Echocardiographic AV-interval optimization in patients with reduced left ventricular function

Background

Power Doppler (PD) has improved diagnostic capabilities of vascular sonography, mainly because it is independent from the angle of insonation. We evaluated this technique in a prospective comparison with conventional imaging, consisting in Duplex and Color Doppler, for the evaluation of Renal Artery (RA) stenosis.

Methods

Sensitivity, specificity and predictive values of PD and conventional imaging were assessed in a blinded fashion on eighteen patients, 9 with angiographic evidence of unilateral RA stenosis (hypertensive patients) and 9 with angiographically normal arteries (control group). PD images were interpreted with an angiography-like criteria.

Results

In the control group both techniques allowed correct visualization of 16 out of the 18 normal arteries (93% specificity). Only in five hypertensive patients RA stenosis was correctly identified with conventional technique (56% sensitivity and 86% negative predictive value); PD was successful in all hypertensive patients (100% sensitivity and negative predictive value), since the operators could obtain in each case of RA stenosis a sharp color signal of the whole vessel with a clear "minus" at the point of narrowing of the lumen. All results were statistically significant (p < 0.01).

Conclusions

This study demonstrates that PD is superior to conventional imaging, in terms of sensitivity and specificity, for the diagnosis of RA stenosis, because it allows a clear visualization of the whole stenotic vascular lumen. Especially if it is used in concert with the other sonographic techniques, PD can enable a more accurate imaging of renovascular disease with results that seem comparable to selective angiography.     

High-throughput liquid chromatography-tandem mass spectrometry determination of bupropion and its metabolites in human, mouse and rat plasma using a monolithic column

In the present work, a high-throughput LC/MS/MS method using a Chromolith RP-18 (50 mm x 4.6 mm) monolithic column was developed and partially validated for the determination of bupropion (BUP), an anti-depressant drug, and its metabolites, hydroxybupropion and threo-hydrobupropion (TB), in human, mouse, and rat plasma. A modern integrated liquid chromatograph and an LC/MS/MS system with a TurboIonSpray (TIS) interface were used for the positive electrospray selected reaction monitoring (SRM) LC/MS analyses. Spiked control plasma calibration standards and quality control (QC) samples were extracted by semi-automated 96-well liquid-liquid extraction (LLE) using ethyl acetate. A mobile phase consisting of 8mM ammonium acetate-acetonitrile (55:45, v/v) delivered isocratically at 5 ml/min, and split post-column to 2 ml/min directed to the TIS, provided the optimum conditions for the chromatographic separation of bupropion and its metabolites within 23s. The isotope-labeled D(6)-bupropion and D(6)-hydroxybupropion were used as internal standards. The method was linear over a concentration range of 0.25-200 ng/ml (bupropion and threo-hydrobupropion), and 1.25-1000 ng/ml (hydroxybupropion). The intra- and inter-day assay accuracy and precision were within 15% for all analytes in each of the biological matrices. The monolithic column performance as a function of column backpressure, peak asymmetry, and retention time reproducibility was adequately maintained over 864 extracted plasma injections.     

Probing the pathways of chylomicron and HDL metabolism using adenovirus-mediated gene transfer

PURPOSE OF THE REVIEW: This review clarifies the functions of key proteins of the chylomicron and the HDL pathways. RECENT FINDINGS: Adenovirus-mediated gene transfer of several apolipoprotein (apo)E forms in mice showed that the amino-terminal 1-185 domain of apoE can direct receptor-mediated lipoprotein clearance in vivo. Clearance is mediated mainly by the LDL receptor. The carboxyl-terminal 261-299 domain of apoE induces hypertriglyceridemia, because of increased VLDL secretion, diminished lipolysis and inefficient VLDL clearance. Truncated apoE forms, including apoE2-202, have a dominant effect in remnant clearance and may have future therapeutic applications for the correction of remnant removal disorders. Permanent expression of apoE and apoA-I following adenoviral gene transfer protected mice from atherosclerosis. Functional assays, protein cross-linking, and adenovirus-mediated gene transfer of apoA-I mutants in apoA-I deficient mice showed that residues 220-231, as well as the central helices of apoA-I, participate in ATP-binding cassette transporter A1-mediated lipid efflux and HDL biogenesis. Following apoA-I gene transfer, an amino-terminal deletion mutant formed spherical alpha-HDL, a double amino- and carboxyl-terminal deletion mutant formed discoidal HDL, and a carboxyl-terminal deletion mutant formed only pre-beta-HDL. The findings support a model of cholesterol efflux that requires direct physical interactions between apoA-I and ATP-binding cassette transporter A1, and can explain Tangier disease and other HDL deficiencies. SUMMARY: New insights are provided into the role of apoE in cholesterol and triglyceride homeostasis, and of apoA-I in the biogenesis of HDL. Clearance of the lipoprotein remnants and increase in HDL synthesis are obvious targets for therapeutic interventions.     

Pathway for the synthesis of mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii. Biochemical and genetic characterization of key enzymes

The biosynthetic pathway for the synthesis of the compatible solute alpha-mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii is proposed based on the activities of purified recombinant mannosyl-3-phosphoglycerate (MPG) synthase and mannosyl-3-phosphoglycerate phosphatase. The former activity was purified from cell extracts, and the N-terminal sequence was used to identify the encoding gene in the completely sequenced P. horikoshii genome. This gene, designated PH0927, and a gene immediately downstream (PH0926) were cloned and overexpressed in Escherichia coli. The recombinant product of gene PH0927 catalyzed the synthesis of alpha-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and d-3-phosphoglycerate retaining the configuration about the anomeric carbon, whereas the recombinant gene product of PH0926 catalyzed the dephosphorylation of mannosyl-3-phosphoglycerate to yield the compatible solute alpha-mannosylglycerate. The MPG synthase and the MPG phosphatase were specific for these substrates. Two genes immediately downstream from mpgs and mpgp were identified as a putative bifunctional phosphomannose isomerase/mannose-1-phosphate-guanylyltransferase (PH0925) and as a putative phosphomannose mutase (PH0923). Genes PH0927, PH0926, PH0925, and PH0923 were contained in an operon-like structure, leading to the hypothesis that these genes were under the control of an unknown osmosensing mechanism that would lead to alpha-mannosylglycerate synthesis. Recombinant MPG synthase had a molecular mass of 45,208 Da, a temperature for optimal activity between 90 and 100 degrees C, and a pH optimum between 6.4 and 7.4; the recombinant MPG phosphatase had a molecular mass of 27,958 Da and optimum activity between 95 and 100 degrees C and between pH 5.2 and 6.4. This is the first report of the characterization of MPG synthase and MPG phosphatase and the elucidation of a pathway for the synthesis of mannosylglycerate in an archaeon.     

Agrin-induced phosphorylation of the acetylcholine receptor regulates cytoskeletal anchoring and clustering

At the developing neuromuscular junction, a motoneuron-derived factor called agrin signals through the muscle-specific kinase receptor to induce postsynaptic aggregation of the acetylcholine receptor (AChR). The agrin signaling pathway involves tyrosine phosphorylation of the AChR beta subunit, and we have tested its role in receptor localization by expressing tagged, tyrosine-minus forms of the beta subunit in mouse Sol8 myotubes. We find that agrin-induced phosphorylation of the beta subunit occurs only on cell surface AChR, and that AChR-containing tyrosine-minus beta subunit is targeted normally to the plasma membrane. Surface AChR that is tyrosine phosphorylated is less detergent extractable than nonphosphorylated AChR, indicating that it is preferentially linked to the cytoskeleton. Consistent with this, we find that agrin treatment reduces the detergent extractability of AChR that contains tagged wild-type beta subunit but not tyrosine-minus beta subunit. In addition, agrin-induced clustering of AChR containing tyrosine-minus beta subunit is reduced in comparison to wild-type receptor. Thus, we find that agrin-induced phosphorylation of AChR beta subunit regulates cytoskeletal anchoring and contributes to the clustering of the AChR, and this is likely to play an important role in the postsynaptic localization of the receptor at the developing synapse.     

Comparative study of in vivo and in vitro phagocytosis including germicidal capacity in Odontaster validus (Koehler, 1906) at 0 degree C

The phagocytosis and germicidal capacity of Saccharomyces cerevisiae by phagocytic amoebocytes (PA) of the Antarctic starfish Odontaster validus were studied in vivo (after incubation periods of 1, 2, and 4 h) and in vitro (after incubation periods of 1, 2, 4, 8, and 12 h) at 0 degree C. The total number of PA and the phagocytic capacity (PC), phagocytic index (PI), and germicidal capacity (GC) of the PA were calculated. Results showed significant variability of the total PA number in different animals. There was a significant increase in PC and no significant differences in PI and GC for different in vitro incubation times. In vivo, experiments showed no significant difference of PC and PI, but there was a significant increase in GC as incubation periods increased. Comparison between in vitro and in vivo results revealed that PI and PC were significantly higher in vitro and that GC was significantly higher in vivo. The present study shows for the first time the phagocytosis and GC of an Antarctic invertebrate in vivo at low temperature (0 degree C), and the results are comparing with the available literature for echinoderms.     

Genomic organization and regulation of the vav proto-oncogene

Vav and vav2 are members of the dbl family of guanine nucleotide exchange factors (GEF) for the rho/rac family of GTP binding proteins. Vav is expressed primarily in hematopoietic cells, while vav2 has a wider tissue distribution. The genomic structure of the human vav proto-oncogene was studied by identifying and sequencing all 27 exons of the gene from overlapping P1 and cosmid clones. The gene spans a 77-kb region on chromosome 19. In contrast, the coding region of vav2 is distributed over 30 exons spanning 227-kb. The overall organization of the exons which encode both proteins was found to be similar. In humans, alternative splicing of exons 6, 16 and 28 generated at least two distinct vav2 mRNA species. Several differences from the original vav cDNA sequence were noted. The most important difference was the identification of amino acid 718 as isoleucine, rather than threonine. This change warrants the reclassification of the vav SH2 domain as a type 3 SH2, instead of a type 2 SH2 as originally proposed by Songyang et al. (Mol. Cell. Biol. 14 (1994) 2777-2785). A series of vav promoter deletions were constructed using the enhanced green fluorescent protein (EGFP) as a reporter gene. A 23-bp segment that included a potential CBF/AML-1 binding site was found to be essential for EGFP expression in U937 cells. The same constructs were not active in HeLa cells, which do not express vav. A potential c-myb DNA binding site within the vav promoter was not required for EGFP expression.     

Construction of a branched chain at C-3 of a hexopyranoside. Synthesis of miharamycin sugar moiety analogs

Synthesis of the conveniently protected epimer at C-3' of the miharamycin sugar moiety was accomplished starting from the corresponding 3,3'-spiroepoxide. Reaction of the epoxide with lithium cyanide, followed by hydrolysis and spontaneous cyclization, afforded the intermediate deoxylactone methyl 4,6-O-benzylidene-3-C-(carboxymethyl)-alpha-D-glucopyranoside-3',2-lacto ne (8). Stereoselective hydroxylation with MoO5 x py x HMPA, reduction with lithium aluminum hydride and cyclization with diethyl azodicarboxylate-triphenylphosphine gave the target molecule methyl 2,3'-anhydro-4,6-O-benzylidene-3-C-[(R)-1,2-dihydroxyethyl]-alpha -D-glucopyranoside (5). Direct reduction of 8 gave other analogs having no C-3' hydroxyl group together with having a C-3' hydroxyl group (hemiacetal). In addition, C-3' epimers were also synthesized through C-3', C-3' dihydroxy analogs. Wittig reaction of an appropriate ketosugar with [(ethoxycarbonyl)methylene]triphenylphosphorane leading to a 7:3 Z/E mixture, followed by hydroxylation with osmium tetroxide, reduction and cyclization afforded the target molecule 5 and the miharamycin sugar moiety methyl 2,3'-anhydro-4,6-O-benzylidene-3-C-[(S)-1,2-dihydroxyethyl]-alpha -D-glucopyranoside. Examination of X-ray data for 5 and its NMR spectroscopy data allowed us to explain a contradiction reported in the literature.     

Thimet oligopeptidase EC 3.4.24.15 is a major liver kininase

Bradykinin (BK) is a potent hepato-portal hypertensive agent although it is efficiently inactivated by the liver. The organ converts angiotensin I to AII, but at a much slower rate than it inactivates BK. We had previously identified EC 3.4.24.15 as an hepatic bradykinin inactivating endopeptidase that hydrolyzes BK at the F5-F6 bond. The aim of this study was to determine the relative importance of BIE, as compared to other kininases, in normal, cirrhotic or inflamed rat livers, as well as in samples of human liver. Using specific substrates and inhibitors we showed that: 1) purified BIE preparation hydrolyzed BK and a BK analogue (BK-Q) with similar efficacy; BK-Q was functionally active since it caused an increase in hepato-portal pressure, as did BK itself. 2) BK degradation in rat serum was performed by ACE since BIE and prolylendopeptidase (PEP) activities were negligible. 3) normal rat liver homogenate contained a large amount of BIE activity which was eliminated by a specific EC 3.4.24.15 inhibitor; ACE and PEP activities were negligible. 4) There was no difference (p>0.05) in BIE activity in the liver homogenates from rats with normal, inflamed or cirrhotic livers. 5) BIE activity was efficiently removed from livers (normal, inflamed or cirrhotic) that were perfused with TritonX-100.6) Human liver had an similar enzymatic pattern although ACE activity was detected. We concluded that in normal, inflamed or cirrhotic rat livers, as well as in the human liver, the bradykinin inactivating endopeptidase (EC 3.4.24.15), and not ACE, is the major hepatic kininase.