Origin and evolution of the chloroplast trnK (matK) intron: a model for evolution of group II intron RNA structures

The trnK intron of plants encodes the matK open reading frame (ORF), which has been used extensively as a phylogenetic marker for classification of plants. Here we examined the evolution of the trnK intron itself as a model for group II intron evolution in plants. Representative trnK intron sequences were compiled from species spanning algae to angiosperms, and four introns were newly sequenced. Phylogenetic analyses showed that the matK ORFs belong to the ML (mitochondrial-like) subclass of group II intron ORFs, indicating that they were derived from a mobile group II intron of the class. RNA structures of the introns were folded and analyzed, which revealed progressive RNA structural deviations and degenerations throughout plant evolution. The data support a model in which plant organellar group II introns were derived from bacterial-like introns that had "standard" RNA structures and were competent for self-splicing and mobility and that subsequently the ribozyme structures degenerated to ultimately become dependent upon host-splicing factors. We propose that the patterns of RNA structure evolution seen for the trnK intron will apply to the other group II introns in plants.     

Role of caveolin-1 in p42/p44 MAP kinase activation and proliferation of human airway smooth muscle

Chronic airways diseases, including asthma, are associated with an increased airway smooth muscle (ASM) mass, which may contribute to chronic airway hyperresponsiveness. Increased muscle mass is due, in part, to increased ASM proliferation, although the precise molecular mechanisms for this response are not completely clear. Caveolae, which are abundant in smooth muscle cells, are membrane microdomains where receptors and signaling effectors can be sequestered. We hypothesized that caveolae and caveolin-1 play an important regulatory role in ASM proliferation. Therefore, we investigated their role in p42/p44 MAPK signaling and proliferation using human ASM cell lines. Disruption of caveolae using methyl-beta-cyclodextrin and small interfering (si)RNA-knockdown of caveolin-1 caused spontaneous p42/p44 MAPK activation; additionally, caveolin-1 siRNA induced ASM proliferation in mitogen deficient conditions, suggesting a key role for caveolae and caveolin-1 in maintaining quiescence. Moreover, caveolin-1 accumulates twofold in myocytes induced to a contractile phenotype compared with proliferating ASM cells. Caveolin-1 siRNA failed to increase PDGF-induced p42/p44 MAPK activation and cell proliferation, however, indicating that PDGF stimulation actively reversed the antimitogenic control by caveolin-1. Notably, the PDGF induced loss of antimitogenic control by caveolin-1 coincided with a marked increase in caveolin-1 phosphorylation. Furthermore, the strong association of PDGF receptor-beta with caveolin-1 that exists in quiescent cells was rapidly and markedly reduced with agonist addition. This suggests a dynamic relationship in which mitogen stimulation actively reverses caveolin-1 suppression of p42/p44 MAPK signal transduction. As such, caveolae and caveolin-1 coordinate PDGF receptor signaling, leading to myocyte proliferation, and inhibit constitutive activity of p42/p44 MAPK to sustain cell quiescence.     

Polycefin, a new prototype of a multifunctional nanoconjugate based on poly(beta-L-malic acid) for drug delivery

A new prototype of nanoconjugate, Polycefin, was synthesized for targeted delivery of antisense oligonucleotides and monoclonal antibodies to brain tumors. The macromolecular carrier contains: 1. biodegradable, nonimmunogenic, nontoxic beta-poly(L-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin alpha4 and beta1 chains of laminin-8, which is specifically overexpressed in glial brain tumors; 3. monoclonal anti-transferrin receptor antibody for specific tissue targeting; 4. oligonucleotide releasing disulfide units; 5. L-valine containing, pH-sensitive membrane disrupting unit(s), 6. protective poly(ethylene glycol); 7. a fluorescent dye (optional). Highly purified modules were conjugated directly with N-hydroxysuccinimidyl ester-activated beta-poly(L-malic acid) at pendant carboxyl groups or at thiol containing spacers via thioether and disulfide bonds. Products were chemically validated by physical, chemical, and functional tests. In vitro experiments using two human glioma cell lines U87MG and T98G demonstrated that Polycefin was delivered into the tumor cells by a receptor-mediated endocytosis mechanism and was able to inhibit the synthesis of laminin-8 alpha4 and beta1 chains at the same time. Inhibition of laminin-8 expression was in agreement with the designed endosomal membrane disruption and drug releasing activity. In vivo imaging showed the accumulation of intravenously injected Polycefin in brain tumor tissue via the antibody-targeted transferrin receptor-mediated endosomal pathway in addition to a less efficient mechanism known for high molecular mass biopolymers as enhanced permeability and retention effect. Polycefin was nontoxic to normal and tumor astrocytes in a wide range of concentrations, accumulated in brain tumor, and could be used for specific targeting of several biomarkers simultaneously.     

Respiratory chain supercomplexes in the plant mitochondrial membrane

The intricate, heavily folded inner membrane of mitochondria houses the respiratory chain complexes. These complexes, together with the ATP synthase complex, are responsible for energy production, which is stored as ATP. The structure of the individual membrane-bound protein components has been well characterized. In particular, the use of Blue-native polyacrylamide gel electrophoresis has been instrumental in recent years in providing evidence that these components are organized into supercomplexes. Single particle electron microscopy studies have enabled a structural characterization of some of the mitochondrial supercomplexes. This has provided the opportunity to define a functional role for these supercomplexes for the first time, in particular for the dimeric ATP synthase complex, which appears to be responsible for the folding of the inner mitochondrial membrane.     

Zinc homeostatic proteins in the CNS are regulated by crosstalk between extracellular and intracellular zinc

Release of Zn²⁺ from presynaptic glutamatergic terminals has long been considered the principle challenge necessitating the existence of zinc homeostatic proteins (ZHP) in the mammalian nervous system. It is now known that neural cells also possess an intracellular zinc pool, termed here [Zn²⁺]_i, which functions in a cell signaling context. A major challenge is characterizing the interaction of these two populations of zinc ions. To assess the relationship of this Zn²⁺ pool to cellular ZHP production, we employed immunofluorescence and immunoblot analysis to compare the expression of ZHP's ZnT‐1 and MT‐I/II in olfactory bulb and hippocampus of wild‐type and ZnT‐3 KO mice, which lack synaptic Zn²⁺. In both areas, the respective distribution and concentration of ZnT‐1 and MT‐I/II were identical in ZnT‐3 KO and control animals. We subsequently examined ZHP content in ZnT‐3 KO and WT mice treated with a membrane‐permeable Zn²⁺ chelator. In both olfactory bulb and hippocampus of the KO mice, the ZHP content was significantly reduced 15 h after chelation of [Zn²⁺]_i compared to WT controls. Our findings support the conclusion that ZHP expression is regulated by crosstalk between synaptic and intracellular pools of Zn²⁺. J. Cell. Physiol. 224: 567–574, 2010. © 2010 Wiley‐Liss, Inc.     

An improved synthesis of 10,11-didehydro Cinchona alkaloids

A revised procedure for the conversion of the four major Cinchona alkaloids (quinine, quinidine, cinchonidine, and cinchonine) into their respective 10,11-didehydro derivatives is described. The reported protocol offers several advantages over a recently published synthetic route. These include (i) enhanced robustness (ii) ready scalability (iii) reduced operational complexity and number of steps (iv) chromatography-free work-up. In addition, toxic solvents were replaced by environmentally less problematic alternatives.     

Effect of lipid composition on buforin II structure and membrane entry

Buforin II is a 21-amino acid polycationic antimicrobial peptide derived from a peptide originally isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. It is hypothesized to target a wide range of bacteria by translocating into cells without membrane permeabilization and binding to nucleic acids. Previous research found that the structure and membrane interactions of buforin II are related to lipid composition. In this study, we used molecular dynamics (MD) simulations along with lipid vesicle experiments to gain insight into how buforin II interacts differently with phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) lipids. Fluorescent spectroscopic measurements agreed with the previous assertion that buforin II does not interact with pure PC vesicles. Nonetheless, the reduced entry of the peptide into anionic PG membranes versus neutral PC membranes during simulations correlates with the experimentally observed reduction in BF2 translocation through pure PG membranes. Simulations showing membrane entry into PC also provide insight into how buforin II may initially penetrate cell membranes. Our MD simulations also allowed us to consider how neutral PE lipids affect the peptide differently than PC. In particular, the peptide had a more helical secondary structure in simulations with PE lipids. A change in structure was also apparent in circular dichroism measurements. PE also reduced membrane entry in simulations, which correlates with decreased translocation in the presence of PE observed in previous studies. Together, these results provide molecular-level insight into how lipid composition can affect buforin II structure and function and will be useful in efforts to design peptides with desired antimicrobial and cell-penetrating properties.     

DNA damage-induced reactive oxygen species (ROS) stress response in Saccharomyces cerevisiae

Cells are exposed to both endogenous and exogenous sources of reactive oxygen species (ROS). At high levels, ROS can lead to impaired physiological function through cellular damage of DNA, proteins, lipids, and other macromolecules, which can lead to certain human pathologies including cancers, neurodegenerative disorders, and cardiovascular disease, as well as aging. We have employed Saccharomyces cerevisiae as a model system to examine the levels and types of ROS that are produced in response to DNA damage in isogenic strains with different DNA repair capacities. We find that when DNA damage is introduced into cells from exogenous or endogenous sources there is an increase in the amount of intracellular ROS which is not directly related to cell death. We have examined the spectrum of ROS in order to elucidate its role in the cellular response to DNA damage. As an independent verification of the DNA damage-induced ROS response, we show that a major activator of the oxidative stress response, Yap1, relocalizes to the nucleus following exposure to the DNA-alkylating agent methyl methanesulfonate. Our results indicate that the DNA damage-induced increase in intracellular ROS levels is a generalized stress response that is likely to function in various signaling pathways.