Tuula K. Torkkeli

Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei

A cDNA encoding for the glucoamylase P enzyme (GAMP) of the fungus Hormoconis resinae was introduced into the cellulolytic filamentous fungus Trichoderma reesei under the control of the promoter of the major cellulase gene (cbh1) of Trichoderma. The transforming vector plasmid used was found to be integrated into the genome of T. reesei at various locations and in multiple copies. The size of the GAMP secreted by Trichoderma varied because of different glycosylation patterns. The best transformant strains secreted about 700 mg/l of active GAMP, which is 20-fold more than obtained with H. resinae.

Uterine and lung uteroglobins in the rabbit. Two similar proteins with differential hormonal regulation.

Previous studies have shown that several rabbit tissues contain proteins which cross-react in the radioimmunoassay for uteroglobin, a progestin-regulated protein in rabbit uterus (Torkkeli et al. (1977) Mol. Cell. Endocrinol. 9, 101-118). In the present study, a uteroglobin-like protein was purified to an apparent homogeneity from an extra-uterine tissue, rabbit lung, by successive chromatographies on hydroxyapatite, Sephadex G-75, SP-Sephadex, DEAE-cellulose and CM-cellulose. The final preparation behaved homogeneously in various polyacrylamide gel electrophoretic systems and in isoelectric focusing. The uteroglobin-like protein isolated from the lung had very similar physicochemical and immunological properties to those of uteroglobin present in the rabbit uterine fluid. The two proteins had: (i) the same molecular weight, of approx. 13 000, with a two subunit structure (each approx. Mr 7000); (ii) identical behavior in polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions; (iii) the same isoelectric point at pH 5.4; (iv) absence of carbohydrate in the molecule; (v) very similar amino acid compositions; (vi) lack of tryptophan among the amino acids; (vii) the same N-terminal amino acid (glycine), and (viii) indistinguishable immunological characteristics. Collectively, these data strongly suggest that uterine and lung uteroglobins are identical proteins. In contrast to the induction of the uterine uteroglobin by steroids with progestational activity, the synthesis of extra-uterine uteroglobins was no affected by these steroid hormones to any major extent. In keeping with the concept that lung is a target tissue for glucocorticoid action, cortisol and dexamethasone were capable of increasing the concentration of lung uteroglobin 3-fold (from 3 to 9 microgram/mg soluble protein). These compounds did not, however, alter the secretion of the uterine protein. Administration of high doses of testosterone and 5alpha-dihydrotestosterone elevated significantly the content of both uterine and lung uteroglobin. Only approx. one-fifth of the adult pulmonary uteroglobin levels were present in lungs of newborn rabbits indicating that developmental changes occur in the lung uteroglobin content.

Changes in cytosol and nuclear progesterone receptor concentrations in the rabbit uterus and their relation to induction of progesterone-regulated uteroglobin

Abstract To study the relation between steroid receptor concentrations and biological response, we measured cytosol and nuclear progesterone receptors from rabbit uterus under different experimental conditions, and compared receptor values with induction of uteroglobin, a progesterone-regulated protein. A 5-day progesterone treatment (1 mg/kg per day) reduced the nuclear receptor content by 40%, slightly elevated cytosol receptor levels and increased uteroglobin content 3000-fold. Estradiol and tamoxifen altered progesterone-induced changes in the receptor and uteroglobin values: cytosol and nuclear receptors rose significantly, but uteroglobin induction declined markedly, when progesterone treatment was supplemented with estradiol or tamoxifen. A 50% inhibition of progesterone action on uteroglobin synthesis was achieved with 1 μg/kg of estradiol per day. Thus under certain conditions, there is a clear disparity between steroid receptor levels and biological response.

Hormonal regulation of uterine blastokinin synthesis and occurrence of blastokinin-like antigens in nonuterine tissues.

Abstract A radioimmunoassay (RIA) for blastokinin from rabbit uterine fluid has been developed using antisera raised in male guinea pigs against purified blastokinin. [ 125 I]Iodoblastokinin was prepared with a chloramine-T method and the separation of bound and free antigens in the RIA was achieved by polyethylene glycol precipitation. Using this technique, blastokininlike antigenicity was detectable in several tissues of female and male rabbits, the highest concentrations being present in the uterine fluid and cytosols derived from endometrium, myometrium and lungs. Blastokinin preparations purified from rabbit uterine fluid and lungs exhibited very similar physical properties. Blastokinin-like antigens in lungs of both female and male rabbits were not influenced by administration of either progestins or human chorionic gonadotropin (hCG), indicating a different control mechanism than that for blastokinin of uterine origin. Administration of hCG for 5 days brought about approximately a 400-fold increase in the immunoreactive blastokinin level in uterine flushings (2600 vs. 6.7 μg/uterus), a 10-fold increase in the endometrium (50 vs. 4 μ/g tissue wet weight) and a 35-fold increase in the myometrium (170 vs. 5gmg/g tissue). Following a single intravenous dose of progesterone, radio immunoassayable blastokinin started to rise in the uterine fluid within 8 h of hormone administration, peaked at 12 h and declined to pretreatment levels after 30 h. A similar time-course of blastokinin induction was observed when medroxyprogesterone acetate, d-norgestrel or norethisterone was given. No rabbit blastokinin-like antigenicity was found by RIA in human uterine flushings collected from normal women during the different phases of the menstrual cycle or following administration of synthetic progestins.

Further studies on the role of estradiol in the induction of progesterone-regulated uteroglobin synthesis in the rabbit uterus

Abstract The role of a continuous presence of estradiol for the induction of uteroglobin, a progester-one-regulated protein in the rabbit uterus, was studied by administration of various doses of estradiol (0.1–2000 μg per day) along with a constant dose of progesterone (2 mg per day) for 5 days to estrous rabbits. Smaller doses of estradiol slightly potentiated the action of progester-one, as judged by measurements of luminal fluid uteroglobin content and of uteroglobin mRNA activity in the uterus. By contrast, higher doses of estradiol almost totally inhibited the action of progesterone. A 50% inhibition of uteroglobin secretion was achieved with a daily dose of 3 μg of estradiol (1.5 μg/kg per day). A similar dose of estradiol was required to decrease by 50% the progesterone-induced accumulation of uteroglobin mRNA activity. The decline in the uteroglobin content in the uterine flushes elicited by estradiol treatment was not due to inhibition of the release of this protein from the endometrial cells, since all the doses of estradiol given along with progesterone diminished uterine tissue uteroglobin content from that achieved with progesterone alone. When estradiol was administered alone to estrous rabbits, a dose-dependent increase was seen in both tissue and luminal fluid uteroglobin levels. The maximal luminal fluid uteroglobin content was about 10-fold higher than that in the control animals, but was only less than 1% of that achieved with a daily dose of 2 mg of progesterone. A half-maximal increase in the uterine fluid uteroglobin concentration was obtained with a daily dose of 3 μg (1.5 μg/kg per day) i.e. with the same dose as that required for a 50% inhibition of progesterone action.

A novel glucoamylase preparation for grain mash saccharification.

SummaryThe capacity to saccharify barley grain mash of Hormoconis resinae glucoamylase P produced by a heterologous host, Trichoderma reesei, was compared with that of Aspergillus niger glucoamylase. The results showed that the glucoamylase P secreted by T. reesei produces more fermentable sugars from mash and thus makes a higher ethanol yield possible in fermentation.

Early changes in rabbit uterine progesterone receptor concentrations and uteroglobin synthesis after progesterone administration

Abstract Uterine cytosol and nuclear receptors were measured at various time intervals after one dose or two consecutive doses of progesterone and the receptor values compared with the course of induction of uteroglobin, a progesterone-regulated uterine protein. The data of this study suggest (i) that progesterone action involves receptor consumption and (ii) that in the rabbit uterus the biologic response is not a direct function of available cytosol or nuclear receptor sites.