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Biomedical subjects

A Germani

Publications and source records attributed to A Germani.

18 recordsLinked to original sources
PubMed2003

The exon 7-spliced Lck isoform in T lymphocytes: a potential regulator of p56lck signaling pathways.

The protein-tyrosine kinase p56lck is the product of the lck gene. It plays a pivotal role in T-lymphocyte activation and thymocyte development, as indicated by the defective immune responses of lck-/- mice. We have demonstrated that an exon 7-deleted lck mRNA is produced by alternative splicing in all human cells expressing the lck gene. We have now looked for the protein encoded by this spliced lck mRNA and attempted to determine the function of the deleted Lck protein. This paper shows that the LckDelta7 protein is present in JCaM1.6 T-cells and we inferred that this isoform accounts for 15% of the total Lck proteins in the parental Jurkat T-cell line. We report that deletion of the first 51 amino-acids (exon 7) of the Lck catalytic domain greatly reduces the kinase activity of the recombinant protein. The residual activity can, nevertheless, be enhanced by adding Mn(2+), whereas this cation has no effect on the activity of the p56lck mutated in its active site (K273E). The enforced production of LckDelta7 protein in transfected Jurkat cells results in slower cell proliferation than does p56lck. These findings suggest that the LckDelta7 protein is a p56lck cell-signaling regulator. This mechanism could be common to both humans and mice, in which we also found the exon 7-spliced lck transcript.

Adenosine Triphosphate↗
PubMed2000

SIAH-1 interacts with alpha-tubulin and degrades the kinesin Kid by the proteasome pathway during mitosis.

SIAH-1, a human homologue of the Drosophila seven in absentia (Sina), has been implicated in ubiquitin-mediated proteolysis of different target proteins through its N-terminal RING finger domain. SIAH-1 is also induced during p53-mediated apoptosis. Furthermore, SIAH-1-transfected breast cancer cell line MCF-7 exhibits an altered mitotic process resulting in multinucleated giant cells. Now, using the two-hybrid system, we identified two new SIAH interacting proteins: Kid (kinesin like DNA binding protein) and alpha-tubulin. We demonstrate that SIAH is involved in the degradation of Kid via the ubiquitin-proteasome pathway. Our results suggest that SIAH-1 but not its N-terminal deletion mutant, affects the mitosis by an enhanced reduction of kinesin levels. Our results imply, for the first time, SIAH-1 in regulating the degradation of proteins directly implicated in the mitotic process.

Cell Cycle↗
PubMed1999

SIAH-1 inhibits cell growth by altering the mitotic process.

SIAH-1, the human homologue of the drosophila seven in absentia gene, is a p53-p21Waf-1 inducible gene. We report that stable transfection with SIAH-1 of the epithelial breast cancer cell line MCF-7 blocks its growth process. The transfectants show a redistribution of SIAH-1 protein within the nucleus, more specifically to the nuclear matrix, associated to dramatic changes in cell morphology and defective mitosis. Multinucleated giant cells (2-12 nuclei in more than 50% cells) were a most striking observation associated with tubulin spindle disorganization and defective cytokinesis. There were also present at high frequency abortive mitotic figures, DNA bridges and persistance of intercellular bridges and midbodies, along with an increased expression of p21Waf-1. These results indicate that the mechanism of growth arrest induced by SIAH-1 in MCF-7 cells involves disorganization of the mitotic program, mainly during nuclei separation and cytokinesis.

Apoptosis↗
PubMed1999

hSiah2 is a new Vav binding protein which inhibits Vav-mediated signaling pathways.

The hematopoietic proto-oncogene vav has been characterized as a Rac1-GDP/GTP exchanger protein which regulates cytoskeletal reorganization as well as signaling pathways leading to the activation of stress-activated protein kinases (SAPK/JNKs). Furthermore, vav overexpression enhances basal and T-cell receptor (TCR)-mediated stimulation of the nuclear factor of activated T cells (NFAT). We report here the interaction between Vav and hSiah2, a mammalian homolog of Drosophila Seven in absentia (Sina) that has been implicated in R7 photoreceptor cell formation during Drosophila eye development via the proteasome degradation pathway. Vav and hSiah2 interact in vitro and in vivo and colocalize in the cytoplasm of hematopoietic cells. The Src homology domain of Vav and the C-terminal region of hSiah2 are required for this interaction. We provide evidence for a negative regulation by hSiah2 of Vav-induced basal and TCR-mediated NFAT-dependent transcription. Overexpression of hSiah2 also inhibits the onco-Vav-induced JNK activation. Although the Vav-interacting domain is located in the C-terminal portion of hSiah2, the N-terminal region of hSiah2 is necessary for the inhibitory role that seems to be independent of the proteasome degradation.

Animals↗
PubMed1998

Vav binding to heterogeneous nuclear ribonucleoprotein (hnRNP) C. Evidence for Vav-hnRNP interactions in an RNA-dependent manner.

The vav proto-oncogene is exclusively expressed in hematopoietic cells and encodes a 95-kDa protein that contains multiple structural domains. Vav is involved in the expansion of T and B cells, in antigen-mediated proliferative responses, and in the induction of intrathymic T cell maturation. It becomes rapidly and transiently tyrosine-phosphorylated upon triggering of a large number of surface receptors and catalyzes GDP/GTP exchange on Rac-1. We now provide evidence for the specific interaction of Vav with heterogeneous nuclear ribonucleoprotein (hnRNP) C. Vav and hnRNP C interact both in vivo and in vitro mediated through the carboxyl Src homology 3 domain of Vav and the proline-rich motif located in the nuclear retention sequence of hnRNP C. More importantly, Vav-hnRNP C complexes are present in living hematopoietic cells and both proteins localize in the nuclei, mainly on perichromatic fibrils but also on clusters of interchromatin granules. The Vav-hnRNP C interaction is regulated by poly(U) RNA, although a basal association is still detected in the absence of RNA. Furthermore, RNA homopolymers differentially alter the binding affinity of Vav to hnRNP C and hnRNP K. We propose that Vav-hnRNP interactions may be established in an RNA-dependent manner.

Cell Line↗
PubMed1998

Intrauterine growth retardation: evidence for the activation of the insulin-like growth factor (IGF)-related growth-promoting machinery and the presence of a cation-independent IGF binding protein-3 proteolytic activity by two months of life.

Thirty-seven children with intrauterine growth retardation (IUGR) were enrolled in a 3-mo longitudinal study. Weight, length, and knee-heel length (by knemometry) were measured at birth and at 7, 14, 30, 60, and 90 d. GH, IGF-I, IGF binding protein (BP)-3, IGFBP-1, and C-peptide were measured at birth and at 2 mo. IGFBP-3 Western immunoblotting and proteolytic activity assay were also performed. Twenty-five newborns with birth weight appropriate for gestational age were chosen as controls. At birth IUGR newborns showed levels of GH and IGFBP-1 significantly higher, and IGF-I, IGFBP-3, and C-peptide significantly lower than control subjects. At 2 mo GH and IGFBP-1 levels decreased, whereas IGF-I, IGFBP-3, and C-peptide rose, attaining the concentrations found in control subjects at birth. Baseline peptide levels as well as their 2-mo variations did not correlate with the gain in weight, supine length, and knee-heel length recorded at 3 mo. Fourteen of nineteen IUGR cord blood samples showed the presence of the intact approximately 42-39-kD IGFBP-3 doublet and the major approximately 29-kD fragment. At 2 mo the IGFBP-3 band pattern was characterized by the predominance of a approximately 18-kD fragment in 6 of 19 tested IUGR infants. The incubation of 2-mo IUGR samples with normal adult serum induced the appearance of the approximately 18-kD band, which was not modified by the addition of EDTA. These results suggest that: 1) the IGF-related growth-promoting mechanism is impaired in IUGR children at birth but is fully restored at 2 mo; 2) the cord blood levels of GH, IGF-I, IGFBP-3, IGFBP-1, and C-peptide are not predictive of the weight and length gain during the first 3 mo of life; 3) IUGR children have at least two different IGFBP-3 proteases, one cation-dependent protease that is present at birth and able to yield the major approximately 29-kD IGFBP-3 fragment and a second one, with a different activation timing, which exhibits cation independence and induces the formation of a approximately 18-kD IGFBP-3 form.

Adult↗
PubMed1997

Uncoupling of p21 induction and MyoD activation results in the failure of irreversible cell cycle arrest in doxorubicin-treated myocytes.

Doxorubicin (Dox, Adriamicin), a potent broad spectrum anthracycline anticancer drug, selectively inhibits muscle specific gene expression in cardiac cells in vivo and prevents terminal differentiation of skeletal muscle cells in vitro. By inducing the expression of the helix-loop-helix (HLH) transcriptional inhibitor ld2, Dox represses the myogenic function of the MyoD family of muscle regulatory factors (MRFs). In many cell types, terminal differentiation is coupled to an irreversible exit from the cell cycle and MyoD plays a critical role in the permanent cell cycle arrest of differentiating myocytes by upregulating the cyclin dependent kinase inhibitor (cdki) p21. Here, we correlate Dox effects on cell cycle with changes of E2F/DP complexes and activity in differentiating C2C12 myocytes. In Dox-treated quiescent myoblasts, which fail to differentiate into myotubes under permissive culture conditions, serum re-stimulation induces cyclin/cdk re-association on the E2F/DP complexes and this correlates with an evident increase in E2F/DP driven transcription and re-entry of myoblasts into the cell cycle. Despite Dox ability to activate the DNA-damage dependent p53/p21 pathway, when induced in the absence of MyoD or other MRFs, p21 fails to maintain the postmitotic state in Dox-treated myocytes induced to differentiate. Thus, uncoupling p21 induction and MyoD activity results in a serum-reversible cell cycle arrest, indicating that MRF specific activation of cdki(s) is required for permanent cell cycle arrest in differentiating muscle cells.

Animals↗
PubMed1995

Enhanced expression of myogenic regulatory genes in aging skeletal muscle.

MyoD, myogenin, myf-5, and MRF4, belonging to the family of basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs), control muscle cell differentiation, in concert with other transcription factors such as MEF-2, yet their role in age-related skeletal muscle alteration has not been addressed. We here report that MyoD and myogenin transcripts are expressed at high levels in the hind limb muscles of newborn mice and their level of expression continuously declines throughout postnatal life to become virtually undetectable in the adult mouse. However, these transcripts are again expressed at high levels in the muscles of older mice. MRF4 transcript, on the other hand, is present at a constant level throughout the life span of the animal. Conversely, the expressions of myf-5 and MEF-2C, components of the autoregulatory loop for the activation of bHLH gene expression, conspicuously increase in adult and senile muscle. In order to establish whether these transcripts are functioning in the aged muscle we investigated the expression of bHLH inhibitory factor Id mRNA showing that it does not present significant changes during aging. Immunofluorescence analysis with an anti-myogenin antibody revealed nuclear accumulation of the protein in the muscle fibers of old, but not of adult, mice. Muscle-specific genes transactivated by MyoD and myogenin such as AChR, MLC, and MCK are also up-regulated during aging, albeit at a lower level. Significant changes in the size and ratio of type I/type II fibers are detectable in senile muscle. These findings show that all members of the MRF family are expressed to a high extent and are likely active in senile muscle. It is conceivable that these changes might operate as a compensatory mechanism in maintaining the expression of differentiated muscle products in senile muscle at a steady-state level.

Aging↗
PubMed1994

TPA-induced differentiation of human rhabdomyosarcoma cells involves dephosphorylation and nuclear accumulation of mutant P53.

Previous studies have shown that human rhabdomyosarcoma cells are induced to differentiate by TPA, in the absence of appreciable alterations of the muscle regulatory genes and their products (1). The question was addressed whether the tumor suppressor p53 could be a target of TPA action in these cells. Genomic analysis by a Polymerase Chain Reaction/Single-Strand Conformation Polymorphism (PCR/SSCP) and direct sequencing indicate the presence of a mutation in exon VII at codon 248 (C to T transition) and a loss of heterozygosity of p53 gene in human rhabdomyosarcoma cell line (RD). It is here shown that transcription of p53 mRNA strongly decreases in RD cells induced to growth arrest and differentiate by TPA treatment. In these cells immunoprecipitation and immunoblot analysis show that both synthesis and total cellular concentration of the protein are also reduced by TPA. Nevertheless nuclear p53 accumulation is at much higher extent, whereas 32P-orthophosphate labelling, followed by immunoprecipitation, demonstrates a decrease of phosphorylation of both cytoplasmic and nuclear p53. These results indicate that TPA causes a number of alterations of mutant p53, likely mediated through a protein kinase C dependent mechanism, which might impair the transforming ability of mutant p53 in growth-arrested and differentiating RD cells.

Base Sequence↗
PubMed1985

Stereometry in very close-range stereophotogrammetry with non-metric cameras for human movement analysis.

In this paper a stereophotogrammetric algorithm based on a black-box approach to the modelling of object to image spaces relationship is proposed. The algorithm is well suited for 'very close-range photogrammetry', with respect to experiments in which the measurement field is 0.5 X 0.5 X 0.5 m or smaller, as in the analysis of a few or small body segments movements. The attainable accuracy is high, better than 0.1% of the observation distance. Non-professional and even different cameras can be used. Consequently an inexpensive experimental set-up can be realized. A very simple, cheap and easily usable calibration object is needed. Computation time for the reconstruction of object-space co-ordinates of point body landmarks is one order of magnitude lower than in the case of the Direct Linear Transformation (DLT) (Abdel Aziz and Karara, Proceedings of the ASP/U1 Symposium on Close-Range Photogrammetry, pp. 1-18. American Society of Photogrammetry, 1971; Marzan and Karara, Proceedings of the Symposium on close-range Photogrammetric Systems, pp. 420-467. American Society of Photogrammetry, 1975). Computation time for calibration is two-fold in respect of the DLT. An example of application to the recording of the movements of the index finger with respect to the metacarpophalangeal joint is given.

Biomechanical Phenomena↗
PubMed1984

Analysis of DNA synthesis rate of cultured cells from flow cytometric data.

The rate of DNA synthesis along S phase is estimated from flow cytometric histograms on the basis of a mathematical model of a cell population. In the absence of loss, the model expresses the population kinetics in terms of DNA synthesis rate, S-phase influx, and population size. A single histogram is sufficient to determine the DNA synthesis rate when the population is in balanced exponential growth. Two suitably chosen histograms are necessary if the S-phase influx is exponential in a time interval longer than the S-phase duration. The analysis procedure was tested on published autoradiographic data and applied to three cultured cell lines (CM-S, 3LL, and M14 cells) that show various patterns of DNA distribution. In each case the cell-cycle fractions, the DNA synthesis rate, and the S-phase duration were obtained.

Animals↗
PubMed1983

A general expression for sequential DNA-fluorescence histograms.

A general expression for time sequences of DNA-fluorescence histograms from flow microfluorometry is given in this paper. Such expression is given in terms of the law of DNA growth along S phase, the flux of cells into S, and the efflux out of M. Special conditions of growth (e.g. the exponential steady-state growth), and the case of blocks in S phase are also analyzed. Some simulations of the expressions obtained are presented.

Cells, Cultured↗
PubMed1983

Estimation of cell DNA synthesis rate from flow-cytometric histograms.

The rate of DNA synthesis along S-phase can be estimated on the basis of a simple model of a cell population, from a single FCM histogram under conditions of balanced exponential growth, and from two histograms suitably spaced in time under conditions of locally exponential S-phase influx. An algorithm that constructs a piecewise constant approximation of the synthesis rate and estimates the phase fractions from a single histogram is presented. The results of the application of the algorithm to a set of computer generated histograms in balanced exponential growth are reported.

Animals↗