Long-lived alphaMUPA transgenic mice show reduced SOD2 expression, enhanced apoptosis and reduced susceptibility to the carcinogen dimethylhydrazine

Calorie restriction (CR) extends the life span of various species through mechanisms that are as yet unclear. Recently, we have reported that mitochondrion-mediated apoptosis was enhanced in alphaMUPA transgenic mice that spontaneously eat less and live longer compared with their wild-type (WT) control mice. To understand the molecular mechanisms underlying the increased apoptosis, we compared alphaMUPA and WT mice for parameters associated with SOD2 (MnSOD), a mitochondrial antioxidant enzyme that converts superoxide radicals into H(2)O(2) and is also known to inhibit apoptosis. The SOD2-related parameters included the levels of SOD2 mRNA, immunoreactivity and enzymatic activity in the liver, lipid oxidation and aconitase activity in isolated liver mitochondria, and the sensitivity of the mice to paraquat, an agent that elicits oxidative stress. In addition, we compared the mice for the levels of SOD2 mRNA after treatment with bacterial lipopolysaccharides (LPS), and for the DNA binding activity of NFkappaB as a marker for the inflammatory state. We extended SOD2 determination to the colon, where we also examined the formation of pre-neoplastic aberrant crypt foci (ACF) following treatment with dimethylhydrazine (DMH), a colonic organotypic carcinogen. Overall, alphaMUPA mice showed reduced basal levels of SOD2 gene expression and activity concomitantly with reduced lipid oxidation, increased aconitase activity and enhanced paraquat sensitivity, while maintaining the capacity to produce high levels of SOD2 in response to the inflammatory stimulus. alphaMUPA mice also showed increased resistance to DMH-induced pre-neoplasia. Collectively, these data are consistent with a model, in which an optimal fine-tuning of SOD2 throughout a long-term regimen of reduced eating could contribute to longevity, at least in the alphaMUPA mice.     

Nitric Oxide Synthase Expression in Macrophages of Histoplasma capsulatum-Infected Mice Is Associated with Splenocyte Apoptosis and Unresponsiveness

Splenic macrophages from Histoplasma capsulatum-infected mice express inducible nitric oxide synthase (iNOS), and the iNOS expression correlates with severity of the infection. We examined whether production of NO is responsible for apoptosis and the anti-lymphoproliferative response of splenocytes from mice infected with H. capsulatum. In situ terminal deoxynucleotidyl transferase nick end labeling revealed apoptotic nuclei in cryosections of spleen from infected but not normal mice. Splenocytes of infected mice were unresponsive to stimulation by either concanavalin A or heat-killed H. capsulatum yeast cells. Splenocyte responsiveness was restored by addition to the medium of N^G-monomethyl-l-arginine, a known inhibitor of NO production. The proliferative response of splenocytes from infected mice was also restored by depletion of macrophages or by replacement with macrophages from normal mice. In addition, expression of iNOS returned to its basal level when the animals had recovered from infection. These results suggest that suppressor cell activity of macrophages is associated with production of NO, which also appears to be an effector molecule for apoptosis of cultured splenocytes from infected mice.

Nitric oxide (NO) has been reported to induce apoptosis in many cells including smooth muscle cells (20), oligodendrocytes (27), pancreatic β cells (11), melanoma cells (35), thymocytes (7), B lymphocytes (4), and macrophages (2). Fehsel et al. recently demonstrated apoptosis in freshly isolated thymocytes after exposure to NO (7). In the same report, they also showed apoptotic foci in close proximity to blood vessels after lipopolysaccharide treatment. Capillary endothelial and dendritic cells adjacent to apoptotic foci stained strongly for inducible nitric oxide synthase (iNOS), suggesting that NO may be the mediator for thymic apoptosis (7). Data from another laboratory also showed that cloned thymic stromal cell monolayers eliminate thymocytes in vitro through production of NO (26). Furthermore, apoptosis has been suggested as a mechanism by which the immune system replenishes itself and maintains homeostasis (30).The dimorphic fungus Histoplasma capsulatum is a facultative intracellular pathogen of the macrophage (32). Although it is not an obligate intracellular pathogen, the organism is found almost exclusively inside host cells during histoplasmosis (5). In our in vitro studies, H. capsulatum exhibits uninhibited growth in normal unstimulated murine macrophages (32). In activated macrophages, either peritoneal macrophages and cells from the Raw 264.7 line stimulated by gamma interferon (IFN-γ) or splenic macrophages stimulated by IFN-γ and lipopolysaccharide, growth of the fungus is inhibited (13, 18, 32). Furthermore, the anti-histoplasma activity of macrophages is dependent on the expression of iNOS and the production of NO (14, 18). However, the significance of NO production in immunoregulation of histoplasmosis is not clearly defined.In this study, we examined whether NO can act as a regulator of apoptosis in lymphoproliferative responses of splenocytes from H. capsulatum-infected mice. We showed that iNOS was induced in splenic macrophages during active infection and the expression of iNOS coincided with active infection. We also observed by in situ terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL) of spleen sections that apoptosis occurred in immune cells in the spleens of infected mice but was minimal in control mice. The link between apoptosis and NO production was established by inclusion of N^G-monomethyl-l-arginine (NMMA) in the culture medium. Inhibition of NO production reduced the amount of apoptosis in splenocyte culture. Thereby, we also confirmed the findings of Zhou et al. (36) that production of NO by splenocytes of H. capsulatum-infected mice suppressed the splenic lymphocyte proliferative response. In addition, we showed that macrophages were mediators of splenocyte unresponsiveness through the NO that they produced and that NO production was associated with apoptotic changes in cultured splenocytes from infected mice.     

Detection of the putative E2 protein of hepatitis C virus in human liver

The question was asked whether a predicted envelope protein, considered to be processed from the polyprotein precursor encoded by the putative E2/NS1 region of the hepatitis C virus (HCV) genome, may be observed in HCV-infected humans. Two polyclonal antibodies against recombinant E2/NS1 proteins were prepared and their reactivity tested against liver extracts from HCV-infected patients by immunoblotting analysis. A band corresponding to a size of 44 kDa was detected in liver extracts from patients who were positive for the HCV-specific antibody anti-C100-3 but not in liver extracts from patients who did not have anti-C100-3 antibody. Additionally, no band was detected using preimmune sera or antisera which had been preabsorbed with recombinant E2/NS1 proteins. Deglycosylation studies demonstrated that the 44 kDa protein was a glycosylated form of a 38 kDa protein which corresponds to the predicted molecular weight of the putative E2/NS1 protein. These results suggest that the 44 kDa protein is a product of the E2/NS1 region. Frequent observation of the 44 kDa band in cases of chronic active hepatitis C suggests a correlation between the expression of this protein and the progression of hepatitis. © 1994 Wiley-Liss, Inc.     

Notes on the large scale preparation and on the properties of anti-lymphocyte serum for use in mice

Mouse spleen and thymus cells have been used in the preparation of horse anti-mouse anti-lymphocyte serum (HAMLS). The cells were used either separately or in a mixture and three types of immunization schedules were used, viz. two-pulse, extended and chronic.

Antisera of marked immunosuppressive activity, as measured by the ability to prolong the life of skin homografts in mice, were obtained using all three schedules, the median survival time being, at best, 27·5 days for the chronic schedule, 26·7 days for the two-pulse schedule and 25·8 days for the extended schedule. The two-pulse and the extended schedules produced non-toxic antisera in a relatively short period of time but were uneconomic in terms of antigen and horses. The chronic schedule was preferred but after 10 weeks the development of unwanted antibodies precluded the further useful immunization of the horses.

     

Slow and fast fiber isoform gene expression is systematically altered in skeletal muscle of the Sox6 mutant, p100H.

We have previously demonstrated that p100H mutant mice, which lack a functional Sox6 gene, exhibit skeletal and cardiac muscle degeneration and develop cardiac conduction abnormalities soon after birth. To understand the role of Sox6 in skeletal muscle development, we identified muscle-specific genes differentially expressed between wild-type and p100H mutant skeletal muscles and investigated their temporal expression in the mutant muscle. We found that, in the mutant skeletal muscle, slow fiber and cardiac isoform genes are expressed at significantly higher levels, whereas fast fiber isoform genes are expressed at significantly lower levels than wild-type. Onset of this aberrant fiber type-specific gene expression in the mutant coincides with the beginning of the secondary myotube formation, at embryonic day 15-16 in mice. Together with our earlier report, demonstrating early postnatal muscle defects in the Sox6 null-p100H mutant, the present results suggest that Sox6 likely plays an important role in muscle development.     

Modulation of level response areas and stimulus selectivity of neurons in cat primary auditory cortex

Sounds commonly occur in sequences, such as in speech. It is therefore important to understand how the occurrence of one sound affects the response to a subsequent sound. We approached this question by determining how a conditioning stimulus alters the response areas of single neurons in the primary auditory cortex (AI) of barbiturate-anesthetized cats. The response areas consisted of responses to stimuli that varied in level at the two ears and delivered at the characteristic frequency of each cell. A binaural conditioning stimulus was then presented > or =50 ms before each of the stimuli comprising the level response area. An effective preceding stimulus alters the shape and severely reduces the size and response magnitude of the level response area. This ability of the preceding stimulus depends on its proximity in the level domain to the level response area, not on its absolute level or on the size of the response it evokes. Preceding stimuli evoke a nonlinear inhibition across the level response area that results in an increased selectivity of a cortical neuron for its preferred binaural stimuli. The selectivity of AI neurons during the processing of a stream of acoustic stimuli is likely to be restricted to a portion of their level response areas apparent in the tone-alone condition. Thus rather than being static, level response areas are fluid; they can vary greatly in extent, shape and response magnitude. The dynamic modulation of the level response area and level selectivity of AI neurons might be related to several tasks confronting the central auditory system.     

A critical look at survival of diabetics with end-stage renal disease. Transplantation versus dialysis therapy

The survival of 100 consecutive patients with diabetic nephropathy after treatment with hemodialysis, peritoneal dialysis, or renal transplantation was reviewed at our institution from 1976 to 1982. Standard actuarial survival analysis revealed an overall survival of 83% and 61% at one and two years, respectively. Coronary angiography was used as a screening procedure for renal transplantation. In the dialysis group, 27 patients were considered acceptable transplant candidates on the basis of the coronary angiography but were not transplanted for other reasons. When the survival analysis was limited to those "transplant candidates" the survival rates were 78%, 51%, and 8% at 1, 2, and 5 years, respectively. In comparison, survival after transplantation was 81%, 67%, and 45%, at 1, 2, and 5 years, respectively. In order to eliminate bias, survival comparisons were subsequently made using the Cox Proportional Hazard Model to take into account the time the transplant patients spent on dialysis prior to renal transplantation. When this analysis was performed, there was no significant difference in survival between transplantation and dialysis for the first two years, but overall survival after five years was significantly better after renal transplantation even when the comparison was limited to acceptable transplant candidates who remained on dialysis (P = .04). Survival for patients with significant coronary disease (greater than 70% stenosis of a coronary vessel or moderate to severe left ventricular dysfunction) was analyzed according to therapeutic modality. Although overall prognosis was poor in this group as a whole (1, 2, and 5 year survivals were 76%, 45%, and 19%, respectively), the cardiac patients had a trend to better survival after renal transplantation than when maintained on dialysis (P = .22). In addition to other factors such as quality of life, rehabilitation, and progression of other diabetic complications, the benefit of renal transplantation on patient survival must be considered when deciding between renal transplantation and maintenance dialysis therapy for diabetic patients with renal failure.     

ASO Author Reflections: National Analysis of Breast Surgery Malpractice Cases: A Teachable Moment?

Annals of Surgical Oncology -