Expression of transforming growth factor alpha in human tissues: Immunohistochemical study and Northern blot analysis

Summary The expression of transforming growth factor alpha (TGF-) was examined in various human tissues and the fetus, using immunohistochemistry and Northern blot analysis. TGF- immunoreactivity was detected mainly in the epithelial cells of the digestive tract, liver, pancreas, kidney, thyroid, adrenal, skin, mammary gland and genital organs. In the digestive tract, epithelial cells with regenerative change or hyperplastic change showed strong immunoreactivity to TGF-. Peripheral nerve, vessels, megakaryocytes and macrophages in the lung and spleen were also positive for TGF-. By Northern blot analysis the expression of TGF- mRNA was confirmed in the digestive tract, salivary gland, thyroid, kidney and mammary gland. In the human fetus, the nerve tissues, liver, adrenal and kidney were positive for TGF-. Strong immunoreactivity to TGF- was observed in the hepatocytes of the fetus. These findings indicate that TGF- is produced by a variety of nonneoplastic cells in both adult and fetal tissues.     

A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae

Summary The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT, and HO genes of Saccharomyces cerevisiae as probes. It was shown that S. exiguus has a DNA sequence homologous with the HO gene of S. cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S. exiguus. However, there is no DNA sequence in S. exiguus that is homologous with the MAT genes of S. cerevisiae.     

β-Subunit expression is required for cAMP-dependent increase of cloned cardiac and vascular calcium channel currents

In contrast to vascular smooth muscle (VSM). cAMP-depehdent phosphorylation increases L-type voltage dependent Ca²⁺-channel (L-VDCC) activity in heart. To investigate whether this difference depends on the tissue-specific ₁-subunit of the L-VDCC or its regulation by other subunits, we used a Xenopus laevis oocyte expression system. Injection of cAMP into oocytes expressing cardiac ₁ or VSM ₁ alone had no effect on L-VDCC activity. However, cAMP increased L-VDCC activity 2-fold in oocytes co-expressing cardiac ₁ or VSM ₁ with the skeletal muscle ß-subunit. These results suggest that the presence of the ß-subunit is required for cAMP-mediated increase of L-VDCC activity and that the characteristics of tissue-specific ß-subunits may explain differential regulation of L-VDCC activity.     

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     

Regulated overproduction of α-amylase by transformation of the amylolytic yeast Schwanniomyces occidentalis

Summary High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S. occidentalis, which we called SwARS1. The SwARS1 fragment is also functional in S. cerevisiae. The average copy number of the plasmids in both yeast species was 5–10 per cell under selective conditions. S. occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned -amylase gene from S. occidentalis secreted about five times more -amylase than cells without additional copies of the -amylase gene. Both the chromosomal copy and the plasmid-carried copies of the -amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.     

Molecular cloning and characterization of a Candida tsukubaensis α-glucosidase gene in the yeast Saccharomyces cerevisiae

Summary The molecular cloning of an -glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cerevisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing -1,2, -1,3, -1,4 and 1,6 linked, as well as aryl and alkyl, d-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an -glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2–4.6, a temperature optimum of 58°C and is readily inactivated at pasteurization temperature (60°C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X--d-glucoside to detect the expression of the cloned -glucosidase in S. cerevisiae transformants, was developed.     

Characterization of a novel α-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae

A highly active -amylase (76 250 Da) secreted by the raw starch-degrading yeast Lipomyces kononenkoae strain IGC4052B was purified and characterized. Using high performance liquid chromatography (HPLC), end-product analysis indicated that the L. kononenkoae -amylase acted by endo-hydrolysis on glucose polymers containing -1,4 and -1,6 bonds, producing mainly maltose, maltotriose and maltotetraose. The following NH₂-terminal amino acids were determined for the purified enzyme: Asp-Cys-Thr-Thr-Val-Thr-Val-Leu-Ser-Ser-Pro-Glu-Ser-Val-Thr-Gly. The L. kononenkoae -amylase-encoding gene (LKA1), previously cloned as a cDNA fragment, was expressed in Saccharomyces cerevisiae under the control of the PGK1 promoter. The native signal sequence efficiently directed the secretion of the glycosylated protein in S. cerevisiae. De-glycosylation of the enzyme indicated that post-translational glycosylation is different in S. cerevisiae from that in L. kononenkoae. Zymogram analysis indicated that glycosylation of the protein in S. cerevisiae had a negative effect on enzyme activity. Southern-blot analysis revealed that there is only a single LKA1 gene present in the genome of L. kononenkoae.     

Interspecific actions of α mating pheromones on the a mating-type cells of three Saccharomyces yeasts

Summary The a mating pheromones synthesized in three Saccharomyces yeasts (S. cerevisiae, S. kluyveri, and S. exiguus) displayed interspecific actions on the a cells of all three species despite the fact that the amino acid sequences of all three pheromones are different. Mating between species, however, did not occur. The interspecifie pheromone — a cell reaction was not necessarily more effective than the interspecific one. Deceased on March 28, 1987     

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.     

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.     

Proteinase — Antiproteinase imbalance in meningitis: Determination of α 1 proteinase inhibitor (α 1PI), elastase — α-1PI complex,and elastase inhibition capacity in cerebrospinal fluid

Summary Mortality and long-term neurologic sequelae are still frequent complications of meningitis despite effective antibiotic treatment. This suggests that pathogen-independent inflammatory mechanisms may play an important role in the course of this illness.Neutrophil granulocytes form the primary immune defense in meningitis. Once activated, these cells release elastase into the cerebrospinal fluid (CSF). Elastase may induce tissue damage if local antiproteinase capacity is low as under normal conditions.To define the relevance of this mechanism we studied 22 patients with meningitis. Concentrations of elastase in complex with the main antiproteinase 1-proteinase inhibitor (elastase- 1PI), 1-proteinase inhibitor ( 1PI), and elastase inhibition capacity (EIC) were measured in CSF of 9 patients with bacterial meningitis (BM), aged 1 month-214 years; 13 patients with non-bacterial meningitis (NBM), aged 1 month–15 years; and 20 patients in whom meningitis was excluded after spinal tap (control group), aged 6 months–15 years. The concentration of elastase- 1PI in the BM group (median 552 g/l) was significantly higher than in either the NBM group (median 30 g/l,p<0.01) or the control group (median 30 g/l,p<0.01). Similarly, the 1PI-concentration in the BM group was significantly higher (median 113 mg/l) than either the NBM group (median 13.7 mg/l,p<0.025) or the control group (median 6.3 mg/l,p<0.001). The concentration of elastase- 1PI shows a significant correlation with the duration of the infectious symptoms before admission to the hospital (r=0.51,p<0.02), but not with the number of neutrophil granulocytesr=0.23, p=0.21).Free elastolytic capacity in CSF could be demonstrated in 4 patients: 1 with BM, 2 with NBM, and 1 with pertussis pneumonia and enzephalitis.The measured insufficiency of the proteinase-antiproteinase system may indicate high-risk patients in need of additional anti-inflammatory therapy, e.g., with corticosteroids, during the initial phase of meningitis.Abbreviations 1PI 1-proteinase inhibitor, 1-antitrypsin - elastase- 1PI complex elastase- 1-proteinase inhibitor complex - EIC elastase inhibition capacity - BM group: bacterial meningitis - NBM group: non-bacterial meningitis - CSF cerebrospinal fluid     

Direct induction of tetraploids or homozygous diploids in the industrial yeast Saccharomyces cerevisiae by hydrostatic pressure

Summary Hydrostatic pressure and a dye plate method were used to investigate the direct induction of tetraploids or homozygous diploids from the industrial diploid or haploid yeast Saccharomyces cerevisiae. Above 200 MPa, hydrostatic pressure greatly inactivated the strains HF399s1 ( haploid), P-540 (a/ diploid), and P-544 (a/ diploid). At the same time, when pressure-treated cells of these strains were spread on a dye plate, some of the visible colonies were stained red/blue or dark blue (variant colonies); the rest stained violet, similar to colonies originating from diploid cells or haploid cells that were not pressure-treated. In addition, above 100 MPa, the formation of variant colonies increased with increasing pressure, and maximized (1x10^-1) at 200 and 250 MPa, respectively. The size of almost all variant cells from P-544, P-540, and HF399s1 was visibly increased compared with that of untreated cells and the measured cellular DNA content of P-540 and HF399s1 was double that of untreated cells. Furthermore, based on random spore analysis and mass-matings, induced variants in the diploid strains were found to be tetraploid with an a/a// genotype at the mating-type locus or, in the haploid strains, homozygous diploid with an / genotype. From these results we conclude that pressure treatment in combination with a dye plate is a useful method for strain improvement by direct induction of tetraploids or homozygous diploids from industrial strains whether diploid or haploids.     

β-Subunits co-determine the sensitivity of rat neuronal nicotinic receptors to antagonists

We have investigated the effect of 4 ganglionic cholinergic antagonists (hexamethonium, mecamylamine, pentolinium, trimetaphan) on rat 32 and 34 neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. Current responses were elicited by fast application of acetylcholine on voltage-clamped oocytes (holding potential Vinh = -80mV). Concentration-inhibition curves were used to get estimates of IC₅₀, the antagonist concentration yielding 50% reduction of the peak current. The K_B's of the antagonists were calculated using estimates of the apparent K_D of acetylcholine. The order of affinity of the antagonists was similar for both receptor subtypes: mecamylamine pentolinium > hexamethonium > trimetaphan. However, 34 neuronal nAChRs were 9 to 22 times more sensitive to each of the 4 antagonists than 32 receptors. These results further underline the importance of the -subunit as co-determinant of the functional properties of neuronal nAChRs.     

Hybridization and cytoduction among yeast cells of the same mating type

Summary Use of a selective system for cytoduction in Saccharomyces cerevisiae allowed us to monitor hybrid formation and to clone the haploid nuclei of cells which have participated in illegitimate matings: a × a, × . Our approach has made it possible to select nuclei with mating-type switches and mutations within the MAT locus. It was shown that matings in × crosses often proceed through nonheritable genetic changes located within chromosome III. We suggest that these non-heritable genetic changes are due to premutational lesions, expressed phenotypically as transient -matingtype. After a mating event these lesions are either repaired or converted to true mutations within the MAT locus.     

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.     

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.     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

Evidence for preferential multiplication of the internal unit in tandem repeats of the mating factor a genes in Saccharomyces yeasts

Summary We have determined DNA sequences of the mating factor a genes of Saccharomyces uvarum and Saccharomyces italicus and compared them to that of the MF1 gene of S. cerevisiae. The DNA sequences of the mating factor genes in both species were almost completely identical to that of the MF1 gene of S. cerevisiae except for the number of tandem repeated units; these latter consisted of a spacer peptide and a mature mating factor and there were three units in S. uvarum and five units in S. italicus compared with four units in the MF1 of S. cerevisiae. From the detailed comparison of DNA sequences of the spacer peptide-mating factor units from these three species, the high sequence homology can be recognized in the internal units of the tandem repeats. This suggests that the internal units might be multiplied preferentially in the tandem repeated units of mating factor genes.     

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.     

Serum interferon activity in inflammatory bowel disease

Serum interferon (IFN) of-class was studied in 64 consecutive patients, 26 with Crohn's disease, 38 with ulcerative colitis, and in 34 healthy volunteers. Detectable IFN- in 10 patients was associated with a moderate to severe activity of chronic inflammatory bowel disease (CIBD). However, 19 of 28 patients (68%) with activity in their disease did not have elevated IFN- levels. The three groups, ulcerative colitis, Crohn's disease, and healthy volunteers did not reveal any statistically significant difference in serum IFN-, as four of 34 healthy controls without intercurrent infections had elevated levels as well. Possible effects of, , and classes of IFN on endogenous arachidonic acid (AA) release and metabolism in human neutrophils was investigated in a substudy. IFN- caused a dose-dependent release of AA from phospholipids and metabolism of a modest fraction of leukotriene B₄ (LTB₄) and 5-hydroxyeicosatetraenoic acid (5-HETE) at doses reaching a maximum of 100 IU/ml. IFN of the and classes did not exert such effects. Addition of complement 5a to cells activated by IFN- caused induction of increased 5-li-poxygenase activity with unchanged release of AA. As only 16% of all CIBD patients had elevated IFN- levels as compared to 12% among the group of healthy volunteers, IFN- does not seem to be of importance for the perpetuation of the inflammatory reaction in ulcerative colitis or Crohn's disease, and other factors may therefore be responsible for activation of the inflammatory cells to production of LTB₄ and 5-HETE.