Phenotypic and epistatic grouping of hypo- and hyper-rec mus mutants in Aspergillus

The mutants musK to musS of Aspergillus nidulans are sensitive to methyl-methanesulfonate (MMS) and several of them are meiotic-defective and alter mitotic recombination frequencies. All were found to be cross-sensitive to 4-nitro-quinoline-N-oxide (4-NQO) but unexpectedly none of them was hypersensitive to -rays and few to UV light. Double mus;uvs mutants were constructed to test for interactions with uvs mutations of the four epistatic groups of Aspergillus, UvsF, UvsC, UvsI, and UvsB. All meiotic-defective mus mutations caused some lethal interactions, usually with uvsF. None of them showed epistasis with UvsF or UvsB group mutants and one, musO, may represent a new group. Three mus mutations that affect recombination were assigned to the UvsC group, namely musN and K, and also musL which is recombination-defective and closely resembles uvsC. While uvsC mutants are mutators and lack UV-mutagenesis, most mus mutants had no effects on mutation. Only musR, which appeared epistatic with uvsI, showed reduced UV-reversion frequencies similar to uvsI. The recombination-proficient mus mutants appeared to be epistatic with more than one group, but in several cases sensitivities were slight and overlaps insufficient to obtain corroborating results with MMS and 4-NQO.     

Influence of adoptively transferred thioglycollate-elicited peritoneal macrophages on metastasis formation in mice with depressed or stimulated NK activity

The effect of thioglycollate-elicited macrophages (TG-M) on natural killer (NK)-cell activity and metastases formation in mice was investigated. Intravenously (i.v.) inoculated TG-M inhibited spleen NK activity of normal mice and abrogated polyinosinic: polycytidylic (poly IC) induced augmentation of NK cell function. TG-M also inhibited the clearance of i.v.-injected radiolabeled B16 melanoma cells from the lungs of normal or poly IC stimulated mice. Formation of experimental B16 melanoma metastases was dramatically increased in mice pretreated with TG-M. Administration of TG-M increased metatasis formation to a greater extent than anti-asialo GM₁ serum, while anti-asGM₁ serum was more efficient than TG-M in depressing spleen NK cell activity. When mice with low NK reactivity (beige mice or mice treated with anti-asialo GM₁ serum) were inoculated with TG-M, there was a substantial additive augmenting effect on metastasis formation in the lungs. Treatment with poly IC elevated NK-cell activity and had profound antimetastatic effects in normal but not in TG-M pretreated mice. The metastasis augmenting effect of TG-M was fully expressed in poly IC-treated mice as well as in athymic nude mice. Inoculation of proteose peptone-elicited macrophages (PM), unlike TG-M, did not depress NK activity or augment metastasis formation in normal or poly IC-treated mice. However, since the inhibition of NK activity in TG-M-treated mice was relatively weak, and a substantial additional increase in metastases was observed in NK-depressed mice after transfusion of TG-M, it seems unlikely that the TG-M-induced inhibition of NK reactivity is entirely responsible for the augmented formation of metastases. Further studies revealed that i.v. inoculation of TG-M, but not PM, induced intravascular inflammatory reactions, and damage to endothelial cells and basement membrane of the lung vasculature. These reactions may contribute to increased tumor cell extravasation and metastasis formation in mice pretreated with TG-M.     

Some of the swi genes of Schizosaccharomyces pombe also have a function in the repair of radiation damage

Summary In Schizosaccharomyces pombe the frequency of mating-type (MT) switching is reduced by mutations in the swi genes. The ten hitherto known swi genes can be subdivided into three classes: Ia, Ib and II. Strains having swi5 (class Ib), swi9 (class II) and swi10 (class II) mutations do not only show reduced MT switching, but also exhibit an increased sensitivity to UV- and -rays. For that reason, 19 previously described rad genes were tested for their effect on MT switching. We found that swi9, rad10, rad16 and rad20 are allelic with each other indicating that the former allocation of these rad mutations to three different genes must have been erroneous. Among the remaining 16 rad genes examined, rad22 seems to be a new class II swi gene. The double mutants swi5 swi9 and swi5 swi10, but not swi9 swi10, are much more sensitive to radiation than the respective single mutants. Thus a cumulative increase in sensitivity occurs only if the mutants belong to different classes; previously the same correlation was found with regard to cumulative effects in MT switching.     

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.     

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.     

BamH1 polymorphism in the Chinese,Malays, and Indians in Singapore and its application in the prenatal diagnosis of β-thalassemia

Summary The distribution of restriction fragment length polymorphism (RFLP) at theBamH1 site of the -globin gene was investigated in the Chinese, Indian, and Malay race in Singapore. The sample comprised of 183 normal individuals and 35 -thalassemia carriers in which 13 were couples with at least one -major child. The results from this study indicate thatBamH1 polymorphism will be informative in 22% of pregnancies at risk for -thalassemia major in Chinese, 19% in Malays and 7% in Indians. In prenatal diagnosis usingBamH1 polymorphism for one -major affected family, the fetus was diagnosed to be normal or -carrier. The validity ofBamH1 polymorphism in the exclusion of -thalassemia major was subsequently confirmed at birth by globin chain biosynthesis.     

Diameter and flow velocity changes of feline small pulmonary vessels in response to sympathetic nerve stimulation

Using an X-ray television system, we measured directly changes in the internal diameter (ID), flow velocity, and volume flow of the small pulmonary vessels (100–500 m ID) in response to electrical sympathetic nerve stimulation (SNS) in anaesthetized cats before and after adrenergic receptor blockade. Flow velocity was obtained by measuring the distance that the leading edge of the contrast medium moved per 0.1 s in the small arteries. Volume flow was obtained from the product of flow velocity and cross-sectional area calculated from the ID of the small arteries. SNS was accolmplished with 10- to 15-V square-wave pulses of 2-ms duration at 20–30 Hz for 20-s periods. In response to SNS, arterial ID decreased significantly by 8–13% in the 200- to 500-m vessels but not in the 100- to 200-m vessels. In the veins, on the other hand, there was no significant ID decrease in any of the 100- to 500-m vessels. After -receptor blockade (phentolamine, 2 mg/kg i.V.), there were significant ID increases (4–9%) in the 100- to 500-m arteries in response to SNS, the maximum increases being in the 100- to 200-m arteries. After -blockade (propranolol, 2 mg/kg i.V.), the ID decrease due to SNS in the 200- to 500-m arteries was enhanced (24–27%) and, in addition, the 100- to 200-m arteries exhibited a significant ID decrease (18%). Combined and -blockade completely abolished the ID decrease due to SNS. In the veins, on the other hand, no ID change occurred even after - or -blockade. The results indicate that SNS selectively constricts 200- to 500-m arteries. The data suggests that SNS has -mediated vasoconstrictor and -mediated vasodilator effects on the 100- to 500-m arteries and that the ID response pattern to SNS depends chiefly on the balance between -mediated vasoconstriction and -mediated vasodilation. Associated with the ID decrease due to SNS, flow velocity was increased by 21%. However, SNS did not affect volume flow, because the increase in velocity was compensated by the reduction in the cross-sectional area (due to the decreased ID).     

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.     

Modification of the Na+ current conducted by the rat skeletal muscle α subunit by coexpression with a human brain β subunit

Sodium channels are multimeric structures composed of and subunits. Oocytes injected with RNA encoding only the subunit express voltage-gated Na⁺ currents. The kinetics of inactivation, however, are abnormal. Co-injection of rat brain and subunits modifies inactivation of I_Na such that it closely resembles endogenous currents [1]. Here we show that a subunit derived from human brain directs the same functional modification of I_Na expressed by a rat skeletal muscle subunit. This implies that functional domains for the interaction of and subunits are highly conserved across both tissues and species.     

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.     

Altered synthesis of interferon-γ and expression of interferon-γ receptor by peripheral blood mononuclear cells from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis

Previously we reported disease-specific interaction between interferon- (IFN-) and interleukin-4 (IL-4) in patients with IgA nephropathy (IgAN), suggesting the existence of unusual T cell behavior in this disease. In the present study, we investigated characteristic synthesis of interferon- (IFN-) and expression of IFN- receptor (IFN-R) in the peripheral blood mononuclear cells (PBMC) from patients with IgAN and other chronic proliferative glomerulonephritis (PGN). Heparinized peripheral blood samples were obtained from 38 patients with chronic mesangial proliferative glomerulonephritis (CGN; including 24 with IgA nephropathy) and 20 healthy controls. PBMC were isolated by gradient centrifugation and fragments were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) for 72 hr. IFN- concentrations in supernatants were evaluated by the enzyme-linked immunosorbent assay (ELISA). Other parts of PBMC pellets were reacted with anti-human IFN-R monoclonal antibody and FITC-labeled anti-mouse second antibody for analysis of IFN-R expression on these cells by FACScan. The remaining PBMC were fractionated into CD4⁺ T cells, CD8⁺ T cells, B cells, NK, cells and macrophages using the MACS cell sorting system. The isolated cells were evaluated for IFN- or IFN-R mRNA expression by the semiquantitative RT-PCR method.In vitro IFN- synthesis was enhanced in patients with CGN, and NK cells were revealed to be responsible for such enhancement. On the other hand, the expression of IFN-R on macrophages was suppressed in CGN patients. These results suggest that impairment of regulation of the IFN- system might be involved in the development of CGN.     

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.     

Block of non-L-, non-N-type Ca2+ channels in rat insulinoma RINm5F cells by ω-agatoxin IVA and ω-conotoxin MVIIC

The high-voltage-activated (HVA) Ba²⁺ currents of rat insulinoma RINm5F cells insensitive to dihydropyridines (DHP) and -conotoxin GVIA (-CTx-GVIA) have been studied for their sensitivity to -agatoxin-IVA (-Aga-IVA) and -CTx-MVIIC. Blockade of HVA currents by -Aga-IVA was partial (mean 24%), reversible and saturated around 350 nM (half block 60 nM). Blockade by -CTx-MVIIC was more potent (mean 45%), partly irreversible and saturated above 3 M. The effects of both toxins were additive with that of nifedipine (5 M) and were more pronounced at positive potentials. -Aga-IVA action was additive with that of -CTx-GVIA (3 M) but was largely prevented by cell pre-treatment with -CTx-MVIIC (3 M). In contrast, -CTx-MVIIC block was attenuated by -CTx-GVIA treatment ( 15%), suggesting that -CTx-MVIIC blocks the N-type ( 15%) and the non-L-, non-N-type channel sensitive to -Aga-IVA ( 30%). Consistent with this, cells deprived of most non-L-type channels by pre-incubation with -CTx-GVIA and -CTx-MVIIC exhibited predominant L-type currents that activated at more negative potentials than in normal cells (-30 mV in 5 mM Ba²⁺) and were effectively depressed by nifedipine (maximal block of 95% from -30mV to +40 mV). Our results suggest that, besides L- and N-type channels, insulin-secreting RINm5F cells possess also a non-L-, non-N-type channel that contributes significantly to the total current ( 30%). Although the pharmacology of this channel is similar to Q-type and ₁ class A channels, its range of activation (>-20 mV) and its slow inactivation time course resemble more that of N- and P-type channels. The channel is therefore referred to as Q-like.     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.     

Trennung der intestinalen β-Galactosidasen beim lactose-toleranten Erwachsenen durch Ultrazentrifugation im Dichtegradienten

Zusammenfassung Die intestinalen-Galactosidasen von 4 lactose-toleranten, erwachsenen Mitteleuropäern wurden im Saugbiopsie-Gewebe nach Solubilisierung mit Triton X-100 in einem linearen Mannitol-Gradienten (5–20%) auf der Ultrazentrifuge bei 4°C und 44000 U/min getrennt. Bei 12stündiger Zentrifugation fanden sich 3 Fraktionen, von denen die beiden schneller sedimenticrenden Lactose spalten. Alle 3 Fraktionen hydrolysieren p-Nitrophenyl--Galactosid. Die 3 isolierten-Galactosidasen entsprechen wahrscheinlich der neutralen Bürstensaum-Lactase, der sauren lysosomalen Lactase und einer cytoplasmatischen Hetero--Galactosidase.     

β subunits determine the time course of desensitization in rat α3 neuronal nicotinic acetylcholine receptors

Standard two-electrode voltage-clamp techniques were used to investigate some of the pharmacological and functional properties of two types of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes after pairwise injection of 34 or 32 mRNAs. Currents of several A amplitude were elicited by fast application of micromolar concentrations of either acetylcholine (ACh) or 1,1-dimethyl-4-piperazine (DMPP). The activation of either receptor type by DMPP showed cooperativity (Hill coefficient, n1.7) with a half-maximal activation concentration (EC₅₀) of 15–30 M. In 34 receptors, ACh displayed cooperativity (n=1.8) but was less efficacious than DMPP, yet its EC₅₀ was about equal to that of DMPP. Finally, in 32 receptors, ACh was much less efficacious and had a much lower EC₅₀. Desensitization induced by either DMPP or ACh was slow in 34 nicotinic ACh receptors but was rapid and extensive in 32 receptors, causing a significant proportion of the response to wane within the first few seconds of agonist application.     

Effect of alteration of peripheral blood flow on the central circulation in man during supine cycling in different ambient temperatures

Summary Whether the alteration of peripheral circulation caused by changing ambient temperature (T_a) affects central circulatory changes in man during supine cycling was investigated in four well-trained men, who exercised at two levels (117.7 or 176.6 W). Exercise metabolic rate (VO₂) in cold (0 C or 10 C) was the same as it was at 20 C, whereas the cardiac output (CO; CO₂ rebreathing technique) and heart rate were significantly lower (e.g., 176.6 W at 0 C, both p<0.01). In heat (30 C or 40 C), the VO₂ reduced with falling CO and mean arterial blood pressure from those at 20 C (e.g., 176.6 W at 40 C, all cases p<0.01), whereas the peak post-exercise calf blood flow (CBF_p) increased (p<0.01). The VO₂ and stroke volume (SV) were inversely proportional to the ratio of CBF_p to CO/kg body weight (CBF_p/CO) (r>–0.78, p<0.001). Total peripheral resistance (TPR) was related to arteriovenous oxygen difference (A-VO₂ difference) (r>0.78, p<0.001). The TPR and A-VO₂ difference decreased as T_a rose, while CBF_p/CO was almost the same. As CBF_p/CO had exceeded 50 and further progressed, however, the two parameters elevated until the same level as that at 0 C. The present results suggest that during moderately prolonged (16–60 min) supine cycling in different T_a's the central circulatory changes are mainly affected by the altered peripheral blood flow in competing between skin and muscle for blood flow.     

Gene activation by copy transposition in mating-type switching of a homothallic fission yeast

Summary Mating-type switching in homothallic clones of the fission yeast, Schizosaccharomyces pombe, appears to follow the same route as previously found for mutations from homothallism to heterothallic strains. A copy of mat2-P is transposed to and inserted at mat1, where it functionally replaces the mat1-M allele, and only the mat1 segment is expressed (!) to determine the actual mating type: mat1-M(!) mat2-P = = mat1-P(!) mat2-P. This phenomenon has hitherto been concealed by the high switch-back rate from to observed in homothallic wild-type strains. It only becomes apparent in the presence of mutant switching genes, which retard the rates of mating-type interconversion and temporarily freeze one or the other state of gene activation at the mat1 segment. Mutations to lowered rates of switching are found to map both inside and outside the mating-type locus. While the internal mutations of this kind exert their effect autonomously in the cis-configuration, the unlinked mutations are recessive to their wild-type alleles.We dedicate this paper to Carsten Bresch. The authors first met in the most stimulating scientific environment C. B. had created at the Southwest Center for Advanced Studies.     

Provisional quality control parameters and interpretive criteria for testing susceptibility ofStreptococcus pneumoniae andHaemophilus influenzae to quinupristin/dalfopristin (RP59500)

Studies were undertaken to select tentative criteria for susceptibility testing of quinupristin/dalfopristin againstStreptococcus pneumoniae andHaemophilus influenzae. Against 612 isolates ofStreptococcus pneumoniae, MICs of quinupristin/dalfopristin were 1.0 g/ml for all but one strain. With a tentative MIC breakpoint of either 1.0 g/ml or 2.0 g/ml for susceptible, a disk diffusion zone diameter breakpoint of 19 mm embraced all but two of the susceptible pneumococci; 16 mm included all strains. ForHaemophilus influenzae, MICs of quinupristin/dalfopristin clustered near the tentative breakpoints; 91.5% of the MICs were 2.0 to 8.0 g/ml. This precluded satisfactory performance of the disk diffusion test in discriminating between resistant and susceptible isolates unless MIC breakpoints are modified for this species: clinical experience will be needed before that can be justified. Based on data from a multilaboratory study, the following quality control limits are proposed forStreptococcus pneumoniae ATCC 49619 when testing quinupristin/dalfopristin: 0.25 to 1.0 g/ml for broth microdilution tests and 19 to 24 mm for disk diffusion tests. For tests ofHaemophilus influenzae ATCC 29247, MIC limits are 2.0 to 16 g/ml; disk tests were very reproducible but are not yet recommended.