Metabolism of androst-4-en-3,17-dione by the filamentous fungus Neurospora crassa

Microbial transformation of androst-4-en-3,17-dione (AD; I) using Neurospora crassa afforded six metabolites; 6beta,14alpha-dihydroxyandrost-4-en-3,17-dione (II), 6beta,9alpha-dihydroxyandrost-4-en-3,17-dione (III), 7alpha-hydroxyandrost-4-en-3,17-dione (IV), 9alpha-hydroxyandrost-4-en-3,17-dione (V), 14alpha-hydroxyandrost-4-en-3,17-dione (VI), and androst-4,6-dien-3,17-dione (VII). The steroid products were assigned by interpretation of their spectral data such as (1)H NMR, (13)C NMR, FTIR, and mass spectroscopy. The characteristic transformations observed were C-6beta, C-7alpha, C-9alpha, C-14alpha hydroxylations, and C6-C7 dehydrogenation. The best fermentation condition was found to be 6-day incubation at 25 degrees C and pH value of 5.0-6.5 according to TLC profiles. Time course study showed the accumulation of V and VI from the third day and IV from the fourth day of the fermentation. Optimum concentration of the substrate, which gave maximum bioconversion efficiency, was 3.5mM in one batch. Biotransformation was completely inhibited in a concentration above 7.0mM.     

Helpful diagnostic markers of steroidogenesis for defining hyperandrogenemia in hirsute women

HYPOTHESIS: Androgen excess carries varied clinical manifestations in women. Although testosterone and dehydroepiandrostendionesulfate (DHEAS) determination is considered useful in diagnostic workup, there is no laboratory definition that sufficiently describes androgen excess. DESIGN: We studied 464 hirsute women with a Ferriman and Gallwey score of at least 8 between 2000 and 2005. Our examination included clinical data, total testosterone (T), sex hormone-binding globulin (SHBG), the free androgen index (FAI), and DHEAS. Additionally, androstendione, 17alpha-hydroxyprogesterone (17OHP), dehydroepiandrostendione (DHEA), and 11-deoxycortisol were determined at baseline and 60min after corticotropin challenge (250microg synacthen). RESULTS: Of 464 women, 77.6% fulfilled the clinical criteria for hyperandrogenemia. Of these 360 women, 78.1% had hyperandrogenic hirsutism. Of these 281 women, 43.4% showed increased stimulation of 17OHP to 250microg of synacthen. Another 37.4% showed adrenal steroid biosynthesis defects other than 21alpha-hydroxylase deficiency, such as defective 11beta-hydroxylation or 3beta-hydroxysteroid dehydrogenase malfunction. The diagnosis of polycystic ovary syndrome was applicable to 12.4%. In addition, our results show that 72% of 281 patients with secondary hirsutism had normal T concentrations, and 55% had a normal FAI. Only 5% of hirsute patients with a normal FAI had elevated DHEAS values. However, 40% showed elevated DHEA levels, while 26% of the women with normal FAI showed androstendione values over the maximal levels in the 79 controls. CONCLUSIONS: Our data suggest that in addition to testosterone and FAI, androstendione and DHEA are significantly helpful parameters in diagnosing hyperandrogenemia in hirsute women. DHEAS was not found to be helpful.     

Genetic basis and mapping of the resistance to Rice yellow mottle virus. III. Analysis of QTL efficiency in introgressed progenies confirmed the hypothesis of complementary epistasis between two resistance QTLs

Our previous studies have hypothesised that a complementary epistasis between a QTL located on chromosome 12 and a QTL located on chromosome 7 was one of the major genetic factors controlling partial resistance to Rice yellow mottle virus (RYMV). We report research undertaken to verify this hypothesis and to introgress the resistant allele of these two QTLs from an upland resistant japonica variety, Azucena, into a lowland susceptible indica variety IR64. Three cycles of molecular marker-assisted back cross breeding were performed using RFLP and microsatellite markers. Resistance to RYMV was evaluated in F₂ and F₃ offspring of the BC₁ and BC₂ generations. Marker-assisted introgression (MAI) was very efficient: in the selected BC₃ progeny the proportion of the recipient genome was close to 95% for the ten non-carrier chromosomes, and the length of the donor chromosome segment surrounding the two QTLs was less than 20 cM. The relevancy of the complementary epistasis genetic model proposed previously was confirmed experimentally: in BC₁ and BC₂ generations only F3 lines having the allele of the resistant parent on QTL₁₂ and QTL₇ show partial resistance to RYMV. Comparison of our experimental process of MAI with the recommendations of analytic and simulation studies pointed out the methodological flexibility of MAI. Our results also confirmed the widely admitted, but rarely verified, assumption that QTL-alleles detected in segregating populations could be treated as units of Mendelian inheritance and that the incorporation of these alleles into elite lines would result in an enhanced performance. The next step will be the design of tools for the routine use of molecular markers in breeding for partial resistance to RYMV and the development of material for the analysis of resistance mechanisms and the structure of a virus resistance gene in rice. Received: 11 August 2000 / Accepted: 20 March 2001     

Towards on-line monitoring of cell growth in microporous scaffolds: Utilization and interpretation of complex permittivity measurements

Here we demonstrate the ability to characterize microporous scaffolds and evaluate cell concentration variation via the utilization and interpretation of complex permittivity measurements (CP), a direct and nondestructive method. Polymer-based microporous scaffolds are of importance to tissue engineering, particularly in the promotion of cell adhesion, proliferation, and differentiation in predefined shapes. Chitosan gel scaffolds were seeded with increasing concentrations of macrophages to simulate cell growth. Complex permittivity measurements were performed using a dielectric probe and a vector network analyzer over a frequency ranging from 200 MHz to 2 GHz. An effective medium theory was applied to interpret the data obtained; respectively, Looyenga and Maxwell-Wagner-Hanai functions were used to retrieve the porosity and the variation of the cell concentration from the CP measurements. Calculated porosities were in agreement with experimental evaluation-porosity ranged from 81-96%. Changes in cell concentration inside the scaffolds upon injection of differing cell concentrations into the scaffold were detected distinguishably. Variations resulting from the cumulative injection of 400-1800 microL of 10(6) cells/mL solution into the scaffold were monitored. Results suggest that CP measurements in combination with an appropriate effective medium approximation can enable on-line monitoring of cell growth within scaffolds.     

Analysis of recombinant acylated pneumococcal surface adhesin A of Streptococcus pneumoniae by mass spectrometry

Streptococcus pneumoniae pneumococcal surface adhesin A (PsaA) is a species-common, immunogenic surface lipoprotein. In this study, the psaA gene was expressed as a nonfusion acylated protein in an Escherichia coli expression system. Yields of pure recombinant PsaA (rPsaA) were 8-10 mg/liter of fermentation culture. Analysis of rPsaA tryptic digests by HPLC-electrospray mass spectrometry (MS) confirmed 98% of the expected protein sequence. GC/MS data demonstrated very similar acylation of native and rPsaA by C12:0-C22:0 fatty acids, with C16 and C18 predominating. Negative ion electrospray MS/MS analysis of the rPsaA lipid anchor released by Pronase-E confirmed that the structure was based on an N-terminal palmitoylcysteine (Pam(3)Cys). Electrospray MS heterogeneity analysis of intact rPsaA indicated that all of the observed heterogeneity could be accounted for by the fatty acid distributions. The availability of well-characterized rPsaA will facilitate the continued research and development of protein-based vaccines for the prevention of pneumococcal disease.     

Use of fluorescent DNA-intercalating dyes in the analysis of DNA via ion-pair reversed-phase denaturing high-performance liquid chromatography

SYBR Green 1 is an asymmetrical cyanine DNA-binding dye that provides an opportunity for increasing the sensitivity of nucleic acid detection when used in conjunction with gel electrophoresis. In this paper, we summarize the general properties and specific uses of SYBR green 1 in ion-pair reversed-phase denaturing high-performance liquid chromatography (IP DHPLC). We describe several applications for the WAVE DHPLC platform that illustrate the generic potential of such intercalating dyes in mutation detection and gene expression profiling. We show that SYBR Green 1 obviates the need to use end-labeled oligodeoxynucleotides for the sensitive detection of nucleic acids during chromatography. Moreover the incorporation of SYBR Green 1 into samples and elution buffers does not impair resolution and has no significant effect on the retention times of DNA fragments compared with dye-free DHPLC.     

A molecular marker for thymocyte-positive selection: selection of CD4 single-positive thymocytes with shorter TCRB CDR3 during T cell development

The generation of the naive T cell repertoire is a direct result of maturation and selection events in the thymus. Although maturation events are judged predominantly on the expression of surface markers, molecular markers, more intimately involved in the selection process, can be informative. We have identified a molecular marker for selection in later stages of maturation in humans. Thymocytes are selected for the expression of TCR beta-chains with shorter CDR3 at the double-positive to single-positive (SP) transition. Here we extend these studies to the mouse and show that the selection phenotype is not related to alpha-chain pairing but is a function of the MHC haplotype. Interestingly, the selection is much more apparent in CD4 SP thymocytes than in CD8 SP cells. This is in contrast to human thymocytes, where the selection is equally apparent in both lineages. The involvement of MHC in the process argues that this is a positive selection stage. The difference in the extent of this selection between the two SP lineages may indicate a class difference in the nature of the TCR-MHC interaction, the role of coreceptors in the selection process, or both.     

6-Methylcryptoacetalide, 6-methyl-epicryptoacetalide and 6-methylcryptotanshinone from Salvia aegyptiaca

From the whole plant of Salvia aegyptiaca, 6-methylcryptoacetalide, 6-methyl-epicryptoacetalide and 6-methylcryptotanshinone have been isolated and characterized, mainly by spectroscopic means. In addition to these novel diterpenoids, the known compounds 3beta-hydroxy-olean-12-en-28-oic acid, 3beta-hydroxy-oleana-11,13(18)-dien-28-oic acid, sitosterol-3beta-glucoside, sitosterol, stigmasterol, 5-hydroxy-7,3',4'-trimethoxyflavone and 5, 6-dihydroxy-7,3',4'-trimethoxyflavone were isolated.