We have isolated a new mutant of Bacillus subtilis temperature sensitive in DNA replication; its properties are those of an initiation mutant. When liquid cultures are shifted to 48 degrees DNA replication is the first macromolecular synthesis that stops, but only after synthesis of the amount of DNA predicted for the completion of one replication round. When spores of the mutant are germinated and shifted to 48 degrees at subsequent times, one round of DNA replication is observed only when the shift occurs between 60 and 100 min; earlier shifts do not allow replication to start, later shifts allow more than one replication. The DNA replicated after a shift to high temperature is enriched in markers close to the terminus. The reinitiation of DNA replication stopped by the high temperature, takes place following a shift to a permissive temperature only if protein synthesis is allowed. Examination of DNA replication following toluene treatment shows that the elongation of DNA chains is not affected at the non-permissive temperature. This mutant is shown by PBS-1 mapping to correspond to a new gene denominated dna P, which is located between the thy A and fur A genes and is distinct from all the mapped dna and rec genes of Bacillus subtilis. The mutation confers to the cells also a deficiency in the ability to be transformed, to be transfected with SPP1 phage DNA, and to survive treatment with methyl-methane sulfonate. These deficiencies, observed at the permissive temperature, are no more temperature dependent than in the parental strain. The ability to perform homologous and heterologous transduction with PBS-1 phage and the sensitivity to ultraviolet radiation or mitomycin C are normal.

The development of SPP1 has been studied in several B. subtilis mutants conditionally defective in initiation of DNA replication. Initiation of SPP1 replication is independent of the host DnaA (replisome organizer), DnaB, DnaC and DnaI products, but requires the DnaG (DNA primase) and the DNA gyrase. Furthermore, SPP1 replication is independent of the DnaK (heat shock) protein. The phage-encoded products required for initiation of SPP1 replication have been genetically characterized. Analysis of the nucleotide sequence (3.292 kilobases) of the region where SPP1 initiation replication mutants map, revealed five open reading frames (orf). We have assigned genes 38, 39 and 40 to three of these orfs, which have the successive order gene 38-gene 39-orf39,1-gene 40-orf41. The direction of transcription of the reading frames, the lengths of the mRNAs as well as the transcription start point, upstream of gene 38 (PE2), were identified. Proteins of 29.9, 14.6 and 46.6 kDa were anticipated from translation of gene 38, gene 39 and gene 40, respectively. The purified G38P and G39P have estimated molecular masses of 31 and 15 kDa. G38P and G39P do not share significant identity with primary protein sequences currently available in protein databases, whereas G40P shares substantial homology with a family of DNA primase-associated DNA helicases. G38P binds specifically to two discrete SPP1 DNA restriction fragments (EcoRI-4 and EcoRI-3). The G38P binding site on EcoRI-4 was localized on a 393 bp DNA segment, which lies within the coding sequence of gene 38. The putative binding site on EcoRI-3 was inferred by DNA sequence homology, it maps in a non-coding segment. G39P, which does not bind to DNA, is able to form a complex with G38P. The organization of the SPP1 genes in the gene 38 to gene 40 interval resembles that one found in the replication origin regions of different Escherichia coli double-stranded DNA phages (lambda, phi 80 and P22). We propose that the conserved gene organization is representative of the replication origin region of a primordial phage.

SPP1-encoded replicative DNA helicase gene 40 product (G40P) is an essential product for phage replication. Hexameric G40P, in the presence of AMP-PNP, preferentially binds unstructured single-stranded (ss)DNA in a sequence-independent manner. The efficiency of ssDNA binding, nucleotide hydrolysis and the unwinding activity of G40P are affected in a different manner by different nucleotide cofactors. Nuclease protection studies suggest that G40P protects the 5' tail of a forked molecule, and the duplex region at the junction against exonuclease attack. G40P does not protect the 3' tail of a forked molecule from exonuclease attack. By using electron microscopy we confirm that the ssDNA transverses the centre of the hexameric ring. Our results show that hexameric G40P DNA helicase encircles the 5' tail, interacts with the duplex DNA at the ss-double-stranded DNA junction and excludes the 3' tail of the forked DNA.

Dystocia and stillbirth are significant causes of female and neonatal death in many species and there is evidence for a genetic component to both traits. Identifying causal mutations affecting these traits through genome wide association studies could reveal the genetic pathways involved and will be a step towards targeted interventions. Norwegian Red cattle are an ideal model breed for such studies as very large numbers of records are available. We conducted a genome wide association study for direct and maternal effects of dystocia and stillbirth using almost 1 million records of these traits. Genotyping costs were minimized by genotyping the sires of the recorded cows, and using daughter averages as phenotypes. A dense marker map containing 17,343 single nucleotide polymorphisms covering all autosomal chromosomes was utilized. The genotyped sires were assigned to one of two groups in an attempt to ensure independence between the groups. Associations were only considered validated if they occurred in both groups. Strong associations were found and validated on chromosomes 4, 5, 6, 9, 12, 20, 22 and 28. The QTL region on chromosome 6 was refined using LDLA analysis. The results showed that this chromosome most probably contains two QTL for direct effect on dystocia and one for direct effect on stillbirth. Several candidate genes may be identified close to these QTL. Of these, a cluster of genes expected to affect bone and cartilage formation (i.e. SPP1, IBSP and MEPE) are of particular interest and we suggest that these genes are screened in candidate gene studies for dystocia and stillbirth in cattle as well as other species.

Vestibular schwannomas are benign neoplasms that arise from the vestibular nerve. The hallmark of these tumors is the biallelic inactivation of neurofibromin 2 (NF2). Transcriptomic alterations, such as the neuregulin 1 (Nrg1)/ErbB2 pathway, have been described in schwannomas. In this study, we performed a whole transcriptome analysis in 31 vestibular schwannomas and 9 control nerves in the Affymetrix Gene 1.0 ST platform, validated by quantitative real-time PCR (qRT-PCR) using TaqMan low density arrays. We performed a mutational analysis of NF2 by PCR/denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (MLPA), as well as a microsatellite marker analysis of the loss of heterozygosity (LOH) of chromosome 22q. The microarray analysis demonstrated that 1,516 genes were deregulated and 48 of the genes were validated by qRT-PCR. At least 2 genetic hits (allelic loss and/or gene mutation) in NF2 were found in 16 tumors, seven cases showed 1 hit and 8 tumors showed no NF2 alteration. MET and associated genes, such as integrin, alpha 4 (ITGA4)/B6, PLEXNB3/SEMA5 and caveolin-1 (CAV1) showed a clear deregulation in vestibular schwannomas. In addition, androgen receptor (AR) downregulation may denote a hormonal effect or cause in this tumor. Furthermore, the osteopontin gene (SPP1), which is involved in merlin protein degradation, was upregulated, which suggests that this mechanism may also exert a pivotal role in schwannoma merlin depletion. Finally, no major differences were observed among tumors of different size, histological type or NF2 status, which suggests that, at the mRNA level, all schwannomas, regardless of their molecular and clinical characteristics, may share common features that can be used in their treatment.

BACKGROUND

Alternative splicing of the osteopontin (opn, spp1) gene generates three protein splicing isoforms (OPN-SI), designated as OPNa, OPNb, and OPNc, which have demonstrated specific roles in different tumor models. This work aims to investigate the roles of each OPN-SI in prostate cancer (PCa) progression by using in vivo and in vitro functional assays.

METHODS

The expression levels of OPN-SI in prostate cell lines were analyzed by qRT-PCR. PC-3 was stably transfected with expression vectors containing OPNa, OPNb, and OPNc, as well as empty vector controls. PC-3 cells overexpressing each construct were analyzed for in vivo tumor growth and in relation to different aspects mimicking tumor progression, such as cell proliferation, migration, invasion, and soft agar colony formation.

RESULTS

OPN-SI are overexpressed in PCa as compared to non-tumoral prostate cell lines. OPNc and OPNb overexpressing cells significantly activated enhanced xenograft tumor growth and PC-3 proliferation, migration, invasion, and soft agar colony formation, as well as the expression of MMP-2, MMP-9, and VEGF. These isoforms also support sustained proliferative survival. We found that both OPNc and OPNb pro-tumorigenic roles are mainly mediated through PI3K signaling. Inhibition of this pathway by using LY294002 specifically inhibited tumor progression features evoked by OPNc and OPNb overexpression.

CONCLUSIONS

Our data provide evidence that both OPNc and OPNb splicing isoforms promote distinct aspects of PCa progression by inducing PI3K signaling. These data give support to strategies aiming to downregulate OPNc and OPNb expression as an approach to inhibit PCa progression.

Novel and traditional eggshell quality measurements were made from up to 2000 commercial pedigree hens for a candidate gene association analysis with organic eggshell matrix genes: ovocleidin-116, osteopontin (SPP1), ovocalyxin-32 (RARRES1), ovotransferrin (LTF), ovalbumin and ovocalyxin-36, as well as key genes in the maintenance and function of the shell gland [estrogen receptor (ESR1) and carbonic anhydrase II (CAII)]. Associations were found for (i) ovalbumin with breaking strength and shell thickness; (ii) ovocleidin-116 with elastic modulus, shell thickness and egg shape; (iii) RARRES1 with mammillary layer thickness; (iv) ESR1 with dynamic stiffness; (v) SPP1 with fracture toughness and (vi) CAII with egg shape. The marker effects are as large as 17% of trait standard deviations and could be used to improve eggshell quality.

The Hedgehog (Hh) signaling pathway affects fetal testis growth. Recently, we described the dynamic cellular production of Hh signaling pathway components in juvenile and adult rodent testes. The Hh signaling is understood to regulate cord formation in the fetal testis, but minimal knowledge exists regarding how Hh signaling impacts the postnatal testis. To investigate this, we employed hanging drop cultures, which are used routinely in embryoid body formation. This approach has the advantage of using small media volume, and we examined its suitability for short-term culture of both murine embryonic gonads and adult testis tubules. The effects of cyclopamine, a specific Hh signaling inhibitor, were examined following culture of Embryonic Day 11.5 urogenital ridges (as control) and adult seminiferous tubule fragments for 24-48 h using histological, cell proliferation, and gene expression analyses. Cultured embryonic testes displayed generally normal cord structure, anti-Müllerian hormone (Amh) expression, and cell proliferation; known Hh target gene expression (Gli1, osteopontin, official symbol Spp1, and Amh) was altered in response to cyclopamine. Cultured adult tubules exhibited some loss of seminiferous epithelium organization over 48 h. Spermatogonia continued to proliferate, however, and no significant loss of viability was noted overall. Addition of cyclopamine significantly affected levels of Gli1, Igfbp6, Ccnd2 (cyclin D2), Ccnb1 (cyclin B1), Spp1, Kit, and Amh mRNAs; these genes have been shown previously to be expressed in Sertoli and germ cells. These novel results identify Hh target genes in the testis and demonstrate this signaling pathway likely affects cell survival and differentiation in the context of normal adult testis.

The Booroola (FecB) mutation in sheep is linked to markers from a region of syntenic homology to human chromosome HSA4q, but the chromosomal location in sheep has not been determined. Analysis of linkage in Booroola half-sib pedigrees and 17 full-sib families identified genetic linkage between platelet-derived growth factor receptor-alpha (PDGFRA) and alpha s1-casein (CSN1S1) at 12 cM (Zmax = 9.14) and between PDGFRA and the microsatellite markers BM143 and OarHH55 (Zmax = 6.28 and 3.83, respectively). The microsatellite markers OarAE101 and BM143 and genes from the linkage group (PDGFRA, SPP1, and EGF) were mapped in a partial sheep x hamster somatic cell hybrid panel. All markers identified bands specific to somatic cell hybrids containing the sheep chromosome t1 (rob6;24) or t1q (chromosome 6). In sheep the casein genes alpha s1 (CSN1S1), alpha s2 (CSN1S2), beta (CSN2), and kappa (CSN3) are tightly linked, and CSN2 has been mapped to sheep chromosome 6q23-q31. We conclude that the Booroola mutation is located within a conserved syntenic group that maps to sheep chromosome 6.

Sox9 is an SRY-related transcription factor required for expression of cartilaginous genes in the developing skeletal system and heart valve structures. In contrast to positively regulating cartilaginous matrix, Sox9 also negatively regulates matrix mineralization associated with bone formation. While the transcriptional activation of Sox9 target genes during chondrogenesis has been characterized, the mechanisms by which Sox9 represses osteogenic processes are not so clear. Using ChIP-on-chip and luciferase assays we show that Sox9 binds and represses transactivation of the osteogenic glycoprotein Spp1. In addition, Sox9 knockdown in post natal mouse heart valve explants and rib chondrocyte cultures promotes Spp1 expression and matrix mineralization, while attenuating expression of cartilage genes Type II Collagen and Cartilage Link Protein. Further, we show that Spp1 is required for matrix mineralization induced by Sox9 knockdown. These studies provide insights into the molecular mechanisms by which Sox9 prevents pathologic matrix mineralization in tissues that must remain cartilaginous.

Bone formation is indirectly influenced by 1,25-dihydroxyvitamin D3 (1,25D3) through the stimulation of calcium uptake in the intestine and re-absorption in the kidneys. Direct effects on osteoblasts and bone formation have also been established. The vitamin D receptor (VDR) is expressed in osteoblasts and 1,25D3 modifies gene expression of various osteoblast differentiation and mineralization-related genes, such as alkaline phosphatase (ALPL), osteocalcin (BGLAP), and osteopontin (SPP1). 1,25D3 is known to stimulate mineralization of human osteoblasts in vitro, and recently it was shown that 1,25D3 induces mineralization via effects in the period preceding mineralization during the pre-mineralization period. For a full understanding of the action of 1,25D3 in osteoblasts it is important to get an integrated network view of the 1,25D3-regulated genes during osteoblast differentiation and mineralization. The current data will be presented and discussed alluding to future studies to fully delineate the 1,25D3 action in osteoblast. Describing and understanding the vitamin D regulatory networks and identifying the dominant players in these networks may help develop novel (personalized) vitamin D-based treatments. The following topics will be discussed in this overview: (1) Bone metabolism and osteoblasts, (2) Vitamin D, bone metabolism and osteoblast function, (3) Vitamin D induced transcriptional networks in the context of osteoblast differentiation and bone formation.

This study investigates the potential of 3D silk scaffolds fabricated using tropical tasar non-mulberry, Antheraea mylitta and mulberry, Bombyx mori silk gland fibroin proteins as substrate for osteogenic and adipogenic differentiation of rat bone marrow cells (BMCs). The scaffolds are mechanically robust and show homogenous pore distribution with high porosity and interconnected pore walls. Low immunogenicity of fabricated silk scaffolds as estimated through TNF alpha release indicates its potential as future biopolymeric graft material. Rat bone marrow cells cultured on scaffolds for 28 days under static conditions in osteogenic and adipogenic media respectively led to induction of differentiation. Proliferation and spreading of fibroblasts and bone marrow cells on silk scaffolds were observed to be dependent on scaffold porosity as revealed through confocal microscopic observations. Histological analysis shows osteogenic differentiation within silk scaffolds resulting in extensive mineralization in the form of deposited nodules as observed through intense Alizarin Red S staining. Similarly, adipogenesis was marked by the presence of lipid droplets within scaffolds on staining with Oil Red O. Real-time PCR studies reveal higher transcript levels for osteopontin (Spp1), osteocalcin (Bglap2) and osteonectin (Sparc) genes under osteogenic conditions. Similarly, upregulated adipogenic gene expression was observed within A. mylitta and B. mori scaffolds under adipogenic conditions for Peroxisome proliferator activated receptor gamma (PPARgamma2), lipoprotein lipase (LPL) and adipocyte binding protein (aP2) genes. The results suggest suitability of silk fibroin protein 3D scaffolds as natural biopolymer for potential bone and adipose tissue engineering applications.

Sustained increase of cardiac workload is known to trigger cardiac remodeling with eventual development of cardiac failure. Compelling evidence points to a critical role of enhanced cardiac Na(+)/H(+) exchanger (NHE1) activity in the underlying pathophysiology. The signaling triggering up-regulation of NHE1 remained, however, ill defined. The present study explored the involvement of the serum- and glucocorticoid-inducible kinase Sgk1 in cardiac remodeling due to transverse aortic constriction (TAC). To this end, experiments were performed in gene targeted mice lacking functional Sgk1 (sgk1 (-/-)) and their wild-type controls (sgk1 (+/+)). Transcript levels have been determined by RT-PCR, cytosolic pH (pH( i )) utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, Na(+)/H(+) exchanger activity by the Na(+)-dependent realkalinization after an ammonium pulse, ejection fraction (%) utilizing cardiac cine magnetic resonance imaging and cardiac glucose uptake by PET imaging. As a result, TAC increased the mRNA expression of Sgk1 in sgk1 (+/+) mice, paralleled by an increase in Nhe1 transcript levels as well as Na(+)/H(+) exchanger activity, all effects virtually abrogated in sgk1 (-/-) mice. In sgk1 (+/+) mice, TAC induced a decrease in Pgc1a mRNA expression, while Spp1 mRNA expression was increased, both effects diminished in the sgk1 (-/-) mice. TAC was followed by a significant increase of heart and lung weight in sgk1 (+/+) mice, an effect significantly blunted in sgk1 (-/-) mice. TAC increased the transcript levels of Anp and Bnp, effects again significantly blunted in sgk1 (-/-) mice. TAC increased transcript levels of Collagen I and III as well as Ctgf mRNA and CTGF protein abundance, effects significantly blunted in sgk1 (-/-) mice. TAC further decreased the ejection fraction in sgk1 (+/+) mice, an effect again attenuated in sgk1 (-/-) mice. Also, cardiac FDG-glucose uptake was increased to a larger extent in sgk1 (+/+) mice than in sgk1 (-/-) mice after TAC. These observations point to an important role for SGK1 in cardiac remodeling and development of heart failure following an excessive work load.

RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation. RUNX2 induced Sp7 expression in Runx2(-/-) calvarial cells. Adenoviral transfer of sh-Sp7 into primary osteoblasts reduced the expression of Alpl, Col1a1, and Bglap2 and mineralization, whereas that of Sp7 reduced Bglap2 expression and mineralization at a late stage of osteoblast differentiation. Sp7 transgenic mice under the control of 2.3 kb Col1a1 promoter showed osteopenia and woven-bone like structure in the cortical bone, which was thin and less mineralized, in a dose-dependent manner. Further, the number of processes in the osteoblasts and osteocytes was reduced. Although the osteoblast density was increased, the bone formation was reduced. The frequency of BrdU incorporation was increased in the osteoblastic cells, while the expression of Col1a1, Spp1, Ibsp, and Bglap2 was reduced. Further, the osteopenia in Sp7 or Runx2 transgenic mice was worsened in Sp7/Runx2 double transgenic mice and the expression of Col1a1 and Bglap2 was reduced. The expression of Sp7 and Runx2 was not increased in Runx2 and Sp7 transgenic mice, respectively. The expression of endogenous Sp7 was increased in Sp7 transgenic mice and Sp7-transduced cells; the introduction of Sp7 activated and sh-Sp7 inhibited Sp7 promoter; and ChIP assay showed the binding of endogenous SP7 in the proximal region of Sp7 promoter. These findings suggest that SP7 and RUNX2 inhibit osteoblast differentiation at a late stage in a manner independent of RUNX2 and SP7, respectively, and SP7 positively regulates its own promoter.

Wnt signaling is implicated in bone formation and activated in breast cancer cells promoting primary and metastatic tumor growth. A compelling question is whether osteogenic miRNAs that increase Wnt activity for bone formation are aberrantly expressed in breast tumor cells to support metastatic bone disease. Here we report that miR-218-5p is highly expressed in bone metastases from breast cancer patients, but is not detected in normal mammary epithelial cells. Furthermore, inhibition of miR-218-5p impaired the growth of bone metastatic MDA-MB-231 cells in the bone microenvironment in vivo. These findings indicate a positive role for miR-218-5p in bone metastasis. Bioinformatic and biochemical analyses revealed a positive correlation between aberrant miR-218-5p expression and activation of Wnt signaling in breast cancer cells. Mechanistically, miR-218-5p targets the Wnt inhibitors Sclerostin (SOST) and sFRP-2, which highly enhances Wnt signaling. In contrast, delivery of antimiR-218-5p decreased Wnt activity and the expression of metastasis-related genes, including bone sialoprotein (BSP/IBSP), osteopontin (OPN/SPP1) and CXCR-4, implicating a Wnt/miR-218-5p regulatory network in bone metastatic breast cancer. Furthermore, miR-218-5p also mediates the Wnt-dependent up-regulation of PTHrP, a key cytokine promoting cancer-induced osteolysis. Antagonizing miR-218-5p reduced the expression of PTHrP and Rankl, inhibited osteoclast differentiation in vitro and in vivo, and prevented the development of osteolytic lesions in a preclinical metastasis model. We conclude that pathological elevation of miR-218-5p in breast cancer cells activates Wnt signaling to enhance metastatic properties of breast cancer cells and cancer-induced osteolytic disease, suggesting that miR-218-5p could be an attractive therapeutic target for preventing disease progression.

The success of vascular intervention including angioplasty, stenting, and arterial bypass remains limited by negative remodeling resulted in lumen restenosis. This study was to characterize the global transcription profile reflecting concurrent events along arterial remodeling and neointima formation in a rat carotid artery balloon-injury model. Expression profiling of injured and control common carotid arteries on days 4, 7, 14 post-injury that mark the major pathohistological progression stages of neointimal formation were recorded on high-density oligonucleotide arrays. A subset of genes from microarray-based data was further studied using quantitative real time RT-PCR and in situ hybridization with sequential arterial samples from days 1 to 28 post-injury. The gene-encoded proteins were validated with Western blot. Besides temporal induction of a large cluster of genes over-represented by cell proliferation and macromolecule metabolism gene ontology categories, a fast-evolving inflammation could be demonstrated by the induction of Tgfb and other anti-inflammatory genes (e.g., C1qtnf3 (C1q and tumor necrosis factor related protein 3 (predicted))) and a shift from type 1 to 2 helper T cell response. The most significant signature of the induced neointimal profile is enrichment of genes functionally related to angiogenesis and extracellular matrix (ECM) remodeling (e.g., Spp1 (secreted phosphoprotein 1), CD44 (CD44 antigen), and Cxcl12 (chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)). Some of the genes represent stress-responsive mesenchymal stromal cell cytokines. This study highlighted mesenchymal stromal cell cytokines-driven inflammatory extracellular matrix remodeling, as target processes for potential clinical therapeutic intervention.

BACKGROUND

We have previously shown that the expression of CBX7 is drastically decreased in several human carcinomas and that its expression progressively decreases with the appearance of a highly malignant phenotype. The aim of our study has been to investigate the mechanism by which the loss of CBX7 expression may contribute to the emergence of a more malignant phenotype.

METHODS

We analyzed the gene expression profile of a thyroid carcinoma cell line after the restoration of CBX7 and, then, analyzed the transcriptional regulation of identified genes. Finally, we evaluated the expression of CBX7 and regulated genes in a panel of thyroid and lung carcinomas.

RESULTS

We found that CBX7 negatively or positively regulates the expression of several genes (such as SPP1, SPINK1, STEAP1, and FOS, FOSB, EGR1, respectively) associated to cancer progression, by interacting with their promoter regions and modulating their transcriptional activity. Quantitative RT-PCR analyses in human thyroid and lung carcinoma tissues revealed a negative correlation between CBX7 and its down-regulated genes, while a positive correlation was observed with up-regulated genes.

CONCLUSIONS

In conclusion, the loss of CBX7 expression might play a critical role in advanced stages of carcinogenesis by deregulating the expression of specific effector genes.

OBJECTIVE

This study aimed to determine the potential impact of type 2 diabetes mellitus on left ventricular dysfunction and the development of calcified aortic valve disease using a dyslipidemic mouse model prone to developing type 2 diabetes mellitus.

RESULTS

When compared with nondiabetic LDLr(-/-)/ApoB(100/100), diabetic LDLr(-/-)/ApoB(100/100)/IGF-II mice exhibited similar dyslipidemia and obesity but developed type 2 diabetes mellitus when fed a high-fat/sucrose/cholesterol diet for 6 months. LDLr(-/-)/ApoB(100/100)/IGF-II mice showed left ventricular hypertrophy versus C57BL6 but not LDLr(-/-)/ApoB(100/100) mice. Transthoracic echocardiography revealed significant reductions in both left ventricular systolic fractional shortening and diastolic function in high-fat/sucrose/cholesterol fed LDLr(-/-)/ApoB(100/100)/IGF-II mice when compared with LDLr(-/-)/ApoB(100/100). Importantly, we found that peak aortic jet velocity was significantly increased in LDLr(-/-)/ApoB(100/100)/IGF-II mice versus LDLr(-/-)/ApoB(100/100) animals on the high-fat/sucrose/cholesterol diet. Microtomography scans and Alizarin red staining indicated calcification in the aortic valves, whereas electron microscopy and energy dispersive x-ray spectroscopy further revealed mineralization of the aortic leaflets and the presence of inflammatory infiltrates in diabetic mice. Studies showed upregulation of hypertrophic genes (anp, bnp, b-mhc) in myocardial tissues and of osteogenic genes (spp1, bglap, runx2) in aortic tissues of diabetic mice.

CONCLUSIONS

We have established the diabetes mellitus -prone LDLr(-/-)/ApoB(100/100)/IGF-II mouse as a new model of calcified aortic valve disease. Our results are consistent with the growing body of clinical evidence that the dysmetabolic state of type 2 diabetes mellitus contributes to early mineralization of the aortic valve and calcified aortic valve disease pathogenesis.

Secreted phosphoprotein 1 (SPP1, commonly referred to as osteopontin and formerly known as bone sialoprotein 1, early T-lymphocyte activation 1) is an extracellular matrix/adhesion molecule that is upregulated in the pregnant uterus of all mammals examined to date. This study focused on the pig, which has true epitheliochorial placentation and exhibits induction of SPP1 mRNA in luminal epithelium (LE) just before conceptus attachment and in glandular epithelium (GE) after Day 30 of pregnancy. The objective of this study was to determine steroid regulation of SPP1 mRNA and protein in porcine uterine epithelium. To examine the effect of estrogen, cyclic gilts were treated daily (Days 11-14) with 5 mg estradiol benzoate (i.m.) and hysterectomized on Day 15. To evaluate the long-term effect of pseudopregnancy, cyclic gilts were given daily injections (Days 11-15) with steroid as above and hysterectomized on Day 90. In situ hybridization showed high expression of SPP1 mRNA only in LE contiguous with apposing conceptus tissue on Day 15 of pregnancy. In contrast, estrogen injection resulted in moderate but uniform SPP1 mRNA in all LE of Day 15 nonpregnant gilts, with expression maintained through Day 90 of pseudopregnancy. SPP1 mRNA also localized to the GE of Day 90 pseudopregnant gilts, similar to expression in late gestation. Consistent with in situ hybridization results, SPP1 protein localized to the apical surface of LE in all estrogen-treated gilts and in the GE on Day 90 of pseudopregnancy. We conclude that, in pregnant pigs, SPP1 is induced by conceptus estrogen in uterine LE and is regulated in GE in a manner coincident with CL/placental progesterone production.

BACKGROUND

In a human T-cell acute lymphoblastic leukemia (T-ALL) cell line (Molt-4), siRNA-mediated suppression of BCL11B expression was shown to inhibit proliferation and induce apoptosis, functions which may be related to genes involved in apoptosis (such as TNFSF10 and BCL2L1) and TGF-β pathways (such as SPP1and CREBBP).

METHODS

The expression levels of the above mentioned genes and their correlation with the BCL11B gene were analyzed in patients with T-ALL using the TaqMan and SYBR Green I real-time polymerase chain reaction technique.

RESULTS

Expression levels of BCL11B, BCL2L1, and CREBBP mRNA in T-ALL patients were significantly higher than those from healthy controls (P<0.05). In T-ALL patients, the BCL11B expression level was negatively correlated with the BCL2L1 expression level (rs=-0.700; P<0.05), and positively correlated with the SPP1 expression level (rs=0.683; P<0.05). In healthy controls, the BCL11B expression level did not correlate with the TNFSF10, BCL2L1, SPP1, or CREBBP expression levels.

CONCLUSIONS

Over-expression of BCL11B might play a role in anti-apoptosis in T-ALL cells through up-regulation of its downstream genes BCL2L1 and CREBBP.

The gene encoding the chicken chemokine 9E3/CEF4 was cloned, sequenced, and mapped; 9E3/CEF4 was the first nonmammalian cytokine cDNA to be cloned and has significant amino acid identity with both human IL8 and human GROalpha. These results show that this cytokine is chicken IL8 and not GROalpha. The exon:intron structure of chicken IL8 corresponds almost exactly to that of human IL8 and differs from those of other known mammalian CXC chemokine genes. Analysis of the predicted amino acid sequence suggests that overall protein structure is conserved between human and chicken IL8, but that the receptor binding sites are not. Genetic distance analysis also suggests that this gene encodes chicken IL8. A number of potential regulatory sequences similar to those found in human IL8 have been identified in the promoter. These include (5'-3') a hepatocyte NF-1 binding site, an NF-kappaB binding site, and a TATAAA box. The human AP-1 binding site and CCAT box are poorly conserved in the promoter of the chicken gene, but there are other potential AP-1 binding sites and a potential CCAT box. The human IRF-1 and octamer binding sites seem to be absent. However, the chicken gene promoter contains a GATA motif not present in the promoter of human IL8. Sequence comparisons also identify conserved regions in the promoter that may function as transcription factor binding sites as yet undescribed in the human IL8 promoter. Promoter sequence polymorphisms have been identified in chicken lines C and 61, but neither lie in any of the regulatory regions mentioned above. Chicken IL8 contains nine repeats of the "instability" motif ATTTA in the 3' untranslated region (UTR) in exon 4. A multiple restriction single-stranded conformational polymorphism was identified which enabled chicken IL8 to be genetically mapped to Chromosome (Chr) 4, linked to SPP1 and ALB1, and thus showing conserved synteny with mouse Chr 5 and human Chr 4. This is the first nonmammalian chemokine gene to be genetically mapped.

The over-expression of NF-κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti-inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF-κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non-steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF-κB inhibitory activity of compound A in H-2K(b) -tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post-natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin-B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL-6, CCL2, IFNγ, TNF and IL-12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild-type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild-type mice. In conclusion, selective glucocorticoid receptor modulation by compound A represents a potential therapeutic strategy to improve dystrophic pathology.

Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2).