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D Giorgi

Publications and source records attributed to D Giorgi.

At least 19 recordsLinked to original sources
PubMed1995

Direct selection of cDNAs using whole chromosomes.

We have developed a method for direct selection of cDNAs using whole chromosomes as target DNA. Double-strand cDNAs were synthesized from human fetal brain polyadenylated mRNAs. Flow-sorted chromosomes 17 and 19 were amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and used to capture ds cDNAs by an improved magnetic bead capture protocol. To demonstrate the capabilities of this method, the selected cDNAs were used as probes in FISH experiments. The selected cDNA populations specifically painted chromosomes 17 or 19 on metaphase spreads. These results demonstrate that it is possible to do chromosome painting using cDNA probes and that this method is a means to rapidly select expressed sequences encoded by any portion of the genome.

Brain↗
PubMed1995

Molecular cloning of a human genomic region containing the H blood group alpha(1,2)fucosyltransferase gene and two H locus-related DNA restriction fragments. Isolation of a candidate for the human Secretor blood group locus.

We have used the human H blood group alpha(1,2)fucosyltransferase (FUT1) cDNA to screen chromosome 19 cosmid libraries in a search for the human Secretor (Se) blood group gene (FUT2). One cosmid has been isolated that contains two distinct segments that cross-hybridize with FUT1. We have assembled a 100-kilobase (kb) cosmid contig, localized to 19q13.3, encompassing FUT1 and the two FUT1-related sequences, termed Sec1 and Sec2, for Secretor candidate 1 and 2. Sec1 and Sec2 are separated by 12 kb and are 65.5 kb and 35 kb apart, respectively, from the FUT1 gene. We used a cosmid-dependent direct cDNA selection method to clone a cDNA corresponding to a transcript that emanates from Sec2. This cDNA detects a 3.35-kb transcript in human tissues known to express the Se locus. Together with sequence and expression data reported in the accompanying article (Kelly, R. J., Rouquier, S., Giorgi, D., Lennon, G. G., and Lowe, J. B. (1995) J. Biol. Chem. 270, 4640-4649), these data demonstrate that Sec2 corresponds to the human Se blood group locus (FUT2). Our results furthermore define the physical relationship between the H and Se loci and confirm a hypothesis that these two loci represent distinct but closely linked alpha(1,2)fucosyltransferase genes.

Animals↗
PubMed1995

Sequence and expression of a candidate for the human Secretor blood group alpha(1,2)fucosyltransferase gene (FUT2). Homozygosity for an enzyme-inactivating nonsense mutation commonly correlates with the non-secretor phenotype.

Synthesis of soluble A, B, H, and Lewis b blood group antigens in humans is determined by the Secretor (Se) (FUT2) blood group locus. Genetic, biochemical, and molecular analyses indicate that this locus corresponds to an alpha(1,2)fucosyltransferase gene distinct from the genetically-linked H blood group alpha(1,2)fucosyltransferase locus. The accompanying paper (Rouquier, S., Lowe, J. B., Kelly, R. J., Fertitta, A. L., Lennon, G. G., and Giorgi, D. (1995) J. Biol. Chem. 270, 4632-4639) describes the molecular cloning and mapping of two human DNA segments that are physically linked to, and cross-hybridize with, the H locus. We present here an analysis of these two new DNA segments. One of these, termed Sec1, is a pseudogene, because translational frameshifts and termination codons interrupt potential open reading frames that would otherwise share primary sequence similarity with the H alpha(1,2)fucosyltransferase. The other DNA segment, termed Sec2, predicts a 332-amino acid-long polypeptide, and a longer isoform, that share 68% sequence identity with the COOH-terminal 292 residues of the human H blood group alpha(1,2)fucosyltransferase. Sec2 encodes an alpha(1,2)fucosyltransferase with catalytic properties that mirror those ascribed to the Secretor locus-encoded alpha(1,2)fucosyltransferase. Approximately 20% of randomly-selected individuals were found to be apparently homozygous for an enzyme-inactivating nonsense allele (Trp143-->ter) at this locus, in correspondence to the frequency of the non-secretor phenotype in most human populations. Furthermore, each of six unrelated non-secretor individuals are also apparently homozygous for this null allele. These results indicate that Sec2 corresponds to the human Secretor blood group locus (FUT2) and indicate that homozygosity for a common nonsense allele is responsible for the nonsecretor phenotype in many non-secretor individuals.

Alleles↗
PubMed1995

A model-based prediction of the impact on reduction in mortality by a breast cancer screening programme in the city of Florence, Italy.

The efficacy of breast cancer screening for women older than 50 years has been shown in several studies. Service screening is now ongoing or planned in several countries in Europe. MISCAN, a computer simulation programme, has been used to analyse data from the Florence District Programme (FDP) breast cancer experience. First, the model was fitted to the screening results for the period 1975-1986. A good correspondence between the model outcomes and the FDP results was achieved. It was then used to predict the impact on mortality of the new starting programme of the city of Florence (63,000 women, 50-69 years old). Assuming a 70% attendance rate, then for the city of Florence, 2563 screen-detected breast cancers are predicted for the period 1991-2020 out of the total number of 9095 breast cancers for all ages (28%). A total of 3720 deaths for breast cancer are expected without screening. An absolute reduction of 472 deaths (13%) is predicted for the whole population. The estimated number of years of life gained by screening until 2020 is 4354. Simulation by MISCAN has previously been a useful support tool for decision-making about screening. The present paper is the first based on a southern European experience. The possibility of applying MISCAN to predict the impact of a national programme in Italy is discussed.

Adult↗
PubMed1995

Accuracy and reliability of frozen section diagnosis in a series of 672 nonpalpable breast lesions.

Frozen section (FS) diagnosis was routinely performed in a large series of nonpalpable breast lesions from 1977 through 1991. The original FS diagnoses of 672 patients were classified in four categories (1 = benign lesion, 2 = in situ carcinoma, 3 = invasive carcinoma, 4 = deferred diagnosis) and compared with the diagnoses obtained at review of the permanent paraffin sections to estimate the accuracy of FS. A review of the mammographic pattern of the lesion was also performed. Frozen section diagnostic conclusion was deferred to permanent paraffin sections in only 22 cases (3.3%). Benign or malignant (grouping in situ and invasive carcinomas) FS diagnoses were accurate in 623 of 650 cases (95.8%). Overall, the prevalence of malignant lesions was 44.8% with a benign/malignant ratio of 1.2. The diagnosis was modified on the basis of permanent sections in 27 cases (4.2%) with three false positives and 24 false negatives. Sensitivity and specificity of FS diagnoses were 91.7 and 99.2%, respectively. When the comparison between FS and histologic diagnoses was analyzed according to the mammographic pattern, sensitivity among patients with microcalcifications as the only alteration was lower (88.8%) than among patients with opacities (94.9%). On the basis of these results, FS is to be considered a feasible and reliable diagnostic procedure in nonpalpable breast lesions, particularly in cases excised because of a mammographic opacity that is identifiable on gross examination of the surgical specimen.

Breast Neoplasms↗
PubMed1991

Rat pancreatic stone protein messenger RNA. Abundant expression in mature exocrine cells, regulation by food content, and sequence identity with the endocrine reg transcript.

We used a cDNA encoding the human pancreatic stone protein (PSP-S), the secretory inhibitor of CaCO3 crystal growth, as a probe for cloning rat PSP-S messenger RNA. Overlapping clones gave a mRNA sequence of 783 nucleotides encoding a preprotein of 165 amino acids including a prepeptide of 21 amino acids. Rat and human PSP-S showed 70% identity, and the mature proteins had the same length. PSP-S mRNA concentration was measured in the pancreas of rats adapted to diets containing 15, 25, or 70% protein. Compared with the 15% protein diet, concentration increased 3 and 12 times with the diets with 25 and 70% protein, respectively, which is 3 times higher than for serine proteases. A complete sequence identity was observed between the rat PSP-S transcript and the reg mRNA described by Terazono et al. (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114), which is expressed in regenerating pancreatic islets but not in mature islets. A specific role of the reg protein in islet regeneration was suggested. We found that PSP-S (reg) mRNA concentration was indeed increased in isolated regenerating islets. Yet, a transient increase was also observed in exocrine tissue during the initial phase of regeneration following pancreatectomy or acute pancreatitis, suggesting increased expression during cell dedifferentiation. It is concluded that, in mature pancreas, expression of the reg/PSP-S gene occurs primarily in acinar cells. The gene product, which encodes a secretory protein inhibiting CaCO3 crystal growth in juice, is unlikely to play a specific role in islet regeneration.

Amino Acid Sequence↗
PubMed1989

Secretory pancreatic stone protein messenger RNA. Nucleotide sequence and expression in chronic calcifying pancreatitis.

The pancreatic stone protein and its secretory form (PSP-S) are inhibitors of CaCO3 crystal growth, possibly involved in the stabilization of pancreatic juice. We have established the structure of PSP-S mRNA and monitored its expression in chronic calcifying pancreatitis (CCP). A cDNA encoding pre-PSP-S has been cloned from a human pancreatic cDNA library. Its nucleotide sequence revealed that it comprised all but the 5' end of PSP-S mRNA, which was obtained by sequencing the first exon of the PSP-S gene. The complete mRNA sequence is 775 nucleotides long, including 5'- and 3'- noncoding regions of 80 and 197 nucleotides, respectively, attached to a poly(A) tail of approximately 125 nucleotides. It encodes a preprotein of 166 amino acids, including a prepeptide of 22 amino acids. No overall sequence homology was found between PSP-S and other pancreatic proteins. Some homology with several serine proteases was observed in the COOH-terminal region, however. The mRNA levels of PSP-S, trypsinogen, chymotrypsinogen, and colipase in CCP and control pancreas were compared. PSP-S mRNA was three times lower in CCP than in control, whereas the others were not altered. It was concluded that PSP-S gene expression is specifically reduced in CCP patients.

Adolescent↗
PubMed1988

Cancer mortality in migrant populations within Italy.

Italian death certification rates from all causes of death, all diseases of the circulatory system, all neoplasms, and cancers of the upper digestive and respiratory tract, stomach, intestines, lung and breast in middle age (45-64 years) were analyzed according to selected geographic areas of birth and residence at death. For total cancer mortality and most neoplasms considered, the rates in middle age were closer to those of place of birth than to those of area of residence, although this pattern was more evident for some sites (e.g., mouth or pharynx, esophagus, larynx, stomach or bladder) than for others (e.g., intestines or breast). In most cases, migration had an adverse effect on cancer rates, and the lowest mortality was reported among stable populations (i.e., those with the same area of birth and death). These findings are discussed in relation to the major migration fluxes within Italy during the current century. Moreover, these analyses give information on the quality of Italian cancer death certification, since the observation that area of birth is often a more important determinant of cancer rates than area of residence provides indirect evidence that cancer death certification in various Italian geographic areas is satisfactorily reliable and consistent.

Adult↗
PubMed1987

Effect of cholinergic agonists and antagonists on gallbladder volume in fasting man.

The effect of direct cholinergic stimulation and blockade on gallbladder volume, determined by real-time ultrasonography (RUS), was evaluated in twenty normal, fasting subjects. Eleven subjects received atropine sulphate or placebo and 9 subjects a series of 3 injection of prostigmine, bethanechol or placebo, randomly assigned, at intervals of 24 h. RUS was performed under basal conditions after fasting for 12 h and every 5 min after drug injection up to 45 min in the atropine study and up to 60 min after prostigmine and bethanechol. There was no significant variation from fasting gallbladder volume after placebo in either group. After atropine sulphate gallbladder volume at first decreased and then significantly increased. With bethanechol and prostigmine, the volume fell significantly to a trough after 30 to 35 min, and then it returned to the basal value within 60 min. It is suggested that cholinergic mediation is involved in maintaining fasting tone in the gallbladder and that cholinergic stimulation causes contraction of the gallbladder by a direct effect.

Adult↗
PubMed1987

Amylase and chymotrypsinogen synthesis and secretion by the anesthetized rat pancreas.

To investigate the origin of nonparallel secretion, pancreatic juice was collected in the anesthetized rat, during infusion of [3H]phenylalanine. In the basal state, the amylase to chymotrypsinogen ratios of activities and of [3H] incorporations were 3.5 and 2.5 times higher than in the homogenate, respectively. Both ratios decreased to the value in the homogenate upon caerulein stimulation (600 ng/kg). Inhibition of protein synthesis by cycloheximide (15 ng/kg) did not alter enzyme secretion ratios, and depressed basal protein output only partly, suggesting similar secretory pathways for basal and stimulated secretion. Finally, when [3H]phenylalanine was given before anesthesia, the amylase to chymotrypsinogen ratio of incorporations was again higher in basal secretion than in homogenate, even when further protein synthesis was blocked by cycloheximide before urethane injection. Hence, basal secretion comes from a pancreatic compartment which is functional, although minor, in the conscious animal, and shows a higher rate of amylase synthesis, compared to chymotrypsinogen, than the rest of the gland. It could consist of a subpopulation of acinar cells.

Amylases↗
PubMed1986

Regulation of concentrations of mRNA for amylase, trypsinogen I and chymotrypsinogen B in rat pancreas by secretagogues.

Regulation of pancreatic gene expression by the secretory stimulants cholecystokinin-pancreozymin, caerulein and pilocarpine was studied in the rat, by using cloned cDNA probes to quantify concentrations of specific mRNAs (amylase, trypsinogen I and chymotrypsinogen B). It is concluded that long-term pancreatic stimulation results in pre-translational regulation of secretory-protein gene expression, with a preferential accumulation of RNA transcripts encoding serine proteinases, compared with that for amylase.

Amylases↗
PubMed1986

Overproduction of the first three enzymes of pyrimidine nucleotide biosynthesis in Drosophila cells resistant to N-phosphonacetyl-L-aspartate.

Drosophila cells were treated in vitro with N-phosphonacetyl-L-aspartate (PALA) which is a specific inhibitor of aspartate transcarbamylase, the second enzyme of the pyrimidine biosynthetic pathway. By stepwise selection using increasing amounts of this inhibitor, PALA-resistant (PALAr) stable clones have been isolated. Enzymatic activities of aspartate transcarbamylase, carbamyl phosphate synthetase and dihydro-orotase, borne by the same multifunctional protein, CAD, are increased 6-12-fold in these resistant clones compared with parental cells. The aspartate transcarbamylase in PALAr cells is shown by physical, kinetic and immunological criteria to be normal. The data from immunotitration and immunoblotting experiments indicate that the increased enzyme activities result from the overproduction of CAD.

Amidohydrolases↗
PubMed1986

Newly synthesized amylase, lipase and serine proteases are transported at different rates in rat pancreas.

The rates of intracellular transport of newly synthesized amylase, lipase, trypsinogen and chymotrypsinogen were compared in the anaesthesized rat pancreas by measuring their rates of secretion while keeping their relative rates of synthesis in a steady state. It is concluded that pancreatic proteins are not transported all at the same rate through the acinar cell. Among the four enzymes studied, proteolytic enzymes are transported faster than lipase, and amylase is the slowest. The relative rates of transport of these enzymes are not altered by secretory stimulation, since similar results were obtained in the basal state and under constant caerulein infusion (300 ng/kg/h).

Amylases↗
PubMed1985

Regulation of proteolytic enzyme activities and mRNA concentrations in rat pancreas by food content.

Regulation by food content of the expression of genes encoding pancreatic proteases was studied in rats fed diets containing 15%, 25% or 70% protein (w/w) (diet I, II and III). Trypsin, chymotrypsin and elastase activities in pancreas were 1.4, 2.8 and 2 times higher in diet III than in diet I whereas carboxypeptidase A level was unchanged. As compared to diet I, the pancreatic concentration of mRNAs encoding trypsinogen I and chymotrypsinogen B, measured by filter hybridization to specific cDNA probes, were found respectively 3.6 and 3.9 times higher in diet III, and 1.9 and 2.6 times higher in diet II. Elastase I mRNA concentration was 1.8 times higher in diet III, but unchanged in diet II. Procarboxypeptidase A mRNA concentration was not affected. It is concluded to a coordinate pre-translational regulation of serine protease genes expression by the protein content of diet, differing however in amplitude and sensitivity among the three species of enzymes studied.

Animals↗
PubMed1985

Pancreatic stone protein. I. Evidence that it is encoded by a pancreatic messenger ribonucleic acid.

We have previously shown that the pancreatic stone protein (PSP) is an inhibitor of calcium carbonate crystal growth and may participate in the stabilization of the normally supersaturated pancreatic juice. Our aim in this study was to determine if PSP is a normal secretory product of the human pancreas by determining if the normal human pancreas contains a messenger RNA coding for PSP. Human pancreatic messenger RNAs were used to direct protein synthesis in a cell-free translation system. Immunoprecipitation of translation products with a monospecific antibody directed against the PSP yielded a product migrating as a single homogeneous band on sodium dodecyl sulfate-polyacrylamide gels. This product has a molecular weight of 16,000, the value expected for pre-PSP. Products selected by immunoprecipitation with antitrypsin-1 antibodies also migrated as a single band, with a molecular weight of 27,000. It is concluded that a messenger RNA coding for pre-PSP, distinct from the messenger RNA coding for pretrypsinogen, is present in human pancreas. These results support the hypothesis that PSP is a molecular entity, and not a degradation product of trypsinogen 1 or another pancreatic protein.

Animals↗
PubMed1985

Human erythrocyte hexokinase deficiency: a new variant with abnormal kinetic properties.

A 14-month-old child who had a haemolytic episode when he was 5 years old, and with psychomotor retardation, was found to have decreased red cell hexokinase activity. The mutant enzyme was characterized by an increased affinity for glucose associated with an increased inhibition constant for glucose-1,6-diphosphate. Affinity for Mg ATP2-, heat stability and pH-optimum were normal. The isozymic pattern of the red cell enzyme was normal but all the molecular forms were present in reduced amounts. The kinetics of decay of hexokinase during cell ageing was also normal. Glucose consumption of the hexokinase deficient cells was 60-65% of the controls while the amount metabolized through the hexose monophosphate shunt was unchanged. Red cell 2,3-diphosphoglycerate and glucose-6-phosphate levels were normal in the proband but reduced in the erythrocytes of his parents, who were heterozygous for the defect but had normal haematological data. Comparison with the 13 previously reported cases of hexokinase deficiency confirms the broad phenotypic variability that characterizes this disorder.

Blood Glucose↗
PubMed1984

Apple II PASCAL programs for molecular biologists.

A collection of PASCAL programs designed for the Apple II microcomputer is presented. These DNA sequence handling and analysis programs are interactive and may be used even by people with no computer experience. The package allows the user to enter a sequence from the keyboard, to modify it, to generate the reverse complement, to create new sequences from parts of other ones, to display or print sequences in various formats. Some analysis tasks are also performed: Translation, searches for restriction sites, for homology with subsequences, either perfect or with an adjustable match percentage. In addition, two programs are also included: The first one allows DNA data sequences generated with a BASIC program under the CP/M operating system to be used with these PASCAL programs. The second one is designed for the automatic assembly of DNA fragments sequences, obtained with the GILBERT-MAXAM or M13 techniques, into a complete sequence.

Amino Acid Sequence↗