Comparative cytogenetics in four species of Palinuridae: B chromosomes, ribosomal genes and telomeric sequences

The evolutionary pathway of Palinuridae (Crustacea, Decapoda) is still controversial, uncertain and unexplored, expecially from a karyological point of view. Here we describe the South African spiny lobster Jasus lalandii karyotype: n and 2n values, heterochromatin distribution, nucleolar organizer region (NOR) location and telomeric repeat structure and location. To compare the genomic and chromosomal organization in Palinuridae we located NORs in Panulirus regius, Palinurus gilchristi and Palinurus mauritanicus: all species showed multiple NORs. In J. lalandii NORs were located on three chromosome pairs, with interindividual polymorphism. In P. regius and in the two Palinurus species NORs were located on two chromosome pairs. In the two last species 45S ribosomal gene loci were also found on B chromosomes. In addition, the nature and location of telomeric repeats were investigated by FISH in J. lalandii, P. gilchristi, P. mauritanicus Palinurus elephas, and P. regius (Palinuridae, Achelata), and in Scyllarus arctus (Scyllaridae, Achelata): all these Achelata species showed the (TTAGG)n pentameric repeats. Furthermore, in J. lalandii these repeats occurred in all the telomeres and in some interstitial chromosomal sites, associated with NORs.     

The amino acid sequence of a protein from wheat kernel closely related to proteins involved in the mechanisms of plant defence

The amino acid sequence of wheatwin1, a monomeric protein of 125 residues isolated from wheat kernel (variety S. Pastore), is reported. Wheatwin1 is highly homologous (95%) to barwin, a protein from barley seed, which was shown to be related to the C-terminal domain of two proteins encoded by the wound-induced geneswin1 andwin2 in potato and to a protein encoded by the same domain of the hevein gene (hev1) in rubber tree. Similarly to barwin, wheatwin1 contains six cysteine residues all linked in disulfide bridges and the N-terminal residue is pyroglutamate. Moreover, structural studies performed on wheatwin1 andwin1 protein by predictive methods demonstrated that these proteins and barwin are closely related in the secondary structure also. The high level of homology found with the product ofwin1,win2, andhev1 genes strongly suggests that barwin and wheatwin1 play a common role in the mechanism of plant defence.     

Red bat fatality: Geographic extents through deuterium and niche models

Bat fatality at wind energy facilities is a conservation issue, but its effect on bat populations is difficult to estimate. We have little understanding of wind turbine effects on bat population persistence, in part because we have poor knowledge of bat migration pathways and hence the source populations for individual fatalities. We used deuterium ratio analysis combined with genetic algorithm for rule-set prediction and the web-based isoscapes modeling, analysis, and prediction in a geographic information system environment as a novel approach. Our objectives were to explore the utility of these methods together and map the geographic extents of eastern red bat (Lasiurus borealis) specimens salvaged in 2008–2010 from a single, 92-km² wind energy facility in Illinois, USA. Results indicate that combining these methods can be successful and support their use with species where ranges may be less well defined. Because of the migratory nature of this species and the range of deuterium values of pixels in our isotope model, we predicted that 18% and 82% of the specimens would have isotope results inside and outside of the wind facility's isocline respectively. We concluded that 71.4% of the specimens had isotope signatures placing them outside the wind facility's isocline. It could be argued that the wide distribution of bat fatalities dilutes the overall effect of those fatalities on the bat species; however, if other facilities show a similar pattern, each facility could have cumulative and far reaching population-level effects. © 2019 The Wildlife Society.     

Hydrolysis of homocysteine thiolactone results in the formation of Protein-Cys-S-S-homocysteinylation

An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys³⁴ of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys³⁴ of albumin and homocysteine attached to lysine residues.     

Hierarchic organization of globular proteins: Experimental studies on thermolysin

Previous studies from this laboratory have shown that the thermolysin fragment 121–316, comprising entirely the“all-α” COOH-terminal structural domain 158–316, as well as fragment 206–316 (fragment FII) are able to refold into a native-like, stable structure independently from the rest of the protein molecule. The present report describes conformational properties of fragments 228–316 and 255–316 obtained by chemical and enzymatic cleavage of fragment FII, respectively. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultra-violet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. Melting curves of the secondary structure of the fragments show cooperativity with a temperature of half-denaturationT _mof 65–66°C. The results of this study provide evidence that it is possible to isolate stable supersecondary structures (folding units) of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain 158–316 of thermolysin.     

Plasma methionine determination by capillary electrophoresis-UV assay: application on patients affected by retinal venous occlusive disease

Methionine is an important amino acid involved in protein synthesis and transmethylation reactions. It is also the precursor of homocysteine and cysteine, two important risk factors for cardiovascular diseases. As homocysteine research has gained impulsion, the evaluation of plasma methionine concentrations has acquired importance. Methionine measurement generally has been performed by HPLC after o-phthalaldehyde derivatization. Its separation from other amino acids is time-consuming. We set up a new specific capillary electrophoresis method in which analyte derivatization was avoided by sample concentration before analysis. Methionine was detected by UV absorbance at 204 nm with a detection limit of 0.5 micromol/L. By a capillary with an effective length of 50 cm filled with 125 mmol/L Tris phosphate buffer at pH 2.3, the separation occurred in less than 14 min. Precision tests indicated a good test repeatability for both migration times (coefficient of variation [CV]<0.3%) and areas (CV<2.0%). Moreover, a good reproducibility of intraassay and interassay tests was obtained (CV<2.9% and CV<3.5%, respectively). The Passing-Bablok regression and the Bland-Altman test for methods comparison suggest that the data obtained by our method and by a reference HPLC assay are similar. Assay performance was evaluated measuring methionine concentrations in retinal venous occlusive disease.     

SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta production

In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca(2+) is maintained at low levels by pumping Ca(2+) into stores in the endoplasmic reticulum (ER) via the sarco ER Ca(2+)-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca(2+), whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca(2+) clearance and exaggerating inositol 1,4,5-trisphosphate-mediated Ca(2+) liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid beta production. Our results point to a physiological role for the presenilins in Ca(2+) signaling via regulation of the SERCA pump.     

Detection of Tissue Factor Antigen and Coagulation Activity in Coronary Artery Thrombi Isolated from Patients with ST-Segment Elevation Acute Myocardial Infarction

Introduction

Although ruptured atherosclerotic plaques have been extensively analyzed, the composition of thrombi causing arterial occlusion in patients with ST-segment elevation acute myocardial infarction has been less thoroughly investigated. We sought to investigate whether coagulant active tissue factor can be retrieved in thrombi of patients with STEMI undergoing primary percutaneous coronary intervention.

Methods

Nineteen patients with ST-segment elevation acute myocardial infarction referred for primary percutaneous coronary intervention were enrolled in this study. Coronary thrombi aspirated from coronary arteries were routinely processed for paraffin embedding and histological evaluation (4 patients) or immediately snap frozen for evaluation of tissue factor activity using a modified aPTT test (15 patients). Immunoprecipitation followed by immunoblotting was also performed in 12 patients.

Results

Thrombi aspirated from coronary arteries showed large and irregular areas of tissue factor staining within platelet aggregates, and in close contact with inflammatory cells. Some platelet aggregates stained positive for tissue factor, whereas others did not. Monocytes consistently stained strongly for tissue factor, neutrophils had a more variable and irregular tissue factor staining, and red blood cells did not demonstrate staining for tissue factor. Median clotting time of plasma samples containing homogenized thrombi incubated with a monoclonal antibody that specifically inhibits tissue factor-mediated coagulation activity (mAb 5G9) were significantly longer than their respective controls (88.9 seconds versus 76.5 seconds, respectively; p<0.001). Tissue factor was also identified by immunoprecipitation in 10 patients, with significant variability among band intensities.

Conclusions

Active tissue factor is present in coronary artery thrombi of patients with ST-segment elevation acute myocardial infarction, suggesting that it contributes to activate the coagulation cascade ensuing in coronary thrombosis.     

Stabilization of Helical Peptides by Mixed Spaced Salt Bridges

Abstract

Whether or not surface salt bridges have a strong stabilizing effect on the native structure in proteins remains uncertain. Previous studies of model peptides have shown that salt bridges spaced at i,i+4 along the chain are more stabilizing than those spaced at i,i+3, with a preference for the order acid-base rather than base-acid from N to C terminus. An analysis of the effect of spacing the ion pairs in short helical peptides is presented, in which acidic and basic side chains spaced two or three residues apart alternate along the chain. The mixed spacing proves to be stabilizing relative to pure spacings. A control peptide in which salt bridges were spaced uniformly three residues apart proved to form a β-sheet structure rather than a-helix. This is due to formation of a silk-like apolar face consisting of alanine side chains; the mesoscopic structure formed by these sheets can be imaged by scanning microscopy.