The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis.

Hepatitis B viruses (hepadnaviruses) replicate their DNA genomes by reverse transcription of an RNA intermediate. Efforts to examine the biochemical mechanism for viral DNA synthesis have been hampered by the failure to solubilize the reverse transcriptase from virions and to express the polymerase in heterologous systems in an enzymatically active form. Here, we demonstrate that the polymerase of a hepadnavirus synthesized in an in vitro translation reaction exhibits reverse transcriptase activity. Furthermore, our results show that the polymerase acts as a primer for DNA synthesis and remains covalently linked to nascent DNA, a feature that is not known to exist in any other RNA-directed DNA polymerases. Priming of DNA synthesis requires viral RNA but occurs independently of other viral components. The ability to express the hepadnavirus reverse transcriptase in an enzymatically active form will allow detailed biochemical and functional analyses of this complex enzyme, and may facilitate the identification of inhibitors required for antiviral therapy.


The pro region of BPTI facilitates folding.

The in vitro folding pathway of bovine pancreatic trypsin inhibitor (BPTI) has been described previously in terms of the disulfide-bonded intermediates that accumulate during folding of the protein. Folding is slow, occurring in hours at pH 7.3, 25 degrees C. In addition, approximately half of the BPTI molecules become trapped as a dead-end, native-like intermediate. In vivo, BPTI is synthesized as a precursor protein that includes a 13 residue amino-terminal pro region. This pro region contains a cysteine residue. We find that, in vitro, both the rate of formation and the yield of properly folded BPTI are increased substantially in a recombinant model of pro-BPTI. The cysteine residue is necessary for this effect. Moreover, a single cysteine residue, tethered to the carboxy-terminal end of BPTI with a flexible linker of repeating Ser-Gly-Gly residues, is sufficient to assist in disulfide formation. Thus, the pro region appears to facilitate folding by providing a tethered, solvent-accessible, intramolecular thiol-disulfide reagent.


Activation of T cells through a T cell-specific epitope of CD45.

The 180- and 190-kDa isoforms of CD45 are preferentially expressed on the helper inducer (memory) subset of CD4 cells. In order to generate monoclonal antibodies against the extracellular domains of these isoforms and determine whether they could regulate the function and activation of these cells, we developed a mAb, anti-4H2D, by immunizing Balb/c mice with an isogenic mouse pre-B cell line expressing the human 190-kDa CD45 isoform. Anti-4H2D reacts with approximately 60% of T cells, 70% of CD4 cells, and 60% of CD8 cells. The CD4 cell population defined by this mAb corresponds functionally and phenotypically to that defined by the CD45RO+CD29+ subset. Western blotting demonstrated that anti-4H2D reacts primarily with the 190-kDa isoform of CD45 and to a minor extent, the 205- and 180-kDa CD45 isoforms. Interestingly, this mAb reacted with only a subpopulation of mature thymocytes and peripheral T cells, despite the fact that the 190-kDa CD45 isoform, as well as CD45RO and CD29, is more widely distributed on cells of hematopoietic origin. The 4H2D epitope was neuraminidase sensitive, indicating that anti-4H2D reacts with a carbohydrate epitope which is present on only a subset of the T cells containing the 190-kDa CD45 isoform epitopes. Functional studies showed that soluble anti-4H2D augmented T cell proliferation induced by the CD2 and CD3 pathways, and treatment of T cells with this mAb up-regulated [Ca2+]i flux induced by both anti-CD2 and anti-CD3 mAbs. These results suggest that the 190-kDa CD45 isoform on human CD4 cells is heterogeneous and that the 190-kDa isoform recognized by anti-4H2D regulates the function and activation of CD4 helper T cells.


IL-1-induced prostacyclin production by cerebral vascular endothelial cells inhibits myelin basic protein-specific lymphocyte proliferation.

Addition of cerebral vascular endothelial cells (EC) to myelin basic protein (MBP) immune lymph node cells (LNC) cultured in the presence of MBP resulted in the inhibition of MBP-specific proliferative responses. Proliferation was not inhibited in cultures containing indomethacin (IM), suggesting a possible role for prostaglandins. Significant levels of 6-KPGF1 alpha, the stable hydrolysate product of PGI2, but not PGE2 were observed in culture (LNC + EC) supernatants but not in supernatants from cultures containing only LNC or EC. The levels of PGI2 release were proportional to the concentration of exogenous EC present in culture and synthesis of PGI2 could be blocked by IM. These results indicate the requirement for coculture in the generation PGI2. Additional experiments indicated that EC were required for the generation of PGI2 and that either macrophages (M phi), or recombinant murine IL-1 were able to replace LNC in cocultures with EC in order to generate PGI2. The ability of IL-1 to stimulate EC-derived PGI2 synthesis was dose dependent with maximal stimulation observed at 50 U/ml IL-1. The IL-1-induced production of PGI2 by EC as well as PGI2 production in cultures containing EC and either LNC or M phi was inhibited by treatment with anti-IL-1 antibody. These results indicate that EC are capable of inhibiting antigen-specific lymphocyte proliferation by producing PGI2, which can be induced by the lymphokine, IL-1.


The pp60c-src in retinal pigment epithelium and its modulation by vasoactive intestinal peptide.

The pp60c-src is present at high level in differentiated retinal pigment epithelium (RPE) in culture. Immunofluorescence microscopy using GD11 (anti-pp60c-src) shows intense staining in the plasma membrane, especially at the cell-cell junctions, and diffuse staining in the cytoplasma. Western blot analysis shows that the majority of the GD11-reactive molecules is localized in the membrane. The pp60c-src does not translocate between membrane and cytoplasma when the RPE was reacted with vasoactive intestinal peptide (VIP), which is previously shown to stimulate phosphorylation of the pp60c-src in the membranes (Biochem. Biophys. Res. Commun. 174, 452-8, 1991). Here, VIP modulation is shown to alter the reactivity of pp60c-src with a monoclonal anti-phosphotyrosine antibody.


Pathogenesis of myelin breakdown in demyelinating diseases: role of proteolytic enzymes.

The mechanism by which the myelin sheath is degraded in demyelinating diseases is unknown. The demonstration of increased activities of both acid (cathepsins B, D, A) and neutral proteinases in tissue from experimental allergic encephalomyelitis (EAE) in animals and multiple sclerosis (MS, plaques) and the disappearance of myelin proteins implicate a role for proteolytic enzyme in myelin breakdown. The degradation of myelin basic protein (MBP) by proteinase yields encephalitogenic peptides and its loss has been found to cause structural alteration of the myelin sheath. This suggests that MBP degradation is an initial step in the breakdown of myelin in demyelinating diseases. A calcium-activated neutral proteinase (calpain), which degrades MBP, was found to increase in activity in MS tissue and cerebrospinal fluid (CSF), and its presence in myelin suggests that myelin may be autodigested in demyelinating disease. The source of increased proteinase activity has been indicated as macrophages, lymphocytes, and proliferative astrocytes (reactive cells). Increased proteinase activity is found in Schwann cells in Wallerian degeneration, and the presence of calpain in myelin-forming oligodendrocytes and Schwann cells suggests that these cells are likely sources of degradative enzymes. The involvement of proteolytic enzymes in the mechanism of myelin breakdown indicates the possible intervention with proteinase inhibitors for beneficial effect.


Transformation of Cochliobolus lunatus with pUT 720 changes the steroid hydroxylating ability of the fungus.

The filamentous fungus Cochliobolus lunatus, a known 11 beta-hydroxylator of steroids, was transformed to bleomycin resistance using the heterologous plasmid pUT 720. This plasmid contains the Sh ble gene expressed under the control of the Aspergillus nidulans gpd and trpC expression signals. The bleomycin-resistant colonies appeared with a frequency of six per microgram of DNA. All colonies were real transformants and no "abortive" growth was observed. In all transformants tested the plasmid molecules became stably integrated into the genome of the host, and one of the plasmid molecules integrated in a site-specific manner. Transformants retained the ability to hydroxylate the steroid ring, but the hydroxy group was inserted at the 15 alpha position.


RNA editing in the mitochondria of a conifer.

A 712-base portion of the mitochondrial gene coxI and the corresponding portion of the coxI transcript were amplified by PCR and by RT-PCR, respectively, from the gymnosperm western red cedar. Sequence comparison of amplified coxI DNA and mRNA revealed 26 C-to-U differences that are best explained by RNA editing of the type known to occur in angiosperms. This finding suggests that mitochondrial RNA-editing of the C-to-U type arose before the divergence of gymnosperms and angiosperms and can be considered a feature common to all higher plants.


Removal of endotoxin from culture supernatant of Bordetella pertussis with aminated poly(gamma-methyl L-glutamate) spherical beads.

Attempts were made to prepare adsorbents having a high affinity for endotoxin in the culture supernatant of Bordetella pertussis. When poly(gamma-methyl L-glutamate) (PMLG) was used as a matrix and amino groups as the ligand, the highest affinity for endotoxin was attained even at a high ionic strength (mu = 0.2-0.4). PMLG beads containing amino groups of about 3.2 meq/g selectively removed endotoxin from the culture supernatant of B. pertussis without affecting the protective antigens. It was demonstrated that 1 ml of the wet adsorbent adsorbed 4.5 mg of endotoxin. The beads of PMLG derivatives, therefore, are considered to be a useful adsorbent for the removal of endotoxin from the pertussis vaccine, affecting neither filamentous hemagglutinin nor pertussis toxin.


Photoactivation of 2-nitrofluorene in vitro and in the rat in vivo. UVA-induced formation of reactive intermediates that bind covalently to RNA and protein.

2-Nitrofluorene (2-NF), an environmental pollutant, can be activated by UV light to reactive intermediates that bind covalently to RNA and protein in vitro: high levels of covalent binding were obtained. This covalent binding was not dependent on the presence of oxygen in the solution and could be decreased by glutathione. Hydrolysis of the in vitro modified RNA and subsequent analysis of the liberated bases by HPLC revealed that approximately 15% of the covalent binding of 2-NF to RNA could be attributed to the formation of a guanosine adduct of nitroreduced 2-NF, N-(deoxyguanosin-8-yl)-2-aminofluorene. Many other adducts are formed of which the structure and mechanism of formation are as yet unknown. The possibility of photoactivation of 2-NF in the rat in vivo was also investigated. Photoactivation increased covalent binding of 2-NF in the skin but not in other organs. The mechanism of the photoactivation of 2-NF is discussed.


Thrombospondin as a mediator of cancer cell adhesion in metastasis.

Thrombospondin (TSP) is a 450 kDa adhesive glycoprotein. It is present in high concentrations in the platelet alpha-granule and can readily be secreted following platelet activation where local concentrations can be increased by 3-4 orders of magnitude. TSP is also synthesized by a variety of other cells and is incorporated into their extracellular matrix. TSP is a homotrimer with a number of functional domains, at least four of which might serve as receptor recognizing regions. The amino-terminal heparin binding domain interacts with heparin, other glycosaminoglycans and glycolipids and likely recognizes specific cell surface proteoglycans. The central disulfide cross-linked region, 210 kDa non-reduced and 70 kDa reduced, contains a peptide motif CSVTCG which is apparently responsible for binding to glycoprotein IV (CD36) with high affinity. Immediately adjacent to the calcium binding region of TSP, which undergoes considerable molecular relaxation in the absence of calcium, is an RGDA sequence. TSP has been demonstrated to bind to integrins of the alpha v beta 3 and alpha IIb beta 3 class. The carboxy-terminal region of TSP also contains at least one binding epitope for a cell receptor. There are 2 well characterized genes for TSP and truncated forms of TSP have been detected which have inhibitory effects on angiogenesis. Finally, TSP can interact with fibrinogen and fibronectin, perhaps on cellular surfaces, which might serve as secondary receptor-like mechanisms for TSP binding and subsequent mediation of cell adhesion.


Effect of cAMP elevating compounds on inhibition of gap junctional communication and induction of morphological transformation in Syrian hamster embryo cells.

An enhancement of the cellular cAMP level has been shown to protect against phorbol ester-induced inhibition of gap junctional intercellular communication (GJIC) and induction of morphological transformation in Syrian hamster embryo (SHE) cells. Cholera toxin, forskolin, 3-isobutyl-1-methylxanthine (IBMX) and theophylline counteracted the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inhibition of GJIC. The enhancement of communication by these compounds was independent of the TPA concentration, as well as whether the cells were treated with TPA prior to or after the cAMP elevating agents. The induced increase in the cAMP level by cholera toxin occurred in the same concentration range as the enhancement of GJIC. With forskolin the effect on GJIC was apparent at concentrations 10 times lower than needed to enhance the cAMP level. IBMX and theophylline were found to enhance GJIC with only a 20% elevation of cAMP. The cAMP elevating compounds also suppressed the response of TPA on induction of transformed morphology in SHE cells.


Purification and analysis of glycoproteins bearing blood group-A determinants from hamster pancreatic ductal adenocarcinomas.

Although the hamster pancreas does not express A, B or H blood group antigens, all hamster pancreatic ductal adenocarcinomas induced by treatment with N-nitrosobis(2-oxopropyl)amine express blood group-A antigen. Thus, the acquisition of blood group-A antigen expression in this system is a cancer-associated alteration. We have purified three major blood group-A antigen bearing glycoproteins (gp120, gp135 and gp150) from hamster pancreatic cancer cell membrane preparations using affinity chromatography on DBA (Dolichos biflorus) agglutinin-agarose. When assayed by immunoblotting, gp120 and gp135 showed strong blood group-A reactivity, which was removed by treating membrane samples with peptide-N-glycosidase F. Blood group-A reactivity was unchanged by treatment of the membrane fractions with endoglycosidases F and H. In addition, these two glycoproteins bearing blood group-A antigen also bound L-PHA (Phaseolus vulgaris leucoagglutinin). These results demonstrate that gp120 and gp135 express blood group-A antigen on Asn-linked multi-antennary complex type glycan structures. The gp150 showed weak blood group-A expression. This is the first demonstration of the neoexpression of cancer-associated blood group-A determinants which reside on Asn-linked glycan structures.


Non-promoting 12-deoxyphorbol 13-esters as potent inhibitors of phorbol 12-myristate 13-acetate-induced acute and chronic biological responses in CD-1 mouse skin.

In previous experiments, pretreatment of CD-1 mouse skin with prostratin (12-deoxyphorbol 13-acetate) inhibited hyperplasia, induction of ornithine decarboxylase and edema in response to acute treatment with phorbol 12-myristate 13-acetate (PMA). We report here that prostratin inhibits biological responses induced by multiple (chronic) PMA treatment. A typical chronic treatment schedule consisted of five applications of 3.2 nmol (2 micrograms) PMA at 48 h intervals. Most effective inhibition could be achieved when the first PMA treatment was preceded 48 h before by a lower dose of prostratin (256 nmol = 100 micrograms) and each PMA treatment was preceded 15 min before by a higher dose (2.56 mumol = 1 mg) of prostratin. Under this schedule hyperplasia was completely blocked, as was keratin K6 expression (a marker of hyperproliferative epidermis), whereas myeloperoxidase activity (a marker of neutrophil granulocyte infiltration) was reduced to 36%. 12-Deoxyphorbol 13-phenylacetate (dPP), a non-promoting 12-deoxyphorbol derivative that binds to protein kinase C with two orders of magnitude higher potency than does prostratin, showed the same pattern of inhibition as did prostratin for a single PMA treatment but with a corresponding two orders of magnitude higher potency. In the case of chronic PMA treatment, however, dPP failed to inhibit hyperplasia fully, though it reduced keratin K6 expression and inflammation. Dissociation of K6 expression from hyperplasia was unexpected, since expression of these two responses was thought to be closely coupled. We conclude that 12-deoxyphorbol 13-monoesters are functional antagonists for a class of protein kinase C-mediated responses closely correlated to tumor promotion.


Studies on the relationship between xanthine oxidase and histamine release during intestinal ischemia-reperfusion.

Recent studies have demonstrated a connection between xanthine oxidase-generated reactive oxygen intermediates and histamine release during ischemia-reperfusion. In the present work, the effect of modulation of the endogenous histamine level on the xanthine oxidase activity was examined during the reperfusion of a canine ileal segment following a 2 hr of complete ischemia. The xanthine oxidase activity and the plasma histamine level peaked simultaneously at the beginning of reperfusion, reaching mean values of 14.9 nmol/ml/min and 12.1 nmol/l, respectively. Pretreatment with aminoguanidine, a blocker of diamine oxidase (histaminase), resulted in significantly higher levels of histamine during reperfusion, but this elevation was not accompanied by a further increase in xanthine oxidase activity. Pretreatment with the mast cell stabilizer cromolyn significantly diminished the rise in plasma histamine level, with an unchanging activity of xanthine oxidase. No significant alteration could be observed in the postocclusive activity of xanthine oxidase following the intra-arterial administration of 0.5, 1, or 5 nmol of histamine during the last 10 min of the ischemic period. These data suggest that the amount of histamine liberated during reperfusion does not result in a further increase in the xanthine oxidase activity. The release of histamine is not a cause, but rather an effect of the elevated activity of intestinal xanthine oxidase.


Prostaglandin D2 relaxes bovine coronary arteries by endothelium-dependent nitric oxide-mediated cGMP formation.

This study investigates the vasomotor activities of prostaglandin (PG) D2 in bovine coronary arteries in relation to endothelial function. Isolated segments of bovine coronary arteries with intact endothelium were concentration-dependently relaxed by PGD2 (0.01-1 microM), a reaction that was blocked by a selective PGD receptor antagonist (BW A868C). There was a tight correlation between PGD2- and acetylcholine-induced relaxations (r = 0.894, n = 96, p < 0.001). Removal of endothelium abolished the PGD2-induced relaxation and unmasked a contractile activity of the compound. Inhibition of endogenous PGI2 formation by indomethacin did not modify these responses, whereas inhibition of endogenous nitric oxide generation by NG-nitro-L-arginine and NG-monomethyl L-arginine (10 or 100 microM) or scavenging of released nitric oxide by oxyhemoglobin (3 microM) considerably (> 50%) antagonized the PGD2-induced relaxation. The vessel relaxation by PGD2 was associated with a threefold to fourfold increase in vascular cGMP. A considerable reduction in vascular cGMP was measured after removal of the endothelium (by 53%) and inhibition of endogenous nitric oxide generation by NG-nitro-L-arginine (by 70%). This also resulted in a complete inhibition of PGD2-induced cGMP accumulation. Similar results were obtained with the stable PGD2 mimetic ZK 110.841, suggesting that these biological activities of PGD2 were due to the compound itself and not caused by any PGD2 metabolite. A slight but significant increase in cAMP was observed in arteries with intact endothelium as well as after removal of endothelium. Because the relaxing effect of PGD2 was strictly endothelium dependent, the observed relaxation cannot be explained by cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)


Proto-oncogene expression in porcine myocardium subjected to ischemia and reperfusion.

The molecular basis of myocardial adaptation to ischemia and reperfusion is poorly understood. It is thought that nuclear proto-oncogenes act as third messengers, converting cytoplasmic signal transduction into long-term changes of gene expression. We studied the expression of six nuclear proto-oncogenes (Egr-1, c-fos, fosB, c-jun, junB, and c-myc) in myocardium subjected to ischemia and reperfusion in anesthetized pigs. Stunning was achieved by two 10-minute left anterior descending coronary artery occlusions separated by 30 minutes of reperfusion. Hearts were excised after the first occlusion, after the first reperfusion, and at 30, 120, 150, and 210 minutes of reperfusion after the second occlusion. Total RNA was prepared from stunned as well as normally perfused myocardial tissue and subjected to Northern blotting. The response of the six nuclear proto-oncogenes varied.fosB gene expression was never detected. The c-myc gene was expressed, but its level was unchanged by ischemia. c-jun expression was slightly increased by ischemia (3.1 +/- 0.6-fold). The c-fos, Egr-1, and junB genes were highly induced, being fivefold to sevenfold higher in experimental than in control tissue. In three animals pretreated with the beta 1-antagonist metoprolol and then subjected to the above experimental protocol, the induction of proto-oncogenes was similar to that in nonblocked controls. Our results show that the myocardial adaptive response to ischemic stress includes the induction of at least four transcription factors that may be further operative in repair processes and angiogenesis.


Nitric oxide synthase in cardiac nerve fibers and neurons of rat and guinea pig heart.

Participation of nitric oxide (NO) in the autonomic innervation of rat and guinea pig hearts was investigated by applying the NADPH diaphorase technique and immunohistochemistry with NO synthase antiserum. We present evidence that NO synthase is localized in cardiac ganglion cells and nerve fibers innervating the sinuatrial and atrioventricular nodes, the myocardium, local neurons, coronary arteries, and pulmonary vessels, suggesting an involvement of NO in neurogenic heart rate regulation, myocardial cell function, neuronal transmission in cardiac ganglia, and coronary as well as pulmonary vasodilation.


Course in the intensive care unit after 'preparatory' pulmonary artery banding and aortopulmonary shunt placement for transposition of the great arteries with low left ventricular pressure.

In patients with transposition of the great arteries with low left ventricular pressure, pulmonary artery banding with aortopulmonary shunt placement has been advocated to "prepare" the left ventricle for systemic work before an arterial switch operation. In 28 patients, this preparatory procedure was performed with one death. A successful arterial switch operation was performed at a median of 7 days later in 24 of 27 survivors; one child had a Senning performed, and two others died. During this interval period, the left ventricular-to-right ventricular pressure ratio increased from 48 +/- 8% to 98 +/- 19%, and left ventricular mass (indexed for body surface area) increased from 46 +/- 17 to 72 +/- 23 g/m2. After the preparatory procedure, the initial postoperative period was frequently characterized by a low-output syndrome of variable length and severity. Prolonged mechanical ventilation, extended inotropic support, and/or a significant metabolic acidosis was present in 21 of 28 patients in the immediate postoperative period. The low-output syndrome is most likely due to a combination of acute (fixed) right ventricular volume overload from the shunt and acute (transient) left ventricular dysfunction from the pulmonary artery band. This low-output syndrome should be anticipated following the preparatory procedure.


Results of a right ventricular outflow patch for pulmonary atresia with intact ventricular septum.

Although overall outcome has improved, pulmonary atresia with intact septum remains a difficult surgical and clinical problem. To determine whether an early right ventricular outflow patch will result in biventricular repair for this lesion, we reviewed the long-term follow-up (5.8 +/- 0.8 years) of 19 newborns who underwent repair between 1979 and 1990. An early right ventricular outflow patch was placed in 15 of 19 newborns; in the remaining four, this was preceded by an aortopulmonary shunt. Prostaglandin E1 infusion postoperatively eliminated the need for shunt in 14 of 15. Coronary sinusoids were ligated in three newborns. Based on right ventricular morphology, the newborns were divided into two groups: group 1 (tripartite, n = 9) and group 2 (bipartite and monopartite, n = 10). Before surgery, group 1 had significantly larger right ventricular volumes (23.6 +/- 3.7 versus 5.2 +/- 1.1 ml/m2, p < 0.002). Five-year survival was 79% for the entire series. Four infants, all group 2, died within 12 months of their initial surgery. Fourteen of 15 survivors (nine group 1 and five group 2) currently are acyanotic and New York Heart Association functional class I. A biventricular repair was achieved in 12 of 15, and three other children are awaiting evaluation. All 15 survivors had significant right ventricular and tricuspid annulus growth. Our data suggest that early placement of a right ventricular outflow patch in infants with pulmonary atresia and intact ventricular septum, regardless of right ventricular anatomy, results in an excellent chance for biventricular repair.


Aprotinin prevents cardiopulmonary bypass-induced platelet dysfunction. A scanning electron microscope study.

Administration of aprotinin during extracorporeal circulation reduces blood loss and improves platelet function. To evaluate the protective effect of aprotinin on platelets, 50 patients undergoing cardiopulmonary bypass were randomized before surgery to one of three groups. Seventeen patients (group A) received continuous high-dose aprotinin (7 x 10(6) KIU) during cardiopulmonary bypass, 17 (group B) received a single bolus of aprotinin in the pump prime (2 x 10(6) KIU), and 16 (group C) received placebo. Scanning electron microscopy was used to evaluate platelet aggregation on extracellular matrix. The platelet function was graded from 1 to 4, with grade 4 being normal aggregation. Immediately after cardiopulmonary bypass, 16 patients in group A (94%) reached preoperative aggregation grade (mean grade, 3.4 +/- 0.7) compared with nine of 17 in group B (52%) (mean grade, 2.9 +/- 1.2), and none in group C (0%) (mean grade, 1.4 +/- 0.5; p < 0.001). Postoperative platelet count did not differ significantly among the three groups. After surgery, group A bled less than groups B and C (395 +/- 120 versus 488 +/- 135 and 780 +/- 408 ml, respectively; p < 0.01). Patients in the aprotinin groups received fewer red blood cell units (0.9 +/- 1.2 and 1.9 +/- 1.2 versus 3.4 +/- 1.9, respectively; p < 0.01) and were exposed to less homologous blood products (1.3 +/- 1.7 and 2.1 +/- 1.1 versus 6.1 +/- 5, respectively; p < 0.001). By preserving platelet function, aprotinin improves postoperative hemostasis in all patients who receive high dose and in most who receive low dose.


Aprotinin and heparin monitoring during cardiopulmonary bypass.

When high-dose aprotinin is used during cardiopulmonary bypass, there is a prolongation of the activated coagulation time (ACT), which is used to monitor heparinization. The aim of this study was to provide guidelines for monitoring heparin levels by the ACT if aprotinin is used during cardiopulmonary bypass. Heparinized blood from six healthy controls and nine patients on cardiopulmonary bypass was aliquoted and mixed with various concentrations of aprotinin. ACTs were performed on these samples. Activated partial thromboplastin times (APTT) were performed on the citrated plasma mixed with varying concentrations of heparin and aprotinin from the same control patients. Prothrombin times (PT) were performed on plasmas mixed with aprotinin. Aprotinin produced a dose-related prolongation of ACT and APTT but had no effect on PT. This effect occurred whatever the activating agent and in the absence of heparin. Aprotinin prolongs the ACT and APTT independently of heparin. If high-dose aprotinin is used during cardiopulmonary bypass, ACTs should be maintained at times > 750 seconds to allow for appropriate levels of heparin.


Development of peptide- and tyrosine hydroxylase-containing neurons in the fetal spinal cord transplanted into the anterior chamber of the eye of adult rats.

Fetal rat spinal cord transplanted into the anterior chamber of the eye of an adult rat was immunohistochemically stained using antisera to substance P (SP), neuropeptide Y (NPY), methionine-enkephalin (ENK), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH), and distributional changes of peptide- and enzyme-containing neurons 1, 2 and 4 weeks after transplantation were investigated. To examine the effect of colchicine on immunoreactivity, unilateral eyes of these adult host rats received intraocular colchicine treatment. Without colchicine treatment, numerous SP- and CGRP-immunoreactive (IR) neurons were observed in the graft 1 week after transplantation, and their immunoreactivity gradually decreased up to 4 weeks after transplantation. NPY-, ENK-and VIP-IR neurons first appeared in the graft 2 weeks after transplantation. Four weeks after transplantation, the immunoreactivity of NPY and ENK decreased significantly, whereas VIP-IR neurons showed the same intensity as that observed at 2 weeks after transplantation. TH-IR neurons, on the other hand, were seen at every stage, but their immunoreactivity was constant all the time. After colchicine treatment, the number of SP-, NPY-, ENK- and CGRP-IR neurons appeared to increase, while that of VIP- and TH-IR neurons did not change significantly. The distribution patterns of the peptide- and enzyme-containing fibers differed from each other. In the analysis of serial sections stained with 5 peptides (SP, NPY, ENK, VIP, CGRP), fibers containing these peptides were found to be densely accumulated in specific areas of the transplanted spinal cord. The present findings demonstrated that most of the peptide- and enzyme-containing neuron systems in the transplanted spinal cord showed similar distribution patterns and development to those in the normal spinal cord, but that some displayed different distribution.


Influence of position along the medial-lateral axis of the superior colliculus on the topographic targeting and survival of retinal axons.

Topographic order in the rat retinocollicular projection emerges from an initially diffuse projection during an early postnatal remodeling period that is coincident with the period of naturally occurring ganglion cell death. Here, we examine the relationship between a retinal axon's position along the medial-lateral axis as it enters the superior colliculus (SC) and its ability to form an appropriately positioned arbor and survive the remodeling period. At E18-E19, prior to map remodeling, axons labeled with focal DiI injections in the periphery of temporal, nasal, superior or inferior retina are widespread along the medial-lateral SC axis. At P12, after remodeling, the distributions of axons remain widespread over the medial-lateral SC axis relative to the positioning of their terminal arborizations, and resemble the distributions labeled at E18-E19, with the exception that the small proportion of axons most widely mispositioned along the medial-lateral SC axis are less frequent. These data indicate that the most widely mispositioned retinal axons are preferentially eliminated, but that a high proportion of retinal axons mispositioned along the medial-lateral axis as they enter the SC can correct their position, form topographically appropriate arbors, and survive the remodeling period.


Expression of thyroid hormone receptors mRNAs in rat cerebral hemisphere neuronal cultures.

We have studied the expression of the alpha and beta thyroid receptors mRNAs (TR-mRNAs) in cerebral hemisphere neuronal cultures, initiated from 15-day-old rat embryos, by northern analysis. In the cultures grown in the absence of L-triiodothyronine (L-T3), the alpha 2 TR-mRNAs were the predominant form of TR-mRNAs and were approximately 8 to 20-fold higher than the levels of the alpha 1 TR-mRNAs, depending on the age of the cultures. The levels of alpha 2 TR-mRNAs significantly increased by 1.8 fold between day 8 and 15 and remained on a plateau value thereafter until day 22. Over the same time period, there were no significant changes on the levels of alpha 1 TR-mRNAs. The ratio alpha 1/alpha 1 + alpha 2 TR-mRNAs decreased between day 8 and 15. The beta 1 TR-mRNAs increased by 8 fold between day 8 and day 22. On day 8, the beta 1 TR-mRNAs were 1.8 fold lower than the levels of the alpha 1 TR-mRNAs while they were 6 fold higher on day 22. L-T3 treatment of the cultures had no effect on the levels of the alpha 1, alpha 2 and beta 1 TR-mRNAs. The differential temporal expression of the alpha 1 and beta 1 TR-mRNAs suggests distinct functions for both types of T3 receptors in neuronal maturation.


Characterization of a novel set of membrane antigens associated with axonal growth. I. Biochemical and functional studies.

Cranin, a prominent 120 kDa laminin-binding protein of cell membranes, was originally identified and characterized by virtue of its ability to bind laminin directly in ligand-blotting assays. We have now raised polyclonal ('3070') and monoclonal antibodies (MAbs 4 and 199) against a partially purified preparation of cranin, and have characterized the properties and expression of the corresponding antigens in further detail. In immunoblots of E14 chick brain membranes, these antibodies all recognized a single major band migrating at approximately 125 kDa, with minor bands at 115 kDa and 170/180 kDa. The major 125 kDa antigen bound to laminin affinity beads in a divalent cation- and conformation-dependent manner. The 125 and 115 kDa bands were also the most prominent HNK-1 positive proteins detected overall within E14 chick brain membranes. MAbs 4 and 199 specifically inhibited the attachment of NG108-15 cells to low, but not high amounts of substratum-bound laminin. While the relation of 3070/MAb 4 antigens to cranin requires further elucidation, these data, together with evidence presented in the companion papers and elsewhere, suggest that the antigens are important in neural development by mediating at least some of the responses of neural cells to laminin--for example, by acting as a laminin receptor guiding axonal outgrowth and neuronal migration, or by involvement in the transport and binding of laminin to the surface of neurons and reactive glial cells that synthesize laminin.


Viral hepatitis in the 1990s, Part III: Hepatitis C, hepatitis E, and other viruses.

Acute hepatitis can be caused by a number of viruses, especially A, B, C, E, delta, Epstein-Barr virus, and cytomegalovirus. Hepatitis A and B have been discussed previously in this series. The virus responsible for most cases of what commonly has been referred to as non-A non-B hepatitis has been tracked, and antibodies to certain proteins of this virus have been identified. This virus is now referred to as hepatitis C. The possible clinical outcomes after acute hepatitis C virus infection are similar to those for hepatitis B virus infection, except that hepatitis C is far more likely to become chronic. Clinical testing for hepatitis C virus infection is in its infancy and has certain limitations. Successful treatment of at least some cases of hepatitis C is possible. Hepatitis E has recently been described, primarily in third-world countries. It causes an acute hepatitis that may be particularly lethal for pregnant women. Herpesviruses may also cause hepatitis, particularly in the newborn or the immunocompromised. Exotic viruses causing acute hepatitis are enumerated.


Two-site immunoradiometric assay of intact salmon calcitonin.

We developed a two-site immunoradiometric assay (IRMA) of salmon calcitonin (SCT) that detects intact SCT(1-32) and not peptide fragments of the hormone. This was accomplished by using monoclonal antibodies prepared against the peptide fragments SCT(1-11) and SCT(11-32). Two antibodies with specificity for each of the peptides were purified from their respective ascites and evaluated in a two-site format wherein one of the antibodies was adsorbed to polystyrene beads and the other was radioiodinated. In this assay format, the antibody pair detected intact SCT(1-32) but did not react with either SCT(1-11) or SCT(11-32). The sensitivity of the assay could be increased by exchanging the antibodies with respect to bead adsorption and radioiodination. Furthermore, by increasing the incubation time and volume of incubation of sample with the polystyrene-bead-adsorbed antibody, the effective detection limit of the assay could be improved. This assay system can be used to detect intact SCT when the presence of fragments of the hormone might otherwise complicate interpretation of assay data.


Clinical evaluation of a pancreatic lipase mass concentration assay.

An immunoactivation assay for determining pancreatic lipase mass concentration was clinically evaluated and compared with results obtained by measuring total amylase and pancreatic amylase activity. A group of 30 patients with pancreatitis was compared with a control group of 32 patients in which this disease was suspected but excluded. Both lipase mass concentration and pancreatic amylase activity exhibit good sensitivity (0.93 each) and specificity (0.94 and 0.97, respectively) at cutoff concentrations of 200 micrograms/L and 200 U/L, respectively. The median increase in lipase mass concentration (37.1 times the upper limit of the reference interval) in the pancreatitis group was higher than that for either total amylase or pancreatic amylase activity (5.94 and 14.5 times, respectively) but showed a similar time to peak value. We conclude that the lipase assay is the method of choice for diagnosing pancreatitis.


Two-site assays of bone gla protein (osteocalcin) demonstrate immunochemical heterogeneity of the intact molecule.

We developed a panel of monoclonal antibodies to human bone gla protein (BGP; osteocalcin) peptides that span the linear sequence of the molecule, specifically BGP 1-12 (N-terminal), BGP 15-30 (midregion), and BGP 38-49 (C-terminal). These antibodies were evaluated in various combinations of two-site formats in studies of serum BGP concentrations. For clinical studies, we selected from a panel of antibodies the two most sensitive antibody pairs for the intact molecule (N-C); we also used a polyclonal RIA based on BGP-C. For the two-site format, we used two N-terminal antibodies, 029 and 052, adsorbed to polystyrene beads, and radioiodinated a C-terminal antibody, 663. The standard for each of the assays was purified human BGP. The following BGP serum concentrations (microgram/L, mean +/- SE) were measured with the various assays: by the 029-663 assay, results for normal subjects were 7 +/- 3, for patients with renal failure 25 +/- 8, and for patients with Paget disease 12 +/-4; by the 052-663 assay, the respective results were 22 +/- 4, 44 +/- 12, and 31 +/- 7; by the polyclonal assay, the results were 3 +/- 0.2, 13 +/- 2, and 5 +/- 1. The two intact (N-C) assays were significantly (P < 0.01) correlated (r = 0.94), but their serum values differed by more than twofold in terms of the same BGP standard. The polyclonal assay significantly correlated with each of the intact assays (r = 0.83, 0.77), but it, too, gave different serum values for BGP. These studies demonstrate the immunochemical heterogeneity of circulating BGP, heterogeneity that is manifest even in immunoassays specific for the same region of the molecule.


Failure of microchromatographic measurement of fetal hemoglobin in beta zero thalassemia-hereditary persistence of fetal hemoglobin.

We report microchromatographic measurement of fetal hemoglobin (HbF) proportions in a 36-year-old African-American multigravida woman. At 34 weeks she delivered a 630-g male infant who subsequently did well. Hemoglobin electrophoresis of the hemolysate revealed nearly 100% HbF without HbA, an extremely unusual naturally occurring sample. Family studies revealed a combination of hereditary persistence of fetal hemoglobin (HPFH) and beta zero-thalassemia minor. Southern blot technique confirmed heterozygous alpha 2 thalassemia and HPFH but failed to identify the beta thalassemic lesion. The absence of HbA and the very high amounts of HbF led us to measure HbF by several methods to confirm the accuracy of microchromatography of HbF at values approaching 100%. HPLC revealed a 14% F1 suggestive of microchromatographic underestimation due to glycated HbF. We conclude that cation-exchange microchromatography and the Betke method of alkali denaturation underestimate HbF values as they approach 100% and do not recommend these procedures in this rare situation.


Differences in the enzymatic nature and the sugar-chain structure of gamma-glutamyl transferase between normal and carcinomatous human kidney and prostate.

The enzymatic and immunological nature, and the sugar chain structure, of gamma-glutamyl transferase (GGT) purified from tissues of benign prostatic hypertrophy (BPH), prostatic carcinoma (PCa) and renal cell carcinoma (RCa), were compared with those of the normal prostate (NP) and kidney (NK). The specific activities of GGTs in NP, NK, BPH, PCa and RCa were 78.9, 22.5, 105, 92.5 and 52.5 mU/mg protein, respectively. The molecular masses of GGTs from BPH, PCa and RCa were 72 kDa, 78 and 108 kDa, and 79 and 105 kDa, respectively. The Michaelis constants (Km), optimum pHs and the inhibition of GGT activities by several chemical compounds, revealed that the GGT from BPH, PCa and RCa was similar to that of normal GGT. Immunologically, the IgG fraction against anti-human seminal plasma GGT fused to the all of the GGTs tested. The sugar chain heterogeneities of the various GGTs, detected by the serial-lectin affinity technique, differed from one another. The sugar chain of GGT from BPH resembled the sugar chain from NP. On the contrary, the sugar chains of GGTs from PCa and RCa were markedly different from those from normal tissues. In the GGT from PCa, multi-antennary complex type sugar chains were more increased than the enzyme of NP. In general, as previously reported, the sugar chains of GGTs from carcinomatous tissues of prostate and kidney had an increased content of bisecting GlcNAc (beta 1-->4) containing complex type sugar chains. Moreover, the reductions of the biantennary complex type sugar chain with fucose linkage and the hybrid type sugar chain were obvious in the GGT from carcinomatous tissues of the prostate and kidney.


Galanin reinstates the growth hormone response to repeated growth hormone-releasing hormone administration in man.

To clarify the mechanism by which galanin, a 29-amino-acid peptide, increases GH secretion in man. We studied the GH-releasing effect of this neurohormone (galanin, 15 micrograms/kg) infused over 60 minutes after 120 minutes of saline, following a previous GHRH bolus (GHRH 1 microgram/kg i.v. at 0 minutes, galanin infused from 120 to 180 minutes) and coadministered with the second of two consecutive GHRH boluses (GHRH every 120 minutes, galanin infused from 120 to 180 minutes). Fourteen healthy male subjects, aged 20-34 years, in two groups (group A, 20-31 years (n = 8); group B, 25-34 years (n = 6)) were studied. Blood samples were drawn every 15 minutes of 255 minutes. Serum GH was measured in duplicate by IRMA. Statistical analysis of the data was carried out by non-parametric ANOVA test. The GH response to galanin infused 120 minutes after saline overlapped with that induced by the neuropeptide infused following previous GHRH bolus (AUC, mean +/- SEM: 317.3 +/- 73.2 vs 326.8 +/- 54.2 micrograms/l/h). The GH-releasing effect of the second GHRH bolus (126.9 +/- 32.3 micrograms/l/h) was lower than that of the first one (503.4 +/- 41.3 micrograms/l/h; P = 0.0002). Galanin markedly enhanced the GH responses to the second GHRH bolus (1118.0 +/- 212.7 micrograms/l/h; P = 0.0002 vs second GHRH bolus alone) so that it did not significantly differ from the first one (710.9 +/- 107.8 micrograms/l/h). Our results show that the GH-releasing effect of galanin is not modified by GHRH pretreatment and that the neuropeptide reinstates the GH response to the repeated GHRH stimulation in man. They suggest that these effects are due to the inhibition of hypothalamic somatostatin release.


Effects of chronic GnRH analogue administration on gonadotrophin and alpha-subunit secretion in post-menopausal women.

To investigate in detail the regulation of LH, FSH, and alpha-subunit secretion by a GnRH agonist analogue under physiological conditions of gonadotrophin elevation. Six normal healthy post-menopausal women. Subjects were given D-Trp6-Pro9-Net-GnRH (GnRHa), 32 micrograms/kg, subcutaneously, daily for 24 days. On days 1, 2, 3, 4, 7, 11, 14, 17, 21, and 24, blood samples before and after GnRHa injection were taken. Sampling was continued off GnRHa twice a week for 4 weeks and then on days 66, 76, and 98. GnRH tests (100 micrograms i.v.) were performed on days 0, 24, and 98. All serum samples were analysed for LH, FSH, and alpha-subunit levels. LH and FSH levels reached a maximum on day 2 after which there was a steady decline to day 24. LH did not begin to rise again until day 44 (20 days off GnRHa), then rose steadily. FSH began to rise earlier, on day 34 (10 days off GnRHa). alpha-Subunit levels also showed maximum elevation on day 2 but remained equally elevated throughout the period of GnRHa administration and then fell rapidly to baseline by day 34. LH, FSH, and alpha-subunit responses to i.v. GnRH were absent on day 24 and were equivalent to baseline on day 98. We conclude that there is a striking dissociation in the regulation of gonadotrophin and alpha-subunit secretion in response to GnRHa in normal post-menopausal women. Gonadotrophin secretion is profoundly suppressed during GnRHa administration and recovers only after a long delay post-treatment, while alpha-subunit is markedly stimulated and recovers rapidly. The difference between this pattern and that seen in patients with pituitary tumours could be useful for diagnosis.


Joint manifestations in gastrointestinal diseases. 2. Whipple's disease, enteric infections, intestinal bypass operations, gluten-sensitive enteropathy, pseudomembranous colitis and collagenous colitis.

This review addresses the clinical picture of rheumatic diseases seen in Whipple's disease, gluten-sensitive enteropathy, pseudomembranous colitis, collagenous colitis and that developing after enteric infections and intestinal bypass operations for morbid obesity. These disorders exemplify the interplay between antigen entrance through the gastrointestinal canal, specific bacterial properties and genetic host factors such as HLA B27. In most cases such as interplay results in formation of circulating immune complexes causing the development of peripheral joint disease.


Phenotypic analysis of lymphocyte populations in the lungs and regional lymphoid tissue of sheep naturally infected with maedi visna virus.

We have analysed the phenotype of lymphocytes in lung and regional lymph node of symptomatic and asymptomatic sheep infected with the ovine lentivirus, maedi visna virus (MVV). Compared to equivalent tissues from age-matched, non-infected controls, MVV-infected sheep show increased numbers of lymphocytes in the lung, both in the bronchus-associated lymphoid tissue (BALT) and in the alveolar septae. Both CD8+ and CD4+ T lymphocyte numbers in alveolar septae were increased, particularly in animals with clinical respiratory disease. The ratio of CD8+ to CD4+ lymphocytes was similar to that in normal lung. In both MVV-infected and uninfected animals a high proportion of pulmonary lymphocytes, particularly in the alveolar septae, did not express the CD5 antigen, suggesting that they were activated. The number of activated cells was higher in infected sheep. Variable numbers of alveolar macrophages containing MVV-core protein were present in alveolar lumina, the majority of positive cells showing morphological evidence of activation. In regional lymphoid tissue there were increased numbers of CD8+ and gamma delta expressing T cells in lymphoid follicles and germinal centres of infected animals. The specificity of these cells is unknown and we could find no evidence for the presence of cells productively infected with the virus in these structures. This study shows that activated T lymphocytes, particularly of the CD8 subset, play a major part in the pathogenesis of MVV-induced pulmonary and regional lymph node lesions.


Passive immunization against tumour necrosis factor-alpha (TNF-alpha) and IL-1 beta protects from LPS enhancing glomerular injury in nephrotoxic nephritis in rats.

Glomerular injury caused by injection of heterologous anti-glomerular basement membrane antibodies (anti-GBM Ab) is increased in rats pretreated with small doses of bacterial lipopolysaccharide (LPS). We have investigated the involvement of tumour necrosis factor-alpha (TNF-alpha), IL-1 alpha and IL-1 beta in this phenomenon by passive immunization against these cytokines. Anti-TNF-alpha or anti-IL-1 beta antibodies given 1.5 h before the induction of nephritis significantly decreased injury in this model, whether assessed by the magnitude of albuminuria, the prevalence of glomerular capillary thrombi or the intensity of glomerular neutrophil infiltrate. Albuminuria in anti-GBM Ab alone was 11 +/- 3, LPS/anti-GBM Ab 87 +/- 22, and anti-TNF-alpha antibodies/LPS/anti-GBM Ab 21 +/- 6 mg/24 h (mean +/- s.e.) P < 0.05. Passive immunization with antibodies to IL-1 beta had a similar effect (anti-GBM Ab, 0.6 +/- 0.1, LPS/anti-GBM Ab, 92 +/- 19, anti-IL-1 beta antibodies/LPS/anti-GBM Ab 39 +/- 8 mg/24 h, P < 0.05). The prevalence of glomerular capillary thrombi was also reduced significantly by these treatments; from 22 +/- 5% to 4 +/- 1% in the case of anti-TNF-alpha antibodies and 28 +/- 5% to 13 +/- 4% with anti-IL-1 beta antibodies. Similarly, the glomerular neutrophil infiltrate was also reduced by these treatments; from 42 +/- 3 to 25 +/- 1 in the case of anti-TNF-alpha and 47 +/- 2 to 30 +/- 1 with anti-IL-1 beta antibodies. In contrast, passive immunization against IL-1 alpha had no effect on either albumin excretion (4 +/- 3, 83 +/- 22 and 77 +/- 24 mg/24 h), glomerular capillary thrombi (2 +/- 1; 19 +/- 5 and 16 +/- 3) or glomerular neutrophil infiltrate (22 +/- 3; 47 +/- 5 and 48 +/- 5 from the three groups respectively). These results demonstrate that enhanced antibody mediated injury in the kidney is modulated by TNF-alpha and IL-1 beta but not by IL-1 alpha.


Analysis of lymphocyte phenotypes in cord blood from early gestation fetuses.

Using cord blood samples obtained from fetuses between 16 and 40 weeks gestation, we have used a lysed whole blood flow cytometric technique to study the natural history of lymphocyte phenotypes known to be highly represented in cord blood at birth. The majority (51.0 +/- 14.7%) of lymphocytes expressed CD45RA, a marker of 'virgin' cells and there was a correlation between increasing percentages of CD45RA+lymphocytes and gestational age (r = 0.44, P < 0.01). Few cells (8.5 +/- 4.2%) expressed the CD45RO marker of primed lymphocytes and very few (1.0 +/- 0.7%) co-expressed CD45RA and RO, indicating little traffic between the two maturation markers. The percentage of B lymphocytes co-expressing CD5 was high in the fetal circulation (55.5 +/- 10.5%) compared with healthy adults (23.2 +/- 14.3%; P < 0.00001) and the level of CD5+ B cells declined with gestational age in an exponential manner (r = -0.45, P < 0.05). Similarly, levels of T lymphocytes expressing the gamma delta T cell receptor (TCR) declined exponentially (r = -0.59, P < 0.005). These results demonstrate that lymphocytes remain almost entirely unprimed before birth. In addition, CD5+ B lymphocytes and TCR-gamma delta+ T lymphocytes decline exponentially towards birth, in a manner suggesting that they may be seeding peripheral sites such as the spleen, skin and mucosae.


Description of a variably expressed and labile epitope of thyroglobulin and its occurrence in different thyroid diseases using a monoclonal antibody.

A new category of epitopes of human thyroglobulin is being described defined by a monoclonal antibody (TuTg 1). The epitope shows a very variable expression in normal individuals as well as in different thyroid diseases. According to gel filtration data the epitope is located at the intact molecule and is very sensitive to tryptic digestion. The monoclonal antibody was used in an IRMA system for plasma measurements in the follow-up of thyroid cancer patients. In individuals with high epitope expression the sensitivity to detect recurrency during T4 treatment was higher than a sensitive commercial TG IRMA.


Generation of angiotensin II from human plasma by tissue kallikrein.

1. Human plasma was incubated with tissue kallikrein from porcine pancreas, dialysed to obtain a fraction with a molecular mass < 10 kDa and further purified by reverse-phase chromatography. 2. Vasopressor activity in the fractions obtained was tested in the isolated perfused rat kidney. 3. In one fraction a strong vasopressor action was found, which was blocked by saralasin and by an angiotensin II antibody. 4. Aprotinin inhibited the formation of vasopressor substances by tissue kallikrein. 5. U.v.-laser desorption/ionization mass spectrometry revealed a molecular mass of 1046 Da in the purified active fraction. 6. It is concluded that tissue kallikrein forms not only kinins, but also angiotensin II, from human plasma under physiological conditions.


[HIV infection and acquired immunodeficiency syndrome. III].

The present third note describes diagnostic procedures, prognosis, prophylaxis, therapy, and forensic medical problems connected with HIV infection and acquired immunodeficiency syndrome (AIDS). All these are topics of the utmost public health importance which have elicited much interest both among scientists and the general public also during the recent international conference held in Florence last June. HIV infection and the associated syndromes are extremely worrying in view of their increasing diffusion and extreme severity. In spite of the wealth of data already available, as yet we know little about this infection. Besides, all attempts at treatment and organization of prevention have proved ineffective, the latter being rendered vain by the extreme geographic spread of the disease, especially in underdeveloped countries where economic resources and individual and collective public health education are lacking. The fact of having successfully informed the medical profession about these complex problems, even though within the limits of the readers of this journal, is a source of satisfaction to us.


Stimulation of actin and myosin synthesis in rat gastrocnemius muscle by clenbuterol; evidence for translational control.

1. A transient rise in fractional rates of protein and actomyosin synthesis was observed in gastrocnemius muscles of rats fed clenbuterol for 1-2 days but the muscle RNA:protein ratio was unchanged, therefore protein synthesis per unit RNA (kRNA) also increased. 2. Myosin heavy and light chains and actin showed increased incorporation of [3H]phenylalanine at 2 days; these changes were proportional to increases in total protein synthesis. 3. The ratios actin mRNA:18S RNA and fast myosin heavy chain mRNA:18S RNA were unaffected by clenbuterol. 4. The data suggest that the clenbuterol-induced increase in muscle protein synthesis involves both translational control and increased tissue RNA.


Effect of an antithyroid agent (propylthiouracil) and L-ascorbic acid on mixed-function oxidase and drug metabolism in hepatic microsomes of chickens.

1. The experiments were undertaken to determine if an antithyroid agent (propylthiouracil, PTU) and/or ascorbic acid (AA) affect mixed-function oxidase (MFO) in hepatic microsomes of male broiler chicks. 2. Feeding PTU increased the MFO activity in a dose-related manner. Addition of AA to the PTU-containing diet further increased the content of cytochromes P-450 and b5, but not the activities of NADPH-cytochrome c and NADH-cytochrome b5 reductase, and the drug-metabolizing enzymes. 3. Supplemental AA induced cytochrome b5 rather than cytochrome P-450.


Neuronal ubiquitin and neurofilament expression in different lysosomal storage disorders.

We studied various lysosomal storage disorders such as Tay-Sachs' disease, Niemann-Pick's disease, and Hunter's disease for their immunoreactivity with antibodies against ubiquitin (Ub) and neurofilaments (NF). We found that in all cases, irrespective of the nature of the storage material or disorder, only a minor proportion of neurons (20-30% at most), as a rule, moderately reacted with the Ub antibody, while the majority of the distended neurons neither expressed Ub nor NF epitopes. These findings suggest that the UB dependent proteolytic pathway may play a secondary role in the lysosomal storage disorders, at least in the advanced stages which are observed at autopsy. It seems that the Ub expression of a minor proportion of neurons should be regarded as an unspecific epiphenomenon rather than as a mechanism of major significance in the basic metabolism of these disorders, in which the inclusions consist of membrane-bound lipid material.


Quinacrine-staining of neurones, and activity of purine nucleosides and nucleotides in marine and terrestrial invertebrates from several phyla.

1. The acridine derivative quinacrine was used to stain nerve fibres in internal organs of 17 species of marine and terrestrial invertebrates from seven different phyla. 2. The majority of preparations showed quinacrine-stained nerve fibres. There was a degree of variation, ranging from a dense network of nerve bundles to single fibres. Quinacrine-positive nerve cell bodies were also observed in some ganglia. 3. Pharmacological experiments were performed on a variety of isolated tissues from the different marine and terrestrial groups, in order to ascertain their sensitivity to adenine nucleosides and nucleotides. 4. Correlation is drawn between the ability of neurones within a tissue to bind quinacrine, and the ability of that tissue to respond to applied adenine nucleosides and nucleotides.


Congestive heart failure: survival trial of antiarrhythmic therapy (CHF STAT). The CHF STAT Investigators.

This study is a prospective, double-masked, randomized, clinical trial to determine the effect of anti-arrhythmic drug therapy on mortality in patients with congestive heart failure and ventricular arrhythmia. Patients will be assigned to receive either amiodarone or placebo. Eligible patients include those with ischemic and nonischemic congestive heart failure (New York Heart Association class III or VI) and with 10 or more ventricular premature beats per hour. All patients must have shortness of breath with minimal exertion or paroxysmal nocturnal dyspnea, a left ventricular internal dimension (LVIDd) by echocardiogram of 55 mm or greater (> or = 55 mm) or a CT ratio of greater than 0.5, and an ejection fraction of 40% of less. Patients will be entered into the study for 2.5 years and followed for an additional 2 years. Drug therapy will be continued for all patients throughout the entire study unless adverse reactions occur that necessitate individualized treatment. The expectation is that 674 patients are to be entered into the study from 25 participating centers. This sample size will allow for the detection of a 33% decrease in 2-year mortality (20% vs. 30%) in the treated patients as compared to those in the placebo group with a power of 0.90 and a two-sided alpha level of 0.05. Intermittent Holter monitoring, radionuclide ventriculograms, pulmonary function tests, echocardiograms, and blood tests, including arterial blood gases, will be required for each patient. The study analysis will address differences in total mortality, cardiac mortality, and sudden cardiac death between patients receiving anti-arrhythmic drug therapy and those receiving placebo. Other factors to be examined include the effects of antiarrhythmic therapy on suppression of arrhythmias, on ejection fraction, and relation of ischemic events to mortality.


Tumor fixation of bleomycin labeled with 57 cobalt before and after cryotherapy of bronchial carcinoma.

The combined effect of cryotherapy and chemotherapy was studied in 12 patients with bronchial carcinoma. Radiolabeled (57 Co) Bleomycin (BLM) was injected intravenously and initial detection was carried out with a gamma-camera. Plasma half-life and clearance of 57 Co-BLM, as well as tumor/normal tissues ratios were calculated. The same protocol was performed 15 days later immediately after cryotherapy. A mean increase of 30% of radiolabeled BLM was found in the tumor area after cryotherapy, and pharmacokinetic data were significantly different after cryotherapy. The vascular component of cryodestruction offers an explanation for these results, with trapping of the anticancer drug in the tumor and immediately surrounding area due to vascular stasis. It seems that chemotherapy may be more effective after cryotherapy, and a multicenter study is in progress to evaluate the association of cryo-chemotherapy in France.


Factors modulating the effect of retinoids on cultured retinal pigment epithelial cell proliferation.

The effect of several naturally-occurring retinoids and 13-cis-retinoic acid on the proliferation of cultured bovine retinal pigment epithelial (RPE) cells was investigated. None of the retinoids tested were toxic to the cultures and all, except retinylpalmitate, inhibited cell proliferation when given for more than 3 days. The relative potencies of the retinoids were; all-trans-retinoic acid greater than 13-cis-retinoic acid greater than all-trans-retinol approximately equal to all-trans-retinaldehyde. Uptake of retinoic acid by cultured RPE cells was 10-fold less than the uptake of retinol. Although retinoic acid-treated cultures showed strong density-dependent growth inhibition, cellular proliferation was inhibited more in sparse cultures than in dense ones. Retinoic acid did not significantly inhibit the proliferation of first passage bovine or rabbit RPE cells, but partially inhibited the proliferation of first passage human RPE cells. The sensitivity of all these cultures to growth inhibition by retinoic acid increased in subsequent subcultures, yet there was no effect of passage number on retinoic acid uptake. This study demonstrates that RPE cell proliferation can be inhibited by retinoic acid but the sensitivity of these cells to the retinoid's effects are modulated by incubation time, in vitro aging, and cell density.


Expression of K12 keratin in alkali-burned rabbit corneas.

The healing of alkali-injured corneas is characterized by the persistence of polymorphonuclear leukocytes (PMN) in tissues and recurrent corneal epithelial defects. It has been suggested that the proteolytic enzymes secreted by PMN may account in part for the recurrent epithelial defects in the alkali-burned corneas. Cytoplasmic keratins, which form intracellular intermediate filaments, participate in the formation of hemidesmosomes and play a key role in the focal adhesion of epithelial cells to the basement membranes. The K3/K12 keratin pair is a major constituent of differentiated and stratified corneal epithelium. We have recently cloned the cDNA encoding the rabbit K12 keratin. In the present study we examined the expression of K12 keratin during the healing of alkali-burned rabbit corneas by slot-blot and in situ hybridization. Our results indicate that in normal cornea K12 keratin is equally expressed in all cell layers of stratified corneal epithelium and suprabasal layers of limbal epithelium, but not in bulbar conjunctival and other epithelia, i.e., lens, iris, and retinal pigment epithelium. The basal cells of the detached regenerating epithelium of the injured cornea express a very low level of K12 keratin. These observations are consistent with the notion that defective expression of K3/K12 keratins may play a role in the abnormal attachment of the regenerating epithelium to the basement membrane.


Rabbits have a circadian rhythm of aqueous humor cyclic AMP.

Rabbits have circadian rhythms of intraocular pressure (IOP) and aqueous flow. The dark phase increases of IOP and flow are associated with increased aqueous cyclic AMP. If the daily rhythm of aqueous cyclic AMP reflects changes of tissue levels of cyclic AMP which mediate one of the mechanisms which control the rhythms of IOP and aqueous flow, then it must be a circadian rhythm as are the rhythms of IOP and flow. A recent report showed no change in aqueous cyclic AMP from 2 hrs before to 2 hrs after the beginning of subjective dark in animals housed in constant dark. Because the increase of intraocular pressure at the same times did persist in constant dark, the authors concluded that a significant portion of the circadian rhythm of IOP is unrelated to cyclic AMP mediated ocular mechanisms. However, if an inhibitor of cyclic nucleotide phosphodiesterase is added to aqueous samples after harvesting by paracentesis, it is possible to show that most of the increase of aqueous cyclic AMP from mid-light phase to mid-dark phase persists in constant dark and is therefore likely to reflect a circadian rhythm of tissue cyclic AMP.


Humoral immune response against the S-antigen/TNF alpha common epitope in rat EAU suppressed by the monoclonal antibody S2D2.

S-antigen (S-Ag)-induced experimental autoimmune uveoretinitis (EAU) in rats can be suppressed by injecting the mouse monoclonal antibody (mAb) S2D2 or a polyclonal rat anti-idiotype S2D2 (anti-Id S2D2) antibody, the internal image of the epitope of S-Ag recognized by mAb S2D2. This epitope located in amino acids 40-50 of bovine S-Ag (peptide S2), displays an homology with a sequence of human tumor necrosis factor alpha (hTNF alpha) (peptide RRAN) which is also recognized by S2D2. (Stiemer et al., this symposium). We show that one injection of S2D2 at the time of immunization with S-Ag suppressed EAU and modulated the production of antibodies against peptides of bovine or human S-Ag containing the S2 epitope and against peptide RRAN. Immunization against anti-Id S2D2 stimulated antibody production to peptide S2 and RRAN and inhibited EAU. These data suggest that disease suppression could be related to the production of antibodies against the S-Ag/TNF alpha common epitope.


Molecular aspects of autoimmunity: a review.

Molecular genetic techniques are being widely applied to the study of autoimmune diseases. Major advances have been made in diabetes, rheumatoid arthritis and coeliac disease. Work on experimental models of autoimmune uveitis suggests that similar advances will follow in this field. The application of molecular genetics to the study of immunology has lead to great advances in our understanding of the anatomy of antigen recognition. This work has lead to the identification of some of the structural determinants of antigen binding by MHC molecules and is helping to explain some MHC-disease associations. More recently, molecular studies of the T cell receptor have characterized patterns of T cell receptor expression in humans and have lead to the identification of regions of the T cell receptor critical for antigen recognition. These techniques will hopefully provide insights into the nature of autoimmunity and permit the identification of targets for disease specific immunotherapies. This review describes attempts to corelate MHC structure and function in the context of autoimmunity and discusses some of the strategies for analyzing T cell receptor usage in autoimmune disease.


Cytokine induction by immunomodulatory epitopes in S-antigen and tumor necrosis factor alpha.

Common epitopes on S-antigen (arrestin), a potent autoantigen inducing experimental autoimmune uveoretinitis (EAU), and on human tumor necrosis factor alpha (hTNF alpha) are revealed with monoclonal antibodies (mAb) to S-antigen, which inhibit EAU induction. The minimal common sequence for mAb recognition is GVxLxD in the S-antigen/hTNF alpha amino acid (aa) sequences. Peptides containing this sequence motif exhibit monocyte activating capacity analogous to the autocrine stimulatory capacity of hTNF alpha itself. In S-antigen this activity is located at epitope S2 (aa residues 40 to 50), corresponding to the peptide PVDGVVLVDPE (peptide S2). In hTNF alpha the monocyte activating capacity correlates to aa residue 31 to 53, corresponding to the peptide RRANALLANGVELRDNQLVVPSE (peptide RRAN). Peptide S2 but not peptide RRAN is competing for mAbs S6H8 and S2D2 binding to S-antigen. Anti-idiotypic antibodies to S2D2 compete with peptide S2 but not peptide RRAN for binding to mAbs S2D2 and S6H8. In human retinal S-antigen epitope S2 is localized at the aa residues 44-54 and is cleaved in the human peptide 4 (aa 31-50). Competition experiments with peptide 4 (aa 31-50) and peptide 5 (aa 41-60) indicate that the C-terminal aa residues VDPD in the epitope S2 play an important role for internal image recognition of the anti-idiotypic antibodies. Peptide S2 and peptide RRAN define common functional structures in the autoantigen and hTNF alpha molecules. The data suggest regulatory functions of the peptides in cytokine expression, network regulation and in autoimmunity.


Unresponsiveness to self-peptides of S-antigen in EAU: an overview of recent results.

Several observations in the characterization of EAU are examined. First, sequences of heterologous S-Ag (bovine S-Ag in LEW rats) which induce strong in vitro T cell proliferative responses are dissociated from sequences which induce EAU. Strong in vitro responses were detected only to nonself peptide homologues. Second, T cells specific for self-sequences of S-Ag are unresponsive in vitro. Third, TCR V beta 8 gene usage is associated with pathogenic T cells. V beta 8.2 bearing hybridomas from a pathogenic line exhibited enhanced reactivity to pathogenic self-peptides, but were unresponsive unless presented Ag on nonirradiated, splenic APC. We propose that these findings reflect self, nonself discrimination of the epitopes on heterologous autoantigen, and examine the hypothesis that TCR containing V beta 8 have enhanced avidity for MHC complexed with autologous sequences, but that these V beta 8 autoreactive T cells appear unresponsive in vitro due to mechanisms of self-tolerance involving superantigen/coligand participation.


Preclinical and clinical study of FK506 in uveitis.

Efficacy of a new immunosuppressive agent, FK506, in refractory uveitis was studied in 8 patients: 5 with Behcet's disease and 3 with idiopathic retinal vasculitis. The agent was given by oral administration every 12 hours. The previous therapy with systemic corticosteroids or immunosuppressive agents including cyclosporine failed to subside uveitis in these cases. During the observation period of 21.6 +/- 7.8 weeks (mean +/- SD) under FK506 at doses with 0.05, 0.1, 0.15 or 0.2 mg/kg/day, the visual acuity was increased in 44% of treated eyes, unchanged in 44% and decreased in 12%. The inflammatory activity in the ocular fundus was improved in 69% and unchanged in 6% of treated eyes. The effects of FK506 on uveitis by the criteria of improvement of visual acuity and uveitis activity was dose-dependent: 0.05 and 0.1 mg/kg/day were ineffective but 0.15 and 0.2 mg/kg/day were effective in most cases. One patient with Behcet's disease converted from cyclosporine developed moderate renal impairment in 4 weeks under FK506 and the therapy was discontinued in 8 weeks, though the uveitis activity as well as visual acuity was markedly improved. Other 7 cases had no side effect of FK506. Although the number of cases was small and observation period was short, the present clinical data indicate that FK506 is effective to treat refractory uveitis.


Lipopolysaccharide receptors and signal transduction pathways in mononuclear phagocytes.

There is little question but that bacterial lipopolysaccharides (LPS) remain one of the most potent stimuli which can affect macrophage activation. Although the precise biochemical mechanisms responsible for this remain to be fully defined, there is now evidence accumulating from a number of laboratories that functional receptors for these bacterial products do exist and may contribute to the initial triggering event. Unfortunately, there is currently no consensus as to which of the candidate receptors identified to date serves as the primary binding target for LPS, and it is possible that the difference in macrophage cell types, LPS probes, and detection systems will all influence the nature of the binding. At the present time, therefore, macromolecules of 96-kDa, 95-kDa (adhesion beta chain), 80-kDa, 65-kDa, and 55-kDa may be considered as possible LPS targets. With the exception of the 96-kDa protein identified by Hampton and his co-workers, there exists some experimental evidence for a functional role for each of the molecules so far identified. It is apparent that the molecular cloning and sequencing and subsequent biochemical characterization of these LPS receptors will be required to determine unequivocally their role in LPS-mediated triggering events. Such information will be invaluable in sorting out the relevant biochemical second signals involved in macrophage activation. Although much new information has recently been accumulated on potential signaling pathways for LPS, the definitive events remain far from unequivocally established. In view of the obvious importance of LPS-macrophage interactions in the overall capacity of the mammalian host to respond appropriately to the potentially hostile prokaryotic environment, a precise delineation of LPS-mediated macrophage activation is critical to our understanding of this important inflammatory mediator cell.


Late-effects after treatment for germ-cell cancer with cisplatin, vinblastine, and bleomycin.

During a 5-year period from 1979 to 1983 all patients in Denmark with metastatic non-seminomatous and extragonadal germ cell cancer were treated with 6 cycles of cisplatin, vinblastine, and bleomycin (PVB). Thirty-nine patients referred to the Finsen Institute accepted a follow-up examination of side-effects 3.5-9 years after chemotherapy. Renal toxicity consisted of an irreversible decrease in glomerular filtration rate (GFR) in 47% of the patients, while the decrease in GFR was fully reversible in 23%. Significant pulmonary toxicity was observed in smokers and consisted of an irreversible decrease in carbon monoxide diffusion capacity to median 72% of the predicted value. Neurotoxicity was the most pronounced long-term side-effect. Nearly all patients had a peripheral sensory neuropathy probably caused by axonal degeneration. A central conduction defect was observed in 88% of the patients by measuring auditory brain-stem potentials. Irreversible high-frequency hearing loss was induced in 39% of the patients. Parasympathetic nerve dysfunction was found in 36% of the examined patients, including 2 patients with impotence. Half of the patients revealed Raynaud's phenomenon (RP), and the mechanism underlying this side-effect was found to be hyperreactivity of the central sympathetic nervous system. Vascular toxicity was found only in terminal arterioles and was not responsible for RP. PVB treatment caused low sperm counts and a subclinical Leydig cell dysfunction in the majority of patients. Azoospermia was observed in 27% of the patients. Six patients had hypertension and this was not related to renal impairment.(ABSTRACT TRUNCATED AT 250 WORDS)


A prospective study of amylase-rich pleural effusions with special reference to amylase isoenzyme analysis.

By analysis of pleural effusions from 200 patients, 25 cases of amylase-rich effusions were identified, for an overall incidence of 13 percent. Four of the 25 patients (16 percent) had evidence of pancreatitis. These patients had higher mean ratios of pleural fluid to serum amylase levels (18 +/- 6.3 [SEM] vs 4.8 +/- 1.3) compared to patients with nonpancreatic diseases (p = 0.003); all four exhibited a predominant pancreatic isoenzyme profile. Of the 21 patients with nonpancreatic amylase-rich effusions, lung cancer was the most commonly associated condition (8 patients). In 14 of the 21 patients in whom an isoenzyme profile was obtained, salivary-type amylase was predominant. Amylase-rich pleural effusions occur frequently, and pleural fluid isoamylase determination is specific for pancreatitis-associated effusions. The finding of a pleural effusion rich in salivary isoamylase should prompt an evaluation for carcinoma (particularly of lung primary), but may also be seen in other pleural inflammatory conditions.


Proteinase/proteinase inhibitor imbalance in sputum sol phases from patients with chronic obstructive pulmonary disease. Suggestions for a key role played by antileukoprotease.

In order to characterize the imbalance between proteinases and proteinase inhibitors in sputum sol phases, we studied 25 patients (mean age, 59 +/- 11 yr) with exacerbated chronic obstructive pulmonary disease (COPD). An aliquot of sputum was used for bacteriologic determinations, and the remainder was centrifuged in order to obtain gel and sol phases. On the basis of the bacteriologic data, patients were divided into colonized patients (14) and noncolonized patients (11). All of the major inhibitors were immunologically detectable in sol phases without a significant difference between colonized and noncolonized patients (alpha 1-proteinase inhibitor [alpha 1-PI], 2.56 microM +/- 0.53 microM and 2.39 microM +/- 0.72 microM; alpha 2-macroglobulin [alpha 2-MG], 0.21 microM +/- 0.07 microM and 0.16 microM +/- 0.05 microM; antileukoprotease (ALP), 1.78 microM +/- 0.57 microM and 1.53 microM +/- 0.6 microM, respectively [mean +/- SE]). With regard to proteinase activities, both free elastase-like and free chymotrypsin-like activities were detectable in the majority of patients (15/25) (0.59 microM +/- 0.15 microM and 0.74 microM +/- 0.15 microM for elastase-like activity [ELA], and 0.010 microM +/- 0.003 microM and 0.017 microM +/- 0.007 microM for chymotrypsin-like activity [CLA], respectively [mean +/- SE]). The inhibitory profile of proteinase activities, performed by means of a panel of inhibitors, allowed us to assign specific activities mainly to neutrophil elastase and cathepsin G (Cat G). Next we looked at the relationships between inhibitors and proteinase activities. We found a significant negative correlation between neutrophil elastase activity and ALP (r = -0.58; p < 0.01). In confirmation of this suggestion, sol phases were divided into samples (15) with detectable ELA (> 0.50 microM) and samples (10) with no detectable ELA (< 0.18 microM). Levels of alpha 1-PI and alpha 2-MG did not differ significantly between the two groups, whereas ALP values were higher in the group with no detectable ELA (3.12 microM +/- 0.69 microM) than in the other group (0.58 microM +/- 0.21 microM; p < 0.001). We conclude that most sputum sol phases from patients with exacerbated COPD have a high burden of free neutrophil elastase and Cat G. Antileukoprotease seems to be the major naturally occurring inhibitor effective in the modulation of proteinase activities in bronchial secretions under these conditions.


Hoechst 33258, distamycin A, and high mobility group protein I (HMG-I) compete for binding to mouse satellite DNA.

The experiments described were designed to test the hypothesis that the (A+T)-specific DNA binding ligands Hoechst 33258 and distamycin A affect the condensation of mouse centromeric heterochromatin by competing for binding to satellite DNA with one or more chromosomal proteins. The studies focused on the nonhistone chromosomal protein HMG-I since its binding properties predict it would be a target for competition. Gel mobility shift assays show that HMG-I forms specific complexes with satellite DNA and that the formation of these complexes is competed for by both Hoechst and distamycin. In addition, methidium propyl EDTA Fe(II) [MPE Fe(II)] footprints of ligand-satellite DNA complexes showed essentially the same protection pattern for both drugs and a similar, but not identical, HMG-I footprint. If these in vitro results reflect the in vivo situation then the incomplete condensation of centromeric heterochromatin observed when mouse cells are grown in the presence of either chemical ligand could be a consequence of competition for binding of HMG-I (and possibly other proteins) to satellite DNA.


Regulation of expression of the interleukin 6 gene: structure and function of the transcription factor NF-IL6.

The interleukin 6 (IL-6) promoter is rapidly and transiently activated by other cytokines, including IL-1 and tumour necrosis factor (TNF), as well as by phorbol esters and cyclic AMP agonists. Studies using promoter mutants suggested that an IL-1-responsive element mapped within the -180 to -123 region of the IL-6 promoter. A nuclear factor (NF-IL6) that recognized a unique sequence containing an inverted repeat, ACATTGCACAATCT, was identified within the region. Direct cloning of the human NF-IL6 revealed its similarity to C/EBP, a liver- and adipose tissue-specific transcription factor. C/EBP and NF-IL6 recognize the same nucleotide sequence, but exhibit distinct patterns of expression. NF-IL6 is expressed at a low level in normal tissues, but is rapidly and drastically induced by bacterial lipopolysaccharide (LPS) or inflammatory cytokines such as IL-1, TNF and IL-6. Recently, NF-IL6 has been shown to be identical to IL-6DBP, the DNA-binding protein which is responsible for IL-6-mediated induction of several acute-phase proteins. Evidence that NF-IL6 DNA-binding activity is increased after IL-6 stimulation without increased NF-IL6 protein synthesis demonstrates the importance of post-translational modification. There are some results indicating that phosphorylation is involved in transcriptional and binding activities of NF-IL6. Taken together, these findings indicate that NF-IL6 may be an important transcription factor on the signal transduction pathways of IL-1 and IL-6.


Peptidergic neurotransmitters in the endocrine hypothalamus.

More than 20 neuropeptides have been localized in the endocrine hypothalamus. They may exert a neurohormonal effect on the pituitary or innervate other neurons (intranuclear, intrahypothalamic or extrahypothalamic) and act as neurotransmitters. Many of the hypothalamic neuropeptides are synthesized as inactive precursors that are activated by proteolysis during axonal transport from the cell body to the synapse. Studies in which the paraventricular nuclei were bilaterally destroyed have shown that the neuroendocrine cells in the hypothalamus show functional plasticity and cells that do not usually make detectable quantities of a particular neuropeptide may be activated to do so. Within the hypothalamic nuclei are dense networks of synaptic connections among neurons synthesizing the same or different neuropeptides. These local circuits may coordinate the activities of peptidergic neurons in a hypothalamic nucleus. Hypothalamic neurons project axons to the median eminence-pituitary stalk and the posterior pituitary, also to nuclei within the hypothalamus and to extrahypothalamic areas such as the lower brainstem. Peptidergic neurons in the hypothalamus can have combined neurohormonal and neurotransmitter activities mediated by axon terminals on portal capillaries and other hypothalamic nuclei. Double labelling immunohistochemistry has been used to demonstrate reciprocal connections between peptidergic neurons in the hypothalamus, such as those synthesizing growth hormone-releasing hormone and somatostatin.


Determination of tumor marker levels in cystic fluid of benign liver cysts.

The levels of tumor markers in cystic fluid and serum were measured in six patients with benign biliary cyst of the liver. AFP in the cystic fluid was lower than the upper normal limit for serum in all cases, and CEA in the cystic fluid was higher than the upper normal limit for serum in one of the six cases. CA19-9, DU-PAN 2, and SPAN 1 in cystic fluid were much higher than the upper normal limit for serum in all cases (more than 100-fold for CA19-9, twofold for DU-PAN 2, and ninefold for SPAN 1). CA19-9, DU-PAN 2, and SPAN 1 in cystic fluid were significantly higher than the levels in the corresponding serum. Positive immunohistochemical staining against CA19-9, DU-PAN 2, and SPAN 1 was observed in the cytoplasm of the epithelial cells of the cyst wall. These results suggested that the high concentrations of CA19-9, DU-PAN 2, and SPAN 1 in the cystic fluid were due to secretion from the epithelial cells in the benign biliary cysts.


Substance P, vasoactive intestinal polypeptide, and gastrin catabolism in canine liver and kidney.

No reports have described the catabolic mechanism of substance P in vivo. We studied the effects of hepatic or renal transit on substance P, vasoactive intestinal polypeptide, and gastrin in anesthetized dogs. It was found that the liver plays a more important role in vasoactive intestinal polypeptide catabolism than the kidney and that the kidney is more important in gastrin catabolism than the liver. Substance P was more rapidly degraded than the other two peptides in both organs. The transrenal substance P loss measured by C-terminal antiserum differed from that measured by N-terminal antiserum, although there was no difference in the liver. This suggested that there were different patterns of cleavage of substance P between the liver and the kidney, and that its C terminal was degraded more strongly than its N terminal in the kidney.


[Metal endoprostheses for endoscopic therapy in malignant bronchial tumors].

Self-expanding metal stents were implanted in the trachea or main bronchus in 12 patients (eleven men, one woman; mean age 60 +/- 8 years) with nonresectable bronchial carcinoma (n = 11) or tracheal metastasis of a hypernephroma (n = 1). They all had pulmonary complications caused by tumour stenoses (group I: severe dyspnoea [n = 6], group II: retention pneumonia [n = 4] or lung abscess [n = 2] after unsuccessful antibiotic treatment). The procedure was undertaken after local anaesthesia with a flexible bronchoscope (in the first three cases still with a rigid bronchoscope under general anaesthesia) under fluoroscopic control. Immediate reduction in dyspnoea occurred in five of the six patients in group I. In five of the six patients in group II antibiotics cured the infection after stent placement. The therapeutic effect was immediate in severe dyspnoea and retention of secretions. The clinical improvement lasted longer in patients with abscess and retention pneumonia than those with dyspnoea (41 +/- 16 vs. 26 +/- 10 days). If strict indications are observed in cases with malignant bronchial stenosis, implantation of self-expanding stents provides rapidly effective, well-tolerated palliation.


[The content of DNA, RNA and wet weight and the RNA:DNA and wet weight:DNA ratio in different tissues during the growth of chickens. 1. Analysis of the brain, the myocardium, the lungs and the liver].

At each 8 chickens of the race "white leghorn" analyses of the body weight as well of the wet weight and the concentration of DNA and of RNA in different tissues from the 2nd to the 203rd day after hatching were performed. On the 2nd day after hatching the DNA-concentration in the brain amounted to 1.50 +/- 0.12, in the heart-muscle to 2.86 +/- 0.19, in the lung to 7.23 +/- 0.19 and in the liver to 2.86 +/- 0.20 mg/g wet weight. The highest content of nuclei in the brain of 1.38 x 10(9) was estimated on the 56th, in the heart-muscle of 1.94 x 10(9) on the 168th day, in the lung of 16.86 x 10(9) on the 112th day and in the liver of 69.81 x 10(9) on the 203rd day. Further the RNA:DNA- and the wet weight:DNA-ratio of the different tissues was calculated.


Differential expression of acidic cytokeratins 18 and 19 during sexual differentiation of the rat gonad.

The expression of cytokeratins (CKs) 8, 18 and 19 was analyzed in male and female rat gonads from the undifferentiated stage (12.5 days of gestation) until two weeks after birth by indirect immunofluorescence, using specific monoclonal antibodies anti-CK 8 (LE41), anti-CK 19 (LP2K) and anti-CK 18 (LE65 and RGE53). In the undifferentiated blastema, the somatic cells were stained for CK 8 and CK 19, whereas no detectable immunoreactivity for CK 18 was obtained. The same staining CK pattern was observed in ovaries, in the somatic cells of ovigerous cords and in primary follicles. The staining was progressively decreasing in growing follicles after one week after birth. At the onset of testicular differentiation, when the first Sertoli cells differentiate in the gonad of 13.5-day old male fetuses, positive staining for CK 18 became evident, in addition to CK 8 and CK 19 expression. In the following days, CK 8, CK 18 and CK 19 were detected in Sertoli cells in the differentiating seminiferous cords, but progressively the reactivity for CK 19 decreased and was no longer observed after 18.5-19.5 days of gestation. In all cases, CKs were found to be coexpressed with vimentin, and germ cells were negative for both vimentin and CKs. The results reported here show first, that CKs are expressed before sexual differentiation in gonadal blastema in which no epithelial organization is observed, and second, that there is a CK 18/CK 19 shift in expression during morphogenesis of the testis which is not observed in the differentiating ovary. Future studies will have to determine whether these differences in CK expression are due to epitope-masking phenomena or to the regulation of CK synthesis.


Characterization of HNK-1 antigens during the formation of the avian enteric nervous system.

During vertebrate embryogenesis, interaction between neural crest cells and the enteric mesenchyme gives rise to the development of the enteric nervous system. In birds, monoclonal antibody HNK-1 is a marker for neural crest cells from the entire rostrocaudal axis. In this study, we aimed to characterize the HNK-1 carrying cells and antigen(s) during the formation of the enteric nervous system in the hindgut. Immunohistological findings showed that HNK-1-positive mesenchymal cells are present in the gut prior to neural crest cell colonization. After neural crest cell colonization this cell type cannot be visualized anymore with the HNK-1 antibody. We characterized the HNK-1 antigens that are present before and after neural crest cell colonization of the hindgut. Immunoblot analysis of plasma membranes from embryonic hindgut revealed a wide array of HNK-1-carrying glycoproteins. We found that two HNK-1 antigens are present in E4 hindgut prior to neural crest cell colonization and that the expression of these antigens disappears after neural crest colonization. These two membrane glycoproteins, G-42 and G-44, have relative molecular masses of 42,000 and 44,000, respectively, and they both have isoelectric points of 5.5 under reducing conditions. We suggest that these HNK-1 antigens and the HNK-1-positive mesenchymal cells have some role in the formation of the enteric nervous system.


Cytochrome P-450 3A enzymes are responsible for biotransformation of FK506 and rapamycin in man and rat.

The hepatic cytochrome P-450 responsible for metabolism of the structurally related macrolides FK506 and rapamycin in humans was identified using in vitro studies. FK506 and rapamycin metabolism was significantly correlated with nifedipine oxidation in human liver microsomes of eight different individuals. Immunoinhibition with anti-P450 3A4 abolished almost all FK506 and rapamycin metabolite formation. Inactivation of P450 3A4 by incubation of human liver microsomes with triacetyl oleandomycin (50 microM) or gestodene (10 microM) inhibited metabolism of FK506 and rapamycin. In liver microsomes from dexamethasone-treated rats FK506 and rapamycin metabolism was increased compared to liver microsomes from uninduced, phenobarbital-, or 3-methylcholanthrene-induced rats. FK506 and rapamycin were metabolized by reconstituted recombinant human liver P450 3A4. It is concluded that in human and rat liver FK506 and rapamycin are metabolized primarily by cytochrome P-450 3A4.


Beta 1-integrin is a maternal protein that is inserted into all newly formed plasma membranes during early Xenopus embryogenesis.

A monoclonal antibody (mAb 8C8) that recognizes the Xenopus beta 1-integrin chain was used to study the appearance, synthesis and distribution of this integrin subunit during the early development of Xenopus. Both the precursor and the mature form of beta 1-integrin are provided maternally. They do not increase significantly in amount until early gastrula when the level of both forms begins to rise gradually. Synthesis of beta 1-integrin from maternal mRNA is observed throughout the pregastrula phase, though it seems to add only little to the total beta 1-integrin of the embryo. Until late blastula only small amounts of precursor are processed into the mature form. Starting with the formation of the first cleavage membrane, mature beta 1-integrin is inserted into the newly formed plasma membranes of all cells. The membrane domains forming the outer surface of the embryo remain devoid of the antigen. The data suggest an as yet unknown function of beta 1-integrin during the cleavage phase.


IGF binding protein-2 gene expression and the location of IGF-I and IGF-II in fetal rat lung.

Binding proteins for the insulin-like growth factors (IGF-BPs) are important modulators of the biological actions of IGF-I and IGF-II. The generation of IGFBPs within developing organs, and their spatial arrangement, may similarly determine IGF action at specific microanatomical sites. In situ hybridization studies with late gestation (days 16, 18 and 20) fetal rat lung using a cDNA probe for IGFBP-2 showed strong gene expression in the fetal lung epithelial structures (alveoli and airways). The sites of IGFBP-2 gene expression were associated with immunoreactive IGF-II at the apical surface of the epithelium. By day 20, there was also some IGFBP-2 gene expression and immunoreactive IGF-II at discrete sites in the mesenchyme. In contrast, immunoreactive IGF-I was found predominantly distributed in a punctate pattern, consistent with its presence in the lumen or walls of small vessels or capillaries, and in a granular, intracellular form in both epithelial and mesenchymal cells. These studies suggest that endogenously generated IGFBP-2 may determine the distribution of IGF-II, principally at the apical surface of lung epithelia. IGF-I does not colocalise with IGF-II peptide or the sites of IGFBP-2 gene expression. We conclude that the spatial distributions of these two related growth factors are separately controlled, to some extent by endogenously generated binding proteins.


Mechanisms and consequences of portal hypertension.

Portal hypertension is characterised by alterations in the splanchnic and systemic circulation resulting in the development of portosystemic collateral channels, the most important of which are found in the lower oesophagus and stomach. The major clinical complication of gastro-oesophageal varices is bleeding and over the last decade there has been considerable interest in the pharmacological management of this complication. The factors currently implicated in the development of gastro-oesophageal varices in patients with cirrhosis include a) increased portal vascular resistance, b) splanchnic and systemic vasodilatation and c) changes in the lower oesophageal venous anatomy [palisade and perforating venous zones]. In a patient with gastrointestinal bleeding, endoscopic examination of the upper gastrointestinal tract will confirm the diagnosis of portal hypertension by confirming the presence of gastro-oesophageal varices. Cirrhosis is the most common aetiological factor for gastro-oesophageal varices, but imaging techniques, including Doppler ultrasound, computerised tomography and venous phase angiography, may be required to exclude extrahepatic portal venous obstruction from the differential diagnosis. Although the pathogenesis of variceal rupture remains unclear, several risk factors for variceal haemorrhage have been identified, including a) increased size, b) high intravariceal-portal pressure, c) increased varix wall tension characterised by the presence of red spots observed at endoscopy (particularly in large varices since wall tension is related to variceal size), and d) poor liver function. Although oesophagitis may be observed at endoscopy, an erosive mechanism is no longer considered to be of pathogenic significance. A high portal pressure in the immediate postbleeding period is now recognised as predictive of rebleeding. Periodic elevations in intravariceal pressure, associated with the release of enhanced endogenous vasoactive compounds, or beta-adrenergic-mediated stress-related increases in portal pressure, may contribute to the rupture mechanism. Consequently, portal hypertension is now being more widely considered as a multiorgan disorder associated with changes in blood flow within both systemic and splanchnic vascular beds. This article reviews the factors currently implicated in the development of portal hypertension and the approach to diagnosis. The pathogenesis of variceal bleeding will also be considered.