Relationship between porotic changes in alveolar bone and spinal osteoporosis.

Epidemiological studies have shown that post-menopausal women who do not use an estrogen supplement have fewer teeth than those who do. We hypothesized that changes in the dentition of post-menopausal women might be due to alveolar bone alterations by estrogen deficiency. To clarify this, we analyzed the microstructural alveolar bone changes in ovariectomized monkeys and compared these with their lumbar bone mineral density. The % of baseline bone mineral density showed a significant decrease in the ovariectomized group as compared with the controls. The second-molar interradicular septa in ovariectomized monkeys showed a significantly decreased nodes number, cortices number, and an increased structural model index value. More pores were seen in the ovariectomized group at the top of the septa. This study demonstrated that, in such monkeys, estrogen deficiency led to fragility of the trabecular structure of the molar alveolar bone, and such fragility was inversely correlated with lumbar bone mineral density.

Ubiquitous factors that interact simultaneously with two distinct cis-elements on the rat aldolase B gene promoter.

The proximal promoter region (nucleotide -202 to -1) of the rat aldolase B gene confers liver-specific transcription, and contains three indispensable protein-binding sites (site A, site B and site C). Site A binds HNF-1 and HNF-3, and site B binds NF-Y and a CCAAT-binding factor AlF-B (Tsutsumi et al. (1989) Mol. Cell. Biol. 9, 4923-4931; Raymondjean et al. (1991) Nucleic Acids Res. 19, 6145-6153), trans-acting factors that interact with site C, however, have not been well characterized. In this study, we identified specific factors that bind site C by two-dimensional gel electrophoretic analyses. The factors interacted with two distinct cis-elements; site C and site B. This observation, together with the fact that site C contains a C/EBP binding motif, implied that the site C-binding factors are members of C/EBP family. However, analyses of their binding characteristics, their relative molecular masses, and their distribution in different cell types showed that the site C binding factors are different from known members of C/EBP family.

Interactions among four subunits of elongation factor 1 from rice embryo.

To establish the subunit construction of elongation factor EF-1, interactions among four non-identical subunits of rice embryo EF-1 (alpha, beta, beta', and gamma) were analyzed with polyacrylamide gel electrophoresis. Complexes beta beta', alpha beta, alpha beta', and beta gamma were formed by mixing the two respective subunits. However, no complex was formed between EF-1 beta' and EF-1 gamma. Complexes containing three subunits like alpha beta beta', alpha beta gamma, and beta beta' gamma, were formed by mixing the three respective subunits. EF-1 was reconstructed when each subunit was added in the following order, beta, beta', gamma, and alpha. The affinity of EF-1 alpha for other subunits was as follows, beta beta' gamma > beta beta' > beta not equal to beta'. Likewise, the affinity of EF-1 gamma for other subunits was: beta beta' gamma > beta >> beta'. Phe-tRNA binding activity of the reconstructed EF-1 was about 90% of that of the native EF-1. From these results, we concluded that rice embryo EF-1 is constructed of equimolar amount of four subunits, alpha, beta, beta' and gamma.

A novel variant of translation elongation factor-1beta: isolation and characterization of the rice gene encoding EF-1beta2.

A rice gene encoding a novel isoform of translation elongation factor-1beta subunit (termed EF-1beta2) was isolated and characterized. The gene comprises of eight exons, and encodes a 226-amino-acid protein. Expression of EF-1beta2 mRNA is abundant in seeds and cultured cells, but is considerably low in the tissues of the rice seedling. Antiserum raised against an EF-1beta2 synthetic peptide detected a protein with a relative molecular mass of about 32 kDa, indicating the EF-1beta2 gene is actually expressed in rice tissues. EF-1beta2 showed a close similarity to the cognate subunits from plant (beta and beta').

Histochemical and autoradiographic studies on elcatonin internalization and intracellular movement in osteoclasts.

The binding sites and chronologic localization of elcatonin (eCT) in osteoclasts were examined by autoradiography using [125I]elcatonin (125I-eCT). In addition to the structural changes induced by calcitonin (CT) reported so far, changes were also observed in the structure of Golgi apparatus. These changes continued until 48-72 h after incubation with eCT. Developed silver grains of 125I-eCT were localized into multinucleated osteoclasts and mononuclear cells that were ultrastructurally defined as "preosteoclasts." The silver grains located on plasma membranes of those cells and were then internalized; they accumulated, especially in the Golgi apparatus, and remained for 48-72 h. A few silver grains were also detected in lysosomes and small vesicles. The decrease in the number of silver grains in the Golgi apparatus accompanied the recovery of osteoclast structures--Golgi apparatus and then ruffled borders. These findings suggest that (1) CT especially inhibits the sorting function of Golgi apparatus in osteoclasts, resulting in prolonged retention of CT in this organelle. (2) The CT in Golgi apparatus may keep its activity and cause the prolonged effect of CT on osteoclast activity.

Localizational alterations of calcium, phosphorus, and calcification-related organics such as proteoglycans and alkaline phosphatase during bone calcification.

To further approach the mechanisms of bone calcification, embryonic rat calvariae were observed at electron microscopic level by the means of fine structures and various cytochemical localizations, including nonspecific proteoglycan (PG) stained by cuprolinic blue (CB), decorin, chondroitin sulfate, hyaluronan, and alkaline phosphatase (ALP), as well as the elemental mapping of calcium (Ca) and phosphorus (P) by energy-filtering transmission electron microscopy (EFTEM). In the calvariae, calcification advanced as the distance from osteoblasts increased. Closer to the osteoblasts, the osteoid was marked by an abundance of CB-positive PGs around collagen fibrils. After crystallization within matrix vesicles, calcified nodules formed and expanded, creating a coherent calcified matrix. The sizes of CB-positive PG-like structures diminished as calcification proceeded. Although small CB-positive structures were accumulated in early stage-calcified nodules, they were localized along the periphery of larger calcified nodules. Cytochemical tests for decorin, chondroitin sulfate, and hyaluronan determined their presence in the areas around collagen fibrils of the osteoid, as well as in and around calcified nodules, whereas ALP was found in the matrix vesicles, as well as in and around the calcified nodules. Ca tended to localize at the PG sites, while P often mapped to the collagen fibril structures, in the uncalcified matrix. In contrast, Ca/P colocalization was visible in and around the calcified nodules, where ALP and smaller CB-positive structures were observed. The difference in the localization patterns of Ca and P in uncalcified areas may limit the local [Ca2+][PO4(3-)] product, leading to the general inhibition of hydroxyapatite crystallization. The downsizing of CB-positive structures suggested enzymatic fragmentation of PGs. Such structural alterations would contribute to the preservation and transport of calcium. ALP possesses the ability to boost local phosphate anion concentration. Therefore, structurally altered PGs and ALP may cooperate in Ca/P colocalization, thus promoting bone calcification.

An ultrastructural study on the multinucleation process of mouse alveolar macrophages induced by 1 alpha,25-dihydroxyvitamin D3.

The multinucleation process of isolated alveolar macrophages induced by 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] was examined using a scanning electron microscope (SEM) and a transmission electron microscope (TEM). At the beginning of culture, most of the macrophages were spherical in shape. During incubation with 1.2 X 10(-8) M 1 alpha,25(OH)2D3, spreading macrophages appeared among the spherical macrophages, and they increased in number. Spreading macrophages extended many cytoplasmic processes toward adjacent macrophages, and interdigitations of these processes between those of neighboring cells were often seen. Two types of cell contact have been observed in the 1 alpha,25(OH)2D3-treated cells. In some, cytoplasmic processes were put into the cytoplasm of the adjacent cells, where clathrinlike structures were observed at the inner membrane of the concave portion. In others, spreading macrophages occasionally came in contact with adjacent cells by a peripheral rim of their cytoplasm with gap junctions. Cytoplasmic continuity was rarely observed at the boundaries between the closely associated cells. The two types of cell contact were also found, though not frequently, in the untreated cells. These results indicate that 1 alpha,25(OH)2D3 promotes multinucleation of alveolar macrophages through spreading forms with the formation of gap junctions and the coated membrane invagination.

Ultrastructure of human skin by a rapid freezing technique: structural preservation and antigenicity.

To investigate the natural fine structure of skin, human skin biopsies were studied by electron microscopy and immunoelectron microscopy using rapid freezing and freeze-substitution techniques. Well-preserved structures were observed within a depth of 20 microns from the slammed surface of the samples. Epidermal cells were always closely located with very narrow intercellular spaces. Keratin filaments were distributed densely and homogeneously without forming bundles in the cytoplasm of epidermal keratinocytes. Langerhans cell granules were composed of a flat vesicular portion and an oval rod portion. The most striking finding was in the dermal-epidermal junction, where the lamina densa was present but no lamina lucida was observed. Further, by immunoelectron microscopy with anti-keratin monoclonal antibody, the 20-microns-deep part of the samples revealed the most significant staining. These findings indicate that the ultrastructure of routinely processed skin samples may be modified during the procedure for conventional chemical fixation and dehydration and that the ultrastructurally most preserved specimens, reflecting the conditions close to natural in vivo structures of the skin, can be obtained by the techniques described in this paper.

Structural analysis of the chloroplastic and cytoplasmic aldolase-encoding genes implicated the occurrence of multiple loci in rice.

The genes AldP and AldC-a, encoding the rice chloroplastic (cp) and cytoplasmic (ct) types of aldolase, respectively, were isolated and sequenced, and their transcription start points (tsp) were determined. Organization of the two genes was found to differ greatly; AldP consisted of six exons while AldC-a consisted of two exons. The deduced amino acid (aa) sequence of AldP contained a cp stromal targeting signal, followed by a sequence that matches the experimentally determined N-terminal sequence of mature AldP. The two enzymes share only 55% aa identity. However, rice AldP had about 73% homology with the cp aldolase of spinach. Also, the homology of AldC-a with maize, spinach and Arabidopsis thaliana cytoplasmic aldolases ranged from 70 to 90%. Southern blot analyses indicated that AldP is encoded at a single locus, whereas the gene encoding the ct counterpart is distributed at three loci on the genome. This feature is quite different from those of maize and spinach, in which only one locus was found for the ct aldolase.

A factor protecting mammalian [75Se]SeCys-tRNA is different from EF-1 alpha.

In Escherichia coli, an elongation factor (EF-Tu-like) specific to SeCys-tRNA, SELB, has been identified; however, a mammalian counterpart of SELB has not been reported to date. We searched for and found this factor in bovine liver extracts using the assay of [75Se]SeCys-tRNA protecting activity against alkaline hydrolysis (SePF activity). We found SePF activity in the protein extracts of the precipitate (microsomal fraction) collected at 150,000 x g from bovine liver. The proteins were separated by Sephacryl S-300 chromatography, and the SePF and EF-1 alpha activities were found in the same fraction, indicating that SePF and EF-1 alpha have the same molecular mass (approximately 50 kDa). We then chromatographed this active fraction using CM-Sephadex C-25 columns. The SePF activity was eluted after the peak of EF-1 alpha activity. This result indicated that this SePF activity was not dependent on EF-1 alpha. In addition to performing these two chromatographies, we investigated pure EF-1 alpha from Bombyx mori but could not detect any SePF activity in B. mori EF-1 alpha. Thus we showed that the SePF activity in bovine liver differs from that of EF-1 alpha in eukaryotes. Therefore the factor protecting [75Se]SeCys-tRNA in bovine liver is not EF-1 alpha and must be a SELB-like factor.

Cloning and sequencing of the cDNA encoding rice elongation factor 1 beta'.

A cDNA clone coding for rice elongation factor 1 beta' (EF-1 beta') was isolated from a rice anther cDNA library. The clone, named RB', was 980 bp long and contained a single open reading frame coding for 223 amino acids; the first 31 amino acids, except for the first methionine, which is absent in the mature protein, are identical to those of the purified protein determined with a protein sequencer. The amino acid sequence of rice EF-1 beta' shows homology to the C-terminal half of Artemia salina EF-1 beta (59%) and human EF-1 beta (63%), but might not have a phosphorylation site for casein kinase II which has been conserved in Artemia saline EF-1 beta and EF-1 delta, human EF-1 beta and silkworm EF-1 beta'.

Isolation and characterization of a rice cDNA encoding the gamma-subunit of translation elongation factor 1B (eEF1Bgamma).

We isolated a rice cDNA clone (refg) encoding the gamma-subunit of translation elongation factor 1B (eEF-1B gamma; the old designation was EF-1 gamma). The refg encodes an open reading frame of 419 amino acids which shows a similarity to the equivalent sequences from animals and yeast. Complex formation analysis, which showed the recombinant protein of refg (His-eEF1B gamma) and formed a complex with GST-eEF-1Bbeta, indicated that the refg encodes rice eEF1B gamma of the eEF1B alphabeta gamma complex. Expression analysis showed that refg mRNA is very abundant in suspension-cultured cells during the exponential phase of growth. A DNA blot analysis indicated that refg is located at a single locus in the rice genome.

Cloning and characterization of the cDNA encoding rice elongation factor 1 beta.

We have cloned and sequenced a cDNA coding for rice elongation factor 1 beta (EF-1 beta). The clone was 1420 bp long and contained an open reading frame coding for 229 amino acids. The overall identity between rice EF-1 beta and rice EF-1 beta' [Matsumoto, S., Oizumi, N., Taira, H. and Ejiri, S. (1992) FEBS Lett. 311, 46-48] is 60% at the amino acid sequence level; a higher percent identical residues (81%) were especially observed in the C-terminal region. Rice EF-1 beta has no conserved phosphorylation site for casein kinase II and no leucine zipper motif, although these motifs are well conserved in EF-1 delta (= beta in plants) subunits of animal EF-1.

Comparison of the effects of intermittent and continuous administration of human parathyroid hormone(1-34) on rat bone.

We compared the effects of synthetic human parathyroid hormone (1-34) (hPTH[1-34]) on rat bones when administered by intermittent injection and by continuous infusion for 4 weeks. Continuous infusion of hPTH(1-34) caused a dose-dependent decrease in the dry weight of the femur and a hyperparathyroidism-like condition in the high-dose group (214 ng/kg/h). In this group, although bone morphology was quite abnormal, the metaphyseal trabecular bone volume increased, whereas the epiphyseal trabecular bone volume tended to decrease, enhancing both bone resorption and formation. The diaphyseal cortical bone of the tibia became markedly porous, and this appeared to be the main cause of the decrease in total bone weight. Conversely, the dry weight of the femur increased in a dose-dependent manner in the intermittent-injection groups. Neither porosity of the cortical bone nor abnormal morphology of the trabecular bone was observed. The volume of the metaphyseal trabecular bone increased while retaining its normal morphology. The epiphyseal trabecular bone formation obviously increased without increase in the number of osteoclasts. In other words, it was postulated that the total weight of bone increased because intermittent injection of hPTH(1-34) increased the trabecular bone volume without loss of the cortical bone volume. These results show that either mode of treatment with hPTH(1-34) continuously accelerated bone formation, whereas the continuous infusion was essential for persistently and markedly enhanced acceleration of bone resorption. It appears that the increase in bone volume with intermittent injection of hPTH(1-34) resulted from the prominent manifestation of its bone-formative action rather than from its bone-resorptive action.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactive localization of transforming growth factor-beta type II receptor-positive cells in rat tibiae.

To identify the target cells of transforming growth factor-beta (TGF-beta) in normal bone tissue, we examined TGF-beta type II receptor expression using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and in situ hybridization in young rat tibiae. In the epiphyseal growth plate, the TGF-beta type II receptor cDNA was detected by RT-PCR and, alternatively, the TGF-beta type II receptor protein and mRNA expression were observed in the chondrocytes in the lower part of the proliferative cell layer, and in maturative and hypertrophic cell layers by immunohistochemistry and in situ hybridization. Of these, proliferative and maturative chondrocytes, in particular, revealed strong mRNA expression. In the cortical bone area, immunoreactivity for the TGF-beta type II receptor was detected in the fibroblastic cells near the osteoblasts on the endosteal surface of cortical bone. In conclusion, our findings suggest that target cells of TGF-beta in normal bone tissue could be considered mainly as extracellular matrix-producing chondrocytes and undifferentiated preosteoblasts, and TGF-beta may affect matrix production and differentiation of these cells.

Changes of cancellous bone mass in rat mandibular condyle following ovariectomy.

Changes in cancellous bone of the rat mandibular condyle following estrogen deficiency were histomorphometrically examined with 120-day-old female Fischer rats. Sixty-four animals were either ovariectomized bilaterally (ovx) or subjected to sham surgery (sham), and eight from each group were killed at 7, 14, 30, and 60 days after surgery. Seven intact animals were killed on day 0. Before killing, tetracycline and calcein were administered to all animals. Following histological observation, bone histomorphometry of the mandibular condyle was done using a confocal laser scanning microscope and an image analyzer. The sampling site was divided into two regions for analysis: (1) a "subchondral region," formed by the region connected to cartilage; and (2) a "central region," formed by the region beneath the former. The changes in these two regions were analyzed separately. In the sham group's condyle, the bone volume of the subchondral and central regions increased with the passage of time, although the bone turnover became low. This bone gain could be due to the effects of growth and the mechanical stimulus by occlusal load. In the subchondral region of the ovx group's condyle, the bone volume decreased significantly at 7 days, but recovered to reach approximately the same value as the sham group from 14 days onward. In the central region of the ovx group's condyle, the bone volume was unchanged, but revealed a significantly lower value than that of the sham group at 60 days (p < 0.01). Thus, ovariectomy inhibited bone gain, which was observed in the sham group's condyle even though there was no bone loss. On the other hand, the trabecular separation in the ovx's condyle of both the subchondral and central regions increased considerably and small marrow cavities interconnected to form a large bone marrow. Therefore, the ovx rat mandibular condyles dynamically altered their structures under the effects of estrogen deficiency and occlusal loads. Consequently, estrogen deficiency induced transient subchondral bone loss and recovery, whereas, in the central region, it inhibited bone gain. This suggests that mechanical loading modulates the normal ovx-induced bone loss found in other parts of the skeleton.

Application of confocal laser scanning microscopy to the observation of bone biopsy specimens.

Iliac bone biopsy specimens from five patients with endstage renal disease were observed by confocal laser scanning microscopy. Images of affected bone specimens were accurately focused, whereas images viewed with conventional light microscopy from thick ground sections were obscure. Especially at high magnifications, fine structures of bone cells, otherwise blurred, were clearly observed with confocal scanning microscopy. From thin-cut sections, images satisfactory for pathological diagnosis were viewed with light microscopy even at high magnification. However, the sections tended to shrink vertically compared with the cross-sectional images of the blocks observed directly by confocal laser scanning microscopy. A three-dimensional image of bone tissue was also constructed from serial optical sections. Confocal laser scanning microscopy is a useful technique for observing bone tissues, and may become essential for the evaluation of bone biopsy specimens.

17 beta-estradiol increases calcium content in fetal mouse parietal bones cultured in serum-free medium only at physiological concentrations.

Using a bone organ culture system that shows mineralization in vitro, we investigated whether 17 beta-estradiol dose-dependently increases calcium content in cultured calvarial bones in serum-free medium. Fetal mouse parietal bones (3 x 3 mm) were cultured in phenol red-free BGJ medium containing phosphate (3-4 mmol/L), calcium (1-1.25 mmol/L), insulin (6 micrograms/ML), and transferrin (6 micrograms/mL) for 4-5 days. Under these culture conditions, the calcium content of the cultured bones (at dissection 34.0 +/- 4.6 micrograms/bone [mean +/- SD], n = 50) increased by 15-20 micrograms during 4-5 days of culture. 17 beta-Estradiol increased the calcium content significantly at 10(-12) to 10(-11) mol/L, but not at lower (10(113) mol/L) or higher (10(-10) to 10(-9) mol/L) concentrations. 17 alpha-Estradiol had no effect. The stimulatory effect of 17 beta-estradiol was completely inhibited by the antiestrogen agent ICI-182,780. The anabolic effect of 17 beta-estradiol was elicited not only in bones from females but also in those from males. 17 beta-Estradiol had no significant effect on 45Ca release from prelabeled parietal bones. Furthermore, light- and electron-microscopic examinations revealed that bone mineralization proceeded through formation of matrix vesicles, without any metastatic or dystrophic calcification. These in vitro findings suggest that 17 beta-estradiol elicits small, but reproducible, direct effects on calcium content in the parietal bones not only in female but also in male fetal mice at physiological-free E2 concentrations (10(-12)-10(-11) mol/L), which is attainable in serum of normal human subjects. In contrast to in vivo studies, pharmacological doses of 17 beta-estradiol had no anabolic effect on parietal bones. The mechanism of such a biphasic effect of estrogens remains to be elucidated.

Reduction in bone formation and elevated bone resorption in ovariectomized rats with special reference to acute inflammation.

Changes in bone modeling and remodeling in the tibia of growing rats within 30 days of ovariectomy (ovx) were evaluated by histomorphometric, mechanical; and biochemical means. Three days after ovx, suppressed bone formation was seen. This was shown by reduced osteoid volume, osteoblast surface, and bone formation rate in the secondary spongiosa, and a reduced longitudinal growth rate in the growth plate. In addition, the alkaline phosphatase and tartrate-resistant acid phosphatase activity in bone marrow supernatants was suppressed in conjunction with elevated serum sialic acid levels, indicating inflammation. Although estrogen deprivation itself may provoke the inflammatory process, the serum sialic acid level in the ovx group returned to the baseline level within 5 days after surgery, while that of estradiol in the ovx group remained consistently lower. This suggests that surgical stress, not estrogen deprivation, is the primary cause of the inflammatory response shortly after ovx. A significant difference (p < 0.01) between the ovx and sham rats was seen in the osteoclast surface, which peaked on day 7 in the ovx rats. On day 14 postovariectomy, the bone formation rate peaked and remained constant until day 30. In the ovx rats, there was a sustained reduction in the serum albumin level until day 30. Estrogen deprivation may be the primary cause of these changes, because both surgical ovx and medical oophorectomy with gonadotropin-releasing hormone agonist (G(nRHa) reduce the serum albumin level. In numerous studies dealing with changes after ovx in rats, we have observed: 1) a transient reduction in bone formation in relation to inflammatory changes evoked by ovx surgery, and 2) a sustained reduction in the serum albumin level for at least 30 days after ovx that is possibly due to estrogen deprivation.

Cellular distribution of thrombomodulin as an early marker for warm ischemic liver injury in porcine liver transplantation: protective effect of prostaglandin I2 analogue and tauroursodeoxycholic acid.

BACKGROUND: Warm ischemia of the graft from non-heart-beating donors is considered a risk factor for posttransplant graft dysfunction. The early administration of cytoprotective agents may help improve graft dysfunction. METHODS: Four groups of 10 pigs each underwent orthotopic liver transplantation. Prostaglandin I2 analogue, OP-41483, was administered intraportally 30 min before warm ischemic insult in donors and after reperfusion in recipients in one group. In the other study group, additional intravenous tauroursodeoxycholic acid (TUDC) was given before the warm ischemic insult in donors and after reperfusion, then maintained continuously until postoperative day (POD) 7. RESULTS: Exposure of liver grafts to warm ischemia resulted in severe congestion with the disappearance of thrombomodulin (Tm) from the sinusoidal endothelial cells (SECs) and smooth muscle cells (SMCs) around biliary epithelial cells (BEpCs) 2 hr after reperfusion, followed by positive immunoreactivity of Tm in BEpCs with hyperbilirubinemia, which was related to high mortality. Combined administration of OP-41483 and TUDC had a protective effect, demonstrated by sustained immunoreactivity of Tm from SECs and SMCs until POD 7, without that reactivity in BEpCs. This was associated with reduced congestion and hyperbilirubinemia, similar to the control group not subjected to warm ischemia. CONCLUSIONS: These findings suggest that negative immunoreactivity of Tm in SECs and SMCs surrounding BEpCs and positive in BEpCs may be an early marker for ischemic liver injury, and that OP-41483 and TUDC may protect against the microcirculatory and biliary derangement.