Evaluation and refinement of tmRNA structure using gene sequences from natural microbial communities

DNA harvested directly from complex natural microbial communities by PCR has been successfully used to predict RNase P RNA structure, and can potentially provide an abundant source of information for structural predictions of other RNAs. In this study, we utilized genetic variation in natural communities to test and refine the secondary and tertiary structural model for the bacterial tmRNA. The variability of proposed tmRNA secondary structures in different organisms and the lack of any predicted tertiary structure suggested that further refinement of the tmRNA could be useful. To increase the phylogenetic representation of tmRNA sequences, and thereby provide additional data for statistical comparative analysis, we amplified, sequenced, and compared tmRNA sequences from natural microbial communities. Using primers designed from gamma proteobacterial sequences, we determined 44 new tmRNA sequences from a variety of environmental DNA samples. Covariation analyses of these sequences, along with sequences from cultured organisms, confirmed most of the proposed tmRNA model but also provided evidence for a new tertiary interaction. This approach of gathering sequence information from natural microbial communities seems generally applicable in RNA structural analysis.     

Escalation and extinction selectivity: morphology versus isotopic reconstruction of bivalve metabolism

Studies that have tested and failed to support the hypothesis that escalated species (e.g., those with predation-resistant adaptations) are more susceptible to elimination during mass extinctions have concentrated on the distribution and degree of morphological defenses in molluscan species. This morphological approach to determining level of escalation in bivalves may be oversimplified because it does not account for metabolic rate, which is an important measure of escalation that is less readily accessible for fossils. Shell growth rates in living bivalves are positively correlated with metabolic rate and thus are potential indicators of level of escalation. To evaluate this approach, we used oxygen isotopes to reconstruct shell growth rates for two bivalve species (Macrocallista marylandica and Glossus markoei) from Miocene-aged sediments of Maryland. Although both species are classified as non-escalated based on morphology, the isotopic data indicate that M. marylandica was a faster-growing species with a higher metabolic rate and G. markoei was a slower-growing species with a lower metabolic rate. Based on these results, we predict that some morphologically non-escalated species in previous tests of extinction selectivity should be reclassified as escalated because of their fast shell growth rates (i.e., high metabolic rates). Studies that evaluate the level of escalation of a fauna should take into account the energetic physiology of taxa to avoid misleading results.     

Evidence for segregation of heterologous GPI-anchored proteins into separate lipid rafts within the plasma membrane

Cholesterol and glycosphingolipid-rich membrane rafts, which are rich in GPI-anchored proteins and are distinct from caveolae, are believed to serve as platforms for signal transduction events and protein recycling. GPI-anchored proteins with diverse functions as well as caveolin may be recovered in a membrane fraction insoluble in cold non-ionic detergent. This study tests for possible heterogeneity in the protein composition of the lipid rafts and detergent-insoluble membrane complexes by examining the two GPI-anchored homologous human folate receptors (FR)-alpha and -beta, the GPI-anchored human placental alkaline phosphatase (PLAP), and caveolin (control) in transfected CHO cells. Both FR and PLAP showed the equal distribution of cell-surface vs. sequestered (recycling) protein typical of GPI-proteins. Quantitative affinity purification of detergent-insoluble complexes using biotinylated folate or specific antibodies demonstrated a strong association of the homologous FR-alpha and FR-beta in the same detergent-insoluble complex and separate complexes containing either PLAP or caveolin. Immunogold localization experiments using antibody crosslinking to produce larger aggregates of GPI-anchored proteins for visualization by electron microscopy also showed a clear separation between FR- and PLAP-rich membrane microdomains. Thus, even though functionally diverse and heterologous GPI-anchored proteins are known to share endocytic and recycling vesicles, they may be segregated in distinct lipid rafts on the basis of their ecto(protein) domains facilitating clustering, compartmentalization and homotypic protein interactions.     

Substantial sulfatide deficiency and ceramide elevation in very early Alzheimer's disease: potential role in disease pathogenesis

In addition to pathology in the gray matter, there are also abnormalities in the white matter in Alzheimer's disease (AD). Sulfatide species are a class of myelin-specific sphingolipids and are involved in certain diseases of the central nervous system. To assess whether sulfatide content in gray and white matter in human subjects is associated with both the presence of Alzheimer's disease (AD) pathology as well as the stage of dementia, we analyzed the sulfatide content of brain tissue lipid extracts by electrospray ionization mass spectrometry from 22 subjects whose cognitive status at time of death varied from no dementia to very severe dementia. All subjects with dementia had AD pathology. The results demonstrate that: (i) sulfatides were depleted up to 93% in gray matter and up to 58% in white matter from all examined brain regions from AD subjects with very mild dementia, whereas all other major classes of lipid (except plasmalogen) in these subjects were not altered in comparison to those from age-matched subjects with no dementia; (ii) there was no apparent deficiency in the biosynthesis of sulfatides in very mild AD subjects as characterized by the examination of galactocerebroside sulfotransferase activities in post-mortem brain tissues; (iii) the content of ceramides (a class of potential degradation products of sulfatides) was elevated more than three-fold in white matter and peaked at the stage of very mild dementia. The findings demonstrate that a marked decrease in sulfatides is associated with AD pathology even in subjects with very mild dementia and that these changes may be linked with early events in the pathological process of AD.     

Macrophage-colony-stimulating factor-induced activation of extracellular-regulated kinase involves phosphatidylinositol 3-kinase and reactive oxygen species in human monocytes

We previously reported that activation of the phosphatidylinositol (PI) 3-kinase pathway was important in M-CSF-induced monocyte survival. Because M-CSF also induces activation of the mitogen-activated protein (MAP) kinase extracellular-regulated kinase (Erk), we focused on dissecting the mechanism used by M-CSF to induce Erk activation in human monocytes. We found that, in addition to the MAP/Erk kinase inhibitor PD098059, the PI 3-kinase inhibitors LY294002 and wortmannin both suppressed Erk activation in M-CSF-treated monocytes, suggesting that 3-phosphorylated products of PI 3-kinase played a role in Erk activation. Investigating the biochemical pathways regulated by PI 3-kinase to activate Erk, we found that, in response to M-CSF, normal human monocytes induced reactive oxygen species (ROS), which were suppressed by the PI 3-kinase inhibitor wortmannin but not by the solvent control DMSO or the MAP/Erk kinase inhibitor PD098059. We next found that, in the absence of M-CSF, ROS could induce Erk activation in human monocytes. Exogenous H(2)O(2) induced Erk activation in human monocytes, which was suppressed by exogenous catalase. To determine whether ROS induced by M-CSF played a role in Erk activation, we found that N-acetylcysteine and diphenyleneiodonium both suppressed Erk activation in M-CSF-treated monocytes. Erk activation by M-CSF also seemed to play a role in cellular survival in monocytes. These data suggest that, in M-CSF-stimulated human monocytes, PI 3-kinase products and ROS production play a role in Erk activation and monocyte survival.     

Comparing beta-carotene,vitamin E and nitric oxide as membrane antioxidants

Singlet oxygen initiates lipid peroxidation via a nonfree radical mechanism by reacting directly with unsaturated lipids to form lipid hydroperoxides (LOOHs). These LOOHs can initiate free radical chain reactions leading to membrane leakage and cell death. Here we compare the ability and mechanism by which three small-molecule membrane antioxidants (beta-carotene, alpha-tocopherol and nitric oxide) inhibit lipid peroxidation in membranes. We demonstrate that beta-carotene provides protection against singlet oxygen-mediated lipid peroxidation, but does not slow free radical-mediated lipid peroxidation. Alpha-Tocopherol does not protect cells from singlet oxygen, but does inhibit free radical formation in cell membranes. Nitric oxide provides no direct protection against singlet oxygen exposure, but is an exceptional chain-breaking antioxidant as evident from its ability to blunt oxygen consumption during free radical-mediated lipid peroxidation. These three small-molecule antioxidants appear to have complementary mechanisms for the protection of cell membranes from detrimental oxidations.     

Enhanced mtDNA repair capacity protects pulmonary artery endothelial cells from oxidant-mediated death

In rat cultured pulmonary arterial (PA), microvascular, and venous endothelial cells (ECs), the rate of mitochondrial (mt) DNA repair is predictive of the severity of xanthine oxidase (XO)-induced mtDNA damage and the sensitivity to XO-mediated cell death. To examine the importance of mtDNA damage and repair more directly, we determined the impact of mitochondrial overexpression of the DNA repair enzyme, Ogg1, on XO-induced mtDNA damage and cell death in PAECs. PAECs were transiently transfected with an Ogg1-mitochondrial targeting sequence construct. Mitochondria-selective overexpression of the transgene product was confirmed microscopically by the observation that immunoreactive Ogg1 colocalized with a mitochondria-specific tracer and, with an oligonucleotide cleavage assay, by a selective enhancement of mitochondrial Ogg1 activity. Overexpression of Ogg1 protected against both XO-induced mtDNA damage, determined by quantitative Southern analysis, and cell death as assessed by trypan blue exclusion and MTS assays. These findings show that mtDNA damage is a direct cause of cell death in XO-treated PAECs.     

Golgi alpha-mannosidase II deficiency in vertebrate systems: implications for asparagine-linked oligosaccharide processing in mammals

The maturation of N-glycans to complex type structures on cellular and secreted proteins is essential for the roles that these structures play in cell adhesion and recognition events in metazoan organisms. Critical steps in the biosynthetic pathway leading from high mannose to complex structures include the trimming of mannose residues by processing mannosidases in the endoplasmic reticulum (ER) and Golgi complex. These exo-mannosidases comprise two separate families of enzymes that are distinguished by enzymatic characteristics and sequence similarity. Members of the Class 2 mannosidase family (glycosylhydrolase family 38) include enzymes involved in trimming reactions in N-glycan maturation in the Golgi complex (Golgi mannosidase II) as well as catabolic enzymes in lysosomes and cytosol. Studies on the biological roles of complex type N-glycans have employed a variety of strategies including the treatment of cells with glycosidase inhibitors, characterization of human patients with enzymatic defects in processing enzymes, and generation of mouse models for the enzyme deficiency by selective gene disruption approaches. Corresponding studies on Golgi mannosidase II have employed swainsonine, an alkaloid natural plant product that causes "locoism", a phenocopy of the lysosomal storage disease, alpha-mannosidosis, as a result of the additional targeting of the broad-specificity lysosomal mannosidase by this compound. The human deficiency in Golgi mannosidase II is characterized by congenital dyserythropoietic anemia with splenomegaly and various additional abnormalities and complications. Mouse models for Golgi mannosidase II deficiency recapitulate many of the pathological features of the human disease and confirm that the unexpectedly mild effects of the enzyme deficiency result from a tissue-specific and glycoprotein substrate-specific alternate pathway for synthesis of complex N-glycans. In addition, the mutant mice develop symptoms of a systemic autoimmune disorder as a consequence of the altered glycosylation. This review will discuss the biochemical features of Golgi mannosidase II and the consequences of its deficiency in mammalian systems as a model for the effects of alterations in vertebrate N-glycan maturation during development.