Biomedical subjects

P Botti

Publications and source records attributed to P Botti.

At least 19 recordsLinked to original sources

PubMed2003

Synthesis of cyclic peptides from unprotected precursors using removable N alpha-(1-(4-methoxyphenyl)-2-mercaptoethyl) auxiliary.

A new method to cyclize unprotected peptides is presented. The method involves the use of a 1-phenyl-2-mercaptoethyl derivative on the N-terminal glycine. This template acts as an auxiliary thiol-containing group in order to drive cyclization with a counterpart thioester moiety on the same molecule. Subsequent facile removal of the derivative generates products with only native peptide structure. The successful, high-yield cyclization of the peptide GSPYSSDTTPA is described.

Chromatography, High Pressure Liquid↗

PubMed2001

Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary.

We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet(7)]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the alpha-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet(7)]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by approximately 45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.

Amino Acid Sequence↗

PubMed2001

Computationally accessible method for estimating free energy changes resulting from site-specific mutations of biomolecules: systematic model building and structural/hydropathic analysis of deoxy and oxy hemoglobins.

A practical computational method for the molecular modeling of free-energy changes associated with protein mutations is reported. The de novo generation, optimization, and thermodynamic analysis of a wide variety of deoxy and oxy hemoglobin mutants are described in detail. Hemoglobin is shown to be an ideal candidate protein for study because both the native deoxy and oxy states have been crystallographically determined, and a large and diverse population of its mutants has been thermodynamically characterized. Noncovalent interactions for all computationally generated hemoglobin mutants are quantitatively examined with the molecular modeling program HINT (Hydropathic INTeractions). HINT scores all biomolecular noncovalent interactions, including hydrogen bonding, acid-base, hydrophobic-hydrophobic, acid-acid, base-base, and hydrophobic-polar, to generate dimer-dimer interface "scores" that are translated into free-energy estimates. Analysis of 23 hemoglobin mutants, in both deoxy and oxy states, indicates that the effects of mutant residues on structurally bound waters (and visa versa) are important for generating accurate free-energy estimates. For several mutants, the addition/elimination of structural waters is key to understanding the thermodynamic consequences of residue mutation. Good agreement is found between calculated and experimental data for deoxy hemoglobin mutants (r = 0.79, slope = 0.78, standard error = 1.4 kcal mol(-1), n = 23). Less accurate estimates were initially obtained for oxy hemoglobin mutants (r = 0.48, slope = 0.47, standard error = 1.4 kcal mol(-1), n = 23). However, the elimination of three outliers from this data set results in a better correlation of r = 0.87 (slope = 0.72, standard error = 0.75, n = 20). These three mutations may significantly perturb the hemoglobin quaternary structure beyond the scope of our structural optimization procedure. The method described is also useful in the examination of residue ionization states in protein structures. Specifically, we find an acidic residue within the native deoxy hemoglobin dimer-dimer interface that may be protonated at physiological pH. The final analysis is a model design of novel hemoglobin mutants that modify cooperative free energy (deltaGc)--the energy barrier between the allosteric transition from deoxy to oxy hemoglobin.

Computational Biology↗

PubMed1999

Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1 rapid communication.

BACKGROUND: Fractalkine is a newly identified T-cell and monocyte/macrophage (Mphi) chemokine with a transmembrane domain and is a cell-surface protein on activated endothelium. It can mediate adhesion of cells expressing the fractalkine receptor CX3CR1. These unique features make fractalkine well suited for leukocyte recruitment in tissues with high blood flow as in the renal glomerulus. METHODS: Fractalkine expression in glomeruli and response of isolated glomerular inflammatory cells to fractalkine were studied in the Wistar-Kyoto (WKY) crescentic glomerulonephritis model. Antibody was used to confirm the proinflammatory role of fractalkine. RESULTS: Fractalkine was markedly induced in the endothelium of nephritic rat glomeruli, and inflammatory leukocytes infiltrating the glomeruli expressed increased levels of CX3CR1. Anti-CX3CR1 antibody treatment dramatically blocked leukocyte infiltration in the glomeruli, prevented crescent formation, and improved renal function. CONCLUSIONS: Fractalkine plays a central role in leukocyte trafficking at the endothelium in the high-flow glomerular circuit and, in turn, implicates CX3CR1 as a prime drug target for therapeutic intervention of endothelium-related inflammatory diseases.

Animals↗

PubMed1998

Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia.

A recently identified chemokine, fractalkine, is a member of the chemokine gene family, which consists principally of secreted, proinflammatory molecules. Fractalkine is distinguished structurally by the presence of a CX3C motif as well as transmembrane spanning and mucin-like domains and shows atypical constitutive expression in a number of nonhematopoietic tissues, including brain. We undertook an extensive characterization of this chemokine and its receptor CX3CR1 in the brain to gain insights into use of chemokine-dependent systems in the central nervous system. Expression of fractalkine in rat brain was found to be widespread and localized principally to neurons. Recombinant rat CX3CR1, as expressed in Chinese hamster ovary cells, specifically bound fractalkine and signaled in the presence of either membrane-anchored or soluble forms of fractalkine protein. Fractalkine stimulated chemotaxis and elevated intracellular calcium levels of microglia; these responses were blocked by anti-CX3CR1 antibodies. After facial motor nerve axotomy, dramatic changes in the levels of CX3CR1 and fractalkine in the facial nucleus were evident. These included increases in the number and perineuronal location of CX3CR1-expressing microglia, decreased levels of motor neuron-expressed fractalkine mRNA, and an alteration in the forms of fractalkine protein expressed. These data describe mechanisms of cellular communication between neurons and microglia, involving fractalkine and CX3CR1, which occur in both normal and pathological states of the central nervous system.

Amino Acid Sequence↗

PubMed1998

Anatomo-radiographic study on the osteogenesis of carpal and tarsal bones in horse fetus.

The aim of this study is to point out the time of appearance of the carpal and tarsal bones in the fetal horse, considering an estimated fetal age, to follow their morphological development through to birth, and to characterize possible abnormal shape and/or delay of their ossification. The right carpal and tarsal region of 140 equine fetuses of both sexes (71 males, 69 females) and different ages (from 70 to 340 days of gestation) were examined radiographically in order to identify the sites of ossification from their earliest appearance. The times of appearance of the sites of ossification of the carpal bones are chronologically stated for each bone.

Animals↗

PubMed1997

Anatomo-radiographic study of prenatal development of bovine fetal teeth.

The aim of this study was to improve the knowledge of the time of appearance of dental germs and their morphological development until birth in bovine fetuses. Skulls and isolated mandibles of 35 Simmenthal bovine fetuses, of both sexes and ages from 97 to 280 days old were examined. The radiographic examination was performed with high definition and mamofilms. The exposure values ranged from 36 kV-6 mAs to 55 kV-12 mAs according to skull dimensions. In this study, the first dental germ was observed at 97 days, identified as the third maxillar premolar tooth. Through the morphological study and accurate description of the lingual and vestibular aspect of the occlusive surface of the teeth, three roots for the third and fourth maxillar premolar teeth and two for the second maxillar premolar tooth were observed. Two roots for the second and third mandibular premolar teeth and three for the fourth mandibular premolar tooth were also observed. The germ of the first mandibular molar tooth was seen at 140 days and the first of the maxillar arch at 280 days.

Animals↗

PubMed1996

Osteogenesis of the fetal bovine skull.

The research was carried out in order to study the osteogenesis of skull bones in bovine fetuses. A total of 24 fetuses were considered. The age of specimens ranged from 52 to 212 days. After silver-nitrate impregnation of the skulls, the specimens were examined radiologically using latero-lateral, latero-medial, and dorso-ventral projections.

Aging↗

PubMed1993

Pulsed magnetic fields improve osteoblast activity during the repair of an experimental osseous defect.

The influence of pulsed low-frequency electromagnetic fields (PEMFs) on bone formation was investigated in studies of the healing process of transcortical holes, bored at the diaphyseal region of metacarpal bones of six adult horses, exposed for 30 days to PEMFs (28 G peak amplitude, 1.3 ms rise time, and 75 Hz repetition rate). A pair of Helmholtz coils, continuously powered by a pulse generator, was applied for 30 days to the left metacarpal bone, through which two holes, of equal diameter and depth, had been bored at the diaphyseal region. Two equal holes, bored at the same level in the right metacarpal and surrounded by an inactive pair of Helmholtz coils, were used as controls. All horses were given an intravenous injection of 25-30 mg/kg of tetracycline chloride on the 15th and again on the 25th day after the operation and were killed 5 days later. The histomorphometric analysis indicated that both the amount of bone formed during 30 days and the mineral apposition rate during 10 days (deduced from the interval between the two tetracycline labels) were significantly greater (p < 0.01 and p < 0.0001, respectively) in the PEMF-treated holes than in the controls. As did a previous investigation, these preliminary findings indicate that PEMFs at low frequency not only stimulate bone repair but also seem to improve the osteogenic phase of the healing process, at least in our experimental conditions.

Animals↗

PubMed1992

HBV and HCV infection in i.v. drug addicts; coinfection with HIV.

A group of 122 drug addict patients were studied to evaluate the incidence of HIV, HBV, HCV infections and of laboratory findings of hepatic damage. Our data show that hepatic damage is more frequent in patients affected by HBV-HCV coinfection than those with HBV or HCV infection alone and that HIV positivity supports HBV-HCV coinfection.

Adolescent↗

PubMed1992

[Tension-free laparoscopic hernioplasty].

Hernia recurrence after traditional "open" hernioplasty is an observed event. The tension undergone by anatomical structures of the area is believed to be responsible in large part for these failures. Thus, techniques involving "tension free" hernioplasty have been developed, some of which involve laparoscopic access. Here an experience is reported regarding laparoscopic hernioplasty carried out to repair groin hernias, first of all on an animal model (pigs: 7 cases) then in the clinical field. The technique chosen was the endo-peritoneal positioning of a PTFE prosthesis. The results revealed several advantages over the traditional methods, basically the possibility of reinforcing the entire inguinal floor at the same time as repairing the hernia, and the decrease in groin area discomfort and less time off from work for the patient.

Abdominal Muscles↗

PubMed1992

[Experimental intestinal laparoscopic resection].

At present, there is a debate in the literature regarding the possibility of extending the laparoscopic approach to intestinal resection. Some techniques have been developed, but certain imperfections and problems remain. This experimental contribution suggests answers to some technical problems arising in the course of a small-bowel laparoscopic resection on a pig.

Anastomosis, Surgical↗

PubMed1992

[Contribution of glutathione to detoxification in alcoholism. Biochemical-clinical studies].

The effects of reduced glutathione (GSH) administration (1.2 g/day and 2.4 g/day intravenously) on erythrocyte glutathione levels, serum gamma-glutamyl transpeptidase activity (GGTP) and urinary glucaric acid elimination were studied in a population of 24 chronic alcoholics voluntarily admitted to a 30 day detoxification protocol in comparison to a 12 patient control group treated only with chlordiazepoxide (initial dose 75-100 mg/day). Glutathione treatment increases dose-dependently and in a significant way erythrocyte glutathione levels and hastens the recovery of serum GGTP and urinary glucaric acid elimination. The relationship between glutathione, GGTP and glucaric acid is discussed, suggesting the possible role of GSH against the oxidative damage of alcohol.

Adult↗

PubMed1991

Electromagnetic stimulation of bone repair: a histomorphometric study.

The effect of pulsing electromagnetic fields (PEMFs) on bone repair was studied in principal metacarpal bones of eight adult male horses: Six horses were treated with PEMFs, and two horses were untreated. In treated horses, Helmholtz coils were applied during a 60-day period to the left metacarpal bones, bored with eight holes of equal diameter and depth, from the middiaphysis toward the distal metaphysis. Eight equal holes bored in the right metacarpal, surrounded by unactivated Helmholtz coils, were taken as controls. The two untreated horses were taken as additional control. The results of computer-assisted histomorphometric analysis indicate that (a) in diaphyseal levels, the amount of bone formed during 60 days is significantly greater (p less than 0.01) in PEMF-treated holes than in contralateral ones and those in control horses; (b) in metaphyseal levels, PEMF-treated holes are sometimes more closed, sometimes less, as compared with contralateral holes and those in control horses; in any case the statistical analysis indicates that the symmetry in the rate of hole repair, found between the two antimeres of control horses, is not appreciable at metaphyseal levels also; (c) there was no statistically significant difference between untreated holes in PEMF-treated horses and holes in control horses, neither at diaphyseal nor at metaphyseal levels. These preliminary findings indicate that PEMFs at low frequency influence the process of bone repair on both diaphysis and metaphysis, and seem to improve the process of bone repair in skeletal regions normally having a lower osteogenetic activity, i.e., in diaphyses as against metaphyses.

Animals↗

PubMed1990

[Acute toxicity of trichloroethylene. Description of a case series at the Autonomous Service of Toxicology of Florence during the 1977-1988 period].

The authors report the number of acute trichloroethylene intoxications admitted to the Toxicological Unit of Florence University from January 1977 to December 1988. The identification of the solvent metabolic pathway allowed to clarify the pathogenesis of hepatorenal dysfunction observed during acute intoxications. Together with gastrointestinal decontamination and cardiac arrhythmia control we have studied the effect of drugs supposed to act as blockers of trichloroethylene metabolism or as inactivators of the hepatotoxic free radical metabolite 2-2(1)-3 trichloroxirane. The prognostic modification related to new therapeutic protocols is reported and discussed.

Acute Disease↗

PubMed1989

Intraarticular sodium hyaluronate injections in the Pond-Nuki experimental model of osteoarthritis in dogs. II. Morphological findings.

The effect of the intraarticular sodium hyaluronate (HA) injection on the osteoarthritic knee joint has been evaluated in dogs using an experimental model of osteoarthritis induced by sectioning the anterior cruciate ligament. Seven weeks after surgery, the damage, graded according to Mankin's scale, was significantly reduced in knee joints treated with HA from the second week postsurgery compared to untreated joints. When intraarticular HA therapy was initiated after the seventh week, osteoarthritis progression was still reduced compared to controls. Both morphology and morphometry showed a beneficial effect of HA on the cartilage response to the damage, as well as a clearcut inhibitory effect on the development of the fibroblastlike cell layer on the articular cartilage in untreated joints. The beneficial effects on the cartilage integrity and response to osteoarthritic damage might be related to a primary effect of HA on the cartilage surface. However, these effects do not exclude the possibility that, in addition, HA might act on the synovial membrane by limiting the synovial reaction.

Animals↗

PubMed1989

Pyrrolidone carboxylic acid in acute and chronic alcoholism. Preclinical and clinical studies.

2-Pyrrolidone-5-carboxylic acid (PCA) is a cyclic derivative of glutamic acid, physiologically present in mammalian tissues. We herein report preclinical pharmacology experiments showing that PCA releases GABA from the cerebral cortex of freely-moving guinea-pigs and displays anti-anxiety effects in a simple approach-avoidance conflict situation in the rat. In clinical pharmacology experiments, PCA significantly shortens the plasma half-life of ethanol during acute intoxication. In chronic alcoholics a treatment with PCA (2g/day per 30 days) significantly hastens the recovery to physiological values of plasma gamma-glutamyl transferase activity and of the urinary excretion of glucaric acid, which are considered suitable markers of the trend of the alcoholic disease. The evidence emerging from preclinical and clinical studies strongly suggests that, by combining central anxiolytic actions with the peripheral recovery of the antioxidant defense system in the liver, PCA should be further investigated as an interesting endogenous molecule possibly helpful in the therapy of alcoholism.

Alcoholic Intoxication↗

PubMed1989

Intraarticular sodium hyaluronate injections in the Pond-Nuki experimental model of osteoarthritis in dogs. I. Biochemical results.

An established anterior cruciate ligament deficiency-induced articular cartilage degeneration was used to evaluate the effects of intrasynovial injection of hyaluronic acid upon cartilage destruction. In this study, proteoglycan solubility under associative and dissociative conditions was compared in two treatment protocols at intervals of seven, 13, and 17 weeks after surgical breakage of the anterior cruciate ligament in 2.5-year-old Beagle dogs. Untreated joints showed a marked increase in both total soluble glycosaminoglycan (GAG measured as uronic acid) and in the associative fraction. In both treated groups, there was a reduced amount of soluble GAG. Cessation of treatment after seven weeks caused gradual regression, with an increasing amount of CaCl2-soluble material in the associative fraction, while inception at seven weeks gave biochemical evidence of reversal, with increasing GAG present in the guanidine-soluble (dissociative) fraction on the insoluble residue. The protective effects of hyaluronic acid suggest the potential clinical application of this therapy in retarding the advance of osteoarthritis.

Animals↗