Recognition of 2-keto-3-deoxyoctonate in bacterial cells and lipopolysaccharides by the sialic acid binding lectin from the horseshoe crab Carcinoscorpiusrotundacauda

The sialic acid binding loctin carcinoscorpin agglutinates $\text{Escharichia}\text{coli}\text{K12}\text{and}\text{Salmonella}\text{minnesots}$ R595 cells. This interaction can be inhibited by the saccharides namely 2-keto-3-deoxyoctonate and the disaccharide D-(N-acetylneuraminyl) (2→6)2-acetamide-2-deoxy-D-galactitol. N-acetylneuraminic acid is shown to be a poor inhibitor. The same behaviour is seen when purified lipopolysaccharides from these two Gram negative bacteria are used. $\text{Vibrio}\text{cholerae}$, a Grum negative bectarium devoid of 2-keto-3-deoxyoctonate and $\text{Staphylococcus}\text{sureus}$ a typical Gram positive bacterium failed to agglutinate in the presence of the lectin. The results suggest that the 2-keto-3-deoxyoctonate residues might represent the physiological substrate for the sialic acid binding lectin from the horseshoa crab.     

Ultrastructural observations on chick bone processed by quick-freezing and freeze-substitution

Summary The ultrastructure of newly formed bone was examined with the use of quick-freezing followed by freeze-substitution. Osteoblasts and young osteocytes were characterized by a smooth cell contour, whereas old osteocytes were irregular in shape. The plasma and intracytoplasmic membranes were clearly identifiable as trilaminar substructures. With the method described herein the tissue is handled in the anhydrous state. Thus mitochondrial granules could be demonstrated in all samples, since their preservation is not affected by non-aqueous solutions. The matrices of intact mitochondria were densely stained with poststaining. The contents of the Golgi complex, rough-surfaced endoplasmic reticulum (RER), nuclear envelope, vesicles, and vacuoles were stained to various degrees. Lacunar spaces were always filled with flocculent and filamentous materials, and the plasma membrane was in direct contact with them. Membrane-bounded matrix vesicles were clearly visible within the osteoid extracellular matrix which was the initial site of mineral crystal deposition. In heavily mineralized bone matrix, the periodic pattern of collagen fibrils was retained, and the electron density of mineralized matrix in freeze-substituted and unstained sections which had been floated on ethylene glycol was greater than that encountered in sections processed in aqueous reagents.     

Different tartrate sensitivity and pH optimum for two isoenzymes of acid phosphatase in osteoclasts. An electron-microscopic enzyme-cytochemical study

Summary By differentiation of substrate specificity, pH optimum range, and sensitivity to various inhibitors, 2 isoenzymes of acid phosphatase in bone cells have been studied at the electron-microscopic level. When p-nitrophenyl phosphate was used for the substrate, the demonstrable enzyme activity was affected by neither tartrate nor sodium fluoride. The reaction product, when incubated at pH 5–6, was detected in all sites along the pathway for the biosynthesis of acid phosphatase in the osteoclast, including the perinuclear space, cisternae of the endoplasmic reticulum, Golgi complex, various vesicles, and vacuoles. In the osteoclasts attached to bone, the enzymatic activity was demonstrated at the extracellular ruffled border and on the eroded bone surface. Reaction products became confined to lysosomes and extracellular ruffled border when incubated at pH 6–7. Unattached osteoclasts showed a similar intracytoplasmic localization of enzyme as the attached ones, except for the absence of the extracellular enzyme activity. The mononuclear, immature type of osteoclast also resembled the mature osteoclast in terms of enzymatic localization. Except for the osteoclasts, the acid p-nitrophenyl phosphatase activity was restricted to lysosomal vesicles in various bone cells, monocytes, and macrophages. Such activity was inhibited by adding 50 mM tartrate to the p-nitrophenyl phosphate medium. When -glycerophosphate or p-nitrocatechol sulfate was the substrate, most of the reaction product was localized intracellularly. Unlike the acid p-nitrophenyl phosphatase, the acid -glycerophosphatase or arylsulfatase activity in osteoclasts and other bone cells was inhibited completely by 10 mM tartrate or 10 mM sodium fluoride. Even preincubation of 100 mM tartrate in the buffer inhibited -glycerophosphatase activity completely, but p-nitrophenyl phosphatase activity was inhibited incompletely. Consequently, our results suggest that acid p-nitrophenyl phosphatase is a useful cytochemical marker for identification of the osteoclast family at electron-microscopic levels of resolution.