Search

Quantitative ultrasound (QUS) is now widely used for evaluating bone in vivo, because obtained ultrasonic wave properties directly reflect the visco-elasticity. Bone tissue is composed of minerals like hydroxyapatite (HAp) and a collagen matrix. HAp crystallites orientation is thus one parameter of bone elasticity. In this study, we experimentally investigated the anisotropy of ultrasonic wave velocity and the HAp crystallites orientation in the axial-radial and axial-tangential planes in detail, using cylindrical specimens obtained from the cortical bone of three bovine femurs. Longitudinal bulk wave propagation was investigated by using a conventional ultrasonic pulse system. We used the one cycle of sinusoidal pulse which was emitted from wide band transmitter. The nominal frequency of the pulse was 1MHz. First, we investigated the anisotropy of longitudinal wave velocity, measuring the anisotropy of velocity in two planes using cylindrical specimens obtained from identical bone areas. The wave velocity changed due to the rotation angle, showing the maximum value in the direction a little off the bone axis. Moreover, X-ray pole figure measurements also indicated that there were small tilts in the HAp crystallites orientation from the bone axis. The tilt angles were similar to those of the highest velocity direction. There were good correlations between velocity and HAp crystallites orientation obtained in different directions. However, a comparatively low correlation was found in posterior bone areas, which shows the stronger effects of bone microstructure. In the radial-tangential plane, where the HAp crystallites hardly ever align, weak anisotropy of velocity was found which seemed to depend on the bone microstructure., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In amphibians, calcium carbonate crystals are present in the endolymphatic sac and the inner ear. The formation of these crystals is considered to be facilitated by a protein called otoconin-22. We examined the spatial and temporal expression of otoconin-22 during the development of the bullfrog (Rana catesbeiana) using RT-PCR, in situ hybridization (ISH), and immunofluorescence techniques. By RT-PCR, otoconin-22 mRNA was first detected in embryos at Shumway stage 20, and this expression pattern continues in late stages. The first otoconin-22 mRNA-positive reaction was detected in stage 22 embryos in the placode of the endolymphatic sac. Otoconin-22 protein was observed in the epithelial cells of the endolymphatic sac at stage 24. On the other hand, a whole-mount ISH technique showed the first expression of otoconin-22 mRNA in the inner ear, in addition to the endolymphatic sac, at the mid-phase of Shumway stage 25. We discuss the role of otoconin-22 in the formation of calcium carbonate crystals in the endolymphatic sac and inner ear.

We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the ultimobranchial gland of the adult bullfrog using immunohistochemical (IHC) and in situ hybridization (ISH) techniques. In the ultimobranchial gland, PC1-immunoreactive cells were columnar, and were present in the follicular epithelium. When serial sections were immunostained with anti-calcitonin, anti-CGRP, anti-PC1, and anti-PC2 sera, PC1 was found only in the calcitonin/CGRP-producing cells. No PC2-immunopositive cells were detected. In the ISH, PC1 mRNA-positive cells were detected in the follicle cells in the ultimobranchial gland. No PC2 mRNA-positive cells were detected. RT-PCR revealed expression of the mRNAs of PC1 and the PC2 in the ultimobranchial gland. However, very little of the PC2 mRNA is probably translated because no PC2 protein was detected either by IHC staining or by Western blotting analysis. We conclude that the main prohormone convertase that is involved in the proteolytic cleavage of procalcitonin in the bullfrog is PC1.

We cloned cDNAs encoding PC1 and PC2 from a cDNA library constructed for the anterior pituitary gland of the bullfrog (Rana catesbeiana) and sequenced them. The bullfrog PC1 cDNA consisted of 2972 base pairs (bp) with an open reading frame of 2208 bp and encoded a protein of 736 amino acids, including a putative signal peptide of 26 amino acids. The protein showed a high homology to R. ridibunda PC1 (95.1%) and mammalian PC1 (72.6%). The bullfrog PC2 cDNA consisted of 2242 bp with an open reading frame of 1914 bp and encoded a protein of 638 amino acids, including a putative signal peptide of 23 amino acids. This protein showed a high homology to R. ridibunda PC2 (95.5%) and mammalian PC2 (84.8%). The catalytic triad of serine proteinases of the subtilisin family was found at Asp-168, His-209, and Ser-383 in the PC1 protein and at Asp-167, His-208, and Ser-384 in the PC2 protein. In situ hybridization staining revealed that PC2 mRNA was detected in corticotrope cells of the tadpoles, but not in those of the adults. In the adult, only PC1 mRNA was detected in the pars distalis but both PC1 and PC2 mRNAs were detected in the pars intermedia. The data also showed that PC1 mRNA was expressed in gonadotrope cells.

Anuran amphibians have a special organ called the endolymphatic sac (ELS), containing many calcium carbonate crystals, which is believed to have a calcium storage function. The major protein of aragonitic otoconia, otoconin-22, which is considered to be involved in the formation of calcium carbonate crystals, has been purified from the saccule of the Xenopus inner ear. In this study, we cloned a cDNA encoding otoconin-22 from the cDNA library constructed for the paravertebral lime sac (PVLS) of the bullfrog, Rana catesbeiana, and sequenced it. The bullfrog otoconin-22 encoded a protein consisting of 147 amino acids, including a signal peptide of 20 amino acids. The protein had cysteine residues identical in a number and position to those conserved among the secretory phospholipase A(2) family. The mRNA of bullfrog otoconin-22 was expressed in the ELS, including the PVLS and inner ear. This study also revealed the presence of calcitonin receptor-like protein in the ELS, with the putative seven-transmembrane domains of the G protein-coupled receptors. The ultimobranchialectomy induced a prominent decrease in the otoconin-22 mRNA levels of the bullfrog PVLS. Supplementation of the ultimobranchialectomized bullfrogs with synthetic salmon calcitonin elicited a significant increase in the mRNA levels of the sac. These findings suggest that calcitonin secreted from the ultimobranchial gland, regulates expression of bullfrog otoconin-22 mRNA via calcitonin receptor-like protein on the ELS, thereby stimulating the formation of calcium carbonate crystals in the lumen of the ELS.

Previously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A(2) (PLA(2)) from a bullfrog pituitary fraction obtained during the purification of thyrotropin (TSH). Subsequently, partial amino acid sequence (N-terminal 45 amino acid residues) analysis revealed this protein to be identical to the N-terminal amino acid sequence of otoconin-22, the major protein of aragonitic otoconia in the Xenopus saccule. In this study we developed an antibody against the N-terminal peptide of the bullfrog protein and applied it for immunocytochemical study of the pituitary and its surrounding tissue. Western blotting analysis showed that this antibody recognizes a 20.4-kD protein that has a molecular mass close to that of otoconin-22. Immunohistochemical reaction with the antibody was not found in any anterior pituitary cells but was intense in the monolayer epithelial cells of the endolymphatic sac surrounding the pituitary gland, which is a major storage site of calcium carbonate in amphibians. An electron microscopic study revealed that the cuboidal cells in the endolymphatic sac contained large, polymorphic secretory granules in their apical cytoplasm. Immunogold particles indicating the presence of a PLA(2)-like protein were observed predominately in these secretory granules. These findings support the view that this PLA(2)-like protein obtained during purification of TSH was derived from the endolymphatic sac adhering to the pituitary and that this protein is a bullfrog otoconin. (J Histochem Cytochem 49:631-637, 2001)

Prohormone convertases PC1 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. There is a report that prohormone convertase exists in the rat anterior pituitary gonadotrophs, where it had previously been considered that proprotein processing does not take place. In addition to luteinizing hormone and follicle-stimulating hormone, rat pituitary gonadotrophs contain chromogranin A (CgA) and secretogranin II (SgII), two members of the family of granin proteins, which have proteolytic sites in their molecules. In the present study we examined whether there is a close correlation between subcellular localization of prohormone convertases and granin proteins. Ultrathin sections of rat anterior pituitary were immunolabeled with anti-PC1 or -PC2 antisera and then stained with immunogold. Immunogold particles for PC1 were exclusively found in large, lucent secretory granules, whereas those for PC2 were seen in both large, lucent and small, dense granules. The double-immunolabeling also demonstrated colocalization of PC2 and SgII in small, dense granules and of PC1, PC2, and CgA in large, lucent granules. These immunocytochemical results suggest that PC2 may be involved in the proteolytic processing of SgII and that both PC1 and PC2 may be necessary to process CgA.

Patients with late stage gynecologic malignancies occasionally develop massive pelvic hemorrhage, and management of the hemorrhage is often difficult. Transcatheter arterial embolization with an absorbable gelatin sponge following the Seldinger method was performed to control hemorrhage in five patients with cancer of the uterine cervix. Pelvic arteriograms of five patients showed no further extravasation and their bleeding ceased. No patients died of pelvic hemorrhage, and all of them eventually died as a result of the original disease within two years of the procedure. As for complications of this procedure, slight fever (3/5) and minimal lumbar pain (2/5) were noticed, which were easily controlled by an indomethacin suppository. Based on these findings, this therapeutic embolization method proved to be useful in the management of massive pelvic hemorrhage in patients with cervical cancer.

Pituitary dysfunction occasionally results from brain tumors or the surgical resection of brain tumors. The authors examined two patients with hypogonadotropic secondary amenorrhea, who had undergone surgical removal of brain tumors. Changes in immunoreactive gonadotropin-releasing hormone (GnRH) secretion are of interest in patients with a gonadotropin and gonadal steroid deficit, because both steroid and pituitary feedback systems are altered by tumors or tumor resection. The authors thus measured GnRH, luteinizing hormone, and follicle-stimulating hormone levels every 15 minutes for 4 hours by radioimmunoassay and investigated qualitative and quantitative changes in the pulsatile patterns of these hormones in two hypogonadotropic hypogonadism patients. They also performed similar multiple measurements of GnRH in two normal cycle women in follicular phase and two postmenopausal women. The concentration of plasma GnRH in two hypopituitarism patients was compared with that in two normal cycle women and two postmenopausal women. The study showed that the peripheral blood level of GnRH was significantly lower in two hypopituitarism patients than in both normal cycle and postmenopausal women, and that the pulsatile frequency was not different among these three groups. These findings suggest that alteration of feedback systems results in a decrease in the blood level of GnRH, and that pulses of GnRH maintain normal fluctuation despite the alteration of the hormonal circumstances in two hypogonadotropic hypogonadism patients.

Macrophage colony-stimulating factor (M-CSF) is a characteristic cytokine that plays an essential role in placenta maintenance, and thrombin-antithrombin III complex (TAT) is a hemostatic marker that is remarkably altered both in normal pregnancy and in preeclampsia. The present study was designed in order to show various levels of M-CSF and TAT in pregnancies. Peripheral blood was collected from 49 subjects, of whom 31 were normal pregnant women consisting of the four groups (namely 10th, 20th, 30th, and 38th weeks of gestation), 13 were preeclamptic pregnant women (37th week of gestation; mean blood pressure, 158/99 mm Hg), and 5 were nonpregnant controls. We compared blood M-CSF and TAT levels among them. Results showed that blood M-CSF and TAT levels increased significantly with gestational age. Furthermore, the ratio of increase in M-CSF was significantly lower than that in TAT in normal pregnant women compared with controls. In contrast, the ratio of increase in M-CSF was significantly higher than that in TAT in preeclamptic women compared with normal pregnant women. These results concerning the ratio of increase in M-CSF and TAT have not been reported. These findings show that M-CSF level increases significantly in preeclampsia even in its earlier stage, exhibiting a systolic blood pressure of less than 160 mm Hg.

The subject was a 29-year-old pregnant woman whose fetuses were a conjoined twin. We diagnosed the fetuses as a conjoined twin by ultrasonography at the 12th week of gestation. The patient and her family wanted living infants and hoped that they were separated surgically. We consulted with the doctors of pediatric surgery and cardiovascular surgery departments. At the 18th week of gestation, the fetuses died in utero and they were delivered transvaginally by labor induction. Both infants were female with 430 g in total body weight and both of them were 21 cm in length. Pathological findings were thoracogastro-pagus with a single heart, a single pair of liver and a single small intestine.

Background: Cisplatin induces nephrotoxicity and this study evaluated the protective effect of fosfomycin on it in 11 gynecological cancer patients., Methods: The N-acetyl-beta-D-glucosaminidase (NAG), beta 2-microglobulin (beta 2MG), creatinine (uCr) and total protein (TP) levels in a 24-hour urine specimen as well as the blood urea nitrogen (BUN) and serum creatinine (sCr) were measured before and after CAPF chemotherapy alone (control) or with fosfomycin., Results: The results were statistically analyzed by using the t-test. NAG, beta 2MG, uCr and TP levels increased significantly after chemotherapy in the control patients, but BUN and sCr levels did not change significantly. The NAG level in the control group was twice as high as in the fosfomycin group 8 days after chemotherapy (p < 0.01). The uCr and TP in control patients increased significantly after chemotherapy when compared to those in patients coad-ministered fosfomycin. There were no significant changes in beta 2MG, BUN and sCr levels., Conclusions: Cisplatin affected the levels of NAG, beta 2MG, uCr and TP without influencing BUN and sCr levels. Fosfomycin, therefore, may be useful as a supplemental treatment for reducing cisplatin nephrotoxicity, especially proximal tubular damage.

Effects of various types of protein kinase inhibitor on the adhesion and spreading of BALB/c mouse 3T3 cells and on the phosphorylation and stability of focal adhesion kinase (FAK) in the cells were studied. Inhibitors of protein tyrosine kinases, methyl 2,5-dihydroxycinnamate and herbimycin A, inhibited tyrosine-phosphorylation of FAK and the adhesion of 3T3 cells to fibronectin. Among inhibitors of serine/threonine kinases tested, calphostin C, a specific inhibitor of protein kinase C, inhibited cell spreading rather than cell adhesion, and it induced the decrease of intracellular FAK within 30 min. Inhibitors of tyrosine kinase, A kinase, G kinase, and myosin light chain kinase did not induce such a rapid and specific decrease of FAK. When calphostin C (20 microM) was added to sub-confluent monolayer cultures, serine-phosphorylation of FAK was inhibited by 67% within 2 h, and decrease in the amount of FAK and rounding up of the cells began after 4 h. Label-chase experiments indicated that about 60% of 35S-labeled FAK degraded within 1-2 h after addition of calphostin C to monolayer cultures. These results indicated that serine-phosphorylation of FAK induced by protein kinase C was important in the regulation of metabolic stability of FAK.