A Novel, Heterozygous, de novo Splicing Variant Affecting the Intracellular Domain of the Growth Hormone Receptor causing a mild short stature.

INTRODUCTION: Although the majority of Growth Hormone insensitivity syndrome (GHIS) cases are classical, the spectrum of clinical phenotypes has expanded to include "atypical" GHIS subjects with milder phenotypes due to very rare heterozygous growth hormone receptor (GHR) mutations with dominant negative effects. CASE PRESENTATION: A 13-year-old pubertal boy presented with short stature (-1.7 SDS) and delayed bone age (11.5 years). His serum IGF-1 was low (16 ng/ml; reference range: 179-540). IGFBP-3 (1.3 mg/L; 3.1-9.5), and ALS (565 mU/ml; 1500-3500) were also low. GH stimulation test was normal, and GHBP markedly elevated (6300pmol/L; 240-3000). Additionally, the boy had insulin resistance and liver steatosis. His final height reached -1.8 SDS, which was 3.0 SDS below his mid-parental height. GHR gene from genomic DNA and established primary fibroblast culture was analyzed and a synonymous heterozygous GHR: c.945G>A variant, in the last nucleotide of exon 9 (encoding intracellular domain of GHR) was identified. In vitro analysis of the GHR cDNA demonstrated a splicing defect, leading to the heterozygous excision of exon 9. The final predicted product was a truncated GHR protein which explained the elevated GHBP levels. CONCLUSION: We describe the first synonymous heterozygous GHR splicing variant in exon 9 encoding part of the intracellular domain of GHR identified in a patient with mild short stature, thus supporting the continuum of genotype-phenotype of GHIS.

Associations between APOE-, COMT Val108/158Met- and BDNF Val66Met polymorphisms and variations in depressive and anxiety symptoms, sense of coherence and vital exhaustion in the real-life setting of mandatory basic military training.

Apolipoprotein E (APOE) ε, catechol-O-methytranferase (COMT) Val108/158Met and brain-derived neurotrophic factor (BDNF) Val66Met single nucleotide polymorphisms (SNPs) were shown to affect stress perception and response. The present study explored possible associations between these SNPs and changes in subclinical anxiety- and depressive symptoms, sense of coherence (SOC) and vital exhaustion (VE) during compulsory basic military training. The study encompassed 179 conscripts of a training base in Greece. The neuropsychiatric assessment was based on the Beck Depression Inventory, the State-Trait Anxiety Inventory, the Antonovsky SOC scale and the Maastricht Questionnaire. It was conducted at three time points of the 19-day basic military training: on day one (baseline), day six (follow-up I) and day 13 (follow-up II). Statistical analyses included Mann-Whitney test, Chi-square test and cross-sectional time series regression models based on the Skillings-Mack statistic. APOE ε4 non-carriers encountered significant changes in anxiety- and depressive symptoms and SOC (in all cases P < 0.001) over the observation period, whilst ε4 carriers did not. The changes in anxiety, depressive symptoms and SOC attained statistical significance in both BDNF Met66 carriers (in all cases P < 0.001) and non-carriers (P = 0.036; < 0.001; < 0.001, respectively) as well as in COMT Met108/158 carriers (P = 0.004; < 0.001; < 0.001, respectively) and non-carriers (P = 0.02; 0.01; 0.021, respectively. Changes over time in VE were not significant (P > 0.05). The observed resistance of APOE ε4 carriers vs non-carriers to changes in anxiety- and depressive symptoms and SOC when exposed to a stressful environment may point to superior coping capacities of healthy young men carrying the ε4 allele.

RANK-c attenuates aggressive properties of ER-negative breast cancer by inhibiting NF-κB activation and EGFR signaling.

The RANK/RANKL axis emerges as a key regulator of breast cancer initiation, progression, and metastasis. RANK-c is a RANK receptor isoform produced through alternative splicing of the TNFRSF11A (RANK) gene and a dominant-negative regulator of RANK-induced nuclear factor-κB (NF-κB) activation. Here we report that RANK-c transcript is expressed in 3.2% of cases in The Cancer Genome Atlas breast cancer cohort evenly between ER-positive and ER-negative cases. Nevertheless, the ratio of RANK to RANK-c (RANK/RANK-c) is increased in ER-negative breast cancer cell lines compared to ER-positive breast cancer cell lines. In addition, forced expression of RANK-c in ER-negative breast cancer cell lines inhibited stimuli-induced NF-κB activation and attenuated migration, invasion, colony formation, and adhesion of cancer cells. Further, RANK-c expression in MDA-MB-231 cells inhibited lung metastasis and colonization in vivo. The RANK-c-mediated inhibition of cancer cell aggressiveness and nuclear factor-κB (NF-κB) activation in breast cancer cells seems to rely on a RANK-c/TNF receptor-associated factor-2 (TRAF2) protein interaction. This was further confirmed by a mutated RANK-c that is unable to interact with TRAF2 and abolishes the ability to attenuate NF-κB activation, migration, and invasion. Additional protein interaction characterization revealed epidermal growth factor receptor (EGFR) as a novel interacting partner for RANK-c in breast cancer cells with a negative effect on EGFR phosphorylation and EGF-dependent downstream signaling pathway activation. Our findings further elucidate the complex molecular biology of the RANKL/RANK system in breast cancer and provide preliminary data for RANK-c as a possible marker for disease progression and aggressiveness.

RANK and EGFR in invasive breast carcinoma.

Breast cancer is the most common malignancy, affecting one in eight women in North America and Europe. The human epidermal growth factor receptor (EGFR) protein comprises a major determinant of normal development but also cancer. RANK receptor (Receptor Activator of Nuclear factor-κB) is a tumor necrosis superfamily member and a binding partner for RANKL, which was recently implicated in breast cancer initiation, progression and metastasis. Here we provide preliminary evidence of a possible interplay between RANK and EGFR signaling in breast cancer. TCGA (cancergenome.nih.gov) publicly available data for EGFR and TNFRSF11A (RANK) genes from breast cancer patients and breast cancer cell lines were retrieved and analyzed. RANK mRNA showed a statistically significant positive correlation (p <0.001) with the mRNA and protein expression of EGFR, but not with ERBB2/3/4. Further analyses of survival data of a group of breast cancer patients (n = 248) from TCGA, revealed an EGFRhi/RANKhi subpopulation that showed a statistically significant (p = 0.001) reduced overall survival when compared to EGFRlow/RANKlow group of patients. Finally, EGFR and RANK combinatorial in vitro analyses revealed a significant upregulation of AKT and ERK signaling after EGF stimulation in cell lines and also an increase of breast cancer cell invasiveness.

Molecular studies of the immunological effects of the sevoflurane preconditioning in the liver and lung in a rat model of liver ischemia/reperfusion injury.

Sevoflurane has been shown to improve ischemia/reperfusion injury (IRI) through several mechanisms, including amelioration of inflammatory response. However, there haven't been any studies considering the potential role of the complement system in sevoflurane-mediated amelioration of ischemia/reperfusion injury. Our purpose was to investigate the molecular mechanisms involved in sevoflurane preconditioning in liver and lung injury induced by liver ischemia-reperfusion (LIR), giving emphasis to the immunological mechanisms. In order to do that, fifty male Wistar rats were randomly allocated in five groups (n=10 each): Animals in group LIR received ketamine and xylazine and were then subjected to ischemia of the right and median hepatic lobe for 45 min and reperfusion for 6h. Group SEVO/LIR received sevoflurane and then LIR was induced, as in group LIR. Animals in group SHAM/LIR were anesthetized with ketamine and xylazine and then laparotomy followed. Group SHAM/SEVO received sevoflurane for 30 min and then laparotomy followed. Finally, in group VEN, animals only received ketamine and xylazine. Our results showed that sevoflurane preconditioning significantly improved liver-biochemical tests (decreased Alanine transaminase (ALT), Alkaline phosphatase (ALP), Aspartate transaminase (AST) and Alkaline phosphatase (ALP) levels) and limited inflammatory cell infiltration in BALF. Additionally, compared with the LIR group, the reduction in plasma C3 was significantly reduced in the SEVO/LIR group. No significant differences were observed in histological examination in the liver and lung. Immunostaining of the liver for Intracellular Adhesion Molecule 1 (ICAM1) however, showed a decrease in ICAM1 levels in the SEVO/LIR group. In the lung, sevoflurane seemed to exert no effect in ICAM1 levels. Caspase 3 (CASP3) levels in the liver and the lung also appeared unaffected by sevoflurane preconditioning. In the SEVO/LIR group, ICAM1 mRNA expression was significantly reduced in the liver. No statistical significantly differences were observed in Complement component 3 (C3), Complement component 5 (C5) and Clusterin (CLU) mRNA levels in the liver or the lung tissue. Summarizing, sevoflurane preconditioning seems to ameliorate LIR-induced injury in the rats, mediated by mechanisms that include ICAM1 and complement C3 down regulation.

Complement C3, C2, and factor B gene polymorphisms and age-related macular degeneration in a Greek cohort study.

PURPOSE: To elucidate whether polymorphisms of C2, C3, and CFB genes are major genetic determinants of age-related macular degeneration (AMD) in a Greek population. METHODS: This was a case-control association study comprising 120 Greek patients with early and late-stage AMD and 140 independent controls of Caucasian origin. All participants were genotyped for rs547154, rs2230199, rs641153, and rs12614 polymorphisms by a combination of PCR and direct DNA sequencing assays. RESULTS: The frequency of the rs2230199 G allele (minor allele) was significantly higher in patients with AMD in comparison with controls (0.34 vs 0.22, p = 0.0031) and similar to the frequency of other reported populations. There was a significant difference in the frequencies of the rs2230199 genotypes among cases and controls (p = 0.0055). rs2230199 was found to be a significant predictor of advanced AMD status (odds ratio 6.41, confidence interval [CI] 2.72-15.09, p<0.0001; area under the curve 0.706, CI 0.61-0.78, p<0.0001]). For the other single nucleotide polymorphism (SNP) loci, the allele and genotype frequencies did not reach statistical significance. The minor allele frequencies in controls and cases were similar and still much lower than the frequencies reported in other populations. CONCLUSIONS: The rs547154, rs641153, and rs12614 SNPs were not associated with AMD development in Greek patients. However, this finding should be viewed with caution as the particular polymorphisms presented with very low frequencies in the Greek population. Finally, the replication of the reported associations of C3 with AMD suggests that the presence of the C3 G allele could serve as a high-risk genetic marker for the development of AMD and the progression of the disease to the advanced clinical stage.

Detection of a latent soluble form of membrane type 1 matrix metalloprotease bound with tissue inhibitor of matrix metalloproteinases-2 in periprosthetic tissues and fluids from loose arthroplasty endoprostheses.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is implicated in pericellular proteolysis, and, together with tissue inhibitor of matrix metalloproteinases-2 (TIMP-2), in the activation of pro-matrix metalloproteinase-2 on the cell surface. It is expressed on the cell surface either activated or as a proenzyme. A soluble form of MT1-MMP (sMT1-MMP) has been previously identified in periprosthetic tissues and fluid of patients with loose arthroplasty endoprostheses. The aim of this study was to examine periprosthetic tissues and fluids from patients with loose arthroplasty endoprostheses, as well as tissues and fluids from patients with other disorders, for the presence of sMT1-MMP, and to investigate its activation state and possible role. With antibody against MT1-MMP, a protein with molecular mass of ~ 57 kDa was detected by western blotting in all samples tested, representing a soluble form of MT1-MMP, which cannot be ascribed to alternative splicing, as northern blotting showed only one transcript. With various biochemical methods, it was shown that this species occurs in a latent form bearing the N-terminal prodomain, and, additionally, it is bound to TIMP-2, which appeared to be bound via its C-terminal domain to a site different from the active site. Cell ELISA and immunohistochemical analysis revealed that, besides fibroblasts, all other cells, such as inflammatory, epithelial, endothelial, giant and cancer cells, express MT1-MMP on their plasma membrane as a proenzyme. Taking into account the proteolytic abilities of MT1-MMP, the latent sMT1-MMP-TIMP-2 complex could be considered as a new interstitial collagenase. However, the exact role, the production mechanism and the cell origin of this complex remain to be elucidated.

Alternative splicing generates a truncated isoform of human TNFRSF11A (RANK) with an altered capacity to activate NF-κB.

Alternative splicing (AS) is a major post-transcriptional modification taking place in all cells. Many members of the TNF receptor superfamily modulate their function through protein isoforms produced by alternative splicing. TNFRSF11A (RANK) gene, through alternative splicing produces multiple isoforms truncated in their intracellular domain, with distinct functions. Here, we report the identification and characterization of a novel human TNFRSF11A (RANK) variant from human normal brain, named RANK-e5a (TNFRSF11A_e5a). The novel variant lacks 42 nucleotides from exon 5, giving rise to a novel shorter form of exon 5, named exon 5a. The incorporation of the novel exon 5a in RANK mRNA does not affect the open reading frame, producing a truncation of thirteen amino acids of the third and fourth TNFR motifs of the extracellular part of the receptor. By western blot analysis and immunofluorescence staining we were able to further characterize the RANK-e5a isoform at the protein level. In addition, we performed an ELISA assay to characterize RANK/RANKL and RANK-e5a/RANKL binding capacities, and we identified a reduced affinity of RANK-e5a to bind RANKL. Finally, when RANK-e5a is stimulated by RANK ligand, its capability to activate NF-κB is reduced compared to the wild type RANK receptor. Overall, our data provide a novel regulatory mechanism for the RANK/RANKL system, at the RANK receptor level.

The molecular identification of factor H and factor I molecules in rainbow trout provides insights into complement C3 regulation.

The complement system in vertebrates plays a crucial role in the elimination of pathogens. To regulate complement on self-tissue and to prevent spontaneous activation and systemic depletion, complement is controlled by both fluid-phase and membrane-bound inhibitors. One such inhibitor, complement factor I (CFI) regulates complement by proteolytic cleavage of components C3b and C4b in the presence of specific cofactors. Complement factor H (CFH), the main cofactor for CFI, regulates the alternative pathway of complement activation by acting in the breakdown of C3b to iC3b. To gain further insight into the origin of C3 regulation in bony fish we have cloned and characterized the CFI and CFH1 cDNAs in the rainbow trout (Oncorhynchus mykiss). In this study we report the primary sequence, the tissue expression profile, the polypeptide domain architecture and the phylogenetic analysis of trout CFI and CFH1 genes. The deduced amino acid sequences of trout CFI and CFH1 polypeptides exhibit 42% and 32% identity with human orthologs, respectively. RNA expression analysis showed that CFI is expressed differentially in trout tissues, while liver is the main source of CFH1 expression. Our data indicate that factor H and I genes have emerged during evolution as early as the divergence of teleost fish.

Molecular cloning and expression of Aven gene in chicken.

Aven was originally identified as a protein that regulates apoptosis by binding to apoptotic regulators, Bcl-xL and Apaf-1. Recently was found that Aven protein is a potent activator of ATM, critical for its DNA damage-induced activation. An Aven cDNA clone was isolated from chicken (Gallus gallus) after screening of a cerebellum cDNA library. The full-length cDNA is 1,430 nt in size, encoding for a polypeptide of 352 amino acid residues. The predicted amino acid sequence of the chicken Aven is 69, 46, 45 and 37% identical to those of zebra finch, human, xenopus and zebrafish orthologs, respectively. Expression analysis reveals that the chicken Aven gene is expressed in the adult brain, heart, intestine, kidney, lung, stomach and spleen, as well as in the whole embryos of 4- and 6-days old. Phylogenetic analysis of the Aven ortholog proteins from various organisms clusters the chicken Aven in the same group with other bird Avens.

Treatment with bioartificial liver improves lung injury in a swine model of partial hepatectomy and ischemia/reperfusion.

PURPOSE: Hepatic ischemia/reperfusion injury can lead to remote lung injury by inducing oxidative stress and inflammation. this study aims to investigate whether support of liver function with a bioartificial liver can attenuate remote lung injury after extended hepatectomy. METHODS: Fourteen domestic pigs were subjected to liver ischemia for 150 minutes and 70-75% hepatectomy. Six hours after initiation of hepatic reperfusion the animals were randomly allocated to a 6-hour treatment with a bioartificial liver (group b, n=7) or observation (group C, n=7). Hemodynamic and metabolic parameters were monitored for 24 hours following reperfusion. Lung biopsies were used for histological, nitrotyrosine and mrNA analysis. RESULTS: Oxygenation gradually deteriorated in group C, but was not significantly impaired in group b. Histological evaluation revealed improvements in alveolar collapse, necrotized pneumonocytes and lymphocyte infiltration in group b. Nitrotyrosine content of the lung was lower in group b compared to group C (55+/-12 vs. 132+/-22 nM/mg protein, p<0.01). Lung mrNA expression of interleukin-6, Stat-3 and E-selectin also decreased in group b. Expression of transforming growth factor-alpha mrNA did not differ between groups. CONCLUSIONS: Application of a bioartificial liver was associated with improvement in several parameters of post-hepatectomy lung injury. the mechanisms appear to involve reduced nitrosative stress and attenuation of the native inflammatory process in the lung.

A novel mutation in the signal transducer and activator of transcription 3 (STAT3) gene, in hyper-IgE syndrome.

The hyper-IgE syndrome (HIES) is a rare primary immunodeficiency characterized by a highly elevated serum IgE, recurrent staphylococcal skin abscesses and cyst-forming pneumonia. Non-immunological abnormalities, including a distinctive facial appearance, hyperextensive joints, scoliosis, fracture following minor trauma, and the retention of primary teeth are also observed in many patients. Recently, it was shown that heterozygous mutations in signal transducer and activator of transcription 3 (STAT3), can cause autosomal-dominant HIES. Here we identify and characterize a novel mutation in the DNA-binding domain of STAT3 in a patient with hyper-IgE syndrome. Sequence analysis revealed a de novo heterozygous transition of a G-to-A, causing a substitution of a glycine residue for an aspartic acid in the translated sequence (G342D). The patient has normal levels of STAT3, which is able to translocate to the nucleus upon IL-6 stimulation. However, enzyme-linked DNA-protein interaction analysis showed that the G342D mutation affects the binding ability of STAT3 to target DNA sequences. In addition, as shown by qRT-PCR, the mutation abrogates the STAT3-dependent transcription of the retinoid-related orphan receptor gammat (ROR gammat) gene, an indispensable transcription factor for the commitment of naive CD4+ T cells to the Th17 lineage. These data suggest that the novel G342D mutation affects the binding of STAT3 on DNA and the STAT3-dependent expression of ROR gammat mRNA, leading to the HIES phenotype.

Cloning of the sixth complement component and, spatial and temporal expression profile of MAC structural and regulatory genes in chicken.

Humoral cytotoxicity results from the assembly of terminal components of complement, called membrane attack complex (MAC), which lead to the formation of pores on pathogen membranes. The complement components involved in MAC formation are C5b, C6, C7, C8alpha, C8beta, C8gamma and C9. Among them, C6 protein interacts with C5b through a metastable binding site to form a soluble C5b-6 dimer in the vicinity of the activating cell. Formation of the MAC is controlled by complement regulatory molecules, such as CD59, vitronectin and clusterin. Here, we report the molecular characterization of the C6 complement component, as well as the spatial and temporal expression profile of MAC structural (C6, C7, C8alpha, C8beta, C8gamma) and regulatory (CD59, vitronectin and clusterin) genes in chicken (Gallus gallus). The deduced polypeptide sequence of chicken C6 consists of 935 amino acid residues and exhibits 81%, 58%, 56% and 44% identity with zebra finch, human, frog and trout orthologs, respectively. The 'domain' architecture of chicken C6 resembles that of mammalian counterparts and the cysteine backbone is also conserved. MAC structural and regulatory genes are expressed in a wide range of adult chicken tissues, with the liver being the major source of their produced transcripts. The developmental expression profile of chicken MAC structural genes shows that their transcripts initially appear in the 12th embryonic day in the liver, exhibiting a pick in the 17th, while no expression was detected in the early whole embryo (day 4 and 6), as well as in the 2-day old neonate chicken liver. On the other hand, MAC regulatory genes are expressed in all the developmental stages investigated.

Complement factor H and LOC387715 gene polymorphisms in a Greek population with age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of severe visual loss among people over 60 years old. The lack of a broadly effective treatment for AMD underscores the need to identify causative biomarkers that could serve as preventive targets. Thus far, two major susceptibility loci for AMD have been identified, CFH T1277C and LOC387715 G270T. The primary goal of the present study was to elucidate whether these polymorphisms are major genetic determinants of AMD in a Greek population. PATIENTS AND METHODS: A clinic-based, case-control association study was conducted, comprising 100 Greek patients with early and late-stage AMD and 115 independent controls of Caucasian origin. All participants underwent clinical examination including best-corrected visual acuity, intraocular pressure, and dilated fundus examination. Moreover, they were genotyped for CFH T1277C and LOC387715 G270T polymorphisms, by direct sequencing and ARMS PCR, respectively. RESULTS: The frequency of the CFH 1277C allele was significantly higher in AMD patients in comparison with controls while the odds ratios (ORs) for AMD were 4.4-5.5. Statistical comparison of early and advanced AMD patients, on the basis of CFH genotype, revealed that the CFH 1277C allele was associated with both subgroups when compared with the controls (P < 0.001). When statistical comparison was performed between early and advanced patients on the basis of CFH genotypic frequencies, the CC genotype was found to be more prevalent in advanced AMD patients (P = 0.008, OR = 2.3). The frequency of the LOC387715 270 T allele was higher in AMD patients in comparison with controls (P < 0.04) while the ORs for AMD were 1.4-2. No statistically significant differences were located between the early AMD patients and controls, on the basis of LOC387715 genotype (P = 0.189). On the contrary, the T270G polymorphism was associated with advanced AMD (P = 0.04). Moreover, the TT genotype was more prevalent in patients with advanced AMD (P = 0.011, OR = 1.7) when compared with early AMD patients. Assessment of the combined contribution of CFH T1277C and LOC387715 G270T SNPs showed an independent manner of action of these polymorphisms in the development of the disease. CONCLUSIONS: The replication of the reported associations of CFH T1277C polymorphism with AMD suggest that the 1277C allele could serve as a high-risk genetic marker for the development of AMD and the progression of the disease to the advanced clinical stage in the Greek population.

Cloning and characterization of the trout perforin.

The pore-forming protein, perforin is one of the effectors of cell-mediated killing. A perforin cDNA clone was isolated from rainbow trout (Oncorhynchus mykiss) after screening of a spleen cDNA library. The full-length cDNA is 2070 bp in size, encoding for a polypeptide of 589 amino acids. The predicted amino acid sequence of the trout perforin is 64, 58 and 40% identical to those of Japanese flounder, zebrafish and human perforins, respectively. Although its membrane attack complex/perforin (MACPF) domain is conserved, trout perforin shows low homology to human and trout terminal complement components (C6, C7, C8 and C9), ranging from 19 to 26% identity. Expression analysis reveals that the trout perforin gene is expressed in the blood, brain, heart, kidney, intestine and spleen. Phylogenetic analysis of proteins which belong to the MACPF superfamily clusters the trout perforin in the same group with other known perforins.

CR3 complement receptor: cloning and characterization in rainbow trout.

The beta 2 integrin CR3 is a leukocyte adhesion heterodimeric glycoprotein which functions both as receptor for iC3b and in several cell-cell and cell-substrate adhesion interactions. In order to elucidate the molecular evolution of the CR3 receptor, here we report the cloning and characterization of its beta2 (CD18) and aM (CD11b) subunits in rainbow trout (Oncorhynchus mykiss). The predicted polypeptide sequences of trout CD18 and CD11b-like exhibit 50, 49, or 61% and 25, 25, or 30% identity with human, mouse, and zebrafish orthologs, respectively. The 'domain' architecture of trout CD18 and CD11b-like subunits retains several characteristics of the mammalian ortholog proteins, such as cysteine-rich regions, N-linked glycosylation sites and several proposed domains and signal sequences (von Willebrand factor type A, Integrin alpha, Integrin B tail, EGF, and Transmembrane domain). The tissue expression profiles of trout CR3 subunits diverge from those of mammalian counterparts, showing the kidney as the main source of the trout CD18 and CD11b-like mRNA transcripts. This is the first report of cloning and characterization of the CR3 receptor in low vertebrates.

Estimation of hydrodynamic shear stresses developed on human osteoblasts cultured on Ti-6Al-4V and strained by four point bending. Effects of mechanical loading to specific gene expression.

The aim of the present investigation was to study the effects of mechanical strain on the orthopedic biomaterial Ti-6Al-4V-osteoblast interface, using an in vitro model. Homogeneous strain was applied to Human Bone Marrow derived Osteoblasts (HBMDOs) cultured on Ti-6Al-4V, at levels which are considered physiological, by a four-point bending mechanostimulatory system. A simple model for the estimation of maximum hydrodynamic shear stresses developed on cell culture layer and induced by nutrient medium flow during mechanical loading, as a function of the geometry of the culture plate and the load characteristics, is proposed. Shear stresses were lower than those which can elicit cell response. Mechanical loading was found that contributes to the regulation of osteoblast differentiation by influencing the expression of the osteoblast-specific transcription factor Cbfa1, both at the mRNA and protein level, and also the osteocalcin expression, whereas osteopontin gene expression was unaffected by mechanical loading at all experimental conditions.

Effects of physiological mechanical strains on the release of growth factors and the expression of differentiation marker genes in human osteoblasts growing on Ti-6Al-4V.

Mechanical loading factors at the bone-implant interface are critical for the osseointegration and clinical success of the implant. The aim of the present investigation was to study the effects of mechanical strain on the orthopedic biomaterial Ti-6Al-4V/osteoblast interface, using an in vitro model. Homogeneous strain was applied to human bone marrow derived osteoblasts (HBMDOs) cultured on Ti-6Al-4V, at physiological levels (strain magnitudes 500 microstrain (microepsilon) and 1000 microepsilon, at frequencies of load application 0.5 Hz and 1 Hz), by a mechanostimulatory system, based on the principle of four-point bending. Semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR) was used to determine the mRNA expression of Cbfa1 and osteocalcin at different loading conditions. The release of growth factors as a response to stretch was also investigated by transferring stretch-conditioned media to nonstretched cells and by measuring their effect on the regulation of DNA synthesis. Mechanical loading was found to contribute to the regulation of osteoblast differentiation by influencing the level of the osteoblast-specific transcription factor Cbfa1, both at the mRNA and protein level, and also the level of osteocalcin, which is regarded as the most osteoblast-specific gene. Both genes were differentially expressed shortly after the application of different mechanical stimuli, in terms of strain frequency, magnitude, and time interval. Media conditioned from mechanically stressed HBMDOs stimulate DNA synthesis more intensely compared to media conditioned from unstressed control cultures, indicating that mechanical strain induces the release of a mitogenic potential that regulates cell proliferation.

Three isoforms of complement properdin factor P in trout: cloning, expression, gene organization and constrained modeling.

Properdin is a plasma glycoprotein and the only known naturally occurring positive regulator of the complement system, stabilizing the alternative pathway convertase (C3bBb). In order to elucidate the molecular evolution of properdin factor P (pfc), here we report the cloning and characterization of three gene isoforms of properdin in rainbow trout (Oncorhynchus mykiss). The predicted polypeptide sequences of trout properdins pfc1, pfc2 and pfc3 (447, 449 and 447 amino acids, respectively) share 78-90% identity to each other, showing the highest identity score (47%) with their zebrafish ortholog protein. The overall identity with human, mouse and xenopus properdin polypeptides is 44%, 42% and 45%, respectively. The 'domain' architecture of trout properdins resembles that of the mammalian counterpart proteins, composed of six thrombospondin repeat type 1 domains (TSR-1-TSR-6). TSR domains of the three trout properdin isoforms seem to adopt the folding pattern of human thrombospondin 1 TSP-1 domains, where each TSP-1 domain forms an antiparallel three-stranded structure that consists of alternative stacked layers of Trp and Arg residues from respective strands capped by disulfide bonds on each end. The trout pfc2 and pfc3 genes are arranged in nine and ten exons, respectively, which span approximately 3.5kb of the genome. In contrast to the expression profile of the properdin gene in mammals, liver is the main source of the trout properdin mRNA transcripts. In a phylogenetic analysis, trout pfc1, pfc2 and pfc3 genes are clustered with their orthologs from other teleost species. This is the first report of three separate genes coding for properdin factor P in a vertebrate species.