Hydration changes upon DNA folding studied by osmotic stress experiments

The thermal stability of nucleic acid structures is perturbed under the conditions that mimic the intracellular environment, typically rich in inert components and under osmotic stress. We now describe the thermodynamic stability of DNA oligonucleotide structures in the presence of high background concentrations of neutral cosolutes. Small cosolutes destabilize the basepair structures, and the DNA structures consisting of the same nearest-neighbor composition show similar thermodynamic parameters in the presence of various types of cosolutes. The osmotic stress experiments reveal that water binding to flexible loops, unstable mismatches, and an abasic site upon DNA folding are almost negligible, whereas the binding to stable mismatch pairs is significant. The studies using the basepair-mimic nucleosides and the peptide nucleic acid suggest that the sugar-phosphate backbone and the integrity of the basepair conformation make important contributions to the binding of water molecules to the DNA bases and helical grooves. The study of the DNA hydration provides the basis for understanding and predicting nucleic acid structures in nonaqueous solvent systems.     

P-glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells.

The expression of a functional P-glycoprotein (P-gp) which pumps drugs out of brain capillary endothelial cells (BCEC) into blood was studied by evaluating the steady-state uptake and efflux of vincristine (VCR) by primary cultured bovine BCEC. The steady-state uptake of VCR was increased in the presence of metabolic inhibitors, and an anti-P-gp monoclonal antibody, MRK16, as well as verapamil and steroid hormones which are known to reverse multidrug resistance in tumor cells. Furthermore, efflux of VCR from BCEC was inhibited by verapamil. By immunohistochemistry, P-gp was localized at the luminal side of the capillary endothelial cells in both gray matter of bovine brain and primary cultured BCEC. These data suggest that P-gp functions as a drug efflux pump at the luminal side of BCEC and regulates the transfer of certain lipophilic drugs from the blood into the brain.     

Characterization of a Calcium-Dependent Protein Kinase of Tobacco Leaves That Is Associated with the Plasma Membrane and Is Inducible by Sucrose

The plasma membrane fraction from leaves of tobacco containsa 54-kDa protein with autophosphorylation activity, and thelevel of this protein increases after feeding of leaves withsucrose [Ohto and Nakamura (1995) Plant Physiol. 109: 973].The 54-kDa autophosphorylation protein could not be releasedfrom the plasma membrane by treatment with salt or alkali butcould be efficiently solubi-lized by 1% sodium deoxycholate(NaDOC). Ion-exchange chromatography of the NaDOC-solubilizedproteins in the presence of octylglucoside separated the 54-kDaautophosphorylation protein into three peaks. The autophosphorylationactivity of the 54-kDa protein in peaks I and II increased afterfeeding of leaves with sucrose. The 54-kDa protein in the peakII fraction was enriched about 125-fold starting from the microsomalmembrane fraction. The 54-kDa protein in this fraction phosphorylatedhistone HIS in a calcium-dependent manner and cross-reactedwith an antibody against a calcium-dependent protein kinase(CDPK) of Arabidopsis thaliana. These results suggest that the54-kDa protein in the peak II fraction is a novel isoform ofCDPK which is associated with the plasma membrane and is inducibleby sucrose. (Received September 29, 1997; Accepted September 1, 1998)     

Improved mRNA electroporation method for Xenopus neurula embryos

Electroporation has led to new approaches to the analysis of gene regulation of the chick embryonic system. However, application of this method to Xenopus, another model organism of embryology, has left many difficulties to be overcome. The specially devised electrodes, the examination of luciferase activities expressed, and the direct visualization of green fluorescence protein allow us to optimize the conditions of electroporation for Xenopusembryos. The use of mRNA rather than DNA improved the expression efficiency 120 times more than for the case of plasmid DNA, and the effect emerged more immediately after electroporation. The noncontact electroporation adopted here caused less damage to cells and tissues than with the needle type electrode, making it practical for efficient application to early embryos. Furthermore, the mRNA electroporation technique is applicable for other systems in which the DNA electroporation has not had any significant effect because of its low expression efficiency.     

Characterization of Dystroglycan-Laminin Interaction in Peripheral Nerve

Abstract: Dystroglycan is encoded by a single gene and cleaved into two proteins, α- and β-dystroglycan, by posttranslational processing. The 120-kDa peripheral nerve isoform of α-dystroglycan binds laminin-2 comprised of the α2, β1, and γ1 chains. In congenital muscular dystrophy and dy mice deficient in laminin α2 chain, peripheral myelination is disturbed, suggesting a role for the dystroglycan-laminin interaction in peripheral myelinogenesis. To begin to test this hypothesis, we have characterized the dystroglycan-laminin interaction in peripheral nerve. We demonstrate that (1) α-dystroglycan is an extracellular peripheral membrane glycoprotein that links β-dystroglycan in the Schwann cell outer membrane with laminin-2 in the endoneurial basal lamina, and (2) dystrophin homologues Dp116 and utrophin are cytoskeletal proteins of the Schwann cell cytoplasm. We also present data that suggest a role for glycosylation of α-dystroglycan in the interaction with laminin.     

Time-dependent dysregulation of autophagy: Implications in aging and mitochondrial homeostasis in the kidney proximal tubule

Autophagy plays an essential role in cellular homeostasis through the quality control of proteins and organelles. Although a time-dependent decline in autophagic activity is believed to be involved in the aging process, the issue remains controversial. We previously demonstrated that autophagy maintains proximal tubular cell homeostasis and protects against kidney injury. Here, we extend that study and examine how autophagy is involved in kidney aging. Unexpectedly, the basal autophagic activity was higher in the aged kidney than that in young kidney; short-term cessation of autophagy in tamoxifen-inducible proximal tubule-specific autophagy-deficient mice increased the accumulation of SQSTM1/p62- and ubiquitin-positive aggregates in the aged kidney. By contrast, autophagic flux in response to metabolic stress was blunted with aging, as demonstrated by the observation that transgenic mice expressing a green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3B fusion construct, showed a drastic increase of GFP-positive puncta in response to starvation in young mice compared to a slight increase observed in aged mice. Finally, proximal tubule-specific autophagy-deficient mice at 24 mo of age exhibited a significant deterioration in kidney function and fibrosis concomitant with mitochondrial dysfunction as well as mitochondrial DNA abnormalities and nuclear DNA damage, all of which are hallmark characteristics of cellular senescence. These results suggest that age-dependent high basal autophagy plays a crucial role in counteracting kidney aging through mitochondrial quality control. Furthermore, a reduced capacity for upregulation of autophagic flux in response to metabolic stress may be associated with age-related kidney diseases.     

Hydration regulates the thermodynamic stability of DNA structures under molecular crowding conditions

Although water is an integral part of DNA structures, the effects of water molecules on various DNA structures which are formed by not only Watson-Crick but also Hoogsteen base pairs are still unclear. Here, we studied quantitatively the effects of molecular crowding on the thermodynamics of a parallel G-quadruplex formation of [d(TG 4)2]4 with Hoogsteen base pairs. It was demonstrated that molecular crowding conditions stabilized the parallel G-quadruplex. Moreover, the plot of stability of the parallel G-quadruplex structure versus water activity suggested that water molecules were released through the G-quadruplex formation. The stabilization of the DNA structures consisting of Hoogsteen base pairs under cell-like conditions may lead to a structural polymorphism of various DNA sequences regulated by water molecules.     

Spleen-Derived Interleukin-10 Downregulates the Severity of High-Fat Diet-Induced Non-Alcoholic Fatty Pancreas Disease

Obesity is associated with systemic low-grade inflammation and is a risk factor for non-alcoholic fatty pancreas disease (NAFPD), but the molecular mechanisms of these associations are not clear. Interleukin (IL)-10, a potent anti-inflammatory cytokine, is released during acute pancreatitis and is known to limit inflammatory responses by downregulating the release of proinflammatory mediators. The origin of IL-10 that suppresses pancreatitis has not been investigated. Since obesity is known to reduce expression of proinflammatory cytokines in the spleen, we examined whether spleen-derived IL-10 regulates NAFPD caused by high-fat (HF) diet-induced obesity. The following investigations were performed: 1) IL-10 induction from spleen was examined in male mice fed a HF diet; 2) triglyceride content, expression of pro- and anti-inflammatory cytokines and infiltration of M1 and M2 macrophages were determined to evaluate ectopic fat accumulation and inflammatory responses in the pancreas of splenectomy (SPX)-treated mice fed HF diet; 3) exogenous IL-10 was systemically administered to SPX-treated obese mice and the resulting pathogenesis caused by SPX was assessed; and 4) IL-10 knockout (IL-10KO) mice were treated with SPX and ectopic fat deposition and inflammatory conditions in the pancreas were investigated. Obesity impaired the ability of the spleen to synthesize cytokines, including IL-10. SPX aggravated fat accumulation and inflammatory responses in the pancreas of HF diet-induced obese mice and these effects were inhibited by systemic administration of IL-10. Moreover, SPX had little effect on fat deposition and inflammatory responses in the pancreas of IL-10KO mice. Our findings indicate that obesity reduces IL-10 production by the spleen and that spleen-derived IL-10 may protect against the development of NAFPD.     

A New Heterotrophic Dinoflagellate from the North‐eastern Pacific,Protoperidinium fukuyoi: Cyst–Theca Relationship,Phylogeny, Distribution and Ecology

The cyst–theca relationship of Protoperidinium fukuyoi n. sp. (Dinoflagellata, Protoperidiniaceae) is established by incubating resting cysts from estuarine sediments off southern Vancouver Island, British Columbia, Canada, and San Pedro Harbor, California, USA. The cysts have a brown‐coloured wall, and are characterized by a saphopylic archeopyle comprising three apical plates, the apical pore plate and canal plate; and acuminate processes typically arranged in linear clusters. We elucidate the phylogenetic relationship of P. fukuyoi through large and small subunit (LSU and SSU) rDNA sequences, and also report the SSU of the cyst‐defined species Islandinium minutum (Harland & Reid) Head et al. 2001. Molecular phylogenetic analysis by SSU rDNA shows that both species are closely related to Protoperidinium americanum (Gran & Braarud 1935) Balech 1974. Large subunit rDNA phylogeny also supports a close relationship between P. fukuyoi and P. americanum. Three subgroups in total are further characterized within the Monovela group. The cyst of P. fukuyoi shows a wide geographical range along the coastal tropical to temperate areas of the North‐east Pacific, its distribution reflecting optimal summer sea‐surface temperatures of ~14–18 °C and salinities of 22–34 psu.