Characterization of tissue transglutaminase in human osteoblast-like cells.

Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5'-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification of proteins by forming intermolecular epsilon(gamma-glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluation of transglutaminase (TG) activity determined using the [1,4 14C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates of MG-63 cells as compared with HOB and HOS cells (p < 0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.

Biodegradable polymer/hydroxyapatite composites: surface analysis and initial attachment of human osteoblasts.

Biodegradable polymer/hydroxyapatite (HA) composites have potential application as bone graft substitutes. Thin films of polymer/HA composites were produced, and the initial attachment of primary human osteoblasts (HOBs) was assessed to investigate the biocompatibility of the materials. Poly(epsilon-caprolactone) (PCL) and poly(L-lactic acid) (PLA) were used as matrix materials for two types of HA particles, 50-microm sintered and submicron nonsintered. Using ESEM, cell morphology on the surfaces of samples was investigated after 90 min, 4 h, and 24 h of cell culture. Cell activity and viability were assessed after 24 h of cell culture using Alamar blue and DNA assays. Surface morphology of the polymer/HA composites and HA exposure were investigated using ESEM and EDXA, respectively. ESEM enabled investigation of both cell and material surface morphology in the hydrated condition. Combined with EDXA it permitted chemical and visual examination of the composite. Differences in HA exposure were observed on the different composite surfaces that affected the morphology of attached cells. In the first 4 h of cell culture, the cells were spread to a higher degree on exposed HA regions of the composites and on PLA than they were on PCL. After 24 h the cells were spread equally on all the samples. The cell activity after 24 h was significantly higher on the polymer/HA composites than on the polymer films. There was no significant difference in the activity of the cells on the various composite materials. However, cells on PCL showed higher activity compared to those on PLA. A polymer surface exhibiting "point exposure" of HA appeared to provide a novel and favorable substrate for primary cell attachment. The cell morphology and activity results indicate a favorable cell/material interaction and suggest that PLA and PCL and their composites with HA may be candidate materials for the reconstruction of bony tissue. Further investigations regarding long-term biomaterial/cell interactions and the effects of acidic degradation products from the biodegradable polymers are required to confirm their utility.

Dynamics of an enzyme polymorphism in the isopod, Sphaeroma rugicauda (Leach). I. Temporal variation in genotype frequencies.

A 27 month study (November, 1978 through January, 1981) of the diallelic enzyme locus, phosphoglucose isomerase (Pgi), in the estuarine isopod Sphaeroma rugicauda (Leach) revealed seasonal changes in the frequencies of the three genotypes (fast/fast, fast/slow and slow/slow). A comparison of genotype frequencies in the newly-released offspring (August) to the frequencies in the same animals ten months later during the breeding season (June), revealed a decline in heterozygote frequency and a corresponding increase in the frequency of the s/s homozygote both of which appeared to be initiated at the onset of winter, a period characterised by low temperatures and salinity. Survivorship experiments in the laboratory suggested that the increase in frequency of the s/s homozygote, at least, could be explained by this genotype being favoured at low temperatures.

Dynamics of an enzyme polymorphism in the isopod, Sphaeroma rugicauda (Leach). II. Sexual, gametic and fecundity selection.

The estuarine isopod Sphaeroma rugicauda (Leach) exhibits a diallelic polymorphism at the locus coding for the enzyme Pgi. An investigation carried out during the animal's breeding season (June-July) in 1979 and 1980 indicated that the animals were mating at random. It also showed the s/s-homozygote females to be the earliest to release offspring and to be the most fecund of the three genotypes (f/f, f/s and s/s). Further, data from the wild population in 1980 and laboratory experiments indicated that male s-gametes were selected against in s/s females, resulting in offspring ratio distortions in f/s (male) X s/s (female) crosses, in favour of heterozygotes. The net effect of these selective pressures did not, however, significantly alter the genotype frequencies (found in the offspring) from expected values.

Colour, sunlight and internal temperatures in the land-snail Cepaea nemoralis (L.).

The literature on the effects of pigmentation on radiant energy absorption and temperature in poikilothermic animals is reviewed. Some of the difficulties encountered in studies of this type are described.Mercury-filled shells and live snails of different colours were exposed to sunlight. Darker-pigmented shells were almost always hotter than lighter shells. Large differences in pigmentation (yellow compared with brown, yellow unbanded compared with yellow banded) led to more pronounced differences in temperatures than did small differences in pigmentation (yellow compared with pink, pink compared with brown).