Partial inhibition of SERCA is responsible for extracellular Ca2+ dependence of AlF-4-induced [Ca2+]i oscillations in rat pancreatic

AlF₄^-is known to generate oscillations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activating G proteins in many cell types. However, in rat pancreatic acinar cells, AlF₄^--evoked [Ca²⁺]_i oscillations were reported to be dependent on extracellular Ca²⁺, which contrasts with the [Ca²⁺]_i oscillations induced by cholecystokinin (CCK). Therefore, we investigated the mechanisms by which AlF₄^- generates extracellular Ca²⁺-dependent [Ca²⁺]_i oscillations in rat pancreatic acinar cells. AlF₄^--induced [Ca²⁺]_i oscillations were stopped rapidly by the removal of extracellular Ca²⁺ and were abolished on the addition of 20 mM caffeine and 2 µM thapsigargin, indicating that Ca²⁺ influx plays a crucial role in maintenance of the oscillations and that an inositol 1,4,5-trisphosphate-sensitive Ca²⁺ store is also required. The amount of Ca²⁺ in the intracellular Ca²⁺ store was decreased as the AlF₄^--induced [Ca²⁺]_i oscillations continued. Measurement of ⁴⁵Ca²⁺ influx into isolated microsomes revealed that AlF₄^-directly inhibited sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). The activity of plasma membrane Ca²⁺-ATPase during AlF₄^- stimulation was not significantly different from that during CCK stimulation. After partial inhibition of SERCA with 1 nM thapsigargin, 20 pM CCK-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺. This study shows that AlF₄^- induces [Ca²⁺]_i oscillations, probably by inositol 1,4,5-trisphosphate production via G protein activation but that these oscillations are strongly dependent on extracellular Ca²⁺ as a result of the partial inhibition of SERCA. cholecystokinin; plasma membrane adenosine 5'-triphosphatase; G proteins; caffeine     

Vibrio harveyi nitroreductase is also a chromate reductase

The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5alpha and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione S-transferase (GST) and E. coli DH5alpha NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5alpha NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The K(m) values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 micro M, respectively. The V(max) values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each V(max)/K(m) value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30 degrees C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to E. coli than Cr(VI).     

An oligosaccharide fraction from Korean mugwort herb suppresses death of the mouse thymocytes in culture by down-regulating the Fas death receptor gene

Korean mugwort herb is a preparation of dried leaves from Artemisia species and has been used as a traditional medicine in Asia. An oligosaccharide fraction, AVF3, purified from the preparation promoted survival of the mouse thymocytes in culture. A mouse gene array study suggests that the AVF3 may modulate Fas/FasL dependent apoptotic cell death and thus has influence on the survival of the thymocytes in culture. RT-PCR analysis confirmed the down-regulation of the Fas gene by the AVF3 treatment, supporting that the AVF3 modulated thymocyte death by suppressing the Fas gene expression.     

Sequential kinetic parameter estimation of anaerobic fluidized bed bioreactor using an optimization technique

Mathematical model parameters for the methanogenic degradation of propylene glycol were estimated in a sequential manner by means of an optimization technique. Model parameters determined from an initial experimental data set using one bioreactor were then verified with the results from a second bioreactor. The proposed methodology is a useful tool to obtain model parameters for continuous flow reactors with completely mixed regime. Abbrevations: S – substrate concentration (mg COD l^–1); S _in – influent substrate concentration (mg COD l^–1); D _L – dilution rate (day^–1); – stoichiometric coefficients (ND); nx – number of microbial species (ND); X _S – fixed biomass concentration (mg biomass l^–1); X _L – suspended biomass concentration of (mg biomass l^–1); k _d – decay rate of biomass (day^–1); b _S – specific detachment rate of biofilm (day^–1); – specific growth rate of biomass (day^–1); _m – maximum specific growth rate of biomass (day^–1); K _S – half saturation constant (mg COD l^–1); K _I – inhibition constant (mg COD l^–1).     

Assessment of genetic diversity of Korean native pig (Sus scrofa) using AFLP markers

In order to assess the genetic diversity and genetic relationships among the six commercial pig breeds including the Korean native pig, we performed an amplified fragment length polymorphism (AFLP) analysis. Applying the three EcoRI/TagI primer combinations to 54 individual pig samples out of six breeds, a total of 186 AFLP bands were generated, 67 (36%) of which were identified as polymorphic bands. From these polymorphic bands, the three estimates (percentage of polymorphic loci, Neis heterozygosity and Shannon index) of genetic diversity, G(ST) estimates, Neis unbiased genetic distance and two indices of genetic similarity were calculated. From all the calculations of genetic diversity, the lowest genetic diversity was exhibited in the Korean native pig, and the highest in the Chinese Yanbian pig. Given the mean G(ST) value (G(ST) = 0.390) across all pigs examined, levels of apparent breed subdivision were considerable. A UPGMA tree of individuals based on Jaccards similarity index showed that the Korean native pig formed a distinct cluster from the other five pigs. In addition, the tree displayed that all the individuals except for six individuals were grouped into their breeds. Principal component analysis based on the binary data matrix of either presence or absence confirmed the distinctness of the Korean native pig from the other pigs. Our results indicate that the Korean native pig has a low level of genetic diversity and is distinct from the five pig breeds, confirming the results from previous microsatellite data. The findings also suggest that AFLP analysis may be a valuable tool for revealing genetic relationships and genetic diversity among different pig breeds.     

Constant-volume hydrogel osmometer: a new device concept for miniature biosensors

A new type of biosensor is proposed that combines the recognition properties of "intelligent" hydrogels with the sensitivity and reliability of microfabricated pressure transducers. In the proposed device, analyte-induced changes in the osmotic swelling pressure of an environmentally responsive hydrogel are measured by confining it within a small implantable enclosure between a rigid semipermeable membrane and the diaphragm of a miniature pressure transducer. Proof-of-principle tests of this device were performed in vitro using pH-sensitive hydrogels, with osmotic deswelling data for the same hydrogels used as a benchmark for comparison. The swelling pressure of the hydrogel was accurately determined from osmotic deswelling measurements against reservoirs of known osmotic stress. Values of swelling pressure vs salt concentration measured with a preliminary version of the sensor agree well with osmotic deswelling results. Through modification of the hydrogel with various enzymes or pendant binding moieties, the sensor has the potential to detect a wide range of biological analytes with good specificity.     

Molecular identification of a novel carnitine transporter specific to human testis. Insights into the mechanism of carnitine recognition

l-Carnitine is an essential component of mitochondrial fatty acid beta-oxidation and plays a pivotal role in the maturation of spermatozoa within the male reproductive tract. Epididymal plasma contains the highest levels of l-carnitine found in the human body, and initiation of sperm motility occurs in parallel to l-carnitine increase in the epididymal lumen. Using a specific carrier, epididymal epithelium secretes l-carnitine into the lumen by an active transport mechanism; however, the structure-activity relationship comprising the carnitine-permeation pathway is poorly understood. We discovered a novel carnitine transporter (CT2) specifically located in human testis. Analyzing the primary structure of CT2 revealed that it is phylogenetically located between the organic cation transporter (OCT/OCTN) and anion transporter (OAT) families. Hence, CT2 represents a novel transporter family. When expressed in Xenopus oocytes, CT2 mediates the high affinity transport of l-carnitine but does not accept mainstream OCT/OCTN cationic or OAT anionic substrates. We synthesized and tested various carnitine-related compounds and investigated the physicochemical properties of substrate recognition by semi-empirical computational chemistry. The data suggest that the quaternary ammonium cation bulkiness and relative hydrophobicity be the most important factors that trigger CT2-substrate interactions. Immunohistochemistry showed that the CT2 protein is located in the luminal membrane of epididymal epithelium and within the Sertoli cells of the testis. The identification of CT2 represents an interesting evolutionary link between OCT/OCTNs and OATs, as well as provides us with an important insight into the maturation of human spermatozoa.