The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. I. Immunizing properties.

The ability of R-type lipopolysaccharide (LPS), isolated from Neisseria gonorrhoeae colony type 4, to protect against infection with N. gonorrhoea colony type 1 (T1 isolates) in the mouse and chicken embryo was investigated. C57 black mice were immunized intraperitoneally with 50 microgram of LPS, and challenged intracerebrally with 10-20 LD50's of N. gonorrhoeae colony type 1. Immunized mice were significantly protected (P less than 0.01 to less than 0.05) against challenge with different T1 isolates of N. gonorrhoeae when compared with non-immunized mice. Mice, injected with succinylated or alkali-treated LPS were not protected against gonococcal challenges. In a second animal model, leghorn hens were immunized intravenously with three injections of 500 microgram of LPS followed by a booster of 2.5 mg 2 weeks later. Embryonated eggs obtained from immunized hens were protected against challenge with 5 x 10(3) - 1 x 10(4) LD50's of three different T1 isolates. When hens were injected with the chemically modified LPS, the embryos were not resistant to gonococcal challenge. The results of this study demonstrate the ability of R-type gonococcal LPS to provide protection against different T1 isolates of N. gonorrhoeae.     

Differential accumulation of proteinase inhibitor I in normal and crown gall tissue of tobacco, tomato, and potato

A proteinase inhibitor (inhibitor I) is induced in crown gall tumors of tobacco (Nicotiana tabacum) initiated through infection with the tumorinducing bacterium, Agrobacterium tumefaciens, strains B6 or CG-14. Uninfected tissues do not contain immunologically detectable quantities of inhibitor I. Inhibitor I synthesis in tobacco crown gall tumors paralleled tumor growth at the average rate of about 4.5 μg of inhibitor I per 200 mg of fresh tissue per day. Infection of variegated tobacco mutant Dp-I with A. tumefaciens strain CG-14 produced tumors with 25% more inhibitor than tumors induced with strain B6. Unlike tobacco, tumors induced by either bacterial strain on potato (Solanum tuberosum) and on tomato (Lycopersicum esculentum) did not accumulate inhibitor I. Consequently, inhibitor I accumulation is modulated by the type of plant host used in spite of familial relatedness (Solanaceae) and the strain of A. tumefaciens used for infection.     

Localization of angiotensin converting enzyme (kininase II). I. Preparation of antibody-hemeoctapeptide conjugates.

Antibodies to pig lung angiotensin converting enzyme (kininase II) were conjugated to a heme-octapeptide (8-microperoxidase, 8-MP) derived from cytochrome c. 8-MP, which has only one reactive amine, was coupled to antibody in a two-step procedure using a bifunctional active ester, bis-succinyl succinate. In the first-step, 8-MP-succinyl succinate, a stable compound which can be stored. In a second step, the remaining active ester was used for coupling to reactive amines of the antibody. The conjugate consists of 1.6-2.3 8-MP moieties per antibody. Using these procedures, the formation of complex polymers is avoided. Each molecule of conjugate possesses both immunoreactivity and peroxidatic activity. The conjugate has been used to localize angiotensin converting enzyme along the plasma membrane and associated caveolae of pig aortic endothelial cells in culture.     

Effects of prostaglandins and other drugs on the cyclic AMP content of cultured bone cells.

Prostaglandins of the E-series (PGE1 and PGE2) may be involved in disease-related, localized loss of bone. E-prostaglandins increase the cyclic AMP content of many cells; and, to determine if their effects on bone are mediated by cyclic AMP, we examined the effects of E-prostaglandins and of other agents on the cyclic AMP content of cultured bone cells. PGE2 produced a rapid, marked and dose-related increase in the cyclic AMP content of confluent monolayers of bone cells isolated from newborn rat calvaria. At 2.8 X 10(-6) M, PGE1 and PGE2 had approximately the same effect, while the effect of PGF2alpha was much less pronounced. In the presence of theophylline, PGE2 had a more marked effect than parathyroid hormone (PTH) and the combination of PGE2 and PTH had a synergistic effect. The divalent, cationic, ionophore, A23187, produced an increase in cellular cyclic AMP and had an additive effect in combination with PGE2. Synthetic salmon calcitonin (CT), which inhibits the bone resorptive effect of PGE2, increased cellular cyclic AMP and had an additive effect in combination with PGE2. A prostaglandin antagonist, SC-19220, partially inhibited the resorptive effect of PGE2 and reduced its effect on cellular cyclic AMP. The calcium antagonist, D600, inhibited the bone resorptive effects of PGE2 but had no effect on increased cellular cyclic AMP produced by PGE2. The marked effect of PGE2 on bone cell cyclic AMP suggests that this action is involved in the mechanism of PGE2-related bone loss. The fact that agents with different effects on PGE2-induced increases in cellular cyclic AMP can inhibit its resorptive actions, suggests that PGE2-induced changes in cyclic AMP may be related less to its resorptive actions than to its inhibitory effect on bone formation.     

The paradoxical effect of fatty acid on steroid-albumin interaction.

Careful investigation of the influence of palmitic and lauric acid on the interaction of progesterone and testosterone with several batches of untreated and defatted bovine and human serum albumins have revealed that, by contrast with published data for studies with progesterone as well as nonsteroid ligands, there is a surprising stimulation rather than inhibition of binding, albeit with a reduction of the apparent number of binding sites in almost all instances. Furthermore, fatty acid tends to minimize or eliminate the well-known differences in affinity between bovine and human albumin for interactions with these two steroids. The values for binding affinity in the interaction of testosterone with these batches of human serum albumin are significantly higher than those previously published by us and other authors and the value for progesterone-bovine albumin interaction is not in accordance with the "polarity rule". Studies of these same interactions by ultraviolet difference spectroscopy give further evidence of the augmentation in binding but, in the case of defatted bovine albumin only, the aromatic difference troughs are indicative of tyrosine perturbation whereas refatted bovine albumin, defatted and refatted human albumin manifest tryptophan perturbation. Quantitative correlation of perturbation with level of bound steroid suggests that fatty acid alters the ratio (possibly hydrogen-bonded to non hydrogen-bonded) of two forms of bound steroid. There is also further evidence that the binding sites for testosterone and progesterone are not identical.     

Resolution and reconstitution of glutamate decarboxylase from cerebellum

Brain glutamate decraboxylase (EC 4.1.1.15) catalyzes the biosynthesis of the postulated neurotransmitter-aminobutyric acid according to the following chemical equation:L-glutamate -aminobutyric acid+CO₂. Hydroxylamine treatment of the decarboxylase at low ionic strength followed by Sephadex gel filtration resolves apoenzyme from cofactor (>90%). Pyridoxal phosphate completely restores activity. Sodium borohydride inactivates the holoenzyme, but not the apoenzyme. This supports the notion that pyridoxal phosphate is bound to the holoenzyme as a Schiff base. Moreover, salicylaldehyde, a reagent which reacts with amino groups, substantially inactivates the apoenzyme but not the holoenzyme. Reconstitution of the bovine cerebellar holoenzyme from apoglutatamate decarboxylase and pyridoxal phosphate occurs in seconds to minutes, which is much faster than that of the decarboxylase isolated fromE. coli. Native holoenzyme, apoenzyme, and reconstituted holoenzyme have identical molecular weights as estimated by Sephadex gel filtration.A preliminary account of this work has been presented (1).     

Alveolar type II cells: studies on the mode of release of lamellar bodies.

There is increasing evidence that type II alveolar cells are capable of synthesizing surface active material like that obtained from the airways. However a number of problems remain to be solved before it can be stated conclusively that type II cells synthesize the surface active material of the terminal airspace. Among these problems is that of secretion. A number of previous studies have given evidence of the release of lamellar bodies by merocrine secretion. In this study morphologic evidence is presented which supports the view that secretion of lamellar bodies is accomplished by exocytosis. At the apical surface of type II cells, sites can be found where the limiting membrane of the lamellar body is clearly fused with the type II cell plasma membrane and an open channel exists between the contents of the lamellar body and the alveolar space. At these sites the lamellar contents extrude into the airspace with consequent loss of the highly compact organization of intracellular lamellar bodies. The intactness and continuity of the membranes can be traced for the full extent of the exocytosis site. Freeze-etch replicas of the membranes of type II cells show depressions which may represent the sites of discharged lamellae. In addition, tongue-like folds are seen which could be explained as the extensions of cytoplasm which surround the releasing lamellar body and which may flap over the exocytosis pit after discharge. Micrographs of the alveolar space show disorganized lamellar whorls which appear to be unravelling to produce tubular myelin. In view of the unusually large size and lipid composition of lamellar bodies, a mechanism involving hydration of mucopolysaccharide contents as an aid to expulsion of lamellar contents is suggested.     

On the Presence of a Transverse System in Tunicate Muscle

Abstract Ultrastructural investigation of the caudal muscle cells in the tadpole larva of the ascidian Dendrodoa grossularia has shown that in this species, a transverse tubular system is present; diad and triad couplings are formed with the sarcoplasmic reticulum. Peripheral couplings of the sarcoplasmic reticulum are infrequent and have only been observed at the longitudinally apposed faces of the caudal muscle cells.