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.     

β-Adrenorecptors on the surface membranes of lymphocytes and macrophages

The effect of -adrenergic stimulators adrenalin and isoproterenol and of the -adrenergic blocker propranolol on lymphocytosis and on the macrophage adhesion inhibition reaction was studied in vivo and in vitro on sensitized and intact guinea pigs and Wistar rats. Adrenalin and isoproterenol were shown to inhibit the reaction of the antigen with both sensitized and intact cells. Propranolol abolishes the protective effect of adrenalin and isoproterenol and restores the sensitivity of the cells to antigen.Research Laboratory of Experimental Immunology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 6, pp. 723–725, June, 1978.     

Comparative immunochemical and physicochemical study of the specific β-globulins of pregnancy

Specific -globulins of pregnancy of rats, rabbits, and guinea pigs and human _1G-globulin were shown to be similar in their physicochemical properties. Their dynamics and distribution in the pregnant organism also were similar. These proteins evidently perform similar functions in man and animals.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. 86, No. 8, pp. 213–216, August, 1978.     

Comparison of two models of bleomycin-induced lung fibrosis in mouse on the level of leucocytes and T cell subpopulations in bronchoalveolar lavage

Bleomycin produces pulmonary fibrosis in mice when given as a single intratracheal instillation or as multiple subcutaneous injections. Both models are associated with a significant deposition of collagen in the lungs but differ in the location of the initial lesions. Intratracheal instillation of bleomycin directs lesions to peribronchial or peribronchiolar sites, whereas subcutaneous injection produces lesions in subpleural and perivascular locations. It was therefore of interest to analyse the bronchoalveolar lavage (BAL) fluid for differences in the cellular composition, especially of intraepithelial T lymphocytes characterised by the expression of the integrin E7.Intracheal instillation of bleomycin induced a 5 to 6 times higher number of neutrophils and lymphocytes in BAL fluid than the subcutaneous model. Furthermore, intratracheal instillation of bleomycin induced the infiltration of eosinophilic neutrophils, a peculiar subtype of neutrophils often found in rodents, which were not found in BAL after subcutaneous injection of bleomycin. In both models of bleomycin injection, however, CD4⁺, CD8⁺, NK, and T lymphocytes were detected with dominance of the CD4⁺ T cell population. Analysis of the expression of the integrin E7 revealed similar numbers of E7⁺ cells in the CD4⁺ and T cell populations in both models but significantly lower numbers of E7⁺ T cells were found in the BAL fluid within the CD8⁺ T cell population after subcutaneous injection of bleomycin compared to intratracheal instillation.These results show that a difference in route of bleomycin administration not only causes changes in the localisation of the lesions but that this variation may be reflected in alterations within the BAL leucocyte population especially within the intraepithelial T lymphocytes.     

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.     

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.     

Antidepressive properties of propranolol

Department of Pharmacology, Stavropol' Medical Institute. (Presented by Academician of the Russian Academy of Medical Sciences D. A. Kharkevich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 10, pp. 372–374, October, 1992.     

Role of adrenergic structures in functional control over the cerebral circulation

An investigation of the cerebral circulation by the thermoelectric method showed that stimulation of the cervical sympathetic nerve leads to considerable changes in the blood supply to the brain. The changes in blood flow are biphasic in character: An initial small increase is followed by a decrease below the original level. Pharmacological analysis with and adrenoblockers showed that the constrictor response of the cerebral vessels is due to excitation of-adrenergic structures and the dilator response to excitation of-adrenergic structures. A possible mechanism of these changes is postulated.Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 1, pp. 9–12, January, 1976.     

Physical activity decreases the number of β-adrenergic receptors on human lymphocytes

Summary On intact human lymphocytes a specific binding site (BS) for¹²⁵I-Cyanopindolol (¹²⁵I-CYP), a derivative of the -blocking drug pindolol, was characterized. Inhibition of binding for catecholamines in the following order of potency: l-isoprenaline > l-adrenaline > l-noradrenaline proves the BS as a ₂-receptor subtype. In 77 healthy persons (36 females, 41 males) the number of BS amounted to 2,639±125 BS/cell without any significant correlation to age (17–86 years) or sex. The dissociation constant (K_D) indicating the affinity of iodocyanopindolol to the BS on intact lymphocytes was K _d =1.9±1.1×10^–10 M. A change of the number of -adrenergic receptors on intact human lymphocytes has been measured previously in asthmatics and in the myocardium of patients with congestive heart failure. We investigated a possible change in the -receptors on lymphocytes by physical and mental activity (stress) in physicians going about their daily routine work. Persons left alone in a hospital room reading or sleeping were defined as inactive controls. The number of BS on intact lymphocytes was significantly higher in inactive persons at 8 a.m. (2,230±482 BS) compared to active persons (1,743±285 BS;P<0.05) and at 1 p.m. (2,394±253 BS vs 1,733±556;P<0.05) but not different at 6 p.m. (1,634±578 BS vs 1,768±588 BS;P<0.1). The K _d remained unchanged under all conditions. The serum noradrenaline and adrenaline levels were also measured during the day. The serum adrenaline levels were higher in the active group than in the inactive group at 8 a.m. (46±11 pg/ml vs 22±8 pg/ml;P<0.01) and at 1 p.m. (36±13 pg/ml vs 13±5 pg/ml;P<0.01) but not at 6 p.m. (37±28 pg/ml vs 13±6 pg/ml;P<0.1). Our data clearly show higher serum adrenaline levels but fewer BS on intact human lymphocytes in active persons than in inactive persons at 8 a.m. and 1 p.m. The high catecholamine serum levels may cause a down-regulation of the number of -receptors in the active persons.Abbreviations ¹²⁵I-CYP ¹²⁵Iodocyanopindolol - BS binding site - K _d dissociation constant - cAMP cyclic adenosine monophosphate With support of Deutsche Forschungsgemeinschaft Kr 679/3-I     

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.     

Differential effects of prednisolone and indomethacin on zymosaninduced inflammation in a modified murine tissue-chamber model

A tissue-chamber model of inflammation in mice has been modified and used to investigate the kinetics of zymosan-induced inflammatory mediators such as tumour necrosis factor (TNF), interleukin-1 (IL-1) and prostaglandin E₂ (PGE₂). In addition, the influx of polymorphonuclear leukocytes (PMN) into the chamber fluid and the granuloma surrounding the chamber was measured by myeloperoxidase (MPO) activity using a new microtitre plate assay. TNF and IL-1 reached peak concentrations at 3 and 6 h respectively after zymosan injection. Intermediate high concentrations of IL-1 were observed until the end of the experiment at 72 h, but TNF concentrations decreased from 24 h to biologically insignificant values. In contrast, exudate PGE₂ and MPO activity increased up to 24 h after zymosan injection and remained high until 72 h. At 6h after zymosan challenge, oral pre-treatment with prednisolone (3 to 30mg/kg) dose-dependently reduced TNF, IL-1 and PGE₂ concentrations while indomethacin (0.3 to 3 mg/kg) significantly attenuated PGE₂, slightly enhanced TNF and had no effect on IL-1 concentrations in the exudate. Both drugs had similar potencies against exudate and tissue MPO activities. Prednisolone inhibited IL-1 at 72 h post-zymosan. Indomethacin was more potent than prednisolone against PGE₂ (ID₅₀ of <0.3 versus 0.6mg/kg). The data obtained confirm the usefulness and reliability of this model in evaluating the effects of anti-inflammatory agents on inflammatory mediators induced by zymosan.     

Effect of amino acid derivatives of β-carboline-3-carboxylate on behavior in rats

Brain Research Institute, All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR O. S. Adrianov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 5, pp. 586–587, May, 1989.     

Leukocyte adhesion dynamics in shear flow

Of the white blood cells that traverse the circulation, the polymorphonuclear leukocytes commonly called neutrophils, are the most numerous, numbering 5000 per microliter of blood. In a sense, these cells are the emergency response unit within the body's circulatory highway since they are the first to be recruited at sites of tissue trauma, infection, or inflammation. The chief function of neutrophils is the capture, phagocytosis, and degradation of foreign invaders. To carry out this critical function, neutrophils sense infection via chemotactic receptors that trigger cellular activation upon ligation of as few as 10–100 bacterial or chemokine peptides. Despite this acute sensitivity to stimuli, neutrophils circulate in healthy individuals largely in a passive state, with a very low efficiency of capture and arrest on quiescent endothelium. Here, we define the efficiency as the fraction of all cells passing by a given length of vessel wall that is captured and achieves firm adhesion. Within seconds of chemotactic signaling, adhesion molecules expressed on the plasma membrane of neutrophils are activated and a rapid boost in the efficiency of stable adhesion is detected. This occurs for both homotypic neutrophil–neutrophil adhesion and neutrophil capture and adhesion on inflamed endothelium. With such large numbers of circulating neutrophils, and with their inherent capability to rapidly adhere to postcapillary venular endothelium, the body has evolved a diverse set of mechanisms to regulate the inflammatory cascade that begins with intracellular signaling and leads to cell arrest and extravasation at sites of tissue insult. In this article we will focus on the interplay between particle–fluid dynamics that involve the shear and normal forces that transport cells transversely and axially within the vessel, and the activation and interaction of adhesion molecules that involve receptor–ligand bond formation that enables the neutrophils to resist wall shear stress and adhere to specific sites of inflammation. © 2002 Biomedical Engineering Society. PAC2002: 8719Tt, 8717-d, 8714Ee, 8380Lz