The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture

Our research group aims to develop an osteochondral composite using type II collagen gel with hydroxyapatite (HAp) deposited on one side. Soaking gels in Ca2+ and phosphate solution is indispensable to HAp deposition, so relationships between cell behavior and Ca2+ concentration were examined in two- and three-dimensional cultures. The present results indicate that 2-4 mM Ca2+ is suitable for proliferation and survival of osteoblasts, whereas slightly higher concentrations (6-8 mM) favor osteoblast differentiation and matrix mineralization in both 2- and 3-dimensional cultures. Higher concentrations (>10 mM) are cytotoxic. Purely from the perspective of calcium deposition, higher concentrations lead to increased accumulation of Ca2+. Culturing cells in phosphate-containing gel in media with Ca2+ also leads to time-dependent formation of HAp in the gel. Considering the viability of embedded cells, culturing scaffolds in media with Ca2+ concentrations around 5mM is useful for both HAp deposition and osteoblast behavior.     

Otx1 function overlaps with Otx2 in development of mouse forebrain and midbrain

Background: We previously reported that the homozygous mutation of Otx2 gene, a mouse cognate of the Drosophila head gap gene orthodenticle , causes failure in the development of the rostral head anterior to rhombomere 3, which may correspond to earlier Otx2 expression in cells destined for the anterior mesoendoderm. At the same time, the Otx2 heterozygous mutation displayed a phenotype characterized as otocephaly, probably related to expression in the anterior neuroectoderm at the subsequent pharyngula stage. Defects were characteristic in the most anterior and posterior regions of Otx2 expression where Otx1 , another mouse cognate of orthodenticle , is not or weakly expressed. They were not found in the region where Otx1 is expressed.
Results: In the present work, Otx1 null mutant mice were generated by gene targeting in embryonic stem cells. No defects were apparent in the regionalization of the early embryonic rostral brain. The newborn brain defects were subtle and most likely related to later Otx1 -unique expression. Otx1 and Otx2 double heterozygous mutant brains, however, exhibited marked defects throughout the fore- and midbrains, where defects were not apparent with a single mutation alone.
Conclusions: Otx1 and Otx2 play synergistic roles in the development of the forebrain and midbrain where both genes are expressed.     

Immunohistochemistry and angiography in adenomatous hyperplasia and small hepatocellular carcinomas

The sinusoidal structure and blood supply of 38 liver nodules less than 2 cm In diameter were Investigated. There were 18 cases of adenomatous hyperplasia (AH) and 20 cases of hepatocetlular carcinoma (HCC). Growth pattern, encapsulation and vascularity were examined, and Immunohistochemistry performed for factor VIII related antigen (factor VIII), type IV collagen (collagen IV), lamlnln and CD68. There were significant differences between AH and small HCC, except for the expression of CD68. There were differences In tumor size, vasculartty and the components of the basement membrane between AH and small, well differentiated HCC. The cases of AH were supplied by the portal system and maintained the sinusoidal structure, but small well-differentiated HCC were supplied by a mixture of portal and arterial vessels. In spite of their small size, moderately and poorly differentiated HCC had capillary and were supplied by branches of the hepatic artery.     

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

Total energy expenditure of elite synchronized swimmers measured by the doubly labeled water method

To determine the daily energy requirement of elite synchronized swimmers during moderate-intensity training, the average daily energy expenditure measured by the doubly labeled water method, was calculated for nine female Japanese national team synchronized swimmers [four senior; mean (SD) 22.5 (1.0) years old, 52.2 (3.6) kg, and five junior; 17.6 (1.1) years old, 52.8 (2.3) kg]. Their total energy expenditure (TEE) was 11.5 (2.8) MJ · day⁻¹ [2738 (672) kcal · day⁻¹]. When compared with estimated energy requirements derived from “Recommended Dietary Allowances for the Japanese”, 12.1 (0.6) MJ · day⁻¹ [2897 (139) kcal · day⁻¹], there was no difference between mean actual and estimated energy requirements. However, there were considerable differences observed on an individual basis. Their energy intake, estimated from 7- day self-reported dietary records, was 8.9 (1.7) MJ · day⁻¹ [2128 (395) kcal · day⁻¹], which was significantly lower than their TEE (P < 0.05). Resting energy expenditure (REE), as determined by indirect calorimetry, was 5.2 (0.3) MJ · day⁻¹ [1247 (75) kcal · day⁻¹]. Their physical activity level (TEE/REE) was 2.18 (0.43). These results demonstrate that the TEE values of elite female synchronized swimmers are not dissimilar to those reported for athletes participating in other sports, especially competitive swimmers during moderate-intensity training. Accepted: 26 May 2000     

Representation of the tensor veli palatini muscle in the trigeminal motor nucleus of the Japanese monkey (Macaca fuscata)

Distribution of motoneurons supplying the tensor veli palatini (TVP) muscle was examined in the Japanese monkey (Macaca fuscata) by the retrograde horseradish peroxidase (HRP) method. Neurons labeled with HRP which was injected into the tensor veli palatini muscle were seen in the ventromedial aspects of the dorsolateral division of the trigeminal motor nucleus, at all rostrocaudal levels of the trigeminal motor nucleus. The vast majority of these TVP motoneurons were distributed around the margin, especially the dorsal margin, of the cluster of motoneurons which innervate the lateral pterygoid muscle.