Insulin-Like Peptides of the Cerebropleural Ganglion of the Mollusc Anodonta cygnea: Isolation, Purification, and Radioligand Analysis

Cerebropleural ganglia from 4000 individuals of the mollusc Anodonta cygnea were submitted to procedures developed for isolation of vertebrate pancreatic insulins: homogenization and extraction, stage-like isoelectrical sedimentation, and ion-exchange chromatography. As a result of purification of the obtained preparation, using high-effective liquid chromatography, there were identified 7 protein peaks differing by time of retention on the reverse-phase sorbent in acetonitryl gradient and designated as insulin-related peptides (IRP), IRP1-IRP7. The material was characterized by the peptide ability to inhibit specific binding of ¹²⁵I-insulin and of insulin-related factor-1 (¹²⁵I-IGF-1) by plasma membranes of the rat liver and brain. The IC₅₀ value of peptide concentration (nM) able to replace 50% of the labeled hormone bound with the receptor amounted in the insulin radioreceptor system for IRP1 to 330, for IRP3 to 130, for IRP4 to 17, for IRP5 to130, for IRP6 to 420 nM. Peptide IRP7 at a maximal concentration (10⁴ ng/ml) replaced less than 50% of labeled hormone, whereas in IRP2 no inhibitory ability was detected under these experimental conditions. The IC₅₀ value in the case of ¹²⁵I-IGF-1 amounted for IRP1, IRP4, and IRP5 to17, for IRP2 to 50, for IRP3 to 83, for IRP6 to 133 nM. IRP7 at a concentration of 10⁴ ng/ml replaced less than 50% of labeled hormone. The same high relative affinity of the peptide IRP4 (12% of activity of standard insulin and IGF-1) to both receptor types is revealed. The results of analysis in two types of hormonal test systems indicate the ability of the insulin-related peptides of the anodonta cerebropleural ganglion to interact with the vertebrate receptor of insulin and IGF-1. This gives grounds to suggest the presence of the metabolic and growth-stimulating properties in these peptides. For the first time, the IGF-1 activity is revealed in insulin-like molecules in invertebrates. Taking into account the chromatographically revealed differences of physicochemical characteristics of individual IRP as well as predominance of their IGF-1-binding properties, there is suggested another organization of the IRP receptor-binding domains in IPR of this mollusc species, as compared with mammalian insulins.     

Role of Ca2+-activated K+ channel in regulation of NaCl reabsorption in thick ascending limb of Henle's loop

1. Reabsorption of NaCl in the thick ascending limb of Henle's loop involves the integrated function of the Na+,K+,Cl- -cotransport system and a Ca2+-activated K+ channel in the luminal membrane with the Na+,K+-pump and a net Cl- conductance in the basolateral membrane. 2. Assay of K+ channel activity after reconstitution into phospholipid vesicles shows that the K+ channel is stimulated by Ca2+ in physiological concentrations and that its activity is regulated by calmodulin and phosphorylation from cAMP dependent protein kinase. 3. For purification luminal plasma membrane vesicles are isolated and solubilized in CHAPS. K+ channel protein is isolated by affinity chromatography on calmodulin columns. The purified protein has high Ca2+-activated K+ channel activity after reconstitution into vesicles. 4. The purified K+ channel consists of two proteins of 51 and 36 kDa. Phosphorylation from cAMP dependent protein kinase stimulates K+ channel activity and labels the 51 kDa band. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca2+ stimulation. 5. Opening of the K+ channel by Ca2+ in physiological concentrations and regulation by calmodulin and phosphorylation by protein kinase may mediate kinetic and hormonal regulation of NaCl transport across the tubule cells in TAL.     

Schistosoma mansoni polypeptides immunogenic in mice vaccinated with radiation-attenuated cercariae

We compared the humoral immune response of mice protected against Schistosoma mansoni by vaccination with radiation-attenuated cercariae to that of patently infected mice, and we identified antigens that elicit a greater, or unique, immune response in the vaccinated mice. These comparisons were based upon radioimmunoprecipitations and immunodepletion of [35S]methionine-labeled schistosomular and adult worm polypeptides, followed by one- and two-dimensional polyacrylamide gel analyses. The humoral responses of patently infected mice and of mice vaccinated once were remarkably similar and were directed against schistosome glycoproteins ranging in molecular size from greater than 300 to less than 10 kDa. Exposing mice to a second vaccination resulted in a marked change in the immune response, to one predominantly directed toward high molecular size glycoproteins. Sequential immunodepletion techniques identified five schistosomular and seven adult worm antigens that showed a greater or unique immunogenicity in vaccinated mice as compared with patently infected mice. These adult worm antigens were purified by preparative sequential immunoaffinity chromatography and used to prepare a polyclonal antiserum, anti-irradiated vaccine. This antiserum bound to the surface of live newly transformed and lung-stage schistosomula, as assessed by immunofluorescence assays, and was reactive with a number of 125I-labeled schistosomular surface polypeptides, including a doublet of 150 kDa that was also recognized by sera of vaccinated mice but not by sera of patently infected mice.     

Central transmural venous pressure and plasma arginine vasopressin during negative pressure breathing in man

After overnight food and fluid restriction, 8 normal healthy males were examined in the upright sitting position before (prestudy), during and after (recovery) negative pressure breathing (NPB) with a pressure (P = difference between airway pressure and barometric pressure) of -9.6 +/- 0.5 to -10.4 +/- 0.4 mm Hg for 30 min. Plasma arginine vasopressin (pAVP) did not change significantly comparing prestudy with 10 and 30 min of NPB or comparing recovery with NPB at 10, 20 or 30 min. However, at 20 min of NBP, pAVP was slightly lower than at prestudy (p less than 0.05). Central venous pressure (CVP) decreased significantly during NPB, and central transmural venous pressure (CVP-P) increased significantly from -0.9 +/- 0.8 mm Hg to 3.8 +/- 0.7, 4.3 +/- 0.7 and 4.5 +/- 0.6 mm Hg (p less than 0.001) after 10, 20 and 30 min, respectively. Systolic, diastolic and mean arterial pressure and heart rate did not change significantly during NPB. Diuresis, natriuresis, kaliuresis, osmotic excretion and clearance were slightly increased during the recovery hour after NPB compared to prestudy, while urine osmolality decreased during NPB (n = 6). However, none of these changes were significant. There was no significant correlation between CVP-P and pAVP. In conclusion, -10 mm Hg NPB for 30 min in upright sitting subjects did not change pAVP consistently, while CVP-P was significantly increased and HR and arterial pressures were unchanged. This lends support to the concept that arterial baroreceptors and not cardiopulmonary mechanoreceptors are of importance in regulating AVP secretion in man.     

Germination,reproduction and interference in the amphicarpic annual Emex spinosa (L.) Campd

Summary A comparison was made of the dimorphic subterranean and aerial achenes of the annual Emex spinosa (L.) Campd. Subterranean achenes were less dormant, had a higher percentage of viability, germinated at a faster rate, were less temperature-dependent in germination and produced larger seedlings than aerial achenes. Subterranean achenes germinated, equally well in light or dark but aerial achenes had a strong light requirement. There was a linear relationship between numbers of weight of aerial achenes and plant size in the field. More resources were allocated to reproduction in plants restricted in size because of environmental conditions. Subterranean achene production was less affected than aerial by density, nitrogen levels, varying field conditions and interference between plants grown, from subterranean and aerial achenes. In mixtures of such plants, those from subterranean achenes had larger leaf area and stems and more aerial achenes. These differences were not apparent in monocultures by the time of harvest. Seed dimorphism is more likely in short-lived fugitive species and the different roles associated with dimorphism (persistence in situ versus dispersal in E. spinosa) are seen as an ecological advantage in such species.     

Further evidence for heterogeneity of glucose-6-phosphate dehydrogenase deficiency in Papua New Guinea

Summary Four new G6PD variants have been characterized in individuals from Papua New Guinea. This study demonstrates that the previously reported Markham variant and the newly characterized Salata variant may be widely distributed in Papua New Guinea. The data presented here together with those of previously published studies demonstrate a degree of heterogeneity of G6PD deficiency that is much higher than that in other regions of the world where G6PD deficiency is common.     

Calcium ion stimulated endogenous protein kinase catalyzed phosphorylation of basic proteins in myelin subfractions and myelin-like membrane fraction from rat brain

Polypeptide composition and endogenous phosphorylation were investigated in the subfractions of rat brain myelin isolated by either discontinuous or continuous sucrose density gradient centrifugation of myelin. Similarly, a myelin-like membrane fraction (SN4) was also studied. Observations were made that strongly indicated the presence of a calcium-stimulated protein kinase in a highly purified myelin preparation and which exclusively phosphorylated myelin basic proteins of the membrane preparation. Adenosine cyclic 3',5'-phosphate (cAMP) stimulated kinase on the other hand was found to be considerably enriched in the myelin-like membrane fraction. Although this latter enzyme is also capable of phosphorylating the basic proteins, its effect was at least 5 times weaker compared to the calcium-stimulated myelin protein kinase. Within the gradient subfractions there appeared a close relation between the amount of basic proteins and their calcium-stimulated phosphorylation; a similar relationship, however, was not obtained in the case of cAMP-dependent phosphorylation of myelin basic proteins. The former (i.e., Ca2+-stimulated phosphorylation) was found to require a protein factor that functionally resembled calmodulin. The results thus raises an interesting possibility of the presence of calmodulin-like proteins and a calcium-stimulated protein kinase in adult myelin membrane from mammalian brain, both of which have been hitherto unrecognized constituents of myelin membranes.