Erythromycin and cycloheximide sensitivities of protein and RNA Synthesis in sporulating cells of Saccharomyces cerevisiae: Environmentally induced modifications controlled by chromosomal and mitochondrial genes

Summary The purpose of the experiments reported below was to examine the response in sporulation medium of the three diploid cell types MAT MAT, MAT MAT (asporogenic diploids) and MAT MAT (sporogenic diploid) to erythromycin, a specific inhibitor of mitochondrial protein synthesis (MPS) in vegetative cultures, and cycloheximide, a specific inhibitor of cytosol protein synthesis (CPS) in vegetative cultures. When MAT MAT diploids are transferred to sporulation medium a significant fraction of total protein synthesis (CPS + MPS) becomes sensitive to erythromycin in contrast to the behavior of MATa MATa and MAT MAT diploids in which the resistance of CPS to erythromycin is maintained. The decompartmentalization of erythromycin sensitivity is thus cell type specific. Erythromycin stimulates total RNA synthesis of MAT MAT cells in sporulation medium but not of MAT MAT and MAT MAT cells. Cycloheximide inhibits protein synthesis and stimulates RNA synthesis in all three diploid cell types. An erythromycin resistant mutant, shown to be due to a mutation of the mitochondrial genome, exhibited only partial resistance of CPS to erythromycin in sporulation medium in the background of the MAT MAT mating type genotype. Total RNA synthesis in this mutant was not stimulated. The results reported indicate that mitochondrial functions during sporulation are not restricted to those involving respiratory metabolism.     

A mutation affecting sexual agglutinability in MATα locus of Saccharomyces cerevisiae

Summary A temperature-sensitive non-agglutinative mutant of Saccharomyces cerevisiae was isolated and characterized. The mutation, sag2, affected sexual agglutination, conjugation and production of -mating pheromone at a restrictive temperature, but not the response to -mating pheromone. Genetic analyses showed that the mutation was recessive and in the MAT locus. The sag2 mutation complemented with mat2 but not with mat1 These results suggest that sag2 is in the MAT1 gene and that at a restrictive temperature the mutation, sag2, inactivated the MAT1 product, a positive regulator of -mating functions. The sag2 mutation is like mat1-5 in its retention of response to -mating pheromone. However, at 25 °C, sag2 cells were competent to mate, whereas mat1-5 cells were not. Hence, sag2 is regarded as a new allele in the MAT1 gene, which we designate mat1-11.     

An α-mating-type-specific mutation causing specific defect in sexual agglutinability in the yeast Saccharomyces cerevisiae

We have identified a recessive -mating-type-specific gene agl causing agglutinability defect without significant effects on other sexual activities. a cells carrying agl showed sexual agglutination with cells but cells carrying agl showed sexual agglutination with neither cells nor a cells. cells carrying agl produced pheromone and responded to a pheromone just like wild cells. cells carrying agl showed a little decreased but significant mating ability when tested on solid media or membrane filter. The agl mutant is different from any -specific ste mutants found so far in many sexual activities. The agl gene is recessive, and unlinked to the mating type locus. Biological significance of the mating type agglutinability is discussed based on the results obtained with the mutant.     

Mating-type suppression of the DNA-repair defect of the yeast rad6Δ mutation requires the activity of genes in the RAD52 epistasis group

The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MAT a rad6 strain can be suppressed by the MAT2 gene carried on a multicopy plasmid. The a1-2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-2 suppression of the rad6 mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 mutant into the RAD52 recombinational repair pathway. The a1-2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

An α-specific gene,SAG1 is required for sexual agglutination in Saccharomyces cerevisiae

Summary Seven -specific mutants specifically defective in sexual agglutinability were isolated. The other mating functions exhibited by these mutants, designated sag mutants, such as the production of pheromone and response to a mating pheromone, were normal. While the MAT sag1 cells did not agglutinate with wild-type a cells, the MAT sag1 cells did, indicating that the SAG1 gene is expressed only in cells. The mutations were semi-dominant and fell into a single complementation group, SAG1, which was mapped near met3 on chromosome X. Complementation analysis showed that sag1 and aga1, the latter being a previously reported -specific mutation, were mutations in the same gene.     

Nachweis von 6 neuen Steroiden im Plasma von menschlichem Placentablut (Nabelsehnurblut)

Zusammenfassung 16-Hydroxyprogesteron, 17-Hydroxyprogesteron, ⁴-Pregnen-20-ol-3-on, ⁴-Pregnen-17, 20-diol-3-on, Adrenosteron und 11-Hydroxyandrostendion wurden in Extrakten von Plasma des menschlichen Placentablutes (Nabelschnurblut) nachgewiesen.     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes: role of the α subunit in agonist sensitivity and desensitization

Neuronal nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus laevis oocytes after nuclear injection of complementary deoxyribonucleic acid (cDNA) expression vectors. The two receptor subtypes 4/n1 and 3/n1 were readily distinguishable from one another by ACh sensitivity and desensitization. 3/n1 receptors showed lower ACh sensitivity and stronger desensitization than 4/n1 receptors. Furthermore, although the current/voltage relationship was very similar in both receptor subtypes, the voltage dependence of desensitization was found to be strikingly different. As the n1 subunit was unchanged, the subunits must be responsible for these functional differences. Symmetric hybrid cDNAs, 43 and 34, were constructed and functional receptors were obtained by co-injection with n1. These hybrid receptors displayed an ACh sensitivity that was mainly defined by the extracellular sequence of the subunit. In contrast, no part of the subunit was found fully to determine desensitization.     

The REC46 gene of Saccharomyces cerevisiae controls mitotic chromosomal stability,recombination and sporulation: cell-type and life cycle stage-specific expression of the rec46-1 mutation

Summary The recessive hyperrecombination mutation rec46-1, isolated by ultraviolet light mutagenesis of the MAT n+1 chromosome VII disomic strain LBW (Esposito et al. 1982), enhances the mitotic rates of spontaneous gene conversion, intergenic recombination and restitution of haploidy (due to chromosomal loss or mitotic nondisjunction) in MAT n+1 chromosome VII disomic strains. The rec46-1 mutation does not prevent HO directed homothallic interconversion of mating types. MATaIMaT ree46-1/rec46-1 diploids exhibit the same degree of hyperrecombinational activity as MAT rec46-1 n+1 chromosome VII disomics with respect to gene conversion and intergenic recombination resulting in prototrophy. When compared to MAT rec46-1 n+1 disomics however, MATa/MAT rec46-1/rec46-1 diploids exhibit a ten fold reduced level of hyperrecombinational activity with respect to intergenic recombination and present no evidence of chromosomal loss or nondisjunction resulting in 2n-1 monosomic segregants. MATaIMAT rec46-1/rec46-1 diploids are sporulation-deficient. The results obtained demonstrate that the REC46 gene product modulates mitotic chromosomal stability and recombination and is essential for sporulation (meiosis and ascospore formation).     

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.     

Purification and genetic control of α-pheromone-inactivating glycoproteins in the yeast Saccharomyces cerevisiae

Summary Two kinds of a-mating-type-specific proteins inactivating pheromone ( factor) were purified from heat shock extract of MATa cells. Their molecular weights were estimated to be 400,000 and 200,000 by gel filtration. Both proteins were detected in MATa SST1 cells but not in MAT SST1, MATa sst1-1 and MATa/MAT SST1/SST1 cells. In addition, the proteins were detected in mat2-1 SST1 cells but not in mat1-2 SST1 cells. From these results, it is concluded that these proteins are synthesized under the control of the SST1 gene and responsible for the Barrier action of MATa cells. The relationship of these proteins to the secreted Barrier protein having a higher molecular weight is discussed.Abbreviations Mr. molecular weight - PBS 10 mM phosphate buffer, pH 5.5 - SDS sodium dodecyl sulfate     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Site of pheromone action and secretion pathway of a sexual agglutination substance during its induction by pheromone a in α cells of Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae mating-type strains carrying the sec1, sec7 and sec18 genes, pheromone a induces agglutination ability at 24 °C, but not at 36 °C. Even when cells were treated at 36 °C, a biologically active agglutination substance was found in cytoplasm although this activity was not detected in the cell surface fraction. These 36 °C-treated cells became agglutinable with a concomitant appearance of agglutination substance activity in the cell surface fraction. The cells remained agglutinable even when the treatment temperature was dropped to 24 °C under conditions where no de novo synthesis of the agglutination substance occurred. These results indicate that the agglutination substance is transported through the yeast secretory pathway and that pheromone a acts at the step of synthesis of the precursor molecule of the a agglutination substance, similar to the case ofthe -pheromone-induction of the a agglutination substance. Differences in the action of the sex pheromones between agglutination ability induction and induction of G₁ arrest or shmooing is discussed.     

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.     

Immunohistochemical studies on S-100 cells in the anterior pituitary gland of Sprague Dawley rats and spontaneous dwarf rats

The S-100 cells in the pituitary glands of adult male Sprague Dawley rats (SDs) and spontaneous dwarf rats (SDRs) were immunohistochemically examined using anti-S-100 and anti-S-100 monoclonal antibodies. The immunoreactive cells against S-100 protein were divided into three subtypes on the basis of their immunore-activity against subunits of S-100 protein: S-100 dominant type (the -type cell), S-100 dominant type (the \-type cell) and immunoreactive against both S-100 and S-100 (the -type cell). In the SD, -type cells represented 26% of the total S-100 immunoreactive cells (S-100 cells) and were localized in the peripheral area of the ventral region of the pituitary gland. This type of cell was observed forming clusters, with more abundant cytoplasm than the -type cell. The proportion of -type cells was 53%. They were diffusely distributed throughout the gland, and their processes were thicker than those of the -type cell. In the SDR, the proportion of -type cells was 55%, and they were observed throughout the gland. In contrast, -type cells totalled 12% and were localized in small areas of the central and peripheral region of the gland. The proportion of -type cells was 21% in the SD and 33% in the SDR and they were observed forming small clusters in both animal groups. The proportion of -type cells compared with the total of S-100-immunoreactive cells was significantly higher (P < 0.05) in the SDR than in the SD, while the proportion of -type cells was markedly lower (P < 0.05).     

Activation of the skeletal α-actin promoter during muscle regeneration

Little is known conerning promoter regulation of genes in regenerating skeletal muscles. In young rats, recovery of muscle mass and protein content is complete within 21 days. During the initial 5–10 days of regeneration, mRNA abundance for IGF-I, myogenin and MyoD have been shown to be dramatically increased. The skeletal -actin promoter contains E box and serum response element (SRE) regulatory regions which are directly or indirectly activated by myogenin (or MyoD) and IGF-I proteins, respectively. We hypothesized that the skeletal -actin promoter activity would increase during muscle regeneration, and that this induction would occur before muscle protein content returned to normal. Total protein content and the percentage content of skeletal -actin protein was diminished at 4 and 8 days and re-accumulation had largely occurred by 16 days post-bupivacaine injection. Skeletal - actin mRNA per whole muscle was decreased at day 8, and thereafter returned to control values. During regeneration at day 8, luciferase activity (a reporter of promoter activity) directed by –424 skeletal -actin and –99 skeletal -actin promoter constructs was increased by 700% and 250% respectively; however, at day 16, skeletal -actin promoter activities were similar to control values. Thus, initial activation of the skeletal - actin promoter is associated with regeneration of skeletal muscle, despite not being sustained during the later stages of regrowth. The proximal SRE of the skeletal -actin promoter was not sufficient to confer a regeneration-induced promoter activation, despite increased serum response factor protein binding to this regulatory element in electrophoretic mobility shift assays. Skeletal -actin promoter induction during regeneration is due to a combination of regulatory elements, at least including the SRE and E box. © Kluwer Academic Publishers.     

Expression ofβ1 integrins in non-neoplastic mammary epithelium,fibroadenoma and carcinoma of the breast

1 Integrins were examined immunohisto-chemically in normal and mastopathic mammary glands, 12 benign tumours and 90 carcinomas of the breast using monoclonal antibodies against1 and1 to6 subunits. When compared with epithelial cells of non-neoplastic mammary glands and of benign tumours, carcinoma cells showed considerable quantitative changes in the pattern of2,3 and6 subunit expression. In contrast, the distribution pattern of1,1,4 and5 antigens corresponded to the situation observed in non-neoplastic mammary gland epithelium in most instances. An abnormal expression of2 was found in 71.0% of the carcinomas ranging from a remarkably low number of2-positive tumour cells in 27.5% of the cases to a complete absence of the2 molecule in 43.5% of the carcinomas. Of the carcinomas 39.9% exhibited quantitative changes in3 expression with an abnormally low content of3-positive neoplastic cells in 15.4% and a complete absence of this molecule in 24.5% of the cases. Expression of6 was abnormal in 73.2% of the carcinomas, consisting in a greater number of6-negative tumour cells in 31.9% and in a complete absence of6 in 41.3% of the tumours. The abnormally low expression/absence of2 and3 subunits correlated with oestrogen receptor negativity (P<0.033 andP<0.04, respectively). In addition, abnormally low expression/absence of2 correlated with poor differentiation of the tumours (P< 0.014). The quantitative changes in the expression pattern of1-associated subunits in breast carcinomas may cause a disturbed cell-cell and/or cell-matrix interaction that increases the invasive and migratory property of the tumour cells.     

Epidermal growth factor,transforming growth factor-α, and epidermal growth factor receptor content in normal and carcinomatous gastric and colonic tissue

Summary Epidermal growth factor (EGF) and transforming growth factor- (TGF-) are polypeptides which bind to the EGF receptor (EGFr) and may play a role in cell growth and carcinogenesis. Our study investigated the content of EGF, TGF-, and EGFr in tumors of the stomach and the colon in comparison with the sourrounding mucosa. EGF was detected in half of the stomach specimens with concentrations between 1 and 9 ng/g weight irrespective of histology. In the colon no EGF was found in the tumor or normal mucosa. In the stomach normal mucosa contained higher TGF- concentrations (mean 22.4 ng/g) than the tumors (mean 11.8 ng/g), but the difference was not statistically significant because of a wide variation in mucosal values. By contrast, the colon mucosa displayed significantly higher TGF- concentrations than the tumor tissues (33 ng/g versus 12 ng/g; P < 0.01). EGFr content in the gastric mucosa was lower compared to gastric carcinoma (48 fmol/g versus 75 fmol/g) yet not significantly different. In contrast, colorectal tumor specimens disclosed significantly higher concentrations than the mucosal tissues (mean of 155 fmol/g versus 80 fmol/g; P < 0.01). In conclusion, TGF- should not be considered a tumorigenic but a physiological growth factor in the stomach and colon. An elevated EGFr content in colorectal tumors in comparison with the normal mucosa could lead to a growth advantage by an autostimulating mechanism.Abbreviations EGF epidermal growth factor - EGFr epidermal growth factor receptor - TGF- transforming growth factor - ROC receiver operating characteristic Dedicated to Prof. Dr. G. Paumgartner on the occasion of his 60th birthday     

Macrophage inflammatory protein-1 alpha expression in non-neoplastic and neoplastic lung tissue

The chemokines are members of a bipartite superfamily of soluble proteins that have been implicated in a wide range of acute and chronic inflammatory processes, as well as other immunoregulatory functions. Macrophage inflammatory protein-1 alpha (MIP-1) belongs to the C-C subfamily of these chemokines and is primarily a potent chemoattractant and activator of monocytes. MIP-1 is also thought to play a role in host defence. We examined the expression of MIP-1 in normal lung, inflammatory lung tissue and lung cancer cells by the immunoperoxidase method using a MIP-1 monoclonal antibody. MIP-1 protein was found to be expressed not only by alveolar macrophages, but also by bronchial ciliated cells, hyperplastic alveolar type II cells and activated fibroblasts surrounding malignant tissue. Of 33 cases of lung cancer, 23 (70%) expressed MIP-1. These observations suggest that lung cancer cells, non-neoplastic alveolar type II cells and fibroblasts can participate in inflammatory cell recruitment via the production of MIP-1. Tumour derived MIP- may also affect the interaction between lung cancer and host inflammatory cells.