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R Giorda

Publications and source records attributed to R Giorda.

At least 37 records · Page 2Linked to original sources
PubMed2000

Inverted duplications are recurrent rearrangements always associated with a distal deletion: description of a new case involving 2q.

We studied the case of a subject with an inverted duplication of 40 cM of 2q33-q37 concurrent with a 10 cM deletion of the distal 2q, the latter not being detectable by cytogenetics. Microsatellite analysis demonstrated the absence of maternal alleles in the deleted region and a double dosage for one of the maternal alleles in the duplication region. We hypothesised that this type of rearrangement occurs at meiosis I, while the two homologues are synapsed for most of their length. The presence of inverted duplicons in the same chromosome arm would favour the partial refolding of one homologue into itself so leading to the intrachromatid synapsis and recombination of the inverted repeats. The arising recombinant chromosome is deleted for the region beyond the most distal repeat and with the chromatids joined together at the level of the region located between the two duplicons. At meiosis II, the two linked chromatids can join the opposite poles provided that a breakage between the two centromeres occurs leading to a duplicated/deleted chromosome and a simply deleted chromosome. This model can be extended to all the so-called inverted duplication cases and to part of the terminal deletions. In fact the finding that, in our invdup(2q), the entire 40 cM duplication region involves only one of the two maternal alleles, indeed indicates that the abnormal crossover occurs between sister chromatids. The phenotype associated with our 2q rearrangement led us to narrow the critical region for the Albright-like syndrome to 10 cM in the subterminal 2q region.

Child↗
PubMed1999

Constitutive expression and role of the TNF family ligands in apoptotic killing of tumor cells by human NK cells.

Natural killer cells mediate spontaneously secretory/necrotic killing against rare leukemia cell lines and a nonsecretory/apoptotic killing against a large variety of tumor cell lines. The molecules involved in nonsecretory/apoptotic killing are largely undefined. In the present study, freshly isolated, nonactivated, human NK cells were shown to express TNF, lymphotoxin (LT)-alpha, LT-beta, Fas ligand (L), CD27L, CD30L, OX40L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), but not CD40L or nerve growth factor. Complementary receptors were demonstrated to be expressed on the cell surface of solid tumor cell lines susceptible to apoptotic killing mediated by NK cells. Individually applied, antagonists of TNF, LT-alpha1beta2, or FasL fully inhibited NK cell-mediated apoptotic killing of tumor cells. On the other hand, recombinant TNF, LT-alpha1beta2, or FasL applied individually or as pairs were not cytotoxic. In contrast, a mixture of the three ligands mediated significant apoptosis in tumor cells. These findings demonstrate that human NK cells constitutively express several of the TNF family ligands and induce apoptosis in tumor cells by simultaneous engagement of at least three of these cytotoxic molecules.

Antigens, CD↗
PubMed1999

Effects of serotonin transporter promoter genotype on platelet serotonin transporter functionality in depressed children and adolescents.

OBJECTIVE: To investigate possible associations between serotonin transporter (5-HTT) promoter genotypic variants (l/l, l/s, and s/s) and differential regulation of platelet 5-HTT functionality parameters in a group of drug-naive depressed children and adolescents and healthy controls. METHOD: Children and adolescents with major depression (n = 18) defined by DSM-III-R criteria and normal controls (n = 21) were assessed both for platelet serotonin functionality and for genotypic variants on 5-HTT promoter region. Four parameters were considered: (1) serotonin uptake rate (Vmax); (2) serotonin dissociation constant (K(m)); (3) paroxetine binding and density of site (Bmax); and (4) paroxetine dissociation constant (Kd). RESULTS: Depressed children had lower Vmax and K(m). Control subjects with l/l genotype had significantly higher Vmax than control subjects with l/s and s/s genotype. Control subjects with l/l genotype also had significantly higher Vmax than their depressed homologs. In contrast, Vmax was not significantly different between depressed and nondepressed subjects who carried the other 2 genotypes. The 5-HTT promoter genotype, diagnoses, or their interaction had no effect on the other serotonin parameters. CONCLUSIONS: While showing a significant decrease of Vmax and K(m) in a group of drug-naive depressed children and adolescents, these data suggest that l/l genotype has a substantial effect on the decrease of Vmax during a depressive episode.

Adolescent↗
PubMed1997

The cytoplasmic domain of rat NKR-P1 receptor interacts with the N-terminal domain of p56(lck) via cysteine residues.

NKR-P1 is a type II transmembrane protein which acts as an activation receptor on natural killer (NK) cells. The cytoplasmic domains of the CD4, CD8 and 4-1BB receptors contain the sequence Cys-X-Cys-Pro which is directly involved in coupling to another pair of cysteines in the N-terminal domain of the src family tyrosine kinase p56(lck). The cytoplasmic domain of NKR-P1 in rodents also contains the Cys-X-Cys-Pro sequence, but the capacity of the receptor to bind p56(lck) is presently unknown. We tested for direct coupling between these proteins using both protein biochemistry and the yeast two-hybrid technique. Immunoprecipitation studies showed that p56(lck) can be co-immunoprecipitated with NKR-P1 from a rat NK tumor cell line. In addition, the cytoplasmic domain of NKR-P1 interacted with the N-terminal domain of p56(lck) in yeast as assessed by reporter gene activation. Integrity of the cysteine pairs in both proteins was critical in mediating the interaction. The experiments suggest that the association of p56(lck) with NKR-P1 is somewhat weaker than the p56(lck) association with CD8alpha, but of much lower avidity than between CD4 and p56(lck). This could reflect a higher activation threshold for the NKR-P1 and CD8 receptors, which are involved in cytolytic responses, compared to CD4 which is involved in T cell helper function.

Amino Acid Sequence↗
PubMed1996

Analysis of the structure and expression of the augmenter of liver regeneration (ALR) gene.

BACKGROUND: The gene encoding the hepatotrophic factor Augmenter of Liver Regeneration (ALR) has recently been cloned in the rat. The availability of the mouse form of ALR would allow the analysis of the role of this factor in the physiology of liver and other organs, while the identification of the human homolog would allow the transfer of the great wealth of information that has been generated in animal models to clinically oriented pilot trials, and eventually the therapeutic application of this information. MATERIALS AND METHODS: Standard molecular biology approaches have been used to determine the genomic structure of the ALR gene in the mouse, and to characterize the ALR transcript and its protein product. The human ALR cDNA was also isolated and the amino acid sequence of the human gene product deduced. The mapping of mouse and human ALR genes on mouse and human chromosomes was then completed. RESULTS: The protein coding portion of the mouse ALR gene is comprised of three exons, the first containing the 5' untranslated sequence and the initial 18 bases after the ATG translation initiation codon, the second exon encompasses 198 bases, and the third exon contains the remaining portion of the protein coding sequence. Rat, mouse, and human ALR genes (and protein products) were found to be highly conserved and preferentially expressed in the testis and in the liver. The ALR gene maps to the mouse chromosome 17, in a region syntenic with human chromosome 16, where the T/t region has also been mapped. CONCLUSIONS: ALR appears to be a protein with important physiologic properties, not exclusively limited to liver regeneration, with roles that are involved in the synthesis or stability of the nuclear and mitochondrial transcripts that are present in actively regenerating cells, particularly the germ cells of the testes.

Amino Acid Sequence↗
PubMed1994

Rat natural killer cell antigen, NKR-P1, related to C-type animal lectins is a carbohydrate-binding protein.

Natural killer receptor protein 1 (NKR-P1, a family of proteins), which is a dimeric transmembrane protein predominantly on rat and murine natural killer cells, contains an extracellular motif related to calcium-dependent animal lectins. The domain architecture of this protein and the finding that its cross-linking with antibody results in activation of natural killer cells make it a promising candidate for a receptor function. We have expressed a full-length NKR-P1 protein of the rat in COS cells and prepared soluble extracellular fragments by controlled proteolysis or by expression of truncated cDNA in bacteria. Dimerization of soluble NKR-P1 is predominantly dependent on the presence of an intact juxta-membrane stalk region and independent of N-glycosylation. Binding and inhibition studies using monosaccharides and neoglycoconjugates indicate that NKR-P1 is a lectin with a preference order of GalNAc > GlcNAc >> Fuc >> Gal > Man. At neutral pH, Ca2+ is tightly associated with the protein such that only a proportion can be removed by 10 mM EGTA. However, NKR-P1 can be decalcified completely at pH 10 with a total loss of carbohydrate binding. After recalcification at pH 8, carbohydrate binding is completely restored. Thus, NKR-P1 differs from other calcium-dependent animal lectins investigated so far in its pattern of monosaccharide recognition and in the tightness of Ca2+ binding.

Animals↗
PubMed1993

Rat class III Fc gamma receptor isoforms differ in IgG subclass-binding specificity and fail to associate productively with rat CD3 zeta.

Several new rat class III Fc gamma R isoforms are described here, extending the genetic complexity of this receptor family and further distinguishing rat CD16 from mouse CD16, represented by only one receptor isoform, and human CD16, represented by only two isoforms. RNase protection assays reveal that three rat tumor cell lines--RBL-1 basophilic leukemia cells, RM-SV1 macrophages, and CRNK-16 NK cells--all coordinately express multiple and probably identical rtFc gamma RIII-related transcripts in similar relative proportions but at significantly different levels. These results indicate that no single isoform predominates in these cell types but that the overall level of rtFc gamma RIII-related transcripts is differentially regulated. Two of the rtFc gamma RIII isoforms found to have extensive amino acid sequence differences in their second extracellular (EC2) domains are shown to bind rat and mouse IgG subclasses differently. This result suggests that the receptor isoform diversity in this species may function as a mechanism for extending the IgG-binding capacity of rat leukocytes. Cloned cDNA for the rat CD3 zeta protein was also isolated in this study and its ability to augment surface expression of class III Fc gamma R was tested by rosetting of cDNA-transfected COS cells. Like the structurally homologous mouse CD3 zeta, rat CD3 zeta fails to promote surface expression of Fc gamma RIII, sharply contrasting the efficient receptor expression produced by human CD3 zeta. Variations in the transmembrane amino acid sequences correlate with the divergent capacities of these CD3 zeta molecules to augment receptor expression. The high levels of CD3 zeta message expressed in rat NK cells may indicate that other unidentified hetero-subunits are required for assembly of rat CD3 zeta into functional CD16 receptors.

Amino Acid Sequence↗
PubMed1993

Non-radioisotopic typing of human leukocyte antigen class II genes on microplates.

We describe a new nonradioisotopic method for HLA class II molecular typing performed in 96-well plates of the same size as those used in enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA). Biotinylated sequence-specific oligonucleotide (SSO) probes are bound to avidin-coated plates. Digoxigenin-labeled PCR-amplified DNA samples are hybridized, washed and detected with a peroxidase conjugated antibody assay. The method was tested by performing a partial HLA DQA1 and DQB1 typing on 69 randomly selected blood samples. The results are completely concordant with a traditional SSO-PCR typing performed on the same samples. This procedure is simple, fast and could be adapted for performance in semi-automated or automated fashion using equipment already available for ELISA and RIA assays.

Avidin↗
PubMed1993

Lyophilization as a method to store samples of whole blood.

We have investigated the feasibility of using lyophilization as a method of preserving whole blood samples for HLA class II molecular typing. Genomic DNA extracted from lyophilized blood appears similar in size to that extracted from fresh or frozen blood and is suitable for both PCR amplification and Southern blot analysis.

Blood Preservation↗
PubMed1992

The T-cell receptor V beta gene usage in tumor-infiltrating lymphocytes and blood of patients with hepatocellular carcinoma.

To determine a possibly restricted T-cell receptor (TCR) repertoire in tumor-infiltrating lymphocytes (TIL) in response to tumor-associated antigens in patients with hepatocellular carcinoma (HCC), freshly isolated TIL (n = 5) and peripheral blood lymphocytes (PBL; n = 6; 3 paired with TIL) were studied for expression of TCR variable (V) beta regions. RNA purified from TIL or PBL was reverse-transcribed into complementary DNA. This complementary DNA was amplified by quantitative polymerase chain reaction with 22 primers specific for 20 TCR V beta gene families and a 3' constant (C) beta primer. As a reference for later quantitation, a fragment of TCR C alpha was coamplified with each V beta region. Using 32P-labeled 3' primers, the percentage of total V beta expression was calculated by measuring the cpm of each of the amplified products. In contrast to PBL of 6 control, healthy individuals, whose range of expression of each TCR V beta gene varied from 0 to 13%, the expression of some V beta genes in HCC TIL was as high as 33%, indicating a restricted TCR V beta usage in HCC TIL. When polymerase chain reaction-amplified complementary DNAs of the V beta 1 or V beta 3 genes obtained from two TIL preparations were cloned and sequenced, the same rearrangements were found in the majority of DNA clones. The particular V beta genes that were over- or underrepresented in TIL varied among the patients. In 3 of 6 PBL and 3 of 5 TIL, the V beta 3 gene was expressed with a relatively high frequency. The V beta 4 gene expression was consistently low in patients' TIL or PBL. In 3 paired PBL and TIL, V beta expression was similar. In 5 of 6 cases, HCC PBL had different TCR V beta frequencies from those seen in normal PBL. This analysis of TCR V beta usage in freshly isolated TIL and in PBL indicated that T-lymphocytes in patients with HCC might have restricted immunological reactivity and that V beta 3-restricted TIL might represent antitumor effector cells.

Amino Acid Sequence↗
PubMed1992

Genomic structure and strain-specific expression of the natural killer cell receptor NKR-P1.

NK cells are able to lyse a variety of virally infected and neoplastic cells in an MHC-unrestricted manner. The cell-surface protein NKR-P1 is thought to play a key role in this process. NKR-P1, initially identified in rat IL-2 activated NK cells, is encoded in the mouse by at least three similar, but not identical, genes. We previously reported the isolation and characterization of three different NKR-P1 cDNA, termed cDNA 2, 34, and 40, from IL-2 activated mouse NK cells. This report describes the structure of the gene encoding NKR-P1 cDNA 2, the smallest of these three cDNA. Gene 2 is composed of six exons spanning approximately 14 kb of genomic DNA. The first exon encodes the N-terminal intracellular domain, and exons 4, 5, and 6 contain the sequences coding for the CRD. This organization is similar to that of other genes that encode C-type animal lectins. The expression of the NKR-P1 genes in A-LAK cells from 13 mouse strains was examined by Northern blot analysis. NKR-P1 expression appears to coincide with that of the NK1.1 Ag. This observation further supports the hypothesis that the NK1.1 Ag is encoded by one of the NKR-P1 genes. Nucleotide sequence analysis of the promoter region of the three NKR-P1 genes in BALB/c and C57BL/6 mice suggests that differences in the level of expression probably do not result from alterations in the upstream regions of these genes, but may be caused by the expression of strain-specific transacting factors.

Animals↗