Comparative biology and pathology of oxidative stress in Alzheimer and other neurodegenerative diseases: beyond damage and response

In this review, we consider comparative aspects of the biology and pathology of oxygen radicals in neurodegenerative disease and how these findings have influenced our concept of oxidative stress. The common definition of oxidative stress is a breach of antioxidant defenses by oxygen radicals leading to damage to critical molecules and disrupted physiology. Inherent in this definition is that oxidative stress is an unstable situation, for if there is net damage, viability of the system decreases with time, leading to disequilibria and death. While this circumstance defines acute conditions, such as stroke and head trauma which result in dysfunction and death, it does not fit physiological situations or chronic diseases closely aligned to normal physiology. Therefore, we propose that oxidative modifications in Alzheimer disease may actually serve as a homeostatic response to stress resulting in a shift of neuronal priority from normal function to basic survival. This phenomenon is comparable to normal physiological conditions of metabolic decrease, such as those seen in hibernation and estivation. Thus, Alzheimer disease could be seen as part of normal aging that includes additional pathology due to inadequate homeostatic response.     

RNA based vaccines

The recognition that CD8(+) T-cell mediated Th1 immune responses were necessary to produce immunity to intracellular and transformed self pathogens led to intense interest in the delivery of nucleic acids, DNA, or RNA encoding candidate antigens, as vaccines. Antigen presenting cells (APC) encounter most protein and vaccine immunogens as extracellular proteins and, thus, present them on major histocompatibility complex (MHC) class II molecules leading to the activation of CD4(+) T cells. Protein antigens encoded by nucleic acids delivered to dendritic cell (DC) are produced inside the cell and, thus, can stimulate MHC class I mediated activation of CD8(+) T-cell immune responses. Unfortunately, DCs are not readily transfected with DNA (Akbari et al., 1999) resulting in the requirement for high concentrations of DNA and repeated immunizations to achieved immune responses. RNA, on the other hand, is readily taken up and expressed by DC, making it an alternative vaccine candidate. In this article, we will discuss immune responses developed, interactions between APC and RNA that activate and dictate DC activation, and preliminary studies using RNA in vivo and in vitro to develop protective immunity.     

Neither agouti-related protein nor neuropeptide Y is critically required for the regulation of energy homeostasis in mice

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp(-/-)) mice to examine the physiological role of AgRP. Agrp(-/-) mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp(-/-) mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp(-/-) mice. As AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, we generated AgRP and NPY double-knockout (Agrp(-/-);Npy(-/-)) mice to determine whether NPY or AgRP plays a compensatory role in Agrp(-/-) or NPY-deficient (Npy(-/-)) mice, respectively. Similarly to mice deficient in either AgRP or NPY, Agrp(-/-);Npy(-/-) mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. Our results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY.     

Hsp110 over-expression increases the immunogenicity of the murine CT26 colon tumor

Several studies have suggested a positive correlation between heat shock protein (hsp) expression and tumor immunogenicity. Independently, many studies have shown that hsp purified from tumors can be used as a tumor-specific vaccine. In this study, we have explored the connection between hsp expression and anti-tumor immunity by transducing murine CT26 colon carcinoma cells with the cDNA of a major hsp, i.e. hsp110. We have shown that over-expression of hsp110 has no effect on CT26 tumor cell growth in vitro, and does not inhibit their anchorage-independent growth capacity. However, in situ, hsp110 over-expressing CT26 tumor (CT26-hsp110) grew at a significantly reduced rate as compared to the wild-type CT26 tumor in immunocompetent mice. Moreover, immunization of mice with inactivated CT26-hsp110 cells significantly inhibited the growth of wild-type CT26 tumor. This immunity was associated with an increased frequency of tumor-specific T cells after vaccination. An in vivo antibody depletion assay demonstrated that inactivated CT26-hsp110 cells elicited anti-tumor responses involving CD8(+) T cells and natural killer (NK) cells, but not CD4(+) T cells. Lastly, the effect of the addition of granulocyte-macrophage colony stimulating factor (GM-CSF) to these vaccine formulations was determined. Mice immunized with irradiated CT26-hsp110 cells combined with GM-CSF-producing bystander cells revealed a complete inhibition of CT26 tumor growth, indicating a synergy between inactivated CT26-hsp110 vaccine activity and GM-CSF. These observations demonstrate that manipulation of hsp110 expression in tumors, specifically when combined with GM-CSF, represents a potentially powerful approach to cancer vaccine formulation.     

A mathematical model for elongation of a peptide chain

A mathematical model is presented for the steps in the elongation process, and the steady-state elongation rate as a function of the amino acid concentrations is found. In addition, the reset sub-process of the elongation process is modeled. The rate of elongation of peptide chains is found to be a function of the concentration of the amino acid to be bound and the concentration of all other amino acids. In addition, the overall elongation rate depends on the concentrations of elongation factors.     

Floral dimorphism, pollination, and self-fertilization in gynodioecious GERANIUM RICHARDSONII (Geraniaceae)

The selective maintenance of gynodioecy depends on the relative fitness of the male-sterile (female) and hermaphroditic morphs. Females may compensate for their loss of male fitness by reallocating resources from male function (pollen production and pollinator attraction) to female function (seeds and fruits), thus increasing seed production. Females may also benefit from their inability to self-fertilize if selfing and inbreeding depression reduce seed quality in hermaphrodites. We investigated how differences in floral resource allocation (flower size) between female and hermaphroditic plants affect two measures of female reproductive success, pollinator visitation and pollen receipt, in gynodioecious populations of Geranium richardsonii in Colorado. Using emasculation treatments in natural populations, we further examined whether selfing by autogamy and geitonogamy comprises a significant proportion of pollen receipt by hermaphrodites. Flowers of female plants are significantly smaller than those of hermaphrodites. The reduction in allocation to pollinator-attracting structures (petals) is correlated with a significant reduction in pollinator visitation to female flowers in artificial arrays. The reduction in attractiveness is further manifested in significantly less pollen being deposited on the stigmas of female flowers in natural populations. Autogamy is rare in these protandrous flowers, and geitonogamy accounts for most of the difference in pollen receipt between hermaphrodites and females. Female success at receiving pollen was negatively frequency dependent on the relative frequency of females in populations. Thus, two of the prerequisites for the maintenance of females in gynodioecious populations, differences in resource allocation between floral morphs and high selfing rates in hermaphrodites, occur in G. richardsonii.     

Context-dependent protein stabilization by methionine-to-leucine substitution shown in T4 lysozyme.

The substitution of methionines with leucines within the interior of a protein is expected to increase stability both because of a more favorable solvent transfer term as well as the reduced entropic cost of holding a leucine side chain in a defined position. Together, these two terms are expected to contribute about 1.4 kcal/mol to protein stability for each Met --> Leu substitution when fully buried. At the same time, this expected beneficial effect may be offset by steric factors due to differences in the shape of leucine and methionine. To investigate the interplay between these factors, all methionines in T4 lysozyme except at the amino-terminus were individually replaced with leucine. Of these mutants, M106L and M120L have stabilities 0.5 kcal/mol higher than wild-type T4 lysozyme, while M6L is significantly destabilized (-2.8 kcal/mol). M102L, described previously, is also destabilized (-0.9 kcal/mol). Based on this limited sample it appears that methionine-to-leucine substitutions can increase protein stability but only in a situation where the methionine side chain is fully or partially buried, yet allows the introduction of the leucine without concomitant steric interference. The variants, together with methionine-to-lysine substitutions at the same sites, follow the general pattern that substitutions at rigid, internal sites tend to be most destabilizing, whereas replacements at more solvent-exposed sites are better tolerated.