Apparent anion imbalance in the fresh water adapted eel

Summary On adapting brackish waterAnguilla anguilla to fresh water it was noted that, while the plasma sodium, magnesium,pCO₂ and pH were held reasonably constant, there was a substantial fall in chloride concentration (–33 mEq). The gradient of the linear correlations between plasma sodium and chloride also fell (brackish water gradient=0.92, fresh water gradient=0.21) indicating that a new pattern of plasma ion interrelationships was being established. Comparison with plasma Na/Cl ion ratios from other fishes suggested that this phenomenon was peculiar toA. anguilla. Corresponding with the very low plasma chloride levels plasma bicarbonate was four to five times that found in other fishes, and this was thought related to the finding that the haematocrit value almost doubled during adaptation to fresh water. In fresh water adapted fish a fall in plasma chloride was associated with a rise in plasma bicarbonate, however the charge compensation effect of this response was only partial as summing the common plasma cations and anions left an anion deficit of about 34 mEq to be accounted for.     

Identification of the milk fat globule membrane proteins. II. Isolation of major proteins from electrophoretic gels and comparison of their amino acid compositions

The fat globule membranes of milk are derived from the apical plasma membrane of the mammary secretory cells. The nature of the membrane proteins, as isolated from cows' milk, has been studied by the use of discontinuous and continuous SDS-gel electrophoresis. Six methods of preparation of milk fat globule membrane suggested by various authors were tested; gel electrophoresis showed that five major bands were present, independent of the method of preparation. The apparent molecular masses of these proteins as determined on SDS-gels (15% T) were 167, 142, 64, 49 and 46 kDa, respectively. The 167 kDa band stained only with periodic acid-Schiff reagent, while the 142 kDa band stained only with Coomassie blue; the last three bands stained with both. Delipidated membranes were extracted stepwise with water, 0.02 M NaCl and 0.6 M NaCl. The 64 kDa band appears to be nearly insoluble, while the bands of 142, 49 and 46 kDa are fractionated by this procedure. The resolution of all of these proteins by electrophoresis was superior to that achieved by molecular sieve chromatography, and so electrophoretic extraction was used to isolate the major proteins. Dansyl chloride derived proteins were used as markers. Amino acid compositions of the recovered proteins were obtained and are compared.     

Kinetics of release of serotonin from isolated secretory granules. I. Amperometric detection of serotonin from electroporated granules.

We developed a method for measuring the efflux of 5-hydroxytryptamine (5-HT, serotonin) from isolated intact granules of the mast cell of the beige mouse. This method combines electroporation of the vesicle membrane with amperometric detection of 5-HT. A single secretory granule is placed between two platinum electrodes (distance approximately 100 microm) and positioned adjacent (<1 microm) to a carbon fiber microelectrode. A short (approximately 30 micros) high-intensity voltage pulse (electric field of approximately 5 kV/cm) is delivered to the electrodes to trigger the mechanical breakdown of the granule membrane, which activates the release of 5-HT. We observed concurrent swelling of the granule matrix with the oxidation of 5-HT at the carbon fiber electrode (overpotential + 650 mV). Similar to the release of secretory products during exocytosis, the oxidation current exhibits a spike-like time course with a noninstantaneous rising phase (time between onset of current and maximum flux, t(max)) with approximately 25% of the molecules released during this period. When the current reaches its maximum, the granule matrix attains its maximum swollen state. We found that the rising phase depends on the initial cross-sectional area of the granule (t(max) approximately 21r2) and reflects the time required for membrane rupture. The average t(1/2)spike of the amperometric spikes was found to be approximately 150 ms, which is 3-7 times faster than the t(1/2) measured during cellular exocytosis.     

Synthesis and aryl hydrocarbon receptor binding properties of radiolabeled polychlorinated dibenzofuran congeners

Microchlorination of 1,4,9[3H]dibenzofuran gave several polychlorinated dibenzofuran (PCDF) products and 2,3,7,8-[3H]tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-[3H]pentachlorodibenzofuran (PeCDF), and 1,2,3,6,7,8-/1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) of high specific activity (57, 34, and 32.5 Ci/mmol, respectively) were purified by preparative high-pressure liquid chromatography. These compounds were investigated as radioligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor protein. Like 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD), the radiolabeled PCDF congeners exhibited saturable binding with the receptor protein and sucrose density gradient analysis of the radiolabeled ligand-receptor complexes gave specific binding peaks with comparable sedimentation profiles. The rank order of radioligand binding affinities (Kd values) was 2,3,7,8-TCDD greater than 2,3,7,8-TCDF greater than 1,2,3,6,7,8-HCDF greater than 1,2,3,7,8-PeCDF and the maximum difference in Kd values for the four radioligands was less than 13-fold (0.44-5.9 nM). The interactions of the PCDF radioligands with the cytosolic receptor all exhibited saturable binding curves and linear Scatchard plots and the slopes of their Hill plots were in the range 1.0-1.1, thus indicating that cooperativity was not a factor in these binding interactions. The relative stabilities and dissociation kinetics of the radioligand-receptor complexes were highly dependent on the structure of the radioligand. The dissociation curves of the 2,3,7,8-[3H]TCDD and PCDF receptor complexes were biphasic and this suggests that there may be a temporal shift in ligand binding affinities. However, the rates of dissociation did not correlate with the rank order of ligand binding affinities. The stabilities of the radioligand-receptor complexes were also dependent on the structures of the radioligands; for example, the 2,3,7,8-[3H]TCDD-receptor complex degraded more rapidly than the PCDF-receptor complex and these relative stabilities were clearly not related to the Kd values or the relative in vivo or in vitro biologic potencies of these halogenated aryl hydrocarbons.     

Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetes

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi.     

Stimulatory role for endogenous opioid peptides on postexercise insulin secretion in rats

Endogenous opioid peptides (EOP) and prior exercise may modulate the stimulatory effect of glucose on insulin secretion. To gain insights into these relationships, we studied male Wistar rats (187-245 g) during sustained hyperglycemia by use of the glucose clamp technique. Four groups of sedentary fed rats (n = 8/group) either ran (Ex) at 24 m/min, 0% grade, or rested (R) for 40 min. Thirty minutes after Ex or R, arterial blood glucose was elevated to and maintained at 11 mM for 2 h by a variable glucose infusion. At the start of Ex or R rats had saline (Sal) or naloxone (Nal, an opioid antagonist) intravenous infusions for 160 min (40 min Ex + 30 min R + 90 min of a 120-min glucose clamp). Steady-state glucose infusion rates (SSGIR) were approximately 55 mg.kg-1.min-1 at the start of the clamp and declined significantly over the 2nd h to approximately 45 mg.kg-1.min-1. No significant differences existed in SSGIR between groups. R-Sal and Ex-Sal groups did not differ in their insulin response to hyperglycemia. In contrast, when all groups were compared at the end of the Nal or Sal infusion, Ex-Nal had the lowest insulin concentration (749 +/- 174 pmol/l), whereas the R-Nal group had the highest (1,581 +/- 216 pmol/l, P less than 0.05). These data suggest a stimulatory role for EOP on insulin secretion that is expressed after a prior stress (Ex). Thus one function of exercise-induced activation of EOP may be to regulate insulin secretion in the immediate postexercise period.     

Fetal lung surfactant lipid synthesis from glycogen during organ culture

The role of fetal lung glycogen as a precursor for lipids during late gestational development was explored by a combination of in vivo labeling with [U-14C]glucose, administered directly to rat fetuses at 18.5 days, and in vitro assessment using an organ explant culture system. Our major objectives were to demonstrate that radioactivity was transferred specifically and preferentially to surfactant lipids, as glycogenolysis occurred, and to determine the molecular distribution of 14C labeling in newly synthesized phosphatidylcholine (PC). Surfactant and residual (non-surfactant) lipids were separated by sucrose density gradient centrifugation, and other subcellular fractions such as microsomes were isolated by subsequent centrifugations. After 72 h of culture, there was a 5.7-fold increase in the concentration of PC in the surfactant fraction, which contributed 8.8% of total PC at the beginning and 29.6% (P less than 0.001) at the end of the 72 h period. The labeling of PC in the surfactant fraction increased markedly during culture, but there was no significant change in the residual fraction or microsomal PC. Hydrolysis of surfactant PC indicated that the radioactivity was predominantly located in the fatty acyl portion of the molecule, both before and after culture; however, PC glycerol labeling also increased significantly during culture. The distribution of PC radioactivity was similar in the residual fraction and microsomes, with the majority of 14C in the fatty acids. Neutral lipid radioactivity also increased significantly in both the surfactant (240%) and residual (136%) fractions. Quantitation of the changes in radioactivity among subcellular components during lung explant culture indicated that the greatest decrease occurred in glycogen, whereas only lipids, particularly those of the surfactant fraction, were found to show significant increases. These results support the hypothesis that glycogen, which accumulates in fetal lung prior to augmented surfactant production, can supply precursors for synthesis of functionally essential pulmonary phospholipids.     

Splenic natural killer-cell activity in mice infected with Leishmania donovani

Several strains of inbred mice were infected with the protozoan parasite Leishmania donovani, and, at several points during the infection, spleens of groups of these mice were tested for natural killer (NK)-cell activity vs lymphoma target cells in vitro and were evaluated for parasite burdens. Generally, elevated followed by normal (compared to uninfected control mice) or subnormal NK responses occurred as the result of infection. Elevated NK responses were not accompanied by high circulating levels of interferon, yet infected mice responded to an injection of an interferon inducer with interferon production as great as control mice. No consistent correlations among susceptibility phenotype to L. donovani infection, spontaneous NK activity phenotype, and infection-induced NK activation/depression patterns were detected among the various strains of mice.     

Tubulin-colchicine complexes differentially poison opposite microtubule ends

The kinetics of radiolabeled guanosine 5'-triphosphate-tubulin dimer addition to preformed microtubule copolymers, containing large numbers of tubulin-colchicine complexes (TCs), were examined at apparent equilibrium. The results indicated that radiolabeled dimer addition to copolymers occurs predominantly by a "treadmilling" reaction, analogous to that described for unpoisoned microtubules, and that some labeled dimer uptake also occurs by equilibrium exchange. The data further showed that TCs decrease the steady-state treadmilling reaction in a concentration-dependent manner. Since microtubule copolymers exhibited a treadmilling reaction, it was possible to differentially radiolabel opposite copolymer ends with [3H]- and [14C]guanine nucleotides and thus to measure the effects of TCs on dimer loss from opposite copolymer ends upon copolymer dilution. Dimer loss from both copolymer ends was inhibited in a concentration-dependent manner, but dimer loss from copolymer net assembly (A) ends (defined under steady-state conditions) was inhibited to a far greater extent than that from the opposite, net disassembly (D) copolymer ends. TCs therefore exhibited a graded, polar poisoning action, with copolymer A-end association and dissociation rate constants being far more susceptible to TC inhibition than those at the opposite copolymer D ends. The potential significance of this TC effect for regulating microtubule spatial orientation in vivo is discussed.