PubMed Health⌕ Search

Biomedical subjects

Luminita Paraoan

Publications and source records attributed to Luminita Paraoan.

11 recordsLinked to original sources
PubMed2006

Trafficking of osteonectin by retinal pigment epithelial cells: evidence for basolateral secretion.

Osteonectin is a glycoprotein that modulates several aspects of cellular behaviour including proliferation and adhesion. The retinal pigment epithelium forms a continuous monolayer of polarised cells immediately bellow the neuroretina, and is integral to the homeostasis of photoreceptor cells. While osteonectin is expressed by normal retinal pigment epithelium in situ, its expression is significantly increased in retinal pigment epithelial cells associated with several common retinal diseases. This pattern of expression implies an important role for osteonectin in the biology of retinal pigment epithelial cells. However, the trafficking, processing, and eventual fate of osteonectin in these cells is not clear at present. Although the theoretical report of a leader sequence within the osteonectin open reading frame and its extracellular presence in some tissues indirectly support secretion of the protein, there is no direct experimental demonstration of the secretion route to date. As a first step towards understanding the role of osteonectin in retinal pigment epithelium, we studied the intracellular distribution and trafficking of the protein in living cells. Here, we present experimental evidence that a precursor osteonectin fusion protein is targeted to the endoplasmic reticulum/Golgi pathway, with a likely basal secretion in retinal pigment epithelial cells. In addition, we show that the precursor osteonectin protein having the leader sequence masked fails to undergo secretion leading to cell death, a phenotype which may be of relevance not only for retinal pathology, but also for other diseases such as the bone disorder known as pseudoachondroplasia that is associated with a lack of osteonectin secretion.

Achondroplasia↗
PubMed2006

Expression of p53-induced apoptosis effector PERP in primary uveal melanomas: downregulation is associated with aggressive type.

Expression of PERP (p53 apoptosis effector related to PMP-22) was investigated in primary uveal melanomas and its variation was analyzed in relation to clinico-pathological and cytogenetical characteristics of these tumors. The transcriptional level of PERP gene was measured by quantitative real-time RT-PCR in 26 uveal melanomas with known chromosomes 3 and 8 status. PERP protein levels were assessed by Western blot analysis of 22 fresh-frozen tumors and by immunohistochemical analysis of 16 paraffin-embedded tumor specimens. Differential expression of PERP was identified in primary choroidal melanoma specimens, both at transcriptional and protein level. Reduced PERP mRNA level was significantly associated with monosomy 3 (two-way ANOVA and t-test, p=0.004) but not with gains in chromosome 8. Transcriptional downregulation of PERP did not present a statistically significant association with ciliary body involvement, size, PAS-positive loops or cell type. Immunoblotting and immunohistochemistry further demonstrated significantly reduced PERP protein level in monosomy 3 melanomas, as compared with disomy 3 tumors. The altered expression of PERP highlighted this apoptosis-specific target of p53 as a possible contributor to apoptosis in uveal melanoma with PERP downregulation being particularly relevant to the aggressive (monosomy 3) type of uveal melanoma. As PERP is a novel type of p53 effector that is likely to stimulate apoptosis through a mechanism distinct from that of Bcl-2-related mitochondrial effectors, further elucidation of its role in uveal melanoma pathogenesis will assist in the design of novel therapeutic approaches aimed at increasing the rate of apoptosis in this tumor.

Adult↗
PubMed2006

A dual Golgi- and mitochondria-localised Ala25Ser precursor cystatin C: an additional tool for characterising intracellular mis-localisation leading to increased AMD susceptibility.

An artificial mutant Ala25Ser precursor cystatin C was created to help elucidate the cause of intracellular mis-localisation of the biochemically related variant B (Ala25Thr) precursor cystatin C to the mitochondria. Homozygotes of variant B precursor cystatin C were reported to carry an increased susceptibility to developing the exudative form of AMD. Ala25Ser precursor cystatin C shows a dual distribution to the Golgi apparatus and to the mitochondria. This localisation is thus intermediary between that of wild-type cystatin C (targeted to ER/Golgi compartment) and that of variant B precursor cystatin C. Furthermore, the level of secretion of Ala25Ser cystatin C by RPE cells is intermediary between wild type and variant B cystatin C. Ala25Ser precursor cystatin C thus represents a biochemical intermediate between the wild type and the AMD-associated cystatin C and as such, is a novel tool for the investigation of the mechanism of intracellular mis-localisation of variant B cystatin C. Our findings further support the hypothesis that substitution of the alanine residue in the penultimate position of precursor cystatin C signal sequence with a less hydrophobic amino acid residue, such as threonine (as in variant B cystatin C) or serine is sufficient to impair the intracellular trafficking and processing of the protein.

Cystatin C↗
PubMed2005

Identification, expression and functional characterization of the GRAL gene.

The glial cell line-derived neurotrophic factor (GDNF) family is a group of neurotrophic factors with diverse biological functions. Members of the GDNF family exert their functions by interacting with a specific GDNF family receptor alpha (GFRalpha) and activation of the cRET. Here we report the identification and characterization of GDNF receptor-alpha-like (GRAL) gene. Sequence analysis indicated that GRAL is a distant homolog of the GFRalpha family, with 30% of its amino acid sequence identical to that of GFRalpha-3. There are two splice variants of GRAL: the full-length form (GRAL-A) represented by a 2080 bp mRNA and a short form (GRAL-B) represented by a 1833 bp mRNA. In adult mouse, GRAL transcripts have been found primarily in the CNS. In the developing mouse brain, the mRNA level of GRAL in the cerebrocortex and hippocampus reached a maximum at birth and declined afterwards. GRAL-A protein was localized predominantly in the plasma membrane. Overexpression of GRAL-A protected PC12 cells and cultured hippocampal neurons from serum starvation-induced cell apoptosis. The neuroprotective effect of GRAL was associated with marked inhibition of the Jun-N-terminal kinase signaling pathway. Our results suggest that GRAL belongs to a superfamily of GFRalpha and might take part in neuroprotection and brain development.

Amino Acid Sequence↗
PubMed2005

Thrombospondin 1, thrombospondin 2 and the eye.

Thrombospondin 1 and thrombospondin 2 (TSP1 and TSP2), which comprise the subgroup A thrombospondins, are matricellular proteins. As matricellular proteins, they modulate interactions between cells and the cellular environment, regulate cell adhesion and typically are expressed during tissue formative processes. In general, TSP1 and TSP2 counter angiogenesis (including tumour angiogenesis) and play important but contrasting roles during cutaneous repair. The two proteins are involved in development, including that of the eye, although evidence suggests that they have their greatest impact during tissue production in the adult. In the normal adult eye, they tend to be found at sites of ongoing matrix synthesis or cell-matrix interactions. At these sites, the two proteins possibly influence cellular differentiation and/or basement membrane deposition. TSP1 is also present in the intraocular fluids and drainage pathway, where it may function in maintaining the anti-angiogenic environment and in intraocular pressure control, respectively. TSP1 could also be involved in ocular immune privilege. Unlike in skin wounds, where TSP1 is derived from the blood and is present only in the early phases of repair, ocular tissue damage appears to lead to protacted TSP1 synthesis by local cells. This response might help suppress angiogenesis in the transparent tissues of the eye and so lessen visual axis opacification following injury. However, TSP2, which is also produced by damaged ophthalmic tissue and may be especially important in matrix organisation, seems to augment contraction in anomalous intraocular fibrosis. Elucidating the roles of TSP1 and TSP2 in ocular physiology and pathobiology may lead to improved therapies for neovascular, neoplastic, reparative and other ophthalmic diseases.

Animals↗
PubMed2005

Cystatin C prevents degeneration of rat nigral dopaminergic neurons: in vitro and in vivo studies.

Destruction of nigrostriatal dopaminergic (DA) pathway triggers various persistent responses, such as inflammation and increased synthesis of neural growth factors, both in striatum and in substantia nigra. The pathological processes involved in such responses are poorly characterized and could contribute to secondary damage and/or regeneration in the central nervous system (CNS). Cystatin C was previously implicated in the process of neurodegeneration. However, its biological role during neurodegeneration is not understood and remains controversial. The present study identified an increased cystatin C mRNA level in the DA-depleted rat striatum, starting from the second week following a 6-OHDA-induced lesion. Immunohistochemical analysis confirmed the increase in cystatin C protein level in the striatum following DA depletion. Double-labeled fluorescence immunohistochemistry revealed that nigrostriatal neurons, astrocytes, and microglia contributed to the elevated level of cystatin C. Exposure to 6-hydroxydopamine, a DA-specific neurotoxin, resulted in DA neurons loss in the fetal mesencephalic cultures, an effect which could be partially reversed by treatment with cystatin C. Moreover, in vivo DA neurons survival study showed that administration of cystatin C in rats with 6-OHDA-induced lesion partially rescued the nigral DA neurons. The results indicate that the 6-OHDA lesioning induced a relatively slow but sustained up-regulation of cystatin C expression and suggest that the inhibitor may exert a neuroprotective action on DA neurons. The findings raise the possibility that cysteine proteinase inhibitors may be new candidates for neuroprotective treatment of Parkinson's disease. Cystatin C may be useful therapeutically in limiting neuropathy in Parkinson's disease.

Animals↗
PubMed2005

Differential expression of angioregulatory matricellular proteins in posterior uveal melanoma.

Metastases from uveal melanoma, the most common primary malignant eye tumour in adults, develop solely via their vascular bed due to the absence of intraocular lymphatics. The present study investigated the expression in this tumour of three matricellular proteins--Secreted Protein Acidic and Rich in Cysteine (SPARC), thrombospondin 1 (TSP1) and thrombospondin 2 (TSP2)--with putative contrasting roles in the regulation of angiogenesis. Immunohistochemical analysis of the three proteins was carried out in paraffin-embedded specimens from 27 posterior uveal melanomas and was corroborated with Western blot analysis of fresh-frozen samples from seven of the tumours. SPARC immunoreactivity was detected in all specimens and defined two categories of tumour: SPARC-rich (21 of 27 specimens) and SPARC-patchy (six of 27 specimens) uveal melanomas. SPARC-rich tumours had a significantly higher proportion of specimen area occupied by blood vessels (P=0.04) and showed a positive association with the presence of epithelioid-type tumoral cells (P=0.101). TSP1 was not detected by either of the methods in any of the tumours analysed. Some immunopositivity for TSP2 was detected in tumour cells in approximately 40% of specimens, but was not associated with survival, tumour vascularity or any other histopathological indices of survival. The pattern of expression of these matricellular proteins in uveal melanoma is consistent with a cooperative mechanism for establishing an enhanced environment favourable to angiogenesis. Interventions inducing TSP1 expression and/or inhibiting SPARC expression may be candidates for therapies directed towards the inhibition of angiogenesis in posterior uveal melanoma.

Adult↗
PubMed2004

Unexpected intracellular localization of the AMD-associated cystatin C variant.

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.

Aging↗
PubMed2003

Fate of cystatin C lacking the leader sequence in RPE cells.

The study made use of transiently transfected RPE cells expressing mature cystatin C (lacking the 26 amino acids leader sequence of the precursor cystatin C) fused to enhanced green fluorescent protein (EGFP) and, in control transfections, EGFP on its own. The findings demonstrate that 'leaderless' cystatin C is not processed through the secretory pathway of RPE cells. Since a polymorphism in the leader sequence has recently been associated with increased risk for development of exudative age-related macular degeneration (AMD), the present findings lend support to the hypothesis that impairment of function of the leader sequence may contribute to the aetiology of exudative AMD.

5' Untranslated Regions↗
PubMed2002

Multi-parameter analysis of the kinetics of NF-kappaB signalling and transcription in single living cells.

Proteins of the NF-kappaB transcription factor family normally reside in the cytoplasm of cells in a complex with IkappaB inhibitor proteins. Stimulation with TNFalpha leads to proteosomal degradation of the IkappaB proteins and nuclear translocation of the NF-kappaB proteins. Expression of p65 and IkappaBalpha fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IkappaBalpha-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression, the half-time of IkappaBalpha-EGFP degradation was reduced and that of p65 translocation was significantly increased when compared with cells expressing the single fluorescent fusion proteins. These results suggest that the ratio of IkappaBalpha and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IkappaBalpha is the true substrate for TNFalpha stimulation in mammalian cells. When cells were treated with the CRM-1-dependent nuclear export inhibitor, leptomycin B (LMB), there was nuclear accumulation of IkappaBalpha-EGFP and p65-dsRed, with IkappaBalpha-EGFP accumulating more rapidly. No NF-kappaB-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IkappaBalpha-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNFalpha, degradation of IkappaBalpha-EGFP was observed in both the cytoplasm and nucleus. A normal transient transcription response was observed in the same cells using luminescence imaging of NF-kappaB-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-kappaB-dependent transcription occur even when nuclear export of NF-kappaB is inhibited. The results provide functional evidence that other factors, such as modification of p65 by phosphorylation, or interaction with other proteins such as transcriptional co-activators/co-repressors, may critically modulate the kinetics of transcription through this signalling pathway.

Active Transport, Cell Nucleus↗