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 Matrix Associated Region Localizes the Human SOCS-1 Gene to Chromosome 16p13.13

The MarFinder algorithm was applied to a newly sequenced segment of 16p13.13 abutting the 3 end of the human PRM1 PRM2 TNP2 locus. A candidate region of matrix attached was identified. Subsequent biophysical analysis showed that this region was attached to the somatic nuclear matrix. Nucleotide sequence analysis also revealed the presence of a CpG island. Data base queries showed that this region contained the SOCS-1 gene. Thus, the SOCS-1 gene is bounded by a somatic MAR and is just 3 of the spermatid-expressed PRM1 PRM2 TNP2 domain at position 16p13.13.     

Polymerase chain reaction-single strand conformation polymorphism analysis of the p53 gene in paraffin-embedded surgical material from human renal cell carcinomas

p53 tumour suppressor gene mutations were studied in 118 renal cell carcinomas using paraffin-embedded surgical material. Optimal results were obtained with analysis of exon lengths between 150 and 200 base pairs for polymerase chain reaction. Single strand conformation polymorphism and sequencing analysis revealed only two point mutations (2/118, 2%): one involving codon 135; TGCTTC (cysteinephenylalanine) and the other codon 175; CGCCAC (argininehistidine). Both of these cases were classified as granular cell subtype on microscopic observation. The data suggest that the p53 tumour suppressor gene is not related to tumour initiation, promotion, or progression of renal cell carcinomas. However, there is the possibility that granular cell type carcinomas may have a different genetic background from clear cell type renal neoplasms.     

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).     

Distribution of C→T and T→C polymorphisms of the urokinase-type plasminogen activator gene in children with type 1 diabetes mellitus and insulin resistance

Abstract We analysed the distribution of genotypes and frequency of alleles of two polymorphisms in the urokinase-type plasminogen activator (uPA) gene: a CT substitution in exon 6 and a TC substitution in intron 7 in 89 children with type 1 diabetes mellitus and insulin resistance compared with 120 non-diabetic control subjects. All genotypes were determined by the allele-specific polymerase chain reaction. We found that the frequency of the T/T homozygote (15%) in the patient group was significantly (P<0.05) higher than in the controls (7%). There were no differences in the distribution of the TC polymorphism between patients and controls, which suggests that this genetic change is probably phenotypically silent. In conclusion, our results indicate that the higher percentage of T/T homozygotes in patients might be associated with T1DM coexisting with insulin resistance.     

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     

Quantal stores of excitatory transmitter in nerve-muscle synapses of crayfish evaluated from high-frequency asynchronous quantal release induced by veratridine or high concentrations of potassium

At single voltage-clamped opener muscle fibres of crayfish claw, 10–100 mol/l veratridine increased within a few seconds the rate of asynchronous quantal release, ñ, of excitatory transmitter from ñ<1 quantum/s to ñ10,000 quanta/s. Thereafter ñ declined exponentially either with a single, (2)50 s, or with two time constants (1)19 s, (2)50 s. In total (t), about 0.3 million quanta were released by veratridine in a single short fibre of about 1 mm length. These values were estimated by means of the noise analysis technique and they agreed with equivalent parameters of release when 100 mmol/l K⁺ were used as release stimulus. Strong quantal release could be elicited only once in a single muscle by veratridine. Furthermore, the effect of veratridine on quantal release could be completely prevented by pretreatment with tetrodotoxin. In another nerve-muscle preparation of crayfish, the abdominal superficial extensor muscle, up to 3 million excitatory quanta could be released by veratridine in a single fibre. In the latter muscle veratridine-induced asynchronous quantal release was strongly dependent on the extracellular concentration of Ca²⁺ whereas in the claw opener dependence of quantal release on extracellular Ca²⁺ was negligible.This investigation was supported by the Deutsche Forschungsgemeinschaft, SFB 220     

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.     

Cognitive mediators of the social influence-exercise adherence relationship: A test of the theory of planned behavior

The purpose of this study was to examine cognitive constructs from the theory of planned behavior (i.e., attitude, perceived behavioral control, and intention) as potential mediators of the relationship between selected social influence constructs (i.e., subjective norm, social support, and cohesion) and adherence to structured exercise classes. Sixty-two participants completed self-administered questionnaires during the fourth week (social influence constructs) and eighth week (cognitive constructs) of a 12-week exercise program. Exercise adherence was monitored during weeks 9 through 12 using perceived intensity and attendance. Pearson correlations indicated that social support correlated with perceived behavioral control, whereas cohesion correlated with attitude. Path analysis supported two distinct paths from social influence to exercise adherence: (a) social support perceived behavioral control intention excersise adherence, and (b) cohesion attitude intention exercise adherence. Discussion focuses on the theoretical importance of these findings, conceptual and measurement issues regarding subjective norm, and suggestions for future research.     

Blockage to exonuclease III digestion in the chromatin of Saccharomyces cerevisiae maps to the in vitro — determined binding site of a trans-acting regulatory factor

Summary A series of transposable element-induced mutations at the HIS4 locus in Saccharomyces cerevisiae have been attributed to the transposition of a Ty element into the 5 regulatory region of this gene. Various Ty-containing His⁺ revertants have been isolated and the HIS4/Ty junction region sequenced. The only difference found in this region between a His^- and a weak His⁺ strain was a single point mutation, an AG transition. The position of Ty remained unaltered. Examination of lacZ fusion plasmids further implicated this AG transition as being reponsible for the altered phenotype, the bp transition representing an allele of a cis-acting regulatory element. Subsequent gel retardation and methylation interference experiments revealed that this AG mutation enabled the binding of a trans-acting factor (TyBf) in vitro. In this paper we show that the TyBf binding site is in a region of chromatin hypersensitive to digestion by DNase I. The binding site is protected in vivo from digestion with exonuclease III, suggesting the presence of a bound protein in His⁺ (on) but not His^- (off) Ty-containing strains. We propose that a trans-acting factor binding in vivo, presumably TyBf, is responsible for the activation of HIS4 expression in these insertion mutants.     

The effect of praziquantel on the pseudophyllidean cestodeBothriocephalus acheilognathi in vitro

AdultBothriocephalus acheilognathi were incubated in solutions containing 0 (control), 0.1, 1.0, 10.0 and 100 g praziquantel per ml (0, 10², 10³, 10⁴ and 10⁵ gl^–1) of 0.9% saline for 5, 15 and 60 min at a temperature of 18°C. The worms contracted immediately upon being placed in the drug. Scanning and transmission electron microscopy revealed considerable tegumental damage particularly in the neck region. Vacuolization and bubbling to the tegument occurred in all of the drug solutions tested. Exposure to drug concentrations of more than 1.0 gml^–1 (10³ gl^–1) praziquantel for 15 min or greater resulted in many of the bubbles bursting and releasing their contents to the exterior. Mature proglottides were distorted and had occasional large swellings resulting in the mass expulsion of eggs. Praziquantel had no ovicidal activity. Exposure to drug concentrations of 100 g (10⁵ gl^–1) praziquantel per ml saline for 24 h was not lethal to the worms.     

Macrophage Stimulator β-(1→3)-D-Carboxymethylglucan Improves the Efficiency of Chemotherapy of Lewis Lung Carcinoma

We studied the effect of macrophage stimulator water-soluble -(13)-D-carboxymethylglucan on the efficiency of cyclophosphamide chemotherapy in Lewis lung carcinoma. Cyclophosphamide inhibited the growth of primary tumor nodes by 57%. The preparation possessed pronounced antimetastatic activity: metastases were found in 40.9% animals. Combination therapy with cyclophosphamide and (13)-beta;-D-glucan inhibited the growth of intramuscular tumors by 75-89% and reduced the incidence of metastases into the lungs by 92-94%. The therapeutic effect was most pronounced after simultaneous administration of these preparations: tumor growth was suppressed by 89.3% and metastases were found in only 7.5% animals (vs. 100% in the control). The potentiating effect of -(13)-D-carboxymethylglucan is related to accumulation of cysteine proteinase inhibitors in the tumor tissue and plasma, but not to changes in blood cell composition.     

Oligoclonal interspecific origin of ‘North Indian’ and ‘Chinese’ sugarcanes

Sugarcanes consist of several groups of complex polyploid forms. The origin of North Indian and Chinese sugarcanes (referred to as S. barberi and S. sinense) was investigated using genomic in-situ hybridization (GISH), detection of species-specific repeated sequences and RFLP. GISH proved their interspecific hybrid origin. Together with the distribution of species-specific repeated sequences and earlier RFLP data, the results show that both taxa are derived from interspecific hybridization between S. officinarum and S. spontaneum and that no other genus has been directly involved. RFLP indicates that the clones are clustered into a few groups, each derived from a single interspecific hybrid that has subsequently undergone a few somatic mutations. These groups correspond quite well with those already defined based on morphological characters and chromosome numbers. However, the calculated genetic similarities do not support the existence of two distinct taxa. The North Indian and Chinese sugarcanes represent a set of horticultural groups rather than established species.     

Spectrum of spontaneous missense mutations causing cyclic AMP-resistance phenotypes in cultured S49 mouse lymphoma cells differs markedly from those of mutations induced by alkylating mutagens

Mutants of S49 mouse lymphoma cells resistant to cytolysis by analogs of cyclic AMP (cAMP) generally have missense mutations in the gene encoding the regulatory subunit of cAMP-dependent protein kinase. We have compared the mutations in 95 spontaneous isolates with those in 60 mutagen-induced isolates by sequence analysis of amplified cDNAs. Twenty-nine single basepair substitutions in 19 codons produced selectable phenotypes. The spontaneous mutant spectrum was dominated by a CpG transition hotspot in the codon for Arg334. This and other nearby CpG sites were found to be methylated in genomic S49 cell DNA by restriction enzyme analyses. Most of the remaining spontaneous mutants had either G-CC-G or T-AG-C transversions, which have been associated with damage caused by oxygen radicals. In contrast, the majority of mutants induced with the alkylating mutagens ethyl methanesulfonate (EMS) and N-methyl-N-nitro-N-nitrosoguanidine had G-CA-T mutations at non-CpG sites; in addition, EMS induced several A-TG-C, A-TT-A, and G-CT-A substitutions. A single ICR191-induced mutant analyzed had a unique A-TG-C lesion. A number of spontaneous and mutagen-induced isolates had closely linked double or triple substitutions, and two isolates had tandem triple substitutions.     

Lack of association of polymorphisms of the lymphotoxin α gene with myocardial infarction in Japanese

Vascular inflammation plays an important role in the development of myocardial infarction (MI). Lymphotoxin (LTA) is a cytokine with multiple functions in regulation of the immune system and inflammatory reactions. The aim of this study was to examine whether polymorphisms of the LTA gene are associated with the risk of MI in Japanese men and women. A case-control association study was performed for the 252AG and 804CA polymorphisms of the LTA gene and the prevalence of MI. The study population comprised 3,689 unrelated Japanese individuals (2,486 men, 1,203 women), including 1891 patients with MI (1,493 men, 398 women) and 1798 control subjects (993 men, 805 women). Among the control subjects 257 individuals (108 men, 149 women) who had none of the conventional risk factors for coronary artery disease (CAD) were defined as low-risk controls. Genotypes for the two polymorphisms were determined with a fluorescence-based allele-specific DNA primer assay system. Among all study subjects the 252AG and 804CA polymorphisms exhibited linkage disequilibrium. No association of either polymorphism with MI was detected in men or in women in comparisons with total control or low-risk control subjects. However, each of the two polymorphisms was associated with the prevalence of type 2 diabetes mellitus both in men with MI and in those without MI in a recessive genetic model. No association was detected between the polymorphisms and other conventional risk factors for CAD. The LTA gene thus does not appear to be a susceptibility locus for MI in Japanese men or women, although it might affect susceptibility to type 2 diabetes in Japanese men.Abbreviations BMI Body mass index - CAD Coronary artery disease - HLA Human leukocyte antigen - LTA Lymphotoxin - MI Myocardial infarction - PCR Polymerase chain reaction - TNF Tumor necrosis factor     

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.     

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.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

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     

The role of acidic residues in the “fusion segment” of influenza A virus hemagglutinin in low-pH-dependent membrane fusion

Summary To clarify the role of acidic amino acid residues in the fusion segment of hemagglutinin (HA) of influenza A virus (H1N1) in pH-dependent membrane fusion, we have constructed and expressed five mutant HA cDNAs in CV-1 cells by SV40-HA virus vectors (SVHA). Fusion activities of the five mutant HAs were examined by lipid mixing and polykaryon formation assays. In spite of the substitution of Gly and Lys for the acidic residues, all the mutants were found to retain their low-pH-dependent fusion activity by lipid mixing assay. Although SVHA-G19(HA₂19D G), –K11 (HA₂11E K) and –K19(HA₂19D K) induced polykaryon formation at low pH as wild type HA did, SVHA-G11(HA₂11E G) induced limited polykaryon formation and SVHA-G11,19 (HA₂11E G, 19D G) did not. The substitution of Gly for Glu at position 11 inhibited widening of the initial fusion pore. However, Lys mutants induced the formation of an initial fusion pore and widened it at low pH where Lys residues might have positive charges. These results suggest that the neutralization of the charges on acidic residues in the fusion segment at low pH is not important for interaction of the fusion segment with the target lipid bilayer or for triggering the membrane fusion.