Phenotypic differences between induced and spontaneous 2μ-plasmid-free segregants of Saccharomyces cerevisiae

Summary The maximum specific growth rates (_max) of 2 -plasmid-free ([cir°]) segregants of three haploid and one diploid strain of Saccharomyces cerevisiae have been determined and compared with the _max of their 2 -plasmid-containing ([cir ⁺]) progenitors. Two classes of [cir°] strains have been examined: those induced by transformation with a 2 -based recombinant plasmid according to the method of Dobson et al. (1980) and those isolated as spontaneous [cir°] segregants from glucose-limited continuous cultures. The _max of the spontaneous [cir°] segregants was not found to differ significantly from that of their [cir ⁺] parents. In all cases, however, the induced [cir°] strains had a _max which was significantly less than that of their [cir ⁺] counterparts. This effect was particularly marked in the case of the diploid strain where a 34% reduction in _max was observed. The implications of these results are discussed in terms of the effect of the transformation process on host yeast cells.     

Otolith orientation and downbeat nystagmus in the normal cat

Upward drift of the eyes in darkness, influenced by whole body orientation, was studied in 12 cats using electromagnetic search coil and electro-oculographic techniques. Animals were positioned stationary with respect to gravity with 0° tilt (upright) or rolled 90° (on side), pitched 90° (on nose or on tail), or inverted 180° (upside down). A downbeat quick-phase nystagmus (slow-phase upward in the cat's orbit) was measured, varying in magnitude with angle of tilt (0.21°/s at 0° tilt; 4.14°/s at 180° tilt). The drift was not present in the light. Upward eye velocities over a range of body orientations in darkness suggest a systematic drive to the eyes which increases with tilt away from upright. The relationship of this behavior to previous models of angular velocity estimation by an otolith-driven central mechanism is discussed.     

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.     

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.     

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.     

Interferon β1a Treatment Modulates TH1 Expression in γδ + T Cells from Relapsing-Remitting Multiple Sclerosis Patients

A paradigm exists that multiple sclerosis is causally related to dysregulation of T_H1 inflammatory cytokines and T_H2 antiinflammatory cytokines. The cytokine source(s) that initiate the imbalances are unknown. In this study, , CD4, and CD8 T cell receptor-positive (TCR+) cells were isolated from the blood of 26 definitive relapsing-remitting multiple sclerosis patients prior to interferon -1a (IFN1a) therapy and following 8–10 weeks of this therapy. The bioactivities of interferon (IFN), interleukin 10 (IL10), and interleukin 12 (IL12) were determined. The concentrations of IFN, IL10, and IL12 from each cell type did not change significantly with IFN1a treatment. The IL10 secreted by TCR+ cells strongly correlated with the IL12 secreted by the same TCR+ cells, supporting the paradigm. Furthermore, IFN1a therapy decreased the TCR+ cell secretion of T_H1 cytokines after 8–10 weeks of therapy.     

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.     

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.     

Adaptation and habituation of the vestibulo-ocular reflex in intact and inferior olive-lesioned rats

Summary The gain of the vestibulo-ocular reflex (VOR) of intact pigmented rats was adaptively modified by training protocols that created a visual-vestibular conflict. For training, head restrained animals were oscillated on a turntable in front of an optokinetic pattern projected onto a cylindrical wall. The optokinetic pattern either moved the same amplitude with the animal (in-phase: 0.05 Hz ± 20°/s) or opposite in direction (out-of-phase: turntable and pattern 0.05 Hz ± 10°/s each). VOR responses were tested in darkness before and after each 8 min training period for a duration of 40 min. During out-of-phase training the gain of compensatory eye movements measured in light was close to 2 from the beginning on and the VOR tested in darkness increased in gain progressively from 0.48 (±0.12) to 0.9 (±0.3; P < 0.05) in 5 out of 7 rats. Two rats did not adapt their VOR gain. Phase values decreased slightly by about 10°. During in-phase stimulation compensatory eye movements were almost completely suppressed (gain close to 0) from the beginning on and the VOR tested in darkness decreased gradually in gain from 0.62 (±0.17) to 0.13 (±0.1; P<0.001) in all 6 trained rats. Phase values decreased in parallel from 151° to 119° (P< 0.01). The effectiveness of the in-phase training paradigm in the absence of compensatory eye movements indicates that retinal image slip is the relevant signal for adaptation. In seven rats with histologically verified almost complete inferior olive (IO) lesions (chemically induced at least 45 days prior to training), out-of-phase and in-phase stimulation evoked compensatory eye movements with gains comparable to those in intact rats. VOR parameters measured in darkness were altered with respect to those of control rats. Gain differed extremely between individuals and phase lag re acceleration was in all IO-lesioned rats larger than in intact rats. The time constant of the VOR in response to table velocity steps was significantly longer (17 s ±4) than in intact rats (11 s ± 3). Training did not alter the gain of the VOR in 5 out of 7 IO-lesioned rats. One rat increased its gain during out-of-phase training in the first, but not during a second training session (and not during in-phase training) and another rat decreased its gain during in-phase training (but not during out-of-phase training). These changes in VOR gain might have occurred by chance rather than by learning. The absence of adaptation in IO-lesioned rats can be explained either by the absence of climbing fiber mediated slip signals in the cerebellar cortex or by lesion-induced secondary changes which result in a long-term reduction of the inhibitory efficacy of Purkinje-cells. In the absence of arousing stimuli VOR responses of intact rats exhibit a strong decrement during table oscillations in darkness. Between trials, with the rat at rest, response magnitude recovered spontaneously. Six out of 8 IO-lesioned rats expressed a very similar modification of their VOR gain. These results indicate that the neural mechanisms responsible for adaptive gain decrease during in-phase training and those responsible for a gain decrease during short-term habituation are different.     

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.     

Decreased serum levels of TGF-β in patients with acutePlasmodium falciparum malaria

Apart from cellular immunity and immunopathology, various cytokines have been implicated in malaria-associated immunosuppression. In this study, serum levels of transforming growth factor- (TGF-) were determined with an enzyme-linked immunosorbent assay in 37 patients with acutePlasmodium falciparum malaria prior to, during, and after therapy and in 17 healthy controls in Bangkok, Thailand. Patients were treated with artesunate and mefloquine. TGF- serum levels were found decreased prior to treatment (14±11 pg/ml versus 63±15 pg/ml in healthy controls;P<0.05). The serum concentrations of TGF- increased after initiation of treatment and were within normal range on day 21. Serum levels of both tumor necrosis factor-ga (TNF-) and soluble TNF-receptor 55 kDa were inversely correlated to serum levels of TGF- (r= –0.667 andr=}-0.592, n=37; respectively,P < 0.05 for both). No correlation between parasitemia and serum levels of TGF- could be found. The results are compatible with a decreased production and release, an enhanced clearance or utilization, or tissue accumulation of TGF- in acuteP. falciparum malaria.     

Analysis of miniature spontaneous inhibitory postsynaptic currents (sIPSCs) from current noise in crayfish opener muscle

Deviating from the normal situation, some crayfish muscle fibres showed spontaneous inhibitory activity: discharge of large inhibitory postsynaptic currents, IPSCs, alternating with long lasting bursts of current noise. Analysis of the bursts of current noise revealed that they are composed of spontaneous miniature unit currents, sIPSCs. In the burst periods the sIPSCs occurred with an average rate of 3.5–10 kHz and had an amplitude of about ã=90 pA at a driving force E=10 mV. The peak conductance _ã=ã/E of the sIPSCs was _ã=9.2 nS±0.5 (S.D.,n=5) for membrane potentials between E=–60 mV and E=–80 mV. _ã seemed to decrease when the membrane was hyperpolarized. The time constants of decay, , of the sIPSCs were identical with of the IPSCs. Further, and its potential dependence agreed with the mean lifetimes of inhibitory postsynaptic channels operated by -aminobutyric acid (GABA) [Dudel et al. 1977, 1980]. Synchronized opening of about 750 inhibitory synaptic channels generates a sIPSC.Analysis of this anomalous bursting inhibitory activity thus yields the size of the inhibitory quantum of transmission, which could not be obtained from IPSCs.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Altered expression of the steroid bioconverting pathway in pAN 7-1 transformants of Cochliobolus lunatus

Summary The filamentous fungus C. lunatus converts progesterone mainly to its 11-hydroxy derivative. C. lunatus transformed with the plasmid pAN 7-1, which contains the E. coli hph gene expressed under the control of the A. nidulans gpd and trpC expression signals, lacks this activity, but exhibits acetyl side chain degradation of progesterone through the reaction scheme progesterone20-hydroxy-progesterone ⁴-androstene-3,17-dione testolactone+testosterone. The main partof this metabolic pathway is not expressed in the non-transformed strain. It was determined that the site-specific integration of the plasmid into the genome directly influences the expression of genes involved in the bioconversion of steroids.     

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.     

Direct observation of sexual competition inDrosophila melanogaster: The mutantWhite in competition with other genotypes

When two types ofDrosophila are in competition, the frequency dependence of mating success routinely is measured in our laboratory by direct observation of mating pairs in Elens-Wattiaux observation chambers. The present experiments concern white mutants (used here as a reference standard) in competition with three other genotypes: wild-type Canton S, giantgt w ^a , and giantgt ^13z /w ⁺ Y/C(1)Dx, y f. From the present observations, the frequency dependence of mating success seems a very common phenomenon: a rare-type sexual advantage exists for females and for males. Sexual activity of male and femalewhite mutants is significantly higher when food is present in the mating chamber. Males of the other strains also are more active in the presence of food. Homogamic matings are more frequent amonggt ^13z /w ⁺ Y/C(1)Dx, y f flies.     

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.     

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     

Chloride channel regulation in the skeletal muscle of normal and myotonic goats

External intercostal muscle biopsies from normal and congenitally myotonic goats were studied in vitro at 30° C using a two-microelectrode square-pulse cable analysis assisted by computer. The resting chloride conductance (G _cl) was estimated from the difference between the mean membrane conductance in chloride-containing and chloride-free bathing media. The protein kinase C (PKC) activator, 4--phorbol-12,13-dibutyrate, (0.1–2.0 M) blocks a maximum of 76% of G _cl in normal goat fibers and induces myotonic hyperexcitability similar to that of congenitally myotonic goat fibers. The G _cl block was partially antagonized by pretreatment with the PKC inhibitor, staurosporine (10 M). The inactive 4-phorbol-12, 13,didecanoate had no effect at 50 M, whereas the active 4- isomer blocked 41% G _cl at 1 M. The nearly absent G _cl of congenitally myotonic goat fibers was not restored by treatment with high concentrations of the PKC inhibitors staurosporine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), or tetrahydropapaveralone (THP). Also, forskolin and cholera toxin, which may increase cyclic adenosine monophosphate (cAMP) levels, or the R(+) clofibric acid enantiomers and taurine, which increase G _cl in normal fibers, were also unable to restore G _cl in myotonic goat fibers. The data suggest that PKC may be a chloride channel regulator in normal goat skeletal muscle fibers, however the molecular defect of congenitally myotonic fibers does not appear to be due to excessive activity of PKC.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.