Incubation In Saline Research Articles

Hypoxia-inducible Factor-1 Deficiency Results in Dysregulated Erythropoiesis Signaling and Iron Homeostasis in Mouse Development

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of genes whose products play critical roles in energy metabolism, erythropoiesis, angiogenesis, and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif1alpha gene, which encodes the hypoxia-responsive alpha subunit of HIF-1, causes cardiac, vascular, and neural malformations resulting in lethality by embryonic day 10.5 (E10.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-1alpha-deficient embryos, as well as decreased hemoglobin content in erythroid colonies from HIF-1alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif1alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif1alpha-/- embryos. The erythropoietic defects in HIF-1alpha-deficient erythroid colonies could not be corrected by cytokines, such as vascular endothelial growth factor and Epo, but were ameliorated by Fe-SIH, a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis, Hif1alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin, Fpn1, Irp1, and frascati. We conclude that dysregulated expression of genes encoding Epo, EpoR, and iron regulatory proteins contributes to defective erythropoiesis in Hif1alpha-/- yolk sacs. These results identify a novel role for HIF-1 in the regulation of iron homeostasis and reveal unexpected regulatory differences in Epo/EpoR signaling in yolk sac and embryonic erythropoiesis.

Incorporation of α-Amylase Enzyme and a Bioactive Filler into Hydrophilic, Partially Degradable, and Bioactive Cements (HDBCs) as a New Approach To Tailor Simultaneously Their Degradation and Bioactive Behavior

Hydrophilic, partially degradable, and bioactive cements (HDBCs) are starch-containing cements intended to degrade partially in the human body and, in so doing, allow for bone ingrowth inside the pores formed during degradation. Therefore, the study of degradation and bioactivity behavior was performed to assess the suitability of the current HDBCs formulations to achieve those aims. The degradation profile of HDBCs was studied under different conditions, including incubation in phosphate-buffered saline (PBS) and PBS supplemented with alpha-amylase at different concentrations. Thermostable alpha-amylase was also added to some formulations to allow control of the degradation rate and its extent. In a second stage the simultaneous phenomena of enzymatic degradation and bioactivity (both in vitro) was studied. We observed that the degradation of starch present in HDBCs can be easily controlled by the amount of alpha-amylase added to the cement and high values of degradation may be achieved if high enough quantities of enzyme are incorporated. However, the maximum degradation extent is much more dependent on the total amount of starch present in the formulation than on the amount of enzyme added to it: for full pore connectivity, the amount of starch should be higher than the percolation threshold for a 3D specimen. Nonetheless, calcium phosphate was able to nucleate and spread in inner pores of the cement, formed due to degradation, if they were interconnected. For a more thorough covering of the pores with calcium phosphates the amount of starch present in HDBCs should be increased to be higher than the percolation threshold.

Specific Recognition of Apoptotic Cells Reveals a Ubiquitous and Unconventional Innate Immunity

The purpose of physiological cell death is the noninflammatory clearance of cells that have become inappropriate or nonfunctional. Consistent with this function, the recognition of apoptotic cells by professional phagocytes, including macrophages and dendritic cells, triggers a set of potent anti-inflammatory responses manifest on multiple levels. The immediate-early inhibition of proinflammatory cytokine gene transcription in the phagocyte is a proximate consequence of recognition of the apoptotic corpse, independent of subsequent engulfment and soluble factor involvement. Here, we show that recognition is linked to a characteristic signature of responses, including MAPK signaling events and the ablation of proinflammatory transcription and cytokine secretion. Specific recognition and response occurs without regard to the origin (species, tissue type, or suicidal stimulus) of the apoptotic cell and does not involve Toll-like receptor signaling. These features mark this as an innate immunity fundamentally distinct from the discrimination of "self" versus "other" considered to be the hallmark of conventional immunity. This profound unconventional innate immune discrimination of effete from live cells is as ubiquitous as apoptotic cell death itself, manifest by professional and nonprofessional phagocytes and nonphagocytic cell types alike. Innate apoptotic immunity provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses dynamically and may act systemically as a powerful physiological regulator of immunity.

Matching Supply with Demand

Insulin production in pancreatic β cells is stepped up in tandem with glucose-dependent insulin secretion. Calcium, which triggers secretion, stimulates the calpain-dependent cleavage of islet cell autoantigen 512 (ICA512), a catalytically inactive member of the receptor tyrosine phosphatase family that is associated with secretory granules. The cleaved cytosolic fragment (ICA512-CCF) translocates to the nucleus, where it stimulates insulin gene transcription. Mziaut et al. stimulated rat insulinoma INS-1 cells with glucose and a depolarizing concentration of KCl and found that a calpain inhibitor attenuated the increase in pro-ICA512 abundance seen after 2 hours of stimulation followed by 4 hours of incubation in glucose-free saline. Moreover, cells expressing an ICA512-CCF-GFP fusion protein showed increased abundance of endogenous ICA512 mRNA and pro-ICA512, as well as increased abundance of mRNA encoding other components of secretory granules. ICA512-CCF-GFP expression enhanced the nuclear abundance of total and phosphorylated STATs (signal transducers and activators of transcription), and glucose only enhanced the growth-hormone dependent nuclear accumulation of STAT5b in cultured pancreatic islets if they expressed ICA512. Both coimmunoprecipitation and fluorescence resonance energy transfer analysis indicated that ICA512 bound to STAT5b; moreover, a fusion protein containing the cytoplasmic domain of ICA512 inhibited the in vitro dephosphorylation of STAT5. ICA512-CCF-GFP enhanced STAT5-dependent transcriptional activity, and STAT5b knockdown led to a decrease in pro-ICA512 abundance. The E3 SUMO ligase PIASy bound to and sumoylated ICA512; PIASy inhibited STAT5-dependent transcriptional activity and an in vitro assay indicated that sumoylation of ICA512 decreased its binding to STAT5. Thus, the data define a retrograde pathway involving STAT5 through which ICA512 regulates production of secretory granule components. H. Mziaut, M. Trajkovski, S. Kersting, A. Ehninger, A. Altkrüger, R. P. Lemaitre, D. Schmidt, H. D. Saeger, M. S. Lee, D. N. Drechsel, S. Müller, M. Solimena, Synergy of glucose and growth hormone signalling in islet cells through ICA512 and STAT5. Nat. Cell Biol. 8 , 435-445 (2006). [PubMed]

Modulated release of bioactive protein from multilayered blended PLGA coatings

The objective of this study was to develop a poly( d,l-lactic-co-glycolic acid) (PLGA)-based coating system for producing biologically-inspired delivery profiles. Protein-loaded microspheres were made from PLGA (50:50) terminated with carboxylic acid groups (PLGA-2A) blended either with more hydrophobic PLGA (50:50) having lauryl ester endcaps (PLGA-LE) or with the more hydrophilic Pluronic F-127 (PF-127). Dense coatings were formed by pressure-sintering the microspheres. Altering hydrophobicity changed the water concentration within coatings, and consequently the time to onset of polymer degradation and protein release was modulated. After blending up to 8% Pluronic, degradation by-products began accumulating immediately upon incubation in saline, whereas, degradation was delayed for up to 14 days with blending of up to 30% PLGA-LE. Primary protein release peaks from one-layer coatings could be created from 7 to 20 days using 8% PF-127 or 30% PLGA-LE blends, respectively. Multilayered coatings of different blends generated several release peaks, with their temporal occurrence remaining approximately the same when layers of other hydrophobicity were added above or below. To allow design of coatings for future use, results were used to construct a model based on Fourier analysis. This polymer blend system and model can be used to mimic temporally varying profiles of protein expression.

Concentric and Eccentric Carved Costal Cartilage

To determine if a systematic, concentric graft-carving technique results in less warping than eccentric carving. Costal cartilage from ribs 3 through 9 were harvested from fresh human cadavers. Concentric dorsal grafts were carved from cartilage obtained from one side of the cadaver (n = 7) and eccentric grafts were carved from the opposite side (n = 11). After carving, grafts were placed in saline sponges (isotonic sodium chloride) for 1 hour, and photographs were then taken from a lateral and a frontal view. Identical photographs were taken after 2 weeks of incubation in saline (36.7 degrees C). The angle of curvature was measured from the photographs. More warping occurred in the eccentric group than the concentric group on lateral view at 1 hour (4.4 degrees vs 0.4 degrees; P<.001) and at 2 weeks (5.8 degrees vs 1.4 degrees; P = .003). Eccentric grafts progressively warped between the 1-hour and 2-week points. Meticulous concentric carving of costal cartilage grafts may minimize the complication of graft warping.

In Vitro Models for Assessing Transcranial Ultrasound-Enhanced Thrombolysis

To the Editor: Contrary to the promising results of recent clinical studies, Pfaffenberger et al conclude from in vitro data that diagnostic ultrasound (US) is not suitable for transcranial enhancement of tissue plasminogen activator (tPA) thrombolysis.1 Their contention is based on surprising control data showing significant clot lysis without tPA and plasminogen and relatively modest increases in clot dissolution with tPA. Interestingly, their reported clot weight loss of 21.8% after 1 hour of incubation in saline alone is much higher than that which they recently reported in another journal using the same methods (2.8%±1.8%2 and 11.0%±3.8%3). Likewise, the effect of tPA alone (27.4%) also differs with earlier results of 19.9%±4.3%3 and 22.7%±9.0%.2 These discrepancies seem important, because a 19.9% effect of tPA alone would have been lower than combined US and recombinant tPA through the skull (26.2%), thus encouraging the authors to positively assess transcranial US thrombolysis. Because clot dissolution is dependent on the amount of plasminogen present and on the amount of tPA available for activation of the plasminogen, little clot lysis should occur during the first 60 minutes of incubation in saline, with or without tPA. To study our hypothesis, we used experimental conditions similar to those of Pfaffenberger et al. 1.5 mL of citrated …

Engineering of volume-stable adipose tissues

Autologous adipose tissues have been clinically used for augmentation of soft tissues lost due to mastectomy or lumpectomy in plastic and reconstructive surgery. However, this therapy has problems of absorption and subsequent volume loss of the implanted adipose tissues. In this study, volume-stable adipose tissues were engineered in vivo using mechanical support structures fabricated from biodegradable synthetic polymers. Dome-shaped mechanical support structures were fabricated by reinforcing poly(glycolic acid) fiber-based matrices with poly( L-lactic acid). The support structures were placed into subcutaneous pockets of athymic mice, and human preadipocytes suspended in fibrin matrix were injected into the space under the support structures (group I). Injection of either fibrin matrix without preadipocytes under the support structures (group II) or fibrin matrix containing preadipocytes into subcutaneous spaces with no support structures (group III) served as controls. Six weeks after implantation, the original implant volume was maintained approximately in groups I and II, whereas, group III showed significant implant shrinkage. The compressive modulus of the mechanical support structures did not change significantly over 6-week incubation in phosphate-buffered saline at 37 °C. Histological analyses of the implants showed regeneration of adipose tissues in group I. In contrast, groups II and III did not show extensive adipose tissue formation. This study demonstrates that volume-stable adipose tissues can be engineered in vivo using mechanical support structures. This technique offers the potential for augmentation of adipose tissues with volume conservation.

Multilayered thin films that sustain the release of functional DNA under physiological conditions.

The development of thin films and coatings that control the release of DNA from the surfaces of materials could have a significant impact on localized approaches to gene therapy. Here, we report multilayered polyelectrolyte assemblies that sustain the release of functional plasmid DNA from the surfaces of model substrates under physiological conditions. Multilayered assemblies consisting of alternating layers of plasmid DNA encoding for enhanced green fluorescent protein (EGFP) and a synthetic degradable polyamine were deposited on planar silicon and quartz substrates using a layer-by-layer fabrication process. Film growth was monitored by ellipsometry and UV spectrophotometry and correlated linearly with the number of polymer and plasmid layers deposited. In general, the thickness of deposited layers was found to be a function of both the pH and the ionic strength of the polyelectrolyte solutions used. Films up to 100 nm thick were investigated in this study. These assemblies erode gradually upon incubation in phosphate-buffered saline at 37 degrees C, as determined by ellipsometry and UV spectrophotometry, and sustain the release of incorporated plasmid into the incubation medium for a period of up to 30 h. Characterization of the released plasmid by agarose gel electrophoresis revealed that the DNA was released in a relaxed, open circular, rather than supercoiled, topology; subsequent cell transfection experiments demonstrated that the released plasmid is transcriptionally viable and promotes the expression of EGFP in the COS-7 cell line. These layered materials could represent an approach to the controlled administration of one or more functional DNA constructs from the surfaces of biomedical materials and devices.

Relationship of messenger RNA reverse transcriptase-polymerase chain reaction signal to Campylobacter spp. viability.

SUMMARY. Discriminating viable from dead cells is of importance in the development of bacterial detection methods. A positive reverse transcriptase-polymerase chain reaction (RT-PCR) amplification signal was tested as a potential predictor of chick colonization. Some researchers have suggested that the presence of messenger RNA (mRNA) may not correlate with cell viability. Chicken colonization by cells that have positive mRNA signal but that are noncultivable would provide a correlation in cell viability and persistence of mRNA. The role of a viable but noncultivable (VBNC) form of Campylobacter spp. for colonization of poultry could be verified by such an mRNA signal. The levels of four strains of Campylobacter spp., previously isolated from poultry feces, declined progressively over time, and loss of cultivability occurred after 6 to 7 wk incubation in phosphate-buffered saline (PBS) at 4 C. Cold-stored, noncultivable and heat-inactivated (60 C for 10 min) Campylobacter spp. produced inconsistent amplified products from RT-PCR assay, depending on the target transcripts and strains used, although all fresh cultures showed mRNA signals. For the most part, signals of mRNA species from VBNC and heat-killed Campylobacter spp. AH-1, AH-2, and CH-3 persisted. RT-PCR amplification of transcripts originating from the tkt and cmp genes and a 256-base pair amplicon (from a previously described putative haem-copper oxidase) provided consistent signals, whereas transcripts from the flaA gene did not. Presumed VBNC and heat-inactivated Campylobacter spp., which produced positive mRNA signal but was not cultivable by conventional culture-based methods, did not establish colonization in the intestine of chicks 7 days after challenge. These results lead us to question the correlation between mRNA durability with cell viability as well as the significance of the VBNC cells in environmental transmission of Campylobacter spp.

Preparation of degradable porous structures based on 1,3-trimethylene carbonate and D,L-lactide (co)polymers for heart tissue engineering.

Biodegradable porous scaffolds for heart tissue engineering were prepared from amorphous elastomeric (co)polymers of 1,3-trimethylene carbonate (TMC) and D,L-lactide (DLLA). Leaching of salt from compression-molded polymer-salt composites allowed the preparation of highly porous structures in a reproducible fashion. By adjusting the salt particle size and the polymer-to-particle weight ratio in the polymer-salt composite preparation the pore size and porosity of the scaffolds could be precisely controlled. The thermal properties of the polymers used for scaffold preparation had a strong effect on the morphology, mechanical properties and dimensional stability of the scaffolds under physiological conditions. Interconnected highly porous structures (porosity, 94%; average pore size, 100 microm) based on a TMC-DLLA copolymer (19:81, mol%) had suitable mechanical properties and displayed adequate cell-material interactions to serve as scaffolds for cardiac cells. This copolymer is noncytotoxic and allows the adhesion and proliferation of cardiomyocytes. During incubation in phosphate-buffered saline at 37 degrees C, these scaffolds were dimensionally stable and the number average molecular weight (Mn) of the polymer decreased gradually from 2.0 x 10(5) to 0.3 x 10(5) in a period up to 4 months. The first signs of mass loss (5%) were detected after 4 months of incubation. The degradation behavior of the porous structures was similar to that of nonporous films with similar composition and can be described by autocatalyzed bulk hydrolysis.

A biodegradable electrical bioconductor made of polypyrrole nanoparticle/poly(D,L-lactide) composite: A preliminary in vitro biostability study.

The electrical stability of a novel polypyrrole (PPy)/poly(D,L-lactide) (PDLLA) composite was studied in vitro and compared with that of PPy-coated polyester fabrics. Specimens were incubated in Ringer's solution at 37 degrees C for up to 8 weeks with or without the circulation of DC current under a constant 100 mV voltage. In situ current variation with incubation time was recorded. The AC volume electrical conductivity of the specimens before and after incubation in phosphate-buffered saline was recorded using a frequency analyzer. Water absorption and weight loss were monitored metrologically. Changes in the oxidation state of incubated PPy were analyzed with X-ray photoelectron spectroscopy. The morphological changes were observed with scanning electron microscopy, and the glass transition temperature of the PDLLA was investigated using differential scanning calorimetry. The PPy/PDLLA composite in Ringer's solution sustained a relatively stable conductivity up to 8 weeks after an initial period of "conditioning." The PPy-coated fabrics experienced a rapid loss of conductivity when subjected to electrical circulation and regained part of it when disconnected. The volume conductivity of the nonincubated PPy/PDLLA membrane behaved as a typical conductor in the low-frequency range. The mechanisms involved in the various electrical behaviours of the PPy/PDLLA composite and PPy-coated fabrics are discussed. In conclusion, the PPy/PDLLA composite was able to deliver a biologically significant electrical current in a simulated biological solution for up to 8 weeks and therefore may be considered as a first-generation synthetic biodegradable bioconductor.

The Shank Family of Postsynaptic Density Proteins Interacts with and Promotes Synaptic Accumulation of the βPIX Guanine Nucleotide Exchange Factor for Rac1 and Cdc42

The Shank/ProSAP family of multidomain proteins is known to play an important role in organizing synaptic multiprotein complexes. Here we report a novel interaction between Shank and beta PIX, a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases. This interaction is mediated by the PDZ domain of Shank and the C-terminal leucine zipper domain and the PDZ domain-binding motif at the extreme C terminus of beta PIX. Shank colocalizes with beta PIX at excitatory synaptic sites in cultured neurons. In brain, Shank forms a complex with beta PIX and beta PIX-associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42. Importantly, overexpression of Shank in cultured neurons promotes synaptic accumulation of beta PIX and PAK. Considering the involvement of Rac1 and PAK in spine dynamics, these results suggest that Shank recruits beta PIX and PAK to spines for the regulation of postsynaptic structure.

Selective in vitro labeling of white blood cells using 99mTc-labeled liposomes

We describe a method by which endocytosis-based radiolabeling of WBC is achieved using 99mTc-liposomes of optimal size and charge, and of a composition that assures both in vitro (whole blood) and intracellular stability of the radiopharmaceutical. In our study, excellent in vitro stability of 99mTc-liposomes with 95% labeling efficiency was observed with >90% stability up to 6 h and a minimum of 85% after 24 h of incubation either in normal saline or serum. Total WBC labeling efficiency using 99mTc-liposomes determined by radio-thin layer chromatographic analysis was 30.6 ± 2.21%, 20.89 ± 1.31% for monocytes and 9.7 ± 1.74% for polymorphonuclears. Negligible activity was bound to red blood cells. The procedure did not affect the cell viability and the separation of the free 99mTc-liposomes from the cells was done by centrifugation.

Microencapsulation of carbon particles used as oxygen sensors in EPR oximetry to stabilize their responsiveness to oxygen in vitro and in vivo

The electron paramagnetic resonance (EPR) spectra of some paramagnetic materials exhibit a pO2 (partial pressure of oxygen)-dependent linewidth. By recording the EPR linewidth in vivo using low-frequency EPR spectrometers, it is possible to measure the partial pressure of oxygen in tissues. It has been found, however, that some of the paramagnetic materials with optimal spectroscopic properties in vitro may lose or change their responsiveness to oxygen in tissues. The aim of this study was to microencapsulate paramagnetic particles by biopolymers in order to stabilize their responsiveness to oxygen. Carbohydrate char particles (Bubinga) were encapsulated with different biopolymers: cellulose acetate or cellulose triacetate, silicone and polyurethane. The performance of the materials was evaluated in vitro and in vivo. X-band EPR spectroscopy was used to test the variation of the calibration curve (EPR linewidth as a function of the pO2) after incubation in saline and after prolonged residence in tissues. The stability of the responsiveness to pO2 in vivo was carried out by L-band EPR spectroscopy using mice that received injection of the oxygen sensors in the muscles. After residence in saline and prolonged residence in tissues, only the calibration curve of the silicone-coated (coating weight of 0.5% (w/w)) paramagnetic materials remained unchanged, while those of oxygen sensors coated with cellulose acetate, cellulose triacetate and polyurethane changed.

Effect of intraocular lens haptic compressibility on the posterior lens capsule after cataract surgery

Purpose: To evaluate the effect of intraocular lens (IOL) haptic compressibility on the posterior capsule after cataract surgery. Setting: Teaching hospital, London, United Kingdom. Methods: In this randomized prospective study, 60 patients had standardized phacoemulsification with in-the-bag placement of a poly(methyl methacrylate) (PMMA) (Storz P497UV) or hydrogel (Storz Hydroview H60M) IOL. Both IOLs had PMMA haptics of identical configuration and length. The IOL haptic compressibility was measured in air and then during incubation in saline at 37°C over 1 month. Digital retroillumination imaging was performed 1, 7, 28, 90, 180, 360, and 720 days postoperatively. The presence and duration of postoperative capsule folds were recorded and correlated with the haptic compressibility measurements, lens epithelial cell (LEC) growth patterns on the posterior capsule at 6 months, and the extent of posterior capsule opacification. Results: On the first postoperative day, 21 patients (88%) in the Hydroview group had posterior capsule folds that persisted in 12 patients (50%) for 2 years. Nineteen patients (68%) in the PMMA group had folds at day 1 ( P = .01), with 1 patient (3%) still having folds at 1 month ( P = .0002) and no patient having folds at 3 months. At 6 months, 11 patients (46%) in the Hydroview group and no patient in the PMMA group had LEC growth in the direction of the folds. The PMMA IOLs showed a greater decrease in haptic compressibility during incubation. Conclusions: Haptic compressibility should be an important consideration in IOL design. The results suggest that to avoid posterior capsule folds, the compressibility should be less than 2.5 mN.

Rapid fusion of granules with neutrophil cell membranes in hypertensive patients may increase vascular damage

In essential hypertension (EHT) the presence of a metabolic syndrome increases the risk of cardiovascular disease. A cell membrane abnormality is implicated but its role in cardiovascular disease is unclear. Neutrophil accumulation, which occurs by β 2-integrin (CD11b/CD18) expression, followed by release of proinflammatory factors from primary vesicles is an important factor in vascular damage. CD11b and CD69 expression on neutrophils from normal controls and EHT patients was determined by fluorescence-activated cell scanning. Neutrophils were activated with phorbol myristate acetate (PMA). Protein kinase C (PKC) and calpain were inhibited with bisindolylmaleimide and E64d, respectively. In EHT patients CD11b was not increased on neutrophils at rest. However, EHT neutrophils more readily expressed CD11b on incubation in phosphate-buffered saline and more cells went on to exocytose primary granules indicated by expression of CD69. Stimulation with PMA caused more rapid activation in EHT neutrophils with expression of CD11b, followed rapidly by exocytosis of primary granules. Bisindolylmaleimide slowed but did not prevent CD11b expression, which, together with primary granule exocytosis, continued to be faster in EHT neutrophils. E64d also slowed but did not prevent either CD11b expression or primary granule exocytosis, but this inhibitor did abolish the difference between NC and EHT neutrophils. The membrane abnormality in EHT may contribute to cardiovascular risk by increasing the rate of vesicle fusion with the cell membrane to increase neutrophil accumulation and release of inflammatory agents at sites of vascular damage. Calpain activation may be the rate-limiting component that is abnormal.

Durability of sodium sulfate‐treated polylysine‐alginate microcapsules

Chelated hollow microcapsules are unstable under in vitro conditions because of their hygroscopic nature. Nongel inducing cations, such as Na+, stabilize the outer membrane of the alginate-polylysine-alginate microcapsules leading to more stable beads. We made different batches of empty capsules with a mean ± SEM diameter of 607 ± 11 microns, and found that within 1 week of incubating these capsules in normal saline at 37°C, they increased to 718 ± 10 microns (p < 0.05, n = 5). In initial experiments, we made different batches of capsules and divided them into two groups. One group was left untreated (control) whereas the other was treated with 6 mM Na2SO4 for 30 min, before incubation in saline at 37°C. Control capsules increased in weight and size, before beginning to melt in less than 1 week. In contrast, treated capsules rapidly lost weight and remained intact during 1 month of follow-up. In perifusion experiments, we found no deleterious effect of sodium sulfate treatment on the function of islets enclosed in the capsules. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res 54: 396–399, 2001

Up- and down-regulation of Helix command-specific 2 (HCS2) gene expression in the nervous system of terrestrial snail Helix lucorum

A novel gene named Helix command-specific 2 (HCS2) was shown to be expressed predominantly in four giant parietal interneurons involved in withdrawal behavior of the terrestrial snail Helix lucorum L. and several single neurons in other ganglia. Decrease in spontaneous electrophysiological activity of neurons in the isolated CNS by 24 h incubation in saline with elevated Mg 2+ concentration significantly decreased the number of HCS2-expressing neurons. Five short-term serotonin applications (each of 10 μM), during a 24 h incubation of the nervous system in saline induced expression of the HCS2 gene in many cells in cerebral, parietal, pleural and pedal ganglia. Dopamine applications under similar conditions were not effective. Application of anisomycin or cycloheximide, known to block protein synthesis, did not prevent the induction of HCS2 expression under serotonin influence. Skin injury elicited a significant increase in the number of HCS2-expressing cells 24 h later in pleural and cerebral ganglia. Incubation of the isolated nervous system preparations for three days in culture medium elicited close to a maximum increase in number of HCS2-expressing cells. Elevation of the normal Mg 2+ concentration in the culture medium significantly decreased the number of cells demonstrating HCS2 expression. Application of the cAMP activator forskolin (10 μM) increased the expression under Mg 2+, indicating that cAMP was involved in the up-regulation of HCS2. Application of thapsigargin (10 μM), known to release Ca 2+ from intracellular stores, was also effective in increasing expression, suggesting participation of Ca 2+ in regulation of HCS2 expression. Cellular groups expressing the HCS2 gene under different conditions seem to be functionally related since it was demonstrated earlier that some neurons constituting these clusters are involved in the withdrawal behavior and the response of the organism to stress stimuli. From these results we suggest that the HCS2 pattern of expression can be down-regulated by a decrease in synaptic activity in the nervous system, and up-regulated by external noxious inputs, as well as the application of neurotransmitters and second messengers known to be involved in the withdrawal behavior and maintenance of isolated ganglia in culture medium. When up-regulated, the HCS2 expression appears, at least in part in neurons, to be involved in the withdrawal behavior.

A new multipurpose dental adhesive for orthodontic use: An in vitro bond-strength study

The purpose of this study was to evaluate the compatibility of a new dental adhesive, High-Q-Bond (HQB) adhesive, for the bonding of orthodontic brackets by determining its bond strength and the mode of bond failure after debonding. Eighty extracted human premolars were divided into 4 groups, 20 in each group. In groups 1 and 2, stainless steel brackets were bonded to etched enamel with HQB and Right-On adhesives respectively. In groups 3 and 4, the same adhesives were used to bond stainless-steel brackets to roughened, old amalgam restorations prepared in the teeth. After 72 hours of incubation in saline solution at 37°C, debonding was performed with a shearing force. The force at bond failure was recorded, and the mode of bond failure was examined. Results showed that when bonding to enamel, both the HQB and the Right-On material achieved adequate bond strength, and no significant difference was found between the two. However, after debonding, the HQB material left no adhesive on the enamel, whereas the Right-On material left significant amounts of adhesive on the enamel. When bonding to amalgam, the HQB material had a significantly higher shear bond strength than did the Right-On adhesive. It is suggested that HQB can be applied for orthodontic use, but further clinical studies are required to evaluate its efficacy. (Am J Orthod Dentofacial Orthop 2000;118:307–10)