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The objective of this study was to optimize the methodology for spectrophotometric determination of sperm concentration in Atlantic croaker Micropogonias undulatus L. milt and to estimate its potential for short-term cold-storage. The spectrophotometric determination of sperm concentration was evaluated using milt samples from six males serially diluted in Hank's balanced salt solution at 200 mOsm kg−1 (HBSS). The predictive power of regression models between sperm concentration and absorbance was determined from 200 to 500 nm and found to be highest within the visible spectrum despite a peak of milt absorbance at 288 nm. Absorbance reading at 400 nm was selected for further analysis to maximize the absorbance of the sample hence the sensitivity of the method while minimizing the impact of potential sample contamination with blood. The standard-curve of correlation between sperm absorbance at 400 nm and concentration was validated and held an accuracy ranging between −7.40% and +4.56% across males. Total sperm motility duration and the proportion of motile spermatozoa were significantly higher in milt samples diluted 1:3 in HBSS than in the undiluted control during up to 30 h of cold-storage.The methodologies investigated in this study can be applied to optimize sperm usage and achieve predictable artificial fertilization protocols in Atlantic croaker. [ABSTRACT FROM AUTHOR]

KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K+ current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubuledependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4-5 h after drug washout. Coexpression studies with a dominant- negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential. [ABSTRACT FROM AUTHOR]

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level. [ABSTRACT FROM AUTHOR]

KCNH2 (hERG1) encodes the α-subunit proteins for the rapidly activating delayed rectifier K+ current (IKr), a major K+ current for cardiac myocyte repolarization. In isolated myocytes IKr frequently is small in amplitude or absent, yet KCNH2 channels and IKr are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and IKr are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myoctyes IKr, slowly activating delayed rectifier K+ current (IKs), and inward rectifier K+ current (IKl), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (IKCNH2) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished IKr; and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of IKCN2 and its mature channel protein over several hours. Thus the channel protein for IKCNH2 and IKr is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low IKr density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells. [ABSTRACT FROM AUTHOR]

KCNH2 (hERG1) encodes the α-subunit proteins for the rapidly activating delayed rectifier K+ current (IKr), a major K+ current for cardiac myocyte repolarization. In isolated myocytes IKr frequently is small in amplitude or absent, yet KCNH2 channels and IKr are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and IKr are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myoctyes IKr, slowly activating delayed rectifier K+ current (IKs), and inward rectifier K+ current (IKl), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (IKCNH2) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished IKr; and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of IKCN2 and its mature channel protein over several hours. Thus the channel protein for IKCNH2 and IKr is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low IKr density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells. [ABSTRACT FROM AUTHOR]

Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome. We previously showed that the HERG N470D mutation expressed as homotetrameric channels causes a protein trafficking defect, and this can be corrected by the HERG channel blocking drug E-4031. The N470D mutant also has been reported to cause dominant negative suppression of HERG current when coexpressed with wild-type channel subunits. The aims of this study were 1) to investigate the molecular mechanism responsible for the dominant negative effect of the N470D mutant co-expressed with wild-type subunits and 2) to test whether the trafficking defective heteromeric channels could be pharmacologically rescued by E-4031. Using a combination of immunoprecipitation and Western blot methods, we showed that N470D mutant and wild-type HERG subunits were physically associated in the endoplasmic reticulum as heteromeric channels. The coassembly resulted in the retention of both wild-type and N470D subunits in the endoplasmic reticulum. Culturing cells in E-4031 increased the cell surface expression of these channels, although with an altered electrophysiological phenotype. These results suggest that the dominant negative effect of the N470D wild-type coassembled channels is caused by retention of heteromeric channels in the endoplasmic reticulum and that the trafficking defect of these channels can be corrected by specific pharmacological strategies. [ABSTRACT FROM AUTHOR]

Presents a study that used the approaches of site-directed mutagenesis and biochemical modification to inhibit asparagine (N)-linked glycosylation and examined the role of glycosylation in the cell surface expression and turnover of human ether- à -go-go-related gene (HERG). Information on HERG; Details of the inhibition of N-linked glycosylation by tunicamycin; Cell surface expression of wild-type HERG and N-linked glycosylation.

[Figure: see text]. [ABSTRACT FROM AUTHOR]

A two-step reaction sequence yielding 2-aminooxazolines under mild conditions was developed. Both electron-rich and electron-deficient styrenes as well as functionalized primary and secondary amides could be used in the reaction to afford 2-aminooxazolines in reasonable yields. [ABSTRACT FROM AUTHOR]

The article discusses research on the putative stem cells found in the regenerated tissues through markers related to mesenchymal stem cells. It references the study "Putative Stem Cells in Regenerating Human Periodontium," by N. H. et al, published in the July 4, 2008 issue of "Journal of Periodontal Research." The researchers explained that the presence of cells with periodontal ligament (PDL) stem cells qualities in the tissue taken from the so-called guided tissue regeneration (GTR)-treated defects indicates the role of stem cells in healing.

Idiopathic ventricular fibrillation (IVF) is an unrefined diagnosis representing a heterogeneous patient group without a structural or genetic definition. IVF treatment is not mechanistic-based due to the lack of experimental patient-models. We sought to create a methodology to assess cellular arrhythmia mechanisms for IVF as a proof-of-concept study. Using IVF patient-specific induced pluripotent stem cell-derived cardiomyocytes, we integrate electrophysiological optical mapping with computational modeling to characterize the cellular phenotype. This approach flips the traditional paradigm using a biophysically detailed computational model to solve the problem inversely. Insight into the cellular mechanisms of this patient's IVF phenotype could also serve as a therapeutic testbed., Competing Interests: Funding Support and Author Disclosures This work was supported by NIH R01HL170521 (LLE, EG), NIH R01HL163987 (Dr Eckhardt), NIH R01HL139738 (Dr Eckhardt), 2R01HL141214 (Dr Glukhov), AHA846898, and AHA 24SCEFIA1255230 (Dr Lang), 24CDA1258695 (Dr Ni). This work was also funded in part by the Gary and Marie Weiner Professor in Cardiovascular Medicine Research (Dr Eckhardt). All authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.