Cloning and sequencing of the dnaK region of Streptomyces coelicolor A3(2)

Abstract The dnaK homologue of Streptomyces coelicolor A3(2) strain M145 has been cloned and sequenced. Nucleotide sequence analysis of a 2.5-kb region revealed an open reading frame (ORF) encoding a predicted DnaK protein of 618 amino acids (Mr = 66 274). The dnaK coding sequence displays extreme codon bias and shows a strong preference for CGY and GGY, for Arg and Gly codons, respectively. The predicted DnaK sequence has a high Lys:Arg ratio which is not typical of streptomycete proteins. The region immediately downstream from dnaK contains an ORF for a GrpE-like protein; the predicted start codon of grpE overlaps the last two codons of dnaK, indicating that the two genes are translationa…

Sordità neurosensoriale non sindromica: genotipi della Cx26 (GJB2) in famiglie siciliane

EVALUATION OF STABILITY AND ENZYMATIC ACTIVITIES OF PROTEOLYTIC ENZYMES USED IN PANCREATIC ISLET TRANSPLANTATION

In pancreatic islets purification, for cell therapy applications, the major enzymes used are obtained from Clostridium hystoliticum; class I and class II collagenases (Coll-G and Coll-H). In a well defined composition Coll-G/Coll-H together enzymes working on hydrophobic amminoacid, the neutral protease (Dispase) or the thermolysin (Thermostable Neutral Protease), are used in Langerhans islets purification. By electrophoresis and gelatin zymography approaches, in combination to densitometry quantitative valuation we have compared in composition, stability and autodigestion processes C. hystoliticum collagenases, Neutral protease and Thermolysin from two different producers, Roche and Serva.…

TheSCH9 protein kinase mRNA contains a long 5′ leader with a small open reading frame

The SCH9 yeast gene, that was previously identified as a suppressor of cdc25 and ras1- ras2-ts temperature-sensitive mutants, encodes a putative protein kinase that positively regulates the progression of yeast cells through the G1 phase of the cell cycle. We have determined the structure of the SCH9 transcription unit, using primer extension and S1 mapping techniques. The corresponding mRNA included an unusually long 5' region of more than 600 nucleotides preceding the major open reading frame (ORF). While the latter corresponded to a protein of 824 amino acids, an upstream open reading frame (uORF) within the 5' leader could potentially encode a 54 amino acid peptide. To investigate the r…

Analisi delle mutazioni del gene Cx26 (GJB2) in famiglie siciliane con sordità neurosensoriale non sindromica

Screening of subtelomeric rearrangements in autistic disorder: identification of a partial trisomy of 13q34 in a patient bearing a 13q;21p translocation.

Within the framework of a FISH screening protocol to detect cryptic subtelomeric rearrangements in autistic disorder (AD), a patient bearing three copies of the subtelomeric portion of the q arm of chromosome 13 has been identified. Beside AD, the patient also has severe mental retardation and displays several dysmorphic features. Further FISH analyses revealed that the trisomy was caused by the translocation of a 13q subtelomeric fragment to the acrocentric tip of one chromosome 21 [46,XY.ish der(21) t(13;21) (q34;p13)(D13S1825+)]. Gene dosage experiments carried out with three multiallelic polymorphisms of the subtelomeric region of chromosome 13q showed that the putative length of the tr…

Population Data of Nine STR Loci From Western Sicily, Italy

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 μg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. …

L’analisi dei microsatelliti nel condensato aereo polmonare di pazienti con patologie tumorali polmonari come nuova metodologia per la diagnosi precoce.

Screening of subtelomeric rearrangements in autism spectrum disorder. Identification of a partial trisomy of 13q in a patient

Screening of subtelomeric rearrangements in autism spectrum disorder. Identification of a partial trisomy of 13q in a patient.

Phosphorylation of an Overexpressed Yeast Ras2 Protein During the G1 Phase of the Cell Cycle

RAS proteins regulate growth and differentiation in evolutionarily distant systems such as vertebrates and yeast (for reviews, see Tamanoi, 1988; Gibbs and Marshall, 1989; Broach and Deschenes, 1990). At the moleular level, a key function of the yeast RAS1 and RAS2 proteins (collectively referred to as RAS) is to positively regulate the production of cyclic AMP at the onset of the G1 phase of the cell cycle (Toda et al., 1985; De Vendittis et al., 1986). At this stage, RAS proteins are transiently activated by the noncovalent binding of a GTP molecule. Reversal of the effect occurs by the hydrolytic splitting of the ’γ-phosphate of GTP, that leaves a functionally inactive RASGDP complex, th…

A new method to valued efficiency of enzyme blend for pancreas tissue digestion.

One of the best successful example of cell therapy is represented by islet transplantation since the '90. However islet isolation methods are not completely standardized yet. More than half of isolation procedures failed to isolate adequate islets for transplantation, due to variable pancreas condition, and to unpredictable enzymatic blend efficiency. Enzymes used for pancreas digestion are purified from Clostridium histolyticum; these enzymes has a broad substrate specificity and potent collagenolytic activity compared to vertebrate collagenases. However, a major obstacle in human islet isolation successful is due to the variability in composition and concentration of the collagenases used…

TIPIZZAZIONE DEL DNA DI DITTERI CAMPIONATI SU UN CORPO RINVENUTO IN SICILIA

L’entomologia forense, come ormai ampiamente noto, viene utilizzata routinariamente nelle indagini forensi, in particolare in quei casi nei quali i classici dati tanatocronologici (ipostasi, rigidità, temperatura corporea, ecc.) non possono essere d’ausilio per l’identificazione dell’epoca della morte. Gli insetti campionati su resti cadaverici, per potere essere utilizzati a fini forensi, necessitano di una precisa identificazione di specie, dato che ciascuna specie si caratterizza per una differente durata del ciclo vitale. Gli insetti che colonizzano il cadavere, soprattutto nelle prime fasi di colonizzazione, hanno una distribuzione abbastanza uniforme in tutto il mondo, tuttavia è poss…

Comparative study on enzymatic activity and molecules stability of some commercial proteolytic enzymes used in pancreatic islet isolation.

In pancreatic islets purification, for cell therapy applications, the major enzymes used are obtained from Clostridium hystoliticum; class I and class II collagenases (COLL G and COLL H). They are used in a defined tissue dissociation enzyme (TDE) mixtures together neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). The TDE mixtures were in part responsible for the success of the Edmonton protocol; however, just to now, people working in islets purification found discrepancy in an application to another one. This variability in application see in the enzymatic blend composition the higher accused, such as the contamination from endotoxines due to extractive production…

COLLAGENASI RICOMBINANTI DI C. HISTOLYTICUM E METODO PER LA LORO PRODUZIONE.

La presente descrizione si riferisce alla produzione di collagenasi ricombinanti, in particolare è qui descritto un metodo per la produzione di collagenasi Col ricombinante di Clostridium histolyticum caratterizzato da una resa maggiore a circa 140mg/l di coltura di detta collagenasi in forma biologicamente attiva, collagenasi prodotte mediante detto metodo, composizioni che le comprendono e loro uso.

L'Analisi dei polimorfismi del DNA tra le metodologie di identificazione dei bovini. Analisi dei rapporti di filiazione in un'allevamento italiano

L'Analisi dei polimorfismi del DNA tra le metodologie di identificazione dei bovini. Analisi dei rapporti di filiazione in un'allevamento italiano. In 22° Convegno Nazionale Genetisti Forensi italiani.

Suggestive evidence for association of D2S2188 marker (2q31.1) with autism in 143 Sicilian (Italian) TRIO families

We have screened 143 Sicilian (Italian) families with one autistic child to verify, by a linkage disequilibrium approach, the involvement of the 2q31.1 region in the cause of the disease in these families. Our study design includes the use of intrafamilial association to prevent a population stratification bias and ethnic homogeneity of the sample. The results of our analysis provided suggestive evidence of the occurrence of transmission disequilibrium between autism and the D2S2188 polymorphism in Sicilian TRIO families, a finding which provides further and independent support to the hypothesis of the existence of a susceptibility gene (or genes) for autism on chromosome 2q.

ANALISI DI POLIMORFISIMI STR NELLA SICILIA OCCIDENTALE

ANALISI DI POLIMORFISIMI STR NELLA SICILIA OCCIDENTALE.

GENETICA UMANA

Lack of association of HOXA1 and HOXB1 mutations and autism in Sicilian (Italian) patients

Lack of association of HOXA1 and HOXB1 mutations and autism in Sicilian (Italian) patients

C. HYSTOLITICUM RECOMBINANT COLLAGENASES AND METHOD FOR THE MANUFACTURE THEREOF

The present invention relates to the production of recombinant collagenases, and in particular describes a method for the production of recombinant clostridium histolyticum collagenases Col characterized by a yield higher than approximately 140 mg/l of culture of said collagenases in soluble, biologically active form, collagenases produced by this method, compositions comprising these collagenases and the use thereof.

Differential gene expression in p53-mediated G(1) arrest of human fibroblasts after gamma-irradiation or N-phosphoacetyl-L-aspartate treatment.

In human fibroblasts, N:-phosphoacetyl-L-aspartate (PALA) and gamma-radiation induce reversible and irreversible p53-mediated G(1) cell cycle arrest, respectively. By coupling the premature chromosome condensation technique to fluorescence in situ hybridization, we found no evidence of DNA damage after PALA treatment. We used representational difference analysis (cDNA-RDA) to study changes in gene expression after PALA treatment and gamma-radiation in normal human fibroblasts. The mammary-derived growth inhibitor (MDGI) gene was expressed in PALA-treated cells. Ectopic MDGI expression arrested PALA-treated but not irradiated RKO cells. Expression of an antisense RNA against MDGI resulted in…

Role of E-selectin in the modulation of metastatic phenotype

Role of E-selectin in the modulation of metastatic phenotype

Role of S128R polymorphism of E-selectin in colon metastasis formation.

The extravasation of cancer cells is a key step of the metastatic cascade. Polymorphisms in genes encoding adhesion molecules can facilitate metastasis by increasing the strength of interaction between tumor and endothelial cells as well as impacting other properties of cancer cells. We investigated the Ser128Arg (a561c at the nucleotide level) polymorphism in the E-selectin gene in patients with metastatic colon cancer and its functional significance. Genotyping for a561c polymorphism was performed on 172 cancer patients and on an age-matched control population. The colon cancer group was divided into groups with (M+) and without observable metastasis (M−). For in vitro functional assays, …

An association analysis to identify genetic variants linked to asthma and rhinoconjunctivitis in a cohort of Sicilian children

Abstract Asthma and rhino-conjunctivitis are common chronic diseases in childhood. In this cross-sectional study, we performed a gene association analysis with current asthma and rhino-conjunctivitis in a cohort of Sicilian children aged 10–15 years. Overall, our findings reveal the importance of different genetic variants at 4p14, 16p12.1, 17q12, 6p12.2 and 17q21.1, identifying possible candidate genes responsible for susceptibility to asthma and rhino-conjunctivitis.

A new method to value efficiency of enzyme blends for pancreatic tissue digestion.

Islet transplantation, since the 90’s, has been resulting to be one of the best successful example of human cell therapy. Nevertheless, islet isolation procedure is not completely standardized; in fact, more than fifty percent of islets procedures don’t arrive to their transplantation. This is due both to the variability of donor’s pancreas and to an unpredictable enzymatic blend efficiency. Enzymes used in pancreas digestion are extracted from Clostridium histolyticum bacteria and digest several substrates. In particular they have strong collagenolytic activity compared to vertebrate collagenases. However, several impediments persist in human islet isolation success probably due to the var…

Genetica e laboratorio di medicina legale

Le basi dell'organizzazione biologica

UNA NUOVA METODICA DI ESTRAZIONE DEL DNA PER USO FORENSE DA REPERTI OSSEI CARBONIZZATI

UNA NUOVA METODICA DI ESTRAZIONE DEL DNA PER USO FORENSE DA REPERTI OSSEI CARBONIZZATI

Role of S128R polymorphism of E-Selectin in colon metastasis formation.

Role of S128R polymorphism of E-Selectin: in colon metastasis formation.

Analysis of the gastrin-releasing peptide receptor gene in Italian patients with autism spectrum disorders

The gastrin-releasing peptide receptor (GRPR) was implicated for the first time in the pathogenesis of Autism spectrum disorders (ASD) by Ishikawa-Brush et al. [Ishikawa-Brush et al. (1997): Hum Mol Genet 6: 1241-1250]. Since this original observation, only one association study [Marui et al. (2004): Brain Dev 26: 5-7] has further investigated, though unsuccessfully, the involvement of the GRPR gene in ASD. With the aim of contributing further information to this topic we have sequenced the entire coding region and the intron/exon junctions of the GRPR gene in 149 Italian autistic patients. The results of this study led to the identification of four novel point mutations, two of which, that…

WITHDRAWN: Corrigendum to ‘Development of an Italian RM Y-STR haplotype database: results of the 2013 GEFI collaborative exercise’ [Forensic. Sci. Int. Genet. 15 (2015) 56-63]

An inconsistency in the nomenclature used for the rapidly mutating (RM) Y-chromosomal short tandem repeat (Y-STR) marker DYS449 was noted in the above paper. In this paper, the DYS449 allele nomenclature introduced by Ballantyne et al. was used, instead of that described by Redd et al. and subsequently adopted by the International RM Y-STR User Group and in the AMPFlSTR® YFiler Plus kit.

Molecular analyses of NLGN3, NLGN4, GRPR genes in a Sicilian autistic population

Molecular analyses of NLGN3, NLGN4, GRPR genes in a Sicilian autistic population.

Analisi di Polimorfismi STR nella Sicilia Occidentale

Development of an Italian RM Y-STR haplotype database: Results of the 2013 GEFI collaborative exercise.

Recently introduced rapidly mutating Y-chromosomal short tandem repeat (RM Y-STR) loci, displaying a multiple-fold higher mutation rate relative to any other Y-STRs, including those conventionally used in forensic casework, have been demonstrated to improve the resolution of male lineage differentiation and to allow male relative separation usually impossible with standard Y-STRs. However, large and geographically-detailed frequency haplotype databases are required to estimate the statistical weight of RM Y-STR haplotype matches if observed in forensic casework. With this in mind, the Italian Working Group (GEFI) of the International Society for Forensic Genetics launched a collaborative ex…

Identification of proteolytic enzymes from Eriphia verrucosa and Palinurus elephas capable to degrade gliadin

In small intestinal disease, coeliac sprue, proline-rich gluten peptides from wheat, rye and barley are relatively resistant to gastrointestinal digestion, and therefore remain in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. Since most serine endopeptidases are unable to hydrolyse proline residues, proline specific proteases may be therapeutic keys in digestive diseases. Partial hydrolysis reduces the risk of allergenic sensitization while total hydrolysis ensures the elimination of the allergenicity of whey protein (Villad´oniga and others 2007). Kimoto and others (1998) reported that 18-, 31-, 37- and 58-kDa wheat allergens were recognized by the …

Analisi molecolare del gene GRPR in una popolazione autistica siciliana

Analisi molecolare del gene GRPR in una popolazione autistica siciliana.

Analisi molecolare di 3 geni (NLGN3, NLGN4, GRPR) localizzati sul cromosoma X in una popolazione autistica siciliana.

Analisi molecolare di 3 geni (NLGN3, NLGN4, GRPR) localizzati sul cromosoma X in una popolazione autistica siciliana..

Il polimorfismo S128R dell’E-Selectina: analisi genotipica e caratterizzazione funzionale nell’interazione cellule tumorale-endotelio.

Mutazioni: tipi, origini, conseguenze

A new method to value efficiency of enzyme blends for pancreas tissue digestion

In pancreatic islets isolation for cell therapy the major enzymes used are obtained from Clostridium hystoliticum: class I and class II collagenases. They are used in a defined tissue dissociation enzyme mixture together with neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). However, just to now, people working in islets production found variable outcomes in isolation procedures mainly due to large variability in enzymatic blend composition and efficacy. Using electrophoresis and gelatin zymography approaches together with densitometry evaluation assays we compared the composition, stability and auto-digestion processes of C. hystoliticum collagenases, Neutral prote…