Sorting nexin 19 regulates the number of dense core vesicles in pancreatic β‐cells

Aims/Introduction: Insulinoma‐associated protein 2 (IA‐2) regulates insulin secretion and the number of dense core vesicles (DCV). However, the mechanism of regulation of DCV number by IA‐2 is unknown. We examined the effect of sorting nexin 19 (SNX19), an IA‐2 interacting protein, on insulin secretion and the number of dense core vesicles (DCV). Materials and Methods: Stable SNX19 knockdown (SNX19KD) MIN6, a mouse pancreatic β‐cell line, and stable SNX19‐reintroduced SNX19KD MIN6 were established. Quantification of DCV, and lysosomes was carried out using electron micrographs. The half‐life of DCV was detected by pulse‐chase experiment. Results: Insulin secretion and content were decreased in stable SNX19KD MIN6 cells compared with those in control MIN6 cells. Electron micrographs showed that DCV number in SNX19KD cells was decreased by approximately 75% and that DCV size was decreased by approximately 40% compared with those in control cells, respectively. Furthermore, when SNX19 was reintroduced in SNX19KD cells, insulin content, insulin secretion and DCV number were increased. The half‐life of DCV was decreased in SNX19KD cells, but was increased in SNX19KD cells in which SNX19 was reintroduced. The number of lysosomes and the activity of lysosome enzyme cathepsin D were increased by approximately threefold in SNX19KD cells compared with those in control cells. In contrast, they were decreased to approximately half to one‐third in SNX19‐reintroduced SNX19KD cells. Conclusions: SNX19 regulates the number of DCV and insulin content by stabilizing DCV in β‐cells. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2011.00138.x, 2012)     

ELEVATED γ-GLOBULIN AND INCREASED ANTIBODY PRODUCTION IN MICE INFECTED WITH LACTIC DEHYDROGENASE VIRUS

Infection of mice with the lactic dehydrogenase virus (LDV) resulted in an elevated level of γ-globulin. Histologic examination of the spleen and lymph nodes revealed that the number of germinal centers was greatly increased. Immunization with human γ-globulin showed that the capacity of the virus-infected animal to produce anti-human γ-globulin was greatly enhanced and that the virus acted as an adjuvant. From these experiments it is concluded that a virus infection (LDV) can affect the immunologic response of the host to a heterologous antigen.     

STUDIES ON THE MULTIPLICATION AND THE PROPERTIES OF THE LACTIC DEHYDROGENASE AGENT

The procedure used to determine the infective titer of the LDH agent, the reproducibility of this assay, and the relationship between virus dose and plasma enzyme activity were described. Multiplication of the LDH agent began within 6 hours after infection and reached 10^10.8 ID₅₀/ml of plasma within 24 hours. The titer rapidly decreased over the next 72 hours but viremia persisted for at least 16 months with titers as high as 10^5.2 ID₅₀/ml. The appearance of the LDH agent in the circulation preceded the first noticeable rise in plasma LDH activity by close to 24 hours. After 10 months, when the plasma titer of the LDH agent had decreased nearly one millionfold, the plasma enzyme LDH had decreased by less than 50 per cent. The LDH agent is inactivated by ether but withstands lyophilization, and freezing and thawing. It is stable at low temperatures. Ultracentrifugation at 105,000 G for 2 hours leaves less than 0.1 per cent of the LDH agent in the supernatant fluid and filtration through gradocol membranes suggesst that the upper size of the LDH agent is about 55 mµ. Spread of the LDH agent from infected to uninfected mice kept in the same cage and transmission from mothers (infected prior to mating) to their offspring was relatively low.     

The tumor suppressor activity of MDA-7/IL-24 is mediated by intracellular protein expression in NSCLC cells.

mda-7/IL-24 (HGMW-approved symbol IL24) is a tumor suppressor gene whose expression is lost during tumor progression. Gene transfer using adenoviral mda-7/IL-24 (Ad-mda7) exhibits minimal toxicity on normal cells while inducing potent apoptosis in a variety of cancer cell lines. Ad-mda7-transduced cells express high levels of MDA-7 protein intracellularly and also secrete a soluble form of MDA-7 protein. In this study, we sought to determine whether the intracellular or secreted MDA-7 protein was responsible for anti-tumor activity in H1299 lung tumor cells. Ad-mda7 transduction of lung tumor cells increased expression of stress-related proteins, including BiP, GADD34, PP2A, caspases 7 and 12, and XBP-1, consistent with activation of the UPR pathway, a key sensor of endoplasmic reticulum (ER)-mediated stress. Blocking secretion of MDA-7 did not inhibit apoptosis, demonstrating that intracellular MDA-7 was responsible for cytotoxicity. Consistent with this result, when applied directly to lung cancer cells, soluble MDA-7 protein exhibited minimal cytotoxic effect. We then generated mda-7 expression constructs using vectors that target the expressed protein to various subcellular compartments, including cytoplasm, nucleus, and ER. Only full-length and ER-targeted MDA-7 elicited cell death in tumor cells. Thus in lung cancer cells, Ad-mda7 activates the UPR stress pathway and induces apoptosis via intracellular MDA-7 expression in the secretory pathway.     

Titer-dependent antagonism of cocaine following active immunization in rhesus monkeys

Immunization may be a useful pharmacokinetic antagonist therapy for cocaine users. Three rhesus monkeys were immunized with a cocaine:bovine serum albumin conjugate in alum and later with complete and incomplete Freund's adjuvants. Monkeys developed cocaine-binding antibodies (as measured by enzyme-linked immunosorbent assay) after immunization with alum; greater antibody titers developed after immunization with Freund's adjuvants. The response rate-decreasing effect of cocaine diminished in proportion to antibody titer; there was no substantial change in the rate-decreasing effect of bupropion. Plasma cocaine concentrations increased in proportion to antibody titer. Immunizations were well tolerated and had no effect on response rates. These data suggest that the antibody response to a cocaine antigen can produce a specific pharmacokinetic shift in cocaine distribution sufficient to antagonize a behavioral effect of the drug, and can do so with minimal side effects.     

Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo

Overexpression of the melanoma differentiation associated gene-7 (mda-7) in vitro results in suppression of lung cancer cell proliferation. However, the ability of MDA-7 to suppress lung cancer in vivo has not been previously demonstrated. In this study, we investigated the possibility of inducing overexpression of the mda-7 gene in human non-small cell lung carcinoma cells in vivo and its effects on tumor growth. Adenovirus-mediated overexpression of MDA-7 in p53-wild-type A549 and p53-null H1299 subcutaneous tumors resulted in significant tumor growth inhibition through induction of apoptosis. In addition, decreased CD31/PECAM expression and upregulation of APO2/TRAIL were observed in tumors expressing MDA-7. In vivo studies correlated well with in vitro inhibition of lung tumor cell proliferation and endothelial cell differentiation mediated by Ad-mda7. These data demonstrate that Ad-mda7 functions as a multi-modality anti-cancer agent, possessing both, pro-apoptotic and anti-angiogenic properties. We demonstrate for the first time the potential therapeutic effects of Ad-mda7 in human lung cancer.     

Interdisciplinary treatment of ankylosed upper central incisors with an anterior vertical ridge defect: A case report

This case report describes the successful 10-years interdisciplinary treatment of ankylosed upper central incisors with an anterior vertical ridge defect. This treatment was challenging as ankylosis was present before the growth spurt. Orthodontic treatment in association with decoronation, a xenogeneic bone graft, an autogenous sub-epithelial connective tissue graft, and implant placement were performed to correct the vertical ridge defect and to re-establish appropriate function, gingival health, and aesthetics. Decoronation performed during the growth spurt was the key to avoiding alveolar ridge deformity.