IgG from antiserum against endogenous mammalian FMRF-NH2-related peptides augments morphine- and stress-induced analgesia in mice

Two mammalian FMRF-NH2-like peptides have been isolated from bovine brain; an octapeptide with the structure Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2) and an octadecapeptide, Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe- NH2 (A-18-F-NH2). In the present study determinations were made of the effects of intracerebroventricular administration of IgG prepared from antisera raised against these peptides on nociception and morphine- and immobilization-induced opioid analgesia in mice. Both F-8-F-NH2-IgG and A-18-F-NH2-IgG antisera increased nociception (thermal response latency) and significantly augmented morphine- and immobilization-induced analgesia in a naloxone reversible manner, with F-8-F-NH2-IgG antisera having a greater effect than A-18-F-NH2-IgG antisera. These results provide further evidence that mammalian FMRF-NH2-like peptides function as endogenous opiate antagonists and have a role in the mediation of antinociception.     

Neurotensin immunoreactivity in the central nucleus of the rat amygdala: an ultrastructural approach

Neurotensin (NT) was demonstrated in the central nucleus of the rat amygdala (CNA) using a modification of the avidin-biotin complex immunohistochemical technique. Electron-dense reaction product (particles were 15-25 nm in diameter) was localized in perikarya, dendrites, axons, and axon terminals. It was found also associated with profiles of rough endoplasmic reticulum, mitochondria, microtubules, and small agranular as well as large granular vesicles. In distal dendrites, the reaction product was associated with microtubules, vesicles, and postsynaptic densities. Axon terminals of three types formed synaptic contracts with NT-immunoreactive neurons in the CNA: one was characterized by numerous round or oval agranular vesicles, the second by numerous pleomorphic vesicles, and the third by agranular vesicles that were loosely distributed and pleomorphic. All three types formed symmetric axosomatic and asymmetric axodendritic contacts. NT-immunoreactive axon terminals containing small round agranular vesicles stood out clearly from the intermingling profiles of immunonegative structures. We found numerous glomeruli, each consisting of a central NT-immunoreactive dendrite surrounded by all three types of axon terminals. We observed that some NT-immunoreactive terminals formed symmetric axoaxonal contacts with each other, providing evidence for the presence of local NT-to-NT circuits, whereas many others synapsed with axon terminals devoid of NT immunoreactivity.     

Enzymatic methods for determining populations of Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091 in pure and mixed cultures

Summary Streptococcus cremoris AM2 is characterized by an aminopeptidase and Leuconostoc lactis CNRZ 1091 by an -galactosidase and a citrate lyase. These strains were grown in pure or mixed cultures, in presence or absence of citrate (15 mM) and at controlled or uncontrolled pH. Cell populations and the activities of the enzymes were measured during microbial growth. Linear correlations were established between the population of S. cremoris AM2 and aminopeptidase activity, and between that of L. lactis CNRZ 1091 and the activities of -galactosidase and citrate lyase. These correlations held regardless of whether the culture was pure or mixed and if the pH was controlled or not. The presence of citrate did not change citrate lyase and aminopeptidase activities, but inhibited the synthesis of the -galactosidase and not its activity. The linear relationships permit the determination of bacterial populations in less than 2 h without counting but by measuring enzyme activities.     

The thiol proteinases from the latex of Carica papaya L. III. The primary structure of chymopapain

The amino-acid sequence of chymopapain is presented. It was isolated from the latex of the fruits from the tropical species Carica papaya L. and is, besides papain and papaya proteinase omega, the third thiol proteinase from this source. The primary structure contains 218 amino-acid residues. It was deduced from sequence analysis of the native enzyme and of peptides obtained by tryptic, chymotryptic, peptic, thermolysinolytic and mild acidic hydrolysis. Out of a total of eight cysteine residues, six are involved in the formation of three disulfide bonds, the location of which has been established with the help of peptic and thermolysinolytic peptides and fragments, obtained by mild acidic hydrolysis. Chymopapain shares 126 identical amino-acid residues (58%) with papain and 141 (65%) with papaya proteinase omega, including the three disulfide bridges and the free cysteine in position 25, required for activity. Except some amino-acid residues in the substrate-binding site, all residues involved in the catalytic mechanism are conserved. The homology between papaya proteinases is discussed.     

Comparison of antigenic properties of the X-potato virus and its coat proteins. Effect of proteolytic cleavage on antigenic characteristics

Antigenic properties of intact potato virus X (PVX) particles and of PVX coat protein (CP) preparations were compared using different modifications of ELISA test. In the competitive ELISA test (reaction in solution) antibodies to intact virus react much stronger with PVX than with CP while antibodies to CP react much stronger with CP than with PVX. In the direct ELISA test (reaction on the solid support) the difference in reactions of antiCP antibodies with PVX and CP is eliminated while the one in reactions of antiPVX antibodies with these antigens remains. No difference was registered in reactivity of PVX absorbed directly on polystyrene or on immunoglobulin-coated wells (sandwich ELISA) to antiCP antibodies.     

The effect of rimantadine on the structure of model and biological membranes

The action of the antiviral drug rimantadine on the structure of bilayer lipid membranes (BLM) and RBC membranes was investigated. Structural changes in BLM were recorded by ionophore conductivity changes and by changes in the third harmonic of capacity current signal due to lateral compression of BLM in an electric field. It was shown that the adsorption of rimantadine on BLM results in an increase in ionophore mobility in bilayer membranes of dioleolyllecithin (DOL) and common lipids of bovine brain (CL) and in a decrease in those of azolectin (A). Relative changes in the third harmonic signal also depend on the membrane composition and have different signs. The results may be explained by the rimantadine action on the lipid bilayer structure: "rigidification" of A-membranes and "fluidization" of BLM from DOL and CL. Structural reorganization of RBC membranes as investigated by the ability of the cells to enter a micropipette (inner diameter greater than or equal to 3 microns) thereby undergoing deformation. It was shown that rimantadine influences RBC deformability due to drug induced inhomogenous mechanical membrane properties. Also, rimantadine accelerated the process of artificially induced aggregation of erythrocytes. The relation of the effects on artificial and biological membranes, and the structural changes in the lipid phase of membrane are discussed.     

Induction of rat jejunal epithelial cell expression of sucrase-isomaltase by glucocorticoids in primary cell culture and in vivo

The cell cycle phase that mediates the induction of intestinal sucrase-isomaltase (SI) expression by glucocorticoids was investigated by measuring migration rates of 3H-DNA-labeled and of SI-containing epithelial cells by autoradiography and indirect immunofluorescent staining after simultaneous administration of [3H]thymidine and cortisone to 12-d-old rat pups. By 24 and 48 h, lead 3H-DNA-labeled cells had migrated 7.8 and 12.4 cell positions higher on the villus than lead cells expressing SI. Cell migration rates from 12 to 24 h and 24 to 48 h were 0.68 and 0.97 cell position/h. Thus, commitment to SI expression occurred in cells 11.5-12.8 h after the S phase, which is calculated to be in the G1 phase. To determine whether committed cells need to replicate to express SI, cell differentiation was examined in primary cultures of crypt cells originating from corticosterone-treated rats. About two-thirds of cultured cells were retarded in the S phase after plating, as judged by no increase of DNA labeling indices, no change in epithelial cell number, and the absence of mitosis (less than 0.01%). The proportion of cells expressing SI increased from 0 to 6-8% between 12 and 24 h, and reached 48% 48 h after plating on collagen-coated dishes. SI expression did not occur in cells plated on glass or plastic surfaces. Pulse labeling with [35S]methionine confirmed that de novo synthesis of SI occurred in cell cultures. Thus, additional cell cycling of committed cells occurring in vivo is not obligatory for the expression of SI.     

Effect of lard and corn oil intake on serum lipids in young men

An experimental diet with lard (30 g/day for 7 days) and corn oil (30 g/day for 7 days) on high carbohydrate (basal diet) was given to four healthy Japanese young men and the effect of diets containing different fat on serum lipids was examined. Serum total cholesterol was increased significantly from a basal diet of 106 +/- 23 to 141 +/- 26 mg/dl on lard diet, and then decreased significantly (p less than 0.05) to 111 +/- 22 mg/dl on corn oil diet. Serum triglycerides increased significantly (p less than 0.01) from 66 +/- 38 to 173 +/- 32 mg/dl on basal diet. Serum HDL-cholesterol was decreased significantly (p less than 0.01) from 41.9 +/- 1.6 to 31.2 +/- 3.8 mg/dl on lard diet and increased significantly (p less than 0.05) to 41.9 +/- 4.6 mg/dl on corn oil diet. Serum HDL-cholesterol fraction was decreased significantly (p less than 0.01) from 41.6 +/- 4.9 to 28.1 +/- 3.2% on basal diets, but increased significantly (p less than 0.05) to 44.3 +/- 3.1% on lard diet, and then decreased to 36.3 +/- 2.5% on corn oil diet. Serum HDL phospholipid fraction decreased significantly (p less than 0.05) from 62.5 +/- 6.7 to 50.7 +/- 1.8% on basal diet and increased significantly (p less than 0.05) to 60.4 +/- 1.0% on lard and corn oil diet. Serum phospholipids did not change by experimental diets. It is concluded that lard and corn oil have different and specific roles in lipid metabolism.     

Tridimensional analysis of the formation of secretory vesicles in the Golgi apparatus of absorptive columnar cells of the mouse colon

The tridimensional structure of the Golgi apparatus has been studied in the absorptive cells of the mouse colon by means of reduced osmium postfixation and phosphatase cytochemistry. In thick sections of tissue impregnated with osmium tetroxide or treated with a technique to demonstrate TPPase activity, the Golgi formed a continuous ribbon-like structure capping the upper pole of the nucleus. Along the longitudinal axis of this ribbon, compact zones made up of superposed flattened saccules alternated with less compact zones which consisted of highly perforated saccules or bridging anastomosed tubules. In the cis-trans axis, the following elements were observed: (1) a cis element consisting of a continuous osmiophilic tubular network; (2) two or three subjacent elements selectively perforated by wells; (3) a trans compartment made up of two or three TPPase-reactive sacculotubular elements, some showing a "peeling-off" configuration. In some regions, the first flattened saccule of this trans compartment displayed discrete ovoid dilatations, located in compact zones and containing a dense granulofibrillar material; in the subjacent elements this material was seen concentrated in nodular swellings, at the intersection of the meshes of anastomosed membranous tubules. 100-300 nm vesicles containing a similar dense granulofilamentous material were observed in the trans Golgi zone and interspersed in the supranuclear cytoplasm between the Golgi zone and the apical surface of the cell. Smaller vesicles 80-100 nm in diameter containing a fine dusty material were also seen in proximity. These morphological observations suggested that at least two kinds of material were segregated in the saccules of the trans compartment and packaged in vesicles of two class sizes that detached from the Golgi stack on its trans aspect.     

Correlation of bacterial viability with uptake of [14C] acetate into phenolic glycolipid-1 ofMycobacterium leprae within Schwannoma cells

The viability ofMycobacterium leprae, maintained within 33B Schwannoma cells, was estimated in terms of incorporation of [¹⁴C] acetate into its specific phenolic glycolipid-1. This measure of viability was correlated with two other assays,viz., fluorescein diacetate/ethidium bromide staining and mouse footpad growth. Observation of a 2-fold increase in the number of intracellularMycobacterium leprae over an experimental period of 12 days also corroborated this contention. Furthermore, on addition of anti-leprosy drugs to these intracellularMycobacterium leprae there was significant decrease in phenolic glycolipid-1 synthesis indicative of loss of viability of the organisms. This study also established the importance of the host cell for active bacillary metabolism, asMycobacterium leprae maintained in cell-free conditions showed no incorporation into phenolic glycolipid-1. Moreover, compromising the host’s protein synthesis capacity with cycloheximide, also led to reduction in bacillary metabolism. As this system measures the metabolic synthesis of a uniqueMycobacterium leprae component, it would be useful for development and screening of compounds acting against specific bacillary targets.