Expression of surface membrane IgG on pokeweed mitogen-reactive anti-tetanus toxoid antibody-producing cells

Anti-tetanus toxoid antibody-producing cells, differentially expressing surface membrane IgM, were analyzed for the additional expression of surface membrane IgG. ⁺ and ^– cells were rosetted with anti--ox red blood cells and separated by density centrifugation into fractions enriched or depleted or ⁺ cells. These B-cell subsets were assayed for the production of IgM and IgG anti-tetanus toxoid antibody and total IgM and IgG. The results indicated that the majority of anti-tetanus toxoid antibody synthesis in the ^– fraction was by ⁺ cells. In the ⁺ fraction, however, both IgM and IgG anti-tetanus toxoid antibody production was detected in the ^+– and ⁺⁺ fraction. The inclusion of isotype-specific antisera during the first 2 days of culture further established that was expressed on the surface of the majority of the precursors for IgG anti-tetanus antibody productionin vitro. Studies performed to determine the culture requirements of ^– and ⁺ cells revealed that production of IgG anti-tetanus toxoid antibody by both cell subsets was dependent on T cells and pokeweed mitogen. However, some ^– cells could produce IgG in the presence of T cells alone.     

Effects of the kar gene on cytoplasmic mixing and mitochondrial genome suppressiveness,and consequences for cytoduction of petite DNA in Saccharomyces cerevisiae

Summary During a series of cytoduction experiments to transfer Saccharomyces cerevisiae mitochondrial genomes from one nuclear background to another, using the karl-1 nuclear fusion mutation, one of the five petite genomes used proved difficult to transfer. This genome, ^- F13, was highly suppressive (90%) in its original nuclear background. Molecular and genetic studies on the putative karl-1 ^–F13 cytoductant were done to discover the nature of this difficulty. They showed that while the ^–F13 was maintained in a karl-l background, zygotes from a mating with a ⁰ strain showed poor cytoplasmic mixing and therefore inefficient ^–F 13 DNA transfer into first zygotic buds. This also caused a reduction of ^–F13 suppressiveness to 20–30% in crosses with different ⁺ strains. The effect was genome specific since another highly suppressive petite in the karl-l background did not show suppressiveness reduction when crossed to ⁺. The nature of suppressiveness modulation is discussed. Since the ^–F13 genome was eventually transferred using a modification of the original scheme, the problems were not caused by the inability of the acceptor nuclear background to maintain the ^–F13 genome.     

Biphasic short-circuit current response to noradrenaline mediated by Ca2+ and cAMP in cultured rat epididymal epithelium

A study was carried out to investigate the short-circuit current (I _sc) response to noradrenaline (NA) and the signal transduction mechanisms involved in cultured rat cauda epididymal epithelium. In normal Krebs-Henseleit solution, NA (10 mol · l^–1) added basolaterally elicited a biphasic I _sc response consisting of a transient spike followed by a second sustained response. The biphasic response was almost abolished by removing ambient Cl^–. Preloading the tissues witha cell-permeant Ca²⁺ chelator, 1,2-bis(2-aminophenoxy) eth-ane-N,N,N,N,-tetraacetic acid acetoxymethyl ester (BAPTA/AM), or pretreating them with thapsigargin (Tg), a microsomal adenosine triphosphatase inhibitor abolished the initial spike in the I _sc response to NA, but had little effect on the second component. Pretreating the tissues with a non-selective -antagonist, nadolol, reduced the second I _sc response in a dose-dependent fashion but the initial spike was not affected. Microfluorimetric studies showed that NA (100 mol · l^–1) elicited single Ca²⁺ spikes in isolated epididymal cells, which could be abolished by prior treatment with Tg. Biochemical assays showed that NA (10 mol · l^–1) increased intracellular cyclic adenosine monophosphate concentration ([cAMP]_i) and the response was abolished by prior treatment with nadolol (50 mol · l^–1). The results showed that NA elicited a biphasic I _sc response mediated by a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a rise in [cAMP]_i. The Ca²⁺-mediated I _sc response had a faster onset and more transient action than the cAMP counterpart. It is suggested that NA released from noradrenergic nerve endings regulates transepithelial Cl^– secretion in the epididymis thereby providing the specialized millieu vital for sperm storage and maturation.     

Action of KCN on intact and refractory platelets

The action of potassium cyanide on ADP-induced aggregation of intact and refractory platelets was investigated. Cyanide (5·10^–4 M) had virtually no effect on the aggregation of intact cells but stimulated aggregation of refractory platelets. The stimulating effect of the inhibitor on aggregation was not connected with liberation of further quantities of ADP from the cells. Differences in the sensitivity of the aggregating power of intact and refractory cells to partial depression of metabolism is discussed in terms of the calcium model of autoregulation of platelet aggregation suggested previously.Research Institute of Child and Adolescent Physiology, Academy of Pedagogic Sciences of the USSR, Moscow. (Presented by Academician V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 8, pp. 940–943, August, 1976.     

Nitrogen metabolite repression in Aspergillus nidulans: A farewell to tamA?

Summary Previous work has established that nitrogen metabolite repression in Aspergillus nidulans is mediated by the positive acting regulatory gene areA. Pateman and Kinghorn (1977) proposed that the gene tamA plays an equally important regulatory role in nitrogen metabolite repression as the result of work with tamA^r-50, an allele leading to inability to utilise nitrogen sources other than ammonium, and tamA^d-1, an allele leading to nitrogen metabolite derepression. Both tamA^r-50 and tamA^d-1 were subsequently lost. We have therefore attempted to reconstruct Pateman and Kinghorn's work with tamA. We propose that tamA^r-50 was in fact a pyroB^– tamA^– double mutation. pyroB^– mutations lead to a block in vitamin B₆ biosynthesis which can be supplemented by extremely high concentrations of ammonium. tamA^– mutations, possibly as the result of a membrane alteration, reduce the concentration of ammonium required to supplement the pyroB^– auxotrophy. There is, however, no evidence that pyroB^– or tamA- mutations, alone or in combination, affect the regulation of the levels of a number of enzymes subject to nitrogen metabolite repression. Reversion of pyroB^– strains constitutes a powerful positive selection technique for obtaining a wide variety of mutations in glnA, the probable structural gene for glutamine synthetase. We suggest that the nitrogen metabolite derepressed phenotype attributed to tamA^d-1 might have resulted from an extremely leaky glnA^– mutation.     

Effect of the factors of space flight on erythropoiesis

After orbital flight for 19–22 days on the satellites Kosmos-605 and Kosmos-782 erythropoiesis of rats was inhibited and the morphology of their megakaryocytes was modified. These changes disappeared by the 25th–27th day after the flight.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 2, pp. 238–240, February, 1977.     

Thalamic inhibition of locomotion induced by mesencephalic stimulation

By stimulation of a region of the thalamus with its center corresponding to Horsley-Clarke coordinates A7, L2, H2, locomotion of the lightly anesthetized cat with an intact brain can be inhibited, whether evoked by stimulation of the subthalamic or of the mesencephalic locomotor region.Laboratory of Physiology of Movements, Institute for Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. (Presented by Academician V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 8, pp. 903–906, August, 1976.     

Confirmation of the hypothetical diffusion barrier in the region of the Ranvier node of myelinated nerve fibers

Experiments were carried out on single nerve fibers with a covered node. The nerve fiber was isolated together with one to three fragments of neighboring fibers adjacent to the node and which were firmly connected to the nodal ends of the myelin sheath by means of numerous connective-tissue membranes, thereby strengthening the node and protecting it against stretching during dissection. In potassium-free solution after-hyperpolarization of the nerve fiber membrane with an amplitude of 2.7 mV develops immediately after the end of the spike. Its origin is connected with maintenance of the increased potassium permeability of the membrane. During repetitive stimulation of the covered node the amplitude of after-hyperpolarization falls successively, as a result of a decrease in the outward potassium current caused by a reduction in the potassium electrochemical gradient. These observations are regarded as confirmation of the hypothesis expressed previously on the existence of a diffusion barrier in the region of the Ranvier node due to accumulation of K⁺ in the juxtamembranous space.Department of Anatomy and Physiology of Man and Animals, Ul'yanovsk Pedagogic Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 11, pp. 517–519, November, 1978.     

Calcium binding to an elastic portion of connectin/titin filaments

-Connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the -connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on -connectin. Purified -connectin was digested by trypsin into 1700- and 400-kDa fragments, which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 × 10⁷ M^–1 and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 M. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of -connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of -connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl₂ and 70 M leupeptin to split connectin filaments into -connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200 ± 33 kDa (mean ± SD), while - and -connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 m at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N₂ line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction–relaxation cycle of skeletal muscle.     

Untersuchungen Mit J131-Markiertemβ-Globulin Zur Frage Der Abstammung Der Liquoreiweisskörper

Zusammenfassung Die Abstammung der-Globuline im Liquor wurde bei 20 Fällen mit den verschiedensten neurologischen Erkrankungen untersucht. — Die spezifische Aktivität der-Globuline war bei normalen und pathologischen Liquors ausnahmslos niedriger als im Serum. Es treten demnach nur einzelne Serum--Globuline in den Liquor über, ein verschieden großer Anteil der Liquor--Globuline wird im Liquorraum gebildet. Die-Fraktion im Liquor besitzt einen Serumanteil, von dem die liquoreigenen oder auch cerebrogenen-Globuline unterschieden werden können. Beziehungen zwischen der Höhe des liquoreigenen-Globulinanteils zu einzelnen Krankheitsgruppen waren nicht herzustellen. Es ließ sich aber zeigen, daß eine Erhöhung des elektrophoretisch ermittelten relativen-Globulingehaltes im Liquor bei pathologischen Fällen nicht — wie bisher angenommen wurde — mit einer Zunahme des cerebrogenen Eiweißes einherzugehen braucht. — Die Bedeutung des liquoreigenen-Globulins ist noch unbekannt, auch ist es nicht möglich zu entscheiden, welche einzelnen Proteine innerhalb der-Fraktion im Liquorraum entstanden sind oder aus dem Serum stammen.Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Character of the receptors of Auerbach's plexus in the stomach and small intestine

Movements of the stomach and small intestine in eight fed dogs with intact vagus nerves were recorded graphically by a balloon method. Subcutaneous injection of a mixture of benzohexonium (0.125–0.5 ml of a 2.5% solution) with atropine (0.125–0.25 ml of a 0.1% solution) or oxyphenonium (0.125–0.25 ml of a 0.1% solution) first inhibits food motor activity and then converts it to periodic. A similar effect after injection of 0.5–1.0 ml of a 0.1% solution of atropine was found in only two dogs and after injection of 1.0 ml of a 0.1% solution of oxyphenonium in only one dog. Since the preservation of periodic contractions after feeding is characteristic of vagotomized dogs, it is concluded that a pharmacological vagotomy was obtained in the animals studied. It was postulated that the number of muscarinic receptors on cells of Auerbach's plexus exceeds the number of nicotinic receptors.Department of Pharmacology and Experimental Pathology of the Digestive Apparatus, Institute of Physiology, T. G. Shevchenko Kiev University. (Presented by Academician of the Academy of Medical Sciences of the USSR, V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 9, pp. 316–319, September, 1977.     

Nucleotide modification of tRNA in. the yeast Saccharomyces cerevisiae is not Affected by the ψ factor which modulates suppression efficiency

Summary We have examined the tRNAs of two related strains of Saccharomyces cerevisiae, ⁺ and ^–, which differ with respect to an extrachromosomal genetic element that modulates the expression of genotypic and phenotypic suppression. Both the pattern of tRNAs synthesized and the level of nucleotide modification of several selected tRNA species were found to be the same in the ⁺ and ^– strains.     

Action of hemolysate of polycythemic animals on proliferations of bonne marrow cells

Erythrocytic inhibitor from polycythemic rats depressed mitotic activity of cells of of the erythroid series in mice by 40%. The inhibitory effect lasted about 12 h. The inhibitor acted on the G₂ period. The points of application of the inhibitor were not only blast forms of the erythron but also hematopoietic stem cells.Department of Pathological Physiology, Moscow Medical Stomatological Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Fedorov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 3, pp. 337–339, March, 1977.     

Effect of adrenergic receptor blocking agents on mitotic activity of the regenerating liver

The -adrenergic blocking drug phentolamine was injected into male rats 1 h before resection of 70% of the liver and again 24 h after the operation. Phentolamine inhibited mitotic activity of the regenerating liver. Two injections of propranolol, a -adrenergic blocking drug, at the same times caused an increase in mitotic activity. It was concluded that adrenalin, which excites -adrenergic receptors, may inhibit regeneration. By its action through -adrenergic receptors, however, adrenalin stimulates this process.Department of Physiology of Animals, N. G. Chernyshevskii Saratov University. Central Scientific-Research Laboratory, Saratov Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 11, pp. 1373–1374, November, 1976.     

Ribonuclease activity of the “myofibrillary” fraction of normal and autolyzed muscle tissue

The separation in a sucrose gradient of the myofibrillary fraction of normal and autolyzed muscle tissue gave 4 components. During post-mortem destruction of the tissue there was observed a slight decrease of the myofibrillary fraction yield and also certain changes in the distribution of protein between different components. Under the selected conditions RNase activity was found in all 4 components. During the course of autolysis enzymatic activity increased in the whole myofibrillary fraction, as well as in the lysosomal-mitochondrial components of myofibrils.Research Laboratory, Ministry of Health of the USSR, Moscow. Translated from Buylleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 5, pp. 533–537, May, 1978.     

Metabolic control of respiratory neuronal activity and the accompanying changes in breathing movements of the rabbit

Summary The property of the neuronal membrane to be permeable to metabolic modifiers of two regulatory enzymes has been utilized to manipulate the spike activity of inspiratory (I) and expiratory-inspiratory (EI) neurons of the bulbar respiratory centre. The neurons have been classified according to their response to lung distension or collapse (- or -type) and to hyperventilation (tonic firing denoted by +, cessation of activity by –). Using extracellular microelectrodes for single unit recording, the medulla oblongata was superfused with a metabolite-containing CSF. The various neuronal sub-types exhibited a differential activating or inhibitory response to one or several metabolic effectors. For example I ⁺ units were activated by 5 mM glucose-6-phosphate (G-6-P) and 3.5 mM 3-phosphoglycerate (3-PGA), which both inhibited I ⁺ neurons, while 5 mM AMP inhibited I ⁺ much more strongly than I ⁺ cells. The spike density of I ^– and I ^– neurons was increased in the presence of 2.5 mM fructose-6-phosphate and 3.5–5 mM AMP, but became reduced by G-6-P. In contrast, 3 mM fructose-1,6-diphosphate and 5 mM 3-PGA activated the I ^– but inhibited the I ^– neurons. The EI units were characteristically activated by 10 mM citrate, which inhibited all I-type neurons. Activations of the I and I neurons led to an accelerated respiratory rate and a higher tidal volume, while the opposite was true for EI neurons. Intravenous injection of metabolites could not duplicate the striking effects under local applications.Supported by the Deutsche Forschungsgemeinschaft, Grant Ch 25/1.     

Myofibril and sarcoplasmic reticulum changes with exercise and growth

Summary The intent of this study was to observe the effects of different treadmill running programs upon selected biochemical properties of soleus muscle from young rats. Young 10 day litter-mates were assigned to endurance (E), sprint (S) and control (C) groups. Each was partitioned into either 21 or 51 day exercising groups and 10 day controls. For C the myofibril ATPase activity at 21 and 51 days were lower than 10 day activity (p0.05). In the 51 day E group ATPase activity (0.378±0.009 mol Pi·mg^–1·min^–1) was greater than at 10 and 21 days (0.307±0.006 and 0.323±0.008 mol Pi·mg^–1·min^–1) (p0.05). No change occurred in the S group from 10 to 21 and 51 days (p0.05). Both the 21 and 51 day S (0.318±0.011 and 0.399±0.010 mol Pi·mg^–1·min^–1) and E (0.323±0.008 and 0.378±0.009 mol Pi·mg^–1·min^–1) groups had higher activity compared to the C group (0.193±0.029 and 0.172±0.031 mol Pi·mg^–1·min^–1) (p0.05). Maturation (10–51 day) resulted in a lowered sarcoplasmic reticulum (SR) yield and Ca²⁺ binding (p0.05) while Ca²⁺ uptake ability did not change (p0.05). SR yield, Ca²⁺ binding and uptake were not altered with S training (p0.05). The E training resulted in greater Ca²⁺ uptake at 51 days compared to C and S (p0.05), with no change in Ca²⁺ binding (p0.05). The data suggest that E training alters the normal development pattern of young rat soleus muscle.Supported by grants A-6449 and A-0425 from the Natural Sciences and Engineering Research Council of Canada     

Properdin and the protein composition of the lymph and blood during resuscitation

Experiments on dogs showed that terminal blood loss followed by resuscitation by injection of autologous blood into the bone marrow causes a regular redistribution of proteins between the blood, limbs, and tissues. Retention of properdin and -globulins is observed in the interstitial tissue and is not abolished by the resuscitation measures; a stress discharge of -globulins is also found from the lymph nodes.Department of Pathological Physiology, N. P. Ogarev Mordovanian University, Saransk. (Presented by Academician of the Academy of Medical Sciences of the USSR P. D. Gorizontov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 7, pp. 24–26, July, 1977.     

Sodium current in single myocardial mouse cells

Morphologically intact single myocardial cells of the adult mouse show a length of 132±20 m, a width of 21±5 , and a height of 10±4 m (all mean ± SD) and are brick-like in shape. A one suction pipette method is used for voltage clamp of those single cells. The determined time constant of capacitive current =35±14 s is very short. Series resistancer _s, membrane resistancer _m, and membrane capacityc _m are calculated to be 192±48 k, 6.1±1.1 M, and 186±92 pF (all mean ± SD), respectively. Assuming the specific unit membrane capacitance of 1 F/cm², a total membrane area of 1.86×10^–4 cm² is determined yielding a specific membrane resistanceR _m of 1,134 cm². Settling time of voltage clamp is 30 s. TTX-block of sodium current is described by 1:1 binding with aK _D value of 1.4×10^–6M. Using a reduced extracellular sodium concentration the maximum Na current is between 25 and 40 nA at voltages between –40 and –30 mV. Currents of between +20 and +30 mV reverse in an outward direction. Inward currents are approximated by a m³h model. The time constant of activation decreases from 0.7 ms at –60 mV to 0.12 ms at +20 mV. The time constant of inactivation falls from 9.1 ms at –60 mV to 0.6 ms at +20 mV.Steady state inactivationh is characterized by the half maximum valueV _H=–76.1±4.3 mV and the slope parameters=–6.3±1.1 mV (mean ± SD). A prepulse duration of 500 ms is essential for real steady state inactivation. Steady state activationm and inactivationh overlap each other defining a maximum window current at –65 mV.     

Evidence for a nucleotide-dependent topoisomerase activity from yeast mitochondria

Yeast mitochondria were found to contain a novel topoisomerase-like activity which required nucleoside di- or tri-phosphates as a cofactor. ADP supported activity as effectively as ATP and the optimal concentration for each was approximately 20 M. None of the other standard ribo- or deoxyrib-onucleotides could fully substitute for either ADP or ATP. The non-hydrolyzable ATP analogs, adenosine-5-0-(3-thiotriphosphate) (ATP--S), adenylyl (, -methylene) (AMP-PCP), and andenyl-imidodiphosphate (AMP-PNP) also supported activity suggesting that the nucleotide cofactor regulated topoisomerase activity rather than serving as an energy donor in the reaction. The mitochondrial topoisomerase activity relaxed both positively and negatively supercoiled DNA. It was not inhibited by concentrations of ethidium bromide up to 2 g/ml nor by either nalidixic or oxolinic acids; novobiocin, coumermycin, and berenil inhibited the activity. Genetic and biochemical analysis of the mitochondrial topoisomerase activity indicated that it was not encoded by the nuclear TOP1, TOP2, and TOP3 genes.