Progesterone transformations by three species ofHumicola

Five isolates belonging to three species of the genusHumicola were tested in this study for their ability to transform progesterone. An oxidative splitting of the side chain of progesterone with the formation of androst-4-ene-3,17-dione, testosterone and testololactone was achieved by all isolates tested ofH. fuscoatra andH. grisea. H. hyalothermophila transformed progesterone to 11α-, 11β-, 17α- and 21-hydroxyprogesterone and a dihydroxyl product (11α, 17α-dihydroxyprogesterone) with the addition of two trihydroxyl products,viz. cortisol and epicortisol. Qualitative and quantitative analysis of the different products obtained when a selective isolate of each species acted on progesterone were conducted. The chromatographic resolution of the mixture products obtained when the selective isolate of each ofH. fuscoatra andH. grisea had acted individually on 1 g progesterone revealed the presence of 25 and 20% unchanged progesterone, 20 and 22% androst-4-ene-3,17-dione, 25 and 23% testosterone and 30 and 35% testololactone, respectively. Seventy-four % of progesterone were bioconverted byH. hyalothermophila into 21-hydroxyprogesterone (6%), 17α-hydroxyprogesterone (5%), 11α-hydroxyprogesterone (11%), 11β-hydroxyprogesterone (12%), 11α,17α-dihydroxyprogesterone (5%), cortisol (21%) and epicortisol (13%). This is the first record of conversion of progesterone to both cortisol and epicortisol byH. hyalothermophila.     

Confirmation of the relationships of Aspergillus egyptiacus and Emericella nidulans using progesterone transformation

Isolates of Emericella nidulans (7 isolates), Aspergillus egyptiacus (7) and A. versicolor (5) were tested for their ability to transform progesterone. Consistent results were obtained between isolates of the same species. Whilst A. egyptiacus and E. nidulans possessed the enzyme system catalysing the transformation of progesterone into 11-α-hydroxyprogesterone, A. versicolor did not. Thus, progesterone transformation could be considered as further diagnostic evidence that A. egyptiacus is related to the Emericella group.     

Progesterone transformation as a diagnostic feature in the classification of the Aspergillus niger group

Fifty-five isolates of nine species and two species varieties (5 isolates each) of the Aspergillus niger group were tested for progesterone transformation. Generally, all isolates of the different species transformed progesterone to 21-hydroxyprogesterone. In addition, the isolates of seven species have the capacity to hydroxylate progesterone to 11α-hydroxyprogesterone. A systematic variation could be observed between the tested species of the Aspergillus niger group with respect to the transformation reactions of progesterone. Comparative biotransformation results showed that essential differences exist between the species of this group, where the different isolates of individual species always produce one or more identical products. This biochemical differentiation may supplement the morphological criteria used in the classification of this group of fungi.     

Continuous hydroxylation of progesterone byAspergillus ochraceus

Summary In this paper are presented the results of experiments undertaken to determine what conditions are necessary for the hydroxylation of progesterone during continuous culture ofAspergillus ochraceus NRRL 405. It was found possible to operate with either one or two stages, but because of the inhibitory effect of progesterone on the growth of the microorganism, optimum yields of 11α-hydroxyprogesterone could be obtained only with a two-stage system in which the organism was grown continuously in the first stage and progesterone was added continuously and hydroxylated in the second stage. In addition it was observed that there was an apparent cycling of the microorganism concentration in the period preceding the steady state. No explanation for this was found, and this problem in dynamics is being investigated further. Details are presented of an isotope dilution method using 4-C¹⁴-progesterone for determining the concentration of progesterone and its derivatives in fermentation broths. This investigation was supported in part by research grant RG-5782(A) from the U.S. Public Health Service. Contribution No. 373 from the Department of Food Technology, Massachusetts Institute of Technology, Cambridge, Mass.     

Plasma Sex Steroid Levels and Steroidogenesis in the Gonad of the Self-fertilizing Fish Rivulus marmoratus

Synopsis The mangrove killifish, Rivulus marmoratus, is the only known self-fertilizing vertebrate. This species is sexually dimorphic; sexually mature individuals are either hermaphrodite or primary and secondary males. Although the mangrove killifish has a unique reproductive strategy, there has been no study on the reproductive endocrinology of this species. Thus we investigated plasma sex steroid hormone levels and steroidogenesis in the gonads of R. marmoratus by enzyme linked immunosorbent assay (ELISA). Plasma 17β-estradiol (E2) and 11-ketotestosterone (11-KT) were detected both in hermaphrodite and in primary male. Ovarian follicles (follicle-enclosed oocytes) from hermaphrodites, which were categorized into early yolk stage and late yolk stage, and testis tissue of primary males were cultured with different concentrations of 17α-hydroxyprogesterone (OHP) or testosterone (T) for 24 h. Production of T, E2, 11-KT and 17α-20 β-dihydroxy-4-pregnen-3-one (17α,20β-P) in the medium from tissue culture were measured by ELISA. Early and late ovarian follicles of hermaphrodites and testis pieces of primary males synchronously secreted E2, 11-KT, and 17α,20β-P following incubation with OHP or T. We conclude that both hermaphrodite and primary male of the mangrove killifish secrete estrogen, androgen, and progestin synchronously.     

Studies on Geobacillus stearothermophilus – Part V: Transformation of 11α-hydroxyprogesterone

The influence of a hydroxyl group on the biotransformation of 11α-hydroxyprogesterone mediated by the thermophile Geobacillus stearothermophilus, was investigated. Bacterial transformation of 11α-hydroxyprogesterone resulted in the formation of previously reported six hydroxylated progesterone metabolites, identified as 11α-hydroxy-5α-pregnane-3, 6, 20-trione 1, 11α, 20α-dihydroxypregnene-3-one 2, 11α, 6β-dihydroxyprogesterone 3, 11α, 6α-dihydroxyprogesterone 4, 11α, 6β, 20α-trihydroxypregnene-3-one 5, 11α, 6α, 20α-trihydroxypregnene-3-one 6. All transformation products were identified through their spectral data and comparison with reference compounds.     

Antimicrobial agents

In a study of the effect of progesterone, its hydroxy derivatives and various sterols on the growth of dermatophytes, it was demonstrated that all the steroid compounds employed inhibited growth of the 51 strains of dermatophytes tested. No significant differences were found in sensitivity to the given steroids, either in dermatophyte strains of the same species, but of different origin, ior in different species of the four genera used (Trichophyton, Microsporum, Epidermophyton andKeratinomyces). Hydroxylation of the progesterone molecule in any except the C-21 position lowered the inhibitory effect. The course of the transformation reaction on the progesterone or cholesterol molecule was likewise of a uniform character from the aspect of species specificity. Progesterone was simultaneously hydroxylated in positions 15α and 15β by all the strains, giving rise to monohydroxy-derivatives as the main metabolites, and cholesterol was oxidized to cholestenone.     

Effect of triterpenoid glycosides on α- and β-amylase activity and total protein content in wheat seedlings

Influence of the aleanolic acid glycosides from Silphium perfoliatum L. (silphioside B, C, E and G) and their progenins on the amylase activity and total protein content in wheat seedlings was studied. Treatment of the Triticum aestivum L. seeds with 1–10 μM water solutions of mono- and diglycosides (mono- and bisdesmosines) elevated the α-amylase and total amylase activities in seedlings. Silphioside E containing three glucose moieties in its molecule did not change α-amylase activity, but it did if bis-triglycoside acetylated carbohydrate (as in silphioside C). Effects of 5–10 μM solutions of the active glycosides was comparable with that of exogenous gibberellin A₃ and 6-benzylaminopurine.     

α-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans

The effect of biomass concentration on the formation of Aspergillus oryzaeα-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of α-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2–12 g dry weight kg⁻¹. When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzaeα-amylase gene (creA) was deleted, no significant decrease in the specific rate of α-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific α-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium. Received: 13 January 2000 / Received revision: 30 June 2000 / Accepted: 1 July 2000     

Induction of aryl hydrocarbon hydroxylase inNeurospora crassa by benzo (α) pyrene

WhenNeurospora crassa is grown on a minimal medium with sucrose as the carbon source, aryl hydrocarbon [benzo(α)pyrene] hydroxylase is induced in the presence of low concentrations of benzo(α)pyrene. Benzo(α)pyrene, a potent precarcinogen, is taken up readily by the growing mycelium and is metabolized by the intracellular enzymes to yield hydroxylated derivatives. Fractionation of the products by high pressure liquid chromatography following extraction in organic solvents revealed the presence of one major product. The purified major product was identified as 3-hydroxybenzo(α)pyrene by mass spectral analysis and by comparison of fluorescence emission and ultraviolet absorption spectra with authentic samples.     

Genetic diversity of chromosomal metallo-β-lactamase genes in clinical isolates of <Emphasis Type="Italic">Elizabethkingia meningoseptica</Emphasis> from Korea

This study was performed to characterize the chromosomal metallo-β-lactamases (MBLs) of Elizabethkingia meningoseptica isolated from Korea and to propose a clustering method of BlaB and GOB MBLs based on their amino acid similarities. Chromosomal MBL genes were amplified by PCR from 31 clinical isolates of E. meningoseptica. These PCR products were then cloned into a vector and electrotransformed into E. coli DH5α. Nucleotide sequencing was performed by the dideoxy chain termination method using PCR products or cloned DNA fragments. Antimicrobial susceptibilities were determined by the agar dilution method. PCR experiments showed that all 31 E. meningoseptica isolates contained both the blaB and the bla _GOB genes. DNA sequence analysis revealed that E. meningoseptica isolates possessed seven types of blaB gene, including five novel variants (blaB-9 to blaB-13) and 11 types of bla _GOB gene, including 10 novel variants (bla _GOB-8 to bla _GOB-17). The most common combination of MBL was BlaB-12 plus GOB-17 (n=19). Minimum inhibitory concentrations of imipenem and meropenem for the electrotransformants harboring novel BlaB and GOB MBLs were two- or four-fold higher than those for the recipient E. coli DH5α. BlaB and GOB MBLs were grouped in three and six clusters including fifteen novel variants, respectively, based on amino acid similarities.     

Based on biochemical and physiological behavior,where isAspergillus egyptiacus better placed?

Physiological and biochemical properties were tested in 45 isolates ofAspergillus egyptiacus (16 isolates),Emericella nidulans (16) andAspergillus versicolor (13). The three fungal species exhibited common and similar features. The big similarity betweenA. egyptiacus andE. nidulans was greater than betweenA. egyptiacus andA. versicolor. It included the inability to produce base either from sodium citrate or lactic acid media, growth at 45 °C (thermophilicity), and production of very similar pigmentations onAspergillus flavus andparasiticus agar.A. egyptiacus is therefore better placed in theAspergillus nidulans-Emericella assemblage.     

Secretory expression in Escherichia coli and Bacillus subtilis of human interferon alpha genes directed by staphylokinase signals

Summary A DNA segment covering the signal sequence coding region, the ribosome binding site, and the promoter of the staphylokinase (sak) 42D gene (Behnke and Gerlach 1987) was cloned into pUC19 to form a portable expression-secretion unit (ESU). Fusion of human interferon α₁ (hIFNα₁) and hybrid hIFNα_1/2 genes to thissak ESU resulted in secretory expression of the two gene products in bothEscherichia coli andBacillus subtilis. While most of the IFNα was exported to the periplasmic space ofE. coli, about 99% was secreted to the culture medium by recombinantB. subtilis strains. The total yield inE. coli was 1.2×10⁵ IU/ml. This level of expression and export led to instability of the recombinant strains that was spontaneously relieved in vivo by inactivation of thesak ESU through insertion of an IS1 element. No such instability was observed withB. subtilis although expression and secretion levels reached even 3×10⁶ IU/ml. Proteolytic degradation of IFNα by extracellular proteases was avoided by a combination of constitutive expression and secretion during the logarithmic growth phase and the use of exoprotease-reduced host strains. The IFNα₁ protein purified fromB. subtilis culture supernatant was correctly processed, carried the expected 11 amino acid N-terminal elongation that resulted from DNA manipulations and proved to be homogenous in Western blotting experiments. The same recombinant plasmid that directed efficient secretion of hIFNα₁ inB. subtilis gave poor yields when introduced intoStreptococcus sanguis.     

Synthesis and transformations of a pyrazole containing <Emphasis Type="Italic">α</Emphasis>,<Emphasis Type="Italic">β</Emphasis>-didehydro-<Emphasis Type="Italic">α</Emphasis>-amino acid derivatives

Summary.  2H-Pyran-2-ones 1 were transformed with various hydrazines into (E)- or (Z)-α,β-didehydro-α-amino acid (DDAA) derivatives 4 (and 7) containing a highly substituted pyrazolyl moiety attached at the β-position. With heterocyclic hydrazines, the products 4 were accompanied also by decarboxylated enamines E-6. In order to separate (E/Z)-mixtures of acids, they were transformed to the corresponding methyl esters 9 and 10 by the application of diazomethane. Catalytic hydrogenation under high pressures with Pd/C as a catalyst resulted in the formation of racemic alanine derivatives 11. Received January 29, 2002 Accepted May 27, 2002 Published online December 18, 2002 RID="*" ID="*"  Dedicated with deep respect to Professor Waldemar Adam on the occasion of his 65^th birthday. Acknowledgements We thank the Ministry of Education, Science and Sport of the Republic of Slovenia for the financial support (P0-0503-103). Dr. B. Kralj and Dr. D. Žigon (Center for Mass Spectroscopy, “Jožef Stefan” Institute, Ljubljana, Slovenia) are gratefully acknowledged for the mass measurements. Authors' address: Prof. Marijan Kočevar, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia, E-mail: marijan.kocevar@uni-lj.si     

Regio- and Stereo-selective Hydroxylation of Abietic Acid Derivatives by Mucor circinelloides and Mortierella isabellina

Mucor circinelloides and Mortierella isabellina hydroxylated dehydroabietic acid (DehA). DehA was converted regio- and stereo-selectively by whole cells of Mr. circinelloides to give 2α-hydroxydehydroabietic acid in a 75% molar conversion yield (11 mM from 14.7 mM DehA) after 72 h in the cultivation medium containing 3% (v/v) Tween 80. With cells of Ma. isabellina, under the same conditions, 20.5 mM (6.5 g l⁻¹) 2–hydroxydehydroabietic acid (α/β=81/19) was formed from 26.4 mM DehA.     

Infraspecific variability ofJuniperus L. (Cupressaceae) based on monoterpenoid compositions

The compositions of leaf monoterpenoids from 11 species in the Juniperus section (Juniperus chinensis var. chinensis,J. virginiana, J. communis var. montana,J. rigida, J. chinensis var. globosa,J. chinensis var. sargentii,J. chinensis ‘Kaizuka Variegata’,J. squamata ‘Wilsonii’,J. x media ‘Shimpake’,J. x media ‘Plamosa Aurea’, andJ. squamata ‘Aloderi’) were comparatively analyzed by GC-MS. Of the 24 compounds identified, α-pinene, myrcene, limonene, terpinolene, and bornyl acetate were common to all, but particular combinations differed remarkably among taxa. The simplest composition (eight compounds) was found in.J, chinensis var. chinensis; the most complex (19 compounds), inJ. x media ‘Shimpake’. Cluster analysis generated four distinctive clades within the Juniperus section. The minimum spanning network revealed thatJ. squamata ‘Wilsonii’ andJ. x media ‘Shimpake’ were most similar in their chemical makeup.     

Occurrence and genetic association of selected virulence factors in clinicalEscherichia coli isolates

Occurrence ofcnf1+ E. coli pathogenic strains among extraintestinalE. coli isolates was evaluated to explain an impact of cytotoxic necrotizing factor type 1 (CNF1) in human infections. A total of 120E. coli isolates were characterized for presence of virulence factorscnf1- andpap- specific sequences by PCR, and the production of α-hemolysin using blood agar-plate test. Different association patterns among the detected virulence factors were obtained by comparison of various groups of clinicalE. coli isolates. These differences probably reflect a potential impact of CNF1 in the colonization of vaginal environment.     

Hemoglobin types in saanen goats and Barbary sheep: Genetic and comparative aspects

By the use of the Immobiline technique at pH ranges 7.0–7.6 and 6.9–7.9, 16 different hemoglobin (Hb) phenotypes were observed in 61 English Saanen goats. They are explained in this breed by a genetic theory of five β-globin genes (A ₄,A ₆,A ₈,E, andD) and two closely linked α-globin loci (′α and ″α) of which the ″α has a variant allele, provisionally called ″α ^X . Family data together with observed and expected Hb frequencies were in agreement with the genetic theory. Among six Barbary sheep there were three Hb phenotypes explained by the occurrence of the β-chain allelesB andC ^na.     

Hydroxylation of steroids by <Emphasis Type="Italic">Curvularia lunata</Emphasis> mycelium in the presence of methyl-β-cyclodextrine

Transformation of 16 Δ⁵-3β-hydroxy- and Δ⁴-3-ketosteroids of androstane and pregnane classes was carried out using Curvularia lunata mycelium suspended in phosphate buffer with methyl-β-cyclodextrine (MCD). As the result, 20 monohydroxy- and dihydroxy-metabolites, whose structure was determined using spectra of proton magnetic resonance and mass-spectra, have been isolated. Hydroxylation of Δ⁵-3β-hydroxy-steroids occurred mostly in the C-7α position whereas hydroxylation of Δ⁴-3-ketosteroids was in the C-11β position. Only androst-4-en-3,17-dione, 9α-hydroxy-androstenedione, and androsta-1,4-diene-3,17-dione were hydroxylated at C-14α position. Besides main 11β-derivatives, the 6β- and 7β-hydroxy-derivatives with yield 10 and 30%, respectively, were isolated during transformation of progesterone and hydroxymethyl pregnadienone. The ratio of MCD to transforming steroid was 1: 1 (mol/mol). Hydrocortisone and 7α-hydroxyandrostenolone with the yield 55 and 77%, respectively, were obtained at the maximal concentrations of cortexolone 20 g/l and androstenolone acetate 10 g/l in the presence of MCD. Absorption of steroids on mycelium, lower speed of their transformation, low concentrations of modifying substrates, and low yield of hydroxyderivatives have been observed in the absence of MCD.