Biomedical subjects

G Grandi

Publications and source records attributed to G Grandi.

At least 55 records · Page 3Linked to original sources

PubMed1995

An operon encoding a novel ABC-type transport system in Bacillus subtilis.

Downstream from the surfactin synthetase operon in Bacillus subtilis a new operon-type structure has been localized which, on the basis of sequence determination, potentially encodes an ABC-type transport system. The 268 amino acid protein, the product of orf1, represents the solute-binding component of the system whereas the orf2 product, a 234 amino acid protein, is the transmembrane component. Finally orf3 potentially encodes a typical 241 amino acid ATP-binding protein involved in energy supply. Comparison of the three proteins with the subunits of other ABC-type systems suggests that this new system is involved in amino acid transport.

ATP-Binding Cassette Transporters↗

PubMed1994

Characterization of the surfactin synthetase multi-enzyme complex.

Three subunits (srfAORF1, srfAORF2 and srfAORF3) of the Bacillus subtilis surfactin synthetase multi-enzyme complex have been identified by SDS-PAGE and Western blot analyses. In accordance with the sequence analysis of the surfactin (srfA) operon, the protein subunits have a molecular mass of 402,000 Da, 401,000 Da and 144,000 Da, respectively. Confirmation of the identity of the proteins was obtained by analysing the total protein content of a number of mutant strains which harbour deletions or insertions either in the srfA promoter or in different positions within the srfA operon. The three subunits were partially purified by means of a series of chromatographic steps including ion-exchange chromatography, hydrophobic chromatography and gel filtration chromatography. The partially purified proteins were used in activity assays to establish their amino-acid recognition specificity. In agreement with previously published results, this analysis showed that srfAORF1 recognizes glutamic acid and Leu, srfAORF2 recognizes Val, aspartic acid and Leu and srfAORF3 recognizes Leu. In addition, the subunits can activate and bind other amino acids, although with lower specificity. In particular, srfAORF1 binds Val, Ile and aspartic acid, srfAORF2 glutamic acid and Ile and srfAORF3 Ile and Val. Competition experiments as well as sequence comparison strongly suggest that the Leu binding sites of the three subunits can accept, beside Leu, Ile and Val. The kinetic parameters of srfAORF3 for Leu, Ile and Val have been determined.

Amino Acid Sequence↗

PubMed1994

The effect of engineering surface loops on the thermal stability of Bacillus subtilis neutral protease.

Using genetic techniques the contribution of surface loops to the thermal stability of Bacillus subtilis neutral protease (NP-sub) was studied. Mutations were designed to make the surface of NP-sub more similar to the surface of more thermostable neutral proteases such as thermolysin (TLN). The mutations included the replacement of an irregular loop by a shorter variant and the introduction of a ten-residue beta-hairpin. In general, these drastic mutations had little effect on the production and activity of NP-sub, indicating the feasibility of major structural rearrangements at the surface of proteins. In the most stable mutant, exhibiting an increase in thermal stability of 1.1 degree C, approximately 10% of the surface of NP-sub was modified. Several NP-sub variants carrying multiple mutations were constructed. Non-additive effects on thermal stability were observed, which were interpreted on the basis of a model for thermal inactivation, that emphasizes the importance of local unfolding processes for thermal stability.

Amino Acid Sequence↗

PubMed1993

A Bacillus subtilis large ORF coding for a polypeptide highly similar to polyketide synthases.

The nucleotide (nt) sequence of 13.6 kb of the outG locus of Bacillus subtilis, which maps at approximately 155 degrees between the genetic markers nrdA and polC, was determined. One putative coding sequence was identified corresponding to a large polypeptide of 4427 amino acids (aa). Structural organization at the nt and aa sequence level and extensive similarities of the deduced product, especially to EryA, suggest that the locus is potentially responsible for the synthesis of a polyketide molecule. The locus has been renamed pksX. Comparison of the deduced product with known fatty acid and polyketide synthases (PKS) suggested the presence of beta-ketosynthase, dehydratase, beta-ketoreductase and acyl-carrier protein domains. Preliminary data obtained with deletion mutants indicate that pksX is not an essential gene.

Amino Acid Sequence↗

PubMed1993

Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis.

The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilobases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.

Amino Acid Isomerases↗

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↗

PubMed1992

Malignant mesothelioma of the pleura in Trieste, Italy.

One hundred and seventy malignant pleural mesotheliomas seen at necropsy at the Institute of Pathological Anatomy of the Trieste University during the period 1968-1987 were reviewed. The series included 153 men and 17 women, aged between 33 and 92 years (median 70 years). Lifetime work histories were obtained from the patients' relatives by personal or telephone interviews in 162 cases. A majority of the male subjects had been employed in "naval" work, 99 people having worked in the ship-building industry, 19 in the navy and merchant marine, and 7 in docks. A variety of trades appeared in the remaining histories. Work histories were indicative of occupational exposure to asbestos in 150 cases. A further 5 patients with negative or insufficient data showed asbestos bodies in routine lung sections and 5 women had a history of domestic exposure. A majority of the patients had had their first exposure before 1950. The intervals between first exposure and death ranged from 14 to 71 years (median 48 years).

Adult↗

PubMed1992

Lack of detectable human T-cell lymphotropic virus type I sequences in samples from multiple sclerosis patients.

Recently, inconclusive results have followed the early data on the possible association between multiple sclerosis (MS) and human T-cell lymphotropic virus type I (HTLV-I) infection. For this reason, we examined this hypothesis using the polymerase chain reaction (PCR) to study samples of differing origin from Italian MS patients. In particular, we developed a systematic analysis of paraffin-embedded brain white matter from histologically defined lesions of 14 MS patients using PCR and primer sets specific for HTLV-I sequences; additionally, cerebrospinal fluids (CSFs) from 12 patients and peripheral blood mononuclear cells (PBMCs) from subjects at the early and late phase of the disease were investigated for free HTLV-I virions and specific proviral sequences, respectively. In agreement with some groups who reported lack of HTLV-I sequences in PBMCs of MS patients but in clear contrast with others, we failed to detect specific viral sequences using this broad approach.

Base Sequence↗

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

Treatment of malignant mesothelioma with epirubicin and ifosfamide: a phase II cooperative study.

From May 1988 to March 1990, 17 consecutive patients with histologically proven malignant mesothelioma were treated with epirubicin 75 mg/sqm i.v. on day 1 and ifosfamide 1.8 gr/sqm/day i.v. from day 1-5. Treatment was repeated every 3 weeks until progression. Fifty-three chemotherapy cycles were administered to the 17 patients treated (median, 3 cycles/patient). No complete responses, 1 partial response, 8 stable diseases and 8 progressions were noted. Toxocity was acceptable and no treatment-related deaths occurred. Actuarial median survival was 6 months. In this study, a combination of full doses of epirubicin and ifosfamide did not prove to be active in malignant mesothelioma.

Adult↗

PubMed1992

Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease.

Oligonucleotide-directed mutagenesis has been used to replace glycine residues by alanine in neutral protease from Bacillus subtilis. One Gly to Ala substitution (G147A) was located in a helical region of the protein, while the other (G189A) was in a loop. The effects of mutational substitutions on the functional, conformational and stability properties of the enzyme have been investigated using enzymatic assays and spectroscopic measurements. Single substitutions of both Gly147 and Gly189 with Ala residues affect the enzyme kinetic properties using synthetic peptides as substrates. When Gly replacements were concurrently introduced at both positions, the kinetic characteristics of the double mutant were roughly intermediate between those of the two single mutants, and similar to those of the wild-type protease. Both mutants G147A and G189A were found to be more stable towards irreversible thermal inactivation/unfolding than the wild-type species. Moreover, the stabilizing effect of the Gly to Ala substitution was roughly additive in the double mutant G147A/G189A, which shows a 3.2 degrees C increase in Tm with respect to the wild-type protein. These findings indicate that the Gly to Ala substitution can be used as a strategy to stabilize globular proteins. The possible mechanisms of protein stabilization are also discussed.

Alanine↗

PubMed1992

Transfection of E. coli with lambda DNA by electroporation.

In the ambit of the B. subtilis genoma sequencing and mapping project, we have set up an electroporation method to transfect E. coli cells with lambda DNA. This methodology presents features that make it preferable to traditional in vitro packaging for some purposes. Here we will illustrate the experimental procedure and the possible applications.

Bacteriophage lambda↗

PubMed1992

[Churg-Strauss vasculitis in a 53-year-old man].

The Authors describe a 53 year old patient, who, after suffering from asthma over the past 10 years, presented with abdominal pain, diarrhoea, dyspnea, petechias on lower limbs, and subsequently developed fever, polyneuritis, pericardial effusion and renal failure. Laboratory showed elevated IgE, presence of antinuclear antibodies to DNA, serum rheumatoid factor and peripheral eosinophilia. The clinical course was suggestive for systemic vasculitis; lung, skin biopsies and renal angiography confirmed this diagnosis: the association with asthma and eosinophilia fulfill the diagnosis of allergic angiitis and granulomatosis (Churg-Strauss syndrome).

Churg-Strauss Syndrome↗

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

Grafting of a calcium-binding loop of thermolysin to Bacillus subtilis neutral protease.

The surface loop which in the Bacillus subtilis neutral protease (NP) extends from amino acid residue 188 to residue 194 was replaced, by site-directed mutagenesis, with the 10-residue segment which in the homologous polypeptide chain of thermolysin (TLN) binds calcium-4 [Matthews, B. W., Weaver, L. H., & Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The mutant NP was isolated to homogeneity, and its structural, functional, calcium-binding, and stability properties were investigated. Proteolytic fragmentation with Staphylococcus aureus V8 protease of mutant NP was used to isolate and analyze the protein fragment encompassing the site of mutation, unambiguously establishing the effective insertion of the new 10-residue segment. Atomic absorption measurements allowed us to demonstrate that mutant NP binds three calcium ions instead of the two ions bound to wild-type NP, showing that indeed the chain segment grafted from TLN to NP maintains its calcium-binding properties. The mutant NP showed kinetic parameters essentially similar to those of the wild-type NP with Z-Phe-Leu-Ala-OH as substrate. The enzyme inactivation of mutant vs wild-type NP was studied as a function of free [Ca2+]. It was found that mutant NP was much less stable than the wild-type NP when enzyme solutions were dialyzed at neutral pH in the presence of [Ca2+] below 10(-3) M. On the other hand, the kinetic thermal stability to irreversible inactivation of mutant NP, when measured in the presence of 0.1 M CaCl2, was found to be increased about 2-fold over that of the wild-type NP. Thus, modulation of enzyme stability by free [Ca2+] in mutant NP correlates with similar findings previously reported for thermolysin. Overall, the results obtained indicate that protein engineering experiments can be used to prepare hybrid proteins on the basis of sequence and function analysis of homologous protein molecules and show the feasibility of engineering metal ion binding sites into proteins.

Amino Acid Sequence↗

PubMed1991

Irrigation for colostomized cancer patients: a rational approach.

Management of a colostomy by irrigation is simple and effective. Five hundred and nine patients having a colostomy for rectal cancer during the last 10 years were assessed by our Rehabilitation Department as potential candidates for irrigation. Fifty-two patients were considered unsuitable and 40 refused to learn the technique. The remaining 417 (82%) patients adopted the irrigation method. Of these, 97% achieved full continence of periods of 24 to 48 h. There was no case of colonic perforation. The median duration of irrigation was 230 days; 31.6% had been irrigating from 1 to 5 years and 10.3% for more than 5 years. There was a reduction of more than 50%/24 h in the number of appliances used by patients. Irrigation is a simple, safe and cost-effective means of managing a colostomy. With careful selection of patients, irrigation should be recommended as a routine part of rehabilitation.

Colostomy↗

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↗

PubMed1991

Production processes of recombinant IL-1 beta from Bacillus subtilis: comparison between intracellular and exocellular expression.

The human IL-1 beta coding sequence derived from a cDNA library was inserted into two different plasmid expression vectors, pSM214 and pSM308, which promote the synthesis of recombinant products intracellularly and exocellularly, respectively. The hybrid constructs pSM261 and pSM320 were obtained. Bacillus subtilis SMS118 was transformed with these plasmids and the recombinant strains were grown in 1 litre bioreactors. Different growth conditions were analyzed to optimize yields both in terms of biomass and IL-1 beta production. In the pSM261-harbouring strain, IL-1 beta was synthesized in the cytoplasm to levels ranging from 1 to 2.7 mg g-1 of cells, corresponding to up to 40 mg l-1 of the culture. In contrast, SMS118(pSM320) was able to secrete 0.27 mg of natural IL-1 beta per g of cells (6.7 mg l-1 of culture). Processes for the purification of IL-1 beta from the supernatant and the biomass of the two cultures were also developed and compared in terms of yield and simplicity of the purification schemes. From our data it turns out that the route of intracellular expression is very efficient and superior to the one which results in secretion of IL-1 beta. This indicates that the use of B. subtilis as a recombinant host in biotechnology is not strictly dependent on its ability to secrete proteins into the culture medium.

Amino Acid Sequence↗