Sample displacement chromatography of Atlantic Salmon (Salmo salar) thrombin

A modified method of sample displacement chromatography (SDC) was used to purify active salmon thrombin on a heparin-coupled matrix to near homogeneity in milligram amounts from 117 ml plasma. This was achieved by combining a low-pressure multi-column affinity chromatography system with non-homogenous sample application in the order of increasing affinity to Heparin Sepharose. The results suggest that this modified method could be useful in protein purification. Some characteristics of salmon thrombin are presented.     

OX1 orexin receptors activate extracellular signal-regulated kinase in Chinese hamster ovary cells via multiple mechanisms: the role of Ca2+ influx in OX1 receptor signaling

Activation of OX1 orexin receptors heterologously expressed in Chinese hamster ovary (CHO) cells led to a rapid, strong, and long-lasting increase in ERK phosphorylation (activation). Dissection of the signal pathways to ERK using multiple inhibitors and dominant-negative constructs indicated involvement of Ras, protein kinase C, phosphoinositide-3-kinase, and Src. Most interestingly, Ca2+ influx appeared central for the ERK response in CHO cells, and the same was indicated in recombinant neuro-2a cells and cultured rat striatal neurons. Detailed investigations in CHO cells showed that inhibition of the receptor- and store-operated Ca2+ influx pathways could fully attenuate the response, whereas inhibition of the store-operated Ca2+ influx pathway alone or the Ca2+ release was ineffective. If the receptor-operated pathway was blocked, an exogenously activated store-operated pathway could take its place and restore the coupling of OX1 receptors to ERK. Further experiments suggested that Ca2+ influx, as such, may not be required for ERK phosphorylation, but that Ca2+, elevated via influx, acts as a switch enabling OX1 receptors to couple to cascades leading to ERK phosphorylation, cAMP elevation, and phospholipase C activation. In conclusion, the data suggest that the primary coupling of orexin receptors to Ca2+ influx allows them to couple to other signal pathways; in the absence of coupling to Ca2+ influx, orexin receptors can act as signal integrators by taking advantage of other Ca2+ influx pathways.     

Notes on inland water molluscs from Egypt (Mollusca: Gastropoda,Bivalvia)

Abstract

A report on two new collections of fresh- and brackish water molluscs in Egypt is filling gaps in the generally known distribution pattern. The recent status of the mollusc fauna of Birket Qarun (Fayum), changed by introduction of marine species into the brackish water lake, is documented.     

Membrane bilayer properties of sphingomyelins with amide-linked 2- or 3-hydroxylated fatty acids

The bilayer properties and interactions with cholesterol of N-acyl hydroxylated sphingomyelins (SM) were examined, and results were compared to nonhydroxylated chain-matched SM. The natural OH(D)-enantiomer of hydroxylated SM (with 16:0 or 22:0 acyl chain lengths) analogs was synthesized. Measuring steady-state diphenylhexatriene anisotropy, we observed that pure 2OH-SM bilayers always showed higher (5-10 °C) gel-liquid transition temperatures (T(m)) compared to their nonhydroxylated chain-matched analogs. Bilayers made from 3OH(D)-palmitoyl SM, however, had lower T(m) (5 °C) than palmitoyl SM. These data show that hydroxylation in a position-dependent manner directly affected SM interactions and gel state stability. From the c-laurdan emission spectra, we could observe that 2OH-palmitoyl SM bilayers showed a redshift in the emission compared to nonhydroxylated palmitoyl SM bilayers, whereas the opposite was true for c-laurdan emission in 3OH-palmitoyl SM bilayers. All hydroxylated SM analogs were able to form sterol-enriched ordered domains in a fluid phospholipid bilayer. 2-Hydroxylation appeared to increase domain thermostability compared to nonhydroxylated SM, whereas 3-hydroxylation appeared to decrease domain stability. When sterol affinity to bilayers containing SM analogs was determined (cholestatrienol partitioning), the affinity for hydroxylated SM analog bilayers was clearly reduced compared to the nonhydroxylated SM bilayers. Our results with hydroxylated SM analogs clearly show that hydroxylation affects interlipid interactions in a position-dependent manner.     

Mangan als Vertreter für Magnesium im Betriebsstoffwechsel und im Baustoffwechsel der Zelle

Ohne Zusammenfassung     

Nitric oxide (NO)--production and regulation of insulin secretion in islets of freely fed and fasted mice

Production of nitric oxide through the action of nitric oxide synthase (NOS) has been detected in the islets of Langerhans. The inducible isoform of NOS (iNOS) is induced by cytokines and might contribute to the development of type-1 diabetes, while the constitutive isoform (cNOS) is thought to be implicated in the physiological regulation of insulin secretion. In the present study we have detected and quantified islet cNOS- and iNOS-derived NO production concomitant with measuring its influence on insulin secretion in the presence of different secretagogues: glucose, L-arginine, L-leucine and α-ketoisocaproic acid (KIC) both during fasting and freely fed conditions. In intact islets from freely fed mice both cNOS- and iNOS-activity was greatly increased by glucose (20 mmol/l). Fasting induced islet iNOS activity at both physiological (7 mmol/l) and high (20 mmol/l) glucose concentrations. NOS blockade increased insulin secretion both during freely fed conditions and after fasting. L-arginine stimulated islet cNOS activity and did not affect islet iNOS activity. l-leucine or KIC, known to enter the TCA cycle without affecting glycolysis, did not affect either islet cNOS- or iNOS activity. Accordingly, insulin secretion stimulated by L-leucine or KIC was unaffected by addition of L-NAME both during feeding and fasting. We conclude that both high glucose concentrations and fasting increase islet total NO production (mostly iNOS derived) which inhibit insulin secretion. The insulin secretagogues L-leucine and KIC, which do not affect glycolysis, do not interfere with the islet NO-NOS system.     

Effect of cooling on intracellular transport and secretion of thyroglobulin

Summary The effect of cooling to 20° C on the intracellular transport and secretion of thyroglobulin was studied by incubating open thyroid follicles isolated from porcine thyroid tissue. Follicles were labeled with ³H-leucine or ³H-galactose and the secretion of labeled thyroglobulin into the incubation medium was followed by chase incubations under various experimental conditions. The observations indicate that the transport of thyroglobulin is inhibited at three sites of the intracellular pathway by cooling to 20° C, i.e., between the RER cisternae and the Golgi cisternae, between the latter and the exocytic vesicles, and between these vesicles and the extracellular space (corresponding to the follicle lumen). The secretion of ³H-leucine-labeled thyroglobulin decreased linearly between 37° and 20° C; within this temperature range the activation energy for secretion, calculated from Arrhenius plots, was found to be 37 kcal/mol. Below 20° C the secretion was scarcely measurable. It is suggested that the three transport blocks at 20° C result mainly from inhibition of membrane fission and fusion due to phase transition in membrane lipids.