Biomedical subjects

C Albanesi

Publications and source records attributed to C Albanesi.

28 records · Page 2Linked to original sources

PubMed1997

Determination of the enantiomeric purity of N-propionyl-6,7-dimethoxy-2-aminotetralin by cyclodextrin-modified micellar electrokinetic chromatography.

N-Propionyl-6,7-dimethoxy-2-aminotetralin (Z12231A), a useful intermediate in the synthesis of dopaminergic agonists, was resolved into its enantiomers by high-performance capillary electrophoresis. Cyclodextrin-modified micellar electrokinetic chromatography was employed with a combination of chiral selectors both in the aqueous phase (a hydroxyalkyl-beta-cyclodextrin) and in the micellar phase (sodium taurodeoxycholate). The absolute amount of the two chiral selectors as well as their ratio were found to be the most critical parameters in order to optimize separation. A resolution factor >3.5 was obtained, allowing a high amount of solute to be loaded in order to improve the detection limit. The reproducibility of the assay was also evaluated.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1996

The same sequence mediates activation of the human urokinase promoter by cAMP in mouse Sertoli cells and by SV40 large T antigen in COS cells.

Cell-specific activation by follicle-stimulating hormone and its intracellular mediator, cAMP, of the human urokinase promoter in mouse Sertoli cells requires overlapping purine-rich and GC-rich sequences between -54 and -42 from the transcriptional start site. We have previously shown that binding of unidentified nuclear factors to these sequences is induced by cAMP stimulation, and that sequences from the enhancerless SV40 replication origin can interfere with the binding, whereas consensus Sp1 binding sites are ineffective. We now show that sequences within the SV40 origin able to compete for the formation of cAMP-induced DNA-protein complexes in Sertoli cell nuclear extracts are binding sites for the SV40 large T antigen. Large T antigen expressed in COS cells binds the cAMP-responsive sequences of the human urokinase gene and transactivates the proximal promoter, thus mimicking the effect of nuclear factors induced by cAMP in Sertoli cells. We show that Egr-1 is one of the factors present in cAMP-induced DNA-protein complexes formed between the human urokinase promoter and Sertoli cell nuclear extracts. However, Egr-1 levels are similar in unstimulated and cAMP-treated Sertoli cells, suggesting that this factor interacts with a different GC-box binding factor, that we have previously shown to be strongly induced by cAMP treatment of Sertoli cells. We propose that SV40 large T antigen in COS cells can mimick the action of heterodimers formed in cAMP stimulated Sertoli cells between Egr-1 and a cell specific cAMP-induced GC-box binding factor.

Animals↗

PubMed1996

A cell- and developmental stage-specific promoter drives the expression of a truncated c-kit protein during mouse spermatid elongation.

In the postnatal testis, the c-kit transmembrane tyrosine-kinase receptor is expressed in type A spermatogonia, and its transcription ceases at the meiotic phase of spermatogenesis. Alternative, shorter c-kit transcripts are expressed in post-meiotic germ cells. These transcripts should encode a truncated version of the c-kit protein, lacking the extracellular, the transmembrane and part of the intracellular tyrosine-kinase domains. The 5' end of the alternative c-kit transcripts maps within an intron of the mouse c-kit gene. We now show that this intron contains a promoter active in nuclear extracts of round spermatids, and that two discrete sequences upstream of the transcriptional start site bind spermatid-specific nuclear factors. Deletion of both these sequences abolishes activity of the promoter in vitro. We have also established that this promoter is functional in vivo, in a tissue-and cell-specific fashion, since intronic sequences drive the expression of the E. coli lacZ reporter gene in transgenic mice specifically in the testis. Transgene expression is confined to haploid germ cells of seminiferous tubules, starting from spermatids at step 9, and disappearing at step 13, indicating that cryptic promoter within the 16th intron of the mouse c-kit gene is active in a short temporal window at the end of the transcriptional phase of spermiogenesis. In agreement with these data, western blot experiments using an antibody directed against the carboxy-terminal portion of the mouse c-kit protein showed that a polypeptide, of the size predicted by the open reading frame of the spermatid-specific c-kit cDNA, accumulates in the latest stages of spermatogenesis and in epididymal spermatozoa. An immunoreactive protein of the same size can be produced in both eukaryotic and prokaryotic artificial expression systems.

Amino Acid Sequence↗

PubMed1994

Expression of the Xist gene in urogenital ridges of midgestation male embryos.

We show by RT-PCR analysis that transcripts from the Xist gene, which is normally expressed from the inactive X chromosome of female somatic cells and postnatal male germ cells, are transiently expressed also in male embryos around the time of testis differentiation, mainly in somatic cells of urogenital ridges. In the postnatal testis, we find that Xist transcripts are mainly localized within the nucleus of haploid spermatids. These findings suggest that inactivation of the X-chromosome might occur not only in adult male germ cells, but also, transiently, in somatic cells of the male urogenital ridge. Both in the embryonal gonad and in differentiating germ cells Xist expression in males overlaps the pattern of expression of the testis determining gene Sry.

Animals↗

PubMed1993

Direct evidence that the mouse sex-determining gene Sry is expressed in the somatic cells of male fetal gonads and in the germ cell line in the adult testis.

Reverse transcriptase polymerase chain reaction (RT-PCR) analysis shows that Sry mRNA is expressed in male fetal urogenital ridges from 12.5 day p.c. embryos, but not in enriched populations of primordial germ cells from the same embryos, indicating that Sry is expressed in the somatic cells of the embryonal gonad at the time of testis determination. We also show that, in the adult male mouse testis, Sry mRNA is expressed at high levels in meiotic and postmeiotic germ cells and, at much lower levels, also in Sertoli cells. Treatment with cyclic adenosine monophosphate (cAMP) analogs of cultured Sertoli cells from postnatal testis completely abolishes Sry mRNA expression.

Animals↗

PubMed1993

Follicle-stimulating hormone induction of steel factor (SLF) mRNA in mouse Sertoli cells and stimulation of DNA synthesis in spermatogonia by soluble SLF.

Follicle-stimulating hormone (FSH) and its intracellular mediator, cAMP, increase the mRNA levels for the Steel factor (SLF, the c-kit ligand) in cultured primary mouse Sertoli cells. The inductive effect of cAMP is more evident in cultures from 13-day-old animals than in cultures from 18-day-old animals. Analysis through the polymerase chain reaction (PCR) indicates that (Bu)2cAMP or FSH treatment increases the levels of the mRNAs for both the potentially soluble form and the transmembrane form of SLF in cultured Sertoli cells. The ratio between mRNAs encoding the potentially soluble form and the transmembrane form of SLF increases during postnatal testis development, and it is higher in cultured Sertoli cells with respect to total testis, suggesting that, under the in vitro conditions, SLF could be produced by Sertoli cells mainly as a soluble factor. Soluble recombinant SLF stimulates, in a dose-dependent fashion, thymidine incorporation in cultures of isolated germ cell populations enriched in the mitotic stages (spermatogonia), independently of the presence of serum, whereas cAMP analogs have no effect. Autoradiographic analysis shows that SLF selectively stimulates DNA synthesis in type A spermatogonia.

Alternative Splicing↗

PubMed1993

Identification of 3',5'-cyclic adenosine monophosphate-inducible nuclear factors binding to the human urokinase promoter in mouse Sertoli cells.

Transcription factors which recognize both the SV40 promoter and the proximal promoter region of the human urokinase-type plasminogen activator (h-uPA) gene are present in nuclear extracts from primary cultures of mouse Sertoli cells; prolonged (more than 12 h) (Bu)2cAMP stimulation of Sertoli cells induces the formation of different specific DNA-protein complexes. A discrete region in the h-uPA promoter, between -54 and -42, is essential for the formation of the cAMP-induced DNA-protein complexes. Mutation of the sequence between -54 and -42 abolishes the response to cAMP of the proximal h-uPA promoter in Sertoli cells. A protein, recognizing a sequence centered around the GC-box present between -48 and -43, is detected by Southwestern analysis, and it is clearly induced by (Bu)2cAMP stimulation. Interaction between this protein and a second factor, recognizing a purine-rich sequence between -53 and -46, partially overlapping the GC-box, is needed for the formation of the cAMP-induced DNA-protein complexes. A preformed complex between the cAMP-induced GC-box-binding factor and the second factor can be detected using nondenaturing conditions during Southwestern analysis.

Animals↗

PubMed1992

A novel c-kit transcript, potentially encoding a truncated receptor, originates within a kit gene intron in mouse spermatids.

We have cloned a novel c-kit mRNA of 3.2 kb expressed in postmeiotic male germ cells. This transcript initiates in the genomic region immediately upstream of the exon coding for the second box of the split c-kit tyrosine kinase domain. The open reading frame (ORF) contains 12 novel amino acids in frame with the C-terminal 190 amino acids of the c-kit protein. It lacks therefore the upstream region in the 5.5-kb c-kit mRNA encoding the extracellular and transmembrane domain, the ATP-binding site and the kinase insert domain present in the c-kit protein.

Amino Acid Sequence↗

PubMed1991

Expression of the mRNA for the ligand of c-kit in mouse Sertoli cells.

The expression of the mRNA for SLF (the c-kit ligand), a product of the "steel" locus, has been investigated in postnatal mouse testis and homogeneous populations of testicular cells. The message was found expressed in postnatal mouse testis but not in germ cells. Studies on primary mouse Sertoli cell cultures from 18 day old mice show that Sertoli cells are the site of SLF mRNA expression in the seminiferous tubules. Treatment of Sertoli cell cultures with cAMP analogs led to a significant increase in the SLF mRNA levels.

Animals↗

PubMed1991

Lens formation from the cornea following implantation into hindlimbs of larval Xenopus laevis: the influence of limb innervation and extent of differentiation.

Corneal fragments of larval Xenopus laevis at stage 48 (according to Nieuwkoop and Faber, '56), were implanted into sham denervated unamputated hindlimbs, denervated unamputated hindlimbs, amputated and sham denervated hindlimbs, and amputated and denervated hindlimbs of larvae at stages 52 and 57. The results show that unamputated limbs at stage 52, either innervated or denervated, manifest a weak capacity to promote the first lens-forming transformations of the outer cornea. This capacity is absent in both limb types at stage 57. After amputation, limbs of both early and late stages form a regenerative blastema and support lens formation from the outer cornea. Denervation of early stage limbs has no appreciable effect on blastema formation and lens-forming transformation of corneal implants. However, denervation of late stage limbs inhibits both processes. These results indicate that the limb tissues of the early stage limbs contain non-neural inductive factors at a low level and that after limb amputation and blastema formation the level of these factors becomes high enough to promote lens formation from implanted cornea, even after denervation. In contrast, the limb tissues of late stage limbs do not contain a suitable level of non-neural inductive factors.

Animals↗