bluuebunny/crossref_metadata_2025 huggingface dataset:
A New Electrochemical Method for the Determination of Proteins with Cupferron‐Cadmium(II) Complex
Authors: Hui Ni , Wang Jiasheng , Sun Wei
Published: February 2010

A new electrochemical method was proposed for the determination of trace amounts of proteins based on the cupferron (Cup) and cadmium(II) complex [Cup‐Cd(II)] as the voltammetric probe. In the selected pH 6.5 Britton–Robinson (B–R) buffer solution, Cup can interact with Cd(II) to form a stable complex of [Cup‐Cd(II)], which had a sensitive linear sweep voltammetric reductive peak at −0.654 V (vs. SCE). The addition of human serum albumin (HSA) into [Cup‐Cd(II)] complex solution could greatly decrease the reductive peak current without the change of the reductive peak potential, which indicated that HSA could interact with [Cup‐Cd(II)] complex to form a supramolecular biocomplex. The interaction mechanism was discussed and the decrease of reductive peak current was proportional to the concentration of HSA, which could be further used for the proteins determination. The optimal conditions of the binding reaction and the electrochemical detection were carefully investigated. Under the optimal conditions a new quantitative determination method for different kinds of proteins such as HSA, bovine serum albumin (BSA) and bovine hemoglobin (BHb) etc. was developed. The proposed method was simple, practical and relatively free from the interferences of coexisting substances, and it was further applied to the samples determination with satisfactory results. The binding constant (βs) and the binding number (m) of HSA with [Cup‐Cd(II)] complex were calculated by the voltammetric data with the results as βs=1.12×106 and m=1.
Electrochemical Redox Behavior of Omeprazole Using a Glassy Carbon Electrode
Authors: Jorge S. M. A. , Pontinha A. D. R. , Oliveira‐Brett A. M.
Published: February 2010

The electrochemical redox behavior of omeprazole (OMZ), a gastric acid pump inhibitor, was investigated at a glassy carbon electrode using cyclic, differential pulse and square‐wave voltammetry over a wide pH range. The pH‐dependent oxidation occurs in two irreversible consecutive charge transfer reactions. Adsorption of the nonelectroactive product was also observed. The first oxidation involves removal of one electron, followed by deprotonation and leads to the formation of a hydroxylated species. The second oxidation process is related to the hydroxyl and amino groups in the benzimidazole moiety. The reduction is irreversible, also pH‐dependent, and occurs in a single step at the sulfoxide group in a diffusion‐controlled mechanism. The diffusion coefficient of omeprazole was calculated to beDOMZ=2.31×10−6 cm2s−1.
Direct Electrochemistry of Glucose Oxidase on Nail‐like Carbon and Its Biosensing for Glucose
Authors: Li Kuangtian , Liu Bin , Zheng Jianbin , Sheng Qinglin , Liu Ruixiao
Published: February 2010

Nail‐like carbon (NLC) was synthesized by a simple hydrothermal method. It was the first time that a novel electrochemical biosensing of glucose was explored based on the glucose oxidase (GOx)‐NLC‐chitosan (CHIT) glassy carbon electrode. Morphology and structure of NLC were characterized by scanning electron microscope; meanwhile the chemical composition was determined by X‐ray diffraction and energy dispersive X‐ray spectroscopy. The cyclic voltammetry of immobilized GOx showed a pair of quasireversible redox peaks with the formal potential (E°′) of −0.458 V and the peak‐to‐peak potential separation was 47 mV at a scan rate of 100 mV s−1. The present biosensor has a linear range of glucose from 0.02 to 1.84 mM (correlation coefficient of 0.9991) and detection limit of 0.01 mM (S/N=3). Compared with the previous reports based on the carbon material biosensor, it has a high sensitivity of 165.5 μA mM−1 cm−2 and low apparent Michaelis–Menten constant of 0.506 mM. Thus, the NLC may have potential applications in the field of bioelectrochemistry, bioelectronics and biofuels.
Improved Performance of an Amperometric Biosensor with Polydiaminonaphthalene on Electrochemically Deposited Au Nanoparticles
Authors: Noh Hui‐Bog , Kumar Pankaj , Biswas Tapan Kumar , Kim Duk‐Soo , Shim Yoon‐Bo
Published: February 2010

The performance of an enzyme sensor fabricated through covalent bond formation on the HRP‐bonded poly(1,8‐diaminonaphthalene) (polyDAN) layer with gold nanoparticles (AuNPs) was applied to catalyze the electrochemical reduction of H2O2. The surface characteristics of the sensor probe were studied using cyclic voltammetry, SEM, XPS, QCM, and impedance spectroscopy. The AuNP‐deposited surface resulted in higher conductivity and sensitivity for H2O2 detection in phosphate buffer solution. A linear calibration plot was obtained in the H2O2 concentration range between 10.0 μM and 25.0 mM with detection limit 5.0±1.25 μM. The lifetime of HRP/polyDAN/AuNP/GC probe was over 70 days without response loss.
Character impact odorants from wild mushroom (Lactarius hatsudake) used in Japanese traditional food
Authors: Miyazawa Mitsuo , Kawauchi Yumi , Matsuda Naoki
Published: February 2010

The components of the volatile oil from wild mushroom (Lactarius hatsudake), used in Japanese traditional food, were analysed and quantified for the first time by capillary GC and GC–MS. Seventy‐six components were separated from the oil and of these 71 components were identified. The main components of the oil were oxidized sesquiterpenes [cis‐isolongifolanone (624.9 μg/100 g), α‐cedrene epoxide (578.7 μg/100 g), humulene epoxide III (453.9 μg/100 g), clovane (425.4 μg/100 g)], aliphatic acids [linoleic acid (585.9 μg/100 g) and palmitoleic acid (333.3 μg/100 g)]. Odour evaluation of the volatile oil from L. hatsudake was also carried out using GC–MS/olfactometry (GC–MS/O) and aroma extract dilution analysis (AEDA), from which it was found that hexanal, 4‐dehydroviridiflorol, myliol and phenylacetaldehyde seem to contribute to the green, spicy and sweet odour of L. hatsudake. Copyright © 2010 John Wiley & Sons, Ltd.
Spectrally accurate method for analysis of stationary flows of second‐order fluids in rough micro‐channels
Authors: Mohammadi A. , Floryan J. M. , Kaloni P. N.
Published: February 2010

A spectral method for the analysis of stationary flows of second‐order fluids in rough micro‐channels is developed. The algorithm employs a fixed computational domain with the boundaries of the flow domain being located inside the computational domain. The physical boundary conditions are enforced using the immersed boundary conditions concept. The algorithm relies on the Fourier expansions in the flow direction and the Chebyshev expansions in the transverse direction. Various tests confirm spectral accuracy of the algorithm. Copyright © 2010 John Wiley & Sons, Ltd.
Stratigraphic separation diagrams as a tool for determining fault geometry in a folded and thrusted region: an example from the Barrandian region, Czech Republic
Authors: Knížek Martin , Melichar Rostislav , Janečka Jiří
Published: February 2010

The stratigraphic separation diagram (SSD) is an often neglected tool of fault analysis, which assists with the interpretation of both the fault‐surface geometry and the mechanism of faulting. As the SSD needs to evaluate displacement or separation along the fault, this particular method can be used only in areas with well‐documented stratigraphic sequences. This paper presents details of some common patterns obtained when using SSDs and also discusses some real examples of two apparently similar faults found in the Barrandian area in the Czech Republic. Although both the Prague and the Tachlovice faults in the Barrandian area seem to be of the same character (the same strike, remarkable stratigraphic separation, similar localization in the Prague Synform), the SSDs of these two faults demonstrate the difference in fault surface geometries and consequently a very distinct mechanism of origin. The Tachlovice Fault is one of the main thrusts in the Prague Synform. It has ramp‐flat geometry and is associated with fault‐related folds. The Prague Fault, on the other hand, was formed later, after the initial folding, and belongs to a group of steep translatory faults. It is shown how SSDs are practical, effective tools for the analysis of these types of faults and associated stratigraphies without the need of 3D seismics or boreholes. Copyright © 2010 John Wiley & Sons, Ltd.
SAR and pharmacophore models for the rhodanine inhibitors of Plasmodium falciparum enoyl‐acyl carrier protein reductase
Authors: Kumar Gyanendra , Banerjee Tanushree , Kapoor Neha , Surolia Namita , Surolia Avadhesha
Published: February 2010

Significance of type II fatty acid synthase pathway in the life cycle of malarial parasite has long been established. Enoyl acyl carrier protein (ACP) reductase of Plasmodium falciparum (PfENR) is the rate determining enzyme of its elongation module. Hence, PfENR has been a target for the development of antimalarials as well as vaccines. Towards this endeavour, we had recently identified rhodanine class of compounds as inhibitors of PfENR. Here, we report a number of new inhibitors belonging to this class. These inhibitors have been divided into two broad subclasses: rhodanine‐furans and rhodanine‐phenyls. The inhibitory activity of all compounds was determined against purified PfENR. IC50 of these compounds were found to be in nanomolar to low‐micromolar range. The structure–activity relationship of both the classes has been explored in detail for the first time. Separate 3D pharmacophore models for this enzyme have been generated for both rhodanine furans and phenyls. The pharmacophore model for rhodanine furan has a Hydrogen bond donor, two Hydrogen bond acceptors, two metal ligators, three hydrophobic, and two aromatic ring features, whereas the pharmacophore model for the phenyl subclass has two hydrogen bond donors, two hydrogen bond acceptor, a metal ligator, two hydrophobic, and two aromatic ring features. These models could be used for in silico screening of compound libraries for PfENR inhibitors. © 2010 IUBMB IUBMB Life, 62(3): 204–213, 2010
Giant cell formation in rabbit long-term bone marrow cultures: Immunological and functional studies
Authors: Dr. Horton M. A. , Rimmer E. F. , Chambers T. J.
Published: February 2010

A method for the long-term culture of rabbit newborn bone marrow has been developed. It is characterized by the rapid appearance of an adherent, adipocyte-containing stromal layer, proliferation of mature myeloid cells, and the formation of numerous, large multinucleate giant cells. By the combined use of morphological, immunological, and functional criteria these giant cells have been characterized as macrophage polykaryons and not osteoclastic giant cells. We conclude that long-term bone marrow culture in the rabbit favors the proliferation, maturation, and fusion of macrophage, but not osteoclast, precursors — new experimental models will have to be developed to enable the developmental biology of osteoclasts to be studied in the rabbit.
Activity of ornithine decarboxylase and creatine kinase in soft and hard tissue of vitamin D-deficient chicks following parenteral application of 1,25-dihydroxyvitamin D3 or 24R,25-dihydroxyvitamin D3
Authors: Ittel Thomas H. , Ross F. P. , Professor Norman Anthony W.
Published: February 2010

We investigated the stimulation of creatine kinase (CK) and ornithine decarboxylase (ODC) by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] in doses ranging from 1.625 to 6500 pmol in 4-week-old vitamin D-deficient chicks. Enzyme activities were monitored for 72 h. 1,25(OH)2D3 but not 24R,25(OH)2D3 enhanced the activity of ODC in duodenum and bone. The time course of ODC activity in bone was biphasic, with an increase after 1 h and a higher peak after 24 h. Diaphyses and epiphyses responded equally well after a dose of 6500 pmol. The kidney, liver, and lung showed 1.5–3.8-fold increase in CK activity following 1,25(OH)2D3, reaching a maximum between 3–5 h. However, sustained stimulation of CK activity could still be demonstrated after 72 h, and the 48-h levels in the lung even exceeded the 5-h values. No change of activity of either enzyme was noted in heart and brain after application of 1,25(OH)2D3. There was no coincidence of stimulation of ODC and CK by 1,25(OH)2D3 in the same tissue, and the dose-responsiveness of both enzymes differed considerably. Near maximum activities of ODC were achieved with 19.5 pmol 1,25(OH)2D3 in duodenum and pancreas, while maximum responses of CK occurred in the liver at 195 pmol and in lung and kidney at 6500 pmol. 24R,25(OH)2D3 failed to produce any consistent effects of either enzyme in all tissues examined. These results, particularly the lack of response to 24R,25(OH)2D3, are different from those reported in rats.
Renal adaptation to phosphorus deprivation: Characterization of early events
Authors: Dr. Levine Barton S. , Ho Letitia D. , Pasiecznik Karen , Coburn Jack W.
Published: February 2010

Renal adaptation to low dietary phosphorus (P) can occur within 4 h. To characterize events preceding adaptation, rats were gavaged with 0.6% P (+P) or 0.03% P (-P) diet and killed 1, 2, or 4 h later. Brush border membrane vesicles were prepared and Na-dependent phosphate (Pi), glucose, and 1-proline transport were measured. In intact rats, 1 h after gavage, serum P in +P was 8.0 ± 0.5 and in -P, 6.1 ± 0.4 mg/dl, p < 0.01. One and 2 h after gavage, Pi uptake was similar between groups; at 4 h, 0.25 min Pi uptake was increased by 59.3% ± 14.8 in -P, p < 0.02, n = 11. In thyroparathyroidectomized rats, Pi uptake increased in -P by 40.1% ± 7.4 compared to +P at 2.5 h after gavage, and by 51.3 ± 9.3 at 4 h, p < 0.025, n = 11. When actinomycin D or cycloheximide were administered both 16 h prior to and at gavage, 0.25 min Pi uptake 4 h after gavage was 59.1% ± 14 and 60.6% ± 19 higher in -P than +P, respectively, p < 0.025. The adaptation was detected only when measured with an inward-directed Na-gradient. Na-dependent glucose and proline uptakes were not changed by -P diet.

These studies demonstrate that early renal adaptation to low dietary P is preceded by a fall in serum P, is independent of parathyroid hormone, and does not require protein synthesis. Early adaptation may represent an increased rate of carrier movement or a change in availability of already synthesized carrier. The signal for adaptation may be the decrease in serum P, or filtered load of phosphorus.
Parathyroid hormone and calcitonin modify inositol phospholipid metabolism in fetal rat limb bones
Authors: Rappaport Mark S. , Dr. Stern Paula H.
Published: February 2010

Inositol-containing phospholipids are believed to be intimately involved in the first steps of cellular signalling by certain hormones and neurotransmitters. We examined whether parathyroid hormone (PTH) and calcitonin (CT), two hormones that affect bone physiology, would elicit changes in inositol-phospholipid metabolism in cultured bone. [3H]inositol readily entered into the tissue phospholipid pool in fetal rat limb bones, and incorporated into phosphatidylinositol (92.9%), phosphatidylinositol-4-P (4.5%), and phosphatidylinositol-4,5-P2 (2.6%). PTH enhanced the incorporation of inositol into PtdIns in limb bones following 2- or 24-h hormone treatments. The effect of PTH was dose dependent (EC50 of 0.3–0.4 nM) and occurred in a concentration range similar to that for hormone-stimulated bone resorption. In contrast, 24-h treatment with CT-inhibited inositol incorporation, also in a dose-dependent manner. Two-hour CT treatment had variable effects on labeling. CT inhibited the stimulatory effect of PTH at both 2 and 24 h. The effects induced by PTH and CT were specific for PtdIns and were independent of the [3H]inositol pool size. These results indicate that inositol-phospholipid turnover can be modified during the action of these hormones on bone tissue. Although the time course of hormone-stimulated inositol incorporation observed here is slower than that found in other tissues, the change in phosphatidylinositol metabolism could mediate delayed effects of PTH or CT. Alternatively, alterations induced by PTH and CT in bone cell membranes, cell populations, or in the mineralized matrix could conceivably result in secondary changes in phosphatidylinositol metabolism.
Skeletal response to dietary calcium in a rat model simulating weightlessness
Authors: Globus Ruth K. , Bikle Daniel D. , Halloran Bernard , Morey-Holton Emily
Published: February 2010

Unweighting the hindlimbs of a rat by tail suspension leads to a decrease in bone in the unweighted hindlimbs, but not in the normally weighted forelimbs. We evaluated whether increments in dietary calcium could prevent this. Growing rats were fed diets ranging in calcium content from 0.1% to 2.4%. After the rats were suspended for two weeks, we found no differences between suspended and control animals fed the same diet with respect to calcium transport or serum levels of calcium, phosphorus, 1,25-dihydroxyvitamin D, and parathyroid hormone. In both groups, increasing dietary calcium reduced active intestinal calcium transport and serum 1,25-dihydroxyvitamin D levels. The calcium content of the tibia and lumbar vertebra (but not the humerus) was reduced in suspended rats compared to control rats fed the same diet. However, increasing dietary calcium increased the calcium content of all bones in both suspended and control animals. The bone formation rate at the tibiofibular junction (measured by double-label tetracycline) was reduced in the suspended animals compared to controls and was not altered by dietary calcium. However, the marrow area of the tibia, an indication of bone resorption, did not differ between suspended and control animals and was equally reduced in both groups when dietary calcium was increased. Our data suggest that the deleterious effects of skeletal unweighting on bone formation cannot be explained by changes in the calciotropic hormones and are not reversed by increments in dietary calcium. However, increasing dietary calcium can increase bone calcium, even in unweighted limbs, by decreasing bone resorption.
Reduced bone mass in calcitonin-deficient rats whether lactating or not
Authors: Dr. Hirsch Philip F. , Hagaman John R.
Published: February 2010

Calcitonin deficiency was produced in lactating and age-matched nonmated rats by thyroidectomy (TX) after transplantation of the parathyroid glands to a thigh muscle. At the end of lactation and a comparable period in the nonlactating rats, this condition resulted in femurs, vertebrae, and tibiae that weighed less than those in the thyroid-intact controls. Furthermore, the femurs of the CT-deficient rats were narrower at midshaft and shorter, indicating reduced bone growth. The reduction in bone mass in CT-deficient rats, although highly significant, was much smaller than that caused by lactation.

Adequate thyroid hormone replacement therapy was provided by giving all the TX rats L-thyroxine (T4) sc or in the drinking water. The body weights of the lactating rats were heavier than those of their nonmated controls but TX had no significant effect on the mean body weight of either group. The previously observed lower concentration of serum calcium in lactating rats than in nonlactating thyroid-intact rats was also seen in TX rats, indicating that CT is not responsible for the relatively low serum calcium during lactation.

Our results showing that the bones of TX rats (with T4 replacement) were smaller and lighter than those from thyroid-intact controls whether lactating or not do not support the concept that CT has a special physiological function to protect the skeleton during lactation.
Differential effect of caffeine administration on calcium and vitamin D metabolism in young and adult rats
Authors: Yeh James K. , Aloia John F.
Published: February 2010

Since coffee drinking may lead to a worsening of calcium balance in humans, we studied the serial changes of serum calcium, PTH, 1,25-dihydroxyvitamin D (1,25(OH)2D) and calcium balance in young and adult rats after daily administration of caffeine for 4 weeks.

In the young rats, there was an increase in urinary calcium and endogenous fecal calcium excretion after four days of caffeine administration that persisted for the duration of the experiment. Serum calcium decreased on the fourth day of caffeine administration and then returned to control levels. In contrast, the serum PTH and 1,25(OH)2D remained unchanged initially, but increased after 2 weeks of caffeine administration. The intestinal absorption coefficient of calcium remained unchanged, instead of declining gradually as observed in the young control group. This finding suggests that the intestinal absorption of calcium was stimulated by the increase in 1,25(OH)2D production after chronic administration of caffeine.

In the adult rat group, an increase in the urinary calcium and endogenous fecal calcium excretion and serum levels of PTH was found after caffeine administration. However, the serum 1,25(OH)2D levels and intestinal absorption coefficient of calcium remained the same as in the adult control group. A decrease in the net balance of calcium occurred as a result of increased calcium excretion.

The current study, using an animal model, supports the suggestion that chronic administration of caffeine could lead to negative calcium balance when there is an impaired ability to increase the efficiency of calcium absorption. Such a situation exists in elderly human subjects, since they have a reduced capacity to synthesize 1,25(OH)2D.
Differential cyclic AMP responses to calcitonin among human ovarian carcinoma cell lines: A calcitonin-responsive line derived from a rare tumor type
Authors: Upchurch Katherine S. , Parker Leroy M. , Scully Robert E. , Krane Stephen M.
Published: February 2010

Several human tumor cell lines have been reported to have specific receptors for calcitonin (CT) and CT-responsive adenylate cyclase. In order to correlate patterns of responsiveness to CT, parathyroid hormone (PTH) and prostaglandin E2 (PGE2) with tumor morphology and intermediate filament protein expression, we examined four human ovarian tumor cell lines (BIN-16, BIN-22, BIN-53, BIN-67) which had been cultured from cells of metastatic foci. In two cell lines (BIN-53 and −16) there were small increases in cAMP content after exposure to CT and in three cell lines (BIN-53, −16, and −22) larger increases with PGE2. There was no cAMP response in any of the cells to PTH. In BIN-67 cells, however, CT induced a striking (> 20-fold) increase in cAMP content. Histologically, the CT-nonresponsive tumor lines were derived from serous adenocarcinomas while the CT-responsive tumor line was from a rare small cell carcinoma. Gel electrophoretic and immunofluorescence microscopic analyses had previously disclosed that the CT-nonresponsive cell lines contained high levels of simple epithelial keratins and no or very low levels of vimentin (characteristic of ovarian surface epithelial cells), while the CT-responsive cell line contained almost exclusively vimentin. Thus, cells cultured from a rare type of ovarian tumor were CT-responsive and were distinguishable from CT-nonresponsive ovarian tumor cells by initial tumor histology and intermediate filament protein expression.
Alterations in hormone-sensitive adenylate cyclase of cloned rat osteosarcoma cells during long-term culture
Authors: Gutierrez Gloria E. , Mundy Gregory R. , Dr. Katz Michael S.
Published: February 2010

The hormone-sensitive adenylate cyclase system of a cloned bone cell line (UMR-106) derived from a rat osteosarcoma was compared in preparations from cells of early passages (< 50) and cells maintained in continuous culture for over two years (late passages). Late passage cells showed greater calcitonin (CT)-stimulated adenylate cyclase activity than did early passages, whereas stimulation by PTH and the β-adrenergic agonist isoproterenol decreased in late passages. Hormone concentrations giving half-maximal stimulation were the same in early and late passages. Stimulation by agents (GTP and fluoride) which act at the stimulatory guanine nucleotide regulatory component (Ns) of adenylate cyclase was equivalent in early and late passages. Forskolin stimulation, which assessed catalytic component (and possibly Ns) activity, was reduced in late passages. These results are consistent with acquisition by cultured UMR-106 cells of CT receptors linked to adenylate cyclase and loss of PTH and β-adrenergic receptors. Alteration of catalytic component (and/or Ns) function may also occur after long-term culture. Since late passage cells appear dedifferentiated by chromosomal analysis and since cAMP may regulate differentiation, altered hormone-sensitive adenylate cyclase may be a marker for and a potential modulator of differentiation occurring in UMR-106 cells over long periods.
Limitations of forearm bone densitometry as an index of vertebral or femoral neck osteopenia
Authors: Pocock Nicholas A. , Dr. Eisman John A. , Yeates Michael G. , Sambrook Phillip N. , Eberl Stefan , Wren Barry G.
Published: February 2010

Osteoporosis of the spine and femoral neck is a major problem in aging populations, but detection prior to a fracture remains a challenge. Forearm bone mineral content has been advocated as a useful screening test in this situation. We have examined the correlation between distal forearm bone mineral content, by single photon absorptiometry, and lumbar vertebral and femoral neck bone mineral density, by dual photon absorptiometry. Eighty women aged 20 to 76 years were studied and significant correlations (p < .001) were found between the measurements on the forearm and at the two axial sites (r = 0.66 and 0.69, respectively). However, forearm bone mineral content was an unreliable predictor of axial bone mineral density. For prediction of lumbar spine osteopenia with a sensitivity of 88%, the false positive rate was 91%. Conversely, to achieve a specificity of 82%, the false negative rate was 65%. Similarly, for prediction of femoral neck osteopenia at a sensitivity of 92%, the false positive rate was 87%, and at a specificity of 90%, the false negative rate was 33%. These data demonstrate that forearm bone densitometry cannot be used as a screening procedure for osteopenia of the lumbar spine or femoral neck.
In vivo hydroxyapatite formation induced by lipids
Authors: Raggio C.L. , Boyan B.D. , Dr. Boskey Adele L.
Published: February 2010

Proteolipids and complexed acidic phospholipids that cause in vitro hydroxyapatite formation, similarly cause hydroxyapatite deposition in 10-μ pore Millipore chambers when implanted in rabbit muscle pouches. The amount of mineral deposited during a 3-week period, based on the calcium and phosphate contents of the chambers, was directly related to the dry weight of the lipid implanted in the chamber. Chambers containing total lipid extract from rabbit bone from which the complexed acidic phospholipids had been removed, acidic phospholipids from which the the proteolipids had been removed, and empty chambers did not accumulate any detectable mineral during the course of the study. Chambers implanted with synthetic hydroxyapatite served as controls for chemical analyses. The presence of hydroxyapatite in the chambers was established 3 weeks after implantation based on electron microscopic, compositional, and wide-angle X-ray diffraction analyses of the deposits. In the cell-free chambers, lipid-induced hydroxyapatite deposition, but not bone matrix formation occurred. This study demonstrates that proteolipids and complexed acidic phospholipids can cause hydroxyapatite mineral deposition in a physiologic environment. To date, these lipids are the only materials isolated from mineralizing tissues, other than reconstituted collagen, that have been shown capable of causing in vivo mineralization in the absence of cells.
The effect of 1,25-dihydroxyvitamin D3 on the cytoskeleton of rat calvaria and rat osteosarcoma (ROS 17/2.8) osteoblastic cells
Authors: Dr. Gronowicz Gloria , Egan J.J. , Rodan G.A.
Published: February 2010

1,25-dihydroxy vitamin D3 produces pronounced shape changes in fetal rat calvaria and osteosarcoma-derived (ROS 17/2.8) osteoblastic cells, characterized by retracting processes and cell rounding followed by aggregation of cells. The 1,25(OH)2D3 effect on ROS 17/2.8 morphology was determined morphometrically on scanning electron micrographs. The hormone effect was found to be dose dependent between 10−12 and 10−9M. The shape changes appeared 12 h after hormone (10−10M) addition and were present in 80% of the ROS 17/2.8 cells and in 50% of the calvaria cells at 72 h. Cycloheximide at 1 μM, inhibited the hormone-dependent change in morphology. The 1,25(OH)2D3 effects were partially mimicked by 10−8M 25(OH)D3 but not by 10−10M 25(OH)D3 or 10−11-10−8M 24,25(OH)2D3. 1,25-dihydroxy vitamin D3 also increased cell proliferation twofold at 14 days in serum-free medium.

1,25(OH)2D3 treatment produced changes in microfilament organization, visualized with rhodamine-conjugated phalloidin. Microfilaments were localized at the terminal attachment points and in the perinuclear region, and few if any, were seen in the retracting processes themselves. Estimation of cytoskeletal actin and myosin by gel electrophoresis of Triton X-100 nonextractable proteins showed a 30% reduction in these proteins in the hormone-treated cells. Microtubules visualized by indirect immunofluorescence showed no major changes in organization. Both colchicine and cytochalasin D altered the hormone-induced shape change, suggesting that both microfilaments and microtubules were required for this process.

Thus, 1,25(OH)2D3 had pronounced effects on cell shape in osteoblastic cells, probably via de novo protein synthesis. These changes lead to rearrangement of the cytoskeleton, primarily the microfilaments.
On the problem of covalent linkages between phosphoproteins and collagen in bovine dentin and bone
Authors: Dr. Glimcher Melvin J. , Lefteriou Beatrice , Kossiva Dora
Published: February 2010

The majority of phosphoproteins in bovine bone and dentin are insoluble in EDTA and guanidine hydrochloride (Gu · HCl) at 2°C. After removal of EDTA and Gu · HCl-soluble proteins at 2°C, collagen α-chains and α-chain polymers were extracted from bovine bone and dentin in Gu · HCl at elevated temperatures and purified by several chromatographic techniques and SDS-PAGE. Small amounts of O-phosphoserine were found in all collagen components. In contrast, O-phosphoserine was not detected in the purified collagen components soluble in EDTA or Gu · HCl at 2°C nor was hydroxyproline detected in the EDTA-soluble phosphoproteins. In contrast, although the vast majority of EDTA-insoluble collagen and phosphoprotein molecules can be readily dissociated by a variety of molecular sieving and ion-exchange chromatographic procedures, a small number are very strongly associated or covalently cross-linked. These results are consistent with the findings that both hydroxyproline and hydroxylysine are present in purified phosphoprotein components released from the EDTA-insoluble tissue by bacterial collagenase. The hydroxylysine/100 hydroxyproline ratios in the phosphoprotein-collagen complexes are much higher than those in dentin or bone collagens.
Serum osteocalcin (BGP) in tumor-associated hypercalcemia
Authors: Dr. Body J.J. , Cleeren A. , Pot M. , Borkowski A.
Published: February 2010

Serum osteocalcin (BGP) is a new marker of bone turnover that reportedly evaluates bone formation. Thus, its measurement could assess the bone formation rate in tumor-associated hypercalcemia. We measured concentrations of BGP and other parameters of bone metabolism in 54 untreated hypercalcemic cancer patients as compared to 109 healthy subjects. Primary tumor sites were breast (19), lung (11), head and neck (6), multiple myeloma (3), kidney (2), and various (11) or multiple (2). Mean BGP levels were higher in the hypercalcemic subjects, 4.6 ± 0.4 (SEM) ng/ml, than in the normal subjects, 3.6 + 0.1 ng/ml (p < .05), and were normalized in the 22 patients who could be reevaluated after successful treatment of hypercalcemia with intravenous aminohydroxypropylidene diphosphonate (APD). There was no correlation of BGP levels with age, sex, or renal function. Compared with the Gaussian distribution in the normal subjects, there was a considerable scatter of the data in hypercalcemic patients, suggesting the existence of defined subgroups with abnormally low or abnormally high values. However, we found no significant relationship of BGP concentrations with tumor site or histology or with bone metastatic involvement. We found also no significant correlation between concentrations of serum BGP and total or ionized calcium, alkaline phosphatase, parameters of bone resorption, and indices of parathyroid function. In summary, serum BGP levels were slightly elevated in tumor-associated hypercalcemia and were normalized after successful treatment of hypercalcemia. More importantly, BGP concentrations varied widely even in the subgroups of patients with hypercalcemia accompanying massive bone metastatic involvement or in the patients without detectable skeletal metastases. Thus, BGP determination cannot help to differentiate between paraneoplastic hypercalcemia and hypercalcemia due to metastatic bone lysis; moreover, our findings suggest great variability of the bone formation rate in tumor-associated hypercalcemia, whether bone metastases are present or not.
A single-day treatment of tumor-induced hypercalcemia by intravenous amino-hydroxypropylidene bisphosphonate
Authors: Dr. Thiébaud D. , Jaeger P. , Jacquet A.F. , Burckhardt P.
Published: February 2010

Twenty patients with malignant hypercalcemia were treated with amino-hydroxypropylidene bisphosphonate (AHPrBP, previously APD), a potent inhibitor of osteoclast-mediated bone resorption. To assess the efficacy of a single-day treatment and determine the optimal dose of this compound, all patients received AHPrBP intravenously over 24 h, but they were divided into two subsequent groups of 10 patients: Group A received a single dose of 60 mg AHPrBP and group B received a single dose of 30 mg. In both groups all patients responded to AHPrBP with a decrease in plasma calcium concentration after a mean time lag of 1 day. Within 6 days, plasma calcium (corrected for serum proteins) fell from 3.24 ± 0.14 to 2.24 ± 0.06 mmol/liter in group A (p < .001), but only from 3.22 ± 0.15 to 2.49 ± 0.10 mmol/liter in group B (p < .005). Whereas in all patients from group A plasma calcium was within the normal range at days 9 and 14, in 4 patients of group B it was still above the normal range at day 9, and in 5 patients at day 14. There was a significant difference in plasma calcium between group A and group B from days 5 to 14 (p < .005). In both groups, urinary calcium excretion fell dramatically and similarly, and plasma phosphate concentration decreased significantly (p < .01) to values slightly below the normal range from days 4 to 6. A similar and significant decrement in TmP/GFR was noted at days 4 and 6 (p < .01) which was related to the rise (p < .01) in plasma parathyroid hormone concentration from days 4 to 9. Urinary hydroxyproline excretion decreased significantly (p < .01) in both groups from days 0 to 9, with a trend to reincrease from day 6 on in group B. We conclude that a single intravenous dose of AHPrBP is effective in the treatment of tumor-induced hypercalcemia; but 60 mg has a more pronounced and prolonged effect than 30 mg, achieving normocalcemia in al patients, while 30 mg normalized plasma calcium only in moderately hypercalcemic patients. The decrease in plasma calcium after a single day infusion of 60 mg is similar to that previously observed during treatment of 30 mg per day during 6 days.
Paget's disease of bone treated in five days with AHPrBP (APD) per Os
Authors: Dr. Thiébaud D. , Jaeger P. , Burckhardt P.
Published: February 2010

Amino-hydroxypropylidene bisphosphonic acid (AHPrBP, previously APD) is a potent inhibitor of bone resorption. Since it remains in bone for a long time, and since it was not found to impair bone mineralization, it could be administered at high dose over a short period of time. Therefore, 11 patients with symptomatic Paget's disease received AHPrBP orally at 1200 mg/day over 5 consecutive days. Controls were performed after 1 month in all patients, 6 months in 8 patients, and one year in 4 patients. Clinical improvement and biochemical remission was observed in all patients, except one with severe disease.

Side effects were negligible. Disease activity at bone scintigram decreased over 6 months. Plasma alkaline phosphatase activity fell progressively and significantly from 210 ± 26 U/I (x̄ ± SEM) to 103 ± 10 U/I after 6 months (nl < 120 U/I).

Urinary excretion of hydroxyproline decreased immediately and became normal (nl < 2.3 μmol/IGF) as a mean at day 5 (from 4.6 ± 0.4 μmol/IGF to 2.1 ± 0.3 μmol/IGF). Thereafter it remained within the normal range (2.0 ± 0.2 μmol/l at day 180). Plasma calcium and phosphate concentrations fell transiently between day 4 and 15, whereas plasma PTH levels increased over this period of time. In conclusion, a short course of AHPrBP given per os at high dose induces a rapid decline in activity and remission of moderate Paget's disease, without significant side effects.
Synthetic parathyroid hormone fragments shortened at the amino terminus stimulate glucose-6-phosphate dehydrogenase activity in the distal renal tubule
Authors: Sakaguchi Kazushige , Fukase Masaaki , Kobayashi Ikuo , Kimura Terutoshi , Sakakibara Shumpei , Katsuragi Shigeo , Morita Kaoru , Noda Toshiharu , Fujita Takuo
Published: February 2010

The cytochemical bioassay, using glucose-6-phosphate dehydrogenase (G6PD) activity in the distal convoluted tubule of a guinea pig as an index, is specific and the most sensitive method of evaluating the biological activity of parathyroid hormone (PTH). Using this method, biological activities of the amino- or carboxylterminal PTH fragments and analogues, human (h) PTH-(3–34), [Tyr34]hPTH-(7–34)amide(NH2), [Tyr34]hPTH-(13–34)NH2, hPTH-(39–84), hPTH-(51–84), hPTH-(69–84), were tested over a concentration range of 10−16 to 10−13M. In addition, the combined effect of these hormones with human or bovine PTH-(1–84) and the effect of dibutyryl ((Bu)2) cAMP were also evaluated.

In the 14-min time-course study, amino-terminal PTH fragments and analogues induced cyclic changes of G6PD activity with shorter cycle lengths in higher concentrations and with constant peak heights regardless of the concentrations. Human and bovine PTH-(1–84) showed the same activity on G6PD activation at 6 min. hPTH-(3–34), [Tyr34]hPTH-(7–34)NH2, and [Tyr34]hPTH-(13–34)NH2 were equipotent with PTH-(1–84) on a molar basis, and none of these analogues inhibited PTH-(1–84) even with doses up to 240 times that of PTH-(1–84). Carboxyl-terminal PTH showed no effect. (Bu)2cAMP mimicked the effect of PTH-(1–84) on G6PD activation in time course and dose response.

We conclude that the amino terminus is not essential for the biological activity of PTH in the cytochemical bioassay.
Healing of rachitic lesions in chicks by 24R,25-dihydroxycholecalciferol administered locally into bone
Authors: Lidor Cobi , Atkin Isaac , Prof. Ornoy Asher , Dekel Samuel , Edelstein Samuel
Published: February 2010

In an attempt to further define the nature of the active metabolite in bone formation, a series of experiments were conducted whereby vitamin D metabolites were administered locally in vivo into the proximal epiphyseal growth plate of the tibiae of rachitic chicks. Local administration of 3 μg of 24,25(OH)2D3in vivo to D-deficient chicks resulted in disappearance of the rachitic lesions in the same leg. Administration of 1 μg 1,25(OH)2D3 in a similar manner failed to show any sign of healing. Injection of 5 μg 25(OH)D3 was followed by recovery from rickets in both the injected right leg and in the vehicle-injected left tibia, although the recovery was more pronounced in the injected leg. Lower doses of 0.3 or 1 μg 24,25(OH)2D3 failed to reverse the rachitic lesions and induced only minimal recovery. These findings suggest that 24,25(OH)2D3 at the higher doses has a direct local effect on cartilage and bone, while 1,25(OH)2D3 has no such effect in chicks. 25(OH)D3 is probably absorbed from the epiphyses into the blood stream and converted into the active metabolites, which were indeed detected in the blood to exert its systemic effects.
Computer modeling and analysis of cross-sectional bone density studies with respect to age and the menopause
Authors: Dr. Sambrook Philip N. , Eisman John A. , Furler Stuart M. , Pocock Nicholas A.
Published: February 2010

Controversy exists about rates of bone mineral loss from the lumbar spine at the menopause. Cross-sectional studies of lumbar bone mineral against age have been interpreted as evidence for or against a menopause-related change in lumbar bone loss, depending on their goodness of fit to either linear or nonlinear equations. To investigate the ability of cross-sectional studies to detect accelerated lumbar bone loss at the menopause, we constructed models of different patterns of loss and evaluated the findings in cross-sectional analyses of computer-simulated populations of normal women. A sample size of >300 was needed to distinguish between linear and nonlinear patterns of bone loss with a power of 90% when the data was analyzed throughout life. Examining for differences between the slopes of the regressions of pre- and postmenopausal women was more useful for detecting nonlinear bone loss under some circumstances. A difference between slopes was apparent in studies containing 100 subjects if lumbar bone density (BMD) was assumed to be unchanged prior to the menopause, but a larger study size was needed (> 1000) if BMD was assumed to fall before the menopause.

We also measured lumbar BMD by dual photon absorptiometry in 141 normal females. When the data was analyzed against age throughout life, a nonlinear pattern of bone loss was found, and comparison of preand postmenopausal subjects showed a significant difference in the slopes of the linear regressions.

Our patient data support the concept of a relatively stable lumbar BMD prior to the menopause with a rapid but transient decline postmenopausally. The modeling studies emphasize the importance of an adequate study size in cross-sectional studies to identify accelerated lumbar bone loss at the menopause.
Synergism between parathyroid hormone and interleukin 1 in stimulating bone resorption in organ culture
Authors: Dr. Dewhirst Floyd E. , Ago Joanne M. , Peros William J. , Stashenko Philip
Published: February 2010

The interaction of interleukin 1 (IL-1), a locally produced factor, and parathyroid hormone (PTH), a systemic factor, in stimulating bone resorption was examined using fetal rat long bone organ culture. Concentrations of IL-1 and PTH, which stimulated little bone resorption when present singly, produced marked resorption when present simultaneously. This synergistic interaction of IL-1 and PTH was not affected by the presence of the prostaglandin synthetase inhibitor indomethacin. Both interleukin 1α and interleukin 1β were capable of producing synergy. Synergy was not produced by sequential exposure of bone to IL-1 and PTH, but required the simultaneous presence of both mediators. The leftward shift in the dose response curve of PTH produced by IL-1 may be an important mechanism controlling localized bone resorption. A role for IL-1 in stimulating bone resorption in pathologic conditions, such as arthritis and periodontal disease, is strengthened by the finding that even low concentrations of IL-1 can produce resorptive effects by synergistic interaction with PTH.
Modulation of 1,25-dihydroxyvitamin D3 receptor binding and action by sodium butyrate in cultured pig kidney cells (LLC-PK1)
Authors: Costa M. Elizabeth , Feldman David
Published: February 2010

We have studied the effects of sodium butyrate (SB) on 1,25(OH)2D3 receptors in the pig kidney cell line (LLC-PK1). In this system, we have shown that 1,25(OH)2D3 induction of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity is dependent on the level of 1,25(OH)2D3 receptors. Treatment of confluent cells with SB (5 mM/48 h) caused an -50% decrease in total receptors, whereas the affinity for 1,25(OH)2D3 was unchanged. At 5 mm, the action of SB on these receptors required more than 24 h to be detected. The effect of the decrease in receptors on the functional response to the hormone was studied by measuring the 1,25(OH)2D3 induction of 24-hydroxylase activity after treatment with SB. The induction of 24-hydroxylase activity at higher doses of hormone paralleled the reduction in receptors and was diminished by 25–50%. At low doses of hormone, the cells appear to be more sensitive to 1,25(OH)2D3 induction, exhibiting an unexplained increase in 24-hydroxylase activity compared to cells not exposed to SB. An additional effect of SB was also noted: SB decreased cell proliferation and inhibited [3H]thymidine incorporation by 75% when added to cells prior to confluence. At confluence, SB caused a less drastic effect on protein and DNA synthesis. Therefore, most binding experiments were conducted at confluence when the SB effect on cell proliferation was less. Other short chain fatty acids in addition to SB were also tested. The action to decrease 1,25(OH)2D3 receptors was more specific upon exposure to SB. We have previously demonstrated up-regulation of 1,25(OH)2D3 receptors in LLC-PK1 cells after treatment with various vitamin D metabolites. After 24–48 h of treatment, 24,25(OH)2D3 induced a 2-5-fold increase in receptors levels. When added together with 24,25(OH)2D3, SB prevented the up-regulation whether measured at 24 or 48 h. The blockade of up-regulation was not observed if SB was added only in the last hour of exposure to 24,25(OH)2D3. In summary, we describe an effect of SB to decrease both 1,25(OH)2D3 receptors and the functional response to hormone. SB was also found to inhibit cell growth and proliferation.
cDNA cloning of alkaline phosphatase from rat osteosarcoma (ROS 17/2.8) cells
Authors: Noda M. , Yoon K. , Thiede M. , Buenaga R. , Weiss M. , Henthorn P. , Harris H. , Rodan G.A.
Published: February 2010

Two cDNA clones of rat alkaline phosphatase (AP) were isolated from a rat osteosarcoma λgt 11 cDNA library (ROS 17/2.8) utilizing a human bone-liver-kidney (BLK) type AP cDNA. These clones contain overlapping DNA sequences of 597 and 520 bp, respectively, corresponding to the 3' noncoding region of AP mRNA. The sequence homology with the human BLK AP cDNA is 61%. In Northern blot analysis the rat cDNA hybridizes to a single band of 2.5 kb mRNA from ROS 17/2.8 and rat liver, under highly stringent conditions. Steady state levels of AP mRNAs measured in several rat osteosarcoma cell lines (ROS 17/2.8, ROS 2/3, ROS 25/1, UMR 106) correlate with the level of AP enzymatic activity in these cells. Dexamethasone, which stimulates AP enzymatic activity in ROS 17/2.8 cells, increases the relative abundance of AP mRNA in a dose-dependent manner. This probe can be used to study AP expression in rat tissues and cells.