Development of a new calcium phosphate cement that contains sodium calcium phosphate

A cement powder consisting of sodium calcium phosphate, Na3Ca6(PO4)5, in addition to tetracalcium phosphate and beta-tricalcium phosphate was prepared by pulverizing blocks of 4 wt% sodium-, 11 wt% carbonate-containing apatite samples that were heated at 1700 degrees C for 5 h. When mixed with 30 wt% malic acid or citric acid at a powder liquid ratio of 3:1, the cement set in 3 or 7 min at room temperature with compressive strength being around 52 or 27 MPa. In HeLa-cell cultures, the cement mixed with malic acid was less cytotoxic than the cement mixed with citric acid, which was far less cytotoxic than a commercial carboxylate cement used as a negative control, suggesting malic acid to be superior to citric acid as a liquid in this regard. Similar findings were also obtained with osteoclasts, of which culture experiments clearly suggested that the number of osteoclasts on the cement mixed with malic acid was significantly greater than that on the cement mixed with citric acid. Since osteoclastic response to substrates could be used as a maker in evaluating their bioresorbability associated with osteoclasts, the above finding may suggest that the cement that is to be mixed with malic acid would be more useful as bone substitutes.     

Enzymatic determination of a molar ratio of free branched-chain amino acids to tyrosine (BTR) and its clinical significance in plasma of patients with various liver diseases

A molar ratio of free branched-chain amino acids to tyrosine (BTR) was determined in the plasma of patients with liver diseases using a new enzymatic method. In addition, clinical significance of BTR was studied by comparing particularly with that of Fischer's ratio (a molar ratio of branched-chain amino acids to aromatic amino acids (tyrosine+phenylalanine], which was obtained by conventional HPLC (Amino acid autoanalyzer, Hitachi 835). Following results were obtained: 1) Enzymatically determined branched-chain amino acids and tyrosine showed significant correlations with respective results obtained by HPLC (r = 0.937, 0.972). 2) Significant correlation was also found between enzymatically determined BTR and Fischer's ratio obtained by HPLC. Changes of BTR in clinical courses were found to be in parallel with those of Fischer's ratio. 3) BTR as well as Fischer's ratio correlated significantly with ICG R15, KICG, prothrombin time (%) and serum albumin level. 4) BTRs in patients with decompensated liver cirrhosis or with fulminant hepatitis were significantly lower than those in patients with acute or chronic hepatitis. In conclusion, new enzymatic assay of branched-chain amino acids and tyrosine as described here is quite simple method, and is also considered to be very useful parameter of the clinical conditions of patients with liver diseases, particularly representing the severity of liver diseases and the protein nutritional status.     

Nucleotide mutations associated with hepatitis B e antigen negativity

One hundred and forty four patients with chronic hepatitis B were tested to identify new mutations associated with hepatitis B e antigen (HBeAg) negativity, using a full genome sequence analysis. All the patients were Chinese and had hepatitis B virus infection of genotype C. Patients with none of the pre-core or core promoter mutations were significantly (P < 0.001) less common in the group with anti-HBe (13%) than in the group with HBeAg (56%). The complete nucleotide sequence was determined in four anti-HBe-positive patients who had neither pre-core nor core promoter mutations and in five HBeAg-positive patients who also had neither of these mutations (the groups were matched for age and sex). Six mutations were found to be significantly more common in the former group than in the latter: G529A (3/4 vs. 0/5), C934A (4/4 vs. 1/5), A1053G (4/4 vs. 1/5), G1915T/A (4/4 vs. 0/5), T2005C/A (4/4 vs. 0/5), and C3026T (3/4 vs. 0/5). Three of the six mutations were significantly more common in the four anti-HBe-positive patients who had neither pre-core nor core promoter mutations, compared to 11 HBeAg-positive patients who had pre-core and core promoter mutations, and also compared to 15 anti-HBe-positive patients who had pre-core and core promoter mutations, suggesting further the specificity of these mutations. Of the six mutations, two resulted in amino acid substitution in the polymerase protein, and one is located near the enhancer I region. The results suggest that the six newly discovered mutations are associated with HBeAg negativity.     

Decreased prolactin level in secretory granules and their increased exocytosis in estrogen-induced pituitary hyperplasia in rats treated with a dopamine agonist.

Treatment with bromocriptine (CB) stimulates the release of secretory granules from human prolactinomas by exocytosis in spite of a remarkable decrease in serum prolactin (PRL) levels. In an attempt to elucidate the reasons for this phenomenon, secretory granules were analyzed in estrogen-induced pituitary hyperplasia in rats which served as a model of human disease. The amount of protein contained in cell fractions rich in secretory granules was the same in CB-treated pituitaries as that in the controls, whereas PRL levels decreased to about a half the level of the controls. Morphometric analysis using point-counting and electron immunocytochemistry revealed that the PRL concentrations in secretory granules decreased to about 50% after CB-treatment. Serum PRL levels estimated by counting the exocytotic secretory granules were remarkably lower in CB-treated rats than in the controls. It was suggested that the composition of secretory granules changed or that nonhormonal constituents in the granules disintegrated more slowly after CB treatment, thus allowing exocytosed granules to be observed more frequently. Additionally CB might also suppress a bypass release of PRL by which PRL is secreted without being packaged into secretory granules.     

Tumor necrosis factor alpha-induced interleukin-8 production via NF-kappaB and phosphatidylinositol 3-kinase/Akt pathways inhibits cell apoptosis in human hepatocytes

Tumor necrosis factor alpha (TNF-alpha) not only induces apoptotic signals but also causes antiapoptotic and regenerative responses in the liver. However, the molecular mechanism(s) of the latter events remains unclear. In the present study, we examined TNF-alpha-induced genes in Hc human normal (unsensitized) hepatocytes by cDNA microarray analysis. Interleukin-8 (IL-8) induction was the most pronounced of the upregulated genes. The IL-8 protein level was also increased. IL-8 belongs to the ELR-CXC chemokine family and appears to exert mitogenic and antiapoptotic functions in other cell systems. IL-8 expression by TNF-alpha was inhibited when two survival signals, nuclear factor kappaB (NF-kappaB) and phosphatidylinositol 3-kinase (PI3K)/Akt, were inhibited by a mutant form of inhibitor of NF-kappaB (IkappaB); by dominant negative (kinase-dead) Akt; or by treatment with LY 294002, an inhibitor of PI3K. TNF-alpha induced apoptosis in Hc cells that were sensitized by inhibition of NF-kappaB and PI3K activation. IL-8 administration protected mice against concanavalin A-induced hepatitis in vivo. IL-8 also rescued the sensitized Hc cells, at least in part, from TNF-alpha-induced apoptosis in vitro. TNF-alpha inhibited DNA synthesis in unsensitized Hc cells in the absence of serum. Exogenous IL-8 reversed, though anti-IL-8 neutralization antibody enhanced, growth inhibition by TNF-alpha. These results indicate that IL-8, the production of which is stimulated by TNF-alpha, inhibits apoptosis of sensitized hepatocytes and releases normal (unsensitized) hepatocytes from growth inhibition induced by TNF-alpha.     

Kinetics of hepatic transport of 4-methylumbelliferone in rats. Analysis by multiple indicator dilution method

Hepatic elimination of 4-methylumbelliferone (4MU), which has been used as a model compound for conjugative metabolism, was studied by means of a multiple indicator dilution (MID) method in the isolated perfused rat liver. Using this method, three intrinsic hepatic clearances, CL _int,inf , CL _{int,eff, and} CL _{int,seq, which represent the influx, efflux, and sequestration processes, respectively, were obtained. When the dose was increased from a low dose (50 g/rat liver) to a high dose (3000 g/rat liver), the hepatic availability of 4MU increased from 0.11 to 0.73. With increasing dose, the} CL _{int,eff value increased approximately two times, while the} CL _{int,seq value decreased to approximately one-third. The remarkable dose dependence of hepatic availability was due to nonlinearity in both} CL _{int,eff and} CL _{int,seq values. However, the} CL_int,inf value was almost independent of dose. The dose-dependent change in CL_int,seq might be explained by the saturation of conjugative metabolism of 4-MU, while the increase in the CL _{int,eff value with increasing dose might be partly explained by the nonlinear tissue binding of 4-MU, since the tissue unbound fraction determined by an ultrafiltration method using liver homogenate increased approximately 1.5 times at higher concentration of 4-MU compared to that at lower concentrations. In addition, based on a comparison of the individual intrinsic clearances, i.e.}, CL _int,inf , CL _{int,eff, and} CL _{int,seq, the major determining process of the apparent hepatic intrinsic clearance of 4MU is thought to be the sequestration process at the high dose. However, at the low dose, the membrane transport process (influx and efflux processes) as well as the sequestration process also determine the apparent hepatic intrinsic clearance}.     

Hypofluorescent spots in indocyanine green angiography of peau d‘orange and fundus

Purpose. Hypofluorescent spots were seen inindocyanine green (ICG) angiography of peau dorangefundus in eyes with angioid streaks. Origin of the hypofluorescentspots were examined with attention to their correlationwith a peau dorange appearance of the central fundususing a computer-assisted image comparison system. Methods. ICG angiography was performed in 5 patientshaving peau dorange appearance of fundus using ascanning laser ophthalmoscope (SLO) and a digitalvideo-fundus camera. The same central fundus areas corresponding to hypofluorescent spots in an ICGangiogram were then digitally identified in afluorescein angiogram and in a red-free picture in all10 eyes of the 5 patients. Monochromatic lightobservation was also performed with a dark fieldobservation using a SLO to see subretinal orintrachoroidal pigment clumping. Results. In no patient, the areas identified withhypofluorescent spots did show relevant changes ina fluorescein angiogram or a red-free picture. SLOexamination revealed not perfusion defect at the sameareas. The dark field observation showed no pigmentclumping at the peripapillary and papillomacularbundle regions where hypofluorescent spots were seen.Conclusions: Hypofluorescent spots seen in ICGangiograms did not show exact consistency with peau dorange changes in their location and shape. Perfusion defects or blocking by pigments were not acause of hypofluorescent spots. The scatteredhypofluorescent spots were considered to be relevantwith irregular affinity of the fundus to ICG dye.     

Expression of cripto-1 in human colorectal adenomas and carcinomas is related to the degree of dysplasia

The expression and localization of cripto-1 (CR-1) and epidermal growth factor receptor (EGFR) were assessed by immunocytochemistry in 41 human colorectal carcinomas, 57 adenomas, 9 hyperplastic polyps and in 98 noninvolved colonic mucosa samples that were adjacent to adenoma and/or carcinoma. Thirty-two (78.0%) and 19 (46%) carcinomas showed staining for CR-1 and EGFR, respectively, whereas 24 (42.0%) and 25 (43.8%) of the adenoma samples were reactive with the anti-CR-1 and anti-EGER antibodies, respectively. Two (22.2%) and 1 (11.1%) of the hyperplastic polyps demonstrated moderate levels of staining with anti-CR-1 and anti-EGFR antibodies. In contrast, none of the normal, noninvolved colonic mucosa samples reacted with the CR-1 antibody, whereas only 1 (1.0%) reacted with the EGFR antibody. Between EGFR and CR-1 expression, there was no significant association within either adenomas or carcinomas. A significant difference in the incidence for CR-1 expression was observed between adenomas and carcinomas (p<0.001). Within adenomas, the frequency of CR-1 was related to the histological degree of atypia. Immunostaining for p53 was also observed in 10 (24%) of the carcinomas, in 10 (17%) of the adenomas and in none of the hyperplastic polyps nor colonic mucosa samples. No statistically significant difference for p53 staining was observed between the adenomas and carcinomas. However, adenomas with moderate atypia exhibited relatively strong positive staining for p53 (p<0.05) compared to either adenomas with mild or severe atypia. A slight trend (p<0.05) for coexpression of p53 and CR-1 was detected in adenomas but not in carcinomas. These data demonstrate that CR-1 is a tumor marker for colon carcinomas and additionally that the expression of CR-1 may be an important factor in the early stages of colon cancer development during the adenomacarcinoma transition.