Steroidogenic Enzyme Gene Expression and Testosterone Production are Developmentally Modulated by Bone Morphogenetic Protein Receptor-1B in Mouse Testis

Bone morphogenetic proteins (BMPs) and receptors (BMPR-1A, BMPR-1B, BMPR-2) have been shown to be vital for female reproduction, while their roles in males are poorly described. Our study was undertaken to specify the function of BMPR-1B in steroidogenic enzyme gene expression, testosterone production and reproductive development in male mice, given that Bmpr1b mRNA is expressed in mouse testis and Bmpr1b knockout results in compromised fertility. Male mice were passively immunized for 6 days with anti-BMPR-1B in the presence or absence of exogenous gonadotrophins. We then measured the effects of anti-BMPR-1B on testicular hydroxysteroid dehydrogenase isoforms (Hsd3b1, Hsd3b6, and Hsd17b3) and aromatase (Cyp19) mRNA expression, testicular and serum testosterone levels, and testis and seminal vesicle weight. In vitro testosterone production in response to anti-BMPR-1B was determined using testicular culture, and Leydig cell culture in the presence or absence of gonadotrophins. In Leydig cell culture the contribution of seminiferous tubules and Leydig cells were examined by preconditioning the media with these testicular constituents. In adult mice, anti-BMPR-1B increased testosterone and Hsd3b1 but decreased Hsd3b6 and Cyp19 mRNA. In adult testicular culture and seminiferous tubule conditioned Leydig cell culture, anti BMPR-1B reduced testosterone, while in normal and Leydig cell conditioned Leydig cell culture it increased testosterone levels. In pubertal mice, anti-BMPR-1B reduced gonadotrophin stimulated seminal vesicle growth. In conclusion, BMPR-1B has specific developmental functions in the autocrine and paracrine regulation of testicular steroidogenic enzyme gene expression and testosterone production in adults and in the development of seminal vesicles during puberty

Proteomic and Microscopic Strategies towards the Analysis of the Cytoskeletal Networks in Major Neuropsychiatric Disorders

Mental health disorders have become worldwide health priorities. It is estimated that in the next 20 years they will account for a 16 trillion United State dollars (US$) loss. Up to now, the underlying pathophysiology of psychiatric disorders remains elusive. Altered cytoskeleton proteins expression that may influence the assembly, organization and maintenance of cytoskeletal integrity has been reported in major depressive disorders, schizophrenia and to some extent bipolar disorders. The use of quantitative proteomics, dynamic microscopy and super-resolution microscopy to investigate disease-specific protein signatures holds great promise to improve our understanding of these disorders. In this review, we present the currently available quantitative proteomic approaches use in neurology, gel-based, stable isotope-labelling and label-free methodologies and evaluate their strengths and limitations. We also reported on enrichment/subfractionation methods that target the cytoskeleton associated proteins and discuss the need of alternative methods for further characterization of the neurocytoskeletal proteome. Finally, we present live cell imaging approaches and emerging dynamic microscopy technology that will provide the tools necessary to investigate protein interactions and their dynamics in the whole cells. While these areas of research are still in their infancy, they offer huge potential towards the understanding of the neuronal network stability and its modification across neuropsychiatric disorders

Cellular responses during morphological transformation in Azospirillum brasilense and its flcA knockout mutant

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA− strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome

Profilin-1 overexpression in MDA-MB-231 breast cancer cells is associated with alterations in proteomics biomarkers of cell proliferation, survival, and motility as revealed by global proteomics analyses

Despite early screening programs and new therapeutic strategies, metastatic breast cancer is still the leading cause of cancer death in women in industrialized countries and regions. There is a need for novel biomarkers of susceptibility, progression, and therapeutic response. Global analyses or systems science approaches with omics technologies offer concrete ways forward in biomarker discovery for breast cancer. Previous studies have shown that expression of profilin-1 (PFN1), a ubiquitously expressed actin-binding protein, is downregulated in invasive and metastatic breast cancer. It has also been reported that PFN1 overexpression can suppress tumorigenic ability and motility/invasiveness of breast cancer cells. To obtain insights into the underlying molecular mechanisms of how elevating PFN1 level induces these phenotypic changes in breast cancer cells, we investigated the alteration in global protein expression profiles of breast cancer cells upon stable overexpression of PFN1 by a combination of three different proteome analysis methods (2-DE, iTRAQ, label-free). Using MDA-MB-231 as a model breast cancer cell line, we provide evidence that PFN1 overexpression is associated with alterations in the expression of proteins that have been functionally linked to <i>cell proliferation</i> (FKPB1A, HDGF, MIF, PRDX1, TXNRD1, LGALS1, STMN1, LASP1, S100A11, S100A6), <i>survival</i> (HSPE1, HSPB1, HSPD1, HSPA5 and PPIA, YWHAZ, CFL1, NME1) and <i>motility</i> (CFL1, CORO1B, PFN2, PLS3, FLNA, FLNB, NME2, ARHGDIB). In view of the pleotropic effects of PFN1 overexpression in breast cancer cells as suggested by these new findings, we propose that PFN1-induced phenotypic changes in cancer cells involve multiple mechanisms. Our data reported here might also offer innovative strategies for identification and validation of novel therapeutic targets and companion diagnostics for persons with, or susceptibility to, breast cancer

qRT-PCR primer sets used in this study.

<p>qRT-PCR primer sets used in this study.</p

Strains and plasmids used in this study.

<p>Amp^r, Km^r, Tet^r indicate resistance to ampicillin, kanamycin, and tetracycline, respectively; CR, Congo red binding; Floc, flocculation.</p><p>Strains and plasmids used in this study.</p

Plant-root binding abilities of <i>A. brasilense</i> strain.

<p>Colonization of wheat root by Sp7 (1 and 4), Sp7-flcAΔ (2 and 5) and Sp72001 (3 and 6). <i>A</i>. <i>brasilense</i> strains harbor the reporter plasmid pLA-lacZ, containing a constitutively expressed <i>lacZ</i> gene and were stained with X-gal. Sp7 (1, 4) has strong binding ability to wheat roots and can be found all over the root surface; Sp7-flcAΔ (2, 5) and Sp72001 (3, 6) lost the ability to bind to wheat roots and could only be found in lateral root emergence areas. Scale bars indicate 50 µm (group A) and 5 µm (group B) (Magnification ×100 in group A and ×1000 in group B).</p

Differential protein expression in Sp7 and Sp7-flcAΔ analyzed by 2-DE.

<p>The left section shows the normalized expression volume of the spot in wild type (Sp7) and <i>flcA</i> knock out strain (Sp7-flcAΔ) under flocculation conditions; the relative fold change is shown above each column (Nx indicates that relative fold change could not be calculated as the protein was only detected in either Sp7 or Sp7-flcAΔ). The right section is a 3D representation of the area of interest as provided by PDquest software. Arrows indicate spots with relatively higher expression.</p

Differential gene expression in Sp7 and Sp7-flcAΔ under both nutrient and flocculation conditions, analyzed by qRT-PCR.

<p>Transcription levels of <i>atpD, clpP, glnA, livK, nark, narL1, nos</i> and <i>phaA</i> were significantly higher in Sp7-flcAΔ than in Sp7 under flocculation conditions. Transcription levels of <i>cheW, galU</i> and <i>gltA</i> were significantly lower in Sp7-flcAΔ than in Sp7 under flocculation conditions. Transcription levels of <i>ahpC, gloA</i>, <i>lpxC</i> and <i>sodA</i> did not differ significantly in Sp7 and Sp7-flcAΔ under either culture condition. * indicates a significant difference between group means (P<0.05). Data is presented as mean ±SEM, relative to expression of two reference genes, n = 4 biological replicates.</p