Bone-specific heparan sulfates induce osteoblast growth arrest and downregulation of retinoblastoma protein

The heparan sulfate (HSs) sugars of the extracellular matrix (ECM) play a key role during both development and wound repair in regulating the flow of growth and adhesive factors across their cell surface receptors. The aim of this study was to assess the structural and functional differences of HS chains extracted from the conditioned media (soluble), cell surface, and ECM of primary human osteoblast cultures, and to analyze their effects on osteoblast cell growth. HS chains from these compartments were characterized through a combination of enzymatic degradation, anion exchange chromatography, and molecular sieving. Although the chains were all approximately the same size, they varied systematically in their sulfate content, suggesting differences in their protein-binding domains. When added to pre-confluent hFOB1.19 osteoblast cultures, HS doses exceeding 500 ng/ml inhibited proliferation, without affecting viability, irrespective of their origin. Furthermore, HS doses of 500 ng/ml also downregulated retinoblastoma, Cyclin A and CDK1 protein expression, indicating that high doses of osteoblast HS negatively regulate cell cycle, resulting in growth arrest; when high doses of HS were withdrawn after a prolonged period, linear cell growth was reestablished. Thus, despite differences in sulfation, HS from either the soluble, cell surface, or matrix compartments of primary human osteoblast cultures are functionally similar with respect to their effects on growth. Binding assays revealed that the HS chains bound TGFbeta1, a known inhibitor of osteoprogenitor growth, at higher affinity than a suite of other bone-related, heparin-binding growth factors. Overcoming such sugar-mediated inhibition may prove important for wound repair.     

Androgen Receptor CAG Repeats Length Polymorphism and the Risk of Polycystic Ovarian Syndrome (PCOS)

Objective

Polycystic ovarian syndrome (PCOS) refers to an inheritable androgen excess disorder characterized by multiple small follicles located at the ovarian periphery. Hyperandrogenism in PCOS, and inverse correlation between androgen receptor (AR) CAG numbers and AR function, led us to hypothesize that CAG length variations may affect PCOS risk.

Methods

CAG repeat region of 169 patients recruited following strictly defined Rotterdam (2003) inclusion criteria and that of 175 ethnically similar control samples, were analyzed. We also conducted a meta-analysis on the data taken from published studies, to generate a pooled estimate on 2194 cases and 2242 controls.

Results

CAG bi-allelic mean length was between 8.5 and 24.5 (mean = 17.43, SD = 2.43) repeats in the controls and between 11 and 24 (mean = 17.39, SD = 2.29) repeats in the cases, without any significant difference between the two groups. Further, comparison of bi-allelic mean and its frequency distribution in three categories (short, moderate and long alleles) did not show any significant difference between controls and various case subgroups. Frequency distribution of bi-allelic mean in two categories (extreme and moderate alleles) showed over-representation of extreme sized alleles in the cases with marginally significant value (50.3% vs. 61.5%, χ² = 4.41; P = 0.036), which turned insignificant upon applying Bonferroni correction for multiple comparisons. X-chromosome inactivation analysis showed no significant difference in the inactivation pattern of CAG alleles or in the comparison of weighed bi-allelic mean between cases and controls. Meta-analysis also showed no significant correlation between CAG length and PCOS risk, except a minor over-representation of short CAG alleles in the cases.

Conclusion

CAG bi-allelic mean length did not differ between controls and cases/case sub-groups nor did the allele distribution. Over-representation of short/extreme-sized alleles in the cases may be a chance finding without any true association with PCOS risk.     

Dietary oils mediate cortisol kinetics and the hepatic mRNA expression profile of stress-responsive genes in gilthead sea bream (Sparus aurata) exposed to crowding stress. Implications on energy homeostasis and stress susceptibility

Juveniles of gilthead sea bream were fed with plant protein-based diets with fish oil (FO diet) or vegetable oils (66VO diet) as dietary lipid sources. No differences in growth performance were found between both groups, and fish with an average body mass of 65–70 g were crowded (90–100 kg/m³) to assess the stress response within the 72 h after the onset of stressor. The rise in plasma cortisol and glucose levels was higher in stressed fish of group 66VO (66VO-S) than in FO group (FO-S), but the former stressed group regained more quickly the cortisol resting values of the corresponding non-stressed diet group. The cell–tissue repair response represented by derlin-1, 75 kDa glucose-regulated protein and 170 kDa glucose-regulated protein was triggered at a lower level in 66VO-S than in FO-S fish. This occurred in concert with a long-lasting up-regulation of glucocorticoid receptors, antioxidant enzymes, enzyme subunits of the mitochondrial respiratory chain, and enzymes involved in tissue fatty acid uptake and β-oxidation. This gene expression pattern allows a metabolic phenotype that is prone to “high power” mitochondria, which would support the replacement of fish oil with vegetable oils when theoretical requirements in essential fatty acids for normal growth are met by diet.     

Oxygen Consumption Constrains Food Intake in Fish Fed Diets Varying in Essential Amino Acid Composition

Compromisation of food intake when confronted with diets deficient in essential amino acids is a common response of fish and other animals, but the underlying physiological factors are poorly understood. We hypothesize that oxygen consumption of fish is a possible physiological factor constraining food intake. To verify, we assessed the food intake and oxygen consumption of rainbow trout fed to satiation with diets which differed in essential amino acid (methionine and lysine) compositions: a balanced vs. an imbalanced amino acid diet. Both diets were tested at two water oxygen levels: hypoxia vs. normoxia. Trout consumed 29% less food under hypoxia compared to normoxia (p<0.001). Under both hypoxia and normoxia trout significantly reduced food intake by 11% and 16% respectively when fed the imbalanced compared to the balanced amino acid diet. Oxygen consumption of the trout per unit body mass remained identical for both diet groups not only under hypoxia but also under normoxia (p>0.05). This difference in food intake between diets under normoxia together with the identical oxygen consumption supports the hypothesis that food intake in fish can be constrained by a set-point value of oxygen consumption, as seen here on a six-week time scale.     

Multiple shoot induction and regeneration of whole plants from cotyledonary node and nodal explants of Sterculia urens Roxb., a gum yielding tree

Plant regeneration from the nodal explants of 1-month-old in vitro grown plants and cotyledonary node explants of 15-days-old seedlings of Sterculia urens is reported. Nodal explants were grown on MS medium supplemented with various growth regulators like BA, KIN and TDZ. For shoot induction 13.3 μM BA, 0.9 μM TDZ and 9.3 μM KIN were found optimum. Among the three growth regulators 0.90 μM TDZ was used for the growth of cotyledonary node explants. An average of 8.6 shoots per node and 11.2 shoots per cotyledonary node were observed in 4 to 5 weeks. These shoots were subsequently rooted in vitro on half strength MS medium containing various concentrations of auxins like IBA and NAA. The best concentrations for rooting of shoots were 19.7 μM IBA and 16.1 μM NAA. Plantlets were acclimatized to ex vitro conditions and established in the field.     

Recombinant Lactobacillus plantarum expressing and secreting heterologous oxalate decarboxylase prevents renal calcium oxalate stone deposition in experimental rats

Background

Calcium oxalate (CaOx) is the major constituent of about 75% of all urinary stone and the secondary hyperoxaluria is a primary risk factor. Current treatment options for the patients with hyperoxaluria and CaOx stone diseases are limited. Oxalate degrading bacteria might have beneficial effects on urinary oxalate excretion resulting from decreased intestinal oxalate concentration and absorption. Thus, the aim of the present study is to examine the in vivo oxalate degrading ability of genetically engineered Lactobacillus plantarum (L. plantarum) that constitutively expressing and secreting heterologous oxalate decarboxylase (OxdC) for prevention of CaOx stone formation in rats. The recombinants strain of L. plantarum that constitutively secreting (WCFS1OxdC) and non-secreting (NC8OxdC) OxdC has been developed by using expression vector pSIP401. The in vivo oxalate degradation ability for this recombinants strain was carried out in a male wistar albino rats. The group I control; groups II, III, IV and V rats were fed with 5% potassium oxalate diet and 14^th day onwards group II, III, IV and V were received esophageal gavage of L. plantarum WCFS1, WCFS1OxdC and NC8OxdC respectively for 2-week period. The urinary and serum biochemistry and histopathology of the kidney were carried out. The experimental data were analyzed using one-way ANOVA followed by Duncan’s multiple-range test.

Results

Recombinants L. plantarum constitutively express and secretes the functional OxdC and could degrade the oxalate up to 70–77% under in vitro. The recombinant bacterial treated rats in groups IV and V showed significant reduction of urinary oxalate, calcium, uric acid, creatinine and serum uric acid, BUN/creatinine ratio compared to group II and III rats (P < 0.05). Oxalate levels in kidney homogenate of groups IV and V were showed significant reduction than group II and III rats (P < 0.05). Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V.

Conclusion

The present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary oxalate excretion and CaOx crystal deposition by increased intestinal oxalate degradation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0086-y) contains supplementary material, which is available to authorized users.     

Albuminuria associated with CD2AP knockout mice is primarily due to dysfunction of the renal degradation pathway processing of filtered albumin

Here we address the assumption that the massive intact albuminuria accompanying mutations of structural components of the slit diaphragm is due to changes in glomerular permeability. The increase in intact albumin excretion rate in Cd2ap knockout mice by >100-fold was not accompanied by equivalent changes in urine flow rate, glomerular filtration rate or increases in dextran plasma clearance rate, which demonstrates that changes in glomerular permeability alone could not account for the increase in intact albumin excretion. The albuminuria could be accounted for by inhibition of the tubule degradation pathway associated with degrading filtered albumin. There are remarkable similarities between these results and all types of podocytopathies in acquired and toxin-induced renal disease, and nephrotic states seen in mice with podocyte mutations.     

Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors

Background

Although numerous mouse models of breast carcinomas have been developed, we do not know the extent to which any faithfully represent clinically significant human phenotypes. To address this need, we characterized mammary tumor gene expression profiles from 13 different murine models using DNA microarrays and compared the resulting data to those from human breast tumors.

Results

Unsupervised hierarchical clustering analysis showed that six models (TgWAP-Myc, TgMMTV-Neu, TgMMTV-PyMT, TgWAP-Int3, TgWAP-Tag, and TgC3(1)-Tag) yielded tumors with distinctive and homogeneous expression patterns within each strain. However, in each of four other models (TgWAP-T ₁₂₁ , TgMMTV-Wnt1, Brca1 ^Co/Co ;TgMMTV-Cre;p53 ^+/- and DMBA-induced), tumors with a variety of histologies and expression profiles developed. In many models, similarities to human breast tumors were recognized, including proliferation and human breast tumor subtype signatures. Significantly, tumors of several models displayed characteristics of human basal-like breast tumors, including two models with induced Brca1 deficiencies. Tumors of other murine models shared features and trended towards significance of gene enrichment with human luminal tumors; however, these murine tumors lacked expression of estrogen receptor (ER) and ER-regulated genes. TgMMTV-Neu tumors did not have a significant gene overlap with the human HER2+/ER- subtype and were more similar to human luminal tumors.

Conclusion

Many of the defining characteristics of human subtypes were conserved among the mouse models. Although no single mouse model recapitulated all the expression features of a given human subtype, these shared expression features provide a common framework for an improved integration of murine mammary tumor models with human breast tumors.     

Temporal and functional changes in glycosaminoglycan expression during osteogenesis

Heparan sulfate proteoglycans (HSPGs) are complex and labile macromolecular moieties on the surfaces of cells that control the activities of a range of extracellular proteins, particularly those driving growth and regeneration. Here, we examine the biosynthesis of heparan sulfate (HS) sugars produced by cultured MC3T3-E1 mouse calvarial pre-osteoblast cells in order to explore the idea that changes in HS activity in turn drive phenotypic development during osteogenesis. Cells grown for 5 days under proliferating conditions were compared to cells grown for 20 days under mineralizing conditions with respect to their phenotype, the forms of HS core protein produced, and their HS sulfotransferase biosynthetic enzyme levels. RQ-PCR data was supported by the results from the purification of day 5 and day 20 HS forms by anionic exchange chromatography. The data show that cells in active growth phases produce more complex forms of sugar than cells that have become relatively quiescent during active mineralization, and that these in turn can differentially influence rates of cell growth when added exogenously back to preosteoblasts.