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Robert Long

Publications and source records attributed to Robert Long.

10 recordsLinked to original sources
PubMed2006

Characterization of coronary atherosclerotic plaque using multicontrast MRI acquired under simulated in vivo conditions.

PURPOSE: To compare coronary atherosclerotic plaque characterization using multicontrast MRI on: 1) freshly excised vessels under simulated in vivo conditions, and 2) preserved vessels. MATERIALS AND METHODS: T1-weighted (T1W), T2-weighted (T2W), proton density-weighted (PDW), and diffusion-weighted (DW) MR images were acquired on 13 freshly excised human coronary arteries from explanted hearts. Vessels were imaged in an MR-compatible tissue culture chamber using a 4.7 Tesla small-bore MR scanner. Eight vessels were then preserved in buffered formalin and rescanned following the same imaging protocol. A three-dimensional spatially penalized fuzzy C-means (3D-SPFCM) technique was applied to classify different plaque constituents. The classification results from vessels under "fresh" and "preserved" conditions were compared with corresponding histological sections. RESULTS: For most plaque constituents, the plaque characterization results show no significant difference between fresh and preserved scans, and little difference between scans and the histological reference standard. In the case of thrombus, apparent signal changes between fresh and preserved images were identified. Overall, MR scans conducted under preserved conditions provided a 1.8% to 17.5% greater signal-to-noise ratio (SNR) than those conducted in the fresh stage. CONCLUSION: Preservation of coronary vessels did not alter the contrast between plaque tissues on multicontrast MRI, and did not significantly change the results of plaque constituent characterization.

Coronary Artery Disease↗
PubMed2005

HIV viral protein R causes atrial cardiomyocyte mitosis, mesenchymal tumor, dysrhythmia, and heart failure.

HIV viral protein R (Vpr) affects the immunocyte cell cycle and circulates as free polypeptide in plasma of AIDS patients. Effects of Vpr on cardiomyocytes were explored using transgenic mice (TG) with Vpr targeted to cardiomyocytes by the alpha-myosin heavy-chain promoter. TG and WT littermate hearts were evaluated histopathologically, ultrastructurally, molecularly via RNA microarray analysis and quantitative RT-PCR, and functionally by cardiac magnetic resonance imaging (MRI) and electrocardiograms (ECG). Six hemizygous lines were created (Vpr(a,b,c,d,e,h)). Vpr RNA was expressed exclusively in myocardium and Vpr mRNA expression correlated with phenotypic changes. Vpr(b) exhibited the highest expression and mortality. TGs developed congestive heart failure ( approximately 8 weeks), abnormal cardiomyocyte nuclei and mitoses ( approximately 12 weeks), and became moribund ( approximately 20 weeks) with atrial mesenchymal tumors. MRI revealed four-chamber dilation, defective contraction, and atrial masses. Pathologically, cardiomegaly and atrial mesenchymal tumors occurred ( approximately 16-20 weeks). ECGs showed prolonged R-R, Q-T, and P-R intervals ( approximately 12 weeks). RNA encoding collagen and bone morphogenic protein 4, 6, and 7 were increased. Vpr targeted to cardiomyocytes caused defective contractility and atrial tumors. Since some Vpr cardiomyocytic effects resemble those found in terminally differentiated immunocytes, some pathogenetic mechanisms may be shared at the subcellular level.

Animals↗
PubMed2005

Transgenic expression of the deoxynucleotide carrier causes mitochondrial damage that is enhanced by NRTIs for AIDS.

Nucleoside reverse transcriptase inhibitors (NRTIs) are antiretrovirals for AIDS with limiting mitochondrial side effects. The mitochondrial deoxynucleotide carrier (DNC) transports phosphorylated nucleosides for mitochondrial DNA replication and can transport phosphorylated NRTIs into mitochondria. Transgenic mice (TG) that exclusively overexpress DNC in the heart tested DNC's role in mitochondrial dysfunction from NRTIs. Two TG lines were created that overexpressed the human DNC gene in murine myocardium. Cardiac and mitochondrial structure and function were examined by magnetic resonance imaging, echocardiography, electrocardiography, transmission electron microscopy, and plasma lactate. Antiretroviral combinations (HAART) that contained NRTIs (stavudine (2', 3'-didehydro-2', 3'-deoxythymidine or d4T)/lamivudine/indinavir; or zidovudine (3' azido-3'-deoxythymidine or AZT)/lamivudine/indinavir; 35 days) were administered to simulate AIDS therapy. In parallel, a HAART combination without NRTIs (nevirapine/efavirenz/indinavir; 35 days) served as an NRTI-sparing, control regimen. Untreated DNC TGs exhibited normal cardiac function but abnormal mitochondrial ultrastructure. HAART that contained NRTIs caused cardiomyopathy in TGs with increased left ventricle mass and volume, heart rate variability, and worse mitochondrial ultrastructural defects. In contrast, treatment with an NRTI-sparing HAART regimen caused no cardiac changes. Data suggest the DNC is integral to mitochondrial homeostasis in vivo and may relate mechanistically to mitochondrial dysfunction in patients treated with HAART regimens that contain NRTIs.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Regioselectivity of taxoid-O-acetyltransferases: heterologous expression and characterization of a new taxadien-5alpha-ol-O-acetyltransferase.

In addition to the anticancer drug Taxol, yew (Taxus) species produce a large variety of other taxane diterpenoids which differ mainly in the type of acyl and aroyl groups appended to the many hydroxyl functions on the taxane core; acetate esters are particularly common. Taxol bears an acetate at C10 and another at C4 thought to originate by intramolecular migration of a C5 acetate function in the process of oxetane ring formation, but many other naturally occurring taxoids bear acetate groups at C1, C2, C7, C9, and C13, in addition to C5 and C10. cDNAs encoding a taxoid 5alpha-O-acetyltransferase (taxadien-5alpha-ol as substrate) and a taxoid 10beta-O-acetyltransferase (10-deacetylbaccatin III as substrate) have been acquired from a recently isolated family of Taxus acyl/aroyltransferase clones. To explore the origins of other acetylated taxoids, the group of recombinant Taxus acyltransferases was investigated with a range of polyhydroxylated taxoids as substrates. From this survey, a new acetyltransferase clone (denoted TAX19) was identified that was capable of acetylating taxadien-5alpha-ol with activity comparable to that of the previously identified 5alpha-O-acetyltransferase (clone TAX1). However, when these two recombinant enzymes were presented with taxadien-triol and tetraol substrates, they exhibited different regiospecificities. The TAX1 enzyme preferentially acetylates the "northern" hemisphere hydroxyls at C9 and C10, whereas the TAX19 enzyme preferentially acetylates the "east-west" pole positions at C5 and C13. The TAX1 enzyme possesses the lowest KM value with taxadien-5alpha-ol (an early pathway metabolite) as substrate, with much higher KM values for the polyhydroxylated taxoid substrates, whereas the TAX19 enzyme possesses lower KM values (than the TAX1 transferase) for all taxoid substrates tested. These results suggest that both TAX1 and TAX19 acyltransferases may function at the early C5 acetylation step of taxoid metabolism, and that the TAX19 acyltransferase, because of its broader specificity for polyhydroxylated taxoids, may also function later in metabolism and be responsible for the production of many other acetylated taxoids.

Acetyl Coenzyme A↗
PubMed2004

FISH diagnosis of the common 57-kb deletion in CTNS causing cystinosis.

Cystinosis is an autosomal recessive lysosomal storage disease caused by mutations in CTNS. The most prevalent CTNS mutation, a 57-kb deletion, occurs in approximately 60% of patients in the United States and northern Europe and removes exons 1-9, most of exon 10, the CTNS promoter region, and all of an adjacent gene of unknown function called CARKL. CTNS codes for the lysosomal cystine transporter, whose absence leads to intracellular cystine accumulation, widespread cellular destruction, renal Fanconi syndrome in infancy, renal glomerular failure in later childhood, and other systemic complications. Because treatment with oral cysteamine can prevent or delay these complications significantly, early and accurate diagnosis is critical. This study describes the generation of fluorescence in situ hybridization (FISH) probes for the 57-kb deletion in CTNS, enabling cytogenetics laboratories to test for this common mutation. The probes would also be able to detect a less frequent 11.7-kb deletion. A blinded study was performed using multiplex PCR analysis as the gold standard to determine the presence or absence of the 57-kb deletion. The FISH probes, evaluated on 12 lymphoblastoid cell lines from singly deleted, doubly deleted, and nondeleted patients, made the correct diagnosis in every case. This appears to be the first FISH-based diagnostic method described for any lysosomal storage disorder. It can assist in the antenatal and perinatal diagnosis of cystinosis and promote earlier salutary therapy with cysteamine.

Alleles↗
PubMed2003

Heparanase degrades syndecan-1 and perlecan heparan sulfate: functional implications for tumor cell invasion.

Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. In this work we demonstrate the following. 1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells. 2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS. 3) Syndecan-1 does not directly inhibit HPSE-1 enzymatic activity. 4) The presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays. 5) Inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggest that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis.

Adenocarcinoma↗
PubMed2002

Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis.

The structural pharmacophore of Taxol, responsible for binding the N terminus of the beta-subunit of tubulin to arrest cell proliferation, comprises, in part, the 13-O-(N-benzoyl-3-phenylisoserinoyl) side chain. To identify the side chain transferase of Taxol biosynthesis, a set of transacylases obtained from an enriched cDNA library (constructed from mRNA isolated from Taxus cuspidata cells induced with methyl jasmonate for Taxol production) was screened. A cDNA clone (designated TAX7) encoding a taxoid C-13 O-phenylpropanoyltransferase was isolated which yielded a recombinant enzyme that catalyzes the selective 13-O-acylation of baccatin III with beta-phenylalanoyl CoA as the acyl donor to form N-debenzoyl-2'-deoxytaxol. This enzymatic product was converted to 2'-deoxytaxol by chemical N-benzoylation, and the identity of this derivative was confirmed by spectrometric analyses. The full-length cDNA has an ORF of 1,335 bases and encodes a 445-aa protein with a calculated molecular weight of 50,546. Evaluation of kinetic parameters revealed K(m) values of 2.4 +/- 0.5 microM and 4.9 +/- 0.3 microM for baccatin III and beta-phenylalanoyl-CoA, respectively. The pH optimum for the recombinant O-(3-amino-3-phenylpropanoyl)transferase is at 6.8. Identification of this clone completes acquisition of the five aroyl/acyltransferases involved in the biosynthesis of Taxol. Application of these transacylase genes in suitable host cells can improve the production yields of Taxol and could enable the preparation of second-generation Taxol analogs possessing greater bioactivity and improved water solubility.

Acyl Coenzyme A↗
PubMed2002

The final acylation step in taxol biosynthesis: cloning of the taxoid C13-side-chain N-benzoyltransferase from Taxus.

The formation of several acyl groups and an amide group of Taxol is catalyzed by regioselective CoA thioester-dependent acyltransferases. Several full-length acyltransferase sequences, obtained from a cDNA library constructed from mRNA isolated from Taxus cuspidata cells induced for Taxol production with methyl jasmonate, were individually expressed in Escherichia coli, from which a cDNA clone encoding a 3'-N-debenzoyl- 2'-deoxytaxol N-benzoyltransferase was identified. This recombinant enzyme catalyzes the stereoselective coupling of the surrogate substrate N-debenzoyl-(3'RS)-2'-deoxytaxol with benzoyl-CoA to form predominantly one 3'-epimer of 2'-deoxytaxol. The product 2'-deoxytaxol was confirmed by radio-HPLC,(1)H-NMR, and chemical ionization-MS. This enzymatic reaction constitutes the final acylation in the Taxol biosynthetic pathway. The full-length cDNA coding for the N-benzoyltransferase has an ORF of 1,323 nucleotides and encodes a 441-residue protein with a calculated molecular weight of 49,040. The recombinant enzyme expressed in E. coli has a pH optimum at 8.0, a k(cat) approximately 1.5 +/- 0.3 s(-1) and K(m) values of 0.42 mM and 0.40 mM for the N-deacylated taxoid and benzoyl-CoA, respectively. In addition to improving the production yields of Taxol in genetically engineered host systems, this enzyme provides a means of attaching modified aroyl groups to taxoid precursors for the purpose of improving drug efficacy.

Aldehyde-Ketone Transferases↗