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Biomedical subjects

M Stefanini

Publications and source records attributed to M Stefanini.

At least 37 records · Page 2Linked to original sources
PubMed2000

Calcium confusion--is the variability in calcium response by Sertoli cells to specific hormones meaningful or simply redundant?

When results of more than ten different studies on hormone-induced calcium signals in Sertoli cells are taken together, a wide variety of responses emerges. The reported changes range from increased concentrations, via no response at all, to decreased calcium concentrations. Minor variations in cell isolation techniques, culture conditions, or techniques for measuring the intracellular calcium could explain some of these differences. However, erratic variations in response are also observed within research groups under very similar experimental conditions. Such 'negative' findings are mainly reported orally and do not further penetrate the scientific community. As hormone-dependent calcium responses evidently may depend very much on the context of the cells, calcium transients would appear to be unreliable bioassay principles with which to detect the primary actions of FSH and effectors such as androgens on Sertoli cells. A more important biological question is whether these sometimes opposed calcium transients are connected with a particular cellular response. To date there is no evidence for such a tight coupling in Sertoli cells, implying that, at least under in vitro conditions, calcium signals might even be redundant altogether. Such calcium variability is probably not unique to Sertoli cells, and the aim of this commentary is to promote an open debate that may help to transform the current state of 'calcium confusion' into a better understanding of the intracellular calcium language.

Animals↗
PubMed1999

Activation of Jun N-terminal kinase/stress-activated protein kinase pathway by tumor necrosis factor alpha leads to intercellular adhesion molecule-1 expression.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine implicated in the pathogenesis of numerous chronic and acute inflammatory conditions. We have previously shown that mouse Sertoli cells respond to TNF-alpha by increasing interleukin-6 production and intercellular adhesion molecule-1 (ICAM-1) expression (1). In this cell type TNF-alpha activates the mitogen-activated protein kinase (MAPK) pathways p42/p44 MAPK, JNK/SAPK, and p38, the last of which is responsible for interleukin-6 production (1). To determine which MAPK signaling pathway is required for TNF-alpha induction of ICAM-1 expression, we have utilized the protein kinase inhibitor dimethylaminopurine, demonstrating that treatment of Sertoli cells with such compound significantly reduced ICAM-1 expression and JNK/SAPK activation. Moreover, dimethylaminopurine treatment increased the expression of MAPK phosphatase-2, providing a possible mechanism of action of this compound. By using agonist antibodies to p55 and to p75 TNF-alpha receptors and both human and mouse TNF-alpha, we demonstrate that both TNF receptors are expressed and that only the p55 receptor is involved in ICAM-1 expression. The p55 receptor activates all of the three pathways, whereas p75 failed to activate any of the MAPKs. Altogether our results demonstrate that TNF-alpha up-regulates ICAM-1 expression through the activation of the JNK/SAPK transduction pathway mediated by the p55 receptor.

Adenine↗
PubMed1999

Cutaneous bleeding related to zinc deficiency in two cases of advanced cancer.

BACKGROUND: One patient with metastatic squamous cell carcinoma of the larynx and one with advanced non-Hodgkin lymphoma, both with poor nutritional status, developed ecchymoses and demonstrated prolonged bleeding time and abnormal aggregation of platelets. METHODS: On the basis of previous studies, the author measured the level of zinc in the serum and urine of these patients and the effect of zinc supplementation on the clinical symptoms and laboratory findings. Also studied were serum and urine zinc levels in 15 newly diagnosed, untreated cancer patients with no evidence of bleeding. RESULTS: The two patients were found to have very low serum and urine zinc levels, which were 6 and 9 microgram/dL and 28 and 22 microgram/24 hours, respectively, compared with normal average values of 70-150 microgram/dL and 150-1200 microgram/24 hours. The mean values of zinc in the serum and urine of the 15 patients newly diagnosed with cancer were 109.18 microgram/dL and 433.8 microgram/24 hours, respectively. Oral administration of zinc without any additional therapy was rapidly followed by control of bleeding, normalization of bleeding time, and platelet aggregation. Discontinuation of zinc caused the return of bleeding and abnormal laboratory findings, which again were corrected with zinc supplementation. CONCLUSIONS: Severe depletion of zinc in two patients with advanced cancer and malnutrition was accompanied by cutaneous bleeding and laboratory findings of platelet dysfunction, both of which were corrected by oral supplementation of zinc and reoccurred on discontinuing therapy. Zinc deficiency should be suspected in patients with advanced cancer demonstrating bleeding when a prolonged bleeding time is the only hemostatic abnormality.

Aged↗
PubMed1999

Cyclic expression of endothelin-converting enzyme-1 mediates the functional regulation of seminiferous tubule contraction.

The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific "stages" of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.

Animals↗
PubMed1999

Alterations in the CSB gene in three Italian patients with the severe form of Cockayne syndrome (CS) but without clinical photosensitivity.

Cockayne syndrome (CS) is a rare autosomal recessive disorder characterized by postnatal growth failure, mental retardation and otherwise clinically heterogeneous features which commonly include cutaneous photosensitivity. Cultured cells from sun-sensitive CS patients are hypersensitive to ultraviolet (UV) light and, following UV irradiation, are unable to restore RNA synthesis rates to normal levels. This has been attributed to a specific deficiency in CS cells in the ability to carry out preferential repair of damage in actively transcribed regions of DNA. We report here a cellular and molecular analysis of three Italian CS patients who were of particular interest because none of them was sun-sensitive, despite showing most of the features of the severe form of CS, including the characteristic cellular sensitivity to UV irradiation. They all were altered in the CSB gene. The genetically related patients CS1PV and CS3PV were homozygous for the C1436T transition resulting in the change Arg453opal. Patient CS2PV was a compound heterozygote for two new causative mutations, insertions of an A at position 1051 and of TGTC at 2053, leading to truncated proteins of 367 and 681 amino acids. These mutations result in severely truncated proteins, as do many of those that we previously identified in several sun-sensitive CS-B patients. These observations confirm that the CSB gene is not essential for viability and cell proliferation, an important issue to be considered in any speculation on the recently proposed additional function of the CSB protein in transcription. Our investigations provide data supporting the notion that other factors, besides the site of the mutation, influence the type and severity of the CS clinical features.

Child, Preschool↗
PubMed1999

Type 4 cyclic adenosine monophosphate-specific phosphodiesterases are expressed in discrete subcellular compartments during rat spermiogenesis.

The type 4 cAMP-specific phosphodiesterases (PDE4) are a family of closely related enzymes with similar catalytic domains and divergent amino- and carboxyl-terminus domains. Multiple PDE proteins with heterogeneous amino termini are derived from each gene. To understand the significance of this heterogeneity, the expression and localization of variants derived from PDE4A and PDE4D genes was investigated during spermatogenesis in the rat. RNase protection analysis with mRNA for testes at different ages of development showed that two transcripts (PDE4D1 and PDE4D2) are expressed at day 10 and 15 of age and become undetectable thereafter. An additional PDE4D transcript appears at day 30 and increased during testid maturation. This latter transcript codes for a long variant of the PDE4D gene and is expressed in germ cells as demonstrated by RNase protection with RNA from isolated pachytene spermatocytes and round spermatids. The presence of a corresponding PDE4D protein with a molecular mass of 98 kDa was established by immunoprecipitation and Western blot analysis with antibodies specific for PDE4D and by immunoaffinity chromatography purification of the 98 kDa variant from isolated germ cells. PDE4A transcripts were also expressed in pachytene spermatocytes and round spermatids. Two polypeptides encoded by these PDE4A transcripts were expressed in pachytene spermatocytes, reached a maximum in round spermatids, and declined thereafter. Immunofluorescence analysis demonstrated a localization of the PDE4D protein in the manchette and in a periacrosomal region of the developing spermatid, a localization confirmed by immunogold electron microscopy. Conversely, the PDE4A was mostly soluble in the cytoplasm of round spermatids. These data demonstrate that PDE4D and PDE4A variants are expressed at different stages and localized in distinct subcellular structures of developing spermatids. Different properties of the mRNAs derived from the two genes and localization signals are responsible for the temporal and spatial expression of the different PDE4 isoenzymes.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Is hyperdiploidy of immature ejaculated germ cells predictive of testis malignancy? A comparative study in healthy normozoospermic, infertile, and testis tumor suffering subjects.

The possibility of diagnosing neoplastic testis pathologies by studying immature germ cells released from the seminiferous epithelium and present in the semen has been reported. It has been suggested that carcinoma in situ (CIS) of the testis and testis tumor may be identified by studying specific surface antigenic determinants or ploidy of chromosome 1 of malignant germ cells recovered from the semen. A noninvasive diagnostic approach of this type would be of great interest if we consider that CIS is supposed to precede the development of testicular germ cell tumors and that the frequency of that preneoplastic condition is increased in specific andrologic pathologies. To evaluate the reliability of this diagnostic approach, we have quantified the presence of immature hyperdiploid germ cells in the seminal fluid of normal healthy subjects, of infertile oligozoospermic patients affected by maldescended testis or left vancocele, and of patients suffering from CIS or testis tumor. Cell ploidy was evaluated on seminal cell fractions highly enriched in immature germ cells, by means of in situ hybridization with a DNA-probe specific for chromosome 1. Our observations indicate that chromosome 1 hyperdiploidy is not necessarily a predictive parameter of testis tumoral pathologies. The percentage of hyperdiploid immature seminal germ cells is in fact increased in nontumoral pathologies associated with infertility, such as cryptorchidism and left varicocele.

Adult↗
PubMed1998

Testicular germ cells of HIV-seropositive asymptomatic men are infected by the virus.

In situ PCR hybridization studies in the testis of infected asymptomatic subjects detected the presence of HIV-1 proviral DNA in the nuclei of germ cells at all stages of differentiation suggesting that HIV-seropositive men produce infected spermatozoa that are released in the genital tract. In all subjects studied spermatogenesis was normal, the presence of provirus was not associated with germ cell damage and a very mild local immune response was observed. The HIV hybridization pattern observed in germ cells supports the hypothesis of a clonal infection. It is suggested the possibility of a direct infection of the germ cells by cell-free virus and that the testis might represent a site of early viral localization, well tolerated because of the immune privilege of this organ.

HIV Seropositivity↗
PubMed1998

Molecular analysis of mutations in the CSB (ERCC6) gene in patients with Cockayne syndrome.

Cockayne syndrome is a multisystem sun-sensitive genetic disorder associated with a specific defect in the ability to perform transcription-coupled repair of active genes after UV irradiation. Two complementation groups (CS-A and CS-B) have been identified, and 80% of patients have been assigned to the CS-B complementation group. We have analyzed the sites of the mutations in the CSB gene in 16 patients, to determine the spectrum of mutations in this gene and to see whether the nature of the mutation correlates with the type and severity of the clinical symptoms. In nine of the patients, the mutations resulted in truncated products in both alleles, whereas, in the other seven, at least one allele contained a single amino acid change. The latter mutations were confined to the C-terminal two-thirds of the protein and were shown to be inactivating by their failure to restore UV-irradiation resistance to hamster UV61 cells, which are known to be defective in the CSB gene. Neither the site nor the nature of the mutation correlated with the severity of the clinical features. Severe truncations were found in different patients with either classical or early-onset forms of the disease.

Alleles↗
PubMed1998

Analysis of mutations in the XPD gene in Italian patients with trichothiodystrophy: site of mutation correlates with repair deficiency, but gene dosage appears to determine clinical severity.

Xeroderma pigmentosum (XP) complementation group D is a heterogeneous group, containing patients with XP alone, rare cases with both XP and Cockayne syndrome, and patients with trichothiodystrophy (TTD). TTD is a rare autosomal recessive multisystem disorder associated, in many patients, with a defect in nucleotide-excision repair; but in contrast to XP patients, TTD patients are not cancer prone. In most of the repair-deficient TTD patients, the defect has been assigned to the XPD gene. The XPD gene product is a subunit of transcription factor TFIIH, which is involved in both DNA repair and transcription. We have determined the mutations and the pattern of inheritance of the XPD alleles in the 11 cases identified in Italy so far, in which the hair abnormalities diagnostic for TTD are associated with different disease severity but similar cellular photosensitivity. We have identified eight causative mutations, of which four have not been described before, either in TTD or XP cases, supporting the hypothesis that the mutations responsible for TTD are different from those found in other pathological phenotypes. Arg112his was the most common alteration in the Italian patients, of whom five were homozygotes and two were heterozygotes, for this mutation. The presence of a specifically mutated XPD allele, irrespective of its homozygous, hemizygous, or heterozygous condition, was always associated with the same degree of cellular UV hypersensitivity. Surprisingly, however, the severity of the clinical symptoms did not correlate with the magnitude of the DNA-repair defect. The most severe clinical features were found in patients who appear to be functionally hemizygous for the mutated allele.

Abnormalities, Multiple↗
PubMed1998

Junctional contacts between Sertoli cells in normal and aspermatogenic rat seminiferous epithelium contain alpha6beta1 integrins, and their formation is controlled by follicle-stimulating hormone.

The distribution of alpha6beta1 integrins at the level of cell-to-cell contacts within the rat seminiferous epithelium was investigated. Double fluorescence experiments using phalloidin staining of actin filaments and anti-integrin subunit antibodies showed that the receptor belongs to the Sertoli cell lateral domains engaged in the characteristic junctional structures known as ectoplasmic specializations (ES), at the level both of inter-Sertoli junctions and of the contacts between Sertoli cells and elongating spermatids. In the seminiferous epithelium of aspermatogenic testes, obtained through X-irradiation in utero (Sertoli-cell-only testes), at the level of inter-Sertoli junctions both ES and alpha6beta1 integrins are present. In order to study the dependence of alpha6beta1 receptors and ES formation upon FSH stimulation during development, 9-day-old testes were grown in organ culture in basal as well as FSH-supplemented conditions. FSH stimulation, which is necessary for the progression of spermatogenesis to early meiotic stages, appears to be required for the development of inter-Sertoli junctional structures containing ES and alpha6beta1 integrins. These observations indicate that the receptor belongs to the inter-Sertoli junctional machinery and that its expression at that level is not dependent on active spermatogenesis but requires FSH stimulation.

Animals↗
PubMed1998

Presence and cellular distribution of HIV in the testes of seropositive subjects: an evaluation by in situ PCR hybridization.

Cellular distribution of HIV-1 proviral DNA has been studied, by in situ PCR hybridization, in the testes of infected men who died at various stages of the disease. In seropositive asymptomatic subjects, HIV-1 proviral DNA was present in the nuclei of germ cells at all stages of their differentiation. The presence of provirus did not induce germ cell damage, was associated with normal spermatogenesis, and was not accompanied by morphologic signs of immune response. The observed HIV hybridization pattern of germ cells suggests clonal infection. Mechanisms responsible for HIV penetration in testicular germ cells remain to be clarified; however, the possibility of a direct infection of the germ cells by cell-free virus is suggested. In the testes of AIDS-deceased men, histologic features of hypoplasia with arrested spermatogenesis were evident, and few infected spermatogonia and spermatocytes were observed. The whole of these data demonstrates that the testis is a site of early viral localization that fails to elicit an immunological response, and that HIV-seropositive men produce infected spermatozoa that are released in the genital tract.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A.

Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB- XP-E cell extracts, but microinjection of the protein into DDB- XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin.

Cells, Cultured↗
PubMed1997

Chromosomal fragility in the cancer-prone disease xeroderma pigmentosum preferentially involves bands relevant for cutaneous carcinogenesis.

Spontaneous and folate-induced chromosomal fragility was analyzed in peripheral blood lymphocytes from 6 patients affected by the cancer-prone disease xeroderma pigmentosum (XP), from the parents of 4 of the patients, and from 12 normal subjects. All XP patients were defective in nucleotide-excision repair; 4 belonged to group C and 1 each to groups A and D. A tendency toward increased spontaneous chromosomal fragility was observed in the XP family members, and lesions indicating substantial chromosomal damage, which were not observed in any healthy donors, were frequently found. The spontaneous lesion sites in lymphocytes from homozygous and heterozygous carriers of XP defects appeared to be significantly associated with those observed in normal skin fibroblasts from the same subjects. These XP spontaneous fragility sites showed a statistically significant association with the rearrangement breakpoints reported in skin pre-tumoral and tumoral lesions from normal and unrelated XP donors. Under conditions of folate deprivation, the chromosomal fragility level, the pattern and the frequency of expression of fragile sites in XP patients and in their parents were similar to normal. However, XP patients generally showed a higher susceptibility to breakage at sites described as mutagen and carcinogen targets.

Adolescent↗
PubMed1997

Xeroderma pigmentosum and trichothiodystrophy are associated with different mutations in the XPD (ERCC2) repair/transcription gene.

The xeroderma pigmentosum group D (XPD) protein has a dual function, both in nucleotide excision repair of DNA damage and in basal transcription. Mutations in the XPD gene can result in three distinct clinical phenotypes, XP, trichothiodystrophy (TTD), and XP with Cockayne syndrome. To determine if the clinical phenotypes of XP and TTD can be attributed to the sites of the mutations, we have identified the mutations in a large group of TTD and XP-D patients. Most sites of mutations differed between XP and TTD, but there are three sites at which the same mutation is found in XP and TTD patients. Since the corresponding patients were all compound heterozygotes with different mutations in the two alleles, the alleles were tested separately in a yeast complementation assay. The mutations which are found in both XP and TTD patients behaved as null alleles, suggesting that the disease phenotype was determined by the other allele. If we eliminate the null mutations, the remaining mutagenic pattern is consistent with the site of the mutation determining the phenotype.

Cell Line↗
PubMed1997

Phenotypic heterogeneity in nucleotide excision repair mutants of rodent complementation groups 1 and 4.

Rodent ultraviolet light (UV)-sensitive mutant cells in complementation groups (CGs) 1 and 4 normally are known for their extraordinary (approximately 80-100 x) sensitivity to mitomycin C (MMC), although some CG1 mutants with reduced MMC sensitivity were previously reported (Stefanini et al. (1987) Cytotechnology 1, 91). We report here new CG1 and CG4 mutants with only 1.6-10 x wild-type MMC sensitivity despite low unscheduled DNA synthesis (UDS) levels. Mutant UV140, in UV CG4, has approximately 3.8 x the UV sensitivity of parental line AA8, approximately 1.6 x wild-type MMC sensitivity, wild-type X-ray and ethyl methanesulfonate (EMS) sensitivity, and is only slightly (approximately 1.4 x) hypermutable to 8-azaadenine resistance by UV light. It has moderately decreased incision of UV-damaged DNA, has moderately decreased removal of (6-4) photoproducts, and is profoundly deficient in UDS after UV. After UV, it shows abnormally decreased DNA synthesis and persistently decreased RNA synthesis. In addition a cell-free extract of this mutant displays strongly reduced nucleotide excision repair synthesis using DNA treated with N-acetoxy-acetyl-amino-fluorene (AAF). The extract selectively fails to complement extracts of group 1 and 4 mutants consistent with the notion that the affected proteins, ERCC1 and ERCC4, are part of the same complex and that mutations in one subunit also affect the other component. Mutant UV212 is a CG1 mutant with approximately 3.3 x wild-type UV and approximately 5-10 x wild-type MMC sensitivity, with profoundly deficient UDS and hypermutability (approximately 5.8 x) by UV. Mutant UV201, probably in CG1, is only slightly (approximately 1.5 x) UV-sensitive and has near wild-type (1.02X) UV mutability. These unusual group 1 and 4 mutants demonstrate that the unique UV and MMC sensitivity phenotypes displayed by these groups can be separated and support the idea that they are the result of distinct repair functions of the corresponding ERCC1 and ERCC4 genes: nucleotide excision repair for UV lesions and a separate repair pathway for removal of interstrand crosslinks.

Acetoxyacetylaminofluorene↗