotid gland showed diurnal variation with two marked peaks at 13 and 21 h. These results indicate that a diurnal variation in the number of rat parotid beta-adrenoceptors, which is presumably regulated by gene expression, is coupled with a change in salivary amylase secretion.


Detection of the granulocyte colony-stimulating factor receptor using biotinylated granulocyte colony-stimulating factor: presence of granulocyte colony-stimulating factor receptor on CD34-positive hematopoietic progenitor cells.

Granulocyte colony-stimulating factor (G-CSF) was linked to NHS-biotin to yield biotinylated G-CSF (b-G-CSF), which retained the ability to stimulate colony formation by normal bone marrow (BM) cells in methylcellulose. The use of streptavidin-phycoerythrin conjugate in conjunction with flow cytometry demonstrated that the binding of biotinylated G-CSF to its receptor is saturable, competitive, and specific. A 100-fold molar excess of unlabeled G-CSF almost completely inhibited the binding of the biotinylated G-CSF to the human leukemia cell line U937, which is known to possess the G-CSF receptor. G-CSF receptors were clearly detected by flow cytometry on adult human peripheral granulocytes and monocytes, but not on lymphocytes. Using this method, the expression of G-CSF receptors on hematopoietic progenitor cells in bone marrow and umbilical cord blood, detected as CD34-positive (CD34+) cells, were examined. A small but significant number of CD34+ cells were detected among the bone marrow mononuclear cells and umbilical-cord-blood mononuclear cells (4.28% +/- 0.31%, 1.09% +/- 0.20%, respectively). The percentage of CD34+ BM mononuclear cells was significantly higher than for cord blood mononuclear cells (P less than 0.01). These CD34+ cells were then analyzed by biotinylated G-CSF binding. CD34+ cells from bone marrow contained 25.8% +/- 7.9% G-CSF receptor positive cells and those from cord blood possessed 29.2% +/- 7.0% of G-CSF receptor-positive cells. The difference was not statistically significant.


Digital indocyanine green videoangiography and choroidal neovascularization.

This report describes a new system for digital indocyanine green videoangiography (ICGV) that provides enhanced imaging of the choroidal circulation. This newly assembled system was used to study a consecutive series of 129 patients with exudative age-related macular degeneration (AMD), and ill-defined or occult choroidal neovascularization (CNV). Overall, 39% of the patients in this study with occult CNV could be reclassified as having well-delineated or so-called classic CNV by virtue of the additional findings provided by ICGV. In this series, ICGV was particularly useful in identifying occult CNV in eyes with a large, serous pigment epithelial detachment (PED) and in eyes with recurrent CNV after previous laser photocoagulation treatment. Some of these patients were selected for laser photocoagulation of the abnormal choroidal vessels in order to evaluate the feasibility of this form of treatment on the basis of combined clinical, fluorescein angiographic, and ICGV findings. The results of this study suggest that ICGV is an important adjunct in the evaluation, classification, and laser treatment of patients with occult CNV secondary to AMD.


[Interferons].

The interferons are a family of proteins that are grouped together on the basis of their antiviral, antiproliferative and immunomodulatory effects. They are divided into three classes: alpha, beta, and gamma, which can be distinguished structurally, biochemically, and antigenically. Clinically they are used for the treatment of malignant tumors and of viral diseases, particularly of chronic viral hepatitis. The present role of interferons as therapeutic agents in the treatment of viral hepatitis will be critically discussed in the lecture.


Replication of human immunodeficiency virus in HL-60 cells.

HL-60 cells carrying the CD4 marker could be productively infected with human immunodeficiency virus (HIV) but did not form syncytia, though 11-14 days p.i., there was a transient decrease in cell multiplication and viability. After prolonged passage, a subpopulation of HL-60 cells was selected. The virus produced differed from the initial input virus grown on CEM cells: the virions lacked knobs, were either empty or had abnormal cores, had a higher ratio p24/gp41,gp110 and were less infectious. After prolonged passage, virus was produced which was fully infectious for CEM but not for fresh HL-60 cells, and which ressembled the input virus with respect to morphology and p24/gp41,gp110 ratio.


Prostatic hyperplasia over 80 grams weight, is transurethral resection an adequate procedure? Results of a prospective study.

A prospective study of transurethral resection with continuous low pressure irrigation was undertaken to find out, whether large prostates can be operated transurethrally without undue risk. Since 1986, the beginning of the study, 121 prostates with adenomas over 80 grams weight (> 80 g group) were resected. The mean age of the patients was 70.1 years, the mean weight of the resected prostate tissue was 104 grams in 66 minutes. The postoperative mortality was below 1% (1 patient). 17 patients (14%) needed blood transfusion, 11 patients had peroperative hemodilution. Even with large prostate, considering the rather high mean age of the patients treated, there is no considerably elevated operating risk, when large prostates are resected transurethrally. It however has to be emphasized, that this is achieved only with continuous low pressure irrigation and the loop of a 28 Fr. standard resectoscope in combination with an automatically regulated electrosurgical unit.


[Role of platelet activating factor and its antagonists in bronchial asthma].

Inhalation of the platelet activating factor (PAF) produces symptoms of bronchial asthma, a disease in which PAF plays an important role. This lipid mediator released by many kinds of cells exerts its effects on blood cells and on cells of the bronchial wall both directly and indirectly. The crucial role played by PAF in the inflammatory cascades explains the current interest in specific PAF antagonists. These antagonists have both bronchodilator and anti-inflammatory properties and act simultaneously as beta-adrenoceptor agonists and corticosteroids. The first clinical trials of PAF antagonists in the treatment of resistant asthma have given results that are interesting but not superior to those obtained with the conventional anti-asthmatic drugs. The indications for PAF antagonists combined with other specific antagonists will soon be determined.


Epitope specificity of antibodies to type II collagen in rheumatoid arthritis and systemic lupus erythematosus.

Antibodies to human type II collagen were examined in the sera of 105 patients with rheumatoid arthritis (RA), 44 patients with systemic lupus erythematosus (SLE) and 11 patients who fulfilled the criteria of both diseases (RA-SLE overlap), using a solid-phase radioimmunoassay (RIA). The frequencies of antibodies to native and denatured human type II collagen were 20% and 27% in RA, 14% and 16% in SLE, and 45% and 36% in RA-SLE overlap. The specificity of the antibodies was further examined by inhibition with native and denatured type II collagen, by immunoblotting on native and denatured type II collagen, and by immunoblotting on cyanogen-bromide derived polypeptides of type II collagen. We could not identify any disease-specific patterns of reactivity. Thus, in the three disease groups the antibody response was polyclonal; there were antibody populations that reacted with native and/or denatured collagen, and epitopes could be assigned to at least three CB peptides, CB10.5, CB11 and CB8.


Plasma interleukin-6 and renin substrate in reactive arthritis, rheumatoid arthritis, and systemic lupus erythematosus.

In order to study the role of interleukin-6 (IL-6) in inflammatory disease we monitored plasma levels of IL-6 and acute phase proteins such as C-reactive protein (CRP) and renin substrate (RS) in patients with reactive arthritis (ReA), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). Venous plasma samples were collected: (1) during the acute phase or exacerbation of the disease, and (2) several months latter during convalescence. Increased mean [95% confidence intervals (CI)] levels of plasma IL-6 were observed in patients with ReA both in the acute phase and later, 229 (177 to 280) ng/l and 197 (134 to 260) ng/l respectively (P less than 0.001 as compared to controls). The corresponding plasma IL-6 levels in RA patients were 283 (223 to 340) ng/l and 183 (151 to 226) ng/l, respectively (P less than 0.001 as compared to controls). Plasma IL-6 levels in SLE patients were not increased. Plasma RS levels were increased in all patient groups, but no significant correlation to IL-6 or CRP levels was observed, whereas plasma IL-6 and CRP levels showed a positive correlation in ReA and RA patients.


Granular basal cell tumor in a Wistar rat.

A granular cell variant of a cutaneous basal cell tumor in a Wistar rat is described. The tumor resembles the variant as described in man and dogs. The granular basal cells contain cytoplasmic PAS positive granules, and immunostained positively with HMW cytokeratins. Ultrastructurally, the cytoplasmic granules were secondary lysosomes.


Ovarian carcinomas express a sperm acrosomal antigen, defined by monoclonal antibody 9H8.

A new monoclonal antibody (MAb 9H8, IgM class) reactive with human ovarian carcinoma has been raised after immunizing C57BL/6 mice with bovine sperm. Immunohistological studies indicated that 20/21 serous ovarian adenocarcinomas expressed 9H8-defined antigen but it was absent in benign ovarian tumors (0/11). 1/11 of breast carcinomas and 5/5 of rectal carcinomas expressed this antigen, although to a considerably lesser degree. Tumors of lung, skin, brain and mesothelium were negative. The antigen was also expressed in embryonic skin, in renal collecting tubule cells and in saliva. In bovine, human and mouse sperm the antigen is confined to the acrosomal region. The molecular weight of this antigen was determined by Western blot analysis and gel filtration. In SDS-PAGE the antigen ran as a broad band barely entering the 7% gel, indicating an apparent molecular weight > 300 kDa. In the absence of detergents and reducing agents this glycoprotein forms larger complexes (> 1,500 kDa) as determined by gel filtration on Sephacryl S300. The epitope contains carbohydrate structures recognized by lectin PNA (peanut agglutinin).


Gene dosage and down-regulation of the alpha-interferon receptor.

The gene coding for the alpha,beta-interferon (alpha,beta-IFN) receptor is localized to chromosome 21. Cells from patients with Down's syndrome (trisomy 21) contain an extra chromosome 21, which results in a 1.5 times increase of dosage for the genes localized to this chromosome. Trisomy 21 cells express more cell-surface alpha-IFN receptors, consistent with the increased gene dosage. Down-regulation of the alpha-IFN receptors in trisomy 21 and normal cells was studied by incubating the cells with alpha-IFN. The alpha-IFN-induced effects showed 1.6 times more internalized cell-surface alpha-IFN receptors in trisomy 21 cells compared with normal cells, but no statistically significant change in the dissociation constants. A close relationship was found between the alpha-IFN receptor number and the biological response expressed as 2',5'-oligoadenylate synthetase activity.


Effect of short-termed pancreatic duct obstruction on the pancreatic subcellular organellar fragility and pancreatic lysosomal enzyme secretion in rabbits.

We studied the effect of short-term (3 h) pancreatic duct obstruction (PDO) on the exocrine pancreas and on the secretion of lysosomal enzymes into the pancreatic juice of rabbits during stimulation by pancreatic secretagogues. The following evaluations were made: serum amylase levels, pancreatic water content, pancreatic amylase, trypsinogen and cathepsin B content, and output of pancreatic enzymes and lysosomal hydrolases when stimulated by secretin and caerulein as well as the distribution of cathepsin B in subcellular fraction. Cellular fragility (LDH leakage from dispersed acini) and subcellular organellar fragility (cathepsin B leakage from lysosomes and malate dehydrogenase leakage from mitochondria) were also evaluated. PDO for 3 h plus secretin infusion caused a significant rise in serum amylase levels, pancreatic water content, and pancreatic amylase and trypsinogen content due to congestion of digestive enzymes during PDO. There was also a redistribution of cathepsin B from the lysosomal fraction to the zymogen fraction and increased cellular and subcellular organellar fragility. In normal rabbits and in those with only secretin infusion, caerulein stimulated the secretion of cathepsin B into pancreatic juice. Just after PDO, the secretion of cathepsin B, amylase and trypsinogen significantly decreased. By 24 h after PDO, the output of cathepsin B stimulated by caerulein and secretin had increased significantly. Amylase and trypsinogen output were also significantly increased at this stage, in both the secretin and caerulein fractions. These results indicate that the secretion of lysosomal enzymes into pancreatic juice is stimulated by gut hormones, such as caerulein, in the normal physiological state and in pathological states, such as PDO. These results also show an important role of increased cellular and subcellular organellar fragility in the pathogenesis of pancreatic injuries induced by PDO and augmented secretion of both lysosomal enzymes and pancreatic digestive enzymes in the recovery stage after PDO and their important roles at this stage. Lysosome enzymes also seem to play some physiological roles in the pancreatic ductal system in normal physiological states as well as their roles in pathological states, because cathepsin B can activate trypsinogen, and trypsin can activate many other enzymes.


Immunolocalization of beta 1 integrins in human gingival epithelium and cultured keratinocytes.

Beta 1 integrins are cell surface receptors for extracellular matrix binding. We have recently shown that these receptors may also play a role in cell-cell binding of human epidermal keratinocytes. In this study we used immunofluorescence and confocal laser scanning microscopy to localize beta 1 integrins in frozen sections of human gingiva and cultures of human gingival keratinocytes. The results show that beta 1 integrin polypeptides, localized by monoclonal and polyclonal antibodies, were detected mainly in the basal layer of the keratinized epithelium. There was also scattered staining in connective tissue fibroblasts, nerves, and blood vessel walls. In the basal layer, the integrins were found around the entire periphery of the basal keratinocytes. Furthermore, confocal laser scanning microscopy (CLSM) revealed that most of the staining was in fact localized in dot-like structures at the lateral cell membranes of neighboring basal cells. In cultured human gingival keratinocytes maintained in low calcium (0.15 mM) conditions beta 1 integrins were localized in several different structures: trails which were left behind when the cells moved in culture, dots underneath the cells, around the nucleus, and in cell-cell contacts. The trails were also found to contain fibronectin and type IV collagen but not laminin. Switching the keratinocytes to high calcium (1.2 mM) conditions induced the formation of cell-cell contacts which were strongly positive for beta 1 integrins. No fibronectin or type IV collagen was found in cell-cell contact sites.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatitis B core antigen-specific interferon gamma production of peripheral blood mononuclear cells during acute exacerbation of chronic hepatitis B.

To examine the relationship between hepatitis B core antigen-specific interferon gamma production and the liver injury, we measured the sequential change in this production by peripheral blood mononuclear cells of seven patients with chronic hepatitis B. Four patients who experienced acute exacerbation showed increased interferon gamma production when the serum alanine aminotransferase level peaked or during the recovery phase. In the three patients who did not experience acute exacerbation, interferon gamma production gradually decreased in one who had a low peak of alanine aminotransferase but did not show significant change in the other two. Increased production of hepatitis B core antigen-specific interferon gamma at the time of acute exacerbation suggests that interferon gamma induced by hepatitis B core antigen plays a role in hepatocellular injury of patients with chronic hepatitis B.