The use of a retroviral vector to identify foetal striatal neurones transplanted into the adult striatum

Summary A retrovirus which encodes -galactosidase was used to infect embryonic rat striatal cells before grafting these cells into the lesioned adult rat striatum. Examination of the grafts after long term survival (8 months) revealed that a few small and large cells expressed large amounts of bacterial -galactosidase activity. The larger diameter cells were identified as neurones by their size, shape and presence of neuronal processes. The identity of the small diameter cell types was not established.     

Transplantation of cerebellar anlagen to hosts with genetic cerebellocortical atrophy

Summary Embryonic cerebellar grafts from genetically normal donors were implanted into the cerebellomedullary cistern of adult Purkinje cell degeneration (pcd) and weaver mutant mice, which are respectively characterized by the selective loss of Purkinje and granule cells. Grafts placed into both mutant recipients exhibited a layered cellular organization reminiscent of the normal cerebellar cortex. Molecular, Purkinje, and granule cell layers were identifiable. Grafted Purkinje cells displayed characteristic cytological features, such as hypolemmal cisterns in association with mitochondria in the perikaryon, and lamellar structures in their axons. The cytological features of granule cell somata in the grafts appeared similar to those of mature granule cells. Electron microscopic examination of the molecular layer of the grafts revealed the presence of parallel fibers, which were not oriented in a parallel fashion; axon terminals of such fibers were often presynaptic to dendritic spines. The number of parallel fibers was markedly reduced in grafts implanted into both mutants compared to the normal cerebellar cortex; however, this phenomenon is commonly seen in cerebellum in tissue culture and in cerebellar transplants into normal hosts. It is concluded, therefore, that the environment of the mutant hosts does not affect the survival of Purkinje or granule cells and that transplantation of solid cerebellar grafts in the neurological mutants studied does not seem to pose any apparent limitations beyond those inherent to the process of cerebellar growth and differentiation outside its normal environment.Dedicated to Herbert von Karajan     

Neural nicotinic acetylcholine responses in solitary mammalian retinal ganglion cells

Using the patch-clamp technique,whole-cell recordings from solitary rat retinal ganglion cells in culture have established the nicotinic nature of the acetylcholine responses in these central neurons. Currents produced by acetylcholine (5–20 mol/l) or nicotine (5–20 mol/l) reversed in polarity near –5 mV and were unaffected by atropine (10 mol/l). Agonist-induced currents were blocked by low doses(2–10 mol/l) of the classical ganglionic antagonists hexamethonium and mecamylamine, as well as by d-tubocurarine and dihydro--erythroidine (the latter two do not discriminate clearly between ganglionic and neuromuscular junction receptors). Treatment with the potent neuromuscular blocking agent -bungarotoxin (10 mol/l) did not affect the cholinergic responses of these cells, while toxin F (0.2 mol/l), a neural nicotinic receptor antagonist, readily abolished acetylcholine-induced currents. Thus, the experiments performed to date show that the nicotinic responses of retinal ganglion cells in the central nervous system share the pharmacology of autonomic ganglion cells in the peripheral nervous system. The ionic current carried by the nicotinic channels was selective for cations, similar to that described for nicotinic channels in other tissues. In addition, single-channel currents elicited by acetylcholine were observed in whole-cell recordings with seals > 5 G as well as in occasional outside-out patches of membrane. These acetylcholine-activated events, which had a unitary conductance of 48 pS and a reversal potential of 0 mV, represent the ion channels that mediate the neural nicotinic responses observed in these experiments on retinal ganglion cells.     

A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae

Summary A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. oryzae strain deficient in orotidine-5-phosphate decarboxylase (pyrG) and the vector pA04-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. oryzae pyrG mutant with circular PA04-2 resulted in the appearance of Pyr⁺ transformants at a frequency of up to 20 per g of DNA, whereas with linear pA04-2 up to 200 transformants per g DNA were obtained. In 75 % of the Pyr⁺ transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pA04-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding -galactosidase, and uidA, encoding -glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.     

Measurement of tumor load and distribution in a model of cancer-induced osteolysis: A necessary precaution when testing novel anti-resorptive therapies

The Arguello model of cancer metastasis to bone has been used extensively to study breast cancer-induced osteolytic disease. The effects of therapy on skeletal disease and on tumour burden in soft organs are traditionally measured using radiography and/or time-consuming histomorphometry, respectively. The purpose of this study was to develop a sensitive and efficient method for evaluating tumour burden in vivo using MDA-231 cells transduced with the E. coli lacZ gene (MDA-231BAG). Osteolysis was measured by radiography and tumour burden was measured histomorphometrically or biochemically. In untreated mice, measurements of tumour burden in bone extracts using human cytokeratin-associated tissue polypeptide antigen (TPA) ELISA or E. coli beta-galactosidase (beta-gal) activity immunoassay reflected the extent of osteolytic disease as measured by radiography; however, tumour load could be detected before onset of osteolysis. When monitoring the effect of therapy (0.2 mg/kg ibandronate/day), radiography alone proved to be insufficient. Mice treated with the bisphosphonate ibandronate from time of inoculation with cancer cells had no radiologically visible signs of osteolysis but significant tumour load was measured in the bone extracts using these assays. Furthermore, beta-gal activity could be used as a measurement of tumour load in soft organs, and unlike other human breast cancer markers expressed by the MDA-231 cells in vitro, beta-gal activity was detected in the serum of mice with progressive disease. In conclusion, we describe an efficient model of breast cancer-induced osteolysis to quantify the effect of therapy on disease load and distribution, which could be beneficial in evaluating novel therapies for the treatment of the disease.     

Impaired monocyte-to-macrophage maturation in patients with lymphadenopathy syndrome

Thein vitro maturation of monocytes from patients with lymphadenopathy syndrome (LAS) was studied by means of enzymatic activity performed during a 7-day incubation period. Monocytes from LAS patients, healthy homosexuals, and healthy heterosexuals were assayed for -galactosidase and -N-acetylglucosaminidase activity on days 3, 5, and 7 of culture. The LAS monocytes had significantly lower (P<0.01) absolute levels of both enzymes compared with controls or healthy homosexual subjects. All three groups showed a linear increase in enzyme activity over time. There was no statistical difference between the slopes of the curves of enzyme activity vs time for the three groups, indicating that the rate of increase in enzyme activity was similar for the groups. These results suggest that monocyte-to-macrophage maturation is impaired in LAS. LAS monocytes are initially less mature than those of healthy homosexuals or heterosexuals but retain their capacity to mature during incubationin vitro.     

Stoffwechsel und Kinetik von β-Acetyldigoxin

Zusammenfassung Die nach oraler Gabe von-Acetyldigoxin bei Menschen untersuchte Pharmakokinetik zeigt gegenüber Digoxin kein unterschiedliches Verhalten. Im Blut findet sich nur Digoxin. Die Ergebnisse der Dünnschichtchromatographie des Urins und Stuhls weichen von denen des Digoxin nicht ab. Lediglich die quantitativ bessere Resorption unterscheidet-Acetyldigoxin vom oral verabfolgten Digoxin.     

Immunoautoradiographic study of species specificity of β-globulins of pregnancy

A comparative immunoautoradiographic analysis was made of human _1-G-globulin and specific pregnancy -globulins of guinea pigs, rats, and rabbits. The antigens tested were found to possess high species specificity.Department of Biochemistry, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. M. Lopukhin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 6, pp. 721–723, June, 1977.     

The dynamics of insulin release from mouse pancreatic islet cells in suspension

Summary The overall dynamics of glucose-induced insulin release was strikingly similar in dispersed cells and intact islets perifused in parallel. Both preparations exhibited a latency of 1–2 min, after which period there was a brisk rise of insulin release followed by a sustained second phase. During the second phase, insulin release from dispersed cells attained a stable plateau rate, whereas the release from intact islets continued to rise. Epinephrine (1 M) inhibited the release in both preparations, but the return to basal rate was faster in the dispersed cells than in the intact islets. The dispersed cells oxidized glucose at a constant rate for at least 60 min; the glucose oxidation was markedly sensitive to changes of the glucose concentration in the range of 3–20 mM.     

The effect of intravenously administered salbutamol on serum potassium in asthmatic and nonasthmatic atopic subjects

The role of adrenergic mechanism in the pathogenesis of allergic disease is controversial. Recent experimental and clinical reports have suggested that -adrenergic blockade impairs and stimulation enhances extrarenal potassium uptake in humans. This led us to study the effect of the intravenous administration of salbutamol, a specific -2-adrenergic agonist, on serum potassium in 9 healthy subjects and in 23 patients with allergic asthma and/or rhinitis. Serum potassium fell significantly and reached a peak decline at the end of venous infusion in all the normal subjects. Seventeen atopic subjects showed a lower or absent serum K⁺ decrement: there was no difference between asthmatic and rhinitic patients. There was no relation among the salbutamol-induced serum potassium decrement, serum glucose increment, blood pressure and heart-rate changes, and nonspecific bronchial reactivity. These findings suggest that -2-adrenergic hyporesponsiveness is present only in some allergic patients.     

Potassium tolerance and bronchial reactivity in asthmatic and nonasthmatic atopic subjects

Abnormal autonomic nervous system responsiveness may contribute to the pathogenesis of allergic diseases. Therefore, we measured the -adrenergic systemic (metabolic) responsiveness by means of acute potassium load in 10 normal healthy subjects and in 19 patients with allergic asthma and/or rhinitis. Ten allergic patients showed a greater potassium increment, as in normal subjects, when potassium was infused in the presence of propranolol. There was no difference between asthmatic and rhinitic patients. We then examined the relation between the response to potassium tolerance and the nonspecific, nonpharmacological bronchial reactivity in response to inhalation of ultrasonically nebulized distilled water. Some allergic patients showed bronchial hyper-reactivity, while others did not show a difference compared with the controls; there was no significant difference between asthmatics and rhinitics, and there was no relation between nonspecific bronchial reactivity and potassium load tolerance. These findings suggest that systemic -adrenergic hyporesponsiveness may be present only in some allergic patients. There is no demonstrable relation among atopic state, nonspecific, nonpharmacological bronchial reactivity, and systemic -adrenergic hyporesponsiveness.     

Statin treatment and a disease-specific pattern of β-amyloid peptides in Alzheimer’s disease

According to the amyloid cascade hypothesis, sporadic Alzheimers disease (AD) is caused by the production and aggregation of -amyloid (A), and the production of A has recently been linked to the metabolism of cholesterol. We have previously published clinical studies where the effect of statin treatment on A production has been investigated. No effect on A was found, which is in disagreement with cell and animal studies. In the present study we investigated the effect of statin treatment on a disease-specific pattern consisting of a C-terminally-truncated quintet of A peptides. Nineteen patients with AD were treated with simvastatin for 12 months and the quintet of A peptides were analysed in cerebrospinal fluid before and after treatment. Also included was a group of 15 untreated patients with AD. We found that the A peptide pattern at baseline was in agreement with earlier findings; however, we did not find any change in the A peptide pattern after statin treatment. We suggest that clinical studies with extended treatment periods are performed where higher dosages of statins are used. We also believe that the pleiotropic effects of statins should be investigated further in order to elucidate the connection between Alzheimers disease and statin treatment.     

Integrin-mediated entry into S phase of human gastric adenocarcinoma cells

The integrins are a family of integral membrane receptors that participate in binding to various extracellular and cell surface proteins during adhesion, migration, and homing of normal and neoplastic cells. In this study, we characterized the involvement of integrins in mediating the growth of an adhesion-dependent gastric adenocarcinoma line, ST2. This line was distinguished and selected for study based on its inability to grow when suspended in soft agar or plated on poly(2-hydroxyethyl methacrylate)-coated dishes. ST2 cells arrested in G0/G1 of the cell cycle when deprived of adhesion to substrate. Using purified matrix components, collagen was found to be highly active in promoting 1 integrin-mediated cell attachment and spreading. Subsequent to spreading on collagen, the cells were released from G0/G1 block and progressed into S phase. Monoclonal antibodies to 2 or 1 integrin blocked the reinduction of both cell spreading and entry into S phase. These studies suggest that during the metastatic process, integrin receptor interaction with the insoluble matrix may be an important step leading to proliferation of some tumors.     

Expression of insulin-like growth factor mitogenic signals in adult soft-tissue sarcomas: significant correlation with malignant potential

The insulin-like growth factor (IGF) signal transduction system involves receptors, ligands and binding proteins (IGFBPs) that have been shown to have mitogenic and distinct anti-apoptotic effects on malignant cell lines of both epithelial and mesenchymal origin. Expression of the IGF signal system might be a mechanism by which human soft-tissue sarcomas (STS) obtain a proliferative advantage over normal adjacent tissues. IGFBP2, one of at least six different binding proteins identified to date, is secreted by most sarcoma cell lines and appears to be involved in cell proliferation and transformation. Circulating levels of this protein are markedly increased in malignancy. We have assessed 46 adult STS specimens of low, intermediate and high pathological grade of malignancy for the immunohistochemical expression of IGFBP2, IGF1, IGF2, IGF1 receptor- and - (IGF1R/). The protein expression was measured by quantitative color video image analysis and semi-quantitative evaluation, and the measurements correlated well (Spearman, P<0.001). Using both methods, significant differences in expression of IGFBP2 among each of the three grades, expression of IGF2 between intermediate and high grade, and expression of IGF1R between low-intermediate and low-high grade were observed (Dunnett test, P<0.05). Multiple regression analysis for both quantitative and semi-quantitative data confirmed the significance of the relationship and independence of the proteins, except IGF2. We concluded that IGFBP2 and IGF1R are independent predictors of the malignant potential of adult STS.     

The effect of beta-adrenergic blockade on leg blood flow with repeated maximal contractions of the triceps surae muscle group in man

Summary The effect of -adrenergic blockade on torque output and leg blood flow was examined in seven healthy young men during repeated maximal isometric voluntary contractions of the triceps surae muscle group. Exercise was performed in either a bent- or straight-leg position during each of four drug treatments: placebo, propranolol, metoprolol, oxprenolol. Contractions were sustained for 5 s with 5 s relaxation for a total of 10 min followed by a 10-min recovery. Leg blood flow was measured during the 5 s relaxation separating contractions using strain gauge plethysmography. Torque output decreased during the 10-min contractions with no differences between the four drug treatments. Leg blood flow was lower with -blockade during the initial stages of exercise and recovery in the bent-leg position but no differences were observed after 3 min exercise or recovery. Leg blood flow in the straight-leg position was not different between any of the four drug treatments, but it was significantly less than in bent-leg exercise. The lower blood flows during the initial stages of exercise in the -blocked conditions probably reflect a slowing of the central cardiovascular response because of ₁-receptor blockade of the heart rather than on the ₂-receptors effects on peripheral vacular resistance. It is concluded that local vasodilator substances released from the working muscle may play a more important role than ₂-receptor stimulation of smooth muscle in skeletal muscle resistance vessels in regulating local muscle blood flow during maximal exercise of the triceps surae muscle group.