Biomedical subjects

M A Iannone

Publications and source records attributed to M A Iannone.

9 recordsLinked to original sources

PubMed2000

Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry.

BACKGROUND: We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres. METHODS: A fluoresceinated oligonucleotide reporter sequence is added to a "capture" probe by OLA. Capture probes are designed to hybridize both to genomic "targets" amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called "ZipCodes". The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNP genotype. RESULTS: Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere-based SNP analysis agreed with genotyping by sequencing in all cases. CONCLUSIONS: Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs.

Chromosomes, Human, Pair 19↗

PubMed2000

Expression, purification, and characterization of the human receptor activator of NF-kappaB ligand (RANKL) extracellular domain.

Receptor activator of NF-kappaB ligand (RANKL) is a type II transmembrane protein found on osteoblasts which functions as a major determinant of osteoclast differentiation and activation. RANKL mediates bone homeostasis through binding to the cognate ligand on osteoclasts, RANK, and a soluble decoy receptor, osteoprotegerin (OPG). We designed a construct encoding the extracellular domain of human RANKL that conformed to reports of native processing. To encourage folding and posttranslational modification of a normally membrane-inserted moiety, we expressed the RANKL truncate as a secreted protein using the signal sequence from OPG in a Trichoplusia ni cell line using a baculovirus expression vector. RANKL was purified by a three-step process including an OPG-Fc affinity column. SDS-PAGE and mass spectral analysis indicated that the protein was >99% pure and glycosylated. Circular dichroism spectra revealed that the protein exhibited structural elements similar to tumor necrosis factor-alpha. By BIAcore analysis, RANKL bound to OPG with an affinity of 6.7 nM. Sedimentation equilibrium analytical ultracentrifugation analyses established that our protein existed as a trimer. We conclude that our expressed human RANKL truncate is folded, is functional, and exhibits self-association consistent with other family members.

Amino Acid Sequence↗

PubMed2000

A microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension.

A rapid, high throughput readout for single-nucleotide polymorphism (SNP) analysis was developed employing single base chain extension and cytometric analysis of an array of fluorescent microspheres. An array of fluorescent microspheres was coupled with uniquely identifying sequences, termed complementary ZipCodes (cZipCodes), which allowed for multiplexing possibilities. For a given assay, querying a polymorphic base involved extending an oligonucleotide containing both a ZipCode and a SNP-specific sequence with a DNA polymerase and a pair of fluoresceinated dideoxynucleotides. To capture the reaction products for analysis, the ZipCode portion of the oligonucleotide was hybridized with its cZipCodes on the microsphere. Flow cytometry was used for microsphere decoding and SNP typing by detecting the fluorescein label captured on the microspheres. In addition to multiplexing capability, the ZipCode system allows multiple sets of SNPs to be analyzed by a limited set of cZipCode-attached microspheres. A standard set of non-cross reactive ZipCodes was established experimentally and the accuracy of the system was validated by comparison with genotypes determined by other technologies. From a total of 58 SNPs, 55 SNPs were successfully analyzed in the first pass using this assay format and all 181 genotypes across the 55 SNPs were correct. These data demonstrate that the microsphere-based single base chain extension (SBCE) method is a sensitive and reliable assay. It can be readily adapted to an automated, high-throughput genotyping system. [Primer sequences used in this study are available as online supplementary materials at www.genome.org.]

DNA, Complementary↗

PubMed1996

Xenogeneic ICAM-1 gene transfer suppresses tumorigenicity and generates protective antitumor immunity.

Tumorigenicity in Fischer rats was not significantly reduced when the rat ICAM-1 gene was overexpressed in the rat tumor cell lines, JM-1 and SST-2. When these rat tumor cell lines were genetically modified with a gene encoding human ICAM-1, tumorigenicity was dramatically reduced. Expression of xenogeneic ICAM-1 did not alter the growth rate, expression of the major histocompatibility complex, nor morphological appearance of the cells. However, it did facilitate a tumor-specific immunological recognition and rejection of the genetically modified tumor cells. This effect resulted in a tumor-specific, long-term protective immunity directed against genetically unmodified tumor cells. Most importantly, administration of tumor cells genetically modified with genes encoding xenogeneic ICAM-1 can facilitate an immunological response to genetically unaltered pre-existing tumors. Transferring splenocytes from animals 'vaccinated' with the xenogeneic ICAM-1 gene altered tumor cells was able to transfer the antitumor response into recipient animals. Furthermore, transfer of CD8+ T lymphocytes produced the same result. These results suggested that tumors specific CD8+ T lymphocytes were activated by the xenogeneic altered tumor cells. THis activation generated the long-term, tumor-specific immunity.

Animals↗

PubMed1991

Ca2+ ionophore-induced cyclic adenosine-3',5'-monophosphate elevation in human neutrophils. A calmodulin-dependent potentiation of adenylate cyclase response to endogenously produced adenosine: comparison to chemotactic agents.

The cyclic adenosine-3',5'-monophosphate (cAMP) elevation caused by exposure of human neutrophils to the Ca2+ ionophore A23187 was prevented when endogenously produced adenosine was either removed by preincubation with adenosine deaminase or blocked from binding to the adenosine receptor by antagonists [theophylline or (E)-4-(1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-9H-purin-8-yl)cinnamic acid]. In the absence of endogenous adenosine, A23187 potentiated the neutrophil cAMP response to 2-chloroadenosine, prostaglandin E1, and isoproterenol. When neutrophil suspensions were preincubated with concentrations of Ro 20-1724, which appeared to maximally inhibit cAMP phosphodiesterase, A23187 was still able to substantially elevate cAMP levels, suggesting that A23187 increases cAMP by amplifying adenylate cyclase responsiveness to the agonist rather than by inhibiting cAMP phosphodiesterase. The ability of A23187 to augment the cAMP elevation caused by 2-chloroadenosine was persistent over a 10-min period. The neutrophil cAMP elevations caused by chemoattractants leukotriene B4, C5a, and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) were all prevented when endogenously produced adenosine was eliminated from the cell suspensions by the addition of adenosine deaminase. The A23187-induced cAMP elevation was inhibited completely by the calmodulin inhibitors chlorpromazine, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, whereas cAMP levels induced by FMLP, leukotriene B4 and C5a were less affected. It appears that A23187 raises cAMP in human neutrophils by a calmodulin-dependent potentiation of adenylate cyclase responsiveness to endogenously produced adenosine while the chemoattractant-induced cAMP elevations (FMLP), leukotriene B4, and C5a), although possibly Ca2+ dependent, are less sensitive to calmodulin inhibitors and may involve additional biochemical events.

2-Chloroadenosine↗

PubMed1991

The effects of heparin cofactor II-derived chemotaxins on neutrophil actin conformation and cyclic AMP levels.

The serine proteinase inhibitor heparin cofactor II (HC) can be cleaved by polymorphonuclear leukocyte (PMN) elastase (LE) to yield potent chemotactic activity for PMN and monocytes. In contrast to the bacterially-derived chemotaxin formyl-Met-Leu-Phe (fMLP), the HC-derived chemotaxin does not stimulate PMN degranulation or oxidative burst activity. We compared the effects of HC-derived chemotaxins to the effects of fMLP on PMN actin conformation and on the cAMP levels. Both the HC chemotaxins and fMLP rapidly induced an increase in F-actin which was similar in magnitude and time-course. However, in contrast to fMLP, HC-derived chemotaxins did not elevate cAMP levels. HC-derived chemotaxins may be useful probes of chemotactic responses, since they do not have the mixed biological activities of fMLP.

Actins↗

PubMed1989

Chemotactic peptide induces cAMP elevation in human neutrophils by amplification of the adenylate cyclase response to endogenously produced adenosine.

The transient increase in human neutrophil cAMP levels induced by the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) is shown to be caused by amplification of adenylate cyclase response to endogenously produced adenosine. The FMLP-stimulated increase in neutrophil cAMP was potentiated markedly by a nonmethylxanthine cAMP phosphodiesterase inhibitor (Ro 20-1724). By inhibiting the degradation of newly formed cAMP, Ro 20-1724 rendered the FMLP-induced cAMP elevation persistent rather than transient. The role of endogenously produced adenosine in this phenomenon is demonstrated by the ability of either adenosine deaminase or theophylline, an adenosine receptor antagonist, to prevent FMLP-stimulated cAMP elevation. The general nature of the FMLP-potentiated cAMP response is indicated by the finding that FMLP-treated neutrophils, in the presence of exogenously supplied adenosine deaminase, exhibited augmented cAMP generation in response to three different types of receptor agonists: 2-chloroadenosine, prostaglandin E1, and L-isoproterenol. Moreover, like the neutrophil cAMP increase caused by FMLP alone, the ability of FMLP to augment cAMP response to 2-chloroadenosine in adenosine deaminase-treated cells was short-lived and declined after 1.0 min of exposure to FMLP. Preincubation of neutrophil suspensions with the adenylate cyclase inhibitor SQ 22,536 completely prevented FMLP-induced cAMP generation. Furthermore, when neutrophil suspensions were preincubated with concentrations of Ro 20-1724, which apparently maximally inhibit cAMP phosphodiesterase, a 30-s incubation with FMLP still resulted in substantially elevated cAMP levels. It therefore appears that FMLP raises cAMP by activating adenylate cyclase rather than inhibiting cAMP phosphodiesterase.

Adenosine↗

PubMed1989

Effects of adenosine on neutrophil polarization induced by N-formyl-methionyl-leucyl-phenylalanine, sodium propionate and colchicine.

Polarization of human neutrophils (a characteristic bipolar shape change) can be induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP); sodium propionate, which causes a rapid acidification of the cytosol; or colchicine, which disrupts microtubules. We have previously reported that adenosine, endogenously produced in human neutrophil suspensions, inhibits FMLP-induced polarization. We report here that endogenously produced adenosine also inhibits sodium propionate-induced polarization but has no effect on colchicine-induced polarization. These results suggest that neutrophil polarization may be a multistep process inducible by compounds that trigger different biochemical events.

Adenosine↗

PubMed1987

Taxol inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced human neutrophil polarization and H2O2 production while decreasing [3H]FMLP binding.

We have studied the effect of taxol on two N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced neutrophil functions and the possible mechanism by which it inhibits these functions. Taxol inhibited FMLP-induced human neutrophil polarization (a characteristic change in neutrophil shape in response to a chemotactic stimulus) and H2O2 generation. Taxol also decreased the specific binding of [3H]FMLP to human neutrophils at 4 degrees C. The decreased binding of FMLP to its receptor may be responsible for the inhibition by taxol of FMLP-induced polarization and H2O2 generation.

Alkaloids↗