Hank's Balanced Saline Solution Research Articles

Sol-gel synthesis of lithium doped mesoporous bioactive glass nanoparticles and tricalcium silicate for restorative dentistry: Comparative investigation of physico-chemical structure, antibacterial susceptibility and biocompatibility.

Introduction: The sol-gel method for production of mesoporous bioactive glass nanoparticles (MBGNs) has been adapted to synthesize tricalcium silicate (TCS) particles which, when formulated with other additives, form the gold standard for dentine-pulp complex regeneration. Comparison of TCS and MBGNs obtained by sol-gel method is critical considering the results of the first ever clinical trials of sol-gel BAG as pulpotomy materials in children. Moreover, although lithium (Li) based glass ceramics have been long used as dental prostheses materials, doping of Li ion into MBGNs for targeted dental applications is yet to be investigated. The fact that lithium chloride benefits pulp regeneration in vitro also makes this a worthwhile undertaking. Therefore, this study aimed to synthesize TCS and MBGNs doped with Li by sol-gel method, and perform comparative characterizations of the obtained particles. Methods: TCS particles and MBGNs containing 0%, 5%, 10% and 20% Li were synthesized and particle morphology and chemical structure determined. Powder concentrations of 15mg/10mL were incubated in artificial saliva (AS), Hank's balanced saline solution (HBSS) and simulated body fluid (SBF), at 37°C for 28days and pH evolution and apatite formation, monitored. Bactericidal effects against S. aureus and E. coli, as well as possible cytotoxicity against MG63 cells were also evaluated through turbidity measurements. Results: MBGNs were confirmed to be mesoporous spheres ranging in size from 123nm to 194nm, while TCS formed irregular nano-structured agglomerates whose size was generally larger and variable. From ICP-OES data, extremely low Li ion incorporation into MBGNs was detected. All particles had an alkalinizing effect on all immersion media, but TCS elevated pH the most. SBF resulted in apatite formation for all particle types as early as 3days, but TCS appears to be the only particle to form apatite in AS at a similar period. Although all particles had an effect on both bacteria, this was pronounced for undoped MBGNs. Whereas all particles are biocompatible, MBGNs showed better antimicrobial properties while TCS particles were associated with greater bioactivity. Conclusion: Synergizing these effects in dental biomaterials may be a worthwhile undertaking and realistic data on bioactive compounds targeting dental application may be obtained by varying the immersion media.

Disregarded determinant role of transfection medium in chitosan mediated siRNA delivery

It is well known that the chitosan (CS) properties such as molecular weight (Mw) and deacetylation degree (DD) are crucial for siRNA delivery. In the present study, we have unintentionally found that the transfection medium has much more substantial effect, which has not been reported before. Specifically, the transfection efficiency was considerably increased in Hank’s Balanced Saline Solution (HBSS), with various Mw (30–150 kD) and DD (85–95%) combinations in different cell types. In addition, even though the CS/siRNA nanoparticles physical parameters such as diameter size and polydisperse index were disturbed by cell culture medium or serum, a relatively high knockdown efficiency could still be obtained in HBSS. Moreover, the highly efficient silencing in HBSS even did not require pre-mixing to formulate the CS/siRNA nanoparticles. In conclusion, our findings have confirmed a neglected but crucial important factor of transfection medium in the chitosan mediated siRNA delivery.

Characterization of short‐term storage of the semen ofTrachelyopterus galeatus(Siluriformes: Auchenipteridae) incorporating dimethylsulphoxide (dmso) to the diluent

The purpose of this study was to evaluate the effect of adding dimethylsulphoxide cryoprotectant (DMSO) to the diluent used with refrigerated semen of Trachelyopterus galeatus. Semen samples were analysed for colour, volume, pH, motility rate, concentration and morphological changes. Semen, cooled to 4°C, were diluted in Hanks Balanced Saline Solution (HBSS) and HBSS + DMSO at concentrations of 2.5%, 5.0% and 10% and evaluated at time intervals of 0; 12 h; 24 h; 36 h; 48 h; 60 h and 72 h. The fresh semen (undiluted) was white in colour with a milky appearance, had pH (6 ± 0), motility rate of 90.6 ± 13.4% and concentration of 2.7 ± 0.7 × 106 cells ml−1. Morphological analysis demonstrated that 70.6 ± 0.65% of sperm were normal, while 29.4 ± 1.5% presented some change, such as a bent tail (13.02 ± 0.32%) or a strongly bent tail (4.45 ± 0.13%). Chilled fresh semen were viable for 48 h when they had a motility rate of 35 ± 8.6%. Treatments in concentrations of 2.5% and 5.0% showed motility rates of 30 ± 4% and 27.5 ± 7.5%, respectively, after 60 h of exposure, and were considered efficient. This protocol is therefore indicated for semen conservation in T. galeatus.

In-Vitro Degradation of Hollow Silica Reinforced Magnesium Syntactic Foams in Different Simulated Body Fluids for Biomedical Applications

This article reports the mechanical and biocorrosion behaviour of hollow silica nanosphere (SiO2) reinforced (0.5–2 vol.%) magnesium (Mg) syntactic foams. Room temperature tensile properties’ characterization suggests that the increased addition of hollow silica nanospheres resulted in a progressive increase in tensile yield strength (TYS) and ultimate tensile strength (UTS) with Mg-2 vol.% SiO2 exhibiting a maximum TYS of 167 MPa and a UTS of 217 MPa. The degradation behaviour of the developed Mg-SiO2 syntactic foams in four different simulated body fluids (SBFs): artificial blood plasma solution (ABPS), phosphate-buffered saline solution (PBS), artificial saliva solution (ASS) and Hanks’ balanced saline solution (HBSS) was investigated by using potentiodynamic polarization studies. Results indicate that corrosion resistance of the Mg-SiO2 syntactic foam decreases with increasing chloride ion concentration of the SBF. Mg-1.0 vol.% SiO2 displayed the best corrosion response and its corrosion susceptibility pertaining to corrosion rate and polarisation curves in different SBF solutions can be ranked in the following order: ABPS > PBS > HBSS > ASS. The surface microstructure demonstrated the presence of a better passivated layer on the syntactic foams compared to pure Mg. The observed increase in corrosion resistance is correlated with intrinsic changes in microstructure due to the presence of hollow silica nanospheres. Further, the effect of corrosive environment on the degradation behaviour of Mg has been elucidated.

Bio-corrosion studies of Fe-based metallic glasses

Abstract Fe-based metallic glasses are known for their high corrosion resistance. Also, the corrosion resistance of metallic glasses are greatly influenced by the alloying elements. In the present study, the bio-corrosion resistance of two Fe-based metallic glasses (Fe32Ni36Cr14P12B6 and Fe67Co18B14Si1) has been studied. The bio-compatibility of these metallic glasses are studied in four simulated body fluids (SBFs), namely artificial saliva solution (ASS), phosphate-buffered saline solution (PBS), artificial blood plasma solution (ABP), and Hank’s balanced saline solution (HBSS). Potentiodynamic polarization studies are performed on both the metallic glasses, for understanding their bio-compatibility.

Highly porous polycaprolactone scaffolds doped with calcium silicate and dicalcium phosphate dihydrate designed for bone regeneration

Polycaprolactone (PCL), dicalcium phosphate dihydrate (DCPD) and/or calcium silicates (CaSi) have been used to prepare highly porous scaffolds by thermally induced phase separation technique (TIPS). Three experimental mineral-doped formulations were prepared (PCL-10CaSi, PCL-5CaSi-5DCPD, PCL-10CaSi-10DCPD); pure PCL scaffolds constituted the control group.Scaffolds were tested for their chemical-physical and biological properties, namely thermal properties by differential scanning calorimetry (DSC), mechanical properties by quasi-static parallel-plates compression testing, porosity by a standard water-absorption method calcium release, alkalinizing activity, surface microchemistry and micromorphology by Environmental Scanning electronic Microscopy (ESEM), apatite-forming ability in Hank Balanced Saline Solution (HBSS) by Energy Dispersive X-ray Spectroscopy (EDX) and micro-Raman, and direct contact cytotoxicity.All mineral-doped scaffolds released calcium and alkalinized the soaking medium, which may favor a good biological (osteogenic) response.ESEM surface micromorphology analyses after soaking in HBSS revealed: pure PCL, PCL-10CaSi and PCL-10CaSi-10DCPD kept similar surface porosity percentages but different pore shape modifications. PCL-5CaSi-5DCPD revealed a significant surface porosity increase despite calcium phosphates nucleation (p < 0.05).Micro-Raman spectroscopy detected the formation of a B-type carbonated apatite (Ap) layer on the surface of PCL-10CaSi-10DCPD aged for 28 days in HBSS; a similar phase (but of lower thickness) formed also on PCL-5CaSi-5DCPD and PCL; the deposit formed on PCL-10CaSi was mainly composed of calcite.All PCL showed bulk open porosity higher than 94%; however, no relevant brittleness was observed in the materials, which retained the possibility to be handled without collapsing.The thermo-mechanical properties showed that the reinforcing and nucleating action of the inorganic fillers CaSi and DCPD improved viscoelastic properties of the scaffolds, as confirmed by the increased value of storage modulus and the slight increase in the crystallization temperature for all the biomaterials.A detrimental effect on the mechanical properties was observed in samples with the highest amount of inorganic particles (PCL-10CaSi-10DCPD).All the scaffolds showed absence of toxicity, in particular PCL-10CaSi-10DCPD.The designed scaffolds are biointeractive (release biologically relevant ions), nucleate apatite, possess high surface and internal open porosity and can be colonized by cells, creating a bone forming osteoblastic microenvironment and appearing interesting materials for bone regeneration purposes.

Sensitivity Of Murine Melanoma Cells B16 To Plasma Needle Treatment

Melanoma is the most dangerous form of skin cancer originated from the pigment-producing melanocytes in the basal layer of the epidermis. Its capability to produce metastasis, along with the development of quimio or radioresistance makes this kind of cancer very dangerous and hard to heal, so is important an early detection as well as the search of new treatments. B16 murine cell line has been used extensively as a model for the study of this type of cancer. Non thermal plasma needle has been effectively used in the inactivation of microorganisms, in the disinfection of several types of tissues and to accelerate the wound healing processes. It also has been tested as an inductor of apoptosis in HepG2 cancer cell line. The observed effects of plasma upon cells has been attributed to the generation of reactive oxygen/nitrogen species (RONS), which can disrupt the plasmatic membrane and react with several biomolecules including DNA. The aim of the present work is to evaluate the sensitivity of the murine melanoma cell line B16 to a helium-generated plasma needle exposure. B16 cells were grown at 37°C and 5% CO2 atmosphere, in minimal essential medium with 10% FBS. Cells were harvested by tripsinization, washed twice with Hanks balanced saline solution and further incubated for at least one hour in MEM at 37°C to recover. Blood samples were obtained from healthy donors by venopunction and lymphocytes were separated by using the Ficoll Hypaque technique. Nucleated cells were collected, washed twice with HBSS, resuspended in RPMI-1640 with 10% FBS and kept at 37°C for at least one hour. Both cell types were resuspended in HBSS and then 200 µl aliquots were distributed in a microwell plate and exposed to plasma generated by a flow of 0.7 LPM of helium through a 13.56 MHz radiofrequency generator at a power of 5 W. The needle was kept at a distance of 2 mm. Cell death was evaluated by the fluorescein diacetate technique. After treatments, cells were stained with a 1:1 fluorescein diacetate (80 µg/ml) and ethidium bromide (50 µg/ml) solution and observed with an epifluorescence microscope. Living cells are stained in green while dead cells are stained in red. Genotoxicity was assessed by means of the comet assay. After treatments, cells were mixed with an equal volume of 1% low melting point agarose, poured on top of fully frosted microscope slides and immerse in cold lysis solution for an hour. Slides were then transferred to an electrophoresis cell, covered with the electrophoresis solution (0.3M NaHO, 0.1 mM Na2EDTA) for 20 minutes to allow DNA unwinding and then a current was applied (20 V, 300 mA, 20 minutes). Slides were stained with 60 µl of a 20 µg/ml ethidium bromide solution and observed under an epifluorescence microscope. Comets were scored by means of the Comet Assay IV Analyzer (Perceptive Instruments Inc.). The results show that the exposure to plasma produced cytotoxicity and genotoxicity on B16 cells in a dose-dependent manner. It was observed that the effect of non-thermal plasma was higher in cells of melanoma of mouse comparing to the effect observed in the lymphocytes.

The α-Subunit Regulates Stability of the Metal Ion at the Ligand-associated Metal Ion-binding Site in β3 Integrins

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β3 integrins αIIbβ3 and αVβ3, but a potential role of this interaction on stability of the metal ion at LIMBS in β3 integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β3 integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded αIIbβ3 versus αVβ3, the result of stronger interactions of LIMBS with αV, which reduce stability of the LIMBS metal ion in αVβ3. Replacing the αIIb-LIMBS interface residue Phe(191) in αIIb (equivalent to Trp(179) in αV) with Trp strengthened this interface and destabilized the metal ion at LIMBS in αIIbβ3; a Trp(179) to Phe mutation in αV produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular αIIbβ3 and a W179/F substitution in αVβ3 reduced and increased, respectively, the apparent affinity of Mn(2+) to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in αVβ3 structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.

Islet Transplantation in a Subarachnoid Cavity Surrounding Olfactory Bulb of Diabetic Rats

Islet Transplantation in a Subarachnoid Cavity Surrounding Olfactory Bulb of Diabetic Rats

Fatigue and tensile properties of radicular dentin substrate

The purpose of this study was to compare the fatigue and tensile strengths of radicular dentin. Forty bovine lower central incisors were used, twenty teeth for the fatigue test and twenty teeth for the tensile test. Bovine teeth were each sectioned into coronal and radicular portions. Dentin slabs of 1mm thickness were prepared along the radicular tooth using a low-speed cutting machine and trimmed into dumbbell-shaped specimens. A dentin slab was harvested from each tooth. Subsequently, fatigue and tensile tests were performed in Hank's balanced saline solution at 37°C. The staircase method was employed to determine fatigue strength and its standard deviation. Fracture surfaces were observed by scanning electron microscopy. Mean fatigue strength and tensile strength were 44.3±5.0 and 84.4±8.3MPa, respectively. The fatigue strength of radicular dentin was significantly lower than the tensile strength. The fatigue strength of radicular dentin was only approximately one half of the tensile strength.

Stability, accumulation and cytotoxicity of an albumin-cisplatin adduct

The accumulation and cytotoxicity of a 10 μmol L⁻¹ equimolar human serum albumin-cisplatin adduct (HSA-Pt) was investigated in suspension Ehrlich Ascites Tumor Cells (EATC) and adherent Ehrlich Lettré Ascites Cells (Lettré). HSA-Pt did not induce apoptosis nor was it taken up by the cells to any significant amount within 24 h incubation. The accumulation and cytotoxicity of HSA-Pt was compared to 10 μmol L⁻¹ cisplatin for which a larger accumulation and cytotoxicity were observed in EATC compared to Lettré. The experiment was performed with cell medium exchange every fourth hour as HSA-Pt and cisplatin were not stable in RPMI-1640 with 10% serum. The stability was determined using size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) and after 4 h new platinum peaks were observed. These findings indicate that before conducting cell experiments, the stability of the compound in the cell medium should be investigated especially when long exposure times are applied. Furthermore, HSA-Pt was found to be stable in Hanks Balanced Saline Solution (HBSS) and in Phosphate Buffered Saline (PBS) at pH 5.3, 6.1 and 7.4. Thus, the shift in pH when HSA-cisplatin passes from blood (pH 7.4) to tumor tissue (pH 5-6) is not capable of releasing cisplatin from HSA.

蛍光 X 線分析法を用いたチタン表面におけるリン酸カルシウム析出量の評価

A quantitative analysis of calcium phosphate (CP) layers deposited on metallic titanium substrates was carried out by X-ray fluorescence spectrometry (XRF) in order to evaluate the osteogenic capability of metallic biomaterials. The titanium substrates were prepared by NaOH and heat treatments, and then, they were soaked in Hanks' balanced saline solution (HBSS) at 310 K, leading to the deposition of a CP layer on the sample surface. The resulting samples were analyzed by XRF, and the amount of Ca and P in the CP layers was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). As a result, calibration curves were obtained for determining the amounts of Ca, P and the CP deposition; the XRF quantification of the CP layers was carried out with good accuracy.

Quantitative Evaluation of Calcium Phosphate on Surface of Titanium by X-ray Fluorescence Analysis

A quantitative analysis of calcium phosphate (CP) layers deposited on metallic titanium substrates was carried out by X-ray fluorescence spectrometry (XRF) in order to evaluate the osteogenic capability of metallic biomaterials. The titanium substrates were prepared by NaOH and heat treatments, and then, they were soaked in Hanks’ balanced saline solution (HBSS) at 310 K, leading to the deposition of a CP layer on the sample surface. The resulting samples were analyzed by XRF, and the amount of Ca and P in the CP layers was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). As a result, calibration curves were obtained for determining the amounts of Ca, P and the CP deposition; the XRF quantification of the CP layers was carried out with good accuracy. [doi:10.2320/matertrans.M2009158]

Effect of fibrinogen on the secretion of interleukin-1beta and - 8 by polymorphonuclear leukocytes

To investigate the effect of one of the acute-phase proteins, fibrinogen, on the release of IL-1beta and -8 by human peripheral polymorphonuclear leukocytes (PMN) and the possible role of fibrinogen during the destruction of periodontium. Peripheral PMN were isolated by discontinuous density gradient centrifuging technique. The freshly isolated PMN were suspended in Hank's balanced saline solution (1 x 10(9)/L) supplemented with 0.5% BSA and 0.1% glucose. The levels of IL-1beta and -8 in the supernatants produced by cultured cells upon the addition of human fibrinogen at different concentrations were measured by ELISA technique. Incubated with human fibrinogen at 2 g/L or 10 g/L for different time periods, human peripheral PMN released significantly greater amount of IL-1beta [(10.41 +/- 0.37) - (35.86 +/- 0.30) ng/L or (22.81 +/- 0.45) - (57.77 +/- 2.08) ng/L] and IL-8 [(93.90 +/- 13.95) - (2045.66 +/- 53.03) ng/L or (115.02 +/- 10.61) - (3858.69 +/- 25.65) ng/L] than PMN without the stimulation of fibrinogen (IL-1beta, P < 0.001, and IL-8, P < or = 0.016). The higher concentration of fibrinogen or the longer treatment time, the higher levels of IL-1beta and -8 were released by PMN (P < 0.001). Fibrinogen induced the secretion of pro-inflammatory cytokines IL-1beta and -8 by PMN and may be involved in magnification of the inflammatory response of periodontium and bone resorption.

CaTiO3 coating on titanium for biomaterial application—Optimum thickness and tissue response

The objectives of this study were to determine the optimum thickness of a CaTiO(3) film for biomaterial applications and to investigate the biocompatibility and bone formation of titanium with a CaTiO(3) film. First, CaTiO(3) films of 10, 20, 30, and 50 nm in thickness were deposited on titanium substrates using radiofrequency magnetron sputtering followed by annealing at 873 K in air for 7.2 ks. The optimum thickness of the CaTiO(3) film for bone formation was determined by comparison with its performance regarding calcium phosphate formation in Hanks' balanced saline solution (HBSS). Regarding calcium phosphate formation, the performance of the specimen with a 50-nm-thick CaTiO(3) film was superior to those of specimens with other thicknesses. A titanium prism with a CaTiO(3) film of 50-nm in thickness was surgically inserted in both soft and hard rat tissues. The biocompatibility of CaTiO(3)-deposited titanium and bone formation on it was investigated by histological observations. A slight inflammatory reaction was observed around the titanium with the 50-nm-thick CaTiO(3) film, while no severe response, such as degeneration and necrosis, was observed in either soft or hard rat tissue. New bone formation on the titanium plate with the CaTiO(3) film was more active than that without the film. The 50-nm-thick CaTiO(3) film has biocompatibility and can facilitate new bone formation in vivo, and, consequently, it is an excellent surface modification method for biomaterial applications.

Calcium phosphates formation on CaTiO3 coated titanium

In this study, performance of calcium phosphate formation of CaTiO3 coating film on Ti in Hanks' balanced saline solution (HBSS) was investigated. CaTiO3 thin films with a thickness of 50 nm were deposited on Ti using radiofrequency (RF) magnetron sputtering. The temperature of Ti substrate was adjusted to room temperature (RT) and 873 K. Thereafter, the specimens deposited at RT were annealed at 873 K in air for 7.2 ks. The films were characterized by grazing incident angle X-ray diffractometry (GI-XRD) and X-ray photoelectron spectroscopy (XPS). After immersion in HBSS for 60 d, on CaTiO3 coated Ti, the formation of hydroxyapatite (HAP) was observed. Furthermore, HAP layer formed was thicker on the specimen on which CaTiO3 film was deposited at RT and annealed than that prepared at 873 K. The major difference between both specimens was the chemical properties of the outermost surface. In summary, CaTiO3 thin film deposited at RT and followed by annealing at 873 K for 7.2 ks in air enhances calcium phosphate formation ability on Ti.

The human ABCG1 gene: identification of LXR response elements that modulate expression in macrophages and liver

The ABC transporter ABCG1 (ATP binding cassette transporter G1), expressed in macrophages, liver, and other tissues, has been implicated in the efflux of cholesterol to high density lipoprotein. The ABCG1 gene is transcriptionally activated by cholesterol loading and activators of liver X receptors (LXRs) and retinoid X receptors (RXRs) through genomic sequences that have not been fully characterized. Here we show that ABCG1 mRNA is induced by LXR agonists in RAW264.7 macrophage cells, HepG2 hepatoma cells, and primary mouse hepatocytes. We identify two evolutionarily highly conserved LXR response elements (LXREs), LXRE-A and LXRE-B, located in the first and second introns of the human ABCG1 gene. Each element conferred robust LXR-agonist responsiveness to ABCG1 promoter-directed luciferase gene constructs in RAW264.7 and HepG2 cells. Overexpression of LXR/RXR activated the ABCG1 promoter in the presence of LXRE-A or LXRE-B sequences. In gel-shift assays, LXR/RXR heterodimers bound to wild-type but not to mutated LXRE-A and LXRE-B sequences. In chromatin immunoprecipitation assays, LXR and RXR were detected at LXRE-A and -B regions of DNA of human THP-1 macrophages. These studies clarify the mechanism of transcriptional upregulation of the ABCG1 gene by oxysterols in macrophages and liver, two key tissues where ABCG1 expression may affect cholesterol balance and atherogenesis.

Silicon Contamination Adsorbed on Pure Titanium Plate during Soaking Test in Hanks' Balanced Saline Solution

Silicon contamination adsorbed on pure Titanium plate during soaking test in Hanks’ balanced saline solution (HBSS) and distilled water was investigated. Amounts of silicon eluted from various materials of vessels were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). The amounts of silicon deposited on the surface of pure titanium plates after soaking were evaluated by XPS. The amounts of eluted silicon in the solutions were dependent on the materials of vessels and pH value of solutions. The surface concentration of silicon on titanium plates reflected the amounts of eluted silicon in solutions. It is possible to reduce the silicon contamination by choosing suitable vessels as containers for solutions. However, in spite of using a vessel with no silicon elution, surface concentration of silicon on titanium plates was detected because of the silicon impurity of solution itself.

RhoG Signals in Parallel with Rac1 and Cdc42

RhoG is a member of the Rho family of small GTPases and shares high sequence identity with Rac1 and Cdc42. Previous studies suggested that RhoG mediates its effects through activation of Rac1 and Cdc42. To further understand the mechanism of RhoG signaling, we studied its potential activation pathways, downstream signaling properties, and functional relationship to Rac1 and Cdc42 in vivo. First, we determined that RhoG was regulated by guanine nucleotide exchange factors that also activate Rac and/or Cdc42. Vav2 (which activates RhoA, Rac1, and Cdc42) and to a lesser degree Dbs (which activates RhoA and Cdc42) activated RhoG in vitro. Thus, RhoG may be activated concurrently with Rac1 and Cdc42. Second, some effectors of Rac/Cdc42 (IQGAP2, MLK-3, PLD1), but not others (e.g. PAKs, POSH, WASP, Par-6, IRSp53), interacted with RhoG in a GTP-dependent manner. Third, consistent with this differential interaction with effectors, activated RhoG stimulated some (JNK and Akt) but not other (SRF and NF-kappaB) downstream signaling targets of activated Rac1 and Cdc42. Finally, transient transduction of a tat-tagged Rac1(17N) dominant-negative fusion protein inhibited the induction of lamellipodia by the Rac-specific activator, Tiam1, but not by activated RhoG. Together, these data argue that RhoG function is mediated by signals independent of Rac1 and Cdc42 activation and instead by direct utilization of a subset of common effectors.

The Role of Soluble Interleukin (IL)‐6 Receptor in Mediating the effects of IL‐6 on Matrix Metalloproteinase‐1 and Tissue Inhibitor of Metalloproteinase‐1 Expression by Gingival Fibroblasts

Interleukin-6 (IL-6) is a multifunctional cytokine thought to play a role in the tissue destruction that characterizes periodontal disease. IL-6 exerts its cellular effects through a cell-surface receptor which also exists in a soluble form (sIL-6r). This study investigated the effects of IL-6 on matrix metalloproteinase (MMP)-1 activity in gingival fibroblast cultures, specifically determining the role of the sIL-6r in mediating these actions. Fibroblasts were grown to confluence, washed in Hank's balanced saline solution (HBSS), and then cultured for 72 hours in serum-free medium supplemented with 0.2% bovine serum albumin, 1 microgram/ml Escherichia coli LPS and containing various combinations of IL-6 and its soluble receptor over the concentration range 0 to 1,000 ng/ml. MMP-1 and tissue inhibitor of MMP (TIMP)-1 protein levels in the conditioned medium were assessed by enzyme-linked immunosorbent assay (ELISA) and collagenolytic activity determined using a 3H-acetylated type I collagen degradation assay. Results indicated that the addition of IL-6 alone to cultures, over the concentration range 0 to 1,000 ng/ml, had no significant effect on MMP-1 protein expression. However, addition of IL-6 in combination with its soluble receptor resulted in a statistically significant, dose-dependent upregulation in MMP-1 expression. The IL-6/sIL-6r combination also induced a significant increase in collagenolytic activity in cultures. IL-6 and sIL-6r, either alone or in combination, had no marked effect on TIMP expression or cell growth. These data strongly suggest that future clinical studies investigating the role of IL-6 in periodontal disease must also determine the levels of sIL-6r within the periodontal tissues.