Autophagy supports survival and phototransduction protein levels in rod photoreceptors.

Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod cell loss is the primary pathologic event.

Stimulation of extracellular signal-regulated kinases and proliferation in rat osteoblastic cells by parathyroid hormone is protein kinase C-dependent.

Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10(-12) to 10(-11) m) is sufficient for substantial increases in ERK activity. Treatment with PTH-(1-34) (10(-8) m), PTH-(1-31), or 8-bromo-cAMP failed to stimulate ERKs, whereas treatment with phorbol 12-myristate 13-acetate, serum, or PTH peptides lacking the N-terminal amino acids stimulated activity. Furthermore, the activation of ERKs was prevented by pretreatment of osteoblastic cells with inhibitors of protein kinase C (GF 109203X) and MEK (PD 98059). Treatment of UMR cells with epidermal growth factor (EGF), but not PTH, promoted tyrosine phosphorylation of the EGF receptor. Transient transfection of UMR cells with p21(N17Ras) did not block activation of ERKs following treatment with low concentrations of PTH. Thus, activation of ERKs and proliferation by PTH is protein kinase C-dependent, but stimulation occurs independently of the EGF receptor and Ras activation.

Expression of the rfa, LPS biosynthesis promoter in Salmonella typhimurium during invasion of intestinal epithelial cells.

Salmonella regulates transcription of many of its genes in response to environmental conditions encountered inside or outside the eukaryotic cells it infects. In this paper, we examined Salmonella typhimurium gene expression within epithelial cells, by using bacterial luciferase as a reporter. We focused on gene expression controlled by Salmonella rfa promoter, using lac promoter as a control. We observed down regulation for both promoters during the initial 2 h of invasion. The decreased levels of luciferase activity appeared to be due to metabolic changes, since we observed similar results with tissue culture medium alone. Gene expression stabilized to a new steady state for the Salmonella rfa promoter, while a lac promoter activity steadily decreased. Bacterial luciferase activity was a good indicator of intracellular numbers and allowed us to detect as few as 1000 bacterial cells/infected monolayer. Both promoters were not dependent on host protein synthesis for expression.

Antibody responses and infertility in mice following oral immunization with attenuated Salmonella typhimurium expressing recombinant murine ZP3.

Ovarian ZP3, the primary sperm receptor, is a major glycoprotein of mouse zona pellucida (ZP). Because antibodies raised against ZP3 block sperm-egg interaction, ZP3 has been considered a candidate immunogen in the development of a contraceptive vaccine. This study explored the possibility of using an attenuated Salmonella typhimurium vaccine strain expressing recombinant ZP3 to elicit an antibody response and infertility in mice. A cDNA sequence generated by the polymerase chain reaction encoding 342 amino acid residues (23-364) of the mouse (m)ZP3 was cloned into an Asd+ vector. An avirulent Salmonella vaccine strain stably expressed the ZP3 polypeptide and colonized the internal organs of mice after oral inoculation. Oral immunization of female BALB/c mice with the recombinant Salmonella vaccine strain expressing mZP3 induced significant levels of anti-native ZP IgG antibodies in serum and IgA antibodies in vaginal secretions. The IgG antibodies thus induced also bound to ZP in vivo. When mated with males, 3 of 6 females immunized with the recombinant Salmonella were infertile. In contrast, none of the mice that received Salmonella containing the vector plasmid produced antibodies to ZP and all were fertile. No ovarian inflammation was observed in the immunized mice at autopsy. The results suggest a potential oral contraceptive vaccine to control populations of rodent vectors of disease and to induce reversible infertility in humans.

Immune responses to Streptococcus sobrinus surface protein antigen A expressed by recombinant Salmonella typhimurium.

In this study, we used a vaccine strain of Salmonella typhimurium to express antigenic determinants of the SpaA antigen of Streptococcus sobrinus, which is involved in the caries-forming process. We cloned either a single repeat (pYA2901) or three tandem repeats (pYA2905) of the 0.48-kb fragment of the spaA gene, which codes for an important component of the SpaA protein, plus a 1.2-kb minor antigenic determinant and measured the resulting immune responses to SpaA in orally immunized BALB/c mice. The single or triple repeat of the spaA gene fragment was inserted into the Asd+ vector pYA292 and was transformed into the S. typhimurium delta cya delta crp vaccine strain chi 4072 containing delta asd in the chromosome. Female BALB/c mice were then orally immunized with two doses of the S. typhimurium containing either of the two SpaA constructs, and the immune responses to the expressed SpaA protein were assessed. Significant serum immunoglobulin G (IgG) anti-SpaA titers were detected in mice immunized with chi 4072(pYA2905) but not chi 4072(pYA2901). Salivary anti-SpaA IgA titers were minimal and were only detected in mice immunized with S. typhimurium expressing the SpaA encoded by pYA2905. Intestinal anti-SpaA IgA titers, however, were detected in both groups of mice, particularly in mice immunized with chi 4072(pYA2905). An oral booster 26 weeks after the initial series of immunizations resulted in increased serum IgG titers in both chi 4072(pYA2901)- and chi 4072(pYA2905)-immunized animals, particularly in the chi 4072(pYA2905)-immunized animals. No anamnestic IgA response was detected in the saliva following the booster immunization.