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Biomedical subjects

G Galli

Publications and source records attributed to G Galli.

At least 127 records · Page 7Linked to original sources
PubMed1993

Regulation of extracellular matrix synthesis by transforming growth factor beta 1 in human fat-storing cells.

BACKGROUND: Fat storing cells (FSC) are nonparenchymal liver cells generally considered the major source of the hepatic extracellular matrix (ECM). Transforming growth factor beta 1 (TGF-beta 1) is a potent regulator of ECM synthesis in various cell types. In this study, the effect of TGF-beta 1 on procollagen types I, III, IV, laminin (Lam), and fibronectin (FN) synthesis in cultured human FSCs was analyzed. METHODS: FSCs were isolated from wedge sections of normal human livers. Morphological studies were performed by immunofluorescence and electron microscopy. ECM components in human FSC cultures were measured by an enzyme-linked immunosorbent assay. The expression of messenger RNA (mRNA) was evaluated by Northern blot and in situ hybridization. RESULTS: Cultured human FSCs displayed numerous fat droplets in the perinuclear zone, and immunoreactivity for vimentin and alpha-smooth muscle actin. A weak nonfibrillar staining was observed by using a polyclonal antidesmin antibody. TGF-beta 1 induced a dose-dependent increase of procollagen I, III, and FN accumulation in human FSC cultures, whereas procollagen IV and Lam production was not affected. Furthermore, TGF-beta 1 increased the expression of alpha 1 (I), alpha 1 (III) procollagen, FN and TGF-beta 1 mRNA in human FSC cultures. CONCLUSIONS: These data indicate that TGF-beta 1 is able to increase the synthesis of procollagen I, III, and FN in cultured human FSCs. Moreover, TGF-beta 1 can induce its own mRNA in the same cells.

Adipose Tissue↗
PubMed1993

Acetaldehyde-protein adducts, but not lactate and pyruvate, stimulate gene transcription of collagen and fibronectin in hepatic fat-storing cells.

Hepatic fibrosis is an important morphological feature of alcohol-induced liver injury. We previously reported that acetaldehyde, but not ethanol can stimulate type I collagen and fibronectin synthesis in cultures of rat fat-storing cells (FSC) by increasing transcription of the specific genes. The effect of lactate and pyruvate was studied on collagen I, III, fibronectin accumulation by cultured rat FSCs and it was investigated whether acetaldehyde could increase procollagen I and fibronectin gene transcription through the formation of protein adducts. Lactate and pyruvate (5, 15 and 25 mmol/l) did not significantly affect collagen I, III and fibronectin production by cultured FSCs. Pyridoxal-phosphate and p-hydroxymecuribenzoate (inhibitors of acetaldehyde-protein adduct formation) blocked the stimulatory effect of acetaldehyde on procollagen I and fibronectin gene transcription. These data suggest that ethanol may act as a liver fibrogenic factor through acetaldehyde, its immediate metabolite, whereas lactate does not seem to play a role. Acetaldehyde might stimulate gene transcription of extracellular matrix components by liver FSCs through the formation of adducts with proteins.

Acetaldehyde↗
PubMed1993

Characterization of the srfA locus of Bacillus subtilis: only the valine-activating domain of srfA is involved in the establishment of genetic competence.

srfA is a locus required for the production of the lipopeptide antibiotic surfactin. This locus is also necessary for efficient sporulation and competence development. Mutations in the 5' portion of the srfA operon affect all three of these processes, whereas mutations in the 3' portion of srfA only affect sporulation and surfactin production. Analysis of the proteins encoded by the srfA locus revealed seven large domains which are likely to be responsible for the activation and binding of the seven amino acids of surfactin. Identification of the amino acid that is activated by the srfA domains was determined by amino acid-dependent pyrophosphate exchange reactions on partially purified cell extracts of strains carrying different srfA mutations. These results indicate colinearity between the order of the domains in the srfA locus and the amino acid sequence of surfactin. The minimal genetic element of srfA required for the establishment of competence was shown to be the 5' region of the second open reading of srfA, which encodes the valine activation domain. This portion of srfA, when cloned on a plasmid, complemented the competence deficiency of a srfA deletion mutant in trans.

Amino Acid Sequence↗
PubMed1993

Quantitative changes of hydroxyacid formation during platelet-neutrophil interaction.

In recent years the interactions between lipoxygenase enzymes present in platelets and leukocytes, resulting in the transcellular biosynthesis of eicosanoids, have been discovered and their relevance in thrombotic and inflammatory disorders recognized. However, attention has focused on the synthesis of novel products, not normally formed by the single-cell population. Less information is available on the changes in hydroxyacid formation during platelet-neutrophil interactions. In this study, we evaluated the quantitative changes of the levels of 12- and 5-hydroxyeicosatetraenoic acids, leukotriene B4, and thromboxane B2 occurring during the incubation of platelet-neutrophil mixed suspensions stimulated with the calcium ionophore A23187. Cell-to-cell interaction resulted in quantitative changes of the level of hydroxyacids; in mixed platelet-neutrophil suspensions, the levels of 12-hydroxyeicosatetraenoic acid were significantly increased with respect to those measured in platelets incubated alone. The amount of 5-hydroxyeicosatetraenoic acid formed by neutrophils was significantly decreased by the presence of platelets in the incubation medium. In addition, a slight but significant reduction in leukotriene B4 synthesis was observed. Concomitantly with these changes, the formation of thromboxane B2 by platelets was modified, indicating that--besides the lipoxygenase pathway--the arachidonic acid metabolism by cyclooxygenase is affected. Our study demonstrates that in mixed platelet-neutrophil suspensions profound quantitative modifications in hydroxyacid and thromboxane synthesis occur, indicating that the overall balance of arachidonic acid products may be altered in pathologic conditions in which early events of multicellular origin have been recognized.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1992

Defibrotide has antiischemic activity in perfused rabbit hearts, preventing tissue Ca++ overloading.

Defibrotide (D), a polydeoxyribonucleotide obtained from mammalian lungs, reduced the ischemic contracture due to low perfusion (0.2 ml/min) of the isovolumic left heart of the rabbit and abolished the irregularity of the rhythm of the heart, thereby restoring the cardiomechanical activity upon reperfusion (20 ml/min). D stimulated the release of PG-like material. Indomethacin infusion completely prevented both the antiischemic activity of D and its ability to increase the generation of prostaglandins in the rabbit heart. Measurement by atomic absorption spectroscopy of calcium content in ischemic heart tissue and its mitochondrial fraction indicated that the ischemic procedure significantly increased tissue calcium content in both. D, Prostacyclin (PGI2) and Nifedipine protected the heart from ischemic ventricular contracture and prevented accumulation of calcium in the heart. The effect of D on preventing Ca++ overload was completely abolished by indomethacin infusion. The results indicate that the beneficial effects of Defibrotide in experimental ischemia are primarily due to a release of Prostaglandin E2 (PGE2) and PGI2, which in turn may inhibit the detrimental effects of calcium overload in myocytes and mitochondria.

Animals↗
PubMed1992

Cystic mesenchymal hamartoma of the liver.

Two cases of multicystic mesenchymal hamartoma of the liver in children are described. In both cases noninvasive imaging techniques (ultrasound, CT or MR, radionuclide scan) showed a bulky cystic mass suggesting a correct preoperative diagnosis, while selective arteriography defined the operability of the tumor.

Child, Preschool↗
PubMed1992

A particle beam-liquid chromatography-mass spectrometry method for the determination of lipoxygenase metabolites of arachidonic acid.

An application of the particle beam-liquid chromatography-mass spectrometry technique to the quantification of hydroxyeicosatetraenoic acids (15-, 12-, and 5-HETE) in biological samples is presented. The acids are extracted with Ethyl acetate and then transformed into pentafluorobenzyl esters, thus increasing the sensitivity of their detection by negative chemical ionization mass spectrometry. Reverse-phase HPLC separation of HETEs is performed in about 10 min with a water-methanol gradient. The procedure shows a detection limit of nearly 0.5 pmol, about one order of magnitude lower than that of the widely used HPLC/UV methods. The quantitative determination is linear (r2 greater than 0.998) for all of the HETEs in the range tested (3-1500 pmol) and a CV lower than 8.5% was observed for repeated analysis of samples. As an application of the method, HETEs formed from endogenous arachidonate were evaluated in extracts obtained from coincubates of platelets and neutrophils stimulated with calcium ionophore A23187.

Adult↗
PubMed1992

Evidence for the presence of 7-hydroperoxycholest-5-en-3 beta-ol in oxidized human LDL.

Low density lipoprotein (LDL) cholesterol is known to be oxidized both in vitro and in vivo giving rise to oxygenated sterols. Conflicting results, however, have been reported concerning both the nature and the relative concentrations of these compounds in oxidized human LDL. We examined the extracts obtained from Cu(2+)-oxidized LDL. Thin layer chromatography analysis showed that the sterol mixture became more complex with reaction time. Analysis of the components by thin layer chromatography and mass spectrometry allowed to establish that 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha OOH and beta OOH) are largely prevalent among the oxysterols at early times of oxidation. These hydroperoxy derivatives have not been previously identified in oxidized LDL. The concentration of 7-hydroperoxycholest-5-en-3 beta-ol decreased with oxidation time with a concomitant increase of cholest-5-en-3 beta, 7 alpha-diol (7 alpha OH), cholest-5-en-3 beta, 7 beta-diol (7 beta OH), cholesta-3,5-dien-7-one (CD) and cholest-5-en-3 beta-ol-7-one (7CO). After 24 h of oxidation a minor component of the LDL sterols was cholestan-3 beta-ol-5,6-oxide (EP).

Cholesterol↗
PubMed1992

Purification of recombinant human growth hormone by isoelectric focusing in a multicompartment electrolyzer with Immobiline membranes.

Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a combination of ion-exchange chromatography and metal ion affinity chromatography. For the last purification step, a multicompartment electrolyzer was used, containing three compartments delimited by isoelectric membranes and two additional anodic and cathodic chambers. The central compartment was situated between two membranes having isoelectric points (pI) of 5.08 (anodic) and of 5.16 (cathodic), i.e. equidistant from the pI value of hGH (pI 5.12). r-hGH was isoelectric between these two membranes and could not leave the central chamber, while more acidic and more cathodic impurities collected in the two lateral chambers under the influence of the electric field. The r-hGH, thus purified, exhibited a single band by isoelectric focusing (IEF) in immobilized pH gradients (IPG) and gave recoveries greater than 90%. The problem of isoelectric precipitation in a practically ion-free environment was alleviated by focusing in 30% glycerol added with 1% neutral detergent (Nonidet-P40). The latter was eliminated by passage through a Q-Sepharose column after collecting the pI 5.12 band from the electrolyzer. Also the pre-hormone (pre-hGH) can be purified in a similar manner (30% glycerol, 1% Nonidet P-40) between two membranes having pIs 4.77 (anodic) and 4.87 (cathodic) (pre-hGH pI 4.82). This paper demonstrates the possibility of purifying by a focusing process also poorly soluble proteins at the pI.

Growth Hormone↗
PubMed1992

Lateral discoid meniscus: treatment and results.

Forty-six patients (47 knees) who had symptomatic discoid lateral menisci were operated on between August 1981 and May 1989. The patients were categorized according to Watanabe's classifications: complete (80.8%), incomplete (10.6%), and Wrisberg type (8.5%). The treatment of the discoid meniscus is based on total or partial meniscectomy, achieved by the percutaneous or arthrometric technique. Of the 47 knees, 30 underwent a follow-up evaluation. Using Ikeuchi's knee scale, 27 cases (90%) had either excellent or good ratings, and 3 (10%) received fair ratings. There were no poor outcomes. The results in the knees treated by partial meniscectomy were better than those in the knees treated with total meniscectomy.

Adolescent↗
PubMed1992

A collaborative trial for the evaluation of blood cholesterol measurement in clinical laboratories in Italy.

A collaborative trial for the evaluation of blood cholesterol measurement in Italy was carried out, with the use of two lyophilized controls, whose target values, respectively 4.42 and 6.21 mmol/l, were established by means of "definitive" methodology (isotope dilution/mass spectrometry). Results from 480 participants showed a somewhat broad dispersion (CV 6.1% and 6.3% respectively), and a definite bias (-0.25 mmol/l and -0.61 mmol/l respectively) with respect to the target values. The different analytical systems were characterized by different combinations of inaccuracy and imprecision; however, the bias observed for the higher concentration sample was a constant finding. The behaviour of the control materials, in comparison with that exhibited by patients' sera, was assessed in a manual enzymatic procedure and in the Kodak Ektachem 700 and Technicon Chem 1 systems. The peculiar property of one control material to behave differently from patients' sera in some analytical systems, i.e. the lack of commutability, was found to be partially responsible for the observed bias in the three methods studied. The importance of testing for commutability of the control materials to be used for the control of accuracy is stressed.

Blood Chemical Analysis↗
PubMed1991

Prochymosin expression in Bacillus subtilis.

Prochymosin (PC) sequence was cloned in Bacillus subtilis using two kinds of plasmid constructions. In plasmid pSM316 the cDNA was inserted to obtain the intracellular expression of the enzyme. The enzyme turned out to be expressed in an insoluble form which could be converted to native enzyme under proper denaturing and refolding conditions. The levels of intracellular expression of PC were further enhanced by modifying the 5' region of the gene in a way that a two-cistron expression system was created. For the PC secretion, the cDNA was fused to the subtilisin leader sequence and expressed under the control of the B. subtilis neutral protease promoter. A properly folded PC was secreted by the cells, although to low levels.

Amino Acid Sequence↗
PubMed1991

Calcitonin gene-related peptide increases the production of glycosaminoglycans but not of collagen type I and III in cultures of rat fat-storing cells.

We investigated whether calcitonin gene-related peptide (CGRP) was able to affect the production of collagen and glycosaminoglycans (GAG) in cultures of rat fat-storing cells (FSC). Rat CGRP (1 nM-1 microM) induced a dose-dependent increase of total GAG production in FSC cultures with an EC50 of 28 nM. One uM human CGRP (8-37) shifted the dose-response curve of rat CGRP to the right (EC50 = 257 nM) without depressing the maximal response. Salmon calcitonin (1 nM-1 microM) did not significantly modify total GAG accumulation in FSC cultures. Collagen type I and III production was not significantly affected by either CGRP or calcitonin in FSC cultures. These findings suggest that peripheral sensory neuropeptides may modulate liver fibrogenesis.

Adipose Tissue↗
PubMed1991

A new human growth hormone production process using a recombinant Bacillus subtilis strain.

We constructed a series of hybrid plasmids which directed the synthesis of different human growth hormone (hGH) precursor sequences in Bacillus subtilis. In addition to the 191 amino acids of the hormone, the precursors had in common an amino-terminal extension characterized by the presence of a methionine at position 1 and of the tetrapeptide Ile-Glu-Gly-Arg preceding the first residue (Phe) of hGH. The sequence between the methionine and the tetrapeptide was specific for each precursor and, because of the presence of charged residues, conferred particular properties to the molecules. Long homopolymeric tail-containing precursors such as MRRRRRRIILM-IEGR appeared insoluble whereas shorter sequences of the type MRR-IEGR and MEELM-IEGR augmented the solubility of the precursors with respect to Met-hGH. The soluble precursors could be easily purified from the bulk proteins taking advantage of the charged residues present on the N-terminal tail. After purification, the natural hGH was obtained by treating the precursors with the protease Factor Xa which cleaves after the arginine residue of the tetrapeptide IEGR. A protocol for the production and purification of authentic hGH from a strain expressing one of these soluble precursors is reported.

Amino Acid Sequence↗