Effects of pH during recombination of human erythrocyte membrane apoprotein and lipid.

The recombinates from human red cell membrane proteins and lipids resulting from dialysis of the components in 2-chloroethanol against aqueous buffers from pH2-12 have been studied by density gradient centrifugation, polyacrylamide gel electrophoresis and freeze-fracture electron microscopy. Between pH 4 and 10 most of the proteins were found in the recombinates whereas below pH 4 and above pH 10 only part of them were recovered in the lipoprotein band after density gradient centrifugation. At low pH, increasing incorporation of the "major glycoprotein" into the recombinates was detected by gel electrophoresis and in parallel increasing amounts of particles were found in the freeze-fracture membrane faces. The necessity of working at low pH values from pH 2-4, however, and a critical evaluation of all the data presently available leads to the conclusion that the 2-choloroethanol technique is not adequate for recombination studies tending to membrane reconsitution.     

Specialized transduction of the ilvD-thyB-ilvA region mediated by Bacillus subtilis bacteriophage SP beta

Specialized transducing SP beta particles were found that carried the Bacillus subtilis genes lying to the left of the prophage attachment site. Three classes of transducing particles were differentiated, depending upon whether they carried ilvA only, thyB and ilvA, or ilvD, thyB, and ilvA. Lysates prepared by the induction of strains that carried both a transducing phage and a plaque-forming phage contained the two particles in a ratio of about 1:3,000. When the transducing particles were used to transduce a phage-sensitive auxotrophic strain to prototrophy, some of the transductants carried only the transducing phage genomes which, by themselves, were defective. One putative nondefective transducing phage (for ilvA only) is also described. SP beta can mediate specialized transduction even in the absence of the major (recE) bacterial recombination system.     

Insertion of bacteriophage SP beta into the citF gene of Bacillus subtilis and specialized transduction of the ilvBC-leu genes.

We isolated a strain of Bacillus subtilis in which the SP beta c2 prophage is inserted into the citF (succinate dehydrogenase) gene. Defective specialized transducing particles for the ilvBC-leu genes were isolated from phage-induced lysates of this lysogen. We isolated a group of phages that differ in the amount of genetic material they carry from this region. Also, we incorporated mutant ilv and leu alleles into the genomes of several transducing phages. Our phage collection enables us to identify the cistron of new ilv and leu mutations by complementation analysis. In this process we discovered a fourth leu cistron, leuD. Characterization of the phages confirmed the published gene order: ilvB-ilvC-leuA-leuC-leuB; leuD lies to the right of leuB.     

A genetic screen in C. elegans reveals roles for KIN17 and PRCC in maintaining 5’ splice site identity

Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5’ GU and ending with 3’ AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and “custody” of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5’ splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5’ splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5’ splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC, and to a lesser extent KIN17, changed alternative 5’ splice site usage at native sites genome-wide, often promoting usage of nearby non-consensus sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.     

Phenotypical features of long Q-T syndrome in transgenic mice expressing human Na-K-ATPase alpha(3)-isoform in hearts

To understand why the adult human heart expresses three isoforms of the sodium pump, we generated transgenic mice (TGM) with 2.3- to 5. 5-fold overexpression of the human alpha(3)-isoform of Na-K-ATPase in the heart. Hearts from the TGM had increased maximal Na-K-ATPase activity and ouabain affinity compared with control hearts, even though the density of Na-K-ATPase pump sites (of all isoforms) was similar to that of control mice. In perfused hearts, contractility both at baseline and in the presence of ouabain tended to be greater in TGM than in controls. Surface electrocardiograms in anesthetized TGM had a steeper dependence of Q-T on sinus cycle length, and Q-T intervals measured during atrial pacing were significantly longer in TGM. Q-T dispersion during sinus rhythm also tended to be longer in TGM. Thus TGM overexpressing human alpha(3)-isoform have several of the phenotypical features of human long Q-T syndrome, despite the absence of previously described mutations in Na(+) or K(+) channels.     

Modified phosphatidylethanolamine as the active component of oxidized low density lipoprotein promoting platelet prothrombinase activity

We analyzed the influence of the atherogenic oxidized low density lipoproteins (LDL) on the activity of the platelet prothrombinase complex, a major contributor to overall thrombin formation in vivo. Platelet dependent thrombin generation was found to be strongly stimulated by in vitro oxidized LDL. The enhancement was additive to that observed with the platelet agonist thrombin. Oxidized LDL increased the platelet binding of annexin-V, suggesting that the augmented surface exposure of aminophospholipids promoted the prothrombinase activity. All of the stimulatory activity of the oxidized LDL could be recovered in the microemulsions prepared from the lipid portion of the modified particles. Phospholipid vesicles were prepared containing the total lipids of the oxidized LDL but lacking specifically in one lipid component. Following the selective removal of the ethanolamine phospholipids (PE) from the LDL lipids, the platelet-dependent thrombin formation was markedly reduced. Vesicles enriched with the isolated PE fraction alone enhanced the thrombin generation. Analyses with autoxidized phospholipids indicated that oxidation products of unsaturated diacyl-PE were mainly responsible for the increased prothrombinase activity. Oxidized LDL and its PE fraction lost their stimulatory activity after treatment with NaCNBH(3), a chemical reductant of Schiff base adducts. Phospholipid vesicles supplemented with synthetic aldehyde-PE adducts largely reproduced the stimulation of the thrombin generation. We conclude that the oxidized LDL particles elicit a pronounced prothrombotic response by increasing the activity of the platelet prothrombinase complex. Specific oxidative modifications of the LDL-associated ethanolamine phospholipids are mainly responsible for this stimulation.     

Implications in the difference of anti-Mi-2 and -p155/140 autoantibody prevalence in two dermatomyositis cohorts from Mexico City and Guadalajara

Introduction

Autoantibodies and clinical manifestations in polymyositis/dermatomyositis (PM/DM) are affected by both genetic and environmental factors. The high prevalence of DM and anti-Mi-2 in Central America is thought to be associated with the high UV index of the area. The prevalences of autoantibodies and the clinical manifestations of PM/DM were evaluated comparing two cohorts in Mexico.

Methods

Ninety-five Mexican patients with PM/DM (66 DM, 29 PM; 67 Mexico City, 28 Guadalajara) were studied. Autoantibodies were characterized by immunoprecipitation using ³⁵S-methionine labeled K562 cell extract. Clinical information was obtained from medical records.

Results

DM represented 69% of PM/DM and anti-Mi-2 was the most common autoantibody (35%), followed by anti-p155/140 (11%); however, anti-Jo-1 was only 4%. The autoantibody profile in adult-onset DM in Mexico City versus Guadalajara showed striking differences: anti-Mi-2 was 59% versus 12% (P = 0.0012) whereas anti-p155/140 was 9% versus 35% (P = 0.02), respectively. A strong association of anti-Mi-2 with DM was confirmed and when clinical features of anti-Mi-2 (+) DM (n = 30) versus anti-Mi-2 (-) DM (n = 36) were compared, the shawl sign (86% versus 64%, P < 0.05) was more common in the anti-Mi-2 (+) group (P = 0.0001). Levels of creatine phosphokinase (CPK) were higher in those who were anti-Mi-2 (+) but they responded well to therapy.

Conclusions

Anti-Mi-2 has a high prevalence in Mexican DM and is associated with the shawl sign and high CPK. The prevalence of anti-Mi-2 and anti-p155/140 was significantly different in Mexico City versus Guadalajara, which have a similar UV index. This suggests roles of factors other than UV in anti-Mi-2 antibody production.     

Rpp25 is a major target of autoantibodies to the Th/To complex as measured by a novel chemiluminescent assay

Introduction

Autoantibodies to the Th/To antigen have been described in systemic sclerosis (SSc) and several proteins of the macromolecular Th/To complex have been reported to react with anti-Th/To antibodies. However, anti-Th/To has not been clinically utilized due to unavailability of commercial tests. The objective of the present study is to evaluate the newly developed ELISA and chemiluminescent immunoassay (CLIA) to measure autoantibodies to Rpp25 (a component of the Th/To complex) using immunoprecipitation (IP) as the reference method.

Methods

The first cohort consisted of 123 SSc patients including 7 anti-Th/To positive samples confirmed by IP. Additional seven anti-Th/To positive samples from non-SSc patients were also tested. For evaluation of the QUANTA Flash Rpp25 CLIA (research use only), 8 anti-Th/To IP positives, a cohort of 70 unselected SSc patients and sera from various disease controls (n = 357) and random healthy individuals (n = 10) were studied.

Results

Anti-Rpp25 antibodies determined by ELISA were found in 11/14 anti-Th/To IP positive but only in 1/156 (0.6%) negative samples resulting in a positive percent agreement of 78.6% (95% confidence interval [CI] 49.2, 95.3%) and a negative percent agreement of 99.4% (95% CI 96.4, 100.0%). To verify the results using a second method, 53 samples were tested by ELISA and CLIA for anti-Rpp25 reactivity and the results were highly correlated (rho = 0.71, 95% CI 0.56, 0.81; P < 0.0001). To define the cutoff of the CLIA, anti-Th/To IP positive and negative sera were tested using the anti-Rpp25 CLIA. At the cutoff selected by receiver operating characteristic (ROC) analysis 8/8 (100.0%) of the anti-Th/To positive sera but only 2/367 (0.5%) of the controls were positive for anti-Rpp25 antibodies. The positive and negative percent agreements were 100.0% (95% CI 63.1, 100.0%) and 99.5% (95% CI 98.0, 99.9%), respectively. In the disease cohorts 2/70 (2.9%) of the SSc patients were positive for anti-Rpp25 antibodies compared to 2/367 (0.5%) of the controls (P = 0.032). ROC analysis showed discrimination between SSc patients and controls with an area under the curve value of 0.732 (95% CI 0.655, 0.809).

Conclusion

Rpp25 is a major target of autoantibodies to the Th/To autoantigen complex. Further studies are needed to evaluate the clinical utility of the new assays.     

7-Oxopyrrolopyridine-derived DPP4 inhibitors-mitigation of CYP and hERG liabilities via introduction of polar functionalities in the active site

Design, synthesis, and SAR of 7-oxopyrrolopyridine-derived DPP4 inhibitors are described. The preferred stereochemistry of these atropisomeric biaryl analogs has been identified as Sa. Compound (+)-3t, with a K(i) against DPP4, DPP8, and DPP9 of 0.37 nM, 2.2, and 5.7 μM, respectively, showed a significant improvement in insulin response after single doses of 3 and 10 μmol/kg in ob/ob mice.