Elucidating the Ramularia eucalypti species complex.

The genus Ramularia includes numerous phytopathogenic species, several of which are economically important. Ramularia eucalypti is currently the only species of this genus known to infect Eucalyptus by causing severe leaf-spotting symptoms on this host. However, several isolates identified as R. eucalypti based on morphology and on nrDNA sequence data of the ITS region have recently been isolated from other plant hosts, from environmental samples and also from human clinical specimens. Identification of closely related species based on morphology is often difficult and the ITS region has previously been shown to be unreliable for species level identification in several genera. In this study we aimed to resolve this species-complex by applying a polyphasic approach involving morphology, multi-gene phylogeny and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Six partial genes (ITS, ACT, TEF1-α, HIS3, GAPDH and RPB2) were amplified and sequenced for a total of 44 isolates representing R. eucalypti s.lat. and closely related species. A multi-gene Bayesian phylogenetic analysis and parsimony analysis were performed, and both the resulting trees showed significant support for separation of seven species in R. eucalypti s.lat., including two previously described (R. eucalypti and R. miae), four novel species here described (R. haroldporteri, R. glennii, R. mali and R. plurivora) and one undescribed Ramularia species (sterile). Additionally, Mycosphaerella nyssicola is newly combined in Ramularia as R. nyssicola. Main mass spectra (MSPs) of several R. eucalypti s.lat. strains were generated using MALDI-TOF MS and were compared through a Principal Component Analysis (PCA) dendogram. The PCA dendrogram supported three clades containing R. plurivora, R. glenni/R. mali and R. eucalypti/R. miae. Although the dendrogram separation of species differed from the phylogenetic analysis, the clinically relevant strains were successfully identified by MALDI-TOF MS.

Sterol carrier protein 2 (non-specific lipid transfer protein) is localized in membranous fractions of Leydig cells and Sertoli cells but not in germ cells.

The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2. Western blot analysis revealed high levels of the protein in the somatic cells of the testis, e.g., Leydig and Sertoli cells whereas it could not be detected in germ cells. This cellular localization of SCP2 was confirmed by Northern blotting. Immunocytochemical techniques revealed that in Leydig cells, immunoreactive proteins were concentrated in peroxisomes. Although SCP2 was also detected in Sertoli cells, a specific subcellular localization could not be shown. SCP2 was absent from germ cells. Analysis of subcellular fractions of Leydig cells showed that SCP2 is membrane bound without detectable amounts in the cytosolic fraction. These results are at variance with data published previously which suggested that in Leydig cells a substantial amount of SCP2 was present in the cytosol and that the distribution between membranes and cytosol was regulated by luteinizing hormone. The present data raise the question in what way SCP2 is involved in cholesterol transport between membranes in steroidogenic cells but also in non-steroidogenic cells.

Measurement of steroidogenesis in rodent Leydig cells: a comparison between pregnenolone and testosterone production.

The efficiency and specificity of inhibition of pregnenolone metabolism in mature, immature rat Leydig cells, mouse and tumour Leydig cells by SU-10603, a 17 alpha-hydroxylase inhibitor and epostane (WIN-32729), a 3 beta-hydroxysteroid dehydrogenase inhibitor, were studied. Metabolism of [14C]pregnenolone by mature rat Leydig cells was inhibited for more than 95% in the presence of 20 microM SU-10603 and 5 microM epostane. The sum of the different steroids produced by Leydig cells from immature rats incubated in the presence of a 5 alpha-reductase inhibitor was only 80% of pregnenolone production in the presence of SU-10603 and epostane. Pregnenolone metabolism could also be inhibited in tumour Leydig cells but not in mouse Leydig cells. Pregnenolone and testosterone production by Leydig cells from mature rats were similar when steroidogenesis is maximally stimulated by luteinizing hormone (LH). However, in the presence of LH and bovine serum albumin (bSA), or 22 R-hydroxycholesterol and bSA, pregnenolone production was 1.7- and 6-fold higher respectively, than testosterone production. The data show that for measuring the steroidogenic activity of Leydig cells estimation of pregnenolone production is more reliable than measuring testosterone production. At high activities of the cholesterol side-chain cleavage (CSCC) the conversion of pregnenolone into testosterone may become the rate-limiting step for testosterone production. Under all conditions the conversion of cholesterol into pregnenolone is the (hormonal regulated) rate-determining step for steroidogenesis.

Luteinizing hormone induction of the cholesterol side-chain cleavage enzyme in cultured immature rat Leydig cells: no role of insulin-like growth factor-I?

Long-term inductive effects of luteinizing hormone (LH) on cholesterol side-chain cleavage (CSCC) enzyme activity were studied, using cultured Leydig cells isolated from 21-day-old rats. Particular reference was given to the role of insulin-like growth factor-I (IGF-I) as an autocrine or paracrine modulator or as an essential extracellular mediator of LH action. The CSCC enzyme activity was measured using an excess of 22(R)-hydroxycholesterol as substrate to saturate the enzyme, and inhibitors of pregnenolone metabolism to concentrate all the products of the enzyme reaction in pregnenolone. The rate of sterol conversion into pregnenolone (CSCC enzyme activity) reflected the amount of cytochrome P-450scc (P-450scc), as was shown by Western blotting. In cells cultured without LH, the CSCC enzyme activity decreased to 10% on day 7 of the culture period. In the presence of various doses of LH ranging from 0.01 to 100 ng/ml, the CSCC enzyme activity also diminished during the first 3 days of culture, but during the following days, the amount of CSCC enzyme was stimulated by LH. In contrast to the absence of any LH effect on the activity of the CSCC enzyme during the first days of the culture, the endogenous steroid production (no added 22(R)-hydroxycholesterol) could be stimulated at least 10-fold by high doses of LH. When LH (1 ng/ml) was added to cells which had been cultured for 7 days without hormones, CSCC enzyme activity was elevated 8-fold after 4 days of exposure of LH. These effects of LH could be mimicked by dbcAMP (0.5 mM). No evidence could be provided that IGF-I plays any role in the LH induction of the CSCC enzyme; neither the addition of exogenous IGF-I or analogs that do not bind to IGF-I binding proteins (IGFBPs) nor the inactivation of endogenous IGF-I action (through binding to IGFBP and antibodies to IGF-I or via masking of IGF-I receptor by antibodies) could influence the LH induced CSCC enzyme activity. The present data raise the question under which conditions IGF-I is capable of modulating Leydig cell steroidogenesis.

Inhibition of the luteinizing hormone-dependent induction of cholesterol side chain cleavage enzyme in immature rat Leydig cells by Sertoli cell products.

The modulation of the luteinizing hormone (LH) induction of cholesterol side chain cleavage (CSCC) enzyme in immature rat Leydig cells was studied using rat Sertoli cell-conditioned medium (SCCM), which stimulates short-term endogenous steroid production. Luteinizing hormone increased the CSCC enzyme activity 10-fold in cells cultured for 7 days in the absence of hormones. This enzyme induction was abolished almost completely in the presence of SCCM. The inhibition was dose dependent (half-maximal effect at 5 mg protein/l) and paralleled by a decrease in the amount of cytochrome P-450scc (P-450scc) enzyme. There were no indications for loss of cell viability. The inhibitory action of SCCM could be localized at the level of adenylate cyclase activation and at steps beyond cyclic adenosine monophosphate production. The inhibition was not specific for Sertoli cell products because conditioned media from different cell lines and media from isolated rat hepatocytes displayed similar effects. Trypsin treatment of SCCM destroyed the activity whereas the bioactivity could resist heating for 5 min at 100 degrees C. Generally occurring (growth) factors, such as epidermal growth factor or tumor necrosis factor alpha, may have contributed to the observed inhibitory effects of SCCM. These inhibitory effects of Sertoli cell products in vitro are in contrast to stimulatory effects of Sertoli cells on Leydig cell steroidogenesis in vivo after FSH administration.