Cryopreservation of cultured periosteum: effect of different cryoprotectants and pre-incubation protocols on cell viability and osteogenic potential

Evidence has accumulated that periosteal cells have a great potential to regenerate bone. We have demonstrated that cultured periosteum (CP) in membrane form is an effective device to regenerate alveolar bone. To increase the availability of CP in a clinical environment, an effective cryopreservation protocol for CP has been developed. In this study, three different cryoprotectants (Me(2)SO, glycerol, and ethylene glycol) were used. The effect on cell viability of pre-incubation temperature, pre-incubation time, and agitation during incubation was investigated. Samples were stored at -196 degrees C for 10 days. Cell viability was assessed by a colorimetric cell viability assay using a tetrazolium salt, and the assay results were confirmed by confocal laser scanning microscopy after staining with a combination of calcein AM and ethidium homodimer-1. The activity of the cells after thawing was assessed by alkaline phosphatase assay. To assess the osteogenic potential of cryopreserved CP, the CP was grafted to calvarial defects in athymic rats. The greatest cell viability was obtained in the group equilibrated at 37 degrees C for 30 min with Me(2)SO, under agitation, showing 63.3 +/- 10.5% recovery. After cryopreservation, the cell growth of surviving cells was identical when Me(2)SO was used as a cryoprotectant. Alkaline phosphatase (ALP) activity was maintained in the groups cryopreserved with Me(2)SO and glycerol. The transplantation experiment showed that the calvarial defects were completely closed by grafting cryopreserved CP, which demonstrates that the osteogenic property of CP was well maintained. An efficient cryopreservation protocol for CP has been developed and this will provide a convenient and effective treatment option for bone regeneration in clinics.     

Varying amounts of different aldehydes present in the cryoprotectants dimethyl sulphoxide and 1,2-propanediol

Using high-pressure liquid chromatography two cryoprotectant solvents, dimethyl sulphoxide (four manufacturers) and 1,2-propanediol (one manufacturer) were investigated for aldehyde content. Fractionation of the aldehydes by high pressure liquid chromatography identified up to 11 aldehydes and two ketones in both cryoprotectant solvents in varying concentrations, which differed between manufacturer and container type. Of the 11 aldehydes identified, formaldehyde and acetylaldehyde were consistently in the greatest concentrations. As the low molecular weight aldehydes identified contain reactive polarised carbonyl groups they represent a potential source of intracellular damage when used in oocyte cryopreservation.     

Cryopreservation of tissue-engineered dermal replacement in Me2SO: Toxicity study and effects of concentration and cooling rates on cell viability

Cryopreservation of tissue-engineered human dermal replacement plays an important role in skin tissue engineering and skin banking. With the inspection of electronic scanning microscope and viability evaluation by Trypan Blue staining assay and the tetrazolium salt, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, this study investigated the toxicity of Me(2)SO to dermal fibroblasts and effects of cryoprotectant concentration and cooling rate on the viability of dermal replacement. The results demonstrated that the Me(2)SO toxicity to fibroblasts was affected by the exposure time, temperature, and concentration. Furthermore adding cryoprotectant solution at low temperature of 4 degrees C significantly reduced the toxic effect on the tissue-engineered dermal equivalent. An optimal cryopreservation protocol consisting of cooling rate at 1 degrees Cmin(-1) in 10% (V/V) Me(2)SO was derived, with the viability of studied dermal equivalent treated by this protocol being 75% of that of fresh control. The micrograph obtained by electronic scanning microscope also confirmed this result.     

Membrane integrity and development of immature murine cumulus-oocyte complexes following slow cooling to -60 degrees C: the effect of immediate rewarming, plunging into LN2 and two-controlled-rate-stage cooling

Cryopreservation of murine germinal vesicle (GV) stage cumulus-oocyte complexes (COCs) has been shown to result in poor development and cumulus cell damage. In an attempt to determine the stage of the cryopreservation protocol at which damage occurs, three cooling profiles were compared: slow-cooling (0.3 degrees C/min) to -60 degrees C (protocol A); slow-cooling to -60 degrees C and plunging to -196 degrees C (protocol B); or slow-cooling to -60 degrees C followed by further cooling at 10 degrees C/min to -150 degrees C, then plunging to -196 degrees C (protocol C). GV-stage COCs were collected from hormone-primed mice by repeated puncturing of ovarian follicles. COCs were exposed to 1.5 M Me(2)SO prior to cooling to -60 or -196 degrees C. Membrane integrity was assessed immediately after thawing using carboxy fluorescein and propidium iodide. A greater proportion of cumulus cells were damaged following protocol B than protocol A. Damage was less extensive following protocol C than following protocol B. For assessment of development, COCs were matured and fertilised in vitro. Morphological normality was significantly reduced following cooling to -60 or -196 degrees C compared with non-cryopreserved controls. Fertilisation of oocytes assessed as normal post-treatment was not significantly different between any of the groups. Development to blastocyst was least from oocytes exposed to protocol B, being significantly worse than for oocytes exposed to protocol A, but not significantly different to protocol C. A protocol comprising two stages of controlled-rate cooling decreased damage to the membranes of cumulus cells but did not significantly improve embryo development.     

Comparative analysis of transcriptional responses to the cryoprotectants, dimethyl sulfoxide and trehalose, which confer tolerance to freeze–thaw stress in Saccharomyces cerevisiae

We have used microarray analysis to monitor the gene expression profile of Saccharomyces cerevisiae BY4743 in the presence of the cryoprotectants, dimethyl sulfoxide (Me₂SO) and trehalose. Analysis of these profiles suggests that both cryoprotectants increased the expression of genes involved in protein synthesis, ribosomal biogenesis, fatty acid biosynthesis, ergosterol biosynthesis, cell wall biosynthesis, and cellular accumulation of low molecular compounds such as glycerol, arginine and proline. Cryoprotectant treatment reduced the expression of genes involved in the β-oxidation of fatty acids. In addition, Me₂SO increased the expression of genes involved in protein refolding and trehalose increased the expression of genes involved in spore formation. This study supported that exposure to cryoprotectants prior to freezing not only reduce the freeze–thaw damage but also provide various process to the recovery from freeze–thaw damage.     

Cryo-responses of two types of large unilamellar vesicles in the presence of non-permeable or permeable cryoprotecting agents

Cryo-responses of two types of large unilamellar vesicles (LUV) that were made from either egg yolk l-α-phosphatidylcholine (EPC) or 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC), in the presence of non-permeable or permeable cryoprotective agents (CPA) was investigated. Partial ternary phase diagrams of CPA–salt–water with specific CPA to salt ratio (R), were constructed to estimate the phase volume of ice and unfrozen matrix of the LUV dispersion, which could aid in understanding the mechanistic actions of CPA. Leakage of both EPC and DPPC LUV was reduced if the sugar concentrations are above 10% (w/w) for disaccharides and 5% (w/w) for monosaccharides. Above these sugar concentrations, non-permeable CPA were more effective in preventing leakage of DPPC LUV than in EPC LUV. Below these sugar concentrations, EPC and DPPC LUV with limited mobility in the remaining unfrozen matrix were more likely to approach and interact with one and another, which were not anticipated when the LUV were completely embedded in the ice matrix. In the presence of Me₂SO or EG, EPC LUV that had been subjected to freezing and thawing processes were protected from leakage. At room temperature, Me₂SO and EG were detrimental to the DPPC LUV. This study suggests that the choice of CPA for cell cryopreservation depends on the type of phospholipids in plasma membranes, which vary in their acyl chain length and gel–liquid crystal phase transition temperature.     

Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution

Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me₂SO) and stored for extended periods at −80 °C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me₂SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at −80 °C. The cryopreserved platelet units (n = 9) were rapidly thawed at 37 °C, reconstituted in 50% SSP+/plasma and stored at 22 °C. Platelet recovery and quality were examined 1 and 24 h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24 h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me₂SO and additive solution produces acceptable in vitro platelet quality.     

Evaluations of bioantioxidants in cryopreservation of umbilical cord blood using natural cryoprotectants and low concentrations of dimethylsulfoxide

Transplantation using hematopoietic stem cells from umbilical cord blood (UCB) is a life-saving treatment option for patients with select oncologic diseases, immunologic diseases, bone marrow failure, and others. Often this transplant modality requires cryopreservation and storage of hematopoietic stem cells (HSC), which need to remain cryopreserved in UCB banks for possible future use. The most widely used cryoprotectant is dimethylsulfoxide (Me₂SO), but at 37 °C, it is toxic to cells and for patients, infusion of cryopreserved HSC with Me₂SO has been associated with side effects. Freezing of cells leads to chemical change of cellular components, which results in physical disruption. Reactive oxygen species (ROS) generation also has been implicated as cause of damage to cells during freezing. We assessed the ability of two bioantioxidants and two disaccharides, to enhance the cryopreservation of UCB. UCB was processed and subjected to cryopreservation in solutions containing different concentrations of Me₂SO, bioantioxidants and disaccharides. Samples were thawed, and then analysed by: flow cytometry analysis, CFU assay and MTT viability assay. In this study, our analyses showed that antioxidants, principally catalase, performed greater preservation of: CD34+ cells, CD123+ cells, colony-forming units and cell viability, all post-thawed, compared with the standard solution of cryopreservation. Our present studies show that the addition of catalase improved the cryopreservation outcome. Catalase may act on reducing levels of ROS, further indicating that accumulation of free radicals indeed leads to death in cryopreserved hematopoietic cells.     

The relationship between the redox state of Q A and photosynthesis in leaves at various carbon-dioxide,oxygen and light regimes

The response of chlorophyll fluorescence elicited by a low-fluence-rate modulated measuring beam to actinic light and to superimposed 1-s pulses from a high-fluence-rate light source was used to measure the redox state of the primary acceptor Q _A of photosystem II in leaves which were photosynthesizing under steady-state conditions. The leaves were exposed to various O₂ and CO₂ concentrations and to different energy fluence rates of actinic light to assess the relationship between rates of photosynthesis and the redox state of Q _A. Both at low and high fluence rates, the redox state of Q _A was little altered when the CO₂ concentration was reduced from saturation to about 600 l·l^-1 although photosynthesis was decreased particularly at high fluence rates. Upon further reduction in CO₂ content the amount of reduced Q _A increased appreciably even at low fluence rates where light limited CO₂ reduction. Both in the presence and in the absence of CO₂, a more reduced Q _A was observed when the O₂ concentration was below 2%. Q _A was almost fully reduced when leaves were exposed to high fluence rates under nitrogen. Even at low fluence rates, Q _A was more reduced in shade leaves of Asarum europaeum and Fagus sylvatica than in leaves of Helianthus annuus and Fagus sylvatica grown under high light. Also, in shade leaves the redox state of Q _A changed more during a transition from air containing 350 l·l^-1 CO₂ to CO₂-free air than in sun leaves. The results are discussed with respect to the energy status and the CO₂-fixation rate of the leaves.Abbreviations and symbols L 1,2 first and second actinic light beam - Q _A primary acceptor of photosystem II - q _Q Q-quenching     

Methylamine,an inhibitor of ammonium oxidation and chemoautotrophic growth in the marine nitrifying bacteriumNitrosococcus oceanus

Methylamine (CH₃NH ₃ ⁺ ) appeared to utilize the same transport mechanism as ammonium (NH ₄ ⁺ ) to enter cells ofNitrosococcus oceanus. Methylamine uptake did not show clear evidence of saturable kinetics and was not fully saturated at 20 mM. Assimilated CH₃NH ₃ ⁺ was not incorporated into macromolecular constituents, but inhibited rates of nitrification, chemoautotrophic CO₂ fixation and growth. The degree of inhibition was dependent on the relative concentrations of NH ₄ ⁺ and CH₃NH ₃ ⁺ . Rates of CO₂ fixation and growth were inhibited four times more than the rate of nitrification.     

A structural study of the hydrated and the dimethylsulfoxide, N,N′-dimethylpropyleneurea, and N,N-dimethylthioformamide solvated iron(II) and iron(III) ions in solution and solid state

The structures of the solvated iron(II) and iron(III) ions have been studied in solution and solid state by extended X-ray absorption fine structure (EXAFS) in three oxygen donor solvents, water, dimethylsulfoxide (Me₂SO), N,N′-dimethylpropyleneurea (DMPU), and one sulfur donor solvent, N,N-dimethylthioformamide (DMTF); these solvents have different coordination and solvation properties. In addition, the structure of hexakis(dimethylsulfoxide)iron(III) perchlorate has been determined crystallographically to support the determination of the corresponding solvate in solution. The hydrated, the dimethylsulfoxide and N,N-dimethylthioformamide solvated iron(II) ions show regular octahedral coordination in both solution and solid state with mean Fe-O, Fe-O, and Fe-S bond distances of 2.10, 2.10, and 2.52 Å, respectively, whereas the N,N′-dimethylpropyleneurea iron(II) solvate is five-coordinated, d(Fe-O) = 2.06 Å. The compounds vary in color from light green (hydrate) to dark orange or red (DMPU). The hydrated iron(III) ion in aqueous solution and the dimethylsulfoxide solvated iron(III) ions in solution and solid state show the expected octahedral coordination, the Fe-O bond distances are 2.00 Å for both, whereas the N,N′-dimethylpropyleneurea iron(III) solvate is found to be five-coordinated with a mean Fe-O bond distance of 1.99 Å. The N,N-dimethylthioformamide solvated iron(III) ion in the solid perchlorate salt is tetrahedrally four-coordinated, the mean Fe-S bond distance is 2.20 Å. Iron(III) is reduced with time to iron(II) in N,N-dimethylthioformamide solution. The compounds vary in color from pale yellow (hydrate) to blackish red (DMPU).     

Effect of Potassium Dihydrogen Phosphate and Bovine Manure Compost on the Degradation of Chlorothalonil in Soil

The effects of potassium dihydrogen phosphate (KH₂PO₄) and bovine manure compost (BMC) on the degradation and metabolism of chlorothalonil were examined in a sandy loam soil under laboratory conditions. In non-sterilized, non-amended soil, chlorothalonil degradation half-life was 8.8 days. However, it was up to 19.0 days in sterilized non-amended soil, suggesting that the degradation rate was about 2-fold slower than that in non-sterilized non-amended soil. Biological mechanisms accounted for 54.7% of chlorothalonil degradation in non-sterilized soil, indicating that the indigenous microorganisms in soil play an important role in the degradation process. In non-sterilized soil, the more acutely toxic metabolite, 4-OH-chlorothalonil (HTI), of chlorothalonil started forming after the second day of incubation. The concentration of HTI reached its maximum level (2.9 μ g g? 1) at 10 days after treatment, and no further degradation of HTI was observed in the following span of 20 days (10–30 days of incubation). However, the degradation rate of chlorothalonil was decreased substantially by the addition of KH₂PO₄ with a half-life of 17.5 days. No formation of HTI was observed before 10 days of incubation and no significant difference of the metabolite concentration at the end of experiment was observed between KH₂PO₄-amended and non-amended treatments, which denoted that the addition of KH₂PO₄ did not reduce the formation of HTI in the longer incubation course. In BMC-amended soil, the degradation rate was about 1.8 times faster than in non-amended soil. At 30 days of incubation, both chlorothalonil and HTI were degraded to a lower level in BMC-amended soil than in non-amended soil. The application of farm litter is a common fertilizing practice in vegetable fields in China, and thus this practice could not only improve soil fertility but also promote the removal of chlorothalonil and its metabolite HTI to further increase safety in crop rotations.     

Expression of prostaglandin E2 receptor subtypes, EP2 and EP4, in the rat hippocampus after cerebral ischemia and ischemic tolerance

We investigated the distribution and time course of expression of two subtypes of prostaglandin E₂ (PGE₂) receptors, EP2 and EP4, in a rat model of cerebral ischemia and ischemic tolerance. Adult male Sprague-Dawley rats were subjected to either lethal global ischemia (10 min) with or without sublethal ischemic preconditioning (3 min), or ischemia only (3 min). A short 3-min cerebral ischemia and a 3-min ischemia followed by a second lethal ischemia enhanced the expression of EP2 and EP4 receptors in CA1 pyramidal neurons of the hippocampus. In tolerance-acquired CA1 neurons, the immunoreactivities of EP2 and EP4 were upregulated after 4 h and 12 h, respectively. The immunoreactivities were most prominent at 3 days and were sustained for at least 14 days, consistent with results of immunoblotting experiments. However, immunoreactivities for these PGE₂ receptors increased in reactive glial cells in the vulnerable CA1 and hilar regions of rats subjected to lethal ischemia without ischemic preconditioning. Most of the EP2 immunoreactivity occurred in microglial cells and some astrocytes, whereas increased immunoreactivity for EP4 was found only in astrocytes. These data suggest that ischemia and the induction of ischemia tolerance have different regulatory effects on the expression of EP2 and EP4 receptors. Moreover, PGE₂ may exert its unique pathophysiological functions in relation to delayed neuronal death and ischemic tolerance induction in the rat hippocampus via specific PGE₂ receptors.This research was supported by a grant (M103KV010019 04K2201 01930) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of the Republic of Korea.     

During a systemic inflammatory response, the effect of non-steroidal anti-inflammatory drugs on seizure susceptibility in the immature brain may depend on the proconvulsant and anticonvulsant mechanisms simultaneously induced by the elevation of parenchymal prostaglandin E2 levels

Clinical evidence from paediatric neurology supports the possibility that a protracted inflammatory state in the central nervous system (CNS) may enhance the predisposition of brain tissue to develop seizures. Consequently, non-steroidal anti-inflammatory drugs (NSAIDs) as well as selective cyclooxygenase-2 (COX-2) inhibitors were expected to positively modulate seizure susceptibility during a systemic inflammatory response. Nevertheless, experimental findings and clinical evidence provide controversial results. As a possible explanation for these apparent discrepancies, it is hypothesised that the amount of prostaglandin E₂ (PGE₂) induced in the immature brain parenchyma during systemic inflammatory response is crucial since PGE₂ plays a dual role. Indeed, on the one hand, this prostaglandin increases seizure susceptibility by stimulation of glutamate release from neurons and astrocytes. On the other hand, however, the same prostaglandin induces a massive release of corticosterone, being this hormone known to inhibit efficiently the seizure susceptibility of the immature brain. Hence, the dose-response curve of any given NSAID/COX-2 inhibitor on seizure susceptibility is expected to show different patterns, depending on the amount of PGE₂ levels produced in the brain parenchyma during the effect of drug. The proposed hypothesis also suggests that mild to moderate increase of PGE₂ levels in the immature brain parenchyma may act as a ‘preconditioning’ stimulus, i.e., it may confer a transient resistance to develop seizure-induced brain injury, besides to efficiently counteract seizure susceptibility.     

Control of nitrate and nitrite assimilation by carbohydrate reserves,adenosine nucleotides and pyridine nucleotides in leaves of Zea mays L. under dark conditions

The rate of in-vivo nitrate reduction by leaf segments of Zea mays L. was found to decline during the second hour of dark anaerobic treatment. On transfer to oxygen the capacity to reduce nitrate under dark conditions was restored. These observations led to the proposal that nitrate reductase is a regulatory enzyme with ADP acting as a negative effector. The effect of ADP on the invitro activity of nitrate reductase and the changes in the in-vivo adenylate pool under dark-N₂ and dark-O₂ were investigated. It was found that ADP inhibited the activity of partially purified nitrate reductase. Similarly, the in-vivo anaerobic inhibition of nitrate reduction was associated with a build-up of ADP in the leaf tissue. Under anaerobic conditions nitrite accumulated and on transfer to oxygen the accumulated nitrite was reduced. To explain this phenomenon the following hypothesis was proposed and tested. Under anaerobic conditions the supply of reducing equivalents for nitrite reduction in the plastid becomes restricted and nitrite accumulates as a consequence. On transfer to oxygen this restriction is removed and nitrite disappears. This capacity to reduce accumulated nitrite was found to be dependent on the carbohydrate status of the leaf tissue.     

Subchronic Treatment with Methamphetamine and Phencyclidine Differentially Alters the Adenosine A1 and A2A Receptors in the Prefrontal Cortex,Hippocampus, and Striatum of the Rat

Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the K_d, but not B_max, values of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) binding to adenosine A₁ receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the K_d and B_max values of [³H]DPCPX binding to adenosine A₁ receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the B_max and K_d values of [³H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([³H]CGS21680) binding to adenosine A_2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [³H]CGS21680 to adenosine A_2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A_2A receptors, but not adenosine A₁ receptors in rat brain.     

Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin

Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.     

25(OH)D3 and 1,25(OH)2D3 serum concentration and breast tissue expression of 1alpha-hydroxylase, 24-hydroxylase and Vitamin D receptor in women with and without breast cancer

1,25(OH)2D3 is an antiproliferative agent that may inhibit proliferation of breast cancer (BC) cells in vitro and BC development in animals. Epidemiological studies have shown a high incidence of BC in people less exposed to solar rays. To unravel the role of Vitamin D3 in BC patients, we have investigated serum levels of 25(OH)D3 and its active form 1,25(OH)2D3 as well as tissue expression of 1alpha-hydroxylase, 24-hydroxylase, and Vitamin D-receptor (VDR), determined by semiquantitative RT-PCR, in 88 Brazilian BC patients and 35 women without cancer (submitted to mammoplasties or resection of benign lesions). Median age of women with and without cancer was 51 and 46 years, respectively, and the majority of BC patients were classified as clinical stage II (67%). Although no differences in 25(OH)D3 serum concentration were found, 1,25(OH)2D3 (40+/-21 pg/ml) levels in BC patients were lower than in women without cancer (53+/-23). Our results indicate that 24-hydroxylase, VDR and 1alpha-hydroxylase mRNA tissue expression is similar in both groups and no correlation between 24-hydroxylase, 1alpha-hydroxylase, and VDR expression in breast tumors was found. A low 1,25(OH)2D3 serum concentration seems to be associated to breast cancer, however, the mechanism involved in this regulation is still unclear.     

Comparison of gas chromatography-mass spectrometry,radioimmunoassay and bioassay for the quantification of gibberellin A9 in norway spruce (Picea abies (L.) Karst.)

Quantitative estimates of gibberellin A₉ in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A₉, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.Abbreviations FW fresh weight - GA₉ gibberellin A₉ - GA₉–Me methylated GA₉ - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MID multiple-ion detection - RIA radioimmunoassay