Immunolocalization of vacuolar system-associated protein-60 (VASAP-60)

We have characterized the localization of the protein termed VASAP-60 in different bovine tissues and cell lines, and have investigated if VASAP-60 interacts with other proteins. Monospecific polyclonal antibodies were raised against distinct fragments of VASAP-60: NH(2) (V(22) to Q(234)), central (A(246) to S(418)), and COOH (L(416) to L(533)). These three antibodies recognized an 88-kDa protein in immunoblotting analysis. The calculated Mr of VASAP-60 derived from its cDNA (60.1 kDa) was significantly lower than its Mr estimated by SDS-PAGE, and this was mainly attributed to the glutamic acid- and aspartic acid-rich composition of its central region (A(246) to S(418)). A 58-kDa proteolytically processed form of VASAP-60 was also identified. Immunocytochemistry demonstrated that VASAP-60 is found predominantly in the perinuclear region, colocalized with calnexin in the endoplasmic reticulum (ER), and partially colocalized with the endocytic marker DAMP. Immunohistochemical localization of VASAP-60 also demonstrated its presence within specialized vesicular structures not related to the ER. Immunoprecipitation using extracts prepared from S(35)Met/Cys metabolically labeled cells demonstrates that VASAP-60 interacts with 116-, 48.5-, and 26.5-kDa proteins. Therefore, VASAP-60 was found to be more widely distributed in the vacuolar system than anticipated, suggesting that VASAP-60 may function in intracellular transport events, rather than being an exclusive component of the quality control mechanism of newly synthesized proteins as thought previously.     

Brain-specific restoration of angiotensin II corrects renal defects seen in angiotensinogen-deficient mice

Mice deficient for angiotensinogen (AGT), or other components of the renin-angiotensin system, show a high rate of neonatal mortality correlated with severe renal abnormalities including hydronephrosis, hypertrophy of renal arteries, and an impaired ability to concentrate urine. Although transgenic replacement of systemic or adipose, but not renal, AGT in AGT-deficient mice has previously been reported to correct some of these renal abnormalities, the tissue target for this complementation has not been defined. In the current study, we have used a novel transgenic strategy to restore the peptide product of the renin-angiotensin system, angiotensin II, exclusively in the brain of AGT-deficient mice and demonstrate that brain-specific angiotensin II can correct the hydronephrosis and partially correct renal dysfunction seen in AGT-deficient mice. Taken together, these results suggest that the renin-angiotensin system affects renal development and function through systemically accessible targets in the brain.     

Pre-sertoli specific gene expression profiling reveals differential expression of Ppt1 and Brd3 genes within the mouse genital ridge at the time of sex determination

In mammals, testis determination is initiated when the SRY gene is expressed in pre-Sertoli cells of the undifferentiated genital ridge. SRY directs the differentiation of these cells into Sertoli cells and initiates the testis differentiation pathway via currently ill-defined mechanisms. Because Sertoli cells are the first somatic cells to differentiate within the developing testis, it is likely that the signals for orchestrating testis determination are expressed within pre-Sertoli cells. We have previously generated a transgenic mouse line that expresses green fluorescent protein under the control of the pig SRY promoter, thus marking pre-Sertoli cells via fluorescence. We have now used suppression-subtractive hybridization (SSH) to construct a normalized cDNA library derived from fluorescence-activated cell sorting (FACS) purified pre-Sertoli cells taken from 12.0 to 12.5 days postcoitum (dpc) fetal transgenic mouse testes. A total of 35 candidate cDNAs for known genes were identified. Detection of Sf1, a gene known for its role in sex determination as well as Vanin-1, Vcp1, Sparc, and Aldh3a1, four genes previously identified in differential screens as gene overexpressed in developing testis compared with ovary, support the biological validity of our experimental model. Whole-mount in situ hybridization was performed on the 35 candidate genes for qualitative differential expression between male and female genital ridges; six were upregulated in the testis and one was upregulated in the ovary. The expression pattern of two genes, Ppt1 and Brd3, were examined in further detail. We conclude that combining transgenically marked fluorescent cell populations with differential expression screening is useful for cell expression profiling in developmental systems such as sex determination and differentiation.     

Genetic manipulation of sex differentiation and phenotype in domestic animals

In mammals, a gene based sex determination system ensures that approximately 50% of offspring will be of the male sex and 50% will be of the female sex. In domestic animal production systems, this ratio is not always ideal. Recent advances in our understanding of the molecular biology of sex determination and differentiation, as well as in the control of gene expression and the direct modification of animal genomes, allows us to consider methods for the direct genetic manipulation of sexual phenotype.     

Rare copy number variations in adults with tetralogy of fallot implicate novel risk gene pathways

Structural genetic changes, especially copy number variants (CNVs), represent a major source of genetic variation contributing to human disease. Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease, but to date little is known about the role of CNVs in the etiology of TOF. Using high-resolution genome-wide microarrays and stringent calling methods, we investigated rare CNVs in a prospectively recruited cohort of 433 unrelated adults with TOF and/or pulmonary atresia at a single centre. We excluded those with recognized syndromes, including 22q11.2 deletion syndrome. We identified candidate genes for TOF based on converging evidence between rare CNVs that overlapped the same gene in unrelated individuals and from pathway analyses comparing rare CNVs in TOF cases to those in epidemiologic controls. Even after excluding the 53 (10.7%) subjects with 22q11.2 deletions, we found that adults with TOF had a greater burden of large rare genic CNVs compared to controls (8.82% vs. 4.33%, p?=?0.0117). Six loci showed evidence for recurrence in TOF or related congenital heart disease, including typical 1q21.1 duplications in four (1.18%) of 340 Caucasian probands. The rare CNVs implicated novel candidate genes of interest for TOF, including PLXNA2, a gene involved in semaphorin signaling. Independent pathway analyses highlighted developmental processes as potential contributors to the pathogenesis of TOF. These results indicate that individually rare CNVs are collectively significant contributors to the genetic burden of TOF. Further, the data provide new evidence for dosage sensitive genes in PLXNA2-semaphorin signaling and related developmental processes in human cardiovascular development, consistent with previous animal models.