Maturation process of secondary ossification centers in the rat and assessment of bone age.

The maturation process from the appearance to the fusion of the secondary ossification centers of extremities was studied in Wistar rats aged 0 to 134 weeks. The examination of the secondary ossification centers made by radiography. The assessment of the stage of development was made in accordance with the criteria proposed by Ohwada and Sutow. The secondary ossification center was found to be take one of the following three types of maturation processes : (1) the acute ossification, (2) the delayed ossification, and (3) the incomplete ossification. No fusion was observed up to 134 weeks in certain epiphyses of the rat. This type of ossification designated as the incomplete ossification may be specific to the mouse and rat. The relative lengths of time required for appearance and fusion in the average prospective life were obtained for the rat. They were compared with those of the mouse and man. The relative length of time necessary for maturity of the secondary ossification centers was shown to be the shortest in the rat and the longest in man. The results suggested that the rat may reach maturity in the bone age at 17 to 21 weeks of age. The rat at this age may be regarded as being adult corresponding to age 17 weeks in mice and 18 to 24 years in man.     

Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques

Summary Feulgen nuclear staining with pararosanilin-SO₂ was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell.The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality beween the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by Böhm et al. (1968) and a reaction with ninhydrin at 37° C for 10 h.The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure.Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.Partly supported by Deutsche Forschungsgemeinschaft (DFG), grant Nr. Bo 395/4     

Process of infection with bacteriophage phi chi 174. XL. Viral DNA replication of phi chi 174 mutants blocked in progeny single-stranded DNA synthesis.

Mutation in several different cistrons of bacteriophage phi chi 174 blocks net progeny single-stranded DNA synthesis at the late period of infection (15). For the study of the functions of these cistrons in single-stranded DNA synthesis, asymmetric replication of replicative form DNA was examined at the late period of infection with amber mutants of these cistrons. While the normal, rapid process of asymmetric single-stranded viral DNA synthesis is blocked at the late period of these mutant infections, an asymmetric synthesis of the viral strand of replicative-form DNA is observed in this period, though at a reduced level, together with degradation of prelabeled viral strand. Some intermediate replicative-form molecules were also detected. Asymmetric synthesis of the viral strand of replicative-form DNA at the late period of phi chi infection is completely inhibited in the presence of a low concentration (35mug/ml) of chloramphenicol (which also blocks net single-stranded viral DNA synthesis). These results are discussed in terms of the possible role of the specific viral proteins for normal single-stranded DNA synthesis.     

Endo-beta-galactosidase of Escherichia freundii. Hydrolysis of pig colonic mucin and milk oligosaccharides by endoglycosidic action.

The purified keratansulfate degrading enzyme from $\text{Eschericia freundii}$ could hydrolyze desialyzed pig colonic mucin and milk oligosaccharides. Desialyzed pig colonic mucin was digested to produce GlcNAcβ(1→3)Gal, GlcNAc-6Sβ(1→3)Gal and resistant polymer. Lacto-N-tetraose and lacto-N-tetraitol were hydrolyzed endoglycosidically to release glucose and sorbitol, respectively. Therefore, this enzyme was found to be an endo-β-galactosidase of rather wide specificity.     

Mechanisms of the acido- and thermo-phily of Cyanidium caldarium Geitler I. Effects of temperature, pH and light intensity on the photosynthetic oxygen evolution of intact and treated cells

Photosynthetic oxygen evolution in an acido- and thermo-philicunicellular alga, Cyanidium caldarium, was measured under variousconditions, using a Clark-type oxygen electrode. 1). Maximum Hill reaction activity with p-benzoquinone as theHill oxidant was obtained at 45°C in a wide pH range from1.0 to 7.0. 2) The pH activity curve showed two peaks at pH3.0 and 7.0. The Hill activity had an optimum at pH 3.0 in cellspreilluminated under strong light (300,000 lux, 30 min, 40°C).Sonication of algal cells abolished the pH 3.0 component ofthe Hill reaction producing an activity maximum at pH 7.0. 3)Endogenous O₂ evolution in the absence of the Hill oxidant,which lasted for several minutes after illumination, had a maximumat pH 7.0. 4) This endogenous O₂ evolution was abolished bysonication. 5) KCN inhibited endogenous O₂ evolution, but notthe Hill reaction in the presence of p-benzoquinone. (Received August 19, 1974; )     

Structures of novel sialylated O-linked oligosaccharides isolated from human erythrocyte glycophorins

The O-linked oligosaccharides attached to human erythrocyte glycophorins were extensively characterized. In addition to the previously described disialylated tetrasaccharide, NeuNAc alpha 2----3Gal beta 1----3 (Neu-NAc alpha 2----6)GalNAcOH and monosialylated trisaccharide, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, novel trisialylated oligosaccharides were isolated. Methylation analysis, fast atom bombardment-mass spectrometry, and enzymatic degradation were used to elucidate the following novel structures: formula; see text: These results suggest that O-linked oligosaccharides with a disialosyl group, NeuNAc alpha 2----8NeuNAc alpha 2----, may be present in various tissues.     

ADAMTSL6β promotes fibrillin‐1 microfibril assembly,which is possibly mediated via binding through the third thrombospondin type I domain to fibrillin‐1

Fibrillin‐1 is the major component of extracellular matrix microfibrils. Microfibrils dysfunction is responsible for the onset of various connective tissue diseases, including Marfan syndrome. Although ADAMTSL (a disintegrin and metalloproteinase with thrombospondin motifs‐like) 6β is one of the fibrillin‐1 binding proteins, the detailed mechanism underlying the involvement of ADAMTSL6β in microfibril formation remains unclear. In this study, we created deletion mutants of ADAMTSL6β and examined their interactions with fibrillin‐1 assembly. Pull‐down assay of the ADAMTSL6β deletion mutants and fibrillin‐1 protein revealed that ADAMTSL6β binds to fibrillin‐1 through the third thrombospondin type I domain. Furthermore, we observed that formation of fibrillin‐1 matrix assembly was enhanced in MG63 cells, expressing full‐length ADAMTSL6β, when compared with that of wild type MG63 cells. While MG63 cells expressing Δ TSP3‐ADAMTSL6β form showed enhanced assembly formation, Δ TSP2‐ADAMTSL6β form did not enhance that, indicating the difference between Δ TSP2‐Δ TSP3 has a critical role for fibrillin‐1 assembly. As the difference of Δ TSP2‐Δ TSP3 is the third thrombospondin type I domain, we concluded that the third thrombospondin type I domain of ADAMTSL6β influence the microfibril formation. Our data are the functional presentation of the biological role of ADAMTSL6β in the process of microfibril formation.