Nuhan Purali

Bone generation on PHBV matrices: an in vitro study

Bone formation was investigated in vitro by culturing rat marrow stromal osteoblasts in biodegradable, macroporous poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) matrices over a period of 60 days. Foams were prepared after solvent evaporation and solute leaching. PHBV solutions with different concentrations were prepared in chloroform: dichloromethane (1:2, v/v). In order to create a matrix with high porosity and uniform pore sizes, sieved sucrose crystals (300-500 microm) were used. PHBV foams were treated with rf-oxygen plasma (100 W 10 min) to modify their surface chemistry and hydrophilicity with the aim of increasing the reattachment of osteoblasts. Osteoblasts were isolated from rat bone marrow and seeded onto PHBV foams. The cell density on and in the foams was determined with MTS assay. MTS results showed that osteoblasts proliferated on PHBV. Twenty-one days after seeding of incubation, growth of osteoblasts on matrices and initiation of mineralization were observed by confocal laser scanning microscopy. Increasing ALP and osteocalcin secretion during 60 days confirmed the osteoblastic phenotype of the derived stromal cells. SEM, histological evaluations and confocal laser scanning microscopy showed that osteoblasts could grow inside the matrices and lead to mineralization. Cells exhibited spindle-like morphology and had a diameter of 10-30 microm. Based on these, it could confidently be stated that PHBV seems to be a promising polymeric matrix material for bone tissue engineering.

A novel desmin mutation leading to autosomal recessive limb-girdle muscular dystrophy: distinct histopathological outcomes compared with desminopathies

Background Autosomal recessive limb girdle muscular dystrophy (LGMD2) is a heterogeneous group of myopathies characterised by progressive muscle weakness involving proximal muscles of the shoulder and pelvic girdles including at least 17 different genetic entities. Additional loci have yet to be identified as there are families which are unlinked to any of the known loci. Here we have investigated a consanguineous family with LGMD2 with two affected individuals in order to identify the causative gene defect. Methods and results We performed genome wide homozygosity mapping and mapped the LGMD2 phenotype to chromosome 2q35–q36.3. DNA sequence analysis of the highly relevant candidate gene DES revealed a homozygous splice site mutation c.1289-2A>G in the two affected family members. Immunofluorescent staining and western blot analysis showed that the expression and the cytoskeletal network formation of mutant desmin were well preserved in skeletal muscle fibres. Unlike autosomal dominant desminopathies, ultrastructural alterations such as disruption of myofibrillar organisation, formation of myofibrillar degradation products and dislocation/aggregation of membranous organelles were not present. This novel splice site mutation results in addition of 16 amino acids within the tail domain of desmin, which has been suggested to interact with lamin B protein. We also detected a specific disruption of desmin-lamin B interaction in the skeletal muscle of the patient by confocal laser scanning microscopy. Conclusions Our study reveals that autosomal recessive mutations in DES cause LGMD2 phenotype without features of myofibrillar myopathy.

Chitosan based delivery systems for mucosal immunization against bovine herpesvirus 1 (BHV-1)

Bovine herpesvirus 1 (BHV-1), is a major pathogen of cattle which causes serious infections, including infectious bovine rhinotracheitis (IBR)/infectious pustular vulvovaginitis (IPV). At present, BHV-1 is still a serious threat to animal health and productivity in Turkey, hence to develop a more efficient and economical vaccine system against BHV-1 is certainly an important necessity. A mucosal vaccination strategy would provide both mucosal and systemic immune responses to protect disease progression and transmission. However, vaccination through mucosal membranes requires adjuvants/delivery systems in order to enhance the immunogenicity of the antigens. Chitosan, which is a biodegradable, biocompatible and bioadhesive natural polysaccharide, has been shown to be promising both as a delivery system and an adjuvant for mucosal vaccination. In this study, microparticles with appropriate size (<10μm), positive surface charge and high loading efficiency (∼95%) were prepared for mucosal delivery of BHV-1, using various types of chitosan with different molecular weight and solubility. Particles were shown to be taken up by the cells, mostly around the nucleus, whereas with aggregates which were bigger in size were adsorbed at the surface. Furthermore, gel formulations with a suitable viscosity which would provide easy application and remain on the mucosa for extended period of time were also developed with a high zeta potential indicating a stable system. Both the BHV-1 loaded microparticle and gel formulations were shown to maintain cell viability and antigen integrity. Chitosan-based formulations are suggested as promising adjuvant/delivery systems for mucosal immunization against BHV-1.

Comparison of Er,Cr:YSGG laser and hand instrumentation on the attachment of periodontal ligament fibroblasts to periodontally diseased root surfaces: an in vitro study.

BACKGROUND This study investigates the effects of erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser irradiation and hand instrumentation on the attachment of periodontal ligament (PDL) fibroblasts to periodontally involved root surfaces. METHODS Twenty-four single-rooted periodontally involved human teeth (test groups), and six healthy premolar teeth extracted for orthodontic reasons (control group) were included in this study. A total of 45 root slices were obtained from all selected teeth and assigned to the following five groups: 1) untreated healthy group (+control); 2) untreated periodontally diseased group (-control); 3) hand instrumentation group (scaled Gracey); 4) laser I, Er,Cr:YSGG laser irradiation setting-I (short pulse); and 5) laser II, Er,Cr:YSGG laser irradiation setting-II (long pulse). All of the root slices were autoclaved in phosphate buffered saline and slices were placed onto cell culture inserts. PDL fibroblasts were placed at the density of 80,000 cells on the root plate (5 x 6 mm) and incubated for 48 hours and transferred to 24-well plates. The attachment PDL fibroblasts on the root plates were observed using confocal microscopy (at 12 hours and on days 3 and 7) and scanning electron microscopy (at 12 hours and day 3). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was performed on day 5 for PDL fibroblast survival. RESULTS 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay shows that whereas laser-treated specimens showed a significantly higher cell density, the Gracey-treated group showed a lower cell density compared to the positive control group (P <0.05). Based on confocal microscopy, apparent reduction was observed in the attachment of PDL cells to the periodontally diseased root surfaces. In the laser and Gracey groups, cells looked well-oriented to the root surfaces. Laser-treated groups provided suitable environment for cell adhesion and growth. Laser I treatment was more favorable for the attachment of PDL compared to scaled Gracey, laser II, and even healthy root surfaces. CONCLUSION The results of the study indicate that short-pulse laser setup (laser I) looks more promising regarding the attachment, spreading, and orientation of PDL cells.

Mutation in TOR1AIP1 encoding LAP1B in a form of muscular dystrophy: A novel gene related to nuclear envelopathies

We performed genome-wide homozygosity mapping and mapped a novel myopathic phenotype to chromosomal region 1q25 in a consanguineous family with three affected individuals manifesting proximal and distal weakness and atrophy, rigid spine and contractures of the proximal and distal interphalangeal hand joints. Additionally, cardiomyopathy and respiratory involvement were noted. DNA sequencing of torsinA-interacting protein 1 (TOR1AIP1) gene encoding lamina-associated polypeptide 1B (LAP1B), showed a homozygous c.186delG mutation that causes a frameshift resulting in a premature stop codon (p.E62fsTer25). We observed that expression of LAP1B was absent in the patient skeletal muscle fibres. Ultrastructural examination showed intact sarcomeric organization but alterations of the nuclear envelope including nuclear fragmentation, chromatin bleb formation and naked chromatin. LAP1B is a type-2 integral membrane protein localized in the inner nuclear membrane that binds to both A- and B-type lamins, and is involved in the regulation of torsinA ATPase. Interestingly, luminal domain-like LAP1 (LULL1)-an endoplasmic reticulum-localized partner of torsinA-was overexpressed in the patients muscle in the absence of LAP1B. Therefore, the findings suggest that LAP1 and LULL1 might have a compensatory effect on each other. This study expands the spectrum of genes associated with nuclear envelopathies and highlights the critical function for LAP1B in striated muscle.

Use of Mesenchymal Stem Cells and Darbepoetin Improve Ischemia-Induced Acute Kidney Injury Outcomes

Background: Interest has recently been focused on the possible role of bone marrow-originating stem cells and the therapeutic role of erythropoietin in the recovery of ischemia-induced acute kidney injury (AKI). The aim of the present study was to compare treatment with mesenchymal stem cells (MSCs) to treatment with darbepoetin-α (DPO) or both concomitantly in a rat model of ischemia/reperfusion (I/R) AKI. Methods: Forty male Sprague-Dawley rats were included, and 28 of them were randomly assigned to controls (treated with serum physiologic) or one of the three treatment groups treated with either DPO, MSCs, or both (MSCs and DPO concomitantly) after the induction of I/R injury. Hematocrit, serum creatinine, and BUN levels were obtained at 0, 24, 48, and 72 h of surgery, and renal tissue was obtained at 72 h after nephrectomy for histological analysis. Tissue injury was quantified by standardized histological scoring systems, using light and electron microscopes. Results: Treatment with MSCs or DPO improved renal function compared with controls. However, the improvement observed in renal function in the MSC/DPO group was better than that in the other groups. Histological analysis demonstrated that tissue injury was significantly decreased in rats in the MSC or DPO groups compared to that of the controls; however the best recovery was observed in rats treated with MSCs and DPO concomitantly. Conclusion: These results suggest that concomitant application of DPO and MSCs may be a potential novel renoprotective therapy for patients after having sustained an ischemic renal insult.

Structure and function relationship in the abdominal stretch receptor organs of the crayfish

The structure/function relationship in the rapidly and slowly adapting stretch receptor organs of the crayfish (Astacus leptodactylus) was investigated using confocal microscopy and neuronal modeling methods. Both receptor muscles were single muscle fibers with structural properties closely related to the function of the receptors. Dendrites of the rapidly adapting neuron terminated in a common pile of nerve endings going in all directions. Dendrites of the slowly adapting neuron terminated in a characteristic T shape in multiple regions of the receptor muscle. The slowly adapting main dendrite, which was on average 2.1 times longer and 21% thinner than the rapidly adapting main dendrite, induced larger voltage attenuation. The somal surface area of the slowly adapting neuron was on average 51% larger than that of the rapidly adapting neuron. Variation in the neuronal geometry was greatest among the slowly adapting neurons. A computational model of a neuron pair demonstrated that the rapidly and the slowly adapting neurons attenuated the dendritic receptor potential like low‐pass filters with cut‐off frequencies at 100 and 20 Hz, respectively. Recurrent dendrites were observed mostly in the slowly adapting neurons. Voltage signals were calculated to be propagated 23% faster in the rapidly adapting axon, which is 51% thicker than the slowly adapting axon. The present findings support the idea that the morphology of the rapidly and the slowly adapting neurons evolved to optimally sense the dynamic and the static features of the mechanical stimulus, respectively. J. Comp. Neurol. 488:369–383, 2005.

Lidocaine diminishes arrhythmia by Leiurus quinquestriatus quinquestriatus venom in rats

The venom from the scorpion Leiurus quinquestriatus quinquestriatus has previously been shown to alter the excitability of the neural and skeletal muscle preparations. The present study was undertaken to explore the effects of the venom in cardiac potential signals at the animal, tissue and the cell level in rats hearts.

Osteogenic differentiation of MC3T3-E1 cells on different titanium surfaces

mRNA expressions related to osteogenic differentiation of MC3T3-E1 cells on electro-polished smooth (S), sandblasted small-grit (SSG) and sandblasted large-grit (SLG) surfaces of titanium alloys were investigated in vitro. Gene expression profiles of cells were evaluated using the RT2 Profiler PCR microarray on day 7. Mineralizing tissue-associated proteins, differentiation factors and extracellular matrix enzymes mRNA expressions were measured using Q-PCR. SLG surface upregulated 23 genes over twofolds and downregulated 3 genes when compared to the S surface. In comparison to the SSG surface, at least a twofold increase in 25 genes was observed in the SLG surface. BSP, OCN, OPN, COL I and ALP mRNA expressions increased in the SLG group when compared to the S and the SSG groups. BMP-2, BMP-6 and TGF-β mRNA expressions increased in both the SSG and the SLG surfaces. MMP-2 and MMP-9 mRNA expressions increased as the surface roughness increased. This study demonstrated that surface roughness of titanium implants has a significant effect on cellular behavior and SLG surface apparently increased gene expressions related to osteogenesis when compared to the S and the SSG surfaces.

Stimulation of GABA release by scorpion venom in an isolated synapse in the crayfish (Astacus leptodactylus).

Effects of various types of scorpion venom on gamma-aminobutyric acid (GABA) release were studied in an isolated synapse in the crayfish. Post-synaptic GABA-induced currents were recorded to monitor the GABA release from the pre-synaptic site. In 20mM tetraethylammonium (TEA) chloride solution the GABA-induced currents increased 71%. Exposing the preparations to Leiurus quinquestriatus hebraeus, Leiurus quinquestriatus quinquestriatus or Tityus serrulatus venom (0.1mg/ml) increased GABA-induced currents 4-5 fold. The effect was present in the presence of tetrodotoxin (TTX) but diminished significantly when verapamil was applied. Exposing the preparations Androctonus australis or Buthus tamulus venom did not affect the GABA-induced currents. The results indicate that stimulation of the GABA release by some of the scorpion venoms may partly be due to a possible block of pre-synaptic potassium channels, but not due to an abnormal increase in sodium channel activation.