Dissociation between Ca2+-ATPase and alkaline phosphatase activities in plasma membranes of rat duodenum

The presence of Ca²⁺-ATPase activities with high-affinity sites for Ca²⁺ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802–803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca²⁺, the substrate specificity of Ca²⁺-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca²⁺-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca²⁺-dependent ATP-hydrolysis was greater than that of ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ at Ca²⁺ concentrations below 25 μM. At 0.2 mM Ca²⁺ the rates of ATP, ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca²⁺, but at 0.2 mM Ca²⁺, Ca²⁺-induced hydrolysis of ADP and AMP was greater than either ATP or $\text{p-}\text{nitrophenylphosphate}$. Alkaline phosphatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca²⁺-stimulated ATP hydrolysis at 1 μM Ca²⁺ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca²⁺, Ca²⁺-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca²⁺-stimulated ATP hydrolysis, either at low or at high Ca²⁺ concentrations. Chlorpromazine fully inhibited Ca²⁺-stimulated ATP hydrolysis in basolateral fragments at 5 μM Ca²⁺, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca²⁺-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca²⁺-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium, is stimulated slightly by low Ca²⁺ concentrations, but this Ca²⁺-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.     

Thin-layer chromatography with agarose gels: A quick,simple method for evaluating liposome size

Thin-layer gels can be made with agarose and used to assess within a few minutes the efficiency with which multilamellar vesicles are converted to small unilamellar ones by sonication. A fluorescent lipid marker or vesicle-encapsulated solute permits continuous monitoring of the chromatography. Advantages over agarose gel column chromatography include speed of analysis, small sample size, the possibility of running multiple samples simultaneously, and direct accessibility to fluorescence microscopy. This approach should also be useful in the study of liposome-lipoprotein interactions and in affinity chromatography of liposomes.     

Antigen-specific helper activity in serum of mice primed with sheep red cells. I. Definition of the test system and comparison with other systems

An adoptive transfer system is described to measure serum helper activity in the primary antibody response to sheep red blood cells (SRBC). Mice injected with a high dose of cyclophosphamide and reconstituted with rabbit anti-thymocyte serum-treated spleen cells were used as recipients. Serum obtained 9 hr after ip injection of normal mice with 2 × 10⁸ SRBC (S(SRBC)) injected i.v. in the recipients caused a significant enhancement of the antibody response to 2 × 10⁷ SRBC. The serum helper activity was not generated in thymectomized animals and could be absorbed from S(SRBC) by normal and formalinized SRBC. The SRBC-specific serum helper activity (SSHA) is heat labile (30 min 56 °C) and shows allogeneic restriction. Another test system described in literature for measuring T-cell help in vivo was less suited to measure SSHA in the response to 2 × 10⁷ SRBC. A system using normal mice injected with 10⁵ SRBC for determining specific immune response-enhancing factor (SIREF), demonstrated SIREF activity in S(SRBC). It did, however, not measure SSHA, as absorption of S(SRBC) with formalinized SRBC did not abolish the activity in that system.     

Model studies on the acriflavine-Feulgen reaction

The specificity and quantitative reliability of the Feulgen-acriflavine-SO2 procedure was tested on polyacrylamide model films containing DNA. Noncovalent binding of acriflavine to DNA was observed when the washing procedure, as used in the classical way, was applied. The noncovalently bound acriflavine could be removed with an extra wash in acid-ethanol. The presence of SO2 in the staining solution has been found to enhance covalent binding significantly. The absorbance of films stained by our Feulgen-acriflavine-SO2 procedure is directly proportional to that obtained by the classical Feulgen-pararosanilline-SO2 procedure. The acriflavine-Feulgen procedure has also been tested using a commercial and a purified dye. The use of purified acriflavine, compared to a commercial sample did not result in a significant difference in the maximum absorbance value of stained DNA nor in the absorption or the fluorescence emission spectra of acriflavin covalently bound to DNA.     

Expression of MHC antigens by mouse thymic dendritic cells.

Thymic epithelial cells express MHC antigens in several different patterns. I-A is present throughout the thymic cortex on dendritic cells. The remainder of the I region and H-2K/D are expressed on dendritic cells apparently only variably in the cortex (at least in some haplotypes). All MHC antigens tested are present in the medulla on epithelial cells; expression on medullary lymphocytes cannot be evaluated. Monoclonal anti-MHC antibodies confirm these results. The significance of these findings to T cell maturation is discussed.     

Copepod ectoparasites of Merlangius merlangus and Platichthys flesus

Seasonal changes in the levels of copepod ectoparasitic infections from Merlangius merlangus and Platichthys flesus were examined and related to the annual migrations of young fish into an estuary. Lernaeocera branchialis (L.) was the only parasite common to both fish species; its life-cycle stages infected the branchial chambers of 96·7% flounders ( P. flesus ) and 40·4% whiting ( M. merlangus ). Infection in the two fish species exhibited similar seasonal fluctuations and a temporal relationship between the parasite's occurrence on its two hosts was apparent. Whiting were also parasitized by Clavella uncinata (Milller); 26·9 % fish were affected. The copepods' distribution was highly localized with the majority restricted to the long gill rakers of the first gill arch. Infection was related to size of whiting up to 12·5 cm; there was little increase beyond this length. In addition to L. branchialis , flounder was also host to Acanthochondria depressa (T. Scott) and Lepeophtheirus pectoralis (Müller). A. depressa showed a preference for the ocular side of the body with its developing stages attached mainly to the second gill arch and the adults located in the posterior pocket of the branchial chamber. L. pectoralis was associated with the paired fins. Factors which could result in such localized distributions are discussed.