Search PubMed⌕ Search

Biomedical subjects

R Orlando

Publications and source records attributed to R Orlando.

At least 37 records · Page 2Linked to original sources
PubMed2000

A protective role of the low density lipoprotein receptor-related protein against amyloid beta-protein toxicity.

In order to delineate the neuroprotective role of the low density lipoprotein receptor-related protein (LRP) against amyloid beta-protein toxicity, studies were performed in C6 cells challenged with amyloid beta-protein in the presence or absence of activated alpha(2)-macroglobulin. Toxicity was assessed via two cell viability assays. We found that this endocytic receptor conferred protection against amyloid beta-protein toxicity in the presence of activated alpha(2)-macroglobulin and its down-regulation via inhibition by receptor-associated protein or transfection of cells with presenilin 1, increased susceptibility to amyloid beta-protein toxicity. Increased surface LRP immunoreactivity in response to amyloid beta-protein challenge was associated with increased translocation of LRP from the endoplasmic reticulum to the surface, rather than from increased mRNA or protein expression. Furthermore, this translocation of LRP to the surface was mediated by a calcium/calmodulin protein kinase II-dependent signaling pathway. These studies provide evidence for a protective role of LRP against amyloid beta-protein toxicity and may explain the aggressive nature of presenilin-1 mutation in familial Alzheimer's disease.

Alzheimer Disease↗
PubMed2000

Delayed recognition of inadvertent gut injury during laparoscopy.

Bowel injuries, which may occur as a result of the insertion of an insufflation needle or trocar, are a rare complication of laparoscopy. They are generally recognized either immediately or a few days after the operation. We present a case of laparoscopic perforation of the small intestine in a patient who had undergone previous pelvic surgery for an ovarian carcinoma. On ultrasound (US), the patient had multiple hepatic lesions resembling hepatic metastases. To confirm the diagnosis, laparoscopy with guided liver biopsy was performed on the grounds that this procedure is regarded as more appropriate than CT- or US-guided hepatic biopsy. Veress needle and trocar insertion were performed at a proper distance from the abdominal scar. However, the abdominal cavity was not visible after the trocar's insertion due to the unexpected presence of adhesions. This precluded the continuation of the procedure. In the following days, the patient experienced only mild abdominal discomfort. However, 2 weeks after laparoscopy, the patient presented signs of peritoneal reaction and underwent laparotomy. Adhesion-fixing jejunal loops to the anterior abdominal wall were discovered at the site of the trocar puncture. Moreover, two hiatuses of these loops were observed and sutured. The follow-up was uneventful. As this case illustrates, laparoscopic bowel injuries remain an unpredictable event. Recognition of this complication may occur several days after the procedure, as the tamponating effect of adhesions on the jejunal hiatus delays the involvement of the peritoneum.

Female↗
PubMed2000

An endogenous sulfated inhibitor of neuronal inositol trisphosphate receptors.

In cerebellum, inositol trisphosphate- (InsP(3)-) gated Ca channels play a key role in learning, though they exhibit a low sensitivity to InsP(3) compared to peripheral tissues. In the present study, the cerebellar InsP(3) receptor is shown to be associated with a novel inhibitor of InsP(3) binding. (3)H-InsP(3) binding studies indicated that this inositol trisphosphate receptor inhibitor (IRI) could completely inhibit InsP(3) binding to the purified cerebellar InsP(3) receptor and acted as a competitive inhibitor. Gel filtration of IRI showed a predominant peak at 6500 Da, though this peak appeared to be an aggregate (with a monomeric molecular mass of approximately 1500 Da). Mass spectrometry of IRI showed a predominant peak at 1635 m/z, consistent with this low molecular mass estimate. The inhibitory activity of IRI was prevented by pretreatment with aryl sulfatase, suggesting the presence of a critical sulfo ester in IRI. IRI was insensitive to proteases and organic extraction but bound to concanavalin A, suggesting that IRI is a sulfated glycan. IRI was present in cerebellum but below the level of detection in aorta. IRI was also present in the neuronal cell line N1E115 (which exhibits a low sensitivity to InsP(3)). We conclude that IRI is a novel endogenous sulfated inhibitor of the InsP(3) receptor that modulates the sensitivity of the InsP(3) receptor and thus may explain the low InsP(3) sensitivity of neurons.

Animals↗
PubMed2000

Tandem mass spectrometric analysis of aspergillus niger pectin methylesterase: mode of action on fully methyl-esterified oligogalacturonates.

The substrate specificity and the mode of action of Aspergillus niger pectin methylesterase (PME) was determined using both fully methyl-esterified oligogalacturonates with degrees of polymerization (DP) 2-6 and chemically synthesized monomethyl trigalacturonates. The enzymic activity on the different substrates and a preliminary characterization of the reaction products were performed by using high-performance anion-exchange chromatography at neutral pH. Electrospray ionization tandem MS (ESI-MS/MS) was used to localize the methyl esters on the (18)O-labelled reaction products during the course of the enzymic reaction. A. niger PME is able to hydrolyse the methyl esters of fully methyl-esterified oligogalacturonates with DP 2, and preferentially hydrolyses the methyl esters located on the internal galacturonate residues, followed by hydrolysis of the methyl esters towards the reducing end. This PME is unable to hydrolyse the methyl ester of the galacturonate moiety at the non-reducing end.

Aspergillus niger↗
PubMed2000

Differentiating alpha- and beta-aspartic acids by electrospray ionization and low-energy tandem mass spectrometry.

Spectra obtained by low-energy electrospray ionization tandem mass spectrometry (ESI-MS/MS) of 34 peptides containing aspartic acids at position n were studied and unambiguously differentiated. beta-Aspartic acid yields an internal rearrangement similar to that of the C-terminal rearrangements of protonated and cationized peptides. As a result of this rearrangement, two different ions containing the N- and the C-terminal ends of the original peptide are formed, namely, the bn-1 + H2O and y"l - n + 1 - 46 ions, respectively, where e is the number of amino acid residues in the peptide. The structure suggested for the y"l - n + 1 - 46 ion is identical to that proposed for the vn ions observed upon high-energy collision-induced dissociation (CID) experiments. The intensity of these ions in the low-energy MS/MS spectra is greatly influenced by the presence and position of basic amino acids within the sequences. Peptides with a basic amino acid residue at position n - 1 with respect to the beta-aspartic acid yield very intense bn-1 + H2O ions, while the y"l - n + 1 - 46 ion was observed mostly in tryptic peptides. Comparison between the high- and low-energy MS/MS spectra of several isopeptides suggests that a metastable fragmentation process is the main contributor to this rearrangement, whereas for long peptides (40 AA) CID plays a more important role. We also found that alpha-aspartic acid containing peptides yield the normal immonium ion at 88 Da, while peptides containing beta-aspartic acid yield an ion at m/z 70, and a mechanism to explain this phenomenon is proposed. Derivatizing isopeptides to form quaternary amines, and performing MS/MS on the sodium adducts of isopeptides, both improve the relative intensity of the bn + 1 + H2O ions. Based on the above findings, it was possible to determine the isomerization sites of two aged recombinant growth proteins.

Amino Acid Sequence↗
PubMed2000

Renal clearance of N(1)-methylnicotinamide: a sensitive marker of the severity of liver dysfunction in cirrhosis.

BACKGROUND/AIMS: Data have appeared suggesting that an impairment of renal tubular secretion is present in liver cirrhosis, even in the absence of a clinically evident renal dysfunction. To address this question, we evaluated the renal clearance of N(1)-methylnicotinamide (NMN), a marker of the renal secretory function, in healthy subjects and patients with liver cirrhosis of increasing severity, but with a normal glomerular filtration rate. METHODS: The renal clearances of endogenous NMN, inulin, and creatinine were measured in 14 normal subjects and in two groups of age-matched cirrhotic patients (10 Child A and 10 Child C). In 6 subjects, 2 per group, the concentration dependence of the NMN clearance was also studied, following an oral nicotinamide load. RESULTS: Contrary to expectations, the renal NMN clearance increased in cirrhotic patients, in relation to the severity of liver disease (r = 0.83 with Pugh's score; p < 0.001). The NMN-to-inulin clearance ratio increased from a control value of 2.2 +/- (SD) 0.4 to 3.1 +/- 0.2 and 5.2 +/- 0.9 in Child A and Child C cirrhotics, respectively (p < 0.001 for all comparisons), indicating that NMN was completely cleared from plasma in the latter patients. Consistently, the analysis of the concentration dependence of the renal NMN clearance revealed the presence of a carrier-mediated reabsorption which apparently was no longer operating in the decompensated patients. Discriminant analysis showed that renal NMN clearance, and NMN-to-creatinine and NMN-to-inulin clearance ratios could all distinguish between the three study groups, with sensitivities and specificities equal or greater than 90%. CONCLUSIONS: Contrary to previous proposals, NMN is not a probe of general validity for renal tubular secretion. In particular, due to an imbalance between secretion and reabsorption, its renal clearance in liver cirrhosis cannot be used to determine the degree of tubular secretion of which an individual is capable. However, renal NMN clearance appears to be a very sensitive marker of the severity of liver dysfunction in cirrhosis. The potentialities of this renal parameter as a diagnostic and prognostic test in liver cirrhosis deserve further study.

Adult↗
PubMed2000

Myocardial ischemia caused by an hydatid cyst of the interventricular septum successfully treated with albendazole.

We report the case of a 33-year-old patient with clinical history of echinococcosis admitted to our Hospital for the appearance of chest pain and electrocardiographic findings of anterior ischemia. The cardiac enzymogram was in the normal range, the chest roentgengram did not show any pathological findings, but two-dimensional echocardiography revealed the presence of a small circular area in the interventricular septum. Transesophageal echocardiography and cardiac nuclear magnetic resonance confirmed the presence of a small hydatid cyst in the middle ventricular septum; in addition, a myocardial scintigraphy revealed an apical stress defect with late reperfusion. Besides cardiologic therapy, the patient was treated with albendazole, an antiparasitic drug, 400 mg bid, for cycles of 28 days with 14 day withdrawal. After two cycles of albendazole therapy, two-dimensional echocardiography showed the absence of the round cystic mass of the interventricular septum previously described. In conclusion, in the case described, long-term therapy with albendazole determined the complete recovery from the illness with the simultaneous disappearance of the cyst and of clinical and electrocardiographic findings of myocardial ischemia.

Adult↗
PubMed2000

Are liver cirrhosis and portal hypertension associated with an increased risk of bleeding during laparoscopy? A retrospective analysis of 1,000 consecutive cases.

Laparoscopy is a relatively safe invasive procedure. However, the rate of bleeding complications during this procedure is still debatable. Moreover, it is not clear whether portal hypertension may increase the risk of this event. The authors analyzed retrospectively the records of 1,000 consecutive patients with chronic liver disease undergoing laparoscopy and guided direct-vision hepatic biopsy, and they examined the rate of bleeding complications from the trocar site after insertion of the Veress needle or after liver biopsy. A total of 400 of 1,000 patients had liver cirrhosis. Of these, 22.7% had splenomegaly, 13.0% had laparoscopic signs of portal hypertension, and 8.2% had esophageal varices. Bleeding occurred in 0.9% of patients from the trocar site, in 0.2% from the biopsy site, and in 0% from the Veress needle site. These figures were independent of the presence of advanced liver disease, with or without portal hypertension. Bleeding complications occur rarely during laparoscopy with guided liver biopsy and do not seem to be related per se to the presence of liver cirrhosis and portal hypertension. If anything, bleeding complications represent an unpredictable event in most cases.

Adolescent↗
PubMed1999

Solid-phase extraction/MALDI-MS: extended ion-pairing surfaces for the on-target cleanup of protein samples.

The surface of a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) target can be covalently modified so that it behaves like a medium for solid-phase extraction (SPE). These modified targets are capable of binding molecules of interest, but not contaminants, from sample solutions placed on them. This allows the analyte to be cleaned up on the probe surface by simply washing the target to remove the contaminants prior to MALDI-MS analysis. A limitation of the current SPE/MALDI-MS targets is that they have a fairly low binding capacity, since the coating on these targets is based upon self-assembled monolayers (SAMs). To overcome this limitation, we have investigated new surface coatings for SPE/MALDI-MS that will have a higher binding capacity than targets modified with SAMs. Here, we describe the development of new SPE/MALDI-MS surfaces that have very high molecular weight (> 300,000) polylysine chains attached to them. Targets modified in this manner are capable of binding peptides/proteins by ion-pairing interactions and have approximately 100 times the binding capacity of the SAM-based targets. Furthermore, these polylysine targets can capture over 60% of a protein from a highly contaminated solution. Consequently, polylysine SPE/MALDI-MS targets offer a practical solution for analyzing very small volumes (< 1 microL) of peptide/protein solutions contaminated with high levels of inorganic salts, buffers, detergents, chaotropic agents, and other solubilizing agents.

Binding Sites↗
PubMed1999

On-target exoglycosidase digestions/MALDI-MS for determining the primary structures of carbohydrate chains.

One method used to determine the primary sequence of oligosaccharides is to digest them with exoglycosidases and analyze the resulting digestion products by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Previous research has demonstrated that these digestions can be performed on the MALDI target. However, the procedure requires the sample to be incubated at elevated temperatures, and complete digestion requires a few hours. We demonstrate new conditions that permit exoglycosidase digestions to be performed on the MALDI target at room temperature within 30 min. Oligosaccharide standards were digested with one or more exoglycosidases to show that the enzymes retain their activity and specificity under these new reaction conditions. Using this method, the primary sequences of carbohydrate chains can be determined in a relatively short amount of time.

Carbohydrate Sequence↗
PubMed1999

Safety and immunogenicity of hepatitis A vaccine in infants: a candidate for inclusion in the childhood vaccination programme.

Forty-eight infants received a single dose (720 ELISA units = 0.5 ml) of inactivated hepatitis A vaccine at the fifth month of age with booster at the 11th month of age, together with the second and third doses of the vaccines compulsory under Italian law (diphtheria, tetanus, oral polio and hepatitis B). Overall, the seroconversion rate was 100%. The anti-HAV geometric mean titre (GMT) reached 3,021 mIU/ml in infants born to anti-HAV-negative mothers, but only 399 mIU/ml in infants born to anti-HAV-positive mothers. Hepatitis A vaccine was immunogenic, safe and well tolerated without significant side-effects. There seems to be no reason for not including it in childhood vaccination programmes particularly in low endemic HAV areas.

Enzyme-Linked Immunosorbent Assay↗
PubMed1999

A new liquid chromatography/tandem mass spectrometric approach for the identification of class I major histocompatibility complex associated peptides that eliminates the need for bioassays.

Cell-surface class I major histocompatibility complex (MHC) molecules present processed self- and nonself-peptides to thymus-derived (T) lymphocytes, allowing the intracellular compartment of cells to be sampled in order to detect infection. Since the class I MHC-peptide complex plays a critical role in cell-mediated immunity, it is important to obtain sequence information on the MHC-associated peptides unique to infected cells as a prelude to the development of vaccines. Here, we outline and test an alternative strategy for identifying the proteins that are processed through the MHC pathway. This new strategy eliminates the necessity of developing and maintaining cytotoxic T lymphocyte (CTL) lines for peptide identification. In this new approach genome sequences from the infecting agent are scanned for stretches of amino acids that match a particular MHC binding motif. Molecular masses from these putative MHC-binding peptide sequences are calculated and compared to those found for peptides isolated from pathogen-infected host cells using liquid chromatography/mass spectrometry (LC/MS). Peptides with masses matching those in the database are then analyzed by tandem mass spectrometry (MS/MS) to determine their identity. Using this approach we were able to confirm the processing and presentation of two Trypanosoma cruzi proteins by the MHC class I pathway. These data suggest that a rigorous approach employing two-dimensional separations in conjunction with MS/MS and bioinformatics is a feasible means of identifying pathogen gene products of immunological interest when a CTL assay is unavailable or unsuccessful.

Animals↗
PubMed1999

Characterization of the glycosylation of recombinant endopolygalacturonase I from Aspergillus niger.

The carbohydrate chains of recombinant endopolygalacturonase I (EPG I) from Aspergillus niger were characterized using a combination of mass spectrometric techniques. High performance liquid chromatography (HPLC) in conjunction with electrospray ionization mass spectrometry was used to separate the components of EPG I liberated by trypsin digestion. In-source collision-induced dissociation (CID) was utilized to fragment the digestion products entering the mass spectrometer, and the generation of carbohydrate fragment ions allowed for the identification of glycopeptides. The masses of the resulting glycans were calculated and entered into a carbohydrate database to search for possible structures. The primary sequences of the carbohydrate chains were confirmed by digesting aliquots of the intact glycopeptide with endo- and exoglycosidases and then analyzing the digestion products using matrix-assisted laser desorption/ionization mass spectrometry. These experiments demonstrated that one of the two N-linked sites of EPG I was occupied by a series of high-mannose structures, the second N-linked site was not occupied, and no O-linked sites were detected.

Amino Acid Sequence↗
PubMed1999

Characterization of the N-linked glycosylation site of recombinant pectate lyase.

Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for potential N-linked glycosylation sites, and one potential site was identified at residue 64. The proteins were analyzed for their ability to bind various lectins as an assay for the presence of carbohydrates. The proteins were then digested with trypsin to facilitate the isolation of the potential glycosylation site. The resulting digestion products were subsequently analyzed by liquid chromatography/mass spectrometry using in-source collision induced dissociation to detect glycopeptides. Once the glycopeptide had been identified, treatment with an endoglycosidase both verified the location of glycosylation and identified the mass of the glycan. The Complex Carbohydrate Structural Database was searched for possible N-linked structures with the same mass, and the suggested primary sequence was confirmed by an exoglycosidase digestion. The data demonstrated that the larger recombinant protein contained a high mannose N-linked structure (Man(5)GlcNAc(2)) attached to N-64, while this site was not occupied in the smaller protein.

Aspergillus nidulans↗
PubMed1999

Aberrant presenilin-1 expression downregulates LDL receptor-related protein (LRP): is LRP central to Alzheimer's disease pathogenesis?

Low density lipoprotein receptor-related protein (LRP) polymorphisms have recently been associated with an increased susceptibility of Alzheimer's disease (AD). Furthermore, LRP has been linked to molecules that confer susceptibility to AD (apolipoprotein E, alpha-2-macroglobulin, amyloid precursor protein), previously with the exception of the presenilins. Here we report that aberrant presenilin-1 expression in vivo and in vitro downregulates LRP. Specifically, transgenic mice overexpressing the M146L or L286V presenilin-1 mutation show decreased levels of LRP expression in neuronal populations where presenilin-1 and LRP are closely colocalized or coexpressed. Moreover, cell lines transfected with presenilin-1 also expressed decreased levels of LRP. These findings suggest that LRP may be central to AD pathogenesis since all proteins genetically associated with AD can now be linked via a single pathway to LRP.

Alzheimer Disease↗