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Biomedical subjects

M Rossi

Publications and source records attributed to M Rossi.

At least 253 records · Page 14Linked to original sources
PubMed1998

The nematode resistance gene Mi of tomato confers resistance against the potato aphid.

Resistance against the aphid Macrosiphum euphorbiae previously was observed in tomato and attributed to a novel gene, designated Meu-1, tightly linked to the nematode resistance gene, Mi. Recent cloning of Mi allowed us to determine whether Meu-1 and Mi are the same gene. We show that Mi is expressed in leaves, that aphid resistance is isolate-specific, and that susceptible tomato transformed with Mi is resistant to the same aphid isolates as the original resistant lines. We conclude that Mi and Meu-1 are the same gene and that Mi mediates resistance against both aphids and nematodes, organisms belonging to different phyla. Mi is the first example of a plant resistance gene active against two such distantly related organisms. Furthermore, it is the first isolate-specific insect resistance gene to be cloned and belongs to the nucleotide-binding, leucine-rich repeat family of resistance genes.

Animals↗
PubMed1998

Structural analysis of ASCUT-1, a protein component of the cuticle of the parasitic nematode Ascaris lumbricoides.

CUT-1 from the intestinal parasitic nematode Ascaris lumbricoides is a protein component of the insoluble residue of the cuticle, cuticlin. It contains the CUT-1-like domain which is shared by members of a novel family of components of extracellular matrices. The structure and the thermal stability of recombinant CUT-1 from A. lumbricoides (ASCUT-1) were investigated by Fourier-transform infrared (FT-IR) and CD spectroscopy. The data revealed that the secondary structure of the protein at 20 degrees C, both as insoluble inclusion bodies or in soluble form, contains about 50% beta structure, 14% alpha-helix and 25% turns. A tendency of A. lumbricoides CUT-1 to form aggregates was documented by FT-IR spectroscopy which showed also that the addition of SDS disrupts these interactions. Near-ultraviolet CD spectra confirmed these data and suggested that phenylalanine residues are probably involved in intermolecular hydrophobic interactions responsible for the tendency of the protein to aggregate. Near-ultraviolet spectra showed also that part of the cysteine residues forms disulphide bridges responsible for the tertiary architecture of the protein. Finally, FT-IR and CD data revealed that ASCUT-1 is very stable at high temperatures. This stability and the tendency of ASCUT-1 to form aggregates suggest that these properties may be important for a protein which is a component of a particularly resistant extracellular matrix such as the nematode cuticle.

Amino Acid Sequence↗
PubMed1998

Advantages in using immobilized thermophilic beta-glycosidase in nonisothermal bioreactors.

Catalytic membranes, obtained by immobilizing thermophilic beta-glycosidase onto nylon supports, were used in a nonisothermal bioreactor to study the effect of temperature gradients on the rate of enzyme reaction. Two experimental approaches were carried out to explain the molecular mechanisms by which the temperature gradients affect enzyme activity. The results showed that the thermophilic enzyme behaved as the mesophilic beta-galactosidase, exhibiting an activity increase which was linearly proportional to the transmembrane temperature difference. The efficiency of the system proposed was determined by calculating two constants, alpha and beta, which represent respectively the percentage increase of enzyme activity when a temperature difference of 1 degrees C or a temperature gradient of 1 degrees C cm-1 were applied across the catalytic membrane. The increase of enzyme activity in nonisothermal bioreactors entailed a proportional reduction of production times. The advantages in using thermophilic enzymes immobilized in nonisothermal bioreactors are also discussed.

Bioreactors↗
PubMed1998

Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily.

We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B'-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11+/-2 microM (mean+/-S.D., n=3) and 6610+/-880 s-1 (mean+/-S.D., n=3) respectively at 70 degreesC and pH7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic Moraxella TA144 lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (+/-)-3-bromo-5-(hydroxymethyl)-Delta2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity-stability-temperature relationship is discussed in relation to those of the homologous members of the HSL group.

Amino Acids↗
PubMed1998

Protein crystal growth in the Advanced Protein Crystallization Facility on the LMS mission: a comparison of Sulfolobus solfataricus alcohol dehydrogenase crystals grown on the ground and in microgravity.

Crystals of alcohol dehydrogenase from Sulfolobus solfataricus were grown in the Advanced Protein Crystallization Facility during the Life and Microgravity Sciences Spacelab mission on the US Space Shuttle. Large diffracting crystals were obtained by dialysis, whereas only poor-quality crystals were obtained by vapour diffusion. The quality of both the microgravity and ground-based crystals was analysed by X-ray diffraction. There was some improvement in terms of size and diffraction resolution limit for the microgravity crystals. However, the twinning observed in the Earth-grown crystals was also present for those grown in microgravity.

Alcohol Dehydrogenase↗
PubMed1998

Characterization of Bifidobacterium strains for use in soymilk fermentation.

Soybean milk, which serves as a base for a variety of beverages, contains raffinose, stachyose, pentanal and n-hexanal; the former two may be responsible for flatulence after fermentation, whilst the latter two for a beany flavour. Twenty-seven strains of Bifidobacterium were analyzed for their alpha-galactosidase activity and the production of lactic and acetic acids to determine their potential for use in the production of fermented soymilk. The behaviour of three strains in soymilk was studied to determine their ability to reduce alpha-D-galactosyl oligosaccharides and produce lactic and acetic acids. They all were able to reduce stachyose and raffinose. Pentanal and n-hexanal were metabolized by Bifidobacterium breve MB233. These data indicate that bifidobacteria can be used for biotechnological processes that employ soymilk as the substrate. A product with low levels of alpha-D-galactosyl oligosaccharides and alkylic aldehydes may be obtained.

Acetic Acid↗
PubMed1998

Multiple forms of DNA polymerase from the thermo-acidophilic eubacterium Bacillus acidocaldarius: purification, biochemical characterization and possible biological role.

Two DNA polymerase isoenzymes, called DpA and DpB on the basis of their elution order from DEAE cellulose, were purified to homogeneity from the thermo-acidophilic eubacterium Bacillus acidocaldarius. The enzymes are weakly acidophilic proteins constituted by a single subunit of 117 and 103 kDa respectively. DpA and DpB differ in thermostability, in thermophilicity, in sensitivity to assay conditions and in resistance to sulphydryl-group blocking agents such as N-ethylmaleimide and p-hydroxymercuriobenzoate. They differ also in synthetic template-primer utilization, in the apparent Km for dNTPs and in processivity. In particular, DpA utilizes more effic iently synthetic templates-primers such as poly(dA).poly(dT), poly(dT). (rA)12-18 and poly(rA).(dT)12-18 and presents a greater tendency to accept dNTP analogues modified in the sugar or in the base ring, such as cytosine beta-d-arabinofuranoside 5'-triphosphate, 2',3'-dideoxyribonucleosides 5'-triphosphate, butylphenyl-dGTP and digoxigenin-conjugated dUTP. In addition, DpA presents an exonuclease activity that preferentially hydrolyses DNA in the 5'-3' direction, whereas DpB lacks this activity. The possible biological role of the enzymes is discussed.

Bacillus↗
PubMed1998

Impaired vasoconstriction of peripheral cutaneous blood flow in Type 1 diabetic patients following food ingestion.

Abnormalities in cutaneous blood flow (CBF) in otherwise healthy subjects with Type 1 diabetes mellitus (DM) have been demonstrated in response to local insults to the skin. To investigate whether defects also occurred in response to a regular daily activity, CBF was measured with laser Doppler flowmetry (LDF), before and 20 min after starting a mixed meal in 13 male Type 1 DM subjects with no clinical evidence of neuropathy, nephropathy or macroangiopathy and compared to 7 non-diabetic controls. Diabetic subjects and controls were of similar age and body mass index (mean +/- SD, 33.7 +/- 7.4 vs 37.1 +/- 9.2 years and 25.2 +/- 2.9 vs 24.5 +/- 2.9 kg m(-2), respectively). In subjects with DM, HbA1c was 8.3 +/- 0.6% (normal range 4-5.5%) and duration of diabetes was 18 (8-38) years, median (range). Following a mixed meal the CBF fell in the controls by 36% (24 to 56), median (range), compared to 3% (-5 to 18) in Type 1 DM subjects, P < 0.0005. These results show there is a normal physiological fall in CBF following food ingestion which is attenuated in Type 1 DM. These abnormalities of vasoconstriction in the peripheral microcirculation are present after 8 years of diabetes and precede the development of clinically apparent neuropathy or vascular disease.

Adult↗
PubMed1998

Affinity purification of mouse monoclonal IgE using a protein A mimetic ligand (TG19318) immobilized on solid supports.

A synthetic ligand (TG19318), deduced from the screening of a combinatorial peptide library, has been previously characterized by our group for its applicability in affinity chromatography for polyclonal and monoclonal IgG purification from crude sources. In this study we have extended the characterization of its recognition properties for other immunoglobulin classes, evaluating its ability to purify mouse monoclonal IgE from ascitic fluid. TG19318 affinity columns proved useful for a very convenient one-step purification of IgE directly from crude ascites, by loading the samples on the columns equilibrated with 50 mM sodium phosphate at pH 7 and eluting and adsorbed IgE by a buffer change to 0.1 M acetic acid. Antibody purity after affinity purification was very high and no albumin traces were detected, as determined by SDS-PAGE analysis. Antibody activity was fully recovered after purification, as determined by immunoassays on antigen-coated plates, and up to 5 mg of IgEs could be purified on a 1 ml column in a single run.

Affinity Labels↗
PubMed1998

Allylamine type xanthone antimycotics.

A number of xanthone derivatives bearing the basic chain of naftifine and butenafine antimycotics in 1, 2, 3, and 4 nuclear positions are described. The in vitro antifungal activity against representative strains of molds and yeasts is reported. Only butenafine xanthone analogues show significant activity against Cryptococcus neoformans, in particular the regioisomer 4d (1.5 micrograms/ml).

Allylamine↗
PubMed1998

Molecular biology of hyperthermophilic Archaea.

The sequences of a number of archaeal genomes have recently been completed, and many more are expected shortly. Consequently, the research of Archaea in general and hyperthermophiles in particular has entered a new phase, with many exciting discoveries to be expected. The wealth of sequence information has already led, and will continue to lead to the identification of many enzymes with unique properties, some of which have potential for industrial applications. Subsequent functional genomics will help reveal fundamental matters such as details concerning the genetic, biochemical and physiological adaptation of extremophiles, and hence give insight into their genomic evolution, polypeptide structure-function relations, and metabolic regulation. In order to optimally exploit many unique features that are now emerging, the development of genetic systems for hyperthermophilic Archaea is an absolute requirement. Such systems would allow the application of this class of Archaea as so-called "cell factories": (i) expression of certain archaeal enzymes for which no suitable conventional (mesophilic bacterial or eukaryal) systems are available, (ii) selection for thermostable variants of potentially interesting enzymes from mesophilic origin, and (iii) the development of in vivo production systems by metabolic engineering. An overview is given of recent insight in the molecular biology of hyperthermophilic Archaea, as well as of a number of promising developments that should result in the generation of suitable genetic systems in the near future.

Archaea↗
PubMed1998

Cell-substrate separation: effect of applied force and temperature.

We measure the change in cell-substrate separation in response to an upward force by combining two relatively new techniques, Electric Cell-substrate Impedance Sensing (ECIS) to measure average cell-substrate separation, and collagen-coated magnetic beads to apply force to the top (dorsal) surface of cells. The collagen-coated ferric oxide beads attach to integrin receptors in the dorsal surfaces of osteoblastlike ROS 17/2.8 cells. Magnetic force is controlled by the position and the number of permanent magnets, applying an average 320 or 560 pN per cell. Comparing model calculations with experimental impedance data, the junctional resistivity of the cell layer and the average distance between the lower (ventral) cell surface and substrate can be determined. The ECIS analysis shows that these forces produce an increase in the distance between the ventral cell surface and the substrate that is in the range of 10 to 25%. At temperatures of 4 degrees, 22 degrees and 37 degrees C, the measured cell surface-substrate distances without magnetic beads are 84 +/- 4, 45 +/- 2 and 38 +/- 2 nm. The force-induced changes at 22 degrees are 11 +/- 3 and 21 +/- 3 nm for 320 and 560 pN, and at 37 degrees they are 5 +/- 2 and 9 +/- 2 nm. The resulting cell-substrate spring constants at 22 degrees and 37 degrees are thus about 28 and 63 pN nm-1 (dyne cm-1). Using a reasonable range for the number for individual integrin-ligand adhesion bonds gives a range for the spring constant of the individual adhesion bond of from about 10(-3) to 10(-1) pN nm-1. These data also provide evidence that the number of adhesion bonds per cell increases with temperature.

Animals↗
PubMed1998

Semi-automatic segmentation of gated blood pool emission tomographic images by watersheds:application to the determination of right and left ejection fractions.

Tomographic multi-gated blood pool scintigraphy (TMUGA) is a widely available method which permits simultaneous assessment of right and left ventricular ejection fractions. However, the widespread clinical use of this technique is impeded by the lack of segmentation methods dedicated to an automatic analysis of ventricular activities. In this study we evaluated how a watershed algorithm succeeds in providing semi-automatic segmentation of ventricular activities in order to measure right and left ejection fractions by TMUGA. The left ejection fractions of 30 patients were evaluated both with TMUGA and with planar multi-gated blood pool scintigraphy (PMUGA). Likewise, the right ejection fractions of 25 patients were evaluated with first-pass scintigraphy (FP) and with TMUGA. The watershed algorithm was applied to the reconstructed slices in order to group together the voxels whose activity came from one specific cardiac cavity. First, the results of the watershed algorithm were compared with manual drawing around left and right ventricles. Left ejection fractions evaluated by TMUGA with the watershed procedure were not significantly different (p=0. 30) from manual outlines whereas a small but significant difference was found for right ejection fractions (p=0.004). Then right and left ejection fractions evaluated by TMUGA (with the semi-automatic segmentation procedure) were compared with the results obtained by FP or PMUGA. Left ventricular ejection fractions evaluated by TMUGA showed an excellent correlation with those evaluated by PMUGA (r=0. 93; SEE=5.93%; slope=0.99; intercept = 4.17%). The measurements of these ejection fractions were significantly higher with TMUGA than with PMUGA (P<0.01). The interoperator variability for the measurement of left ejection fractions by TMUGA was 4.6%. Right ventricular ejection fractions evaluated by TMUGA showed a good correlation with those evaluated by FP (r = 0.81; SEE = 6.68%; slope = 1.00; intercept = 0.85%) and were not significantly different (P = 0.42). The interoperator variability with TMUGA was 6.7% for the right ventricle. Thus, the watershed algorithm proposed is an efficient segmentation tool for the semi-automatic analysis of right and left ventricular ejection fractions by TMUGA. Further studies are necessary to check whether this procedure can be used to evaluate ventricular volumes and cardiac outflow.

Algorithms↗
PubMed1998

Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems.

Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the beta-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems.

Abscisic Acid↗
PubMed1998

Domain organization and biochemical features of Sulfolobus solfataricus DNA polymerase.

DNA polymerase from Sulfolobus solfataricus, strain MT4 (Sso DNA pol), was one of the first archaeal DNA polymerases to be isolated and characterized. Its encoding gene was cloned and sequenced, indicating that Sso DNA pol belongs to family B of DNA polymerases. By limited proteolysis experiments carried out on the recombinant homogeneous protein, we were able to demonstrate that the enzyme has a modular organization of its associated catalytic functions (DNA polymerase and 3'-5' exonuclease). Indeed, the synthetic function was ascribed to the enzyme C-terminal portion, whereas the N-terminal half was found to be responsible for the exonucleolytic activity. In addition, partial proteolysis studies were utilized to map conformational changes on DNA binding by comparing the cleavage map in the absence or presence of nucleic acid ligands. This analysis allowed us to identify two segments of the Sso DNA pol amino acid chain affected by structural modifications following nucleic acid binding: region 1 and region 2, in the middle and at the C-terminal end of the protein chain, respectively. Site-directed mutagenesis studies will be performed to better investigate the role of these two protein segments in DNA substrate interaction.

Catalytic Domain↗
PubMed1998

Enzymes from Sulfolobus shibatae for the production of trehalose and glucose from starch.

Enzymes that convert starch and dextrins to alpha,alpha-trehalose and glucose were found in cell homogenates of the hyperthermophilic acidophilic archaeon Sulfolobus shibatae DMS 5389. Three enzymes were purified and characterized. The first, the S. shibatae trehalosyl dextrin-forming enzyme (SsTDFE), transformed starch and dextrins to the corresponding trehalosyl derivatives with an intramolecular transglycosylation process that converted the glucosidic linkage at the reducing end from alpha-1,4 to alpha-1,1. The second, the S. shibatae trehalose-forming enzyme (SsTFE), hydrolyzed the alpha-1,4 linkage adjacent to the alpha-1,1 bond of trehalosyl dextrins, forming trehalose and lower molecular weight dextrins. These two enzymes had molecular masses of 80 kDa and 65 kDa, respectively, and showed the highest activities at pH 4.5. The apparent optimal temperature for activity was 70 degrees C for SsTDFE and 85 degrees C for SsTFE. The third enzyme identified was an alpha-glycosidase (Ss alpha Gly), which catalyzed the hydrolysis of the alpha-1,4 glucosidic linkages in starch and dextrins, releasing glucose in a stepwise manner from the nonreducing end of the polysaccharide chain. The enzyme had a molecular mass of 313 kDa and showed the highest activity at pH 5.5 and at 85 degrees C.

Glucose↗
PubMed1998

Antihypertensive efficacy of angiotensin converting enzyme inhibition and aspirin counteraction.

OBJECTIVE: Blockade of bradykinin breakdown and enhancement of prostaglandin release probably participate in the antihypertensive activity of angiotensin converting enzyme (ACE) inhibitors. Cyclooxygenase blockers may attenuate the efficacy of ACE inhibitors by interfering with prostaglandin synthesis, and patients taking aspirin may not benefit from ACE inhibition. This study was designed to evaluate the incidence of the counteractive phenomenon and to define minimal aspirin dosage that causes an antagonistic effect. METHODS: These were 26 patients with mild to moderate hypertension (group 1) and 26 patients with severe untreated primary hypertension (group 2). Enalapril (20 mg twice a day) was used as a single drug in group 1 and was added to the combination of long-acting nifedipine (30 mg/day) and atenolol (50 mg/day) in group 2. Aspirin was tested at doses of 100 and 300 mg/day, and an attenuation of more than 20% of the mean blood pressure decrease produced by enalapril was the criteria that defined antagonism. RESULTS: The 100 mg dose was ineffective. However, 300 mg aspirin had an antagonistic effect in 57% of patients in group 1 and 50% of patients in group 2: mean arterial pressure was lowered by 63% and 91% less, respectively. Results were independent of the drug administration order. In "responders," aspirin significantly attenuated the renin rise associated with ACE inhibition. CONCLUSIONS: These findings suggest that a number of ACE-inhibited patients are susceptible to 300 mg/day aspirin, regardless of hypertension severity. Antagonism may be mediated through prostaglandin inhibition according to predominance, in an individual patient, of prostaglandin activation (also as a renin secretory stimulus) or angiotensin blockade by enalapril.

Adult↗