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Biomedical subjects

A Luini

Publications and source records attributed to A Luini.

At least 19 recordsLinked to original sources
PubMed1999

CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid.

Membrane fission is essential in intracellular transport. Acyl-coenzyme As (acyl-CoAs) are important in lipid remodelling and are required for fission of COPI-coated vesicles. Here we show that CtBP/BARS, a protein that functions in the dynamics of Golgi tubules, is an essential component of the fission machinery operating at Golgi tubular networks, including Golgi compartments involved in protein transport and sorting. CtBP/BARS-induced fission was preceded by the formation of constricted sites in Golgi tubules, whose extreme curvature is likely to involve local changes in the membrane lipid composition. We find that CtBP/BARS uses acyl-CoA to selectively catalyse the acylation of lysophosphatidic acid to phosphatidic acid both in pure lipidic systems and in Golgi membranes, and that this reaction is essential for fission. Our results indicate a key role for lipid metabolic pathways in membrane fission.

Acyl Coenzyme A↗
PubMed1999

PDMP blocks the BFA-induced ADP-ribosylation of BARS-50 in isolated Golgi membranes.

We reported that an inhibitor of sphingolipid biosynthesis, D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), blocks brefeldin A (BFA)-induced retrograde membrane transport from the Golgi complex to the endoplasmic reticulum (ER) (Kok et al., 1998, J. Cell Biol. 142, 25-38). We now show that PDMP partially blocks the BFA-induced ADP-ribosylation of the cytosolic protein BARS-50. Moreover, PDMP does not interfere with the BFA-induced inhibition of the binding of ADP-ribosylation factor (ARF) and the coatomer component beta-coat protein to Golgi membranes. These results are consistent with a role of ADP-ribosylation in the action of BFA and with the involvement of BARS-50 in the regulation of membrane trafficking.

Adenosine Diphosphate↗
PubMed1999

Molecular cloning and functional characterization of brefeldin A-ADP-ribosylated substrate. A novel protein involved in the maintenance of the Golgi structure.

Brefeldin A (BFA) is a fungal metabolite that disassembles the Golgi apparatus into tubular networks and causes the dissociation of coatomer proteins from Golgi membranes. We have previously shown that an additional effect of BFA is to stimulate the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa (brefeldin A-ADP-riboslyated substrate (BARS)) and that this effect greatly facilitates the Golgi-disassembling activity of the toxin. In this study, BARS has been purified from rat brain cytosol and microsequenced, and the BARS cDNA has been cloned. BARS shares high homology with two known proteins, C-terminal-binding protein 1 (CtBP1) and CtBP2. It is therefore a third member of the CtBP family. The role of BARS in Golgi disassembly by BFA was verified in permeabilized cells. In the presence of dialyzed cytosol that had been previously depleted of BARS or treated with an anti-BARS antibody, BFA potently disassembled the Golgi. However, in cytosol complemented with purified BARS, or even in control cytosols containing physiological levels of BARS, the action of BFA on Golgi disassembly was strongly inhibited. These results suggest that BARS exerts a negative control on Golgi tubulation, with important consequences for the structure and function of the Golgi complex.

ADP-Ribosylation Factors↗
PubMed1999

Intraoperative examination of axillary sentinel lymph nodes in breast carcinoma patients.

BACKGROUND: Routine histologic examination of axillary sentinel lymph nodes predicts axillary lymph node status and may spare patients with breast carcinoma axillary lymph node dissection. To avoid the need for two separate surgical sessions, the results of sentinel lymph node examination should be available intraoperatively. However, routine frozen-section examination of sentinel lymph nodes is liable to yield false-negative results. This study was conducted to ascertain whether extensive intraoperative examination of sentinel lymph nodes by frozen section examination would attain a sensitivity comparable to that obtained by routine histologic examination without intraoperative frozen section examination. METHODS: In a consecutive series of 155 clinically lymph node negative breast carcinoma patients, the axillary sentinel lymph nodes were examined intraoperatively, before complete axillary lymph node dissection. The frozen sentinel lymph nodes were sectioned subserially at 50-microm intervals. For each level, one section was stained with hematoxylin and eosin and the other section immunostained for cytokeratins using a rapid immunocytochemical assay. RESULTS: Sentinel lymph node metastases were detected in 70 of the 155 patients (45%). In 37 cases the sentinel lymph nodes were the only axillary lymph nodes with metastases. Immunocytochemistry did not increase the sensitivity of the examination. Five patients had metastases in the nonsentinel axillary lymph nodes despite having negative sentinel lymph nodes. The general concordance between sentinel and axillary lymph node status was 96.7%; the negative predictive value of intraoperative sentinel lymph node examination was 94.1%. CONCLUSIONS: The intraoperative examination of axillary sentinel lymph nodes is effective in predicting the axillary lymph node status of breast carcinoma patients and may be instrumental in deciding whether to spare patients axillary lymph node dissection.

Axilla↗
PubMed1999

Morphological and biochemical analysis of the secretory pathway in melanoma cells with distinct metastatic potential.

In this report, we have investigated whether alterations of the morphological and functional aspects of the biosecretory membrane system are associated with the metastatic potential of tumor cells. To this end, we have analyzed the morphology of the Golgi complex, the cytoskeleton organization and membrane trafficking steps of the secretory pathway in two human melanoma A375 cell line variants with low (A375-P) and high metastatic (A375-MM) potential. Immunofluorescence analysis showed that in A375-P cells, the Golgi complex showed a collapsed morphology. Conversely, in A375-MM cells, the Golgi complex presented a reticular and extended morphology. At the ultrastructural level, the Golgi complex of A375-P cells was fragmented and cisternae were swollen. When the cytoskeleton was analyzed, the microtubular network appeared normal in both cell variants, whereas actin stress fibers were largely absent in A375-P, but not in A375-MM cells. In addition, the F-actin content in A375-P cells was significantly lower than in A375-MM cells. These morphological differences in A375-P cells were accompanied by acceleration and an increase in the endoplasmic reticulum to Golgi and the trans-Golgi network to cell surface membrane transport, respectively. Our results indicate that in human A375 melanoma cells, metastatic potential correlates with a well-structured morphofunctional organization of the Golgi complex and actin cytoskeleton.

Biological Transport↗
PubMed1999

Sentinel lymph node biopsy and axillary dissection in breast cancer: results in a large series.

BACKGROUND: Axillary lymph node dissection is an established component of the surgical treatment of breast cancer, and is an important procedure in cancer staging; however, it is associated with unpleasant side effects. We have investigated a radioactive tracer-guided procedure that facilitates identification, removal, and pathologic examination of the sentinel lymph node (i.e., the lymph node first receiving lymphatic fluid from the area of the breast containing the tumor) to predict the status of the axilla and to assess the safety of foregoing axillary dissection if the sentinel lymph node shows no involvement. METHODS: We injected 5-10 MBq of 99mTc-labeled colloidal particles of human albumin peritumorally in 376 consecutive patients with breast cancer who were enrolled at the European Institute of Oncology during the period from March 1996 through March 1998. The sentinel lymph node in each case was visualized by lymphoscintigraphy, and its general location was marked on the overlying skin. During breast surgery, the sentinel lymph node was identified for removal by monitoring the acoustic signal from a hand-held gamma ray-detecting probe. Total axillary dissection was then carried out. The pathologic status of the sentinel lymph node was compared with that of the whole axilla. RESULTS: The sentinel lymph node was identified in 371 (98.7%) of the 376 patients and accurately predicted the state of the axilla in 359 (95.5%) of the patients, with 12 false-negative findings (6.7%; 95% confidence interval = 3.5%-11.4%) among a total of 180 patients with positive axillary lymph nodes. CONCLUSIONS: Sentinel lymph node biopsy using a gamma ray-detecting probe allows staging of the axilla with high accuracy in patients with primary breast cancer. A randomized trial is necessary to determine whether axillary dissection may be avoided in those patients with an uninvolved sentinel lymph node.

Albumins↗
PubMed1999

Morphological changes in the Golgi complex correlate with actin cytoskeleton rearrangements.

In this report we have studied the morphological changes of the Golgi complex (GC) that specifically accompany F-actin reorganizations. In starved rat RBL-2H3 tumor mast cells, the GC, that was visualized at immunofluorescence level with antibodies raised against the Golgi-resident proteins giantin, mannosidase II, or TGN-38, showed a compacted morphology with a supranuclear positioning. Concomitant to membrane ruffle formation induced by epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA), and stress fiber formation induced by lysophosphatidic acid (LPA), specific GC morphological changes were observed. When cells were stimulated with EGF or PMA, the compacted GC morphology was transformed into a reticular network that was extended towards the cell periphery. When cells were incubated with LPA, the GC acquired a characteristic ring-shaped morphology. Brefeldin A (BFA) did not affect the PMA- or LPA-induced membrane ruffling and stress fiber formation, respectively, indicating that actin rearrangements occurred independent of the presence of the GC. Upon BFA removal, the presence of PMA or LPA during the recovery process induced the GC to acquire the morphological appearance described above for each agent. Moreover, the PMA- but not the LPA-induced GC rearrangements were sensitive to the actin perturbing agents cytochalasin D and jasplakinolide. When cells were preincubated with the phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin or LY294002, the PMA-induced GC morphological changes were inhibited but not membrane ruffles. Finally, the PMA-induced increase in the post-Golgi transport of glycosaminoglycans to the cell surface was not altered by cytochalasin D or jasplakinolide. Altogether, these data suggest that: (1) the shape of the GC is influenced by the 3D arrangement of actin microfilaments; (2) PI3K regulates the association of the GC with actin microfilaments; and (3) actin microfilaments are not essential for the post-Golgi transport to the plasma membrane.

Actins↗
PubMed1999

Cyclosporin A, but not FK506, increases arachidonic acid release and inhibits proliferation of pituitary corticotrope tumor cells.

The selective immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) are used in the prevention of allogenic transplant rejection and in the therapy of chronic autoimmune inflammatory pathologies. Chronic treatment with CsA leads to secondary functional and trophic alterations of multiple organs and cell systems among which endocrine ones, through insofar uncharacterized mechanisms. With the recent use of FK506 there have been reports of an improved therapeutic efficacy and a reduction of side-effects, as compared to CsA. An intriguing hypothesis is that toxic damage could be due to a systemic CsA activation of arachidonic acid (AA) metabolism, through pathways as yet only partially characterized. The side-effects of both drugs have been poorly studied on cells from tissues other than blood or kidney. We have thus proceeded to study their action on AA release in corticotropic AtT-20/D16-16 cells. The results obtained are as follows: 1) during incubation times > or =12 h, basal AA release is increased by CsA, but not FK506; the acute effect (10 min) of melittin, a PLA2 activator, is significantly potentiated starting from a 30 min pretreatment with CsA but not FK506; manoalide, a PLA2 inhibitor, antagonizes the melittin potentiation of AA release by CsA whereas the inhibition of the melittin stimulus by glucocorticoids is antagonized both by CsA and FK506. 2) during longer (>2 d) incubation times, cell growth is inhibited by CsA but not FK506. These results indicate a role for CsA, not apparent for FK506, in the activation of PLA2 and in the inhibition of cell growth. They also suggest that CsA does not have a direct (i.e. not mediated by the immune system) therapeutic effect in inflammatory processes.

Animals↗
PubMed1999

ARF mediates recruitment of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex.

The small GTPase ADP-ribosylation factor (ARF) regulates the structure and function of the Golgi complex through mechanisms that are understood only in part, and which include an ability to control the assembly of coat complexes and phospholipase D (PLD). Here we describe a new property of ARF, the ability to recruit phosphatidylinositol-4-OH kinase-beta and a still unidentified phosphatidylinositol-4-phosphate-5-OH kinase to the Golgi complex, resulting in a potent stimulation of synthesis of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate; this ability is independent of its activities on coat proteins and PLD. Phosphatidylinositol-4-OH kinase-beta is required for the structural integrity of the Golgi complex: transfection of a dominant-negative mutant of the kinase markedly alters the organization of the organelle.

1-Phosphatidylinositol 4-Kinase↗
PubMed1999

Comparison of radioguided excision with wire localization of occult breast lesions.

BACKGROUND: Clinically occult breast lesions are found with increasing frequency as a result of the widespread use of mammographic screening. METHODS: This study reports a new localization technique in which a small quantity of 99mTc-labelled colloidal albumin is inoculated directly into the lesion under stereotactic-radiographic or ultrasonographic guidance. Correct positioning of the inoculum is checked by scintigraphy. A gamma ray detection probe is then used to locate the lesion and guide its surgical removal. The results of this method in 30 patients were compared with those obtained using the established hook wire method in another 30 patients. RESULTS: In the wire localization group, the mean(s.d.) distance from the lesion centre to the specimen margin was 24.5(0.4) (range 15-45) mm. For the radioguided group the figures were 13.8(0.3) (range 5-25) mm. The mean(s.d.) lesion concentricity (difference between the maximum and minimum distance from the lesion border to specimen margin) was 6.2(0.4) (range 2-15) mm in the wire localization group and 3.8(0.2) (range 1-10) mm in the radioguided group. CONCLUSION: Use of a gamma probe allowed rapid, easy and accurate removal of occult breast lesions. In comparison to the hook wire method, radioguided removal allows reduced excision volume and better lesion centring within the specimen.

Biopsy↗
PubMed1999

Radiation protection in radioguided surgery of breast cancer.

The protocols for sentinel lymph node biopsy and radioguided occult lesion localization could potentially be of great value in the management of breast cancer patients. Both involve the injection of a 99Tcm-labelled radiopharmaceutical close to or into the lesion, localization of the sentinel lymph node or occult lesion by scintigraphy, and surgical removal with the aid of a hand-held gamma-ray detector. We present dosimetric data on patients and hospital personnel involved in these procedures. For evaluation of radiation protection, we measured the absorbed dose and air kerma rate. Activity levels in excised tissues and surgical instruments were also determined. For patients, the mean absorbed dose to the abdomen was 0.45 mGy, which is low compared to doses received from other diagnostic examinations. For surgeons after 100 operations, the mean absorbed dose to the hands was 0.45 mGy and the mean effective dose 0.09 mSv. Absorbed doses to all hospital personnel involved in the procedures were very low compared to recommended annual limits stipulated by the International Commission on Radiological Protection. We conclude that these procedures, performed according to protocols laid down by the European Institute of Oncology, Milan, are safe from the point of view of radiological protection and that only routine precautions are necessary.

Breast Neoplasms↗
PubMed1999

Importance of the level of axillary involvement in relation to traditional variables in the prognosis of breast cancer.

Survival in breast cancer correlates with the presence of metastatic lymph nodes, so that removal and pathological examination of the axillary nodes provides the most important prognostic information and basis for planning subsequent therapy. However as the size of primary tumours at diagnosis is decreasing, the likelihood of axillary involvement is also declining, so that the indications for axillary dissection are undergoing radical revision. To definitively establish the value of removing all three axillary lymph node levels (as defined by Berg) in node positive breast cancer, retrospective analysis of a large series receiving complete dissection was carried out. consecutive breast cancer patients (n=1003) with positive axillary nodes were analyzed: all received identical axillary treatment and the three levels were tagged with metal disks to facilitate recognition and pathological examination. Follow-up (mean 97 months) was exceptionally complete. The length of disease-free and overall survival were taken as the primary endpoints. The variables considered in the statistical analysis were tumour size, number of metastatic nodes, axillary invasion by level (the three classic levels), perilymphnodal invasion and age. By univariate analysis, overall and disease-free survival decreased significantly as tumour diameter, number of involved lymph nodes, and involvement by axillary level increased. Multivariate analysis assessing the relative importance of these variables when all were considered together found that they were all important independent predictive factors for survival. This study confirms the importance of tumour size and number of metastatic axillary nodes as predictors of outcome in breast cancer. In addition, the level of axillary invasion as a third independent factor of equal importance to the established indicators was identified. When axillary dissection is performed it should be complete, and all three Berg levels tagged separately, so that involvement by level can be ascertained. This provides additional important prognostic information on which to base subsequent treatment decisions.

Adult↗
PubMed1999

Role of brefeldin A-dependent ADP-ribosylation in the control of intracellular membrane transport.

The fungal toxin brefeldin A (BFA) dissociates coat proteins from Golgi membranes, causes the rapid disassembly of the Golgi complex and potently stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa. These proteins have been identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a novel guanine nucleotide binding protein (BARS-50), respectively. The role of ADP-ribosylation in mediating the effects of BFA on the structure and function of the Golgi complex was analyzed by several approaches including the use of selective pharmacological blockers of the reaction and the use of ADP-ribosylated cytosol and/or enriched preparations of the BFA-induced ADP-ribosylation substrates, GAPDH and BARS-50. A series of blockers of the BFA-dependent ADP-ribosylation reaction identified in our laboratory inhibited the effects of BFA on Golgi morphology and, with similar potency, the ADP-ribosylation of BARS-50 and GAPDH. In permeabilized RBL cells, the BFA-dependent disassembly of the Golgi complex required NAD+ and cytosol. Cytosol that had been previously ADP-ribosylated (namely, it contained ADP-ribosylated GAPDH and BARS-50), was instead sufficient to sustain the Golgi disassembly induced by BFA. Taken together, these results indicate that an ADP-ribosylation reaction is part of the mechanism of action of BFA and it might intervene in the control of the structure and function of the Golgi complex.

Adenosine Diphosphate Ribose↗
PubMed1998

Procollagen traverses the Golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation.

Newly synthesized procollagen type I (PC) assembles into 300 nm rigid, rod-like triple helices in the lumen of the endoplasmic reticulum. This oligomeric complex moves to the Golgi and forms large electron-dense aggregates. We have monitored the transport of PC along the secretory pathway. We show that PC moves across the Golgi stacks without ever leaving the lumen of the Golgi cisternae. During transport from the endoplasmic reticulum to the Golgi, PC is found within tubular-saccular structures greater than 300 nm in length. Thus, supermolecular cargoes such as PC do not utilize the conventional vesicle-mediated transport to traverse the Golgi stacks. Our results imply that PC moves in the anterograde direction across the Golgi complex by a process involving progressive maturation of Golgi cisternae.

2,2'-Dipyridyl↗
PubMed1998

Constitutive transport between the trans-Golgi network and the plasma membrane according to the maturation model. A hypothesis.

Here we examine the application of the cisternal/carrier maturation model to describe transport of cargo proteins from the Golgi apparatus to the plasma membrane. Interpretation of the available evidence in the light of carrier maturation suggests that the transport intermediates between these stations are large pleiomorphic carriers formed by maturation of the trans-Golgi compartment, rather than vesicles, as would be postulated by the vesicular shuttle model. Mature carriers move along microtubules towards the plasma membrane via a microtubule/(kinesin)-based motor system. The maturation and vesicular transport models are compared in terms of consistency with the available literature.

Biological Transport↗
PubMed1998

ADP ribosylation factor regulates spectrin binding to the Golgi complex.

Homologues of two major components of the well-characterized erythrocyte plasma-membrane-skeleton, spectrin (a not-yet-cloned isoform, betaI Sigma* spectrin) and ankyrin (AnkG119 and an approximately 195-kDa ankyrin), associate with the Golgi complex. ADP ribosylation factor (ARF) is a small G protein that controls the architecture and dynamics of the Golgi by mechanisms that remain incompletely understood. We find that activated ARF stimulates the in vitro association of betaI Sigma* spectrin with a Golgi fraction, that the Golgi-associated betaI Sigma* spectrin contains epitopes characteristic of the betaI Sigma2 spectrin pleckstrin homology (PH) domain known to bind phosphatidylinositol 4,5-bisphosphate (PtdInsP2), and that ARF recruits betaI Sigma* spectrin by inducing increased PtdInsP2 levels in the Golgi. The stimulation of spectrin binding by ARF is independent of its ability to stimulate phospholipase D or to recruit coat proteins (COP)-I and can be blocked by agents that sequester PtdInsP2. We postulate that a PH domain within betaI Sigma* Golgi spectrin binds PtdInsP2 and acts as a regulated docking site for spectrin on the Golgi. Agents that block the binding of spectrin to the Golgi, either by blocking the PH domain interaction or a constitutive Golgi binding site within spectrin's membrane association domain I, inhibit the transport of vesicular stomatitis virus G protein from endoplasmic reticulum to the medial compartment of the Golgi complex. Collectively, these results suggest that the Golgi-spectrin skeleton plays a central role in regulating the structure and function of this organelle.

ADP-Ribosylation Factors↗