Transplantation of skin fibroblasts expressing BMP-2 promotes bone repair more effectively than those expressing Runx2.

We investigated the osteogenic potential of skin fibroblasts that overexpressed BMP-2 or Runx2 by using adenoviral vectors. In in vitro experiments, skin fibroblasts infected with adenovirus vector encoding BMP-2 (AdBMP-2) released substantial levels of BMP-2 proteins into culture media, and those infected with adenovirus vector encoding Runx2 (AdRunx2) produced its protein. Transduction of BMP-2 or Runx2, respectively, increased alkaline phosphatase (ALP) activity and induced expression of mRNAs of ALP, osteocalcin, and osterix in skin fibroblasts. In in vivo experiments, we investigated the bone induction activity by transplantation of a complex composed of carrier [poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS)] and skin fibroblasts (PGS/SF complex). Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2-induced ectopic bone formation when transplanted into the subfascia of back muscle, unlike those infected with AdRunx2. Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2 into craniotomy defects induced bone formation from 2 weeks after transplantation, and almost all PGS was replaced by newly synthesized bone at 6 weeks. To investigate the fate of the transplanted cells, we transplanted skin fibroblasts isolated from green fluorescence protein transgenic mice into craniotomy defects. Transplantation of these skin fibroblasts transfected with AdBMP-2 generated green fluorescence protein-positive osteoblasts and osteocytes, indicating that the transplanted skin fibroblasts differentiated into osteoblastic lineage cells during bone repair. In contrast, transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdRunx2 induced a few ALP-positive cells at 1 week after transplantation, but their number decreased depending on time after transplantation. In addition, transplantation of these complexes was insufficient to induce bone repair. Taken together, our results suggest that skin fibroblasts expressing BMP-2 are more suitable for cell-mediated therapy of bone repair than those expressing Runx2.

Isolation and characterization of a mesenchymal cell line that differentiates into osteoblasts in response to BMP-2 from calvariae of GFP transgenic mice.

We established the clonal mesenchymal cell line, GFP-C3 (C3), which differentiates into osteoblasts in response to BMP-2 from calvariae of newborn green fluorescence protein (GFP) transgenic mice. This cell line cultured with control medium expressed low levels of alkaline phosphatase (ALP) activity and osterix mRNA and undetectable ALP and osteocalcin mRNA. Incubation of these cells with rhBMP-2 increased ALP activity dose-dependently and induced substantial levels of ALP, osteocalcin and osterix mRNA expression. C3 cells infected with adenovirus vector encoding BMP-2 (AdBMP-2) or Runx2 (AdRunx2) showed greatly increased ALP mRNA expression in a time-dependent fashion. Transduction with AdRunx2-induced expression of ALP and osteocalcin mRNA, but not osterix mRNA by day 3. Transduction with AdBMP-2 induced apparent expression of ALP and osterix mRNA by day 1 after transduction, but induced only weak expression of osteocalcin mRNA day 3 after transduction. Transplantation of C3 cells transduced with AdBMP-2 into back subfascia in wild-type mice with a complex of poly-d,l-lactic-co-glycolic acid/gelatin sponge (PGS) generated ectopic bone formation involving GFP-positive osteoblasts and osteocytes 2 weeks after transplantation. C3 cells transduced with AdRunx2 or AdLacZ failed to induce ectopic bone formation. Transplantation of C3 cells transduced with AdBMP-2 into craniotomy defects in wild-type mice using PGS as a carrier induced bone formation 2 weeks after transplantation, and replaced defects 4 weeks after transplantation. C3 cells transduced with AdRunx2 failed to induce bone repair after transplantation into craniotomy defects. These results indicate that C3 cells retain differentiation potential into osteoblasts in response to BMP-2. They are useful tools for analyzing the process of osteoblast differentiation in vivo after transplantation.

Functional expression of intestinal dipeptide/beta-lactam antibiotic transporter in Xenopus laevis oocytes.

An intestinal active transport system specific to small peptides and peptide-like drugs such as beta-lactam antibiotics was functionally expressed in Xenopus laevis oocytes after microinjection of messenger RNA (mRNA) derived from rat intestinal mucosal cells. The transport activity was evaluated by measuring the uptake of a tripeptide-like cephalosporin antibiotic, ceftibuten, which has high affinity for the intestinal peptide/H+ co-transporter and is resistant to peptidases. Ceftibuten transport in mRNA-injected oocytes was pH dependent (a proton gradient is the driving force), stereo selective (uptake of the cis-isomer of ceftibuten was about 4-fold higher than that of the trans-isomer), saturable and temperature dependent. Furthermore, various dipeptides showed cis-inhibitory and trans-stimulatory effects on the uptake of ceftibuten by mRNA-injected oocytes, suggesting that ceftibuten and dipeptides are transported by a common carrier protein. These results are in accordance with the functional properties of native proton-coupled peptide transporter previously clarified by studies with isolated intestinal brush-border membrane vesicles and other experimental systems. A protein with a molecular mass of about 130 kDa expressed in the membrane of mRNA-injected oocytes was identified as the transport protein by specific labeling with a photoreactive beta-lactam antibiotic, [3H]benzylpenicillin, followed by SDS-PAGE analysis of the radiolabeled protein. Furthermore, an experiment with mRNA size-fractionated by sucrose density gradient centrifugation indicated that the peptide transporter is encoded by mRNA of between 1.8 and 3.6 kb. These results, obtained using a heterologous gene expression technique, confirm that intestinal absorption of beta-lactam antibiotics occurs through a carrier-mediated mechanism and show that biologically stable beta-lactam antibiotics can be useful probes for molecular analysis of intestinal peptide transporter.

A case of chronic expanding hematoma in the tensor fascia lata.

We describe a patient in whom a chronic expanding hematoma was pathologically confirmed by examination of the resected specimen. It developed gradually in the left lateral thigh, which had been bruised 8 months before, but had produced no mass-related symptoms initially. Computed tomography (CT) showed the mass to be homogeneous with capsule formation, whereas ultrasonography showed it to resemble a multilocular cyst. The lesion was treated by complete excision of a perifascial mass. Grossly, a well-circumscribed, fusiform mass with a soft cystic center and a fibrous pseudocapsule was observed. Histologically, the mass was composed of necrotic debris, fibrin and blood clots. This was surrounded by a rim of hyalinized fibrous tissue with a chronic inflammatory infiltrate and granulation tissue with sprouting new capillaries. CT was unable to discriminate the chronic expanding hematoma from other soft tissue masses, ultrasonography being needed to reveal its characteristics in this case.

[Neoadjuvant chemotherapy with tegafur suppository for rectal cancer--effects of tegafur on nuclear DNA content of cancer cells--Tochigi Colorectal Cancer Study Group].

To assess the significance of neoadjuvant chemotherapy for large bowel cancer, we investigated an effect of tegafur on cancer cell kinetics by DNA content flow cytometric analysis. The pathologic specimens analyzed in this study were obtained from 28 patients with rectal cancer who had received tegafur suppositories prior to surgery. Mean cumulative doses administered were 13.4 g, and mean duration was 12.7 days. In 24 of the 28 specimens, the DNA ploidy and DNA index of the cancer cells were essentially unchanged after the chemotherapy, and DNA aneuploidy was present in 67%. The proportion of cells in S-phase in DNA aneuploid tumors was significantly higher than in DNA diploid tumors. The proportion of cells in G0G1-phase was inversely correlated to that in S-phase. After the chemotherapy, the proportions of cells in S-phase increased both in DNA diploid and aneuploid tumors, although a significantly higher mean value was found only for the latter (p < 0.05). In the DNA aneuploid tumors, there was a higher frequency of responders confirmed by histological evaluation (44%) as compared to the DNA diploid tumors (25%), and a tendency for increases in S-phase fraction of the responders to be greater than in non-responders. These results suggest that DNA flow cytometry-derived parameters may conceivably be useful in predicting the antitumor effect of 5-FU and its derivatives against large bowel cancer.

[Study on the concentrations of 5-fluorouracil (5-FU), tegafur (ET) and uracil in bile: comparison of UFT or FT].

The serum and bile tegafur (FT), 5-fluorouracil (5-FU) and uracil levels after administration of UFT were assessed in 13 cases of malignant biliary tumor accompanied by biliary obstruction in comparison with FT alone. The serum and bile FT and 5-FU levels showed almost the same transition pattern in both groups, reaching to the plateau in 1-2 weeks and revealing cumulative effect by continual administration. Correlation was obtained between serum and bile levels except for 5-FU level in UFT group (p less than 0.05). Correlation between 5-FU and uracil was obtained in the serum in both groups (p less than 0.05), but no effect of uracil was observed. In bile, correlation was seen only in UFT group (p less than 0.05), and the effect of uracil was observed in bile 5-FU level.

A rapid measurement of delta 5-3 beta-hydroxysteroid dehydrogenase activity coupled with delta 4-delta 5 isomerase by a high pressure liquid chromatography.

By a high pressure liquid chromatography, progesterone (lambda max = 240 nm, epsilon 240 = 17,000) was separable from the other delta 4-3-oxosteroids. Relationship between the amount of progesterone and the area of the peak corresponding to progesterone was established as linear at least between 0-2.0 g. Progesterone is formed from pregnenolone by delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta r-delta 5 isomerase. Because pregnenolone is devoid of absorption oat 240 nm, the enzyme activity was measurable by the high pressure liquid chromatography from the amount of progesterone produced from pregnenolone in vitro in the presence of nicotinamide adenine dinucleotide. As an example of its application, the dehydrogenase activities in the ovaries of the immature rats treated with gonadotropins were measured by the above method. The estimates obtained by this method were agreeable with those by thin layer chromatography, employing radioactive pregnenolone as the substrate.

Successful surgical treatment of secondary Kwashiorkor after total gastrectomy: report of a case.

We report herein the case of a 56-year-old woman who developed secondary Kwashiorkor 9 years after undergoing a total gastrectomy for early gastric cancer. Until she began developing the symptoms of Kwashiorkor, including general fatigue, edema of the face and extremities, anemia, alopecia, and weight loss, she had been leading a normal life post-gastrectomy. Her symptoms were alleviated by total parenteral nutrition (TPN) therapy, but reappeared soon after TPN therapy was discontinued. Therefore, she required several subsequent courses of TPN. In an attempt to permanently resolve the ongoing Kwashiorkor symptoms, reconstructive surgery involving transposition of the jejunum from the previous Graham method to the interposition method was performed 10 years after the initial gastrectomy. After the second operation, her malnutrition was completely alleviated, and she has been in good health for the 8 years since. To our knowledge, there has been no other report of the symptoms of secondary Kwashiorkor after total gastrectomy being alleviated by altering the procedure of reconstruction of the intestinal tract. Thus, we recommend surgical treatment to alter the digestive continuity to a more physiological pathway for selected patients with secondary Kwashiorkor syndrome.

Expression of the Na+ dependent uridine transport system of rabbit small intestine: studies with mRNA-injected Xenopus laevis oocytes.

Xenopus laevis oocytes were used as an expression system to prove and characterize the carrier-mediated transport of uridine in the small intestine. Significant Na+ dependency was observed for the uptake of [3H]uridine by Xenopus laevis oocytes injected with poly(A)+RNA prepared from rabbit small intestinal mucosa. By contrast, the uptake of [3H]uridine was negligible in water-injected oocytes. There was no significant difference in the Na+ dependent uptake rates of [3H]uridine among oocytes expressed by using mRNA prepared by three different methods. The uptake of [3H]uridine by mRNA-injected oocytes was enhanced by increasing the culturing time after mRNA injection. Concentration dependency for uridine transport was observed with the Michaelis constant of 8.27 microM, which was comparable to that reported in the study using the brush-border membrane vesicles from rabbit small intestine (6.4 microM). Furthermore, the uptake of [3H]uridine was significantly inhibited by adenosine and thymidine, but not by adenine and uracil. Consequently, the transport system of uridine expressed in mRNA-injected oocytes is clarified to be similar to that functioning in the brush-border membrane of the small intestine.

Protein crystallization by using porous glass substrate.

The effects of a commercially available porous glass substrate (Corning Porous Glass No.7930) on the heterogeneous nucleation of proteins [hen egg-white lysozyme (HEWL), thaumatin and apoferritin] have been investigated in order to develop an improved method to facilitate the nucleation of protein crystals. It was found that the porous glass substrate could promote the nucleation at lower supersaturations. The induction time for nucleation decreased, and the crystals obtained from porous glass substrates were larger than those from normal glass substrates. Many pores and channels of 10-100 nm in diameter were observed on the porous glass surface by atomic force microscopy (AFM). It is believed that these pores and channels are crucial for facilitating the nucleation process in this work.