Biosynthesis and secretion of the third component of complement by human endothelial cells in vitro.

The third component of complement (C3) is synthesized and released by cultured human capillary endothelial cells. After the incubation of cells in a methionine-free medium containing 35S-methionine for 48 hr, culture supernatants were immunoprecipitated with anti-human C3 serum. SDS solubilization and 2-ME reduction of the immunoprecipitates, followed by separation with SDS-polyacrylamide gel electrophoresis and autoradiography, revealed two major bands comparable to those of the alpha and beta chains of human C3. The content of C3 in the culture medium harvested at different time-intervals was determined by ELISA. The C3 secretion rate was about 250 ng/10(6) cells/5 days in the primary culture medium and 75 ng/10(6) cells/5 days after seven passages. The capillary endothelial cells described here have factor VIIIRAg specific for endothelial cells, and exhibit ring formation resembling capillary lumina, but they lack Weibel-Palade bodies.     

Synaptonemal complex analysis in spermatocytes and oocytes of rainbow trout,Oncorhynchus mykiss (Pisces,Salmonidae): the process of autosome and sex chromosome synapsis

The surface-spreading synaptonemal complex (SC) technique was employed to analyze spermatocytes and oocytes of rainbow trout in order to visualize the process of autosome and sex chromosome synapsis in this species. The structure of lateral elements (LEs) of the SC and the chromosome synapsis process at the stages of leptotene, zygotene and pachytene are described. Comparative analysis of SCs of spermatocytes and oocytes showed a difference in the synaptic process, i.e. in spermatocytes all LEs were synapsed before the appearance of centromeric regions in the biarmed elements, while in the oocytes some fully synapsed LEs, including the centromeric region of the biarmed elements, were found together with fully or partially unsynapsed LEs. In males the sex chromosome synapsis starts only after all autosomes have synapsed. Irregular synapses involving three or four LEs were found in 3.4% of the cells analyzed in mid or late zygotene. Multivalents were found in males and females. Some aspects of initial meiotic development and their implications in rainbow trout cytogenetics, genetics and evolution are discussed.     

Influence of culture method on the proliferation and osteogenic differentiation of human adipo-stromal cells in nonwoven fabrics

The initial attachment, proliferation, and osteogenic differentiation of stromal cells from human fat tissue were investigated in three-dimensional nonwoven fabrics prepared from polyethylene terephthalate (PET) fiber with different diameters. The largest number of cells initially attached was observed in the nonwoven fabrics prepared from PET fiber with a diameter of 22.0 microm, irrespective of fabric porosity. The number of cells attached was larger and the cells were distributed more homogeneously in the fabrics by the agitated seeding method than by the static seeding method. The culture method depended on the time profile of cell proliferation. Cell proliferation improved in the following order: stirred (spinner flask) culture method > agitated culture method > static culture method. In addition, cells proliferated homogeneously in fabrics by the stirred culture method. When evaluated as a measurement of cell osteogenic differentiation, the activity of alkaline phosphatase (ALP) was not influenced by the diameter of fabrics. The static culture method tended to enable cells to enhance ALP activity, in contrast with the stirred and agitated culture methods. It is concluded that fabric fiber diameter and culture method greatly affected the proliferation and differentiation of cells in nonwoven fabrics.     

De novo formation of adipose tissue by controlled release of basic fibroblast growth factor

De novo adipogenesis at the implanted site of a basement membrane extract (Matrigel) was induced through controlled release of basic fibroblast growth factor (bFGF). bFGF was incorporated into biodegradable gelatin microspheres for its controlled release. When the mixture of Matrigel and bFGF-incorporated gelatin microspheres was implanted subcutaneously into the back of mice, a clearly visible fat pad was formed at the implanted site 6 weeks later. Histologic examination revealed that the de novo formation of adipose tissue accompanied with angiogenesis was observed in the implanted Matrigel at bFGF doses of 0.01, 0.1, and 1 microg/site, the lower and higher doses being less effective. The de novo formation induced by the bFGF-incorporated microspheres was significantly higher than that induced by free bFGF of the same dose. The mRNA of a lipogenesis marker protein, glycerophosphate dehydrogenase, was detected in the formed adipose tissues, biochemically indicating de novo adipogenesis. Free bFGF, the bFGF-incorporated gelatin microspheres, or Marigel alone and bFGF-free gelatin microspheres with or without Matrigel did not induce formation of adipose tissue. This de novo adipogenesis by mixture of Matrigel and the bFGF-incorporated gelatin microspheres will provide a new idea for tissue engineering of adipose tissue.     

Cartilage regeneration using slow release of bone morphogenetic protein-2 from a gelatin sponge to treat experimental canine tracheomalacia: a preliminary report

We investigated whether saber sheath-type tracheomalacia could be treated by the slow release of bone morphogenetic protein (BMP)-2 from a gelatin sponge. A 1 cm gap was made in the middle portion of each of 10 consecutive tracheal cartilage rings in the canine cervix (control group, n = 3), then a gelatin sponge containing 12 microg of BMP-2 solution was implanted in the gap (12 microg group, n = 3). In another group (120 microg + P group, n = 3), the implanted gelatin sponge contained 120 microg of BMP-2 solution, and the gap was covered with periosteum. All of the control dogs developed saber sheath-type tracheomalacia, whereas tracheomalacia was not observed in the 12 microg and 120 microg + P groups. In the 12 microg group, fibrous cartilage was observed at the ends of the cartilage stumps. In the 120 microg + P group, newly formed bone and cartilage were observed to form a bridge between the cartilage stumps. The regeneration of cartilage or bone induced by the slow release of BMP-2 from a gelatin sponge might be useful for treatment of tracheomalacia.     

Fate of odontoblasts and blood capillaries in the incisal region of the rat incisor pulp

Transmission electron microscopy of thin sections of the rat incisor pulp revealed that in the middle region of the incisor there were fenestrated capillaries in the “predentinal plexus” and that this region contained the tallest odontoblasts. The odontoblasts gradually became shortened in the incisal part of this region: the fenestrated capillaries in the predentinal plexus changed to continuous type capillaries. Almost all the odontoblasts had degenerated near the incisal end of the tooth. The predentinal plexus disappeared in this region, but the “subodontoblastic capillary plexus” persisted. In a specific region just beneath the worn incisal end, numerous macrophages and polymorphonuclear neutrophils appeared and scavenged the degenerating cells, possibly including the odontoblasts. © 1993 Wiley-Liss, Inc.     

Comparison of chemosensitivity tests: clonogenic assay versus MTT assay

When the development of chemotherapeutic agents reaches the clinical trial stage, it is necessary to perform drug sensitivity tests quickly in order to select the most promising agents for the treatment of cancer. In order to assess the possibility of using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a substitute for the human tumor clonogenic assay (HTCA), we evaluated the correlation between the results obtained by these 2 assays in 5 human lung cancer cell lines. The correlation coefficient between the results of the HTCA and the MTT assay was 0.673, indicating a relatively good correlation. The correlation was most prominent in platinum analogues (r = 0.939) and good in anthracyclines/anthracenedione (r = 0.611). However, no significant correlation was observed in vinca alkaloids, etoposide, irinotecan, SN-38 (an active metabolite of irinotecan), and rhizoxin. The results of the MTT assay showed a high degree of correlation with those of the HTCA in predicting the sensitivity of cancer cell lines to platinum analogues, and anthracyclines/anthracenedione. These results suggest that the MTT assay may be more convenient and quickly performed than the HTCA and can replace HTCA in evaluating the effects of anticancer agents, especially the platinum analogues and anthracyclines/anthracenedione.     

Adult-onset type II citrullinemia and idiopathic neonatal hepatitis caused by citrin deficiency: involvement of the aspartate glutamate carrier for urea synthesis and maintenance of the urea cycle

Citrin is a mitochondrial aspartate glutamate carrier primarily expressed in the liver, heart, and kidney. We found that adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin. In this report, we describe the frequency of SLC25A13 mutations, the roles of citrin as a member of the urea cycle and as a member of the malate-aspartate shuttle, the relationship between its functions and symptoms of citrin deficiency, and therapeutic issues.     

Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis

In the prespermatogenesis period, male germ cells (gonocytes) begin to reproliferate and move to the basement membrane of the seminiferous tubule. Although these two events-reproliferation and relocation-are important for establishment of spermatogenesis, they have not been greatly analyzed both in a mechanical and in an endocrine or paracrine aspect. In this study, the relationship between reproliferation and relocation of gonocytes was examined, using the thymidine analog bromodeoxyuridine (BrdU) labeling method and transmission electron microscopy (TEM). BrdU was injected into the fetuses [day 13.5 post coitus (dpc) to 18.5 dpc] and pups [day 0. 5 post partum (dpp) to 6.5 dpp] of C57BL/6J mice. Two hours later, BrdU positive gonocytes were examined immunohistochemically and these data were analyzed. TEM and LM observation was carried out as well. Gonocytes began to relocate on the basement membrane from 18.5 dpc (1.4%) while BrdU-labeled gonocytes were first detected on 1.5 dpp (13.6%). Relocated BrdU-negative gonocytes were recognized from 18.5 dpc (1.4%), and relocated BrdU-labeled gonocytes were recognized from 1.5 dpp (8.4%). On the other hand, non-relocated BrdU-labeled gonocytes were detected from 1.5 dpp (5.2%). Gonocyte relocation began 2 days earlier than reproliferation during the late fetal period. After birth, the two events occurred at random. These results indicate that the reproliferation of the gonocyte does not correlate with relocation. The two events may be regulated by different mechanisms.