Impairment of rat postnatal lung alveolar development by glucocorticoids: involvement of the p21CIP1 and p27KIP1 cyclin-dependent kinase inhibitors

It has been shown that glucocorticoids accelerate lung development by limiting alveolar formation resulting from a premature maturation of the alveolar septa. Based on these data, the aim of the present work was to analyze the influence of dexamethasone on cell cycle control mechanisms during postnatal lung development. Cell proliferation is regulated by a network of signaling pathways that converge to the key regulator of cell cycle machinery: the cyclin-dependent kinase (CDK) system. The activity of the various cyclin/CDK complexes can be modulated by the levels of the cyclins and their CDKs, and by expression of specific CDK inhibitors (CKIs). In the present study, newborn rats were given a 4-d treatment with dexamethasone (0.1-0.01 microg/g body weight dexamethasone sodium phosphate daily on d 1-4), or saline. Morphologically, the treatment caused a significant thinning of the septa and an acceleration of lung maturation on d 4. Study of cyclin/CDK system at d 1-36 documented a transient down-regulation of cyclin/CDK complex activities at d 4 in the dexamethasone-treated animals. Analysis of the mechanisms involved suggested a role for the CKIs p21CIP1 and p27KIP1. Indeed, we observed an increase in p21CIP1 and p27KIP1 protein levels on d 4 in the dexamethasone-treated animals. By contrast, no variations in either cyclin and CDK expression, or cyclin/CDK complex formation could be documented. We conclude that glucocorticoids may accelerate lung maturation by influencing cell cycle control mechanisms, mainly through impairment of G1 cyclin/CDK complex activation.

Influence of high-conductivity buffer composition on field-enhanced sample injection coupled to sweeping in CE

The aim of this work was to clarify the mechanism taking place in field-enhanced sample injection coupled to sweeping and micellar EKC (FESI-Sweep-MEKC), with the utilization of two acidic high-conductivity buffers (HCBs), phosphoric acid or sodium phosphate buffer, in view of maximizing sensitivity enhancements. Using cationic model compounds in acidic media, a chemometric approach and simulations with SIMUL5 were implemented. Experimental design first enabled to identify the significant factors and their potential interactions. Simulation demonstrates the formation of moving boundaries during sample injection, which originate at the initial sample/HCB and HCB/buffer discontinuities and gradually change the compositions of HCB and BGE. With sodium phosphate buffer, the HCB conductivity increased during the injection, leading to a more efficient preconcentration by staking (about 1.6 times) than with phosphoric acid alone, for which conductivity decreased during injection. For the same injection time at constant voltage, however, a lower amount of analytes was injected with sodium phosphate buffer than with phosphoric acid. Consequently sensitivity enhancements were lower for the whole FESI-Sweep-MEKC process. This is why, in order to maximize sensitivity enhancements, it is proposed to work with sodium phosphate buffer as HCB and to use constant current during sample injection.

Microbial community, biomarker concentrations and their isotopic signature in sediment of Gullfaks and Tommeliten methane seeps in the Northern North Sea

Gullfaks is one of the four major Norwegian oil and gas fields, located in the northeastern edge of the North Sea Plateau. Tommeliten lies in the greater Ekofisk area in the central North Sea. During the cruises HE 208 and AL 267 several seep locations of the North Sea were visited. At the Heincke seep at Gullfaks, sediments were sampled in May 2004 (HE 208) using a video-guided multiple corer system (MUC; Octopus, Kiel). The samples were recovered from an area densely covered with bacterial mats where gas ebullition was observed. The coarse sands limited MUC penetration depth to maximal 30 centimeters and the highly permeable sands did not allow for a high-resolution, vertical subsampling because of pore water loss. The gas flare mapping and videographic observation at Tommeliten indicated an area of gas emission with a few small patches of bacterial mats with diameters <50 cm from most of which a single stream of gas bubbles emerged. The patches were spaced apart by 10-100 m. Sampling of sediments covered by bacterial mats was only possible with 3 small push cores (3.8 cm diameter) mounted to ROV Cherokee. These cores were sampled in 3 cm intervals. Lipid biomarker extraction from 10 -17 g wet sediment was carried out as described in detail elsewhere (Elvert et al., 2003; doi:10.1080/01490450303894). Briefly, defined concentrations of cholestane, nonadecanol and nonadecanolic acid with known delta 13C-values were added to the sediments prior to extraction as internal standards for the hydrocarbon, alcohol and fatty acid fraction, respectively. Total lipid extracts were obtained from the sediment by ultrasonification with organic solvents of decreasing polarity. Esterified fatty acids (FAs) were cleaved from the glycerol head group by saponification with methanolic KOH solution. From this mixture, the neutral fraction was extracted with hexane. After subsequent acidification, FAs were extracted with hexane. For analysis, FAs were methylated using BF3 in methanol yielding fatty acid methyl esters (FAMES). The fixation for total cell counts and CARD-FISH were performed on-board directly after sampling. For both methods, sediments were fixed in formaldehyde solution. After two hours, aliquots for CARD-FISH staining were washed with 1* PBS (10mmol/l sodium phosphate solution, 130mmol/l NaCl, adjusted to a pH of 7.2) and finally stored in a 1:1 PBS:ethanol solution at -20°C until further processing. Samples for total cell counts were stored in formalin at 4°C until analysis. For sandy samples, the total cell count/CARD-FISH protocol was optimized to separate sand particles from the cells. Cells were dislodged from sediment grains and brought into solution with the supernatant by sonicating each sample onice for 2 minutes at 50W. This procedure was repeated four times and supernatants were combined. The sediment samples were brought to a final dilution of 1:2000 to 1:4000 and filtered onto 0.2µm GTTP filters (Millipore, Eschbonn, Germany).

Trends in lipoplex physical properties dependent on cationic lipid structure, vehicle and complexation procedure do not correlate with biological activity

Using a group of structurally related cytofectins, the effects of different vehicle constituents and mixing techniques on the physical properties and biological activity of lipoplexes were systematically examined. Physical properties were examined using a combination of dye accessibility assays, centrifugation, gel electrophoresis and dynamic light scattering. Biological activity was examined using in vitro transfection. Lipoplexes were formulated using two injection vehicles commonly used for in vivo delivery (PBS pH 7.2 and 0.9% saline), and a sodium phosphate vehicle previously shown to enhance the biological activity of naked pDNA and lipoplex formulations. Phosphate was found to be unique in its effect on lipoplexes. Specifically, the accessible pDNA in lipoplexes formulated with cytofectins containing a γ-amine substitution in the headgroup was dependent on alkyl side chain length and sodium phosphate concentration, but the same effects were not observed when using cytofectins containing a β-OH headgroup substitution. The physicochemical features of the phosphate anion, which give rise to this effect in γ-amine cytofectins, were deduced using a series of phosphate analogs. The effects of the formulation vehicle on transfection were found to be cell type-dependent; however, of the formulation variables examined, the liposome/pDNA mixing method had the greatest effect on transgene expression in vitro. Thus, though predictive physical structure relationships involving the vehicle and cytofectin components of the lipoplex were uncovered, they did not extrapolate to trends in biological activity.

In vivo suppression of the renal Na+/Pi cotransporter by antisense oligonucleotides.

A 20-mer phosphorothioate oligonucleotide (AS1) was designed to hybridize to the message for the rat kidney sodium phosphate cotransporter NaPi-2 close to the translation initiation site. Single intravenous doses of this oligonucleotide were given to rats maintained on a low phosphorus diet to increase NaPi-2 expression. At 3 days after oligonucleotide infusion, rats receiving 2.5 micromol of AS1 exhibited a reduction in renal NaPi-2 to cyclophilin mRNA ratio by 40% +/- 17%, and rats receiving 7.5 micromol of AS1 exhibited a reduction in NaPi-2 to cyclophilin mRNA ratio by 46% +/- 21%. Reversed-sequence AS1 was without effect. The higher dose of 7.5 micromol of AS1 also reduced the rate of phosphate uptake into renal brush border membrane vesicles and the expression of NaPi-2 protein detected by Western blotting in these vesicles. Reversed sequence AS1 was again without effect on these parameters. These results suggest that systemically infused oligonucleotides can exert antisense effects in the renal proximal tubule.

Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica.

Ovine pulmonary surfactant is bactericidal for Pasteurella haemolytica when surfactant and bacteria mixtures are incubated with normal ovine serum. To isolate this component, surfactant (1 mg/ml) was centrifuged at 100,000 x gav, and the supernatant was fractionated by HPLC. Fractions were eluted with acetonitrile (10-100%)/0.1% trifluoracetic acid and tested for bactericidal activity. Amino acid and sequence analysis of three bactericidal fractions showed that fraction 2 contained H-GDDDDDD-OH, fraction 3 contained H-DDDDDDD-OH, and fraction 6 contained H-GADDDDD-OH. Peptides in 0.14 M NaCl/10 microM ZnCl2 (zinc saline solution) induced killing of P. haemolytica and other bacteria comparable to defensins and beta-defensins [minimal bactericidal concentration (MBC)50 range, 0.01-0.06 mM] but not in 0.14 M NaCl/10 mM sodium phosphate buffer, pH 7.2/0.5 mM CaCl2/0.15 mM MgCl2 (MBC50 range, 2.8-11.5 mM). Bactericidal activity resided in the core aspartate hexapeptide homopolymeric region, and MBC50 values of aspartate dipeptide-to-heptapeptide homopolymers were inversely proportional to the number of aspartate residues in the peptide. P. haemolytica incubated with H-DDDDDD-OH in zinc saline solution was killed within 30 min. Ultrastructurally, cells contained flocculated intracellular constituents. In contrast to cationic defensins and beta-defensins, surfactant-associated anionic peptides are smaller in size, opposite in charge, and are bactericidal in zinc saline solution. They are members of another class of peptide antibiotics containing aspartate, which when present in pulmonary secretions may help clear bacteria as a part of the innate pulmonary defense system.

Avaliação de microtécnicas de extração para análise de lamotrigina em plasma de pacientes epilépticos por eletroforese capilar

A lamotrigina (LTG) é um fármaco pertencente à classe das feniltriazinas utilizado no tratamento de crises epilépticas generalizadas e focais e no tratamento adjunto da epilepsia refratária. Devido à alta variabilidade interindividual, às interações medicamentosas e aos efeitos adversos apresentados durante a administração da LTG, a monitorização terapêutica nos pacientes que fazem uso deste fármaco é necessária para ajuste de dose individual e evitar os efeitos adversos. Assim, o objetivo deste trabalho foi a avaliação de duas técnicas de microextração: a microextração em fase líquida com fibras ocas (HF-LPME) e a microextração líquido-líquido dispersiva (DLLME) para análise da lamotrigina em amostras de plasma de pacientes epilépticos. Primeiramente foram definidas as condições eletroforéticas: foi utilizado um capilar de sílica fundida de 75 ?m de diâmetro interno e 50 cm de comprimento efetivo. O eletrólito de corrida (BGE) foi composto por ácido 2-morfolinoetanosulfônico (MES), na concentração de 130 mmol L-1 e pH 5,0. As análises foram realizadas à temperatura de 20°C e tensão de 15 kV. A amostra foi injetada hidrodinamicamente (0,5 psi por 10 s) e a detecção foi feita em 214 nm. Nestas condições a LTG e o padrão interno (PI), lidocaína, puderam ser analisados em menos de 7 minutos. A HF-LPME foi avaliada no modo de 3 fases, usando 500 ?L de plasma e 3,5 mL de solução fosfato de sódio 50 mmol L-1 pH 9,0 como fase doadora. O solvente utilizado para impregnar a fibra foi o 1-octanol. Como fase aceptora foram utilizados 60 ?L de solução de ácido clorídrico pH 4,0. Para avaliação da DLLME, foi necessária uma etapa de pré-tratamento da amostra (500 ?L de plasma) com 1 mL de acetonitrila. Após isto, 1,3 mL do sobrenadante foram adicionados a 4 mL de solução fosfato de sódio 50 mmol L-1 pH 9,0 e 120 ?L de clorofórmio (solvente extrator) foram injetados nesta amostra aquosa e 165 ?L de fase sedimentada foram recuperados. As características de desempenho analítico para ambos os métodos foram avaliadas, sendo obtida linearidade na faixa de concentração plasmática de 1-20 ?g/mL e limite inferior de quantificação (LIQ) de 1 ?g mL-1. Os ensaios de precisão e exatidão apresentaram valores de acordo com os guias oficiais. Além disso, os métodos foram seletivos, não apresentaram efeito residual e as amostras foram estáveis. Os valores de recuperação foram de 54,3 e 23% para HF-LPME e DLLME, respectivamente. Os métodos validados foram aplicados com sucesso em amostras de plasma de pacientes epilépticos em tratamento com a LTG. Além disso, as duas técnicas foram comparadas e a HF-LPME apresentou vantagens em relação à DLLME, mostrando ser uma técnica promissora para análise de matrizes complexas, com reduzido consumo de solvente orgânico e possibilidade de automação.

Controlling Long-Range Ordered Self-assembly of Solid-Binding Peptide Monolayers on Atomically Flat Layered Materials

Thesis (Master's)--University of Washington, 2016-06

Gelatinization of starch in mixed sugar systems

Sugars affect the gelatinization of starch, with the effect varying significantly between sugars. Since many food products contain a mixture of sugar sources, it is important to understand how their mixtures affect starch gelatinization. In a Rapid Visco Analyser study of maize starch gelatinization, changing proportions in binary mixtures of refined sugars saw a largely proportionate change in starch gelatinization properties. However, binary mixture of pure sugars and honey, or a model honey system (the main sugars in honey) and honey responded differently. Generally, replacing 25% or 50% of the refined sugar or model honey system with honey gave a large change in starch gelatinization properties, while further increases in honey level had little further effect. Differences between honey and buffered model honey system (either gluconic acid, or a mixture of citric acid and di-sodium phosphate) showed the sensitivity of starch gelatinization to the composition of the nonsaccharide component. (c) 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Design and construction of an ultrasonic probe for use in a Cryo-Magnet NMR Spectrometer

SINNMR (Sonically Induced Narrowing of the Nuclear Magnetic Resonance spectra of solids), is a novel technique that is being developed to enable the routine study of solids by nuclear magnetic resonance spectroscopy. SINNMR aims to narrow the broad resonances that are characteristic of solid state NMR by inducing rapid incoherent motion of solid particles suspended in a support medium, using high frequency ultrasound in the range 2-10 MHz. The width of the normal broad resonances from solids are due to incomplete averaging of several components of the total spin Hamiltonian caused by restrictions placed on molecular motion within a solid. At present Magic Angle Spinning (MAS) NMR is the classical solid state technique used to reduce line broadening, but: this has associated problems, not least of which is the appearance of many spinning side bands which confuse the spectra. It is hoped that SlNNMR will offer a simple alternative, particularly as it does not reveal spinning sidebands The fundamental question concerning whether the use of ultrasound within a cryo-magnet will cause quenching has been investigated with success, as even under the most extreme conditions of power, frequency and irradiator time, the magnet does not quench. The objective of this work is to design and construct a SINNMR probe for use in a super conducting cryo-magnet NMR spectrometer. A cell for such a probe has been constructed and incorporated into an adapted high resolution broadband probe. It has been proved that the cell is capable of causing cavitation, up to 10 MHz, by running a series of ultrasonic reactions within it and observing the reaction products. It was found that the ultrasound was causing the sample to be heated to unacceptable temperatures and this necessitated the incorporation of temperature stabilisation devices. Work has been performed on the investigation of the narrowing of the solid state 23Na spectrum of tri-sodium phosphate using high frequency ultrasound. Work has also been completed on the signal enhancement and T1 reduction of a liquid mixture and a pure compound using ultrasound. Some preliminary "bench" experiments have been completed on a novel ultrasonic device designed to help minimise sample heating. The concept involves passing the ultrasound through a temperature stabilised, liquid filled funnel that has a drum skin on the end that will enable the passage of ultrasound into the sample. Bench experiments have proved that acoustic attenuation is low and that cavitation in the liquid beyond the device is still possible.

Microstructural analysis of surface and interface zones in concrete

Several of OPC paste and concrete specimens, with different mix proportions, were cast against CPF and impermeable formwork (IF) and the profiles of pore structure, microhardness and scratch hardness of the cover zone were established. The chloride ingress and the depth of carbonation of the surface zone of concrete cast against CPF and IF were investigated. The main mechanisms controlling the ECR processes and the factors affecting such treatment were critically reviewed. Subsequently, as a means of restoring passivation of steel embedded in carbonated concrete, such HCP specimens were subjected to ECR. The influence of ECR on the chemistry of the pore solution and the microstructure of the surface and the steel/cement past interface zones were also studied. The main findings of this investigation were as follows: (a) The thickness of the microstructure gradient of cover concrete is significantly decreased with increasing period of water curing but is relatively unaffected by curing temperature, w/e ratio and the use of cement replacement materials. (b) The scratch hardness technique was shown to be potentially useful for characterising the microstructure and microhardness gradients of the surface zone. (c) A relationship between the microstructure gradient and mass transport properties of the surface zone was established. (d) The use of CPF resulted in a significant reduction in porosity of both the cement paste matrix and the aggregate/cement paste transition zone, and a marked improvement in the resistance of the surface zone to carbonation and the ingress of chloride ions. (e) The ECR treatment resulted in a marked densification of the pore structure and in changes to the pore solution chemistry and the cement phases of near-surface and steel/cement paste transition zones. This effect was more pronounced with current density, period of treatment and particularly with the use of sodium phosphate as an electrolyte.

Synthesis and characterization of photochromic indolyl substituted fulgides and fulgimides

The fulgide and fulgimide family constitutes an important class of organic photochromic compounds. The ability of fulgides and fulgimides to interconvert between two key forms by irradiation of different wavelength of light has made them promising material in optical memory devices, optical switches and sensors, and specialty dyes and inks. Thermal stability and hydrolytic stability of fulgides and fulgimides are essential for their practical applications. A deuterated trifluoromethyl indolylfulgide was synthesized based on the synthetic pathway of the proteo trifluoromethyl indolylfulgide using commercially available deuterated starting materials. Deuteration of the isopropylidene group improved the thermal stability of the indolylfulgide by a factor of 7. ^ Fulgimides are the most important fulgide derivatives. Fulgimides improve the hydrolytic stability of fulgides by replacing the succinic anhydride ring with a succinimide ring. A novel trifluoromethyl N-ethoxycarbonylmethyl indolylfulgimide was synthesized from trifluoromethyl indolylfulgide. The trifluoromethyl indolylfulgide was synthesized on a large scale in five steps with an overall yield of 18%. The indolylfulgide was then converted to indolylfulgimide by aminolysis follow by dehydration. The N-ethoxycarbonylmethyl indolylfulgimide showed enhanced hydrolytic stability and photochemical stability in 70/30 ethanol/water. ^ Three novel aqueous soluble fulgimides, trifluoromethyl carboxylic acid indolylfulgimide, dicarboxylic acid indolylfulgimide, and H-carboxylic acid indolylfulgimide, were synthesized. In sodium phosphate buffer (pH 7.4) at 37 ºC, an unusual hydrolysis of the trifluoromethyl group of the closed form of the carboxylic acid indolylfulgimide resulted in the dicarboxylic acid indolylfulgimide which has an additional carboxylic acid group. The closed form of dicarboxylic acid indolylfulgimide was further decarboxylated to generate H-carboxylic acid indolylfulgimide which was not photochromic. The trifluoromethyl dicarboxylic acid indolylfulgimide is the most robust fulgimide yet reported in aqueous solution. ^ A novel aqueous soluble methyl carboxylic acid indolylfulgimide was synthesized from methyl indolylfulgide. The methyl indolylfulgide was synthesized in five steps with an overall yield of 21%. The methyl carboxylic acid indolylfulgimide was synthesized by aminolysis follow by dehydration. The methyl carboxylic acid indolylfulgimide is expected to have improved thermal and photochemical stability in aqueous solutions relative to the trifluoromethyl analog.^

Atividades biológicas de extratos salinos de sementes de plantas da caatinga contra Aedes Aegypti e investigação da participação de proteínas bioativas

Dengue fever, currently the most important arbovirus, is transmitted by the bite of the Aedes aegypti mosquito. Given the absence of a prophylactic vaccine, the disease can only be controlled by combating the vector insect. However, increasing reports of resistance and environmental damage caused by insecticides have led to the urgent search for new safer alternatives. Twenty - um plant s eed extracts from the Caatinga were prepared , tested and characterized . Sodium phosphate ( 50 mM pH 8.0) was used as extractor. All extracts showed larvicidal and ovipositional deterrence activity . Extracts of D. grandiflora, E. contortisiliquum, A. cearenses , C. ferrea and C. retusa were able to attract females for posture when in low co ncentration . In the attractive concentrations, the CE of E. contortisiliquum and A. cearenses were able to kill 52% and 100% of the larvae respectively . The extracts of A. cearenses , P. viridiflora, E. velutina, M. urundeuva and S. brasiliensis were also pupicides, while extracts of P. viridiflora, E. velutina, E. contortisiliquum , A. cearenses, A. colubrina, D. grandiflora , B. cheilantha , S. spectabilis, C. pyramidalis, M. regnelli e G. americana displayed adulticidal activity. All extracts were toxic to C. dubia zooplankton . The EB of E. velutina and E. contortisiliquum did not affect the viability of fibroblasts . In all extracts were identified at least two potential insecticidal proteins such as enzyme inhibitors, lectins and chitin - binding proteins and components of secondary metabolism . Considering all bioassays , the extracts from A. cearenses, P. viridiflora, E. contortisiliquum , S. brasiliensis, E. velutina and M. urundeuva were considered the most promising . The E. contortisiliquum extracts was the only one who did not show pupicida activity, indicating that its mechanism of action larvicide and adulticidal is related only to the ingesti on of toxic compounds by insect , so it was selected to be fragmenting. As observed for the CE , th e protein fractions of E. contortisiliquum also showed larvicidal activity, highlighting that F2 showed higher larvicidal activity and lower en vironmental toxicity than the CE source. The reduction in the proteolytic activity of larvae fed with crude extra ct and fractions of E. contortisiliquum suggest ed that the trypsin inhibitors ( ITEc) would be resp onsible for larvicidal activity . However the increase in the purification of this inhibitor resulted in loss of larvicidal activity , but the absence of trypsin inhibitor reduced the effectiveness of the fractions , indicating that the ITEC contributes to the larvicidal activity of this extract. Not been observed larvicidal activity and adulticide in rich fraction vicilin, nor evidence of the contribution o f this molecule for the larvicidal activity of the extract. The results show the potential of seeds from plant extracts of Caatinga as a source of active molecules against insects A. aegypti at different stages of its development cycle, since they are comp osed of different active compounds, including protein nature, which act on different mechanisms should result in the death of insec

Microbial community composition in sediments of the Hydrate Ridge (Cascadian Margin) collected during RV SONNE cruises SO143 (1999) and SO148-1 (2000)

Cold seep environments such as sediments above outcropping hydrate at Hydrate Ridge (Cascadia margin off Oregon) are characterized by methane venting, high sulfide fluxes caused by the anaerobic oxidation of methane, and the presence of chemosynthetic communities. This investigation deals with the diversity and distribution of sulfate-reducing bacteria, some of which are directly involved in the anaerobic oxidation of methane as syntrophic partners of the methanotrophic archaea. The composition and activity of the microbial communities at methane vented and nonvented sediments are compared by quantitative methods including total cell counts, fluorescence in situ hybridization (FISH). Bacteria involved in the degradation of particulate organic carbon (POC) are as active and diverse as at other productive margin sites of similar water depths. The availability of methane supports a two orders of magnitude higher microbial biomass (up to 9.6×10**10cells/cm**3). Sediment samples were obtained during RV SONNE cruises SO143-2 and SO148-1 at the crest of southern Hydrate Ridge at the Cascadia convergent margin off the coast of Oregon. Sediment cores of 20 - 40 cm length were obtained using a video-guided multiple corer from gas hydrate bearing sediments and from reference sites not enriched in methane in the surface sediments. Samples for total cell counts were obtained from 1 cm core slices, fixed with 2% formaldehyde and stored cold (4°C) and the quantification of aggregates was done via epifluorescence microscopy after staining the sediments with Acridine Orange Direct Counts (AODC) according to the method of Meyer- Reil (1983, doi:10.1007/BF00395813). Total cell counts were defined as the sum of single cells plus the aggregated cells in the syntrophic consortia. DAPI staining was used to measure ANME2/DSS aggregate sizes via epifluorescence microscopy of FISH-treated samples. For FISH, subsamples of sediment cores were sliced into 1 cm intervals and fixed for 2-3 h with 3% formaldehyde (final concentration), washed twice with 1×PBS (10 mM sodium phosphate; 130 mM NaCl), and finally stored in 1×PBS/EtOH (1:1) at -20°C.

Sodium-dependent phosphate transporters in osteoclast differentiation and function.

Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies.