Phagocytosis of different matrix components by different cell types at bone-forming sites in cultured mouse calvariae

Summary Sites of bone formation on fragments of parietal bone of fetal-mice cultured for 10 days were examined by electron microscopy after addition of either ruthenium red or ferrocyanide to the postfixation fluid. Osteoclasts, osteoblast-like cells, and macrophages were the principal active cells at these formation sites. The mononuclear cells (osteoblast-like cells and macrophages) in the osteoid tissue showed evidence of having incorporated elements of calcified tissue. Osteoblast-like cells had phagocytized collagen fibrils and calcified bone matrix. This occurred more frequently in the calcifying area. Mononuclear macrophages showed not only phagocytosis and digestion of cellular debris and bone spicules in the osteoid, but also active incorporation of calcified bone matrix that had been detached from its surroundings by its pseudopod-like projections from long cytoplasmic processes. Collagen fibrils were seldom observed within the macrophages. These observations suggest that in our culture system osteoblast-like cells and macrophages at bone formation sites have a phagocytic role in bone remodeling.This study was supported in part by a grant from the Ministry of Education. Science and Culture of Japan (No. 59771321)     

Secular trend in skeletal maturation in southern Chinese girls in Hong Kong

Southern Chinese girls aged 11 years 9 months to 12 years 3 months in Hong Kong have a mean skeletal age of 12.57 years assessed from the left hand and wrist radiographs by the Greulich and Pyle Atlas Method. Significant secular trend of earlier skeletal maturation was demonstrated with p less than or equal to 0.001. Such difference was contributed by improved socio-economic, nutritional and socio-hygienic conditions during the past decades.     

Inward current generated by Na-Ca exchange during the action potential in single atrial cells of the rabbit.

To investigate the underlying ionic mechanism of the late plateau phase of the action potential in rabbit atrium the whole-cell patch-clamp technique with intracellular perfusion was used. We recorded the inward current during repolarizations following a brief 2 ms depolarizing pulse to +40 mV from a holding potential of between -70 and -80 mV. The development of this current coincides with the onset of the late plateau phase of the action potential. Peak activation of the current occurs about 10 ms from the beginning of the depolarizing pulse, and it decays spontaneously with a slow timecourse. Its voltage dependency from -40 mV to +40 mV shows very steep activation (-40 to -20 mV) and shows almost the same maximum magnitude between -10 mV and +40 mV. This behaviour is quite different from that of the calcium current. The inward current and the late plateau phase of the action potential were both abolished by the application of 5 mM EGTA, 1 microM ryanodine and by reducing the Na+ gradient. The fully activated current-voltage relation of the inward current was plotted as the difference current before and after treatment with Ryanodine, Diltiazem, 20 mM Na+ inside or 30% Na+ outside and shows an exponential voltage dependence with the largest magnitude of the current occurring at negative potentials. The current-voltage (I-V) curve was well fitted by the Na-Ca exchange equation, i = A exp (-(1 - r)EF/RT). The results suggest that the inward current contributes to the generation of the late plateau phase of the rabbit atrial action potential, and is activated by intracellular calcium released from the sarcoplasmic reticulum. Sarcoplasmic reticulum calcium release appears to be triggered both by the membrane voltage and by the calcium current. It is concluded that the inward current is generated by Na-Ca exchange.     

Removal and recovery of Cu(II) from industrial effluent by immobilized cells of Pseudomonas putida II-11

A copper [Cu(II)]-accumulating strain, Pseudomonas putida II-11, isolated from electroplating effluent removed a significantly high amount of Cu(II) from growth medium and buffer. A laboratory-scale fixed bed reactor with cells of P. putida II-11 immobilized in polyacrylamide gel was constructed. The adsorption of Cu(II) by the immobilized cells was pH-dependent. Maximum removal of Cu(II) by the immobilized cells was at pH 8.0. The presence of Cr(IV), Ni(II) and Zn(II) did not significantly inhibit Cu(II) uptake whereas the presence of Pb(II) reduced Cu(II) uptake by fivefold. The presence of borate, carbonate, chloride and sulphate did not significantly inhibit Cu(II) uptake. The Cu(II) removal capacity of the bioreactor with immobilized cells did not change significantly when operated at retention times greater than 3 min. More than 90% of Cu(II) adsorbed on immobilized cells could be recovered by eluting with 0.1 m HCl. The bioreactor could be used for at least five loading-elution cycles without loss of Cu(II) removal capacity. The feasibility of using this bioreactor to remove and recover Cu(II) from electroplating effluent is discussed. Correspondence to: P. K. Wong     

Strain improvement of Claviceps purpurea by protoplast fusion without introducing auxotrophic markers

Protoplasts of morphologically and biochemically different Claviceps purpurea strains producing ergotoxins were fused without introducing selective auxotrophic markers. Fused strains thus obtained differed significantly in biosynthetic activity and morphology from the prototrophic isolates obtained after fusion of the same parent strains marked by auxotrophy. Comparison of the two types of fused strains showed about tenfold higher alkaloid production in fusants obtained from prototrophic strains. Selected stable prototrophic isolates also showed a significant productivity improvement in comparison with the original parent strains. Correspondence to: M. Didek-Brumec     

Recombination-deficient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages

Mutations in the genes recA and recBC were constructed in the virulent Salmonella typhimurium strain 14028s. Both the recA and recBC mutants were attenuated in mice. The mutants were also sensitive to killing by macrophages in vitro. The recombination mutants were no longer macrophage sensitive in a variant line of J774 macrophage-like cells that fail to generate superoxide. This suggests that repair of DNA damage by Salmonella is necessary for full virulence in vivo and that the oxidative burst of phagocytes is one source of such DNA damage.     

Carbohydrate nutrition of claviceps purpurea for alkaloid production related to the osmolality of media

Summary The effects of most frequently used carbohydrates and osmolalities of media on the growth and productivity ofClaviceps purpurea in submerged cultures were investigated. The maximum alkaloid yield was obtained with 20% mannitol as well as with 10% mannitol when 2% NaCl was added to the medium and the osmolality about 1 osmol/kg H₂O was established at the end of fermentation. Increased or decreased osmolality of the medium had a negative influence on fungal growth and alkaloid production. Almost the same effect was observed with sucrose as with mannitol, whereas with glucose only maximal growth was achieved.     

Eukaryotic DNA replication.

The past decade has witnessed an exciting evolution in our understanding of eukaryotic DNA replication at the molecular level. Progress has been particularly rapid within the last few years due to the convergence of research on a variety of cell types, from yeast to human, encompassing disciplines ranging from clinical immunology to the molecular biology of viruses. New eukaryotic DNA replicases and accessory proteins have been purified and characterized, and some have been cloned and sequenced. In vitro systems for the replication of viral DNA have been developed, allowing the identification and purification of several mammalian replication proteins. In this review we focus on DNA polymerases alpha and delta and the polymerase accessory proteins, their physical and functional properties, as well as their roles in eukaryotic DNA replication.     

Characterization of a Trypanosoma cruzi C3 binding protein with functional and genetic similarities to the human complement regulatory protein, decay-accelerating factor.

Evasion of the complement system by microorganisms is an essential event in the establishment of infection. In the case of Trypanosoma cruzi, the causative agent of Chagas disease, resistance to complement-mediated lysis is a developmentally regulated characteristic. Infectious trypomastigotes are resistant to complement-mediated lysis in the absence of immune antibodies, whereas the insect forms (epimastigotes) are sensitive to lysis via the alternative complement pathway. We have purified a developmentally regulated, trypomastigote glycoprotein, gp160, and shown that it has complement regulatory activity. The T. cruzi gp160 restricts complement activation by binding the complement component C3b and inhibiting C3 convertase formation. The protein is anchored in the parasite membrane via a glycosyl phosphatidylinositol linkage, similar to the human complement regulatory protein, decay-accelerating factor. Using anti-gp160 antibodies we have isolated a bacteriophage lgt11 clone expressing a portion of the gp160 gene that shares significant DNA sequence homology with the human DAF gene. These results provide functional, biochemical, and genetic evidence that the T. cruzi gp160 is a member of the C3/C4 binding family of complement regulatory proteins, and that gp160 may provide the infectious trypomastigotes with a means of evading the destructive effects of complement.