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.     

Molecular and cytogenetic organization of the 5S ribosomal DNA array in chicken (Gallus gallus)

The 5S ribosomal (r) RNA genes encode a small (120-bp) highly-conserved component of the large ribosomal subunit. The objective of the present research was to study the molecular and cytogenetic organization of the chicken 5S rDNA. A predominant 2.2-kb gene (5S) consisting of a coding and intergenic spacer (IGS) region was identified in ten research and commercial populations. A variant gene repeat of 0.6kb (5S) was observed in some of the populations. Genetic linkage analysis and cytogenetic localization by fluorescence in-situ hybridization assigned the 5S rDNA to chromosome 9. The 5S rDNA array was determined to be 80.2±7.0kb upon electrophoretic sizing following EcoRV digestion. Sequence analysis of 5S IGS regions revealed considerable conservation between chicken subspecies (98.4% identity) as well as homology with vertebrate Pol III promoter and regulatory sequence motifs. Minor intraindividual sequence variation within 1000bp of IGS was observed in four cloned Red Jungle Fowl (Gallus gallus gallus) 5S repeats (95.5% identity in this region). Sequence comparisons between IGS regions of 5S and 5S genes indicated two short continuous (>20bp) and many short non-continuous homologous regions as well as other conserved features such as promoter and termination motifs.     

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.     

High frequency recombination and the expression of genes cloned on chimeric yeast plasmids: Identification of a fragment of 2-μm circle essential for transformation

Summary We have carried out experiments aimed at explaining the observed variations in transformation frequencies when Saccharomyces cerevisiae or Saccharomyces carlbergensis are transformed with chimeric plasmids that contain one of 4 possible EcoRI fragments of the yeast 2-m circle. These plasmids fall into 2 classes when used to transform 2 different yeast his3 auxotrophs, one (strain LL20) harbours indigenous 2-m circle, and the other (strain YF233) is devoid of this plasmid. Hybrid plasmids containing either the 2.4 mega-dalton (mD) R-form EcoRI fragment (pYF88) or the l.4 mD L-form EcoRI fragment (pYF177) of 2-m circle transform either of the two hosts at a high frequency (50,000 colonies per Mg in LL20 and 10,000 colonies per g in YF233). Hybrid plasmids containing the 1.5 mD R-form EcoRI fragment (pYF87) or the 2.5 mD L-form EcoRI fragment (pYF178) of the 2-m circle transform LL20 at a reduced frequency (6,000–16,000 colonies per g) and YF233 at extremely low frequencies (1–5 colonies per g). All plasmids retrieved from strain YF233 that had been transformed with pYF88 or pYF177 were identical to the original transforming plasmid. Of the plasmids retrieved from strain LL20 that had been transformed with pYF87 and pYF178, approximately half had acquired an extra copy of the 2-m circle. Of the plasmids retrieved from strain LL20 that had been transformed with pYF88 and pYF177, an average of only approximately 13% had acquired an extra copy of 2-m circle. Taken together, these observations indicate that the transformation of yeast by a plasmid lacking the ability to replicate (pYF87 and pYF1780) occurs by the recombinational acquisition of 1 copy of the host 2-m circle, which serves to supply the incoming plasmid with missing essential sequences. A comparison of 2-m circle DNA fragments carried by pYF88 and pYF177 indicates that the region of 2-m circle required for high frequency transformation is a 1.2 mD segment that is common to the 2.4 mD R-form and 1.4 ml) L-form EcoRI fragments. This region extends from the EcoRI cut site adjacent to the PstI site, through to the end of the inverted repeat. However, the inverted repeat sequence alone is not sufficient to bestow high frequency transformation of yeast.     

Photobehavioral differentiation in natural populations ofDrosophila pseudoobscura andDrosophila persimilis

The photoresponses of natural populations ofD. pseudoobscura andD. persimilis, occurring sympatrically, are measured in two environmental conditions (at rest and disturbed). Comparisons of the responses, intraspecifically and interspecifically, lead to the following conclusions. These must be considered within the confines of the operational nature of the measurement of laboratory photoresponses. (1) Within each species population, significant nonenvironmental differentiation has been allowed or produced by selection in the at rest photoresponse. No significant nonenvironmental differentiation is found in the photoresponse measured in a disturbed condition. (2) Within each species population, a higher mean disturbed photoresponse has been favored. The intensities or patterns of selection acting on these two photoresponses have differed such that more intrapopulation differentiation has been allowed or produced in the at rest photoresponse. (3) A higher mean photoresponse has been favored inD. persimilis for both conditions. The intensities or patterns of selection acting between these two species populations on the at rest photoresponse have differed such that more intrapopulation differentiation has been allowed or produced inD. persimilis. (4) Comparisons of this study with one on intraspecific and interspecific differentiaiton in wing length lead to the conclusion that the selective differences inferred above have acted at a level more specifically attuned to photobehavior.     

Activation of atrial muscarinic K+ channels by low concentrations ofβγ subunits of rat brain G protein

Effects of G protein subunits from rat brain on cardiac K⁺ channel was examined in single atrial cells of guinea-pig, using patch clamp techniques. We found that 10 pM concentration of rat brain subunits preparation could activate the atrial muscarine receptor-gated K⁺ channel (I_K.ACh). Neither the detergent, CHAPS, used to suspend nor the boiled preparation activated I_K.ACh. Furthermore, preincubation of subunits preparation in Mg²⁺-free solution, which easily inactivated -GTP-S, did not affect -activation of I_K.ACh. We concluded, therefore, that subunits themselves can activate I_K.ACh.Supported by the grants from the Ministry of Education, Culture and Science of Japan and from the Calcium Signal Workshop on Cardiovascular Systems     

Intraspecific variation in the structural organization and redundancy of chloroplast ribosomal DNA cistrons in Euglena gracilis

Summary Chloroplast DNAs from six different laboratory collections of Euglena gracilis strain Z and var. bacillaris were analyzed with restriction endonucleases EcoRI and Bam HI. The most variable portion of the organelle genome is the region containing the ribosomal cistrons. Intraspecific differences occur in both ribosomal DNA cistron number (one or three) and structural organization among those strains designated as strain Z and bacillaris. One culture previously designated as Z is most likely bacillaris.     

Molecular organisation of an A mating type factor of the basidiomycete fungus Coprinus cinereus

Summary The A3 and A3 genes, which together constitute the A42 mating type factor of Coprinus cinereus, were isolated from a cosmid genomic library by walking 50 kb, a map distance of 0.5 units, from the closely linked metabolic gene pab-1. Cosmid clones having A gene function were identified by transformation into compatible A6 (22) and A5 (11) host cells where either 3 or 3 was expected to elicit the A factor — regulated development of unfused clamp cells. DNAs were digested with various enzymes before transformation in order to identify the smallest fragments containing an active 3 or 3 gene. Two non-overlapping fragments were identified as containing the 3 and 3 genes respectively. Southern hybridisation analyses showed that these two cloned genes had no detectable sequence homology, and that there was little or no hybridisation to the and gene alleles that constitute the A5 and A6 factors. 3 and 3 were shown to be less than 2.0 kb apart and embedded in a DNA sequence extending over 9.0 kb which was unique to our A42 strain and may contain a third A factor gene.     

Neuron Activity in the Prefrontal Cortex of the Brain in Rats with Different Typological Characteristics in Conditions of Emotional Stimulation

Male Wistar rats were separated according to the emotional resonance method (groups of animals avoiding (altruists) and not avoiding (egotists) the pain cries of partner rats) and neuron activity in the prefrontal areas of the cortex was studied in the right and left hemispheres. Assessments were made of changes in the frequency of nerve cell spike activity (in relation to the baseline activity of neurons in sated animals) in rats subjected to one day of food deprivation and after electrical stimulation of emotionally positive (lateral hypothalamus) and negative (tegmentum of the midbrain) brain structures and after exposure to the pain cries of partner rats. The results of these experiments revealed a series of differences in the cell activities of the two groups of rats. In conditions of hunger, the discharge frequency in the altruists was higher than that in egotists. Cortical neuron responses to positive stimulation were greater than those to negative stimulation in rats of both groups. Intracerebral stimulation produced significantly greater increases in discharge frequency in neurons of both prefrontal areas of the cortex in altruists than in egotists. In both groups of rats, neurons in the right hemisphere responded to emotionally negative stimulation with significantly greater activation than cells in the left hemisphere, while activity in the left hemisphere was greater in conditions of emotionally positive stimulation. Altruists showed significantly greater neuron responses during exposure to pain cries from victim rats in both the right and left hemispheres. The responses of egotists to victim cries were not significantly different from baseline activity levels.     

Pattern of antinuclear autoantibodies in diseases with systemic connective tissue lesions

Several types of antinuclear antibodies (ANA) were discovered by the fluorescent antibody method in diseases accompanied by systemic lesions of connective tissue and also in certain other diseases and in clinically healthy blood donors, depending on the character of fluorescence in the nuclei. Diffuse fluorescence of nuclei, diffuse fluorescence without fluorescence of the nucleoli, annular fluorescence, fluorescence in the form of granules, selective fluorescence of nucleoli, and fluorescnece in the form of long, thin, interweaving bands with simultaneous fluorescence in the region of the nuclear membrane were distinguished. The last type of ANA was observed only in various forms of lupus erythematosus, and the character of fluorescence in the nuclei differed from the reticular and filamentous types of fluorescence described previously.Department of Pathological Anatomy and Department of Skin Diseases, N. I. Pirogov Second Moscow 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. 84, No. 7, pp. 75–78, July, 1977.     

The penetration of the salivary glands ofRhodnius prolixus byTrypanosoma rangeli

Ultrastructural studies of the mechanism of penetration of the salivary gland of the reduviid bugRhodnius prolixus byTrypanosoma rangeli showed that trypanosomes from the haemocoele penetrate the outer membranes of the gland flagellum foremost, disrupting the inner layers, to pass between the muscle cells to reach the gland cell basement membrane. This latter is also penetrated flagellum foremost, the parasite invaginating the gland cell plasmalemma beneath, to create a vacuole in which the trypanosome crosses the gland cells to reach the central lumen, often only losing its containing vacuole just before leaving the cell.The structure of the outer membranes surrounding the salivary gland appeared similar to, and often actually part of, the basement membrane of the gland cells. These outer membranes were found to enclose large numbers of multinuleate giant form trypanosomes, whose significance is as yet unknown, but could perhaps represent a stage in the life cycle of the parasite where genetic interchange could take place.     

Theβ−tubulin genes ofDrosophila auraria are arranged in a cluster

When the 1-, 2- and 3-tubulin-specific DNAs fromDrosophila melanogaster were used as probes to recognize tubulin-specific sequences in the chromosomes ofDrosophila auraria, they were found to hybridize to the same polytene band in region 32C of the 2L polytene chromosome. Three overlapping clones were isolated from a EMBL 3 genomic library ofD. auraria, and they all contain -tubulin-specific sequences based on hybridization and partial-sequencing experiments of subcloned fragments. These clones hybridize in situ to the same polytene chromosome band in region 32C and they represent an approximately 35-kb fragment of genomic DNA.     

Acute ethanol consumption synergizes with trauma to increase monocyte tumor necrosis factor α production late postinjury

The hypothesis that acute ethanol uptake plus trauma can synergize to increase immunosuppression was tested. We found that, unlike non-alcohol-exposed patients, patients with acute alcohol use prior to trauma have a transient decrease in monocyte tumor necrosis factor (TNF) production during the very early postinjury (0–3 days) period. However, TNF production by these alcoholexposed patients' monocytes (MØ) became hyperelevated late postinjury (>9 days). Consequently, these massively elevated MØ TNF levels can contribute to posttrauma immunosuppression after acute alcohol use. We also demonstrate that normal monocyte activation with the superantigen,Staphylococcus enterotoxin B (SEB), results in a preferential induction of cellassociated MØ TNF production, described as characteristic of immunosuppressed trauma patients. Acutein vitro ethanol treatment down-regulated the elevated TNF production by trauma patients' MØ after either SEB, muramyl-dipeptide (MDP), interferon- plus MDP, or lipopolysaccharide (LPS) stimulation. Both SEB- and LPS-induced TNF mRNA induction was inhibited by acute alcohol treatment in normal MØ, indicating that ethanol can regulate cytokine gene expression. An additional immunosuppressive effect of acute ethanol's stimulation was suggested by its induction of elevated transforming growth factor production in trauma patients' activated MØ.     

A simple method for measurement of inulin in nanoliter samples using glass capillaries

Summary A simple method using glass capillaries instead of microcuvettes for measurement of inulin in nanoliter samples is given. Inulin was determined with anthron reagent (5 or 10 nl samples +3 l anthron reagent). Glass capillary tubes (o.d.=1 mm, i.d.=0.68 mm, length=150 mm) in which the chemical reaction took place during incubation at 56°C were directly introduced into the optical system of a Zeiss spectrophotometer PMQ II with sphere attachment and objective.Extinction was measured vertically to the axis of the capillary. The changes of extinction of 20 different capillaries with the blank at different positions was only 1.13×10^–3. The exactness of measurement in the concentration range of 100 200 400 750 1500 3000 mg-% inulin was for 5nl/3 l: 19.8 11.0 6.7 4.7 3.0 2.2%. 10nl/3 l: 13.0 8.4 5.1 3.9%.This method of measurement may also be applicable for other colorimetric reactions with nanoliter samples.This work was supported by Fonds zur Förderung der wissenschaftlichen Forschung.     

Candida albicans induces the release of inflammatory mediators from human peripheral blood monocytes

Candida albicans (C. albicans) is a major nosocomial pathogen. We examined arachidonic acid (AA) and cytokine production by monocytes stimulated with C. albicans. [¹⁴C]-AA labeled monocytes released 8.9 ±2.3% of the incorporated AA following stimulation with live C. albicans (C. albicans: monocyte of 161) (P=0.0002). Prior studies indicate that soluble-mannans and-glucans antagonize mannose and-glucan receptors, respectively. Preincubation of monocytes with-mannan (100g/ml) caused 45.8 ±5.7% inhibition of [¹⁴C]-AA release, whereas-glucan (100g/ml) yielded 43.7 ±6.0% inhibition (P<0.05 for each compared to control). Additionally, monocytes stimulated with C. albicans also released interleukin-1 (IL-1), tumor necrosis factor- (TNF), interleukin-6 (IL-6) and interleukin-8 (IL-8). However, a-mannan or-glucan failed to inhibit IL-1 release. These data indicate that C. albicans induces monocytes to release AA and inflammatory cytokines. Furthermore, AA, but not cytokine liberation, is partially mediated by a-mannan and-glucan components of the fungus.     

A Proposed Heuristic for Communicating Heritability Estimates to the General Public, with Obesity as an Example

It is well established that many continuously distributed traits have a heritable component. However, it is often difficult to communicate to the general public the meaning of quantitative estimates of heritability. To address this problem, the present paper introduces a heuristic for communicating heritability to nonscientific audiences. This heuristic involves adopting an extremely simplified model of inheritance and artificially (and somewhat arbitrarily) defining a cutoffs of low environmental risk and affectation status. Using body weight and obesity as an example, we present a table which gives estimates of the proportion of obese persons who are genetically obese assuming varying levels of environmental risk for obesity and relative body weight scores for defining obesity. The resulting statistic may prove useful for lay audiences in understanding a heritability estimate.     

Coronary blood flow in rats native to simulated high altitude and in rats exposed to it later in life

Summary In rats exposed to a simulated high altitude of 3500 m for their whole prenatal and postnatal life a severe cardiac hypertrophy develops. In rats born and first staying 5 weeks at sea level and then being exposed to simulated high altitude, only a unilateral right cardiac hypertrophy occurs. In both groups nutritional coronary blood flow was estimated in left ventricle, right ventricle, and septum and was compared with control animals of similar age. Coronary blood flow was measured at hypoxia in all groups. At first cardiac output was determined by the Fick principle, then⁸⁶Rb was applied and the animals were killed after 55 sec. Activity of⁸⁶Rb was measured in both cardiac ventricles and septum and the fractional uptake was calculated. According to Sapirstein (1956, 1958) the distribution of⁸⁶Rb follows the distribution of cardiac output and from both these data the nutritional blood flow to the parts of the heart may be estimated.Cardiac output was similar in rats exposed to simulated high altitude later in life (newcomers) and in control animals, but it was significantly lower in rats born in the low pressure chamber (natives).Fractions of cardiac output supplying cardiac ventricles and septum in rats from both hypoxic groups were significantly higher than in control animals. In the natives they were significantly higher than in the newcomers. The fractions of cardiac output in both newcomers and natives remained significantly higher than those of the control animals, also when calculated per gram of heart tissue.Nutritional coronary blood flow (in ml/min) was higher in both ventricles and septum of the newcomers and in the right ventricle of the natives, and lower in the septum of the natives, when compared with control animals. Coronary blood flow per gram of heart tissue (in ml/min·g) was significantly higher in all cardiac parts of the newcomers, but it was about the same in all cardiac parts of the natives when compared with controls.The importance of observed changes concerning myocardial tissue oxygenation is analyzed by using Krogh's cylindrical tissue model.Presented in part at the XXVIth International Congress of Physiological Sciences, New Delhi, India, October 20–26, 1974.     

Kinetic and pharmacological properties of high- and low-threshold calcium channels in primary cultures of rat hippocampal neurons

The kinetic, permeability and pharmacological properties of Ca currents were investigated in primary cultures of rat hippocampal neurons. The low-voltage-activated (LVA) Ca current turned on positive to –60mV and fully inactivated in a voltage-dependent way. This current was depressed by nickel (Ni, 40 M) and amiloride (500 M) and was insensitive to -conotoxin (-CgTx) (4 M) and to the Ca agonist Bay K 8644 (5 M). The high-voltage-activated (HVA) Ca current turned on positive to –40 mV and inactivated slowly and incompletely. This current was much less sensitive than the LVA current to Ni and amiloride but more sensitive to cadmium. CgTx blocked only partially this current (about 50%) in an irreversible way. Bay K 8644 had a clear agonistic action almost exclusively on the -CgTx-resistant HVA current component. The present results suggest that the HVA channels, quite homogeneous for their kinetic properties and sensitivity to holding potentials, can be pharmacologically separated in two classes: (i) -CgTx-sensitive and Bay-K-8644-insensitive (-S/BK-I) and (ii) -CgTx-insensitive and Bay-K-8644-sensitive (-I/BK-S), the latter displaying a stronger Cadependent inactivation.     

Ca-dependent K channels in smooth muscle cells permeabilized by β-escin recorded using the cell-attached patch-clamp technique

Using the cell-attached patch-clamp technique, the activity of single, Ca-dependent K channels was recorded in single smooth muscle cells permeabilized by -escin. The conductance and the relationship between the open probability of the channels and pCa recorded in permeabilized cells were very similar to those obtained in excised inside-out patches. At pCa 7, application of 30 M acetylcholine (ACh) or 0.1 M substance P (SP) together with 1 mM guanosine 5-trisphosphate to permeabilized cells elicited transient bursts of channel openings similar to those which occur in intact cells. Transient activation was also observed when 2–30 M inositol trisphosphate (IP₃) was applied to permeabilized cells. This single channel activity was inhibited by pretreatment with low-molecular-weight heparin at 50–100 g/ml. Channel activity at pCa 7.0 was greatly enhanced by 200 M cyclic adenosine monophosphate. These results provide direct evidence that single Ca-dependent K channel activity is regulated by the transmitters ACh and SP, as well as a second messenger, IP₃, via the release of intracellular Ca from intracellular sites which are blocked by heparin. This novel approach is valuable in elucidating second messenger mechanisms involved in the regulation of single channel activity by transmitters and autacoids, since permeabilization by -escin preserves the entire system of receptor-operated signal transduction and allows intracellular application of second messengers at fixed concentrations.     

Effects of ketamine on GABA-evoked excitability of peripheral nerve

Summary The effects of the dissociative anaesthetic, ketamine on GABA-evoked changes in the excitability of myelinated fibers of dorsal and ventral roots of isolated bullfrog sciatic nerves were examined. Ketamine alone (0.01–1000 M) evoked small increases (<5%) in dorsal root fiber excitability, as reflected in the half-maximal A-fiber compound action potential when concentrations were >10 M; with 0.1 M even larger increases (10%) were elicited in the ventral root fibers. As well, the increases evoked by 10 M ketamine were followed by graded decreases. 0.1 and 10 M ketamine, concentrations which by themselves had small or no effect, produced a dose-dependent depression of the large increases in excitability which are induced by activation of GABA receptors. In the presence of ketamine GABA concentration-response curves of the dorsal root fibers showed depression of the maximal response, while those of the ventral root fibers were shifted to the right. This apparent antagonism of GABA responses by ketamine may arise from blockade of receptor-mediated effects (e.g. K⁺/Cl^- currents and/or secondary depolarization from K⁺ accumulation), but could also be caused by a selective potentiation of hyperpolarizing receptors.