[THe clinical aspects of benign prostatic hypertrophy: the times are changing]. To determine if the incidence of benign prostatic hypertrophy (BPH) had increased in the general population and to reevaluate the therapeutic approaches, we reviewed and compared the clinical records, surgical procedures and therapeutic results achieved in 198 patients that had undergone surgery in 1980 (52 patients) and 1990 (146 patients). We observed that the number of BPH procedures had increased in 1990. These, however, had been performed earlier in the course of the disease, in younger patients with more severe, but better controlled pathologies.


Modulation of multidrug resistance by immunosuppressive agents: cyclosporin analogues, FK506 and mizoribine. It has recently been reported that an immunosuppressive agent, cyclosporin A, shows a potent overcoming effect on multidrug resistance (MDR). We studied the presence of such a modulating effect of cyclosporin analogues and other immunosuppressive agents, FK506 and mizoribine, in human multidrug-resistant ovarian cancer cells (TAOV/A0.2). The intensity of the overcoming effect of cyclosporin analogues against adriamycin resistance was found to be in the other of cyclosporin D greater than A greater than C greater than H. It was found that cyclosporin D, which has relatively weak immunosuppressive activity, overcame adriamycin resistance in the multidrug-resistant ovarian cancer cells to a remarkable degree. On the other hand, it was found that FK506, a new potent immunosuppressant, could also distinctly modulate adriamycin-resistance. It was found that FK506 conferred chemosensitization upon adriamycin with reincreasing intracellular adriamycin accumulation in MDR cells which was far less than the parent strain. However, in the case of mizoribine, no modulation of drug resistance existed. Such modulation was not necessarily accompanied by immunosuppressive activity and the two functions were thought to be based on different mechanisms.


Infants at risk for schizophrenia: sequelae of a genetic neurointegrative defect. A review and replication analysis of pandysmaturation in the Jerusalem Infant Development Study. A 1975 report stated that a schizophrenic genotype may be manifested in infants by a neurointegrative defect called pandysmaturation. Recent evidence supports this: (1) 12 studies found delayed development in schizophrenics' infants and in preschizophrenics; (2) "blind" psychometric evaluations favored an adult schizotypal disorder in four to six of seven high-risk subjects with pandysmaturation in the New York study; and (3) finally, in a partial replication of this method using the Jerusalem data, blind diagnoses of "probable" and "possible" pandysmaturation were significantly related to a parental diagnosis of schizophrenia and to cognitive and motor neurointegrative deficits at 10 years. Obstetrical complications were unrelated to diagnosis, pandysmaturation, or outcome in the overall sample. However, we found a small subgroup of schizophrenic offspring in whom the most severe motor deficits at follow-up were related to obstetrical complications, pandysmaturation, and low birth weight.


[The potencies of substance P, substance P fragments, and compound 48/80 for granulocyte infiltration in mouse skin]. To determine whether the N-terminal or C-terminal peptide of substance P (SP) induces granulocyte infiltration in mouse skin, we examined the potencies of SP, the N-terminal peptides SP1-4 and SP1-9, the C-terminal peptides SP4-11 and SP6-11, and a mast cell degranulating agent compound 48/80 in inducing granulocyte (neutrophil and eosinophil) infiltration in the skin of BALB/c mice. The subcutaneous administration of SP (10(-7)-10(-5) M) caused granulocyte infiltration in mouse skin in a concentration-dependent fashion 6 h after the injection. SP1-9 (10(-5)-10(-4) M) also caused granulocyte infiltration in the skin which was associated with mast cell degranulation. However, SP1-4, SP4-11 and SP6-11 (up to 10(-4) M) induced neither granulocyte infiltration nor mast cell degranulation. In addition, compound 48/80 (0.5-50 micrograms/ml) also induced granulocyte infiltration of mouse skin with a concentration-dependent increase in mast cell degranulation. These results indicate that SP induces granulocyte infiltration of mouse skin through mast cell degranulation induced by the N-terminal peptide.


Modulation of differentiation-related responses in human colon carcinoma cells by protein kinase inhibitor H-7. 1-(isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a potent inhibitor of protein kinases, has been used as a tool to examine the role of protein kinases in a variety of cellular functions. Contingent on the cell type, H-7 has been reported either to inhibit or to promote differentiation. The biological effects of H-7 on human colon adenocarcinoma cells have not been reported. In this study we investigated the effects of H-7 on differentiation - related parameters such as cellular morphology, proliferation, the expression of carcinoembryonic antigen (CEA), fibronectin and cytokeratins in human adenocarcinoma cell lines HCT116 and SW480. H-7 induced pronounced morphological alterations in both cell lines. It induced fibronectin expression and down-modulated CEA expression and secretion in the SW480 cells, but not in the HCT116 cells. Expression of acidic keratins was not affected by H-7 treatment in both cell lines. However, the expression of basic keratins were down-modulated in the HCT116 cells and enhanced in the SW480 cells. These studies showed that the protein kinase inhibitor, H-7, modulated phenotypic properties in human colon adenocarcinoma cells. Alterations in phenotypic properties and their significance in regard to the induction of differentiation are discussed.


The NADPH-oxidase-associated H+ channel is opened by arachidonate. The H+ channel associated with the generation of O2.- by NADPH oxidase and the oxidase itself must both be activated in response to stimuli (e.g. phorbol esters, chemotactic peptides, certain fatty acids). We have investigated the effects of membrane potential, an imposed pH gradient and a combination of the two (the protonmotive force) on the H+ conductivity of the cytoplast membrane. H+ conductivity was observed only in the presence of arachidonate and not in its absence. In the presence of arachidonate, H+ movement was determined by the protonmotive force. The effect of arachidonate was probably on a channel, since this fatty acid did not significantly increase the H+ permeability of artificial phospholipid membranes. It appears, therefore, that arachidonate is required both for the activation of O2.- production and the associated H(+)-channel-mediated efflux.


Antigenic heterogeneity in Mycoplasma iowae demonstrated with monoclonal antibodies. Western blots of proteins of 14 Mycoplasma iowae strains and isolates resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were probed with three monoclonal antibodies (MAbs), MI6, MI7, and MI8. MAb MI6 reacted with one or more antigens with apparent molecular weights of 60,000, 70,000, and 94,000. In three strains (N-PHN-D13, R-D2497, and K 1805), antigens located on a single peptide band were recognized, while in others additional epitopes at different molecular-weight positions were revealed. A similar pattern was observed with MAb MI7, although it reacted with fewer antigens than did MAb MI6 and failed to recognize antigens in strains N-PHN-D13 and R-D2497. MAb MI8 reacted with an antigen at an apparent molecular-weight position of 28,000 in four of the 14 strains and isolates. The diverse reaction patterns observed with the MAbs in the 14 M. iowae strains and isolates confirms the occurrence of antigenic variation within this species. Antigenic variation in M. iowae may be pivotal in determining host-parasite interactions, pathogenesis, and the outcome of disease.


Polypeptide N-acetylgalactosaminyltransferase activity in tracheal epithelial microsomes. Pig tracheal epithelium, a site of extensive mucin biosynthesis, contained polypeptide N-acetylgalactosaminyltransferase activity directed towards L-threonine residues. The enzyme preparation was broadly similar in properties to preparations from other tissues, e.g. pig and bovine submaxillary glands, bovine colostrum, BW5147 mouse lymphoma and baby-hamster kidney cells. Enzyme was membrane-bound and was released from microsomal preparations by extraction with Triton X-100. Extracted enzyme had a pH optimum of 7.5, had a requirement for Mn2+ (10 mM) and was inhibited by Na2EDTA. The Km for UDP-N-acetylgalactosamine was 110 microM and that for an octapeptide acceptor (VTPRTPPP) was 3.0 mM at 37 degrees C. Using a range of synthetic peptides of known structure related to TPPP it was established that L-threonine residues were specifically O-glycosylated probably in the alpha-configuration. Synthetic peptides containing the TPPP sequence required a peptide length of five or more for significant acceptor activity. In VTPRTPPP the two threonine residues were similarly glycosylated, as revealed by tryptic cleavage of the glycosylated product and separation of the 3H-labelled fragments. The enzyme preparation also specifically catalysed the transfer of N-acetylgalactosaminyl residues from UDP-N-acetyl[1-3H]galactosamine to bovine submaxillary mucin core protein and to myelin basic protein.


A coagglutination assay with monoclonal antibodies for rapid laboratory identification of Mycoplasma synoviae. An agglutinating monoclonal antibody (MAb S2) specific for a 55,000-molecular-weight surface protein of Mycoplasma synoviae was developed by fusion of spleen cells from immunized BALB/c mice with P3X63 Ag8.653 myeloma cells. Immunogold labeling experiments confirmed the cell surface location of the MAb-recognized antigen. MAb S2-coated Staphylococcus aureus was used in a rapid slide coagglutination assay. Eleven M. synoviae strains, including the type strain WVU 1853, coagglutinated with MAb-coated S. aureus, but five M. gallisepticum strains (PG31, S6, R, F, and A5969) did not.


Ca(2+)-dependent changes in the mitochondrial energetics in single dissociated mouse sensory neurons. Depolarization of neurons promotes Ca2+ influx through voltage-activated channels, raising the intracellular Ca2+ concentration ([Ca2+]i). The consequences of such changes in [Ca2+]i for mitochondrial function were assessed in single, freshly dissociated mammalian neurons. Microfluorimetric techniques were used to measure [Ca2+]i, mitochondrial membrane potential [delta psi m, Rhodamine 123 (Rh 123) fluorescence], NAD(P)H/NAD(P)+ autofluorescence and flavoprotein autofluorescence combined with whole-cell voltage-clamp techniques. Brief (100-500 ms) depolarization of the cell membrane by high K+ or by voltage commands raised [Ca2+]i and depolarized delta psi m. The change in delta psi m was dependent on extracellular Ca2+. Under voltage-clamp control of the cell membrane, the voltage-dependence of the change in Rh 123 fluorescence reflected that of the Ca2+ current. The response was reduced by Ca2+ buffers introduced into the cell. The behaviour of this signal is thus consistent with a mitochondrial response to raised [Ca2+]i and does not reflect the change in cell membrane potential per se. Similar stimuli caused a rapid decrease of NAD(P)H autofluorescence, followed by an increase which could last several minutes. Flavoprotein fluorescence increased transiently, followed by a decrease lasting for several minutes. These signals indicate an initial oxidation of NAD(P)H and FADH, followed by a prolonged increase in the reduced state of both coenzymes. All these changes were dependent on extracellular [Ca2+]. Raising [Ca2+]i again during the period of NAD+ reduction caused an oxidizing response. Ruthenium Red applied to the cells (i) reduced both the Ca2+ current and the depolarization-induced [Ca2+]i transient and (ii) directly quenched Rh 123 fluorescence. When introduced into the cells with patch pipettes, it prevented the changes in autofluorescence without interfering with the Ca2+ conductance. Oligomycin blocked neither the response of delta psi m nor of NADH autofluorescence, suggesting that the signals do not reflect a response to falling ATP/ADP.Pi ratios as a consequence of the high [Ca2+]i. The changes in NADH autofluorescence were sustained in the presence of iodoacetic acid with pyruvate as substrate. Thus brief physiological elevations of [Ca2+]i depolarize delta psi m, probably through Ca2+ cycling across the mitochondrial inner membrane. The changes in autofluorescence are consistent with (i) increased respiration which could result from the depolarization of delta psi m, followed rapidly by (ii) increased activity of the Ca(2+)-dependent intramitochondrial enzymes. Changes in [Ca2+]i within a physiological range may thus promote significant and long-lasting changes in mitochondrial energy production.


Identification of the insulin-like growth factor binding proteins 5 and 6 (IGFBP-5 and 6) in human breast cancer cells. Four estrogen receptor-positive (ER+) [MCF-7, T47D, ZR75 and BT474] and 3 ER- [Hs578T, MDA-MB-468 and MDA-MB-231] human breast cancer cell lines were examined for expression of the IGFBP-5 and IGFBP-6 genes. Northern blot analysis revealed that all cell lines, except MDA-MB-231, expressed IGFBP-5 mRNA. IGFBP-6 mRNA, however, was expressed only by the ER- cell lines. Western immunoblotting indicated that the previously unidentified 31-kDa and 32-kDa IGF binding species secreted by these cell lines are IGFBP-5. The levels of IGFBP-4 and IGFBP-5 were increased in MCF-7 cells by estradiol and IGF-I, respectively, indicating that these BPs may contribute to the growth stimulatory response to these mitogens.


GMP-140 (P-selectin) inhibits human neutrophil activation by lipopolysaccharide: analysis by proton magnetic resonance spectroscopy. Proton magnetic resonance spectroscopy has been used to monitor the effect of GMP-140 on the stimulation of human peripheral blood neutrophils. Stimulation of neutrophils by lipopolysaccharide gives rise to a high resolution lipid spectrum from the intact cells. Fluid phase GMP-140, which prevents adhesion and development of inflammatory responses of neutrophils, was found to inhibit these changes in the lipid spectrum by up to 40%. Anti-GMP-140 Fab fragments reversed this effect while non-immune Fab fragments did not affect the observed inhibition by GMP-140.


Stem cell factor has histamine releasing activity in rat connective tissue-type mast cells. Stem cell factor (SCF) was documented to be involved in the growth of mast cells controlled by fibroblasts. We tested the effect of recombinant rat SCF on degranulation from rat peritoneal mast cells (connective tissue-type mast cells: CTMC). SCF induced histamine release (approximately 20% of total histamine content) in a dose-dependent fashion. The release response was relatively rapid and reached a maximum within 5 min. The release showed total dependence on the presence of extracellular phosphatidylserine (PTS). These results reveal that SCF has histamine releasing activity in CTMC.


Structure-activity analyses of HIV-1 reverse transcriptase. HIV-1 reverse transcriptase is a dimeric enzyme which can exist in both homodimeric (p66/p66) and heterodimeric (p66/p51) forms. The monomeric subunits are catalytically inert. However, during DNA synthesis by the dimeric enzyme, only one subunit (p66) appears to carry out the catalysis, while the second subunit serves only a supportive role. In the case of the p66/p66 homodimers, we find that both the subunits are catalytically competent as judged by the observation that a) primer binding occurs to both subunits and b) catalytically inert dimers can be partially activated by replacement of one of the two inactive p66 subunits.


Lipopolysaccharide and serum cause the translocation of G-protein to the membrane and prime neutrophils via CD14. Lipopolysaccharide (LPS) in combination with human serum, in the absence of a second stimulus, causes an increase in the amount of the alpha -subunit (Gi alpha 2) of the guanine nucleotide binding protein Gi2 associated with the membrane. The LPS-serum complex also primes human neutrophils for O2- production in response to stimulation by the chemotactic factor fMet-Leu-Phe. Added serum factor is essential for priming at low concentrations of LPS. In the presence of serum, significant potentiation can be observed at LPS concentration as low as 0.1 ng/ml. The priming is dose and time dependent. Furthermore, the observed actions of the LPS-serum complex are not reversible since they cannot be overcome by washing. Monoclonal antibody against CD14 inhibits both the direct and priming actions of the LPS-serum complex. On the other hand, neither the antibody against CD11b nor the antibody against TNF-alpha inhibits the action of this complex.


Inhibitors of glycoprotein processing act at an early stage of myogenesis. The glycoprotein processing inhibitors bromoconduritol and N-methyl-1-deoxynojirimycin inhibit myoblast fusion and differentiation, suggesting the critical involvement of one or more glycoproteins in the control of skeletal myogenesis. In the present study we have examined the effect of inhibitors of glycoprotein processing on the expression of the muscle-specific regulatory factor myogenin. Glucosidase inhibitors, but not the mannosidase inhibitor 1-deoxymannojirimycin, inhibited the accumulation of myogenin mRNA in myoblasts, and immunoblotting confirmed that this was reflected in reduced accumulation of myogenin protein. The results indicate that the glycoprotein(s) critically involved in the control of myoblast differentiation act at an early stage in this process by modulating expression of the myogenic regulatory factor myogenin.


Protein kinase C mRNA levels and activity in reconstituted normal human epidermis: relationships to cell differentiation. Although keratinocytes are a major target of phorbol ester actions, the activity and the expression of the eight cloned protein kinase C (PKC) isoenzymes have not been studied in detail in human epidermis. Starting from normal human keratinocytes, we reconstituted in culture a multilayered epithelial tissue which presents many hystological, biochemical, and molecular features of the authentic epidermis and we used it as a model to investigate the PKC activity and mRNA levels. We found that i) PKC activity is higher in differentiated than in non-differentiated cells; ii) the mRNA levels of PKC delta and -eta/L, while are differently affected by spontaneous keratinocyte differentiation, are down-regulated during phorbol esters-induced cell differentiation. Our findings could represent a basis to investigate the involvement of PKC isoforms in the keratinocyte differentiation process.


Role of CFTR in lysosome acidification.

Role of CFTR in lysosome acidification. The role of CFTR in lysosome acidification was examined in CFPAC-1 pancreatic adenocarcinoma cells with the delta F508 mutation that were transduced with a retroviral vector (PLJ-CFPAC) or with the normal CFTR gene (CFTR-CFPAC). Steady-state lysosomal pHi in intact cells was lower in PLJ-CFPAC cells than CFTR-CFPAC cells (3.55 vs 3.80) and was not affected by cAMP or forskolin. Initial rates of ATP-dependent acidification of isolated lysosomes and steady-state ATP-dependent pHi were similar in both cell lines over a range of chloride concentrations and were not altered when cells were exposed to cAMP or to forskolin prior to preparation of lysosomes. These observations suggest that CFTR plays no role in acidification of lysosomes, possibly due to limited permeability of lysosomal membranes to chloride.


Differential regulation of thyrotropin-releasing hormone receptor mRNA levels by thyroid hormone in vivo and in vitro (GH3 cells). We studied the effect of thyroid status on thyrotropin-releasing hormone receptor (TRH-R) mRNA levels both in vivo and in vitro (GH3 cells) using a cloned rat TRH-R cDNA by RT-PCR. Experimental hypothyroid rats were produced by total thyroidectomy and were then killed 7 days after the operation. TRH receptor binding in the anterior pituitary and serum TSH level were elevated approximately 2-fold and 8-fold, respectively, in 7 day thyroidectomized rats. TRH-R mRNA levels in hypothyroid rats were also increased significantly compared with those of normal rats. In GH3 cells, however, no significant change of TRH-R mRNA level was observed between cultures treated with triiodothyronine (T3, 10(-9) and 10(-7) M) and the untreated group. The present data indicate that 1) the in vivo effects of thyroid status on TRH-R mRNA levels differ from the in vitro one, and that 2) the down regulation of TRH-R binding by thyroid hormone in GH3 cells may be mediated by translational or post-translational mechanisms.


Evidence for a fourth rat isoform of the plasma membrane calcium pump in the kidney. This study was conducted to identify plasma membrane Ca(2+)-transporting ATPases present in rat kidney. Characterization of the cDNAs of the plasma membrane Ca(2+)-ATPases revealed a family of proteins with regions of highly conserved amino acid sequence. To examine the extent of the diversity of rat renal plasma membrane Ca(2+)-ATPases, we used the polymerase chain reaction to detect additional gene products in rat kidney mRNA that shared these conserved regions. Sequences corresponding to three previously known rat plasma membrane Ca(2+)-ATPases were obtained. In addition, we found sequence corresponding to a new putative plasma membrane Ca(2+)-ATPase. Our results demonstrate that the rat kidney contains at least four different plasma membrane Ca(2+)-ATPases and the complexity of this multigene family is greater than previously thought.


Further analysis of a transcript nested within the actin 5C gene of Drosophila melanogaster. Previously we uncovered a 0.45-kb transcript within the 3' end transcribed untranslated region (3'UTR) of actin gene at 5C3-4 (act5C) of Drosophila melanogaster. We report here that its sequence bears no similarity to the known DNA or protein sequences. This and act5C transcripts are loaded on different polyribosomal classes. Gel retardation experiments performed with this fragment and several others from act5C reveal no DNA binding activity. The 0.45-kb transcript, initially isolated from different developmental stages of D. melanogaster embryogenesis, is also expressed in Drosophila Kc tissue culture cells, which will be used in transformation experiments designed to identify regulatory features of the nested gene and its possible interaction at some level with its "host" act5C gene.


Inhibition by mepacrine and amylase secretion from intact and permeabilized rat pancreatic acini. In intact rat pancreatic acini, the phospholipase A2 inhibitor mepacrine did not affect basal amylase release but dose-dependently inhibited the carbachol (IC50 65 microM) and CCK-8 (IC50 210 microM)-stimulated amylase release. In permeabilized acini, mepacrine shifted the dose-response curve for calcium to the right by a factor 2 and inhibited the release of amylase stimulated by GTPrS. From these results we conclude that carbachol, CCK-8 and GTPrS probably activate a phospholipase A2 closely coupled to exocytosis.


Pleiotrophin gene expression is highly restricted and is regulated by platelet-derived growth factor. Pleiotrophin (PTN) is a growth and neurite extension promoting polypeptide which is highly expressed in brain and in tissues derived from mesenchyme. The PTN gene is developmentally regulated and is closely related to the MK and RI-HB genes, both of which are developmentally regulated and induced by retinoic acid. We now have screened 17 cell lines and report that expression of the PTN gene in these cells is restricted to embryo fibroblasts and intestinal smooth muscle cells. However, NIH 3T3 cells stimulated by the platelet-derived growth factor (PDGF) express a marked increase in levels of PTN mRNA whereas retinoic acid failed to increase levels of PTN mRNA in NIH 3T3 cells or in F9 embryonal carcinoma cells within 72 hours of exposure. The results suggest that expression of the PTN gene is highly restricted and that the PTN gene is a new member of the PDGF-induced cytokine family.


Neurotrophic factor-like activity in Drosophila. The NGF-family of neurotrophic factors are structurally similar peptides with related functional properties. So far, this family of neurotrophic factors has only been identified in the vertebrate nervous system. We have determined that cultured Drosophila embryonic cells produce and secrete into medium, an activity which stimulates neurite outgrowth of embryonic chick sensory ganglia. This Drosophila activity can be blocked by antibodies to mouse NGF, indicating an immunological relationship between the Drosophila factor, mouse NGF and possibly other vertebrate neurotrophic factors. Addition of mouse NGF to Drosophila embryonic cells in culture results in increased cell number and enrichment of the neuronal phenotype, indicating that Drosophila cells have the ability to respond to the vertebrate factor. In addition, poly(A)+RNA extracted from Drosophila contains a single 1.4 Kb band which cross-hybridizes with a mouse NGF cRNA probe. These results indicate that vertebrate neurotrophic factor-like functions may operate in a genetically defined invertebrate species.


Neuropeptides and inflammation. A somatostatin analog as a selective antagonist of neutrophil activation by substance P. Substance P and somatostatin are neuropeptides found in peripheral sensory nerves. In vitro, these have opposing effects on inflammatory cells. We compared the effects of these peptides on the activation of neutrophils. Neutrophils were isolated from healthy volunteers, and chemotaxis, superoxide anion generation, aggregation, and changes in cytosolic calcium and GTPase activity were measured in the presence of substance P, somatostatin, and the chemoattractant FMLP. Substance P was an effective chemoattractant, 20% as potent as FMLP at equimolar concentrations. Substance P also stimulated GTPase activity in neutrophil plasma membranes. Somatostatin did not activate neutrophils; however, it effectively inhibited neutrophil chemotaxis and GTPase activity provoked by substance P, but not by FMLP. These studies demonstrate that substance P can effectively stimulate chemotaxis, possibly via effects on a GTP-binding protein distinct from that triggered by FMLP, and that somatostatin is a selective antagonist of substance P. The biochemical specificities of these peptides on cells may modulate neurogenic inflammation at the local level.


Synovial fluid analysis of two groups of proteoglycan epitopes distinguishes early and late cartilage lesions. To investigate whether fragmentation of proteoglycans in arthritis results in domains that have different levels of release from cartilage at different stages of the disease. Two regions of the proteoglycan, the hyaluronan-binding region and the glycosaminoglycan-rich region of the core protein, were measured, by immunoassay, in knee joint synovial fluids of patients with rheumatoid arthritis or reactive arthritis. Synovial fluid concentrations of the glycosaminoglycan-rich region were highest in rheumatoid arthritis patients who had little cartilage damage as determined by radiography, whereas release of the hyaluronan-binding region predominated in patients with advanced cartilage destruction. In reactive arthritis, release of the glycosaminoglycan-rich region predominated. These findings indicate that the hyaluronan-binding region is initially retained in the tissue during the development of cartilage destruction. The combined analysis of these markers offers a new avenue for assessment of the degree of cartilage damage in arthritis.


Isolation and culture of synovial microvascular endothelial cells. Characterization and assessment of adhesion molecule expression. In vitro studies investigating the role of the synovial endothelium in the pathogenesis of rheumatoid arthritis (RA) have, until recently, been performed using cultured endothelial cells of nonsynovial macrovascular origin. In an attempt to more correctly model in vivo conditions, a method for the isolation and culture of synovial microvascular endothelial cells (SMEC) has been developed. SMEC were isolated, primarily, by the use of lectin-coated (Ulex europaeus agglutinin type I), magnetizable polystyrene beads. Isolated cells exhibit classic endothelial "cobblestone" morphology, express von Willebrand factor, metabolize acetylated low-density lipoprotein, and exhibit a cytokine (interleukin-1)-mediated expression of endothelial leukocyte adhesion molecule type 1 (ELAM-1) and intercellular adhesion molecule type 1 (ICAM-1). ELAM-1 levels were significantly elevated in SMEC, compared with human umbilical vein endothelial cells, over a range of interleukin-1 concentrations. This increased expression of ELAM-1 by SMEC may be a potentiating step in the pathogenesis of RA.


Interferon-alpha in lupus psychosis.

Interferon-alpha in lupus psychosis. Since the level of interferon-alpha (IFN alpha) is increased in the sera of patients with active systemic lupus erythematosus (SLE) and is detectable in the cerebrospinal fluid (CSF) of some SLE patients with neuropsychiatric manifestations, we investigated the contribution of IFN alpha to the pathogenesis of the neuropsychiatric manifestations of SLE. IFN alpha levels were quantitated by radio-immunoassay in CSF and serum samples from 17 SLE patients with neuropsychiatric manifestations and 28 patients with SLE alone or SLE and other neurologic disorders. Levels of IFN alpha were increased in the CSF of 5 of 6 patients with lupus psychosis, and in 4 of these 5 patients, the levels in CSF were higher than those in serum. IFN alpha levels decreased when the manifestation of lupus psychosis subsided. In contrast, IFN alpha levels in CSF samples from patients with seizures alone were not increased. One patient with lupus psychosis died of complications of generalized seizures resulting from the SLE. At autopsy, we investigated whether IFN alpha protein or messenger RNA was detectable in the subject's brain. IFN alpha protein was immunohistochemically demonstrated in the neurons and in the microglia (focal accumulation), features not present in the brain tissues of subjects who died of other diseases. These findings support the hypothesis that IFN alpha, possibly synthesized in the brain, is the cause of the manifestation of psychosis in patients with SLE.


Breast self examination: use of a visual reminder to increase practice. 1. Breast cancer, the most common type of cancer affecting women and the second leading cause of cancer death in women, will affect more than 10% of the female population of this country. 2. Breast self examination (BSE), known to be an effective component of a three part breast health program which includes physical examination and mammography, is not practiced consistently by American women. 3. A convenient memory aid serving as a visual stimulus, combined with appropriate educational materials, is effective in increasing both the knowledge of breast health and the frequency of BSE practice.


The effect of low molecular weight dextran on haemodynamics and respiratory function during endotoxin-induced shock. The effects of low molecular weight dextran (LMWD) infusion, on gas exchange and haemodynamics were evaluated in sheep during endotoxin shock. The infusion of LMWD was started after signs of shock and lung injury were evident. After a stabilization period 10 micrograms kg-1 E. Coli endotoxin was infused i.v.. Endotoxin infusion resulted in an marked increase in pulmonary artery pressure (PAP) and decrease in mean arterial pressure (MAP), respiratory compliance, arterial oxygen tension (PaO2) and oxygen delivery index (DO2l). After 3 h MAP, PaO2, DO2l and compliance improved significantly in LMWD treated animals. The PAP had also decreased significantly in the LMWD-treated animals, but remained high in the controls (P less than 0.01). It was concluded that LMWD infusion improves haemodynamics and gas-exchange in sheep during endotoxin shock.


Molecular structure and granulocyte/macrophage colony-stimulating factor activity. Granulocyte/macrophage colony-stimulating factor (GM-CSF) plays a critical role in myeloid differentiation and in several immune and inflammatory processes. GM-CSF binds to specific cellular receptors (GM-CSFR) which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design and such design depends on a molecular understanding of ligand-receptor interactions. We present our initial studies evaluating the potential active sites of the molecule. The sites on the GM-CSF molecule that were studied represent two alpha-helices predicted to be critical for GM-CSF activity, as implicated by human-murine chimeric molecule studies. These helices are predicted to be adjacent in native GM-CSF. Peptides corresponding to amino acids 17-31 and 78-99 of GM-CSF were synthesized and cross-linked to one another in two different orientations. The ability of anti-GM-CSF to bind the individual and complexed peptides was evaluated by both ELISA and radioimmunoassay. Significant binding to all peptides was demonstrated. A preferred orientation of the two peptides was apparent, and this agreed with the predicted model structures. Antibodies were developed against the coupled peptides, and these demonstrated significant cross-reactivity with recombinant human GM-CSF. Additionally, analyses of anti-peptide antisera binding studies predict these two amino acid sequences to lie in parallel planes to one another in the native human GM-CSF molecule.


Structural aspects of recognition motifs contributing to autoimmune responses. Sequence analysis of autoimmune-associated antibodies has suggested a structural relatedness between genes used to encode autoantibodies and those encoding unrelated antibodies without autoreactive specificities. Subsequently, the basis for cross-reactive idiotypes across germ-line lineages, as well as conserved interspecies cross-reactivities of autoantigens among serologically similar antibodies, may result from evolutionary duplication of particular types of recognition motifs. As a first step toward elucidating structural recognition principles underlying possible cross-reactive epitopes involved in autoimmune pathologies, structural features of selected motifs associated with native ligand binding are examined for their inherent occurrence in antibody and T-cell receptor repertoires. This analysis considers the putative recognition features representative of common motif subsets shared with loop structures in CDR2 and FR3 regions of antibodies such as charge-2x-charge-x-charge or hydrogen bond donor (acceptor)-2x-charge-x-hydrogen bond donor (acceptor) type motifs, where x is any residue that can participate in maintaining a loop conformation. Such tracts encoded in the CRD2 and FR3 regions of heavy chains of antibodies and T-cell receptors (TCRs) associated with autoimmune dysfunction, with non-autoreactive antibodies, and with native host proteins. Such evolutionarily conserved motifs may be targets for complementary interactions involving autoantibodies and receptors.


Infection of human brain cells by HIV-1: restricted virus production in chronically infected human glial cell lines. To study expression of HIV-1 in human glial cell lines. Chronically HIV-1-infected glial cell lines were established to evade potential artefacts resulting from unphysiological viral entry (i.e., transfection). These cell lines were used to study viral expression and regulation. Chronically infected glial cell lines were established by terminal dilution cloning of human glioma cells exposed to HIV-1. Virus production and expression were assayed by measuring reverse transcriptase activity, p24-antigen levels and syncytia-inducing capacity in C8166 target cells (extracellular), or by indirect immunoperoxidase staining, immunoblot analysis, and p24- and Nef-antigen-capture enzyme-linked immunosorbent assays (intracellular). HIV-long terminal repeat (LTR)-dependent expression of the chloramphenicol acetyltransferase reporter gene was determined in transient transfection assays. Culture supernatant from chronically HIV-1-infected glial cells contained only low levels of virus compared with chronically HIV-infected fibroblasts and T-lymphoma cells. Detailed study of HIV-antigen expression in representative glial cell line TH4-7-5 indicated the presence of all major structural proteins, albeit at low levels, and of Vif, Tat, Rev and Nef. Intracellular levels of Nef exceeded p24-antigen levels by approximately 10-fold. Virus was recovered from TH4-7-5 cells by cocultivation with blood-derived target cells, indicating that low-level virus production is not due to defective provirus. Prominent negative regulatory element (NRE)-mediated suppression of exogenous HIV-LTR activity was observed in TH4-7-5 cells and was unequalled by chronically HIV-producing fibroblast cells or by uninfected fibroblast and glial cells. Our results suggest that restricted virus production by chronically infected glial cells involves LTR-mediated regulation of virus expression.


Phenylethanoid and lignan glycosides from Verbascum thapsus. Verbascum thapsus afforded, in addition to three known phenylethanoid glycosides and four lignan ones, five new phenylethanoid glycosides and one new lignan glycoside. Structures of the compounds were elucidated by spectroscopic methods and chemical evidence.


The KA-2 subunit of excitatory amino acid receptors shows widespread expression in brain and forms ion channels with distantly related subunits. A new ionotropic glutamate receptor subunit termed KA-2, cloned from rat brain cDNA, exhibits high affinity for [3H]kainate (KD approximately 15 nM). KA-2 mRNA is widely expressed in embryonic and adult brain. Homomeric KA-2 expression does not generate agonist-sensitive channels, but currents are observed when KA-2 is coexpressed with GluR5 or GluR6 subunits. Specifically, coexpression of GluR5(R) and KA-2 produces channel activity, whereas homomeric expression of either subunit does not. Currents through heteromeric GluR5(Q)/KA-2 channels show more rapid desensitization and different current-voltage relations when compared with GluR5(Q) currents. GluR6/KA-2 channels are gated by AMPA, which fails to gate homomeric GluR6 receptor channels. These results suggest possible in vivo partnership relations for high affinity kainate receptors.


[Clinical biochemical and genetic aspects of peroxisome-deficient disorders]. Peroxisome-deficient disorders including Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease are characterized by hypotonia, psychomotor delay, hepatomegaly and dysmorphism. Multiple peroxisomal enzymes are deficient in these disorders probably due to the defect of transport machinery of enzymes. Defects of beta-oxidation enzymes causes an accumulation of very-long-chain fatty acids, which is closely related to the pathogenesis. Catalase, a marker enzyme of peroxisome, is distributed in the cytosol. Immunocytochemical staining of peroxisomes using anti-catalase is a useful tool for prenatal and postnatal diagnosis. Although the primary etiology of peroxisomal deficiency has not been determined, genetic heterogeneity was clarified by complementation studies. At least 8 genes are involved in the formation of functional peroxisomes.


Cyclic alternating chemotherapy of high-grade malignant non-Hodgkin lymphomas with VIM-Bleo and CHOP. Between 1986 and 1988, 81 patients with high grade malignant non-Hodgkin lymphoma according to the Kiel classification were treated with the VIM-Bleo/CHOP-regimen: etoposide 100 mg/m2 intravenously on days 1-3, ifosfamide 1.5 g/m2 intravenously days 1-5 with mesna for prophylaxis of cystitis, methotrexate 30 mg/m2 intravenously on days 3, bleomycin 10 mg intravenously on days 8 and 15, cyclophosphamide 750 mg/m2 day 22, doxorubicin 50 mg/m2 day 22, vincristine 1.4 mg/m2 on day 22, and prednisolone 100 mg postoperatively on days 1-5 and 22-26. Cycles were repeated four times beginning on day 43. Regions with bulky disease were irradiated after chemotherapy. 36 patients (44%) had stage II, 12 (15%) stage III and 33 (41%) stage IV disease. B-symptoms were present in 49% of patients. Serum lactate dehydrogenase activity was elevated in 53%. Overall, 59 patients (73%) achieved a complete and 14 (17%) a partial remission. 8 (9%) had stable or progressive disease. After a median follow up of 30 months thus far, probability of long-term relapse free survival is 66% for patients in complete remission. Overall survival is 72% at 24 months. Toxicity from treatment was very low with leukopenia being the main side effect. Major infections were observed in only 2% of cycles with one treatment related death. VIM-Bleo/CHOP is a well tolerated regimen with remission rates in the range of other, more toxic regimens. However, cyclic alternating treatment did not improve results as compared with repeated treatment with a single standard protocol.


A dose-escalation study of recombinant interferon-alpha in patients with a metastatic carcinoid tumour. The efficacy of interferon alpha-2b in doses up to 12 x 10(6) IU three times weekly was studied in 21 patients with a metastatic carcinoid tumour. Of these 21 patients, 19 were evaluable for response. Patients were treated with escalating dosages of interferon alpha-2b: 3 x 10(6) IU, 6 x 10(6) IU and 12 x 10(6) IU. The escalation was performed every 8 weeks when no objective tumour regression was observed. Patients were also evaluated for biochemical response and symptomatic improvement. One objective tumour regression was observed. Of the 15 patients with elevated 5-hydroxyindole acetic acid (5-HIAA) excretion, 5 (33%) had a more than 50% decrease in 5-HIAA excretion. Relief of symptoms occurred in 11 patients (58%). This improvement was already apparent during the initial 8 weeks of treatment. Increasing the dose to 6 or 12 x 10(6) IU interferon alpha-2b did not result in further symptomatic improvement. In contrast toxicity was considerable with the higher dosages of interferon alpha-2b. It is concluded that low dose interferon alpha-2b (3 x 10(6) IU) three times weekly is as effective as higher dosages of interferon alpha-2b at ameliorating symptoms of the carcinoid syndrome.


POMB/ACE chemotherapy in non-seminomatous germ cell tumours: outcome and importance of dose intensity. This study reports the outcome of POMB/ACE (cisplatin, vincristine, methotrexate, bleomycin, actinomycin D, cyclophosphamide, etoposide) chemotherapy in 53 male patients with metastatic non-seminomatous germ cell tumour (NSGCT) treated between 1983 and 1989 in one centre. The overall complete response (CR) rate was 62% [95% confidence interval (CI) 49-75%), and for patients with large or very large volume disease (L/VL, MRC criteria), the CR rate was 56% (95% CI 41-71%). The overall 5 year survival was 61%, and for L/VL volume disease 67%. Comparison with previous studies suggests that POMB/ACE chemotherapy is not superior to BEP, even in patients with adverse prognostic factors. Increased average relative dose intensity and increased relative dose intensity of cisplatin over the first seven courses were not associated with improved survival. However, in patients receiving a relative dose intensity of etoposide greater than or equal to 0.75, survival at 5 years was significantly improved compared with those in whom this parameter was less than 0.75 (79% vs. 44%, P less than 0.05), suggesting that dose intensity of etoposide may be an important determinant of outcome in the chemotherapy of metastatic NSGCT.


Colony stimulating factor-1 stimulates Ishikawa cell proliferation and lipocortin II synthesis. Proliferation of the Ishikawa human endometrial adenocarcinoma cell line is under the concerted control of oestrogen and progesterone. Here we demonstrate that Ishikawa cells express colony stimulating factor-1 (CSF-1), CSF-1 receptor mRNA and are growth stimulated by CSF-1 treatment. An early event associated with CSF-1 treatment is the induction of lipocortin II synthesis, a protein whose expression is also under oestrogen and progesterone control. However, neither CSF-1 or CSF-1 receptor mRNA appear to be modulated by oestrogen or progesterone.


Immunocytochemical determination of estrogen and progesterone receptors in human endometrial adenocarcinoma cells (Ishikawa cells). The regulation of both estrogen and progesterone receptor levels in human endometrial adenocarcinoma cells of the Ishikawa line was investigated immunocytochemically by using monoclonal antibodies. Positive staining for estrogen and progesterone receptors was observed in the nuclei of Ishikawa cells. Intercellular heterogeneity in receptor content was evident from the presence of receptor-positive or -negative cells and from differences in staining intensity of positive cells. Quantitative analysis was performed by scoring the staining intensity and the proportion of positively stained cells. The time and dose-dependent stimulatory effect of estradiol added to culture media on progesterone receptor levels was studied by applying both immunocytochemical and biochemical methods. Estradiol at 10 nM (optimal concentration) increased the intensity score for PR from an initial value of 10.1 to 78.3 after 72 h incubation, and the proportion of the positive staining cells from 6.7 to 42.7%. Promegestone (R5020) was effective at 1 microM concentration in decreasing the intensity score for ER from 31.1 to 14.6 after 72 h exposure and the proportion of positive cells from 19.0 to 11.4%.


Requirement of certain epitope specificities of glycosylation inhibiting factor for the suppression of in vivo IgE and IgG antibody responses. The ovalbumin (OVA)-specific T cell hybridoma 71B1, which constitutively secretes glycosylation inhibiting factor (GIF) and is specific for the immunogenic epitope represented by amino acids 323-339 in the OVA molecules, failed to form GIF having affinity for nominal antigen upon stimulation with OVA-pulsed antigen-presenting cells (APC). However, the GIF produced by the antigen-stimulated 71B1 cells bound to the mAb 14-12, which is specific for the antigen-binding chain of effector type suppressor T cell factor (TseF), and to mAb specific for TCR. The GIF constitutively released from unstimulated 71B1 cells failed to bind to any of these antibodies. Gel filtration of GIF preparations showed that the 14-12+ GIF from the antigen-stimulated 71B1 cells are composed of 80-100 and 25-35 kDa species, while the GIF from unstimulated cells was 12-15 kDa. Reduction and alkylation treatment of the GIF from the antigen-stimulated cells resulted in the disappearance of the 80-100 and 25-35 kDa GIF, which was accompanied by the formation of the 12-15 kDa GIF. Thus, the GIF from the antigen-stimulated 71B1 cells was similar to the previously described OVA-binding GIF from the 231F1 cells with respect to their antigenic structures and molecular size, and both factors appear to be composed of the 14-12+ polypeptide chain and 12-15 kDa non-specific GIF. However, the GIF from the antigen-stimulated 71B1 cells lacked affinity for the native OVA or synthetic peptide 323-339, and failed to suppress the in vivo antibody response to dinitrophenyl (DNP)-OVA. In contrast, the OVA-binding GIF has affinity for native OVA and the peptide 307-317, to which the cell source of the factor is specific, and suppressed the in vivo anti-hapten antibody response to DNP-OVA. The results suggest that formation of antigen-specific TsF is confined to T cells with certain epitope specificities. It was also found that the OVA-binding GIF failed to suppress the in vivo anti-hapten antibody response to DNP-conjugates of urea-denatured OVA (UD-OVA), which does not bind OVA-binding GIF. However, APC pulsed with UD-OVA appear to express the epitope 307-317 for which the OVA-binding GIF has affinity. The results collectively suggest that the affinity of GIF for an immunizing antigen, rather than processed antigen, is required for immunosuppression.


Role of endogenous peptide in human alloreactive cytotoxic T cell responses. The T x B hybrid 174 x CEM.T2 (T2) has been shown to be defective in the processing of proteins for presentation by MHC class I molecules. It continues, however, to express significant quantities of HLA-A2.1, suggesting that this class I molecule is expressed either in a largely peptide-free form or in association with a small subset of peptides. In this paper T2 was used in conjunction with limiting dilution analysis to provide a direct estimate of the fraction of alloreactive cytotoxic T lymphocytes (CTLs) that were dependent upon the presence of peptides for their recognition of HLA-A2.1. Alloreactive cytotoxic T cell lines generated by stimulation with HLA-A2.1 expressing peripheral blood lymphocytes recognized T2 poorly. Split-well analysis of 240 clonal limiting dilution cultures demonstrated that this reflected the existence of two subpopulations. An average 85% of HLA-A2.1 specific CTLs recognized HLA-A2.1 on normal cells but not on T2. The remainder recognized HLA-A2.1 on both T2 and normal targets. CTL lines with the latter specificity could be generated by using T2 as a stimulator cell. Using target cells that either expressed a lower density of HLA-A2.1 or that expressed HLA-A2 molecules that had been mutated to affect CD8 binding, no significant differences in avidity between T2-reactive and T2-unreactive CTLs were seen. Thus the failure of the majority of alloreactive CTLs to recognize T2 is not a consequence of the lower level of HLA-A2.1 surface expression on this cell, but is instead due to the absence of appropriate epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)


IL-4-induced expression of germline gamma 1 transcripts in B cells following cognate interactions with T helper cells. T cell-dependent B cell activation and the induction of isotype switching require antigen and direct contact with helper T (Th) cells. During activation, B cells can switch from the expression of IgM to that of IgG, IgE or IgA, depending on the lymphokines secreted by the Th cell with which they interact. Studies of lipopolysaccharide (LPS)-activated B cells have suggested that lymphokines regulate isotype switching via a transcriptional mechanism that increases the accessibility of downstream CH genes to a switch recombinase(s). To assess the roles of T cell contact and lymphokines in isotype switching, we have examined the accessibility model for the regulation of isotype switching to IgG1 in the context of cognate interactions between Th cells and normal B cells. We demonstrate that Th2 cells that secrete IL-4 can induce expression of germline gamma 1 transcripts in B cells. The steady-state level of germline gamma 1 transcripts induced by Th2 cells is enhanced as compared with the level induced by IL-4 alone or Il-4 and LPS also alters the relative usage of the germline gamma 1 transcription initiation sites. Enhanced expression of germline gamma 1 transcripts requires direct contact between T and B cells suggesting a role for T cell contact-mediated signals in regulating the accessibility of switch regions.


Modulation of IL-4 induced germline epsilon RNA synthesis in human B cells by tumor necrosis factor-alpha, anti-CD40 monoclonal antibodies or transforming growth factor-beta correlates with levels of IgE production. To determine the role of germline epsilon transcription in IgE synthesis, the effects of cytokines on germline epsilon RNA synthesis in IL-4 dependent epsilon switching in B cells was investigated. Induction of germline epsilon transcription in highly purified B cells seems to be a specific property of IL-4, since none of the other cytokines tested [IL-1 alpha, beta, IL-2, IL-3, IL-5, IL-6, IL-7, IL-9, IL-10, G-CSF, GM-CSF, M-CSF, IFN-gamma, IFN-alpha, tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta] were effective. TGF-beta, IFN-gamma, and IFN-alpha inhibit IL-4 dependent IgE synthesis, but only TGF-beta blocked germline epsilon RNA synthesis in purified B cells, indicating that this may be the mechanism by which TGF-beta inhibits IgE synthesis, and that IFN-gamma and IFN-alpha act on other stages of the regulatory process resulting in IgE production. IL-5, IL-6, and TNF-alpha enhance IL-4 dependent IgE synthesis, but only TNF-alpha enhanced IL-4 induced germline epsilon RNA synthesis. Finally, anti-CD40 mAbs and the non-IL-4 producing CD4+ T cell clone A3, which in the presence of IL-4 induce IgE synthesis by purified B cells, both strongly enhanced germline epsilon transcription. These data, together with the observation that epsilon switching in cultures initiated with single sIgM+, sIgE- B cells in all instances was preceded by germline epsilon RNA synthesis, indicate that there is a strong relationship between germline epsilon transcription and IgE synthesis.


Determination of nucleic acid backbone conformation by 1H NMR. In DNA or RNA duplexes, the six-bond C3'-O3'-P-O5'-C5'-C4'-C3' backbone linkage connecting adjacent residues contains six torsion angles (epsilon, zeta, alpha, beta, gamma, delta) but only four protons. This seriously limits the ability to define the backbone conformation by NMR using purely 1H-1H distance geometry (DG) methods. The problem is further compounded by the inability to assign two of the four backbone protons, namely the poorly resolved H5' and H5'' protons, and invariably leads to DG structures with poorly defined backbone conformations. We have developed and tested a reliable method to constrain the beta, gamma, and epsilon (and indirectly alpha and zeta) backbone torsion angles by lower-bound NOE distances to unassigned H5'/H5'' resonances combined with either 1H line widths or the conservative use of sigma J measurements; the method relies only on 1H 2-D NMR data, does not involve any structural assumptions, and leads to much improved backbone convergence among DG structures. The C4'-C5' torsion angle gamma is constrained by lower-bound NOE distances from H2' and from H6/H8 to any H5'/H5'', as well as by sigma JH4, coupling measurements in the 3.9-4.4 ppm region; delta is constrained by H1'-H4' NOE distances and by H3'-H4' and H3'-H2'' J couplings in COSY data; epsilon is partially constrained by H3' line width and/or further constrained by subtracting the minimum possible sigma JH3'-H from the observed sigma JH3' (COSY) to arrive at the maximum possible JH3'-P, which is then converted to H3'-P distance bounds. The angle beta is partially constrained via H5'-P and H5''-P distance bounds consistent with the maximum H5'-P and H5''-P J couplings derived from the observed H5' and H5'' line widths, while alpha and zeta are indirectly constrained by lower distance bounds on the observed (n)H1' to (n + 1)H5'/H5'' NOEs combined with the prior partial constraints on beta, gamma, delta, and epsilon. The combined effects of these additional constraints in determining distance geometry structures have been demonstrated using a 12-base duplex, [d(GCCGTTAACGGC)]2. Coordinate RMSDs per atom between structures refined with these constraints from random-embedded DG structures, from ideal A-DNA, and from B-DNA starting structures were less than 0.4 A for the central 8 base pairs indicating good convergence. All backbone angles for the central 8 base pairs are very well constrained with less than 10 degrees variation in any of the 48 torsion angles.


Conformation and dynamics of an Fab'-bound peptide by isotope-edited NMR spectroscopy. The dynamics and conformation of the peptide antigen MHKDFLEKIGGL bound to the Fab' fragment of the monoclonal antipeptide antibody B13A2, raised against a peptide from myohemerythrin, have been investigated by isotope-edited NMR techniques. The peptides were labeled with 15N (98%) or 13C (99%) at the backbone of individual amino acid residues. Well-resolved amide proton and nitrogen backbone resonances were obtained and assigned for eight of the 12 residues of this bound peptide. Significant resonance line width and chemical shift differences were observed. The 15N and 1H line width variations are attributed to differential backbone mobilities among the bound peptide residues which are consistent with the previously mapped epitope of this peptide antigen. Local structural information was obtained from isotope-directed NOE studies. The approximate distances associated with the experimental NOEs were estimated on the basis of a theoretical NOE analysis involving the relative integrated intensities of the NOE and source peaks. In this way, the sequential NH-NH NOEs obtained for seven of the Fab'-bound peptide residues were shown to correspond to interproton separations of approximately 3 A or less. Such short distances indicate that the backbone dihedral angles of these residues are in the alpha rather than the beta region of phi,psi conformational space; the peptide most likely adopts a helical conformation from F5 to G11 within the antibody combining site. The significance of these results with respect to the type and extent of conformational information obtainable from studies of high molecular weight systems is discussed.


Heterogeneous expression of blood group A-determinant in a human gastric cancer cell line derived from a blood group A individual. The human gastric cancer cell line MKN 45 was derived from the tumour of a blood group A individual, and was known to express large quantities of blood group A-antigen. Using immunofluorescence we found the MKN 45 cells, donated from the Japanese Cancer Research Resources Bank, consisted of A-antigen positive cells (18%) and A-antigen negative cells (82%). After limiting dilution, wild type and mutant cells were cloned with regard to the expression of a cell surface A-antigen. ELISA was used to detect A-antigen in the cell extract of the wild type cells, but none was evident in those of the mutant cells. However, blood group A-gene-specified transferase activity of the mutant cells was comparable to that of the wild type cells.


In vitro selection of RNAs that undergo autolytic cleavage with Pb2+. An in vitro selection method has been developed to obtain RNA molecules that specifically undergo autolytic cleavage reactions by Pb2+ ion. The method utilizes a circular RNA intermediate which is regenerated following the cleavage reaction to allow amplification and multiple cycles of selection. Pb2+ is known to catalyze a specific cleavage reaction between U17 and G18 of yeast tRNA(Phe). Starting from pools of RNA molecules which have a random distribution of sequences at nine or ten selected positions in the sequence of yeast tRNA(Phe), we have isolated many RNA molecules that undergo rapid and specific self-cleavage with Pb2+ at a variety of different sites. Terminal truncation experiments suggest that most of these self-cleaving RNA molecules do not fold like tRNA. However, two of the variants are cleaved rapidly with Pb2+ at U17 even though they lack the highly conserved nucleotides G18 and G19. Both specific mutations and terminal truncation experiments suggest that the D and T loops of these two variants interact in a manner similar to that of tRNA(Phe) despite the absence of the G18U55 and G19C56 tertiary interactions. A model for an alternate tertiary interaction involving a U17U55 pair is presented. This model may be relevant to the structure of about 100 mitochondrial tRNAs that also lack G18 and G19. The selection method presented here can be directly applied to isolate catalytic RNAs that undergo cleavage in the presence of other metal ions, modified nucleotides, or sequence-specific nucleases.


Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Calcineurin, a Ca2+, calmodulin-dependent protein phosphatase, was recently found to bind with high affinity to two different immunosuppressant binding proteins (immunophilins) with absolute dependence on the presence of the immunosuppressants FK506 or cyclosporin A (CsA) [Liu et al. (1991) Cell 66, 807-815]. The binding affinities of the immunophilin-drug complexes toward calcineurin and the stoichiometry of the resultant multimeric complexes have now been determined, and structural elements of FK506, CsA, and calcineurin that are critical for mediating their interactions have been identified. Analogues of FK506 (FK520, FK523, 15-O-demethyl-FK520) and CsA (MeBm2t1-CsA and MeAla6-CsA) whose affinities for their cognate immunophilins do not correlate with their immunosuppressive activities have been prepared and evaluated in biochemical and cellular assays. We demonstrate a strong correlation between the ability of these analogues, when bound to their immunophilins, to inhibit the phosphatase activity of calcineurin and their ability to inhibit transcriptional activation by NF-AT, a T cell specific transcription factor that regulates IL-2 gene synthesis in human T cells. In addition, FKBP-FK506 and CyP-CsA do not inhibit members of the PP1, PP2A, and PP2C classes of serine/threonine phosphatases. These data suggest that calcineurin is the relevant cellular target of these immunosuppressive agents and is involved in Ca(2+)-dependent signal transduction pathways in, among others, T cells and mast cells.


Sequential dephosphorylation of a multiply phosphorylated insulin receptor peptide by protein tyrosine phosphatases. The question of whether protein tyrosine phosphatases (PTPases) dephosphorylate a multiply phosphorylated peptide in a random or ordered manner was investigated using the synthetic triphosphotyrosyl peptide TRDIY(P)ETDY(P)Y(P)RK, corresponding to the major sites of autophosphorylation of the insulin receptor, as a substrate for four purified PTPases. All four enzymes dephosphorylated the triphospho peptide to produce diphospho, monophospho, and nonphosphorylated forms. Partially dephosphorylated peptides were separated by reverse-phase HPLC, and the di- and monophospho peptides were collected and analyzed by solid-phase sequencing to determine the order of dephosphorylation of the three sites by each of the PTPases. The quantitative analysis of the signals for derivatives of tyrosine and phosphotyrosine generated at positions 5, 9, and 10 of the peptide showed that the low molecular weight human placental PTPase 1B preferentially dephosphorylated the two phosphotyrosines at positions 9 and 10 whereas the integral membrane enzyme CD45 (from human spleen) and the bacterially expressed rat LAR preferentially dephosphorylated the phosphotyrosine at position 5. A second low molecular weight enzyme, termed TCPTPase, did not display any specificity for a particular phosphotyrosyl residue. These results demonstrate that different PTPases exhibit a characteristic pattern of dephosphorylation of the triphospho peptide model substrate, raising the possibility that features in the primary structure surrounding the dephosphorylation site may contribute to substrate specificity.


Errors in RNA NOESY distance measurements in chimeric and hybrid duplexes: differences in RNA and DNA proton relaxation. Nuclear magnetic resonance experiments reveal that the base H8/H6 protons of oligoribonucleotides (RNA) have T1 relaxation times that are distinctly longer than those of oligodeoxyribonucleotides (DNA). Similarly, the T1 values for the RNA H1' protons are approximately twice those of the corresponding DNA H1' protons. These relaxation differences persist in single duplexes containing covalently linked RNA and DNA segments and cause serious overestimation of distances involving RNA protons in typical NOESY spectra collected with a duty cycle of 2-3 s. NMR and circular dichroism experiments indicate that the segments of RNA maintain their A-form geometry even in the interior of DNA-RNA-DNA chimeric duplexes, suggesting that the relaxation times are correlated with the type of helix topology. The difference in local proton density is the major cause of the longer nonselective T1s of RNA compared to DNA, although small differences in internal motion cannot be completely ruled out. Fortunately, any internal motion differences that might exist are shown to be too small to affect cross-relaxation rates, and therefore reliable distance data can be obtained from time-dependent NOESY data sets provided an adequately long relaxation delay is used. In hybrid or chimeric RNA-DNA duplexes, if the longer RNA relaxation times are not taken into account in the recycle delay of NOESY pulse sequences, serious errors in measuring RNA proton distances are introduced.


Involvement of the alpha chain in fibrin clot formation. Effect of monoclonal antibodies. Murine monoclonal antibodies 9C3, 7B1, and 9E9 have been obtained using native human fibrinogen as the antigen. The antibodies reacted with the epitopes in the COOH-terminal domain of the A alpha chain. Fragmentation of the A alpha chain with plasmin, and, as in the case of the 9E9 epitope, with V8 protease, followed by isolation of the smallest reacting peptides, allowed the localization of the epitopes for 9C3, 7B1, and 9E9 to the amino acid sequences of alpha 240-268, alpha 425-440, and alpha 541-574, respectively. All three monoclonal antibodies strongly inhibited the rate of fibrin polymer assembly from monomers, both in the purified system and in the human plasma. The mechanism of this strong inhibition implied a rapid formation of fibrin protofibrils, followed by capping with IgG molecules of protofibrils containing approximately ten monomers. These observations demonstrated that certain regions in the COOH terminus of the alpha chain may play an important role in the assembly of a fibrin clot, presumably being involved in lateral aggregation of protofibrils.


Luminescent/paramagnetic probes for detecting order in biological assemblies: transformation of luminescent probes into pi-radicals by photochemical reduction. The spectroscopic methods of fluorescence polarization and electron paramagnetic resonance (EPR) are used to study order and orientation of extrinsically labeled protein elements of ordered biological systems. These methods generate complementary information about the order of the system, but a consistent quantitative interpretation of the related data is complicated because the signals arise from different donors. We introduce a new method that allows us to detect both signals from the same donor. Unsubstituted xanthene dyes (eosin, erythrosin, and fluorescein) were irradiated by laser light at their absorption maximum in the presence of different reducing agents. Due to photochemical reduction, the quinoidal structure of the xanthene ring is transformed into a semiquinone, and a pi-radical is formed having a characteristic EPR signal of an unpaired electron spin with proton hyperfine interactions. A strong EPR signal is observed from the dye in solution or when specifically attached to a protein following irradiation in the presence of dithiothreitol or cysteine. We applied this technique to the study of skeletal muscle fibers. The fluorescent dye (iodoacetamido)fluorescein was covalently attached to the reactive thiol of the myosin molecule in muscle fibers. Fluorescence polarization and EPR spectroscopy were performed on the labeled fibers in rigor. Both signals indicate a highly ordered system characteristic of cross-bridges bound to actin. Our use of the same signal donor for fluorescence and EPR studies of probe order is a promising new technique for the study of order in protein elements of biological assemblies.


A new way to monitor by-pass restorations of electron transport in inhibited chloroplasts by cyclic electron flow cofactors--a study by modulated fluorimetry. Inhibition of electron transport in broken chloroplasts by DBMIB, under light-limiting conditions, is shown to be bypassed by PMS in a manner similar to the known effects of the phenylenediamine derivatives TMPD and DAD. These bypasses were demonstrated and further studied by modulated fluorimetry, monitoring DBMIB inhibition by the shift of the steady-state fluorescence towards the Fm level and the release of inhibition by a reverse shift together with establishment of a quenching effect by background far-red light. Comparative studies were also made with electron transport blocked by DCMU or BNT. A weak bypass by TMPD and a weaker one by PMS of the block created by DCMU was observed by modulated fluorimetry. The block created by BNT is similarly shown to be bypassed by TMPD but hardly or not at all by PMS. Bypass effects persisted even in the presence of ascorbate. It appears that, following reduction of the different cofactors by ascorbate in the stroma side, illumination caused the accumulation of a pool of oxidized cofactor molecules in the lumen, which is able to mediate electron transport between reduced plastoquinone and plastocyanin or P-700. The existence and the size of this pool were found to depend largely on the internal pH at the lumen, presenting an artificial system in which electron flow is controlled by the lumenal pH. The bypassing electron transport in the presence of DBMIB presumably avoids the participation of the cytochrome b6f complex. During its occurrence, there is also a strong imbalance in the activities of the two photosystems for linear electron flow, in favor of PS II. These experiments may thus serve to establish an in vitro model system for a future investigation of effects related to changes in the imbalance between the two photosystems and its regulation. Furthermore, this experimental system may also be utilized to study the role of the internal lumenal pH in control of photosynthesis.


Toxin pharmacology of the ATP-induced hyperpolarization in Madin-Darby canine kidney cells. The effects of Leiurus quinquestriatus hebraeus (LQH) venom, mamba venom, Buthus tamulus (BT) venom, purified apamin and synthetic charybdotoxin on the membrane hyperpolarization induced by extracellular ATP were examined in Madin-Darby canine kidney cells. For this we used a membrane potential probe (bisoxonol) to determine the potential variations. The relation between bisoxonal fluorescence and membrane potential was established by treating Madin-Darby canine kidney cells suspended in solutions containing various external sodium concentrations with gramicidin. Extracellular ATP induced a rapid hyperpolarization that was blocked by LQH venom and synthetic charybdotoxin. BT venom also blocked the response but at a much higher concentration than that of LQH. Mamba venom (Dendroaspis polylepis) and apamin did not modify the ATP-induced hyperpolarization. We concluded that the ATP induced hyperpolarization was due to the augmentation of the potassium conductance probably through Ca(2+)-activated K+ channels sensitive to charybdotoxin but not to mamba venom. The interaction previously described between charybdotoxin and dendrotoxin (the main toxin of mamba venom) was not observed in our case.


Electrophoresis and silver staining of rat urinary proteins including alpha 2u-globulin (rat n II). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining have been used to monitor the fractionation of rat urinary proteins by Amicon Centriprep C30/C10 ultrafiltration and to assess the purification of alpha 2u-globulin, a laboratory animal allergen, precipitated by exhaustive dialysis. Factors influencing the protein composition of rat urine have been identified. Dialysis procedures have been evaluated for precipitation of alpha 2u-globulin and ultrafiltration used for further purification of the allergen.


The germinative zone produces the most cortical astrocytes after neuronal migration in the developing mammalian brain. The origin of astrocytes of the mouse neocortex during the fetal and early postnatal periods as determined by immunocytological, autoradiographic, electron microscopic and antimitotic methods is described. Most astrocytes destined for the white matter and the infragranular cortical layers are derived from the transformation of radial glial cells between P0 and P10 with an inside-out pattern. This cell metamorphosis is not directly preceded by mitosis and involves the activation of the radial glial lysosomal apparatus. In opposition to recent hypotheses, our findings suggest that most astrocytes destined for the supragranular cortical layers are produced in the germinative zone after the migration of the infragranular neurons and themselves migrate afterwards to the upper cortex between E16 and the first postnatal days. These astrocytes do not display an intermediate stage of the radial glial cell and do not participate in the pattern of appearance of the deeper astrocytes. This second step of astrocytogenesis is a condition for normal cytoarchitectonic development and the maintenance of the supragranular layers, since the deprivation of the astrocytic equipment of the supragranular layers by an antimitotic drug drastically reduces the number of supragranular neurons.


Synthesis and non-thrombogenicity of heparinoid polyurethaneureas. Unsaturated polyurethaneureas were synthesized from 4,4'-diphenylmethane diisocyanate, 1,2-diaminopropane and polybutadiene containing hydroxyl groups at both ends of a chain. The polyurethaneureas were cast as a film and subsequently treated with N-chlorosulphonyl isocyanate to produce sulphamate and carboxylate groups on the film surface. The level of in vitro non-thrombogenicity increased with increasing degree of modification. The high blood compatibility is attributed to the low level of platelet stimulation and to heparinoid activity. It was observed that platelet stimulation depends on the surface structure of the original polyurethaneura, and that the heparinoid activity is related with the presence of sulphamate and carboxylate groups.


Pharmacodynamics of pentamorphone during coronary artery bypass grafting in humans. Pentamorphone is a new, highly potent opioid reported to have minimal cardiovascular effects in humans and a high therapeutic index in animals. Pentamorphone was injected intravenously (IV) as the sole anesthetic in 10 patients with left ventricular ejection fractions greater than 0.35 who were undergoing elective coronary artery bypass grafting (CABG). After premedication with lorazepam, 40 micrograms/kg, and establishment of hemodynamic monitoring, pentamorphone was infused at a rate of 2 micrograms/kg/min until unconsciousness occurred (5.1 +/- 1.6 micrograms/kg). Anesthetic induction was accompanied by an average 30% decrease in systolic, diastolic, and mean arterial pressure (MAP), a 19% decrease in heart rate (HR), but no change in cardiac output (CO) or pulmonary artery occlusion pressure. Five patients had a MAP less than 60 mm Hg after induction. Following incision, blood pressure, pulmonary artery occlusion pressure, and CO were unchanged from baseline but HR remained significantly lower. Despite additional pentamorphone (total dose 9.6 +/- 1.8 micrograms/kg), 6 patients required thiopental and/or enflurane to control hypertension intraoperatively. When pentamorphone is used as the sole IV anesthetic in lorazepam-premedicated patients with normal or mildly impaired ventricular function, there is a high incidence of hypotension during induction, and poor control of hemodynamic responses to stimulation. Pentamorphone, 10 micrograms/kg, does not seem to offer any significant advantage over opioids currently used for anesthesia in patients undergoing CABG.


Immunosuppressive effect of FK506 on experimental allergic neuritis in Lewis rats: change of T cell subsets. The effect of a new immunosuppressant, FK506, on experimental allergic neuritis (EAN) was examined in Lewis rats. EAN was induced by inoculation with bovine peripheral myelin. The EAN rats were divided into two groups. FK506-treated EAN rats were prophylactically administered FK506 by injection into the peritoneal cavity at a dosage of 5.0 mg/kg/day for 13 days beginning the day after inoculation. The control EAN rats were injected with only saline solution. FK506 prevented the development of EAN, histologically and clinically. In FK506-treated EAN rats, flow cytometric analysis of T cell subsets of the lymph nodes showed a significant decrease in W3/13 positive T cells on the 14th and 21st day and a decrease in W3/25 positive T cells on the 21st day after inoculation when compared with the control EAN rats. The percentage of OX-8 positive T cells were not significantly different between the two groups. Our results suggest that FK506 prevented the development of EAN by decreasing W3/13 and W3/25 positive T cells.


A case of amylase-producing lung cancer. A case of lung cancer with hyperamylasemia was studied. Small cell carcinoma was diagnosed histologically. The salivary gland and pancreas had no clinical involvement in the hyperamylasemia. Saliva-type amylase was dominant as observed from amylase isozyme patterns in the serum and tumorous tissue. Levels of amylase were higher in tumorous tissue than in normal lung tissue. Immunohistochemical study showed amylase localized in tumor cells. Observation of the ultrastructure revealed electron-dense granules in the cytoplasm of the tumor cells. Findings suggested that amylase was being produced by the lung cancer in this case.


Age-related variation in urinary flow variables and flow curve patterns in elderly males. A group of randomly selected males, over 50 years of age, was examined with regard to history and urinary flow rate. Originally, the group had comprised 93 men without voiding problems, 15 who, although suffering from voiding problems, had not yet contacted their doctor, and 4 who had contacted their doctor because of voiding problems. Five years later the data were re-examined and 61 men were fully investigated again; in the remaining 51 patients only the history was updated. The 2 sets of data were evaluated separately as 2 cross-sectional investigations and as paired data sets by means of non-parametric statistical analysis. All uroflow variables were considered, i.e. Qmax, Qave, Qmax-time, Q "corrected", volume and the ratio Qmax/Qmax-time. In addition, the flow curve configuration was classified as normal or abnormal. When compared as 2 cross-sectional investigations, the latter confirmed the results of the first. Qmax, Qave, Q "corrected" and volume decreased with advancing age, but no correlation could be demonstrated between Qmax-time and age. The flow curve pattern altered, so that a normal flow curve was seldom seem at an advanced age. Uroflow variables tended towards the abnormal with advancing years and symptoms increased concurrently. These changes were largely accepted by the elderly males. Thus the decision to operate for benign prostatic hyperplasia should be based on both history and urinary flow.


Urinary flow rate in benign prostatic hypertrophy in relation to the degree of obstruction of the vesical outlet. Forty men with bladder outflow obstruction from benign prostatic hypertrophy were investigated urodynamically by medium-fill water cystometry and pressure flow study. The most obstructed patients proved to be unstable. A positive link was found between the degree of obstruction, as assessed by the opening pressure (i.e. the detrusor pressure needed to start micturition), and the maximum mechanical power generated by the contracting bladder during voiding. What seems most striking, is that neither detrusor contraction velocity nor the urine flow rate fully mirrored the degree of obstruction--i.e. the opening pressure and the maximum contraction speed did not show a negative correlation, but rather a weak (though insignificant) positive link, and the flow rate decreased only slightly (not significantly) when obstruction increased. Such a surprising observation could be explained by the fact that prostatic obstruction produces both collagen infiltration into the bladder muscle (this affecting the spread of the depolarisation wave) and, at the same time, denervation supersensitivity. The latter actually yields a decrease in electrical resistance between the detrusor smooth muscle cells (and thereby an enhanced synchronisation of the detrusor contraction), which might be only partly cancelled by the effects of collagenosis.


Identification of metastatic disease by T category, gleason score and serum PSA level in patients with carcinoma of the prostate. Pre-operative serum prostate specific antigen (Tandem-R assay), T category, Gleason score and the metastatic (M1) status of a consecutive series of 60 patients with newly diagnosed carcinoma of the prostate were studied prospectively. The results revealed that, of these variables, pre-operative serum PSA (greater than 100 ng/ml) was the single most important indicator of metastatic disease, with 100% predictive value. With this alone, 83.3% of M1 disease could be correctly identified. For the remaining 17%, however, we advocate a high index of suspicion if the tumour is T3-T4 category on digital rectal examination (predictive value = 71.4%) and has a high grade with a Gleason score 8-10 (predictive value = 81%).


An immunohistologic study of the epithelial components of 81 cases of thymoma. Eighty-one cases of thymoma were studied immunohistologically with the use of three mouse monoclonal antibodies: one was specific for subcapsular-cortical, one for intra-cortical, and one for medullary epithelial cells. Twenty-eight (60.9%) of 46 polygonal cell thymomas were of the cortical type and 1 (2.2%) was of the medullary type. Ten (55.6%) of 18 spindle cell thymomas and 7 (41.2%) of 17 mixed cell thymomas were of the medullary type, and 1 (5.6%) of 18 spindle cell thymomas was of the cortical type. Fourteen (17.3%) of 81 thymomas were composed of epithelial cells that were triple positive immunologically; although these are unusual, they also may be present in the normal thymus. Based on these findings, triple-positive epithelium in the normal thymus consists of common stem cells that can differentiate into subcapsular-cortical, intra-cortical, and medullary epithelium; these cells may be the target cells for tumorigenesis. Epithelium in polygonal cell thymoma tends to differentiate into cortical epithelium, whereas epithelium in spindle and mixed cell thymomas differentiates into medullary epithelium.


Establishment of human squamous carcinoma cell lines highly and minimally sensitive to bleomycin and analysis of factors involved in the sensitivity. Human squamous carcinoma cell lines that were highly and minimally sensitive to bleomycin were established from clinical specimens and designated as SCCKN and SCCTF, respectively. Although these cell lines showed a similar growth doubling time in vitro, SCCTF was approximately ten times less sensitive to bleomycin than SCCKN. The bleomycin high and low sensitivities were stable even at the 70-cell passage level in vitro. In addition, nude mouse tumors produced by SCCTF were less sensitive to bleomycin that those produced by SCCKN, and the ratio of the mean tumor weight in bleomycin-treated mice to that in control mice was 89.2% in SCCTF and 18.8% in SCCKN. As compared with SCCKN, SCCTF also was less sensitive to peplomycin (5-fold), mitomycin C (2.3-fold), cis-diamine dichloroplatinum (2.5-fold), and vincristine (6.5-fold). Analyses of low bleomycin sensitivity showed that SCCTF had an approximately 20% decreased cellular accumulation and retention of bleomycin, 1.2-fold increase of bleomycin hydrolase activity, elevated DNA repair activity, and increased poly(adenosine diphosphate-ribose) polymerase activity as compared with SCCKN.


Binding parameters and idiotypic profile of the whole immunoglobulin and Fab' fragments of murine monoclonal antibody to distinct determinants of the human high molecular weight-melanoma associated antigen. The nonspecific accumulation of radioactivity in bone marrow, liver, and spleen in patients with melanoma injected with radiolabeled whole IgG of anti-high molecular weight-melanoma associated antigen (HMW-MAA) monoclonal antibody (mAb) hampers the application of immunoscintigraphy to visualize melanoma lesions. This nonspecific background can be reduced by utilizing fragments which do not contain the Fc portion of anti-HMW-MAA mAb. Since the in vivo targeting to melanoma lesions of radiolabeled F(ab')2 and Fab' fragments of anti-HMW-MAA mAb is critically dependent on their binding parameters, we have analyzed the effect of fragmentation on the binding characteristics of the anti-HMW-MAA mAbs 225.28, 763.74, and TP41.2. The three mAbs recognize distinct and spatially distant determinants with a heterogeneous distribution on the pool of HMW-MAA molecules synthesized by melanoma cells. The determinant recognized by mAb TP41.2 is detectable on a markedly smaller population of HMW-MAA molecules than those recognized by mAbs 225.28 and 763.74. 125I-labeled F(ab')2 fragments of the three mAbs displayed an immunoreactive fraction similar to that of the whole IgG, while Fab' fragments displayed a lower one. Fragmentation of mAbs 225.28 and TP41.2 to F(ab')2 produced a 2- and 1.5-fold reduction in their association constants but did not cause a significant change in that of mAb 763.74. Cleavage of F(ab')2 fragments to Fab' fragments produced 2-, 40-, and 7-fold reductions in the association constants of mAbs 225.28, 763.74, and TP41.2, respectively. These changes qualitatively fit the predictions of theory for univalent and bivalent mAb binding, since mAbs 763.74 and TP41.2 appear to show bivalent binding to melanoma cells and mAb 225.28 to show univalent binding. The affinity constant of IgG, F(ab')2 and Fab' fragments of mAbs 225.28, 763.74, and TP41.2 displays an inverse relationship with the extent of their time-dependent release from the membrane of melanoma cells. Since no endocytosis of mAb could be detected, the latter results suggest that radioactivity remains bound to melanoma cells in vivo for a longer time following injection of F(ab')2 fragments than following that of Fab' fragments of each of the anti-HMW-MAA mAb tested. Radiolabeled Fab' fragments of mAbs 763.74 and TP41.2 displayed a marked reduction in their reactivity with some of the antiidiotypic mAb tested. The loss of some idiotopes is likely to be caused by changes in the conformation of the molecules associated with the fragmentation of IgG and by damage during the iodination procedure.


Tumor selective reactivity of a monoclonal antibody prepared against a recombinant peptide derived from the DF3 human breast carcinoma-associated antigen. The DF3 antigen is a member of a family of high molecular weight glycoproteins aberrantly expressed in malignant mammary epithelium. We have generated a monoclonal antibody (MAb), designated DF3-P, against a recombinant DF3/beta-galactosidase fusion protein. Characterization of this MAb has demonstrated reactivity with immature precursors of DF3 antigen and not with the secreted form. These findings are in contrast to those obtained with MAb DF3, a previously described antibody with predominant reactivity against the mature glycoprotein. The finding that deglycosylation of secreted DF3 antigen with neuraminidase and endo-alpha-N-acetylgalactosaminidase is associated with increased MAb DF3-P reactivity provided additional support for the selectivity of this antibody against the protein core. Epitope mapping studies demonstrate that both the DF3-P and DF3 epitopes are located at a TRPAPGS domain in the 20-amino acid tandem repeat. The results of competition studies with synthetic peptides indicate that the proline in this domain is involved in both epitopes, while the potential glycosylation sites at threonine and serine may contribute to the differential reactivity of MAbs DF3 and DF3-P. Taken together, these findings suggest that both antibodies react with a similar epitope that is modified by the presence of carbohydrate moieties. The results of immunoperoxidase staining studies further demonstrate that while MAb DF3-P reacts with formalin-fixed sections of breast carcinomas, this antibody exhibits little if any reactivity with normal mammary epithelium. Selective expression of the DF3-P epitope in malignant breast cells may be useful in identifying this transformed phenotype.


Tumor-inhibitory monoclonal antibodies to the HER-2/Neu receptor induce differentiation of human breast cancer cells. The HER-2/neu protooncogene (also called erbB-2) encodes a tyrosine kinase receptor for a polypeptide growth-regulatory molecule. Amplification and overexpression of the gene have been frequently observed in human adenocarcinomas and correlated with poor prognosis. To explore the potential of antibody therapy directed at the HER-2/Neu receptor, we have raised a panel of murine monoclonal antibodies to the human protein, and tested their effect on the tumorigenic growth of HER-2/neu-transfected fibroblasts in athymic mice. We previously reported that the i.p. injected antibodies either inhibited or accelerated the tumorigenic growth of HER-2/neu transfectants in athymic mice. Here we report that these opposing effects were induced also by i.v. injected antibodies, they lasted over 7 weeks, and were probably mediated by distinct epitopes on the receptor molecule. To understand the cellular mechanisms underlying antibody-induced tumor inhibition, we tested the effect of the monoclonal antibodies on various cultured human breast cancer cells. Our analysis revealed that the tumor-inhibitory antibodies specifically induced phenotypic cellular differentiation that included growth arrest at late S or early G2 phase of the cell cycle, markedly altered cytoplasm and nuclear morphology, synthesis and secretion of milk components (casein and lipids), and translocation of the HER-2/Neu protein to cytoplasmic and perinuclear sites. The extent of cellular differentiation by various antibodies could be correlated with their tumor-inhibitory potential, whereas a tumor-stimulatory monoclonal antibody or control immunoglobulin were completely inactive with respect to cellular differentiation. Taken together, our in vivo and in vitro studies correlate the tumor inhibitory potential of monoclonal antibodies to HER-2/Neu with their capacity to induce cellular differentiation in vitro. This observation may hold promise for immunotherapy of cancers that express the HER-2/neu oncogene.


Computed tomography of sclerosing hepatocellular carcinoma. We reviewed computed tomography (CT) in eight patients with sclerosing hepatocellular carcinoma who underwent resection. Dynamic CT was performed 25-40 s (rapid phase) and 5-10 min (late phase) after injection of contrast medium. Most of the lesions were round and homogeneous. Tumors tended to show density equal to the liver. The relative density of the tumors in the rapid phase, however, tended to be higher than in other phases. A peripheral low-density area was absent. Dynamic CT was thus important in the diagnosis of sclerosing hepatocellular carcinoma.


In vitro adsorption of a hydrophobic mutagen to gastrointestinal mucus glycoprotein (mucin) and dietary fibre. The adsorption of mutagens by some dietary fibres has been suggested as one mechanism by which dietary fibres protect against colorectal cancer. It is thought that these dietary fibres carry the mutagen out of the digestive tract, decreasing the effective mutagen concentration to which epithelial cells are exposed. The ability of gastrointestinal mucin to alter the extent to which the hydrophobic mutagen 1,8-dinitropyrene (DNP) adsorbs in vitro onto the insoluble dietary fibre alpha-cellulose, was investigated. It was found that crude and purified human ileal mucins themselves adsorbed DNP and decreased the adsorption of DNP onto alpha-cellulose. Purified mucin which had been treated with trypsin also adsorbed DNP. These studies suggest that in the digestive tract there would be competition for the adsorption of DNP between mucin and insoluble dietary fibres, such as alpha-cellulose. This factor must be considered in predictions about the distribution of hydrophobic, mutagenic carcinogens in the digestive tract and their role in the etiology of colorectal cancer.


Non-immunological release of histamine from rat mast cells elicited by antineoplastic agents. We studied the histamine-releasing activity of several antineoplastic drugs on rat pleural and peritoneal mast cells. The drugs tested included the nitrogen mustards cyclophosphamide and ifosfamide, the nitrosourea carmustine, the triazene dacarbazine, the folic acid analogue methotrexate, the pyrimidine analogue cytarabine and fluorouracil, the vinca alkaloids vinblastine, vincristine and Vinorelbine, the epipodophyllotoxins etoposide and teniposide, and the enzyme L-asparaginase. Methotrexate, carmustine, fluorouracil, vinblastine and vincristine failed to elicit histamine release on rat mast cells. All of the other drugs evoked histamine release in both the presence and the absence of extracellular calcium, but ifosfamide, cytarabine and asparaginase induced a much lower release in the absence of this cation. The response elicited by cytarabine and etoposide was much higher in pleural than in peritoneal mast cells. These results indicate that some antineoplastic drugs may directly activate the release of histamine, which could contribute to some of their secondary effects.


Improved diagnosis of carcinoid tumors by measurement of platelet serotonin. Carcinoid patients are diagnosed biochemically on the basis of increased urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA); urinary and platelet serotonin concentrations are considered to provide complementary information. Using established HPLC methods with fluorometric detection, we evaluated the clinical usefulness of measurements of urinary 5-HIAA and urinary, plasma, and platelet serotonin in 30 consecutive patients with histologically proven carcinoid tumors of fore-, mid-, and hindgut origin before treatment. Ten patients showed no signs of serotonin overproduction; 14 had increased concentrations of urinary 5-HIAA and platelet serotonin; and platelet serotonin, but not urinary 5-HIAA, was increased in 6. None had increased urinary 5-HIAA excretion without an increase in platelet serotonin content. In cases with high rates of tumor serotonin secretion, platelet serotonin reached a maximum and did not correlate with serotonin secretion rate, whereas urinary 5-HIAA was correlated. Increased platelet serotonin was correlated with increased plasma serotonin and with occurrence of carcinoid syndrome. Increased urinary serotonin, allegedly caused by increases in circulating 5-hydroxytryptophan, almost invariably coincided with increased platelet serotonin, but not necessarily with above-normal urinary 5-HIAA excretion. From these results and long-term monitoring of three patients during treatment, we conclude that platelet serotonin is more sensitive than urinary 5-HIAA for detecting carcinoids that secrete only small amounts of serotonin.


[Etiopathogenesis and therapeutic trends in adult T-cell leukemia associated with HTLV-I retrovirus]. HTLV-I (Human T-cell leukemia virus type I) has been the first human retrovirus identified and then associated with a definite pathological entity, a leukemic syndrome that specifically affects mature T-lymphocytes (ATL, adult T-cell leukemia), expressing CD3+, CD4+, CD8-, CD11- phenotype. This form of leukemia/lymphoma is endemic in southwestern islands of Japan, although at present the number of HTLV-I seropositive individuals has greatly increased, with a worldwide diffusion, following the expansion wave of the AIDS-associated HIV retrovirus. In fact, double seropositivity for both HIV and HTLV is frequently found among intravenous drug users. Although ATL leukemia or lymphoma occurs with a low frequency among HTLV-I seropositive individuals, it is likely that the evolution from a latent phase of infection to acute leukemia could be favoured by depression of immunosurveillance levels in the host. Therefore, special attention is required to prevent the diffusion of this retrovirus in adults, taking into consideration that newborn babies from seropositive mothers have to be considered at high risk for development of HTLV-I associated disease, on the basis of their immature immunocompetence.


Association of genetic alterations of c-myc, c-fos, and c-Ha-ras proto-oncogenes in colorectal tumors. Frequency and clinical significance. Using Northern and dot-blot analysis we examined normal and tumor tissue from 29 patients with colorectal carcinomas for the expression and amplification of c-myc, c-fos and c-Ha-ras proto-oncogenes. Overexpression of c-myc (6/24), c-fos (4/24), and c-Ha-ras (9/23) was found. For the c-fos proto-oncogene we also have observed decreased levels of expression in 13 percent (3/24) of the cases analyzed. Gene amplification appeared to be a rare event in these tumors and was found in 3/29 (10 percent) tumors for c-myc and in 1/29 (3 percent) for c-fos proto-oncogene. Curves for overall survival and for disease-free survival failed to show a significant tendency in these parameters to be poorer in tumors with alterations of gene expression for any of the proto-oncogenes analyzed. Despite the biologic importance of these genetic alterations in the etiology of colorectal tumors, levels of c-myc, c-fos, and c-Ha-ras gene expression separately or together cannot be considered as prognostic factors for clinical outcome of the disease.


Supravital staining of synovial fluid with Testsimplets. We explored the use of Testsimplet (TS) in synovial fluid (SF) analysis. TS is a glass slide coated with a dry mixture of methylene blue and cresyl violet, which in contact with one drop of SF provides a stained fresh preparation. We applied the TS to the study of 159 SFs of patients with different rheumatic diseases. In those SFs of patients with crystal-associated diseases, the crystal search was performed both on unstained preparations and with TS. TS was as good as the Wright's and Papanicolaou stain in characterizing SF cells, lupus erythematosus cells, and detection of occasional bacteria. TS allowed a better visualization of Reiter's cells, cartilage fragments, synovial villi, fat droplets, and fibrin. Crystals were identified in every TS of those patients with crystal-associated diseases. TS is a rapid and reproducible method of SF supravital staining. Crystals are well preserved for simultaneous examination with compensated polarized light.