To investigate the physiological role of arcuate nucleus cocaine- and amphetamine- regulated transcript in energy homeostasis

Cocaine- and amphetamine- regulated transcript (CART) was originally identified as a mRNA transcript upregulated in rats in response to administration of cocaine and amphetamine. CART is widely expressed in the central nervous system (CNS) with high levels of expression in hypothalamic nuclei such as the arcuate nucleus (ARC). CART was initially thought to act as an anorectic peptide since it is coexpressed with the anorectic neuropeptide pro-opiomelanocortin (POMC) in the ARC. In addition, intracerebroventricular (ICV) administration of CART (55-102) peptide inhibits feeding and administration of anti-CART antibody results in stimulation of feeding. However, subsequent studies have suggested CART may also act as an orexigen since injection of CART (55-102) specifically into the ARC and ventromedial nucleus (VMN) of the hypothalamus results in a significant increase in food intake. These data suggest CART acts through both anorectic and orexigenic circuits. Given the importance of the hypothalamus in the regulation of energy homeostasis, and the role of the ARC in integrating peripheral signals, it is essential to elucidate the role of ARC derived CART. In order to elucidate CART’s true physiological role in the ARC I used a combination of genetic approaches. I generated a recombinant Adeno-associated virus (rAAV) expressing CART antisense (CART-AS) and a transgenic mouse model which utilises the POMC promoter to drive expression of CART-AS. In the transgenic CART-AS model mice exhibited a significantly higher body weight relative to control animals, no significant difference in food intake was observed. In addition, mice expressing the CART-AS transgene demonstrated a reduction in uncoupling protein-1 (UCP- 1) mRNA expression in brown adipose tissue (BAT) which is suggestive of decreased thermogenesis. This may explain the observed increase in body weight in the transgenic mice. Bilateral intra-ARC injections of rAAV-CART-AS resulted in a significant increase in cumulative food intake and body weight gain compared to control animals. There was no significant difference in activity or metabolism levels. The data presented in my thesis provides an important contribution to understanding the role of CART within the ARC. The results from my genetic studies appear to suggest that ARC derived CART has an anorectic role

Analysis of the Essential Functions of the C-terminal Protein/Protein Interaction Domain of Saccharomyces cerevisiae pol epsilon and Its Unexpected Ability to Support Growth in the Absence of the DNA Polymerase Domain

As first observed by Wittenberg (Kesti, T., Flick, K., Keranen, S., Syvaoja, J. E., and Wittenburg, C. (1999) Mol. Cell 3, 679-685), we find that deletion mutants lacking the entire N-terminal DNA polymerase domain of yeast pol epsilon are viable. However, we now show that point mutations in DNA polymerase catalytic residues of pol epsilon are lethal. Taken together, the phenotypes of the deletion and the point mutants suggest that the polymerase of pol epsilon may normally participate in DNA replication but that another polymerase can substitute in its complete absence. Substitution is inefficient because the deletion mutants have serious defects in DNA replication. This observation raises the question of what is the essential function of the C-terminal half of pol epsilon . We show that the ability of the C-terminal half of the polymerase to support growth is disrupted by mutations in the cysteine-rich region, which disrupts both dimerization of the POL2 gene product and interaction with the essential DPB2 subunit, suggesting that this region plays an important architectural role at the replication fork even in the absence of the polymerase function. Finally, the S phase checkpoint, with respect to both induction of RNR3 transcription and cell cycle arrest, is intact in cells where replication is supported only by the C-terminal half of pol epsilon , but it is disrupted in mutants affecting the cysteine-rich region, suggesting that this domain directly affects the checkpoint rather than acting through the N-terminal polymerase active site

Role of the Putative Zinc Finger Domain of Saccharomyces cerevisiae DNA Polymerase epsilon in DNA Replication and the S/M Checkpoint Pathway

It has been proposed that C-terminal motifs of the catalytic subunit of budding yeast polymerase (pol) epsilon (POL2) couple DNA replication to the S/M checkpoint (Navas, T. A., Zheng, Z., and Elledge, S. J. (1995) Cell 80, 29-39). Scanning deletion analysis of the C terminus reveals that 20 amino acid residues between two putative C-terminal zinc fingers are essential for DNA replication and for an intact S/M cell cycle checkpoint. All mutations affecting the inter-zinc finger amino acids or the zinc fingers themselves are sensitive to methylmethane sulfonate and have reduced ability to induce RNR3, showing that the mutants are defective in the transcriptional response to DNA damage as well as the cell cycle response. The mutations affect the assembly of the pol epsilon holoenzyme. Two-hybrid assays show that the POL2 subunit interacts with itself, and that the replication and checkpoint mutants are specifically defective in the interaction, suggesting (but not proving) that direct or indirect dimerization may be important for the normal functions of pol epsilon . The POL2 C terminus is sufficient for interaction with DPB2, the essential and phylogenetically conserved subunit of pol epsilon , but not for interaction with DPB3. Neither Dpb3p nor Dpb2p homodimerizes in the two-hybrid assay

Subunit interactions within the Saccharomyces cerevisiae DNA polymerase ε (pol ε) complex - Demonstration of a dimeric pol ε

Saccharomyces cerevisiae DNA polymerase epsilon (pol ε) is essential for chromosomal replication. A major form of pol ε purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay. The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits. We propose that Pol2pzDpb2p and Dpb3pzDpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes. We present biochemical evidence to support the proposal that pol ε may be dimeric in vivo. Gel filtration of the Pol2pzDpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2pzDpb2p. Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p. This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication

Barriers to Offering Vasectomy at Publicly Funded Family Planning Organizations in Texas

Few publicly funded family planning clinics in the United States offer vasectomy, but little is known about the reasons this method is not more widely available at these sources of care. Between February 2012 and February 2015, three waves of in-depth interviews were conducted with program administrators at 54 family planning organizations in Texas. Participants described their organization’s vasectomy service model and factors that influenced how frequently vasectomy was provided. Interview transcripts were coded and analyzed using a theme-based approach. Service models and barriers to providing vasectomy were compared by organization type (e.g., women’s health center, public health clinic) and receipt of Title X funding. Two thirds of organizations did not offer vasectomy on-site or pay for referrals with family planning funding; nine organizations frequently provided vasectomy. Organizations did not widely offer vasectomy because they could not find providers that would accept the low reimbursement for the procedure or because they lacked funding for men’s reproductive health care. Respondents often did not perceive men’s reproductive health care as a service priority and commented that men, especially Latinos, had limited interest in vasectomy. Although organizations of all types reported barriers, women’s health centers and Title X-funded organizations more frequently offered vasectomy by conducting tailored outreach to men and vasectomy providers. A combination of factors operating at the health systems and provider level influence the availability of vasectomy at publicly funded family planning organizations in Texas. Multilevel approaches that address key barriers to vasectomy provision would help organizations offer comprehensive contraceptive services.Population Research Cente

Regulatory T Cells GATA Have It

The factors that control regulatory T (Treg) cell homeostasis and function are still being defined. In this issue of Immunity, Wang et al. (2011) demonstrate that the Th2 cell-associated transcription factor GATA-3 helps control Foxp3 expression in Treg cells and is required for their proper functional activity in vivo