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Biomedical subjects

M Lorenzi

Publications and source records attributed to M Lorenzi.

At least 19 recordsLinked to original sources
PubMed2001

Phytotoxic protein PcF, purification, characterization, and cDNA sequencing of a novel hydroxyproline-containing factor secreted by the strawberry pathogen Phytophthora cactorum.

A novel protein factor, named PcF, has been isolated from the culture filtrate of Phytophthora cactorum strain P381 using a highly sensitive leaf necrosis bioassay with tomato seedlings. Isolated PcF protein alone induced leaf necrosis on its host strawberry plant. The primary structure and cDNA sequence of this novel phytotoxic protein was determined, and BLAST searches of Swiss-Prot, EMBL, and GenBank(TM)/EBI data banks showed that PcF shared no significant homology with other known sequences. The 52-residue PcF protein, which contains a 4-hydroxyproline residue along with three S-S bridges, exhibits a high content of acidic sidechains, accounting for its isoelectric point of 4.4. The molecular mass of isolated PcF is 5,622 +/- 0.5 Da as determined by mass spectrometry and matches that calculated from the deduced amino acid sequence with cDNA sequencing. The cDNA sequence indicates that PcF is first produced as a larger precursor, comprising an additional N-terminal, 21-residue secretory signal peptide. Maturation of this protein involves the hydroxylation of proline 49, a feature that is unique among other known secreted fungal phytopathogenic proteins.

Amino Acid Sequence↗
PubMed2001

Early cellular and molecular changes induced by diabetes in the retina.

For several decades the pathobiology of diabetic retinopathy has been the object of conjecture and hypotheses. Indeed, very little was known about the cellular events triggered by diabetes in the retina and about the processes underlying the microangiopathy. In the last few years there has been a concerted effort to acquire such information, and the work has targeted not just the retinal vessels but more comprehensively the retina. The picture emerging is one of multiplicity: multiple cell types in the retina are affected early by diabetes and multiple processes are operative in the microangiopathy. The main abnormalities captured to date are altered expression of several genes, apoptosis, microthrombosis, and proinflammatory changes. The new information needs to be integrated into temporal and mechanistic sequences and further expanded but it is beginning to provide a framework for the cellular dimension of diabetic retinopathy.

Animals↗
PubMed2001

IGF-I mRNA and signaling in the diabetic retina.

IGF-I promotes the survival of multiple cell types by activating the IGF-I receptor (IGF-IR), which signals downstream to a serine/threonine kinase termed Akt. Because in diabetes vascular and neural cells of the retina undergo accelerated apoptosis, we examined IGF-I synthesis and signaling in the human and rat diabetic retina. In retinas obtained postmortem from six donors aged 64 +/- 8 years with a diabetes duration of 7 +/- 5 years, IGF-I mRNA levels were threefold lower than in the retinas of six age-matched nondiabetic donors (P = 0.005). In the retinas of rats with 2 months' duration of streptozotocin-induced diabetes, IGF-I mRNA levels were similar to those of control rats, but after 5 months of diabetes they failed to increase to the levels recorded in age-matched controls (P < 0.02). Retinal IGF-I expression was not altered by hypophysectomy, proving to be growth-hormone independent. IGF-IR levels were modestly increased in the human diabetic retinas (P = 0.02 vs. nondiabetic retinas) and were unchanged in the diabetic rats. Phosphorylation of the IGF-IR could be measured only in the rat retina, and was not decreased in the diabetic rats (94 +/- 18% of control values). In the same diabetic rats, phosphorylation of Akt was 123 +/- 21% of control values. There was not yet evidence of increased apoptosis of retinal microvascular cells after 5 months of streptozotocin-induced diabetes. Hence, in the retina of diabetic rats, as in the retina of diabetic human donors, IGF-I mRNA levels are substantially lower than in age-matched nondiabetic controls, whereas IGF-IR activation and signaling are not affected, at least for some time. This finding suggests that in the diabetic retina, the activation of the IGF-IR is modulated by influences that compensate for, or are compensated by, decreased IGF-I synthesis.

Aged↗
PubMed2001

Increased prevalence of microthromboses in retinal capillaries of diabetic individuals.

In diabetic retinopathy, capillary nonperfusion and eventual obliteration can lead to retinal ischemia and sight-threatening neovascularization. The occurrence of retinal microthrombosis in human diabetes has long been suspected and occasionally observed but never systematically demonstrated. We used trypsin digestion to isolate the intact vascular network from retinas obtained postmortem from nine diabetic donors (age 64 +/- 11 years, duration of diabetes 6 +/- 4 years; mean +/- SD) and eight age-matched nondiabetic donors. Topographically matched sectors (each one-sixth of retina) of diabetic and nondiabetic retinas were tested sequentially with antibodies to fibrin cross-linking factor XIII and platelet glycoprotein (GP)-IIIa to identify fibrin-platelet thrombi. In some trypsin digests, we also examined vascular cell apoptosis. The retina from a nondiabetic donor, 24 years of age, who had died of trauma, was used to exclude confounding influences caused by the postmortem period. When compared with those of nondiabetic donors, the retinas of diabetic donors showed double the number of capillary segments with colocalized immunostaining for factor XIII and GPIIIa (P = 0.02). The total area of the positive segments was fourfold greater in the diabetic than in the nondiabetic donors (P = 0.02) and correlated with the duration of diabetes (r = 0.71, P < 0.05). Large thrombi were six times more frequent in the diabetic donors (P = 0.03), and there was a significant topographical association of microthrombosis with apoptotic cells in both diabetic and nondiabetic vessels (P = 0.0001). Hence, diabetes of short duration was found to be associated with a greater than normal number and size of platelet-fibrin thrombi in the retinal capillaries. These thrombi can contribute to capillary obliteration and retinal ischemia and may be a practical target for early drug intervention.

Adult↗
PubMed2001

The surgical treatment of pancreatic pseudocysts. Personal experience.

BACKGROUND: This study is aimed at contributing to defining a correct therapeutic management of pancreatic pseudocysts (PPCs): indications for treatment, operative timing and technical approach. METHODS: A retrospective analysis of 28 patients affected by PPC, 22 males (78.5%) and 6 females (21.5%), with a mean age of 52 years (range 17-76) has been performed. The diagnosis was realised by clinical assessment and US (ultrasonography) or CT (computerized tomography) scanning. The treatment consisted in surgical drainage (internal or external) or percutaneous drainage with US guidance: the cystojejunostomy with a Roux-en-Y loop was the first choice technique. RESULTS: Twenty-two patients (78.5%) under-went a surgical procedure: 19 cystojejunostomies with a Roux-en-Y loop and 3 external drainages. The mean interval between acute pancreatic event and elective surgery was 9 weeks (range 5-21). Perioperative morbidity and mortality was respectively 22.7% (5/22) and 13.6% (3/22). In 4 cases a percutaneous drainage with US-guidance, without morbidity and mortality was performed, but 2 patients required a successive surgical operation for lack of resolution of the PPC. The last 2 patients of this series recovered spontaneously. CONCLUSIONS: Currently the cystojejunostomy with a Roux-en-Y loop remains the first choice technique for an elective and definitive treatment of PPCs: other techniques (endoscopic internal drainage, surgical or percutaneous external drainage) should be limited to complicated PPCs or to high surgical risk patients. A waiting period of 4-6 weeks following the acute pancreatic event is considered the minimal time necessary before the elective treatment.

Adolescent↗
PubMed2001

Successful surgical repair of a ruptured abdominal aortic aneurysm in a nonagenarian.

Mortality due to the surgical treatment of ruptured abdominal aortic aneurysms (RAAA) is high, more than 40%, especially in elderly patients. The literature concerning RAAA in very elderly patients is analyzed by Internet research (Medline). Only rare examples of emergency surgical management in nonagenarian patients are reported, and even fewer reports of successfully operated patients. A case report of a successful surgical repair of RAAA in a nonagenarian is presented. After ultrasound (US) and CT scans, the patient (in good condition as regards blood pressure, respiratory, cardiac and renal function) underwent uneventful aneurysmectomy and reconstruction of the aorta and common iliac arteries by means of a bifurcated prosthesis. The length of hospitalization was 30 days and the patient is still alive and in good condition four years and two months after the operation. The advisability of emergency surgical repair in these patients, questionable because of the excessive surgical risk and poor survival benefit, is discussed. However many other factors affect the outcome of emergency repair (mainly cardiac, respiratory and renal function), independently of age. The authors conclude that age per se does not limit the indication for or success of surgery in very elderly patients.

Age Factors↗
PubMed2000

Bax is increased in the retina of diabetic subjects and is associated with pericyte apoptosis in vivo and in vitro.

Diabetes of even short duration accelerates the death of capillary cells and neurons in the inner retina by a process consistent with apoptosis. We examined whether the process is accompanied by changes in the expression of endogenous regulators of apoptosis. In postmortem retinas of 18 diabetic donors (age 67 +/- 6 years, diabetes duration 9 +/- 4 years) the levels of pro-apoptotic Bax were slightly, but significantly, increased when compared with levels in 20 age-matched nondiabetic donors (P = 0.04). In both groups, Bax localized to vascular and neural cells of the inner retina. Neither pro-apoptotic Bcl-X(S), nor pro-survival Bcl-X(L) appeared affected by diabetes. The levels of these molecules could not be accurately quantitated in lysates of retinal vessels because of variable degrees of glial contamination. However, studies in situ showed in several pericytes, the outer cells of retinal capillaries, intense Bax staining often in conjunction with DNA fragmentation. Bovine retinal pericytes exposed in vitro to high glucose levels for 5 weeks showed elevated levels of Bax (P = 0.03) and increased frequency of annexin V binding, indicative of early apoptosis. Hence, human diabetes selectively alters the expression of Bax in the retina and retinal vascular pericytes at the same time as it causes increased rates of apoptosis. The identical program induced by high glucose in vitro implicates hyperglycemia as a causative factor in vivo, and provides a model for establishing the role of Bax in the accelerated death of retinal cells induced by diabetes.

Aged↗
PubMed2000

[Severe acute pancreatitis. Clinical, diagnostic, and therapeutic features].

BACKGROUND: The aim of this study is to define the actual role of surgical therapy in severe acute necrotizing pancreatitis. METHODS: A retrospective analysis has been carried out on the surgical treatment of severe acute pancreatitis at the Institute of General Surgery and Surgical Specialties, University of Siena (Italy). From January 1980 to December 1997, 230 patients affected by acute pancreatitis were admitted to institution: 24 patients affected by severe disease (necrotizing pancreatitis: clinical and radiological diagnosis, by CT-scan) was choosen for this study. Of 24 patients, 15 were males and 9 females, with mean age of 55 years (range 30-80). In all cases, surgical procedure consisted in pancreatic necrosectomy, multiple abdominal and retroperitoneal drainage and closed management; operated patients with biliary pancreatitis underwent colecystectomy and, if necessary, common biliary duct drainage. RESULTS: The patients underwent surgical procedure, but the remaining 14 were treated by intensive medical care: mortality in these two groups was respectively 40% (4 cases) and 21% (3 cases). CONCLUSION: The conclusion is drawn that intensive medical care is the first therapeutic approach in severe acute pancreatitis, reserving surgery only to selected cases, as those affected by pancreatic infectes necrosis or those who get worse despite of conservative therapy. As to surgical technique, closed procedures vs open or semiopen, and conservative surgery (necrosectomy, multiple drainage and abdominal washing) vs anatomical resection are preferred.

Adult↗
PubMed2000

Response of capillary cell death to aminoguanidine predicts the development of retinopathy: comparison of diabetes and galactosemia.

PURPOSE: To examine the relationship between early retinal capillary cell apoptosis and late histologic lesions of diabetic retinopathy and to compare the effects of aminoguanidine (AMG) on the retinopathies caused by diabetes and galactose feeding. METHODS: Rats with alloxan-induced diabetes and rats fed a 30% galactose diet (known to induce diabetic-like retinopathy) were assigned randomly to receive diet with (2.5 g/kg diet) or without AMG. After 6 to 8 months of diabetes or galactosemia, retinal trypsin digests were prepared, and capillary cell apoptosis was quantitated using the Tdt-mediated dUTP nick-end labeling (TUNEL) reaction in association with morphologic evidence of nuclear fragmentation. At 18 months duration, pericyte ghosts and acellular capillaries were quantitated in the isolated vasculature. Several advanced glycation end products (AGEs) were measured at 4 months of study and at 18 months of study by established methods to assess biochemical effects of AMG. RESULTS: As expected, both diabetic and galactosemic rats showed increased frequency of TUNEL-positive capillary cells at 6 to 8 months and vascular lesions characteristic of retinopathy at 18 months. AMG inhibited both the early apoptosis and late histopathology in the diabetic rats, but neither of these abnormalities in the galactosemic rats. In contrast to its preventative effect on retinopathy in the diabetic rats, AMG showed no inhibitory effect on levels of hemoglobin AGE, or tail collagen pentosidine, fluorescence, and thermal breaking time. Diabetes of 4 months' duration did not cause a detectable increase in retinal levels of several AGEs. CONCLUSIONS: The frequency of early apoptosis in retinal microvascular cells predicted the development of the histologic lesions of retinopathy in diabetes as well as in galactosemia. The beneficial effect of AMG on retinal lesions in diabetes is exerted on pathways that are either not operative or are less important in galactosemia and that may not relate to the accumulation of AGEs.

Animals↗
PubMed1999

Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.

Amino Acid Sequence↗
PubMed1999

Adult colonic intussusception caused by malignant tumor of the transverse colon.

Adult colonic intussusception is rare and often originates from neoplasms. In emergency situations it can be difficult to diagnose. Our aim was to show how the integration of readily available diagnostic means in emergency situations can help in making a correct diagnosis of this disease. A 68-year old male patient presented with vomiting and abdominal pain. The abdomen was distended, with pain to palpation in the left quadrants without a mass. Plain radiographs of the abdomen showed a large amount of gas in the small bowel and in the right and transverse colon. A barium enema demonstrated an endoluminal filling defect in the descending colon. Abdominal ultrasonography revealed the presence of intraperitoneal fluid and thickened left colonic wall at the site of the lesion, with an aspect of a "double ring" consistent with intussusception. A solid formation was also revealed at a point distal to the thickened colonic wall. At emergency laparotomy an approximately 8-cm-long mass was palpable through the left colon. A colostomy was fashioned, and subsequently colonoscopy revealed the presence of a left colon tumor. At the subsequent operation an invagination of the left transverse colon into the descending colon was confirmed. The left transverse and descending colon were resected with high ligation of the left colic artery. Macroscopic examination of the invaginating head showed a vegetating transverse colon neoplasm. We conclude that in emergency settings the association of readily available diagnostic means such as plain abdominal radiography, water soluble contrast enema and ultrasonography may yield reliable information for diagnosing colonic intussusception.

Adenocarcinoma, Mucinous↗
PubMed1998

Müller cell changes in human diabetic retinopathy.

Vascular cells may not be the only cells affected by diabetes in the retina. In particular, abnormalities of the b-wave of the electroretinogram in diabetic patients with absent or minimal microangiopathy have pointed to possible dysfunction of Müller cells, the principal glia of the retina. In this study, we sought evidence for diabetes-induced Müller cell abnormalities by testing the expression of three proteins (Bcl-2, glutamine synthetase [GS], and glial fibrillar acidic protein [GFAP]) that are solely or predominantly expressed in Müller cells and show a reproducible pattern of changes in the context of retinal injuries or degenerations. Retinas obtained postmortem from a total of 14 donors aged 65 +/- 6 years with 10 +/- 4 years of diabetes and histological evidence of microangiopathy and 18 age-matched nondiabetic donors were examined by immunohistochemistry and immunoblotting. The typical Müller cell pattern of Bcl-2 and GS immunostaining was similar for both intensity and distribution in the nondiabetic and diabetic retinas, as were the levels of the two proteins. In contrast, GFAP staining, largely confined to the most proximal retina in the nondiabetic donors, was in most diabetic retinas present along the entire length of the Müller cell processes, throughout the outer retina. Accordingly, the level of GFAP was increased in the diabetic retinas (161 +/- 106 densitometric units/microg protein vs. 55 +/- 45 in the nondiabetic retinas, P = 0.03). These data provide evidence for selective biosynthetic changes of Müller glial cells in diabetes. Because Müller cells produce factors capable of modulating blood flow, vascular permeability, and cell survival, and their processes surround all blood vessels in the retina, a possible role of these cells in the pathogenesis of retinal microangiopathy deserves to be investigated.

Aged↗
PubMed1998

Expression of vascular endothelial growth factor in the human retina and in nonproliferative diabetic retinopathy.

Vascular endothelial growth factor (VEGF)/vascular permeability factor is a likely angiogenic mediator in proliferative diabetic retinopathy, and its role is under scrutiny in the pathogenesis of the capillary leakage characteristic of background diabetic retinopathy. To examine whether the diabetic milieu induces or increases retinal VEGF expression in humans, we examined retinas from nondiabetic eye donors and donors with 9 +/- 5 years of diabetes and documented microangiopathy. To identify possible confounding effects of the postmortem period, we also studied the postmortem stability of the VEGF transcript and the expression of the VEGF protein in rat retinas. In both human and rat retina we detected by Northern analysis a 4.2-kb VEGF mRNA species and by reverse transcriptase polymerase chain reaction the transcripts encoding VEGF165 (the most abundant), VEGF121, and VEGF189. By in situ hybridization and immunohistochemistry VEGF mRNA and protein co-localized at the ganglion cell, inner nuclear, and outer plexiform layers and in the walls of the blood vessels (where mRNA was scarce). The protein was additionally detected in photoreceptors. The abundance and distribution of VEGF mRNA and protein were not altered in the diabetic retinas, indicating that the diabetic environment is not sufficient to increase retinal VEGF expression. The demonstration that VEGF is constitutively expressed in the adult retina and is localized to discrete neural cells and their processes proposes a role for the cytokine in retinal homeostasis and/or function.

Aged↗
PubMed1997

Cytoskeletal changes induced by excess extracellular matrix impair endothelial cell replication.

Thickening of basement membranes is an early and characteristic feature of diabetic vessels, but its consequences on the properties of vascular cells remain undefined. We investigated whether and how excess extracellular matrix (ECM) alters the replication of vascular endothelial cells in vitro. To test the effects of endogenous excess matrix, human umbilical vein endothelial cells (HUVEC) were plated on ECM produced under culture conditions (high ambient glucose) that increase ECM synthesis. Four of six HUVEC isolates plated on such ECM yielded a lower cell number (68 +/- 18%) than cells plated on control ECM. Growth inhibition was observed in HUVEC cultured on elevated concentrations (10 and 50 microg/ml) of exogenous fibronectin, when compared with HUVEC plated on tissue culture plastic or 0.25, 1.0, and 5.0 microg/ml fibronectin; the decreased replication was attributable to delayed transit through the G1 phase of the cell cycle. HUVEC grown on both 1 and 10 microg/ml fibronectin exhibited a modest upregulation of the fibronectin-specific integrin receptor alpha5beta1, and increased attachment to fibronectin substratum. However, unique to the HUVEC plated on growth-inhibitory concentrations of fibronectin was a redistribution in situ of integrins and vinculin to form more numerous focal adhesions, and an increased polymerization of cytoskeletal actin to form stress fibers. Concentrations (0.01 microg/ml) of cytochalasin D intended to prevent excess actin polymerization prevented the growth inhibition. Thus, excess ECM hampers endothelial cell replication in vitro through increased cell-ECM adhesion and attendant cytoskeletal rearrangements. These phenotypic changes provide probes to test whether cell-ECM interactions are altered in diabetic vessels in a direction that may compromise orderly endothelial cell renewal and its antithrombogenic function.

Actins↗
PubMed1997

Mesangial cell abnormalities in spontaneously hypertensive rats before the onset of hypertension.

To identify kidney biosynthetic abnormalities that may precede the onset of hypertension, we studied the expression of fibronectin (FN) and collagen IV (Coll IV) in young SHR (4 weeks of age) whose systolic blood pressure was normal and similar to that of age-matched control WKY rats. In isolated glomeruli the level of FN protein assessed by immunoblotting tended to be lower in the SHR than in the WKY rats. By Northern analysis the FN/actin mRNA ratio was significantly lower in glomeruli from SHR (0.56 +/- 0.47) than in glomeruli from WKY rats (2.0 +/- 0.8). These abnormalities were maintained in vitro since the expression of FN was significantly lower in SHR than in WKY cultured mesangial cells (FN/actin mRNA ratio = 0.84 +/- 0.46 vs. 1.9 +/- 0.7, P = 0.029). No differences in Coll IV mRNA or protein levels were observed in SHR glomeruli and mesangial cells when compared with WKY rats. The levels of aortic FN and Coll IV mRNAs were not different in SHR and WKY rats. In addition, mesangial cells from SHR showed a significantly higher growth rate than those from WKY. The biosynthetic and proliferative abnormalities observed in the SHR mesangial cells appear to reflect genetic characteristics, and could provide novel insights into cellular mechanisms linking the genetics of hypertension with predisposition to glomerular pathology.

Actins↗
PubMed1997

Visual and verbal short-term memory deficits in childhood leukemia survivors after intrathecal chemotherapy.

Assessed survivors of childhood lymphoblastic leukemia (ALL) treated with intrathecal chemotherapy, using the Wide Range Assessment of Memory and Learning (WRAML), compared to controls without cancer, matched as closely as possible in age, SES, and gender. Mild, but consistent, deficits were found in both visual-spatial and verbal single-trial memory tasks. In multitrial learning, only visual-spatial tasks resulted in deficient scores, while verbal learning was within the normal range. IQ results indicated scores 10-20 points lower in the ALL group. Memory results are related to deficits in strategic planning and attentional distractiveness. The WRAML may be a useful clinical tool to evaluate differential memory deficits in children with ALL.

Adolescent↗
PubMed1996

Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy.

To reconstruct the mechanisms for the vasoobliteration that transforms diabetic retinopathy into an ischemic retinopathy, we compared the occurrence of cell death in situ in retinal microvessels of diabetic and nondiabetic individuals. Trypsin digests and sections prepared from the retinas of seven patients (age 67 +/- 7 yr) with .9 +/- 4 yr of diabetes and eight age- and sex-matched nondiabetic controls were studied with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction which detects preferentially apoptotic DNA fragmentation. The count of total TUNEL+ nuclei was significantly greater in the microvessels of diabetic (13 +/- 12 per one-sixth of retina) than control subjects (1.3 +/- 1.4, P = 0.0016), as were the counts of TUNEL+ pericytes and endothelial cells (P < 0.006). The neural retinas from both diabetic and nondiabetic subjects were uniformly TUNEL-. Retinal microvessels of rats with short duration of experimental diabetes or galactosemia and absent or minimal morphological changes of retinopathy, showed TUNEL+ pericytes and endothelial cells, which were absent in control rats. These findings indicate that (a) diabetes and galactosemia lead to accelerated death in situ of both retinal pericytes and endothelial cells; (b) the event is specific for vascular cells; (c) it precedes histological evidence of retinopathy; and (d) it can be induced by isolated hyperhexosemia. A cycle of accelerated death and renewal of endothelial cells may contribute to vascular architectural changes and, upon exhaustion of replicative life span, to capillary obliteration.

Aged↗