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Biomedical subjects

D Bosco

Publications and source records attributed to D Bosco.

At least 19 recordsLinked to original sources
PubMed2000

The long-duration response to L-dopa in the treatment of early PD.

OBJECTIVE: To investigate the long-duration response (LDR) to L-dopa resulting from different regimens of L-dopa. BACKGROUND: In clinical practice, L-dopa is usually administered without considering the LDR due to the drug. Moreover, it has not been established whether in early PD a multiple daily intake of small doses of L-dopa may induce a sustained LDR. METHODS: Twenty-four patients with early PD underwent a double-blind, crossover trial, comparing three different 15-day treatment periods with L-dopa: treatment A (250 mg every 24 hours); treatment B (250 mg every 8 hours); and treatment C (125 mg every 8 hours). After completion, 20 patients underwent a subsequent open-label randomized trial with prolonged treatments (250 mg every 24 hours or 125 mg every 8 hours) up to 3 months. LDR was measured at the end of each treatment. RESULTS: All patients achieved a sustained LDR after treatments A and B, whereas only 17% of patients reached a sustained LDR after treatment C. Overall, the LDRs resulting from treatments A and B had similar magnitude and were larger than the LDR deriving from treatment C. After 3 months of prolonged treatments, only three of 10 patients treated with 125 mg every 8 hours increased their LDR, whereas all 10 patients treated with 250 mg every 24 hours had a maximal and stable LDR. CONCLUSIONS: Sustained LDR to L-dopa is dependent on the amount of the single doses of the drug. A regimen scheduling small, divided doses during the day, as done in clinical practice, is a questionable therapy for the achievement of a sustained LDR.

Aged↗
PubMed2000

Glucokinase and glucokinase regulatory protein: mutual dependence for nuclear localization.

Conditional expression of the glucokinase regulatory protein in insulinoma cells, under control of the reverse tetracycline-dependent transactivator, was used to investigate whether expression of this protein de novo would alter the intracellular distribution of glucokinase. The regulatory protein, which was undetectable in the basal state, could be induced by doxycycline to levels comparable to those of liver and was detected mostly in the nucleus. Concomitantly, glucokinase accumulated in the nucleus. Human embryonic kidney cells were transiently transfected to express glucokinase and the regulatory protein, either separately or together. Each protein localized predominantly to the cytoplasm when expressed alone. On co-expression, however, both proteins localized virtually entirely to the nucleus. The enzymic activity of glucokinase was not required for promoting nuclear import of the two proteins, as shown with a glucose-phosphorylation-deficient mutant. Finally, in embryonic kidney cells expressing the regulatory protein alone, treatment with leptomycin B resulted in a partial redistribution of the protein from the cytoplasm to the nucleus, suggesting that this protein can shuttle between the two compartments.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Lupus anticoagulant positivity in insulin dependent diabetic patients: an additional risk factor in the pathogenesis of diabetic retinopathy?

AIMS: To investigate whether lupus anticoagulant (LA) positivity, a marker of endothelial dysfunction, might be relevant to the pathogenesis of diabetic retinopathy (DR). METHODS: 32 IDDM patients were examined for LA, fibrinogen, prothrombin (PT), PTT, prothrombin degradation products (F1+2), and activated protein C (APC). RESULTS: APC decreased and F1+2 increased significantly in LA positive but not in LA negative patients; 60% of LA positive and 18% of LA negative subjects had DR. PT, PTT, and fibrinogen levels were insignificant. CONCLUSION: These preliminary findings suggest that LA positivity could represent an additional risk factor for DR, acting as a link between the immunological and haemostatic systems.

Adult↗
PubMed2000

Junctional communication of pancreatic beta cells contributes to the control of insulin secretion and glucose tolerance.

Proper insulin secretion requires the coordinated functioning of the numerous beta cells that form pancreatic islets. This coordination depends on a network of communication mechanisms whereby beta cells interact with extracellular signals and adjacent cells via connexin channels. To assess whether connexin-dependent communication plays a role in vivo, we have developed transgenic mice in which connexin 32 (Cx32), one of the vertebrate connexins found in the pancreas, is expressed in beta cells. We show that the altered beta-cell coupling that results from this expression causes reduced insulin secretion in response to physiologically relevant concentrations of glucose and abnormal tolerance to the sugar. These alterations were observed in spite of normal numbers of islets, increased insulin content, and preserved secretory response to glucose by individual beta cells. Moreover, glucose-stimulated islets showed improved electrical synchronization of these cells and increased cytosolic levels of Ca(2+). The results show that connexins contribute to the control of beta cells in vivo and that their excess is detrimental for insulin secretion.

Animals↗
PubMed2000

Importance of cell-matrix interactions in rat islet beta-cell secretion in vitro: role of alpha6beta1 integrin.

It has long been recognized that islet cell function is rapidly altered in vitro, but can be maintained, at least in part, when cells are layered on defined extracellular matrices. The present work addresses the influence of short-term cell-matrix interactions on islet beta-cell function and provides first insight into the molecular basis of these interactions. When primary rat beta-cells were allowed to attach to a matrix produced by a rat carcinoma cell line (804G), there was an increased insulin secretory response to secretagogues. This change was the result of an increase in the proportion of actively secreting beta-cells and in the amount of insulin secreted per active cell, as shown using the reverse hemolytic plaque assay. In turn, the spreading or flattening of beta-cells on this matrix was enhanced by secretagogues, and flattened cells secreted more insulin than rounded cells. Using indirect immunofluorescence, it was found that 1)alpha6beta1 integrins are present at the surface of islet cells in situ, 2) alpha6beta1 expression is heterogeneous among purified beta-cells and is upregulated by insulin secretagogues, 3) alpha6beta1 expression is higher in spreading cells, and 4) anti-alpha6beta1-specific antibodies decrease spreading. These observations demonstrate that islet cell-matrix interactions can improve the sensitivity of insulin cells to glucose and are mediated, at least in part, by alpha6beta1 integrins, suggesting that outside-in signaling through alpha6beta1 integrin plays a major role in the regulation of beta-cell function.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Pharmacodynamics of the long-duration response to levodopa in PD.

OBJECTIVE: To determine the latency, magnitude, and duration of the long-duration response (LDR) to levodopa in PD in relationship to the administration of levodopa at different interdose intervals. METHODS: In six patients with PD, two different 15-day treatment regimens were used in which the drug was administered with interdose intervals of 24 or 8 hours. RESULTS: The LDR built up within a few days with either regimen, but a faster rate of administering levodopa shortened the latency to the appearance of a sustained LDR. Once a sustained response had been reached, the magnitude of the LDR showed a stable ceiling effect that was independent of the levodopa schedule. After discontinuation of treatment, the decay of the LDR was similar for both regimens. CONCLUSIONS: The system underlying the LDR to levodopa may be completely saturated when a sustained response has been fully developed. The intervals between doses of levodopa shorter than the interval effective to reach a sustained LDR should not be used in the clinical management of patients with PD because the antiparkinsonian benefit deriving from the LDR is already maximal and briefer intervals do not provide a greater benefit.

Aged↗
PubMed1999

Loss of long-duration response to levodopa over time in PD: implications for wearing-off.

OBJECTIVE: To determine the modifications of the long-duration response to levodopa in PD over a 1-year period. BACKGROUND: The development of predictable motor fluctuations in PD has been attributed mainly to modifications over time of the short-duration response to levodopa, whereas the role of the long-duration response has not been widely investigated. METHODS: In 17 patients with PD the authors examined prospectively both the short-duration response and the long-duration response to levodopa under standardized conditions on two different occasions separated by a period of approximately 1 year (11.7 +/- 3.6 months). RESULTS: At the end of the follow-up period, the short-duration response increased in magnitude but did not change significantly in duration. A total of 24% of patients lost the long-duration response 1 year after their first examination, but a sustained long-duration response could be reestablished by shortening the interdose interval for levodopa intake. Moreover, the duration of the long-duration response after discontinuation of treatment became significantly shorter during 1 year. CONCLUSION: Modifications of the long-duration response may have a pivotal role in generating a fluctuating response, and suggest that therapeutic strategies based on maintenance of the long-duration response should be sought to avoid the appearance of motor fluctuations.

Aged↗
PubMed1999

Loss of alpha 1 connexin does not alter the prenatal differentiation of pancreatic beta cells and leads to the identification of another islet cell connexin.

Connexin alpha 1, also referred to as Cx43, has thus far been the only gap junction protein identified between the hormone-producing cells of pancreatic islets. To investigate whether loss of this connexin affects the development of endocrine pancreas and the differentiation of insulin-producing beta cells, we have taken advantage of a transgenic line in which the gene coding for connexin alpha 1 had been functionally deleted by homologous recombination. Analysis of pancreas at embryonal day 19.5 (E 19.5) after immunostaining for the four main types of islet hormones, showed that islet cell development was similar in homozygous transgenic mice that completely lacked alpha 1 connexin, in mice that were heterozygous for the transgene, and in age-matched controls with a genetic background similar to that of the transgenic animals. In particular, the three animal groups featured beta cells that had a similar insulin content and ultrastructural organization, including the presence of typical gap junction plaques on the membrane. However, quantitative analysis of freeze-fractured membranes showed that these plaques were less frequent in the transgenic mice lacking alpha 1 connexin. This finding prompted us to revisit the connexin pattern of normal pancreatic beta cells. Using RT-PCR amplification and primers specific for nine of the mammalian connexins, we have found that normal rat and mouse pancreas contain six connexin transcripts, including one that codes for alpha 6 connexin, a protein also referred to as Cx45. This transcript was also identified in isolated pancreatic islets, in FACS-purified suspensions of primary beta cells and in the insulin-producing cells of an experimental tumor. Using antibodies, we found that connexin alpha 6 is expressed by the latter cells, as well as by pancreatic fibroblasts and epithelial duct cells. The data show that pancreatic islets have a normal prenatal development in mice that no longer express alpha 1 connexin. They further provide evidence that normal and tumoral insulin-producing cells natively coexpress connexins alpha 1 and alpha 6.

Animals↗
PubMed1999

Effects of TNF-alpha/colchicine combined treatment on Burkitt lymphoma cells: molecular and ultrastructural changes.

Tumour necrosis factor alpha (TNF-alpha) kills Daudi cells (Human Burkitt Lymphoma), inducing either necrosis or apoptosis without DNA fragmentation. Therefore, we were interested in studying the molecular and ultrastructural events occurring when the nucleus is more accessible and cells are blocked in mitosis, following colchicine treatment. In fact, as early as after 1 h treatment a typical ladder pattern was shown by means of DNA gel electrophoresis. In parallel the quantitative analysis of the different morphological patterns observed gave evidence of an increased percentage of primary necrosis after 6 h treatment, and a higher incidence of cells in late apoptosis as well as in secondary necrosis after 24 h treatment. Our findings show that Daudi cells respond to the combined treatment with an increased formation of micronuclei and nuclear alterations which follow a number of early mitochondrial changes and result in enhanced cell death. These data imply that TNF-alpha-induced apoptosis of Daudi cells can be triggered by mitochondrial changes and is somehow related to microtubule organization.

Apoptosis↗
PubMed1999

Immunocytochemical localization of phospholipase C isozymes in cord blood and adult T-lymphocytes.

The response of T-cells to peptide antigen plus major histocompatibility complex (MHC) consists of a series of cellular events collectively called T-cell activation. An essential component of this pathway is phospholipase C (PLC)gamma1, whose hydrolytic activity increases rapidly after binding of ligands to the T-cell receptor (TCR) and consequent activation of tyrosine kinases. Recent studies also suggest a GTP binding protein-dependent activation of PLCbeta during the early steps of T-cell activation. On the basis of these findings, we first checked the expression of PLC isoforms by Western blotting and by confocal and electron microscopy techniques, and then we looked for the phosphoinositide breakdown induced by CD3 engagement in cord and adult T-lymphocytes. Our results indicated that PLCbeta1 was almost exclusively expressed in cord T-cells, whereas PLCbeta2 was more strongly represented in the adult. The amount of PLCgamma1 was found to be larger in the adult than in cord cells. No significant differences were found in PLCgamma2 and delta2 expression. PLCdelta1 was scarcely detectable. On CD3 stimulation, adult lymphocytes gave rise, as expected, to a dramatic increase in phosphoinositide breakdown, whereas in cord cells this response was scarcely detected. These results indicate that a shift in PLC expression occurs in the postnatal period and that this change is associated with induction of the capability to respond to CD3 engagement with phosphoinositide hydrolysis.

Adult↗
PubMed1999

[Angiodysplasia of the duodenum with arteriovenous shunts, sub-serosal prevalence and interstitial elastosis; peritonitis and rupture of sub-serous dysplasic vessels and hemoperitoneum. Description of a case and review of the literature].

We present a case report of a 74 year old man, affected by angiodysplasia of the duodenum, which developed in peritonitis and hemoperitoneum. After undergoing two surgical interventions, it is 14 days since he died. First of all, the importance of this case is due to the extreme rarity of the angiodysplasia of the duodenum, specially when it becomes worse in peritonitis and hemoperitoneum, this case having been described only one time till now, then the localization mainly sub-serousal and lastly the presence of a component, characterized by an elastotic nature, disposed in islets and interstitial widespread also among the glands, which we have first described in literature.

Aged↗
PubMed1999

Expression of bcl-2, c-erbB-2, p53, and p21 (waf1-cip1) protein in thyroid carcinomas.

The study was carried out on 53 patients who had thyroid cancer with various degree of differentiation. We studied the expression of bcl-2, a-erbB-2, p53, and p21 ras protein. The protein encoded by bCL-2 proto-oncogene is implicated in the prolongation of cell survival by blocking programmed cell death, i.e. apoptosis. The role of p53 and bcl-2 genes in the regulation of apoptosis has important implications in oncogenesis. Wild-type p53 is thought to promote apoptosis, whilst mutant p53 has a similar effect on apoptosis as bcl-2 that is inhibition of programmed cell kinase activity. C-erb-2 protein overexpression is currently being evaluated as a potential risk factor in breast cancer patients? The ras gene family codes for a 21 kD protein (p21), which binds guanine nucleotides and possesses GTPase activity. Through this mechanism, the ras p21 protein participates in the control of cell proliferation, possibly as a signal transducer from cell surface receptors to the nucleus. Activation of ras genes has been implicated in neoplastic transformation of cells. The aim of our study is to evaluate the expression of these markers in thyroid carcinomas. All immunohistochemical study was performed in paraffin-embedded tissues pathology specimen. Any well differentiated tumor in our study was positive for bcl-2 protein. C-erb-2 immunostaining was present in tumor samples in 60% of cases. In most cases, specific membrane staining as well as a weak cytoplasmic positivity of tumor cells were seen. Immunoreactivity for p53 was positive only in 10% of cases. By immunostaining, p21 protein was expressed in 55% of the 53 tumors tested, with different degree of expression. Only some poorly differentiated tumours were positive for bcl-2, furthermore all markers tested were strongly positive in these tumours. In conclusion, our results indicate that bcl-2, c-erbB-2, p53, and p21 ras protein are differently expressed in thyroid carcinomas in relation to the degree of aggressiveness and differentiation.

Adenocarcinoma, Follicular↗
PubMed1998

Enhanced secretion of amylase from exocrine pancreas of connexin32-deficient mice.

To determine whether junctional communication between pancreatic acinar cells contributes to their secretory function in vivo, we have compared wild-type mice, which express the gap junctional proteins connexin32 (Cx32) and connexin26, to mice deficient for the Cx32 gene. Pancreatic acinar cells from Cx32 (-/-) mice failed to express Cx32 as evidenced by reverse transcription-PCR and immunolabeling and showed a marked reduction (4.8- and 25-fold, respectively) in the number and size of gap junctions. Dye transfer studies showed that the extent of intercellular communication was inhibited in Cx32 (-/-) acini. However, electrical coupling was detected by dual patch clamp recording in Cx32 (-/-) acinar cell pairs. Although wild-type and Cx32 (-/-) acini were similarly stimulated to release amylase by carbamylcholine, Cx32 (-/-) acini showed a twofold increase of their basal secretion. This effect was caused by an increase in the proportion of secreting acini, as detected with a reverse hemolytic plaque assay. Blood measurements further revealed that Cx32 (-/-) mice had elevated basal levels of circulating amylase. The results, which demonstrate an inverse relationship between the extent of acinar cell coupling and basal amylase secretion in vivo, support the view that the physiological recruitment of secretory acinar cells is regulated by gap junction mediated intercellular communication.

Amylases↗
PubMed1998

Oral focal mucinosis of the tongue.

Oral focal mucinosis (OFM) is an uncommon clinicopathological entity which is considered to be the oral counterpart of cutaneous focal mucinosis and cutaneous myxoid cyst. It is comprised of a clinically elevated mass with a histological feature of localised areas of myxomatous connective tissue. The present study adds a rare case of OFM of the tongue to the literature, and we present a review of the most characteristic oral myxomatous lesions.

Aged↗
PubMed1998

Upregulation of connexin 26 between keratinocytes of psoriatic lesions.

To assess whether the expression of connexins (Cx) by keratinocytes is altered under conditions of abnormal epidermal differentiation, we have compared Cx26, Cx32, Cx37, Cx40, and Cx43 in the epidermis of 11 psoriatic patients who had not been treated for at least 1 mo and of seven healthy individuals. In all samples of fully mature psoriatic plaques, we have observed a massive expression of Cx26, as judged at both the transcript level (northern blot) and the protein level (immunofluorescence). This protein became consistently detected between keratinocytes of the basal and granular layers at the periphery of psoriatic plaques and in all layers of fully developed psoriatic epidermis, except in regions of parakeratosis. None or a minimal amount of Cx26 was observed in both control and nonlesional regions of psoriatic epidermis. Psoriatic plaques also contained Cx43, the prominent gap junction protein in the interfollicular epidermis of normal human skin. The levels of this protein appeared to be slightly higher in psoriatic than in control skin, as judged at both the transcript level (northern blot) and the protein level (immunofluorescence). Three other connexins (Cx32, Cx37, and Cx40), which are not observed in control interfollicular epidermis, were not induced in either nonlesional or lesional regions of psoriatic skin. The data indicate that selective changes in the normal expression of connexins by keratinocytes are associated with the changes in the proliferation and differentiation program that these cells undergo in psoriasis.

Adult↗
PubMed1998

Sorting human beta-cells consequent to targeted expression of green fluorescent protein.

Pancreatic islets of Langerhans are composed of four major endocrine cell types with a smaller number of nonendocrine cells. To study the molecular constituents and function of just one subpopulation of islet cells, it is necessary to sort them from the other cell types. While rat beta-cells can be sorted by autofluorescence-activated flow cytometry, this has not proved possible on a routine and reproducible basis for human beta-cells. In the present study, we have selectively labeled human beta-cells with green fluorescent protein (GFP), allowing for their sorting by flow cytometry. Human islet cells were infected with replication-defective (attenuated) recombinant adenovirus expressing GFP driven by the rat insulin I promoter (Ad-RIP-GFP) for targeted expression in beta-cells, or beta-galactosidase driven by the promiscuous cytomegalovirus (CMV) promoter (Ad-CMV-beta-gal) as control. Whereas the majority of islet cells can be infected by adenovirus, as shown by control infection with Ad-CMV-beta-gal, increased fluorescence after infection with Ad-RIP-GFP was limited to insulin-containing beta-cells. Infection of islet cells with Ad-RIP-GFP resulted reproducibly in the appearance of a population of intensely fluorescent cells, when analyzed by flow cytometry. These cells were sorted using a fluorescence-activated cell sorter (FACS) and shown by immunofluorescence to consist of >95% beta-cells. The targeted expression of GFP thus allows for preparation of human beta-cells purified close to homogeneity. This method should be readily applicable in any laboratory with FACS capability.

Adenoviridae↗
PubMed1998

Ultrastructural changes in rat cornea after prolonged hypobaric hypoxia.

A study was performed to evaluate the effect of prolonged environmental hypobaric hypoxia on the ultrastructure of rat cornea. 60-day-old female Wistar rats were exposed to a simulated altitude of 5,500 m (350 mmHg) and pO2 of 76 mmHg for 30 days. Control rats were exposed to atmospheric sea level pressure (760 mmHg) and pO2 of 150 mmHg, for the same period. Ultrastructural analysis of the corneal epithelium did not reveal any relevant difference between control and treated rats. In contrast, the corneal stroma of rats subjected to hypoxia showed vascularization with advanced vessel differentiation and signs of active proliferation. The endothelium of hypoxic cornea showed swollen mitochondria and large empty cytoplasmic areas. The endothelial intercellular junctions could hardly be identified in the hypoxic condition. Nevertheless, the most evident change in hypoxic cornea was in Descemet's membrane, which was considerably thickened, to approximately twice that of the control specimen. These results suggest that environmental hypobaric hypoxia induces marked alterations in the corneal stroma and endothelium morphology, probably related to reduced oxygen tension in the aqueous humor, consequent to hypoxemia.

Animals↗