Factors affecting the thermodynamic stability of small asymmetric internal loops in RNA

Optical melting experiments were used to determine the thermodynamic parameters for oligoribonucleotides containing small asymmetric internal loops. The results show a broad range of thermodynamic stabilities, which depend on loop size, asymmetry, sequence, closing base pairs, and length of helix stems. Imino proton NMR experiments provide evidence for possible hydrogen bonding in GA and UU mismatches in some asymmetric loops. The stabilizing effects of GA, GG, and UU mismatches on the thermodynamic stability of internal loops vary depending on the size and asymmetry of the loop. The dependence of loop stability on Watson-Crick closing base pairs may be explained by an account of hydrogen bonds. Models are presented for approximating the free energy increments of 2 x 3 and 1 x 3 internal loops.     

Importance of habitat patch size for occupancy and density of aspen-associated saproxylic beetles

In Fennoscandian boreal forests, aspen (Populus tremula) is one of the most important tree species for biodiversity. In this study we explore how occupancy and density of beetles associated with dead aspen are related to habitat patch size and connectedness in a 45,000 ha boreal managed forest landscape in central Sweden. Patch size was estimated as amount of breeding substrate and connectedness as crown cover of living aspen in the surrounding landscape. The beetles were sampled by sieving of bark or by inspection of species-characteristic galleries in 56 patches with dead aspen. Six of nine aspen-associated species (Xylotrechus rusticus, Ptilinus fuscus, Mycetophagus fulvicollis, Cyphaea curtula, Homalota plana and Endomychus coccineus) showed a positive significant relationship between habitat patch size and occupancy. For all these species, except C. curtula, there was also a significant positive relationship between patch size and density. Connectedness was not retained as a significant variable in the analyses. Species not defined as aspen-associated constituted a significantly larger proportion of the total density of individuals of saproxylic beetles in smaller habitat patches than in larger patches. Richness of aspen-associated species was positively related to habitat patch size. Efforts in the managed forest should be directed towards preserving and creating larger patches of living and dead aspen trees and increasing the amount of aspen at the landscape level.     

Acyl-coenzyme A binding protein expression alters liver fatty acyl-coenzyme A metabolism

Although studies in vitro and in yeast suggest that acyl-CoA binding protein ACBP may modulate long-chain fatty acyl-CoA (LCFA-CoA) distribution, its physiological function in mammals is unresolved. To address this issue, the effect of ACBP on liver LCFA-CoA pool size, acyl chain composition, distribution, and transacylation into more complex lipids was examined in transgenic mice expressing a higher level of ACBP. While ACBP transgenic mice did not exhibit altered body or liver weight, liver LCFA-CoA pool size increased by 69%, preferentially in saturated and polyunsaturated, but not monounsaturated, LCFA-CoAs. Intracellular LCFA-CoA distribution was also altered such that the ratio of LCFA-CoA content in (membranes, organelles)/cytosol increased 2.7-fold, especially in microsomes but not mitochondria. The increased distribution of specific LCFA-CoAs to the membrane/organelle and microsomal fractions followed the same order as the relative LCFA-CoA binding affinity exhibited by murine recombinant ACBP: saturated > monounsaturated > polyunsaturated C14-C22 LCFA-CoAs. Consistent with the altered microsomal LCFA-CoA level and distribution, enzymatic activity of liver microsomal glycerol-3-phosphate acyltransferase (GPAT) increased 4-fold, liver mass of phospholipid and triacylglyceride increased nearly 2-fold, and relative content of monounsaturated C18:1 fatty acid increased 44% in liver phospholipids. These effects were not due to the ACBP transgene altering the protein levels of liver microsomal acyltransferase enzymes such as GPAT, lysophosphatidic acid acyltransferase (LAT), or acyl-CoA cholesterol acyltransferase 2 (ACAT-2). Thus, these data show for the first time in a physiological context that ACBP expression may play a role in LCFA-CoA metabolism.     

Liver and intestinal fatty acid binding proteins in control and TGFβ1 gene targeted deficient mice

The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP liver fatty acid binding protein - I-FABP intestinal fatty acid binding protein - TGF1 transforming growth factor beta-1 - TNF- tumor necrosis factor- - MIP- macrophage inflammatory protein- - PMSF phenylmethyl sulfonyl fluoride - PBS phosphate buffered saline     

The influence of dolichols on fluidity of mouse synaptic plasma membranes

Dolichols are isoprenologues which constitute an important component of biological membranes. However, an understanding of the effects of dolichols on the organization and dynamics of biological membranes has not been forthcoming. The experiments reported here are aimed at understanding the effects of dolichols on the physical properties of mouse brain synaptic plasma membranes. The effect of dolichols incorporated into mouse brain synaptic plasma membranes on fluorescent and electron spin resonance probes sensing the hydrophobic core differed from that of probes reporting closer to the surface of membrane bilayers. Dolichols significantly (P less than 0.01) lowered the polarization, limiting anisotropy, and order parameter of diphenylhexatriene in synaptic plasma membranes and liposomes extracted from synaptic plasma membranes, without changing the rotational relaxation time. Similarly, dolichol increased the fluidity reported by 16-doxylstearic acid in synaptic plasma membranes or liposomes extracted from synaptic plasma membranes. In contrast, dolichols exerted no effect on those properties for trans-parinaric acid or 5-doxylstearic acid in synaptic plasma membranes or liposomes derived therefrom. Dolichols can dramatically alter the structure and dynamics of lipid motion in synaptic plasma membranes and these effects are dependent on the location of the probe in the membrane.     

The specificity of methyl transferases involved in trans mycolic acid biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Trans mycolic acid content is directly related to cell wall fluidity and permeability in mycobacteria. Carbon-13 NMR spectroscopy of mycolic acids isolated from Mycobacterium tuberculosis (MTB) and Mycobacterium smegmatis (MSM) fed 13C-labeled precursor molecules was used to probe the biosynthetic pathways that modify mycolic acids. Heteronuclear correlation spectroscopy (HMQC) of ketomycolic acid from MTB allowed assignment of the complete 13C-NMR spectrum. Incorporation patterns from [1-13C]-acetate and [2-13C]-acetate feeding experiments suggested that the mero chain and alpha branch of mycolic acids are both synthesized by standard fatty acid biosynthetic reactions. [13C-methyl]-L-methionine was used to specifically label carbon atoms derived from the action of the methyl transferases involved in meromycolate modification. To enrich for trans mycolic acids a strain of MTB overexpressing the mma1 gene was labeled. Carbon-carbon coupling was observed in mycolate samples doubly labeled with 13C-acetate and [13C-methyl]-L-methionine and this information was used to assess positional specificity of methyl transfer. In MTB such methyl groups were found to occur exclusively on carbons derived from the 2 position of acetate, while in MSM they occurred only on carbons derived from the 1 position. These results suggest that the MSM methyltransferase MMAS-1 operates in an inverted manner to that of MTB.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.     

Increased Resistance to Extracellular Cation Block by Mutation of the Pore Domain of the Arabidopsis Inward-rectifying K+ Channel KAT1

Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K⁺ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root hair cells. K⁺ channel blockers are potent tools for studying the physiological functions and structural properties of K⁺ channels. In the present study the structural and biophysical mechanisms of Cs⁺ and TEA⁺ block of a cloned Arabidopsis inward-rectifying K⁺ channel (KAT1) were analyzed. Effects of the channel blockers Cs⁺ and TEA⁺ were characterized both extracellularly and intracellularly. Both external Cs⁺ and TEA⁺ block KAT1 currents. A mutant of KAT1 (``m2KAT1'; H267T, E269V) was produced by site-directed mutagenesis of two amino acid residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs⁺ and TEA⁺ than the wild-type K⁺ channel. Internal TEA⁺ and Cs⁺ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K⁺ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested for outward-rectifying K⁺ channels. Received: 31 July 1995/Revised: 5 January 1996     

Relief of Autoinhibition Enhances Vta1 Activation of Vps4 via the Vps4 Stimulatory Element

The endosomal sorting complexes required for transport (ESCRTs) impact multiple cellular processes including multivesicular body sorting, abscission, and viral budding. The AAA-ATPase Vps4 is required for ESCRT function, and its full activity is dependent upon the co-factor Vta1. The Vta1 carboxyl-terminal Vta1 SBP1 Lip5 (VSL) domain stimulates Vps4 function by facilitating oligomerization of Vps4 into its active state. Here we report the identification of the Vps4 stimulatory element (VSE) within Vta1 that is required for additional stimulation of Vps4 activity in vitro and in vivo. VSE activity is autoinhibited in a manner dependent upon the unstructured linker region joining the amino-terminal microtubule interacting and trafficking domains and the carboxyl-terminal VSL domain. The VSE is also required for Vta1-mediated Vps4 stimulation by ESCRT-III subunits Vps60 and Did2. These results suggest that ESCRT-III binding to the Vta1 microtubule interacting and trafficking domains relieves linker region autoinhibition of the VSE to produce maximal activation of Vps4 during ESCRT function.