The physiology of GABAB receptors in the vertebrate retina. Writing a chapter on retinal GABAB receptors is premature, as evidenced by the paucity of citations more than two years old. Despite that, this area of retinal pharmacology has made significant strides and, although it is a story without an ending, it has had an exciting beginning. To date, the experiments indicate that horizontal cell feedback to cones is mediated, at least in part, by the GABAB receptor system which probably regulates a potassium conductance. In the inner retina, GABAB receptors are found on bipolar cells, amacrines, and ganglion cells. Here, the actions are a subtle regulation of channel conductance, but the effects are a dramatic reorganization of a fundamental coding property of the retina, namely the distinction between tonic and phasic responses to light. In both the distal and proximal retina, the GABAB receptor does not appear to work alone, but instead works in concert with the GABAA receptor. The full significance of these interactions has yet to be determined. Although the discovery of the GABAB receptor has led to the resolution of several retinal mysteries, the case is far from closed. At this juncture, what can be said is that the GABAB receptor represents a unique and ubiquitous system that reveals the power of regulating calcium and potassium conductances, as opposed to the more familiar properties of the glutamate/acetylcholine regulated cationic conductances or the GABAA/glycine controlled chloride channels.


Dexamethasone inhibition of cyclooxygenase expression in bovine term placenta. Since both prostaglandin (PG) F2 alpha and corticosteroids are elevated in mammals before the onset of parturition, we studied the effect of the synthetic corticosteroid dexamethasone on PGF2 alpha accumulation and cyclooxygenase (prostaglandin synthase, PGS) expression in the bovine fetal placenta. Cultures were prepared from cotyledons at different stages of gestation. The effect of dexamethasone on PGF2 alpha accumulation and PGS expression was determined by radioimmunoassay and [35S]methionine metabolic labeling followed by immunoprecipitation with specific anti-cyclooxygenase antibodies, respectively. Data demonstrate that in fetal placental cells at term, both PGF2 alpha accumulation and cyclooxygenase expression are significantly inhibited after 18 hours of dexamethasone treatment (150 nM). In contrast, neither first nor second trimester cells were sensitive to dexamethasone treatment. Dexamethasone inhibition of PGF2 alpha synthesis in fetal cells at term was abolished in the presence of RNA or protein synthesis inhibitors (actinomycin D or puromycin, 10 micrograms/ml each). Neither progesterone nor 17 beta-estradiol accumulation were affected by dexamethasone treatment at any stage of gestation. Data suggest that corticosteroids play a role in parturition through PGF2 alpha synthesis regulation by fetal placental cells. Since abnormalities during parturition e.g. retained placenta, are common following dexamethasone induction of labor in cows, we postulate that the local inhibition of PGF2 alpha accumulation by cotyledon cells after corticosteroid administration, may be involved in placental retention.


Comparison of Iloprost, Cicaprost and prostacyclin effects on cyclic AMP metabolism in intact platelets. We have compared the effects of prostacyclin (PGI2) and its stable analogs, Iloprost and Cicaprost, on cyclic AMP metabolism in intact platelets. All three compounds show similar but not identical patterns of prostaglandin concentration-dependent cyclic AMP formation. All three compounds apparently stimulate and inhibit cyclic AMP formation with different concentration dependencies, indicating the presence of distinct stimulatory and inhibitory receptors. Differences in response can be accounted for by slight differences in affinity of stimulatory and inhibitory receptors for the prostaglandins, by the fact that Iloprost contains almost 50% of a relatively inactive isomer, and by the fact that PGI2 is labile in aqueous solution, with a half-life on the order of a few minutes. We conclude 1) stimulation and inhibition of adenylate cyclase is not due to separate effects of 16S- and 16R-stereoisomers of Iloprost because similar patterns were obtained with a single isomeric form of Cicaprost and with authentic PGI2; 2) prostaglandin induced inhibition of adenylate cyclase is readily reversible because inhibition disappears when PGI2 concentration decays below saturation of the inhibitory receptor; 3) the potency of prostaglandins in stimulating platelet adenylate cyclase must be viewed in terms of their effects on both stimulatory and inhibitory receptors.


[Chemo-radiotherapy in advanced head and neck tumors. Personal experience]. From January 1981 through December 1983, 49 untreated patients with locally advanced head and neck cancers were randomized in two groups to receive different radiochemotherapy regimens. Group A, including 29 cases, received 4 cycles of induction chemotherapy with Bleomycin, Methotrexate and Hydroxyurea before definitive external radiotherapy (60 Gy); group B, including 20 patients, received the same total dose of radiotherapy but the 4 cycles of chemotherapy, as described above, were administered between the 20- and the 40-Gy doses. Both groups were compared with a control group treated in the same period with radiotherapy (60 Gy) alone. The response to treatment was evaluated at the end of chemotherapy or radiotherapy alone and at the end of combined regimens. Long-term survival rates were analyzed for all groups relative to complete tumor response, disease-free interval and time to disease progression. In our experience the radio-chemotherapy combination, according to the described schedules, failed to improve both local control and overall survival; the comparison with the control group does not suggest that induction or intercalated chemotherapy can increase long-term survival even if initial complete and partial response rates are high.


[Indications for bone marrow graft in severe hemoglobinopathies]. Allogenic bone marrow transplantation (ABMT) is the only curative approach for sickle cell anemia and major beta-thalassemia. In sickle cell anemia, ABMT can be proposed for severe clinical disease. In major beta-thalassemia, it must be proposed to young patients who have an HLA identical familial donor.


[Bile duct endoprostheses in tumor disease: when is the expense justified?]. Percutaneous transhepatic catheters should be reserved for the minority of palliatively treated tumor patients because of the chronic complications and the discomfort to the patients. Plastic biliary endoprostheses relief malignant obstructive jaundice in most patients sufficiently. Late complications such as occlusion and displacement occur relatively often. The major advantages of expandable metal stents are the relative ease and the low rate of complications during the insertion procedure. The wide lumen and the small surface ensure good flow of bile and reduce the risk of occlusion and cholangitis. However, further development is necessary and the very expensive devices should be restricted to those patients with long life expectancy.


[Female contraception by a normal dose progestogen after 40 years of age. Possible association of nomegestrol--17-beta-estradiol acetate by percutaneous route]. Although fertility declines with age, the use of an effective contraceptive remains necessary in women over 40. Endocrine disorders, which are common in this age group, may also often require control. Conventional estroprogestogens, even those of the latest generation, cannot be used in women with a high cardiovascular risk, since age cannot be totally excluded as a possible risk factor. The contraceptive use of derivatives of 17-hydroxyprogesterone and 19-norprogesterone offer a promising alternative, despite the absence of any exhaustive investigation particularly in situations in which the blood level of estradiol has to be reduced. There are, however, some women who respond to this type of contraception by menstrual cycle irregularities, and sometimes by low blood levels of estradiol, regardless of the drug used. A preliminary study is described in which 5 mg of nomegestrol acetate was combined with 17-beta-estradiol by transcutaneous route and which has so-far demonstrated sustained contraceptive efficacy as well as excellent clinical and metabolic safety.


Effect of ultrasound guided core biopsy of prostate on serum concentration of prostate specific antigen and acid phosphatase activity. Forty-three patients had their serum concentrations of prostate specific antigen and activity of tartrate inhibited acid phosphatase measured before and after digital rectal examination, transrectal ultrasonography and transrectal core biopsy. Transrectal core biopsy significantly increased the values for both tumor markers but rectal examination and ultrasonography without biopsy had no such effect. The measurements returned to normal within one week of biopsy in all but four patients who still had slightly increased concentrations of prostate specific antigen. We recommend that the concentration of prostate specific antigen and activity of tartrate inhibited acid phosphatase are checked before biopsy of the prostate is carried on.


Deficient hippocampal long-term potentiation in alpha-calcium-calmodulin kinase II mutant mice. As a first step in a program to use genetically altered mice in the study of memory mechanisms, mutant mice were produced that do not express the alpha-calcium-calmodulin-dependent kinase II (alpha-CaMKII). The alpha-CaMKII is highly enriched in postsynaptic densities of hippocampus and neocortex and may be involved in the regulation of long-term potentiation (LTP). Such mutant mice exhibited mostly normal behaviors and presented no obvious neuroanatomical defects. Whole cell recordings reveal that postsynaptic mechanisms, including N-methyl-D-aspartate (NMDA) receptor function, are intact. Despite normal postsynaptic mechanisms, these mice are deficient in their ability to produce LTP and are therefore a suitable model for studying the relation between LTP and learning processes.


Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas. Immature T cells and some T cell hybridomas undergo apoptotic cell death when activated through the T cell receptor complex, a phenomenon that is probably related to antigen induced negative selection of developing T cells. This activation-induced apoptosis depends on active protein and RNA synthesis in the dying cells, although none of the genes required for this process have previously been identified. Antisense oligonucleotides corresponding to c-myc block the constitutive expression of c-Myc protein in T cell hybridomas and interfere with all aspects of activation-induced apoptosis without affecting lymphokine production in these cells. These data indicate that c-myc expression is a necessary component of activation-induced apoptosis.


Nitric oxide: a physiologic mediator of penile erection. Nitric oxide (NO) is a cytotoxic agent of macrophages, a messenger molecule of neurons, and a vasodilator produced by endothelial cells. NO synthase, the synthetic enzyme for NO, was localized to rat penile neurons innervating the corpora cavernosa and to neuronal plexuses in the adventitial layer of penile arteries. Small doses of NO synthase inhibitors abolished electrophysiologically induced penile erections. These results establish NO as a physiologic mediator of erectile function.


Six new cases of a caterpillar-induced bleeding syndrome. We describe six new cases of a hemorrhagic diathesis induced by contact with Lonomia achelous caterpillars. Onset of clinical bleeding varied between a few hours and 10 days post-exposure. Laboratory coagulation tests showed prolonged PT, PTT and ThT; normal platelets and a marked decrease of fibrinogen, factor V, plasminogen and factor XIII (including its subunits A and S). Factors VII, II and alfa 2 anti-plasmin were variably affected. In addition, activation of the fibrinolytic system and the generation of a procoagulant effect could also be demonstrated. Two cases developed severe hemorrhagic diathesis and one of them died of a cerebral hemorrhage. Different aspects of this rare syndrome are discussed in relation to its complex physiopathology and the variability observed in all clinical and laboratory manifestations. Therapeutic recommendations and some possible hazards following replacement transfusions are also considered.


Human vascular endothelial cells catabolise exogenous glycosaminoglycans by a novel route. Heparin and other anticoagulant glycosaminoglycans were radiolabelled with 125I and their catabolism by human vascular endothelial cells in culture was studied. Heparin, heparan sulphate and pentosan polysulphate were associated with the cellular fraction and incorporated into the subendothelial matrix, but dermatan sulphate was not found in either fraction. High molecular weight, fully desulphated carbohydrate chains were major catabolic products of all those glycosaminoglycans which were taken up by the cells. Pentosan polysulphate was not degraded further, but the catabolism of heparan sulphate, and to a lesser extent that of heparin, also yielded small oligosaccharides. Thus the first step in catabolism of exogenous glycosaminoglycans by human vascular endothelial cells appears to be complete desulphation, which destroys their biological activity, followed by depolymerisation of the carbohydrate chain. This alternative to the sequential action of lysosomal exoenzymes is dependent upon binding to the cell; thus dermatan sulphate, which is not associated with the cellular fraction, is not catabolised.


Characterization of a factor VIII immunogenic site using factor VIII synthetic peptide 1687-1695 and rabbit anti-peptide antibodies. A 9 amino acid peptide, Ser-Pro-Arg-Ser-Phe-Gln-Lys-Lys-Thr, corresponding to the clotting factor VIII (FVIII) sequence Ser1687-Thr1695, was synthesized in order to analyze a site on FVIII to which antibody inhibitors of FVIII may be directed. This sequence contained a thrombin cleavage site. It was predicted to be immunogenic because a Hopp-Woods hydrophilicity analysis of the amino acid sequence of FVIII showed it to be very hydrophilic, and it contained a proline. The HPLC-purified peptide was cleaved by thrombin at Arg1689-Ser1690, as determined by amino acid sequencing of the cleavage product. Thrombin which had been treated with a specific chloromethyl ketone inhibitor, did not cleave the peptide. Two rabbits immunized with the peptide/keyhole limpet hemocyanin conjugate generated FVIII inhibitory sera with titers of 5.4 and 4.8 Bethesda units. These rabbit anti-peptide antibodies reacted with a peptide/-BSA conjugate on immunodot blot analyses and with native, affinity-purified FVIII in Western blots. In competitive immunoradiometric assays, cryosupernatants of 38/82 patients with FVIII inhibitors reacted with the synthetic peptide. We conclude that FVIII peptide Ser1687-Thr1695 is cleaved by thrombin at the same peptide bond which is cleaved in FVIII, and the peptide contains a site to which patients' inhibitory antibodies can be directed.


[Is cancer pain therapy insufficient?].

[Is cancer pain therapy insufficient?]. Pain from cancer is said to be poorly relieved. To examine the relevance of this statement in our hospital we registered morphine consumption used for cancer pain relief from 1983 to 1990. The use of morphine increased 20-fold during this period, corresponding to a 12-fold increase per patient. A probable explanation of this development is that cancer pain now receives more attention and use of morphine has become more acceptable. New methods such as subcutaneous continuous morphine administration from portable pumps, and the introduction of slow-release morphine, have improved and simplified the treatment. Probably several Norwegian hospitals still lag behind in this respect. Consequently much pain remains unrelieved. We suggest that hospitals evaluate their pain relief programmes. If these are found inadequate, the service should be reinforced. This would probably lead to earlier rehabilitation and improved quality of life for the patient.


[Morphine-antiemetics mixtures for continuous subcutaneous infusion in terminal cancer]. Simultaneous pain, nausea and vomiting are not uncommon in terminal suffering requiring treatment with various compounds of analgesics and antiemetics. At Baerum Hospital the pump reservoirs for continuous, subcutaneous drug delivery are routinely filled by the hospital pharmacist. We examined the physico-chemical stability of various concentrations of mixtures of morphine-metoclopramide and morphine-metoclopramide-haloperidol at 25 degrees C. We found good stability for at least seven days. Addition of haloperidol seems to reduce stability. Plain morphine-haloperidol solutions are unstable. Split products were not found in any of the mixtures. We also examined the osmolality of current clinical compounds, focusing on local irritant effect at the infusion site. All solutions except for one with a high concentration of haloperidol were found to be close to isoosmolarl.


[Continuous drug infusion in terminal cancer]. Today's technology provides portable pumps which facilitate continuous infusion of drugs to relieve suffering in terminal disease. Subcutaneous and epidural infusion is now frequently used in our hospital. The most common indications are gastrointestinal obstruction, impaired absorption of drugs, refractory side effects of oral medication or poor compliance because good pain relief is no longer possible orally. During the last days of life, this method may be the only possible approach to good comfort and relief from terminal agitation and anxiety. Of the patients referred to the advisory group for seriously ill and dying in 1990, 64% received subcutaneous infusions and 15% epidural infusions during the last days or weeks of life. Continuous infusion of drugs from portable pumps has become an almost indispensible method of treatment in an ordinary clinic.


In vivo assessment of neovascularization and incorporation of prosthetic vascular biografts. Neovascularization plays a major role in prosthetic vascular graft healing. New developments in manufacturing biomaterials have encouraged the use of biological and biosynthetic materials for both arterial replacement and bypass procedures. We have analyzed in vivo the process of neovascularization and incorporation of biolized bovine carotid artery (Solco P), a biological material, and the composite of ovine collagen and polyester mesh (Omniflow), a biosynthetic material. The synthetic fabric polytetrafluorethylene (e-PTFE), which is widely used in cardiovascular surgery, served as control. Using the dorsal skinfold chamber of the Syrian golden hamster as site for implantation (n = 15), angiogenesis and neovascularization were analyzed quantitatively by means of intravital fluorescence microscopy. In each chamber a piece (approximately 1 mm2) of each of the three vascular grafts was implanted. Five days after implantation neovascularization was ascertained in 90% of the Omniflow grafts, while only 40% of the PTFE and 20% of the Solco P implants revealed new ingrowing microvessels. On day 10 the density of newly formed microvessels was significantly higher (p less than 0.01) in Omniflow grafts (263.5 +/- 24.1 cm-1) as compared to PTFE (134.2 +/- 20.8 cm-1) and Solco P (153.2 +/- 32.0 cm-1). In addition, the biosynthetic composite revealed a larger extension of neovascularization into the perigraft tissue, and 12 days after implantation these grafts were most tightly incorporated.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of mitochondrial Ca2+ release diminishes the effectiveness of methyl mercury to release acetylcholine from synaptosomes. The interaction of methyl mercury (MeHg) with nerve-terminal mitochondria as a potential mechanism for its effects on the release of acetylcholine (ACh) was studied using rat brain synaptosomes. The primary goal was to assess the relative contribution of extracellular Ca2+ and Ca2+ released from nerve-terminal mitochondria to the previously described stimulatory effects of MeHg on spontaneous release of ACh. A secondary goal was to address possible mechanisms by which MeHg might interact with nerve-terminal mitochondria to elicit Ca2+ discharge and subsequent release of ACh. MeHg depressed the high-affinity uptake of [3H]choline into synaptosomes by approximately 25 and 45% when synaptosomes were incubated with [3H]choline in the presence of 10 and 100 microM MeHg, respectively. In Ca(2+)-containing solutions, 10 and 100 microM MeHg increased the release of [3H]ACh from [3H]choline-loaded synaptosomes by 10 and 30%, respectively; this effect was maximal at 10 sec. Excluding Ca2+ from the reaction medium diminished the effectiveness of both 10 and 100 microM MeHg for inducing [3H]ACh release by about 30 and 25%, respectively, from that of Ca(2+)-containing solutions; however, significant increases still occurred in nominally Ca(2+)-free solutions. Ruthenium red (RR), an inhibitor of mitochondrial Ca2+ transport, was tested for its ability to disrupt MeHg-induced release. RR alone increased [3H]ACh release by 8-10 and 10-13% at 20 and 60 microM, respectively. RR-induced release was attenuated only slightly in Ca(2+)-free solutions. Preincubation of [3H]choline-loaded synaptosomes with RR reduced the stimulatory effect of MeHg on release of [3H]ACh both in the presence and in the absence of Ca2+. The fluorescent potentiometric carbocyanine dye diS-C2(5) was used to assess the ability of RR to prevent MeHg-induced depolarization of intrasynaptosomal mitochondria. RR (20 microM) itself did not depolarize the mitochondrial membrane potential, nor did it prevent MeHg from depolarizing the mitochondria. The results indicate that extracellular Ca2+ contributes only partially to MeHg-induced spontaneous release of ACh. The results with RR suggest that MeHg interacts with mitochondria to induce release of bound intraterminal Ca2+ stores, resulting ultimately in stimulated release of ACh. The ability of RR to prevent release of mitochondrial Ca2+ and, subsequently, ACh is not due to prevention of access of MeHg to the mitochondria, nor to stabilization of the mitochondrial membrane. Finally, MeHg reduces choline uptake into nerve terminals. Thus, MeHg could interfere with cholinergic neurotransmission by affecting the regulatory step in ACh synthesis and by increasing the spontaneous release of transmitter.(ABSTRACT TRUNCATED AT 400 WORDS)


An 'anatomical cascade hypothesis' for Alzheimer's disease. The molecular mechanisms of the cytopathology of Alzheimer's disease are very rapidly being elucidated. However, the factors that restrict the effects of this disease to specific neuroanatomical systems are less well understood. In this brief article a possible hypothesis is outlined to explain this apparent specific localization.


Dopamine receptor interactions: some implications for the treatment of Parkinson's disease. Since the discovery that L-DOPA could alleviate the symptoms of Parkinson's disease, it has been assumed that the striatum is the site of action of the dopamine formed from L-DOPA. However, for the past 15 years, evidence has accumulated to suggest that dopamine is also released by the dendrites of dopamine neurons in the substantia nigra and D1 dopamine receptors in this region of the brain appear to play an important role in the actions of L-DOPA. Activation of D1 receptors in the substantia nigra may, in part, explain some of the synergistic effects of D1 and D2 agonists in animal models for Parkinson's disease. These effects are discussed in light of recent studies suggesting that dopamine, acting on D1 and D2 dopamine receptor subtypes, activates distinct efferent pathways from the striatum. Clinical studies suggest that these findings may have important implications for the treatment of Parkinson's disease.


How complex is the nicotinic receptor system of insects? In insects, nicotinic acetylcholine receptors (nAChRs) are confined to the nervous system. It is a long-standing open question whether the insect nicotinic cholinergic receptor system is less complex than that of the vertebrate nervous system. Simplicity can be conceived in two ways. (1) Fewer receptor subtypes may exist. (2) Single receptors may have a more primitive (homo-oligomeric) quaternary structure. Recent approaches to the molecular cloning of insect nAChRs may contribute valuable new information to this issue. Thus, the identification of multiple genes encoding proteins similar to vertebrate nAChR subunits implicates a remarkable heterogeneity for these receptors. The discovery of putatively non-ligand-binding subunits hints to the existence of vertebrate-like hetero-oligomeric nAChRs. However, the simultaneous occurrence of homo-oligomeric receptors must still be considered.


Eye regionalization and spectral tuning of retinal pigments in insects. The spatial and spectral properties of an eye can often be directly linked to the behaviour and habitat of the animal. In a honey bee (Apis mellifera) society, the drones use the well-developed dorsal part of the eye to detect the queen against the sky during her nuptial flight. Recently it has become clear that the dorsal area of the drone's eye serves its task by cleverly combining a number of optical mechanisms, thus achieving a high spatial acuity as well as a high sensitivity precisely in the wavelength range of interest--the ultraviolet to blue range. Since the various optical specializations in the drone eye have now been recognized, they can be traced in the eyes of other species: thus, the drone eye serves as a model to give a better understanding of the relationship between structure and function of compound eyes in particular, but also of visual systems in general.


Temporal coding in the visual cortex: new vistas on integration in the nervous system. Although our knowledge of the cellular components of the cortex is accumulating rapidly, we are still largely ignorant about how distributed neuronal activity can be integrated to contribute to unified perception and behaviour. In the visual system, it is still unresolved how responses of feature-detecting neurons can be bound into representations of perceptual objects. Recent crosscorrelation studies show that visual cortical neurons synchronize their responses depending on how coherent features are in the visual field. These results support the hypothesis that temporal correlation of neuronal discharges may serve to bind distributed neuronal activity into unique representations. Furthermore, these studies indicate that neuronal responses with an oscillatory temporal structure may be particularly advantageous as carrier signals for such a temporal coding mechanism. Based on these recent findings, it is suggested here that binding of neuronal activity by a temporal code may provide a solution to the problem of integration in distributed neuronal networks.


Local circuits for the control of leg movements in an insect. To produce behaviour that is adaptive, local circuits in the CNS must transform mechanosensory signals from receptors on the body into changes in movement. Substantial insights into the mechanisms underlying these transformations can be obtained by analysing the local circuits of animals from which intracellular recordings can be made from identified neurones during behavior, thus allowing the complete pathways between inputs and outputs to be followed. In the locust (Schistocerca gregaria) these circuits contain both non-spiking and spiking local neurones so that it is possible to elucidate two basic issues of neuronal integration: (1) the operation of the reflex circuitry that must adjust locomotion, and (2) the integrative role of local circuits that use graded interactions in complex neuropil, perhaps even involving compartmentalized neurones.


[Hematopoietic growth factors in primary and therapy-related bone marrow insufficiency]. This investigation is retrospective and comprises 20 patients with bone-marrow insufficiency. During the period 1.4.1988-1.3.1991, these patients were treated with erythropoietin (Epo), the granulocyte-macrophage-colony-stimulating factor (GM-CSF) or the granulocyte-colony-stimulating factor (G-CSF). Thirteen patients had primary bone-marrow insufficiency: six had the myelodysplastic syndrome, three had primary myelofibrosis, two aplastic anemia and two myelomatosis. On account of dominating symptoms of anemia, five patients received Epo while eight received GM-CSF as part of an extensive clinical trial of this preparation. Seven patients with relapse of the haematological malignant disease had bone-marrow insufficiency and pancytopenia secondary to intensive chemotherapy/irradiation: four of these patients received GM-CSF and two received G-CSF with the object of increasing bone-marrow regeneration and to render further chemotherapy possible. One patient received GM-CSF with the object of improving bone-marrow function after autologous bone-marrow transplantation. Treatment with Epo for ten months combined with treatment with interferon for six months resulted in normalization of the haemoglobin concentration in one patient with bone-marrow insufficiency on account of primary myelofibrosis. Treatment with Epo for briefer periods in lower doses was without effect in four other patients with primary bone-marrow insufficiency. Treatment with GM-CSF and G-CSF resulted in neutrophil leukocytosis in 12 out of 15 patients (80%) and, in six out of 14 patients (43%), increased marrow cellularity was demonstrated by means of histological examination of the bone-marrow. One patient showed normal haemoglobin levels during treatment with GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)


Pulmonary inflammation associated with aspirated meconium and epithelial cells in calves. "Meconium aspiration syndrome" is a condition resulting in respiratory distress and the occasional death of newborn human beings. A retrospective study was conducted on 52 calves that were submitted for postmortem examination to the Atlantic Veterinary College, Charlottetown, Prince Edward Island, Canada. These calves died of infectious and noninfectious diseases within the first 2 weeks of life due to a variety of causes. The most common cause of death was infectious enteric disease. Histologic examination of lungs revealed that 42.5% of calves had evidence of meconium, squamous cells, or keratin in the lung. There was considerable variation in the magnitude of histologic changes in lungs containing aspirated material. Typically, affected lungs had only a few inconspicuous pieces of meconium, keratin, and squamous cells within bronchoalveolar spaces. Sporadically, the entire lumen of airways was obliterated by aspirated material. Lungs with aspirated material had a mild but diffuse alveolitis characterized by exudation of a few neutrophils, macrophages, and occasional multinucleated giant cells. Obstruction of small airways and focal atelectasis were also observed. Similar lesions have been reported in human meconium aspiration syndrome. It is concluded that histologic changes similar to those of human meconium aspiration syndrome occur commonly in calves that die within 2 weeks of birth. Further studies involving healthy age-matched calves are required to evaluate the clinicopathologic significance of meconium aspiration in this species.


Genetic susceptibility of indigenous chicks to subgroup A Rous sarcoma virus inoculated via the chorioallantoic membrane. An investigation was made using chicks of two Indian indigenous breeds of fowl, Kadaknath and Aseel, to ascertain genetic resistance to infection by Rous sarcoma virus of subgroup A. A standard inoculation dose of 0.2 ml virus containing 1000 pock forming units ml-1 was injected via the chorioallantoic membrane (CAM) into the 11-day-old embryos that were subsequently hatched. The sensitivity of the two indigenous breeds was compared with the highly susceptible exotic White Leghorn (WL) strain maintained in the laboratory. The Kadaknath breed was about three-fold and Assel, about six-fold less sensitive than the WL strain, indicating superiority of the indigenous breeds over the exotic breed of fowl. Most of the CAM-susceptible chicks died of liver tumour (LT) and most of the CAM-resistant chicks survived. However, conversely associated tumour phenotype subclass chicks, i.e. CAM-susceptible LT-negative chicks that survived and CAM-resistant LT-positive chicks that died, occurred consistently in the three breeds of fowl. Nevertheless, the overall survival potential of Kadaknath chicks measured up to 8 weeks post-hatching was greater than that of Aseel chicks. Neither transformation of embryonic tissue prior to hatching nor the visceral metastasis including liver conformed with the degree of CAM-infection as measured by number of pocks on CAMs.


National Hospital Discharge Survey.

National Hospital Discharge Survey. This report presents statistics on the utilization of non-Federal short-stay hospitals based on data collected through the National Hospital Discharge Survey from a national sample of hospital records of discharged inpatients. Estimates are provided by the demographic characteristics of patients discharged, geographic region of hospitals, conditions diagnosed, and surgical and nonsurgical procedures performed. Measurements of hospital use include frequency, rate, percent of discharges and days of care, and average length of stay.


Antioxidant and pro-oxidant properties of active rosemary constituents: carnosol and carnosic acid. 1. Carnosol and carnosic acid have been suggested to account for over 90% of the antioxidant properties of rosemary extract. 2. Purified carnosol and carnosic acid are powerful inhibitors of lipid peroxidation in microsomal and liposomal systems, more effective than propyl gallate. 3. Carnosol and carnosic acid are good scavengers of peroxyl radicals (CCl3O2.) generated by pulse radiolysis, with calculated rate constants of 1-3 x 10(6) M-1 s-1 and 2.7 x 10(7) M-1 s-1 respectively. 4. Carnosic acid reacted with HOCl in such a way as to protect the protein alpha 1-antiproteinase against inactivation. 5. Both carnosol and carnosic acid stimulated DNA damage in the bleomycin assay but they scavenged hydroxyl radicals in the deoxyribose assay. The calculated rate constants for reaction with .OH in the deoxyribose system for carnosol and carnosic acid were 8.7 x 10(10) M-1 s-1 and 5.9 x 10(10) M-1 s-1 respectively. 6. Carnosic acid appears to scavenge H2O2, but it could also act as a substrate for the peroxidase system. 7. Carnosic acid and carnosol reduce cytochrome c but with a rate constant significantly lower than that of O2(-.).


[Clinical importance of organ-specific antibodies in ulcerative colitis and Crohn disease]. In 479 patients with chronic inflammatory intestinal diseases and other abdominal diseases autoantibodies against intestinal goblet cells and exocrine pancreas were determined by indirect immunofluorescence. In ulcerative colitis 17% had serum antibodies against intestinal goblet cells, in Crohn's disease 26% against exocrine pancreas. Autoantibody prevalence and level of the titre were dependent on the inflammatory activity of both diseases but independent on the therapy. In Crohn's disease high level of titres of antibodies against exocrine pancreas we found in cases with multiple complications. Antibodies titres against intestinal goblet cells in ulcerative colitis and against exocrine pancreas in Crohn's disease exceeding 1:100 improved the diagnostic decision. Besides clinical states, endoscopic and histological findings a positive result of the antibody investigations had to be considered for final diagnosis.


Clinical and electrophysiologic studies of R61748 (transcainide): a new class Ic antiarrhythmic drug. The electrophysiology and antiarrhythmic efficacy of R61748, a 4-OH metabolite of R54718 (transcainide), have been studied in patients and animal experiments. The results indicated that R61748 (1 mg/l) decreased the rate of rise of the transmembrane action potential (Vmax) by 40% and shortened the action potential duration (APD) by 10% in guinea-pig right ventricular papillary muscle. ERP/APD increased by 10%. The effect was dose-dependent. Clinical electrophysiologic studies (EPS) in 6 patients with ventricular premature beats showed that R61748 (0.1 mg/kg) prolonged PA duration by 30%, AH interval by 13% and HV duration by 24%. ECG revealed prolonged P duration by 16%, P-R interval by 16% and QRS duration by 17%. R61748 has also been used in 14 patients with idiopathic premature ventricular contraction (PVCs). PVCs were abolished in 6 patients and decreased more than 93% in 2 patients. The average reduction of PVCs was 80%. R61748 (0.075 mg/kg) abolished an episode of repetitive ventricular tachycardia in 1 patient. It is concluded that R61748 is a potent new class Ic antiarrhythmic agent with slow kinetics. R61748 is the 4-OH metabolite of R54718 (transcainide) (Stroobandt et al., 1987) which is a new class Ic antiarrhythmic agent. Up to now no reports have been published about R61748. This paper presents the initial studies of cellular and clinical electrophysiologic effects and the evaluation of antiarrhythmic efficacy of R61748.


Reaction time in cancer patients receiving peripherally acting analgesics alone or in combination with opioids. Continuous Reaction Time (CRT) was measured in cancer patients receiving peripherally acting analgesics either alone (n = 16) or in combination with opioids (n = 16). Comparison was performed matching the patients from each group for age and performance status. Statistically significant prolongations of CRT and higher sedation scores were seen in the opioid group, while performance status did not have any influence on CRT.


Systematic use of aprotinin in cardiac surgery: influence on total homologous exposure and hospital cost. To assess the impact of systematic use of aprotinin, 115 consecutive adults undergoing cardiac surgery were randomly allocated with a sealed envelope technique. Treated (T) patients (n = 58) received 2.10(6) Kallikrein Inactivating Units (KIU) before incision, 2.10(6) prior to bypass, and 5.10(5) KIU.hr-1 for 5 hrs, whereas control (C) cases (n = 57) received nothing. Surgeons, perfusionists, ICU and ward physicians were blinded. Postoperative blood loss decreased from 1198 ml (C) to 698 ml (T) (p less than 0.001). Total transfusional needs were 7.25 (C) and 4.9 (T) units (p less than 0.01), where from 65% were autologous in group T, versus 51% in group C (p less than 0.02). Total homologous exposure decreased from 4.5 (C) to 2.7 (T) units on the average, from 3 to 1 units as a median (p less than 0.01). Multiple Stepwise Regression Analysis showed treatment as the most important variable influencing postoperative blood loss, but duration and type of procedures were more important to explain transfusion needs. Both groups were comparable for other pre- and intra-operative variables. For coronary operations (n = 75), aprotinin showed the strongest negative association with blood loss, the number of arterial conduits being the second influencing variable. No evidence was found for increased early graft thrombosis. The average hospital bill was 9% lower in the treated group, an unexplained finding needing independent confirmation.


Whipple's disease: the value of upper gastrointestinal endoscopy for the diagnosis and follow-up. Due to the systemic nature of Whipple's disease the clinical presentation may be highly variable. The diagnosis may therefore be unduly delayed. If untreated, Whipple's disease is still potentially lethal. In contrast, the endoscopic findings as they are observed in the postbulbar small intestine and the light microscopic picture of small intestinal biopsies are almost pathognomonic. Out of a group of 18 patients (14 male, 4 female, mean age = 45 yrs), 12 patients were diagnosed using upper gastrointestinal endoscopy and duodenal biopsy, while six patients were diagnosed only by a small intestinal capsule biopsy. The clinical history prior to diagnosis lasted from 1 m to 22 yrs (mean = 3 yrs 6 m) in the first group and from 4 yrs to 21 yrs (mean = 12 yrs 6 m) in the second group. The endoscopic findings at the time of diagnosis were: oesophagitis (1/12), erosive gastritis (4/12), atrophic gastritis (2/12), severe erosive bulbitis (3/10), pathognomonic post-bulbar duodenal lesions (9/12). In 20% of the patients the endoscopic lesions had disappeared 6 m after antibiotics while the lesions had disappeared in all cases 9 m after therapy, despite the fact that PAS positive macrophages remained present in the endoscopic biopsies for years. All patients were treated with antibiotics (8 tetracycline alone, 4 tetracycline, streptomycin and penicillin, 6 trimethoprim). Five patients (27%-4 of the tetracycline group) relapsed within 2 to 20 yrs after the initial diagnosis. Three of these patients (3/5) had typical duodenal lesions on endoscopy at that time.


Immunocytochemical localization of alpha-fetoprotein in the developing and carbon tetrachloride-treated rat liver. The immunocytochemical localization of alpha-fetoprotein (AFP)-producing cells was observed in pre- and postnatal and carbon tetrachloride (CCl4)-treated rat livers in comparison with that of albumin (ALB)-producing cells. According to immunoblotting data, considerable numbers of AFP-positive hepatocytes were observed in the differentiating liver between prenatal day 19 and postnatal day 0 (6 h after birth). Analyses by serial section profiles of these cells revealed that certain AFP-positive hepatocytes are also stained with ALB antiserum. Immunoelectron microscopy of the AFP-producing cells revealed that immunoreactive gold particles are preferentially localized in rough endoplasmic cisternae, Golgi apparatus and Golgi-derived vesicles near the cell surface. In addition, the release of the content of the Golgi-derived vesicles into the differentiating bile canaliculi as well as into the space of Disse by exocytosis is apparent. In CCl4-treated rat liver, immunoreactions to AFP are localized exclusively in newly formed hepatocytes of the regenerative tissue. These AFP-positive cells have not established the hepatic cell cords, and the adjacent ones are conjugated to each other mainly by simple attachment devices as in the case of those in pre- and postnatal rat liver.


Endoscopic palliation of malignant dysphagia. In patients with obstructive tumors of the esophagus and who cannot undergo curative surgery, endoscopic palliative methods can provide a quick relief of dysphagia with subsequent improvement of nutritional status. The technical success rate is as high as 95% for intubation and 100% for laser therapy. Both methods have their specific indications. Intubation is often able to provide quick and definitive palliation while laser therapy requires several sessions and relapses of obstruction are frequent. On the other hand, intubation carries a high risk of perforation (8.7%), and laser appears to be safer (1.7% perforation). Both methods can be combined in many instances, and their association with afterloading or external beam radiation therapy can be beneficial. Even if the overall survival rate is only 6 to 7 months, the improvement of the quality of life is the priority goal of these methods.


Different effects of fractionated irradiation on potassium efflux and exocytotic amylase release. Irradiation of head-neck cancer influences the salivary glands with dryness and discomfort for the patients as a consequence. In the present study we used in vitro secretory models and morphological characterization of rat parotid gland. Irradiation was given as a 5-day schedule with total doses from 20 Gy to 45 Gy. Electrolyte secretion (86Rb as indicator for potassium) caused by noradrenaline was decreased in correlation to irradiation dose delivered compared to controlateral control glands 10 days following irradiation. Noradrenaline stimulated exocytotic amylase release was not at all affected, and there were no signs of quantitative morphological alterations following irradiation compared to control glands. The results indicate that there are differences in radiation sensitivity for the two different secretory processes in the salivary glands; the structures regulating electrolyte and fluid secretion seem to be more vulnerable to irradiation than those regulating exocytosis.


Percutaneous endoscopic gastrostomy.

Percutaneous endoscopic gastrostomy. From March 87 to March 92, fifty eight patients were referred to our department for percutaneous endoscopic gastrostomy (PEG). The modality of the feeding tube insertion is described. The most common indications for placement were neurologic disorders in 62% of the cases (n = 36) and malignant diseases in 32% (n = 19). The success rate of the technique was 98.3% (n = 57). No procedure-related mortality was observed. A low rate of major complication (1.7%) and minor complication (10.5%) was noted. Feeding tubes were removed in 21% of patients (n = 12); none of them with malignant disease. Survival curve analysis demonstrated that 50% of patients died within 3 months of PEG placement. Such results raise questions about the selection of patients undergoing PEG. Our experience of patients undergoing PEG. Our experience suggests that PEG is easy and safe, even in debilitated patients, having an acceptable life expectancy.


Effect of the substrate on neurofilament phosphorylation in mixed cultures of rat embryo spinal cord and dorsal root ganglia. The effect of the substrate on neurofilament phosphorylation was studied in primary cultures of spinal cord and dorsal root ganglia dissociated from 15-day-old rat embryos. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites. (Primaria dishes have a modified plastic surface with a net positive charge). On both polylysine and Primaria substrates, spinal cord neurons were stained with neurofilament monoclonal antibodies reacting with phosphorylated epitopes appearing early in rat embryo development, i.e. soon after neurofilament expression. Conversely, immunoreactivity with antibodies recognizing late phosphorylation events was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, only occurred after several days in culture, at a time when non-neuronal cells (mainly astrocytes) had formed a confluent monolayer.


Endoscopic stenting for biliary strictures. A consensus is growing among units that have an experience in both endoscopic and percutaneous stenting techniques that the endoscopic approach of malignant biliary strictures is more comfortable for the patient and provides less complications. This article describes endoscopic biliary drainage in different malignant stenosis of the bile ducts and delineates the respective indications of percutaneous and endoscopic techniques together with the possible combination of these two methods in selected cases. It also tackles the question of the medical surgical approach of the patients, which might, thanks to a better selection, reduce the morbidity and mortality associated with surgery. The indications of biliary stenting in benign strictures, namely post operative or chronic pancreatitis associated biliary stenoses, are also discussed. Recently, new materials became available for endoscopic and percutaneous biliary drainage, and particularly metallic self expanding stents which might provide a better palliation among these patients. If these stents fulfill their promise on longer follow-up, they may replace the conventional stenting devices.


Increase in endogenous fibrinolysis and platelet activity during exercise in young volunteers. The influence of physical exercise and intake of oral contraceptives on endogenous fibrinolytic activity and platelet aggregation behaviour was studied in young healthy women and compared to an age matched group of male volunteers. Physical exercise beyond the anaerobic threshold significantly increased the activity of tissue-plasminogen activator (t-PA) in male and female volunteers from initial values of 1.6 +/- 0.1 and 1.8 +/- 0.2 IU/ml to 5.5 +/- 1.0 and 5.3 +/- 0.9 IU/ml (P less than 0.01), respectively. In women taking low-dose estrogen oral contraceptives t-PA increased from 1.5 +/- 0.2 to 3.8 +/- 0.5 (P less than 0.01). There were no major alterations in plasminogen activator inhibitor - I (PAI-I) plasma activities. Platelet activity during exercise was significantly enhanced in male test persons indicated by a significant decrease in the ED50 values for ADP. In contrast, in women ED50 values were basically unaltered, irrespective of the intake of oral contraceptives.


Effect of prostacyclin analogue iloprost on haemodynamic parameters during femoro-crural reconstruction. A randomised placebo-controlled trial was conducted to investigate the effects of iloprost, a stable prostacyclin analogue, on peripheral resistance, graft blood flow and mean systemic arterial blood pressure (MABP) during femoro-crural arterial bypass. 3000ng of iloprost, infused into the graft, produced an immediate drop in peripheral resistance by a mean (range) of 40% (4-80%) (P less than 0.01, Wilcoxon). Decreased peripheral resistance persisted to 20 minutes. Graft flow during the same period increased by 52% (-7 to 294%) (P less than 0.01). A transient maximal drop in mean MABP of 22% from 83-65 mmHg occurred 5 minutes post iloprost infusion but caused no detrimental effects and recovered to baseline values by 10 mins. Iloprost produces an immediate decrease in peripheral resistance associated with a prolonged increase in graft blood flow. This may reduce graft failure in the early postoperative period.


Fibrinolytic enhancement with a prostaglandin analogue iloprost. Earlier workers have suggested a possible fibrinolytic effect using prostaglandin or prostaglandin analogues. Despite using maximum tolerated doses of the prostaglandin analogue Iloprost, we have been unable to demonstrate any significant effect on fibrinolysis in patients with stage IV peripheral vascular disease (Fontaine classification).


Cardioprotection by molsidomine and iloprost in myocardial ischemia in anaesthetized cats. In ten anaesthetized open chest cats the ligation of left descending coronary artery (LLDCA) resulted in 80 percent of mortality with survival time of 40.9 +/- 8.6 min. The death of animals was preceded by premature ventricular contractions which occurred with frequency 7.3 +/- 0.6/min after 2.1 +/- 0.3 min following LLDCA. Molsidomine as a dose of 20 micrograms/kg given i.v. to 10 cats 15 min before LLDCA affected none of the above characteristics of acute myocardial ischemia. The pretreatment with iloprost (2 micrograms/kg given i.v. to 10 cats) diminished frequency of premature ventricular contractions and elongated a period of time required for their occurrence after LLDCA, however, iloprost was also unable to diminish mortality of LLDCA cats. Only a combined administration of molsidomine and iloprost significantly diminished mortality among LLDCA cats down to 20 percent and doubled their survival time as compared to the controls. It is concluded that NO-donors (molsidomine) exert permissive effect towards the cardioprotective action of prostacyclin analogues (iloprost) in cats with myocardial ischemia. LLDCA cats pretreated with molsidomine + iloprost enjoyed a more complete cardioprotection than those pretreated with a high dose (3000 units/kg i.v.) of superoxide dismutase.


Iloprost in cardiopulmonary bypass procedures. The inhibition of platelet aggregation during cardiopulmonary bypass and effects on post-operative placebo-controlled study of 145 patients. Significant preservation of platelet numbers and function were shown without significant haemodynamic problems, but no effect on cerebral deficits could be found. The use of iloprost in patients with severe thrombocytopenia seems justified, but the clinical benefits from its use in routine cardiopulmonary bypass remain to be shown.


Iloprost preserves endothelial function against cyclosporin A and sensitises microvessels towards endothelium-dependent and -independent vasodilatation. The effects of iloprost have been investigated on the endothelial damage produced by cyclosporin-A (Cy-A) and on the microvascular reactivity to sodium nitroprusside (SNP), acetylcholine (ACh) and isoprenaline (ISO) following iloprost treatment i.e. in the absence of effective plasma levels, using the hamster cheek pouch. It has been shown that iloprost protects the microvascular arteriolar endothelium from functional damage by Cy-A and that iloprost infusion for 12-14 hr sensitizes the arterioles to ACh, SNP and ISO, an effect which is evident 5 hr but not 24 hr following treatment and in the case of ACh is abolished by indomethacin.


Effects of iloprost and factors affecting outcome in patients with severe inoperable lower limb ischaemia. U.K. Severe Limb Ischaemia Study Group. The effects of i.v. iloprost given for 14-28 days on six month outcome in patients with severe inoperable lower limb ischaemia were investigated in a double-blind placebo controlled study. More iloprost patients (64%) survived with a viable limb than placebo patients (42%). Iloprost improved prognosis in all subgroups of patients, but patients with lower presenting ankle Doppler pressures had a worse outcome than patients with higher pressures.


Pharmacokinetics of intragraft iloprost infusion during femoro-crural reconstruction. Pharmacokinetics of a single intra-graft infusion of 3000ng iloprost during femoro-crural bypass surgery have been investigated. Plasma iloprost concentrations, biphasic half-lives, and total drug clearances were found to occur within ranges demonstrated for IV infusion dose regimens.


Synergistic platelet inhibitory effect of the phosphodiesterase inhibitor piroximone and iloprost. Platelet activity is regulated through synthesis and degradation of the intracellular second messengers cAMP or cGMP. The antiplatelet effect of the phosphodiesterase (PDE) III inhibitor Piroximone (PIR) was studied in vitro in platelet rich plasma. ADP induced aggregation was inhibited by PIR with an IC50 of 67 +/- 43 microM. The inhibitory effect was time and dose dependent. The antiaggregatory effects in vivo were studied in anaesthetised rats. Reduction of platelet count following injection of 100 micrograms/kg bw collagen was measured after bolus injection of PIR and vehicle. Piroximone bolus 2 mg/kg bw resulted in a 50% inhibition of platelet aggregation in rats. Cyclic AMP levels in washed platelets rose time and dose dependently after PIR. Coincubation of PDE III inhibitor PIR and adenylate cyclase activator Iloprost (ILO) resulted in a significant synergistic enhancement of the antiaggregatory effect. The PDE III inhibitor PIR exerted an effective inhibition of platelet aggregation in vivo and in vitro. The inhibitory effects in vitro were synergistically augmented by the prostacyclin analog Iloprost. These platelet inhibitory effects might be of clinical importance.


Effect of prostaglandins and capsaicin on gastric vascular flow and mucosal injury in endothelin-1-treated rats. Infusion of endothelin-1 reduced vascular flow in the isolated perfused rat stomach. Concurrent infusion of iloprost and capsaicin, respectively, did not counteract the flow-reduction caused by endothelin-1. Infusion of prostaglandin (PG)F2 alpha caused vasoconstriction and significantly augmented the endothelin-1-induced flow reduction. In vivo, i.v. infusion of endothelin-1 (50 pmol/kg//min for 10 min) did not cause gastric mucosal damage, but enhanced injury produced by intragastric instillation of 20% ethanol. Intragastric administration of iloprost or PGF2 alpha prevented the pro-ulcerogenic effect of endothelin-1. Similarly, stimulation of afferent sensory neurons by intragastric capsaicin (0.5 mg/kg) protected against damage caused by endothelin-1 and 20% ethanol. Functional ablation of afferent sensory neurons by s.c. administration of 125 mg/kg capsaicin markedly enhanced gastric mucosal damage by intraluminal 20% ethanol. This damage was, however, not further increased by infusion of endothelin-1. These findings show that protection against the proulcerogenic effect of endothelin-1 can occur without antagonism of vasoconstriction. The findings also show that parameters involved in protection such as afferent sensory neurons do not contribute to the pro-ulcerogenic effects of endothelin-1 suggesting that protection against and potentiation of damage rely on different mechanisms.


[A clinical evaluation on postprostatectomy incontinence]. Postprostatectomy incontinence was evaluated in 193 patients, who underwent retropubic prostatectomy (RPP) or transurethral resection of the prostate (TURP) during the past 5 years from 1985 to 1989 at the Kyoto Prefectural University of Medicine. The occurrence rate of postprostatectomy incontinence was 17.2% in the patients who had undergone RPP and 33.3% of those who had undergone TURP. Although postprostatectomy incontinence disappeared within one week in half of the patients, postprostatectomy incontinence remained for more than one month in 4.9% of the patients who had undergone RPP and 3.3% of those who had undergone TURP. The incidence of postprostatectomy incontinence tended to be high in the patients of advanced ages. Postprostatectomy incontinence recognized in this series was classified into stress incontinence and urgency incontinence groups. An analysis of the results of cystometry performed before the operation showed that the volume of maximum desire of voiding in the urgency incontinence group was smaller than that in the group without postprostatectomy incontinence. Furthermore, supposing the urgency degree as a new parameter, which is defined as a ratio of first desire of voiding to maximum desire of voiding, the urgency degree of the urgency incontinence group was higher than that of the group without postprostatectomy incontinence. Patients with an urgency degree higher than 70% were found to have a high probability of urgency incontinence. These findings suggested that advanced age was the risk factor of postprostatectomy incontinence and that a small bladder capacity and high urgency degree were the high risk factors of postprostatectomy urgency incontinence.


An immunohistochemical and prognostic analysis of cytokeratin expression in malignant uveal melanoma. A group of 52 patients with malignant uveal melanoma treated by primary enucleation in 1977-1979 was studied to determine the frequency of immunoreactivity for cytokeratins (CK) in primary and metastatic melanoma, the CK types present, and the prognostic significance of CK expression. By immunohistochemistry, monoclonal antibody (MAb) V9 to vimentin reacted with all 52 formalin-fixed, paraffin-embedded primary tumors and all 31 metastases from 11 patients. MAb CAM 5.2 to CK 8 and 18 reacted with 20 and MAb CY-90 to CK 18 with 25 primary melanomas, whereas MAb KS-B17.2 and MAb CK5 to CK 18 labeled 8 and 6 tumors, respectively. Antibodies to CK 13 and CK 19 each labeled single cells in one specimen, and other CK types were not detected. In 6 primary melanomas, only a few tumor cells were immunopositive for CK 8 and 18, but in 17 cases up to one quarter, and in 2 tumors more than one quarter, of them were labeled. The positive cells were spindle, epithelioid, or intermediate in shape, and tended to be more frequent in mixed than in spindle cell melanomas. MAbs CAM 5.2 and CY-90 did not react with any of the 16 liver metastases, but labeled 7 of 15 other metastases. Metastases were somewhat more common when the primary tumor was immunoreactive for CK 8 and 18, apparently because CKs were more frequent in mixed cell melanomas. Although CK expression is of diagnostic significance and can denote low levels of epithelioid differentiation, it is not an independent prognostic factor in malignant uveal melanoma.


Alcoholic liver disease. Parenchyma to stroma relationship in fibrosis and cirrhosis as revealed by three-dimensional reconstruction and immunohistochemistry. Severe ethanol-induced liver damage is characterized by fibrous dissociation of liver cell plates leading to many apparently isolated hepatocytes. Three-dimensional reconstruction, however, revealed hepatocytes that were surrounded by connective tissue as endpoints of "parenchymal pillars" or in association with liver cell plates and bile ductules. Double immunofluorescence studies displayed the expression of cytokeratin (CK) 7 in bile ducts, including bile ductules, but also in some hepatocytes still organized in liver cell plates. The other bile duct, typical CK, namely CK 19, was only detectable in few hepatocytes. However, the expression of CK 7 and/or CK 19 was less frequent in hepatocytes that were closely associated with bile ductules. CK 7 and CK 19 were also found in some, but not all, Mallory bodies. These observations indicate that the expression of these two CKs is neither related to a transformation of hepatocytes to bile duct-like structures ("ductal metaplasia") nor to the formation of Mallory bodies. Furthermore, double immunofluorescence studies revealed small groups of hepatocytes and bile ductules that were encircled by basement membrane material, thus suggesting the formation of "secretory units."


Immunohistochemical distribution of lipoprotein epitopes in xanthomata from patients with familial hypercholesterolemia. Human xanthomas derived from four subjects with familial hypercholesterolemia (3 homozygotes and 1 heterozygote) were studied by immunohistochemical methods to determine the presence and distribution of lipoproteins, which have been implicated in the pathogenesis of atherosclerosis. Oxidatively modified low-density lipoprotein (OxLDL) epitopes detected with anti-OxLDL monoclonal antibodies, appeared to have a similar distribution in xanthomata to that of macrophages, detected by a cell-specific monoclonal antibody. Double antibody labeling with both an anti-macrophage antibody and an anti-OxLDL antibody demonstrated that OxLDL epitopes are associated with macrophages and occurred intracellularly. Low-density lipoprotein (LDL) epitopes were detected extracellularly, with a distribution that was different from that of OxLDL. In addition, apo(a) epitopes detected by an apo(a) specific monoclonal antibody, had a distribution similar to that of LDL in the dermis and subcutaneous tissues. The observed epitope distribution of LDL, OxLDL, or apo(a) was the same regardless of the method of treatment of the patients from whom the xanthomas were obtained (probucol, simvastatin, LDL apheresis). These findings suggest that OxLDL is likely to play a pathogenetic role in the lipid accumulation by macrophages in xanthomas, and suggest that Lp(a) also may play a role in their pathogenesis.


Monitoring of mite Dermatophagoides farinae allergen-specific IgG and IgG subclass distribution in patients on immunotherapy. House dust mite D. farinae and Der f II-specific IgG and IgG subclass responses were evaluated in 32 adults with perennial rhinitis undergoing immunotherapy for 1 year by means of IgG-RAST and ELISA. The ELISA method, which is based on subclass-specific monoclonal antibodies, could detect 0.5-1.5 ng/ml of specific antibodies. D. farinae and Der f II-specific IgG4 antibodies increased continuously as immunotherapy proceeded, while total IgG and IgG1 antibodies reached a plateau value 6 months after the start of immunotherapy, followed by a slow decrease during maintenance therapy. During the early phase of treatment the concentration of IgG1 and IgG4 antibodies rose, even though the increase of IgG4 antibodies dominated. The positive clinical outcome as measured by the decrease in conjunctival sensitivity was associated with an increased ratio of specific IgG4/IgG1 antibodies as well as the magnitude of the IgG4 subclass response. Quantitation of IgG subclass antibodies in patients undergoing immunotherapy may be of some clinical value, but the clinical usefulness needs to be demonstrated for each type of allergen and possibly also for each assay system.


Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This article describes a general method for detecting pegylated proteins directly after SDS-PAGE. The proteins to which polyethylene glycol (PEG) molecules are attached are stained with a barium iodide solution. The staining is based on the formation of a barium iodide complex with PEG. The described method combines a specific staining of PEG molecules with the high resolution of the SDS-PAGE method. It is shown that pegylated protein is detectable on SDS-PAGE as well as on IEF at concentrations that are not detectable by Coomassie protein staining. This paper also describes the determination of the molecular weight of pegylated hirudin by calibrating SDS-PAGE with polyethylene glycol of different molecular weight. Under the conditions used, PEG showed linear mobility during electrophoresis. However, the use of nonpegylated proteins as standards resulted in incorrect molecular weight values due to the lower mobility of the pegylated protein during electrophoresis. The method described might reflect a general method for determining molecular weight of pegylated proteins.


Laser-induced fluorometric determination of albumin using longwave absorbing molecular probes. A novel fluorescence assay for HSA is described. It is based on longwave-absorbing probes that selectively bind to HSA to form fluorescent complexes. The two probes reported here (Albumin Blue 633 and Albumin Blue 670) are tailored to match the lines of the 633-nm HeNe laser and the 670-nm diode laser, respectively. In both cases, the strong laser-induced fluorescence of the HSA/probe complexes makes the assay sensitive to HSA at trace levels. Detection limits of 0.2 mg/liter were obtained. In addition to its sensitivity, the assay is highly selective for HSA in that the response to other serum proteins is weaker by a factor of at least 100. It also works well on urine, and no significant interferences could be located. Potential interferents are bovine serum albumin and some detergents. Parameters of the probe--HSA interaction were obtained from a Scatchard evaluation. The high specificity and sensitivity of the assay are discussed in terms of molecular geometry of the probes. A photophysical mechanism that leads to the effect is proposed. The assay presents a promising alternative to immunological determinations of HSA since it is less expensive and much more quickly performed.


A microtiter assay for determining protein, acetylcholinesterase activity, and G418 (Neomycin) resistance in cultured cells. The Coomassie brilliant blue assay for the determination of protein has been extended to rapidly and conveniently measure the protein concentration of cells growing in culture in a 96-well microtiter format. Modifications of the standard assay include sodium hydroxide to solubilize the cells and ovalbumin, instead of bovine serum albumin, as a protein standard. The procedure allows a large number of small samples to be assayed simultaneously. Two examples of its use, enzyme-specific activity and drug resistance, are shown. An assay for acetylcholinesterase activity in the same culture plate is demonstrated. G418, an inhibitor of cell protein synthesis, is frequently used to select for cells transfected with the neomycin resistance gene. The required concentration of G418 can be easily determined with this protein assay.


Polymerase chain reaction-aided analysis of gene expression in frozen tissue sections. A simple method for the detection and localization of mRNA in single frozen breast biopsy tissue sections is described. Several extraction procedures were compared. Resuspending sections, which could be left at 0 or -70 degrees C for up to 20 min in H2O containing RNAse inhibitor, optimally released RNA with minimal DNA contamination. Reverse transcription followed by polymerase chain reaction amplification using specific primers yielded products visible by ethidium bromide staining (abundant sequences) or after Southern blotting (low copy message). We found that it was possible, by microdissection, to separate stromal and tumor cells and demonstrated differential expression of several genes in the two populations. With 40 cycles of amplification, dissected stromal and tumor tissue both yielded products encoding glyceraldehyde 3'-phosphate dehydrogenase but only the tumor cells gave products with primers specific for either keratin 19, heat shock protein 89 alpha or the fig oncogene, which encodes one of the fibroblast growth factor receptors that we have recently found to be expressed in breast cancers. With refinement of the dissection technique this offers a very sensitive analytical tool for measuring and defining the cellular sites of synthesis of low-abundance message, requiring only single histological sections.


Replication footprint analysis of cucumber mosaic virus electroporated into tomato protoplasts. Total RNA extracted from cucumber mosaic virus (CMV) strains WT, with its associated satellite CARNA 5 (CMV-associated RNA 5), was successfully electroporated into isolated tomato protoplasts. At various time intervals samples were extracted for total nucleic acids and analyzed by semidenaturing polyacrylamide gel electrophoresis (PAGE). Sequence-specific hybridization probes were used for the detection of viral and satellite RNAs following Northern transfer. The resulting PAGE patterns and/or autoradiographs depict the proportional presence of viral and satellite RNAs in the extracts over time and have been referred to as "replication footprint profiles" (RFPs) of specific CMV/CARNA 5 combinations. The effective isolation and infection of tomato protoplasts, combined with the ability to follow virus/satellite titers during the infection by RFP analysis, yield results similar to those of infected plants and reduces experiments of 21 or more days in whole plants to less than 72 h in protoplasts.


An assay for acidic peptide substrates of protein kinases. The assay of acidic peptides as substrates for protein kinases has not been as easy to perform as testing basic peptides or polypeptides. We have developed a simple, rapid, and cost-effective procedure that allows the design and testing of potential peptide substrates without the constraints imposed by the phosphocellulose filter paper method (the need to incorporate positively charged residues into the peptide sequence). The technique combines the chelation of 32Pi by acid molybdate with PEI-cellulose chromatography. In this way the migration of 32P-labeled Pi, ATP, and protein are impeded while phosphopeptide is eluted in 1.5 ml from a 0.25-ml disposable column. In order to validate the assay we used two angiotensin II analogues as peptide substrates for the protein tyrosine kinase pp60c-src. The assay results using the new procedure were compared to those of the phosphocellulose filter paper technique. We also demonstrated the use of this method to test linear and cyclic peptides that could not be assayed with the phosphocellulose paper technique. This assay will aid those who are attempting to determine the substrate specificity of protein kinases.


Epidural patient-controlled analgesia: an alternative to intravenous patient-controlled analgesia for pain relief after cesarean delivery. Epidural administration of an opioid analgesic by means of a patient-controlled analgesia (PCA) system was compared with conventional intravenous PCA for pain relief after cesarean delivery. One hundred seventeen healthy women were randomly assigned to receive hydromorphone either intravenously (IV-PCA) or epidurally (EPI-PCA) after cesarean delivery with epidural bupivacaine for operative anesthesia. The hydromorphone requirements were 3.4 and 4.2 times more in the IV-PCA group on the first (P less than 0.01) and second (P less than 0.01) postoperative days, respectively. The mean number (+/- SD) of PCA demands during the first 24 h after the operation was 105 (+/- 88) for the IV-PCA group and 33 (+/- 48) for the EPI-PCA group (P less than 0.01). This difference was also significant 24-48 h after surgery. Although the EPI-PCA group utilized significantly less opioid medication, pain and sedation scores were similar in the two treatment groups; however, a significantly larger percentage of patients in the IV-PCA group (46% vs 22%) stated that they felt drowsy during the first postoperative day. Pruritus was reported more frequently in the EPI-PCA (67%) than in the IV-PCA (33%) group. Nausea was experienced by only 10% of patients in the IV-PCA and 6% in the EPI-PCA group. There was no evidence of postoperative respiratory depression, with minimal oxygen saturation values of 93% (+/- 3%) and 94% (+/- 1%) in the IV-PCA and EPI-PCA groups, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Evaluation of a new systolic time interval, the Q-V peak: effects of heart rate, contractile state, and loading conditions in dogs. The authors investigated the effects of alterations in heart rate, contractility, and loading conditions on a newly defined systolic time interval, the Q-V peak, in 46 anesthetized dogs. The Q-V peak was measured as the time from the beginning of the electrocardiographic Q wave to the moment at which the blood flow rate reached its peak in the ascending aorta as determined with an electromagnetic flowmeter. The Q-V peak did not change significantly as the heart rate was varied by atrial pacing between 70 and 110 beats/minute. The Q-V peak shortened when the contractility was augmented with dobutamine (p = 0.0001) and was prolonged when it was depressed with propranolol (p = 0.0001). However, the Q-V peak did not change significantly when the left ventricular end-diastolic pressure or the mean aortic blood pressure was increased to 130% or decreased to 70% of the baseline values. These findings suggest that one may also evaluate left ventricular performance by measuring the time to systole, which the authors define as the Q-V peak.


The giant cell fibroma: a review of 116 cases. A survey of 4342 oral pathology reports accumulated over a five-year period was performed. Diagnoses were 1090 irritation fibromas and 116 giant cell fibromas. A statistical comparison was then made between the giant cell fibromas and the irritation fibromas to determine if there were any differences between these two lesions with respect to sex or race predilection, age distribution, or location in the oral cavity. Finally, various staining techniques were performed on the giant cell fibromas in an attempt to ascertain the origin of the giant cells present in these lesions. The results will be discussed in this paper.


Species differences in 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat, monkey, and human prostate microsomes. The 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes appears to be catalyzed by a single, high-affinity cytochrome P450 enzyme. In the present study we have examined the hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by prostate microsomes from cynomolgus monkeys and from normal subjects and patients with benign prostatic hyperplasia. Our results suggest that although rat, monkey, and human prostate microsomes catalyze the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol, these pathways of oxidation in monkeys and humans are not catalyzed by a single cytochrome P450 enzyme. The ratio of the three metabolites was not uniform among prostate microsomal samples from individual humans or monkeys. The 6 alpha-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol varied independently of both the 7 alpha- and 7 beta-hydroxylation, which varied in unison. The 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by monkey prostate microsomes appeared to be differentially affected by in vivo treatment of monkeys with beta-naphthoflavone or dexamethasone. Treatment of a monkey with dexamethasone appeared to cause a 2.5-fold increase in both the 7 alpha- and the 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol without increasing the 6 alpha-hydroxylation. The 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by human and monkey prostate microsomes, but not the 6 alpha-hydroxylation, was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase. Similarly, the 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by human prostate microsomes, but not the 6 alpha-hydroxylation, was markedly inhibited (greater than 85%) by equimolar concentrations of the imidazole-containing antimycotic drugs ketoconazole, clotrimazole, and miconazole. These results suggest that the 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by monkey and human prostate microsomes is catalyzed by a cytochrome P450 enzyme, whereas the 6 alpha-hydroxylation is catalyzed by a different enzyme which may or may not be a cytochrome P450 monooxygenase. The hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by prostate microsomes from normal human subjects was quantitatively and qualitatively similar to its hydroxylation by prostate microsomes from patients with benign prostatic hyperplasia.(ABSTRACT TRUNCATED AT 400 WORDS)


Antigenic properties of keratan sulfate: influence of antigen structure, monoclonal antibodies, and antibody valency. The influence of (a) antigen structure, (b) type of monoclonal antibody, and (c) antibody bivalency on the immunochemical detection and quantification of keratan sulfate (KS) from aggrecan has been studied. Apparent KS epitope levels were determined by immunoglobulin G (IgG)-enzyme-linked immunosorbent assay (ELISA) in preparations of human aggrecan and in a defined series of lower molecular weight proteoglycan preparations generated by proteolytic and alkali treatment of aggrecan. Gel filtration chromatography showed KS epitope to be preferentially detected in the higher molecular weight fragments of the preparations. In single KS chains the epitope was detected in the chains of higher M(r). The ability of the proteoglycan to inhibit in the IgG-ELISA decreased with a reduction in proteoglycan fragment size, ranging between 6- and 260-fold, depending on the antibody used. This was considered to be a cooperative binding effect. With most antibodies, the sensitivity of the IgG-ELISA (represented by the steepness of the inhibition slope) was also reduced with smaller inhibitor sizes. The lowest limit of detectability (the amount of KS required to generate 20% inhibition) varied by up to 60-fold depending on the antibody used. The use of monovalent Fab fragments instead of the whole IgG anti-KS antibody in the ELISA showed that the bivalency of the antibody also affected the quantitation of the assay. In the Fab-ELISA the assay was found to have an increased detectability (by 9.5-fold with aggrecan as the inhibitor), and the proteoglycan fragments and aggrecan all generated parallel inhibition curves. Although the Fab-ELISA was somewhat influenced by the structural presentation of the KS, this was not apparent for small fragments and single chains. Thus the effects of cooperative binding and antibody valency could be overcome and quantitative data could be obtained for all samples, using papain-digested samples and the Fab-ELISA. Application of this assay to analysis of body fluids showed the KS-containing fragments in synovial fluid, serum, and urine were of different sizes and could be quantified.


Evidence that swainsonine pretreatment of rats leads to the formation of autophagic vacuoles and endosomes with decreased capacity to mature to, or fuse with, active lysosomes. The plant toxin swainsonine causes a variety of biochemical and morphological changes in animal tissues. In rat liver there is an extensive vacuolization which is not accompanied by an accumulation of oligosaccharide. In investigating this proliferation of autophagic vacuoles we have found that swainsonine administration leads to a shift in the density of liver lysosomes as indicated by the distribution of several lysosomal glycosidases in sucrose gradients. Whereas most of these activities are normally found in low density fractions, only a minor portion occurring in high density fractions, the reverse distribution is observed after the administration of microgram doses of swainsonine. Two promoters of the accumulation of autophagic vacuoles, vinblastine and chloroquine, caused the expected increase in very light vacuoles as measured by localization of two acid hydrolases. However, this effect of the two agents was blocked by swainsonine pretreatment. Moreover, swainsonine decreased the degradation of endocytosed asialofetuin and increased the retention of the glycoprotein in very light fractions. These results suggest that vesicle movement and/or fusion is inhibited by the pretreatment with the toxin. That the effects are mediated by a change in vacuolar membrane is suggested by the finding that lysosomes prepared from the livers of swainsonine-fed rats are much more fragile than control lysosomes, more so in metrizamide solutions than in sucrose solutions. The swainsonine may exert its effect through its known ability to alter the biosynthesis of complex glycoproteins, which are abundant and distinctive in lysosomal membranes.


Epitope mapping of monoclonal antibodies to the Escherichia coli F1 ATPase alpha subunit in relation to activity effects and location in the enzyme complex based on cryoelectron microscopy. The interaction of Escherichia coli F1 ATPase (ECF1) with several different monoclonal antibodies (mAbs) specific for the alpha subunit has been examined. The epitopes for each of the mAbs have been localized by using molecular biological approaches to generate fragments of the alpha subunit. The binding of several of the mAbs has also been examined by cryoelectron microscopy of ECF1 Fab complexes. One of the mAbs, alpha II, bound in the region Asn 109-Val 153 without affecting ATPase activity. Most of the mAbs bound in the C-terminal third of the alpha subunit. MAb alpha 1 bound between residues Gln 443 and Trp 513. This mAb activated ATPase activity and was visualized in cryoelectron microscopy, superimposed on the alpha subunit, indicating that the epitope was on the top or bottom of ECF1 in the hexagonal projection. Other mAbs to the C-terminus, including alpha D which also activated the enzyme, reacted between Gly 371 and Trp 513 but failed to bind to small overlapping fragments within this sequence. The epitopes for these mAbs are probably formed by the folded polypeptide which occurs only in Western analysis when long stretches of the alpha subunit are present, suggesting that the C-terminus of alpha is a self-folding domain. In cryoelectron microscopy, Fab fragments for alpha D were seen extending from the sides of the ECF1 complex in hexagonal projection.


Effect of bufalin and pregnanes on vasoreactivity of human resistance arteries. Endogenous Na/K ATPase inhibitory activity has been implicated in salt and water homeostasis in mammals and amphibians. Recent interest has focused on endogenous cardiac glycosides, some progesterone derivatives (pregnanes) and the amphibian bufodienolides. This study has examined the effects of non-planar and planar pregnanes and the bufodienolide bufalin on vasoreactivity of human resistance arteries. Bufalin and a non-planar pregnane caused concentration-dependent potentiation of the tone of submaximally pre-contracted arteries and inhibited endothelium-dependent relaxation, whereas a planar pregnane affected neither response. The relative potency of the compounds studied suggest the results do not simply reflect degrees of Na/K ATPase inhibition. The active compounds may be important in the regulation of vascular tone.


Angiogenesis and rheumatoid arthritis: pathogenic and therapeutic implications. Rheumatoid arthritis can be considered as one of the family of 'angiogenesis dependent diseases'. Angiogenesis in rheumatoid arthritis is controlled by a variety of factors found in the synovial fluid and pannus tissue. Modulation of the angiogenic component of the disease may alter the pathogenesis of the condition, and subsequent cartilage and joint destruction, by reducing the area of the endothelium in the pannus and restricting pannus growth. Current therapeutic strategies exert, to varying extents, an inhibitory effect on the angiogenic process. In particular, the mode of action of the slow acting antirheumatic drugs may be due to their effect on the angiogenic response. The development of novel angiostatic treatments for chronic inflammatory joint disease may lead to a new therapeutic approach in controlling disease progression.


Two novel monoclonal antibodies to fibronectin that recognize the Hep II and CS-1 regions respectively: their differential effect on lymphocyte adhesion. We have obtained two new mAbs to the carboxy-terminal region of fibronectin, namely P3D4 and P1F11, and have studied their binding sites and their ability to block lymphocyte adhesion to fibronectin. ELISA and Western blot analyses showed that P3D4 reacts with both fibronectin chains and both Hep II-containing fragments (58 kDa and 38 kDa). P1F11, raised against the synthetic peptide CS-1, reacted with the 38 kDa fragment and with a 190 kDa fragment derived from the A chain of fibronectin. P1F11 did not react with the 58 kDa fragment thus clearly establishing that 58 kDa comes from the B chain of fibronectin and lacks the CS-1 sequence. mAbs P3D4 and P1F11 were used to evaluate the contribution of the Hep II and CS-1 sites in cell attachment to fibronectin. P3D4 effectively inhibited B cell adhesion to 38 kDa, 58 kDa and fibronectin; P1F11 however produced only limited inhibition, suggesting that lymphocyte interaction with Hep II may modulate further binding to the CS-1 site.


P-selectin (CD62) binds to subpopulations of human memory T lymphocytes and natural killer cells. P-selectin (CD62) is a Ca(2+)-dependent lectin expressed on activated platelets and endothelium. Although P-selectin is known to function as a receptor for myeloid cells, previous studies indicated that P-selectin also bound to a subset of lymphocytes. Using a multi-color immunofluorescence assay we found that purified P-selectin bound to 12.2 +/- 4.1% of peripheral blood lymphocytes and that P-selectin could mediate adhesion of activated platelets to lymphocytes. A subpopulation of CD4+, CD8+, and CD16+ lymphocytes bound P-selectin. There was a marked preference for P-selectin binding to memory cells (CD45RO+) in both the CD4+ and CD8+ populations. Binding to all cell types was Ca(2+)-dependent and blocked by pretreatment of the cells with sialidase. These data suggest that P-selectin may play a role in the recruitment of specific lymphocyte populations to sites of inflammation.


cDNA sequence analysis of CP94: rat lens fiber cell beaded-filament structural protein shows homology to cytokeratins. To study the molecular structure of the gene responsible for a lens fiber cell beaded-filament structural protein of 94kDa (CP94), we isolated its specific cDNA from a rat lens cDNA library by use of anti-mouse CP94 antiserum. The expressed fusion protein kept the epitopes specific against anti-chick CP97 as well as anti-mouse CP94 antibody, and the size was estimated as 190-200kDa, indicating that the cDNA insert of the clone seemed to encode a polypeptide with 80-90kDa in appearance. Northern analysis indicated that CP94 mRNA is expressed only in the lens, and not in the brain, skin, heart, kidney, lung, and liver, and the size was estimated to 2.1-2.3kb. In a lens of inherited microphthalmic mouse, Elo, a trace amount of mRNA with the size closely similar to that of rat mRNA was observed. The entire compiled sequence (1,873bp) showed an open reading frame covering the sequence of 533 amino acids totalling 58,857Da. No sequence homologous to the entire CP94 was found among the entries of any nucleotide and amino acid sequence databases; but with respect to a limited amino acid sequence of N-side region of CP94, a significant homology with cytokeratins was found.


Cloning, expression, and characterization of a gene encoding the human angiotensin II type 1A receptor. The human angiotensin II (AII) type 1a receptor gene and its upstream control sequence has been cloned from a human leukocyte genomic library. The promoter element CAAT and TATA sequences were found at -602 and -538, respectively, upstream from the translational initiation site. The deduced protein sequence is homologous to rat and bovine AT1a receptors (94.7% and 95.3% identity). The expressed gene exhibited high-affinity AII and Dup753 binding and was functionally coupled to inositol phosphate turnover. Northern analysis of human tissues showed AT1 receptor mRNA expression in placenta, lung, heart, liver, and kidney. Using 5' untranslated and coding sequence as probes in a Southern blot analysis, it was established that another AT1 subtype exists in the human genome.


Human insulin-like growth factor binding protein-6 is O-glycosylated. Insulin-like growth factor binding protein-6 is abundant in cerebrospinal fluid and has a marked preferential binding affinity for IGF-II over IGF-I. The present study demonstrates that IGFBP-6 is O-glycosylated but not N-glycosylated. Carbohydrate analysis revealed the presence of approximately 20-30 carbohydrate residues/molecule. Galactosamine, galactose and sialic acid were most abundant, with glucosamine and fucose present in lower concentrations. Mannose was not detected. Enzymatic deglycosylation did not alter the high affinity of IGF binding protein-6 for IGF-II (Ka 4.4 +/- 2.2 x 10(11) M-1) or its preference for IGF-II over IGF-I. Glycosylation of IGFBP-6 may affect its secretion, in vivo stability or localization, but does not affect its ligand binding properties.


Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum. The objective of this study was to determine whether a constitutive isoform of nitric oxide (NO) synthase is present in rabbit corpus cavernosum that could account for the involvement of the L-arginine-NO pathway in neurogenically-elicited relaxation of the corpus cavernosum and, therefore, penile erection. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NO(x) (NO+nitrite [NO2-]+nitrate [NO3-]) by chemiluminescence after reduction of NO(x) to NO by acidic vanadium (III). Equimolar quantities of NO plus citrulline were generated from L-arginine and the formation of both products was time-dependent at 37 degrees C. NO synthase activity was distributed almost entirely to the cytosolic fraction. Enzymatic activity was completely dependent on NADPH, calmodulin, and calcium. Addition of tetrahydrobiopterin increased NO synthase activity by about 30 percent. The NO synthase inhibitor NG-nitro-L-arginine, abolished enzymatic activity. The Km for L-arginine was 17 microM and the Vmax of the reaction was 18 pmol/min/mg protein. These observations indicate that a cytosolic, constitutive isoform of NO synthase, like that found in brain neuronal tissue, is present in rabbit corpus cavernosum.


Action of endothelin-1 on rat astrocytes through the ETB receptor. We investigated the effect of ET-1 on the state of rat cerebral astrocytes (AC) differentiation. AC ceased to proliferate and changed into its differentiated state by treatment with dibutyryl cyclic AMP (DBcAMP). The cell growth activity in DBcAMP-treated AC was stimulated by ET-1 in a dose-dependent manner. Over similar dose ranges, ET-1 suppressed the glutamine synthetase activity in DBcAMP-treated AC. The molar potency of ET-1 in this action was at least 3 orders of magnitude higher than that in mitogenic action in AC under the proliferative state previously reported. Northern blot analysis revealed that ETB receptor mRNA level in DBcAMP-treated AC was markedly higher than that in AC untreated with DBcAMP. Consistently, binding studies showed that the Bmax value for [125I]ET-1 in DBcAMP-treated AC was 16 times higher than that in AC untreated with DBcAMP. These results suggest that ET-1 potently induced a retraction of the differentiation state of AC from fully the specialized state and that the high responsiveness of differentiated AC to ET-1 was partly attributed to the high level expression of the ETB receptor.


Epinephrine induces association of pp60src with Gi alpha in human platelets. Using specific antibodies against the alpha subunit of the inhibitory GTP-binding protein Gi, we analyzed the association of Gi alpha with other cellular components in human platelets. Three tyrosine phosphorylated proteins with molecular mass of 63, 58, and 55 kDa were specifically associated with Gi alpha in resting platelets. Stimulation of platelets with epinephrine, but not with thrombin, induced an increase of the reactivity of the 63- and 55-kDa proteins to anti-phosphotyrosine antibodies on western blotting. By in vitro kinase assay we found that epinephrine induced the association of kinase activity with Gi alpha and that the 63-kDa protein was phosphorylated by this activity. The association of kinase activity with Gi alpha in epinephrine-stimulated platelets paralleled the association of pp60src with Gi alpha, as detected by western blotting analysis using specific anti-pp60src monoclonal antibodies. The interaction of pp60src with Gi alpha may play a role in the mechanism of platelet activation by epinephrine or in the epinephrine-induced potentiation of the action of other platelet agonists.


Nitric oxide mediates tumor necrosis factor-alpha cytotoxicity in endothelial cells. Tumor necrosis factor alpha (TNF-alpha) exerts multiple actions on endothelial cells including among others the expression of pro-coagulant activity and adhesion molecules, and secretion of cytokines. We now show that TNF-alpha induces a time- and dose-dependent cytotoxic effect on cultured bovine aortic endothelial cells. This TNF-induced cytotoxicity, which is preceded by increased production of nitric oxide (NO), is significantly decreased by the NO synthase inhibitor N-iminoethyl-L-ornithine (L-NIO). Dexamethasone, which prevents the expression of cytokine-induced NO synthase in endothelial cells, also inhibits TNF-alpha-dependent cytotoxicity. The results indicate that NO is involved in the cytotoxic effect of TNF-alpha on endothelial cells.


A gradient in expression of the Escherichia coli heat-stable enterotoxin receptor exists along the villus-to-crypt axis of rat small intestine. Binding of Escherichia coli heat-stable enterotoxin to its receptor is critical to the initiation of toxin-induced secretion and diarrheal disease; it is also likely, however, that this receptor binds an endogenous ligand. In order to characterize the expression of the heat-stable enterotoxin receptor in the small intestine, we isolated epithelial cells from villus tip to crypt in rat jejunum and ileum. Binding of radiolabeled toxin was maximal in the villus preparations and gradually decreased along the villus-to-crypt axis, paralleling the decline of sucrase activity. Northern blots of total RNA identified a single heat stable enterotoxin receptor transcript (3.8 kb), predominantly in the villus cell fractions. In situ hybridization demonstrated clear signal in the villus cells with no apparent signal in the crypt cells, lamina propria or muscularis. Expression of this receptor was greatest after enterocytes leave the proliferative cycle and enter villi. This pattern of gene and protein expression may reflect a role of this receptor in binding endogenous ligands which in turn may regulate intestinal ion flux along the villus-to-crypt axis.


Glucagonlike peptide-1(7-36)amide suppresses glucagon secretion and decreases cyclic AMP concentration in cultured In-R1-G9 cells. We previously reported that GLP-1(7-36)amide had glucagonostatic action as well as insulinotropic action in the perfused rat pancreas. In this study, we examined the effect of GLP-1(7-36)amide on glucagon secretion and cAMP concentration in glucagon-secreting cell line, In-R1-G9. GLP-1(7-36)amide (1nM) significantly suppressed glucagon secretion and decreased cAMP concentration in the cells. GLP-1(1-37) did not affect glucagon secretion. It is suggested that inhibitory effect of GLP-1(7-36)amide on glucagon secretion is at least partly mediated by adenylate cyclase system.


Cloning and expression of rat preproendothelin-3 cDNA. We report here the cloning and expression of a rat full-length cDNA encoding preproendothelin-3 (preproET-3). The predicted rat preproET-3 consisted of 167 amino acid residues. As in other ET-family peptides, the mature rat ET-3 was predicted to be produced through unusual processing from a 41-residue intermediate, the big ET-3 in rat. Transient transfection of COS-7 cells with the cloned preproET-3 cDNA resulted in the production of mature ET-3 and this production was inhibited by phosphoramidon, a metaloprotease inhibitor. This suggested that a phosphoramidon sensitive mechanism was involved in the production of ET-3 in the transfected COS-7 cells. Northern blot analysis showed that an approximately 3.0-kb rat preproET-3 mRNA was expressed in rat tissues, including the eye ball, submandibular gland, brain, kidney, jejunum, stomach and spleen. A 2.0-kb and a 3.3-kb mRNA were also detected in the eye ball and small intestine, respectively. The distinct distribution of rat preproET-3 mRNA from that of preproET-1 mRNA suggested that ET-1 and ET-3 played different roles.