Human and mouse introns are linked to the same processes and functions through each genome's most frequent non-conserved motifs

We identified the most frequent, variable-length DNA sequence motifs in the human and mouse genomes and sub-selected those with multiple recurrences in the intergenic and intronic regions and at least one additional exonic instance in the corresponding genome. We discovered that these motifs have virtually no overlap with intronic sequences that are conserved between human and mouse, and thus are genome-specific. Moreover, we found that these motifs span a substantial fraction of previously uncharacterized human and mouse intronic space. Surprisingly, we found that these genome-specific motifs are over-represented in the introns of genes belonging to the same biological processes and molecular functions in both the human and mouse genomes even though the underlying sequences are not conserved between the two genomes. In fact, the processes and functions that are linked to these genome-specific sequence-motifs are distinct from the processes and functions which are associated with intronic regions that are conserved between human and mouse. The findings show that intronic regions from different genomes are linked to the same processes and functions in the absence of underlying sequence conservation. We highlight the ramifications of this observation with a concrete example that involves the microsatellite instability gene MLH1.     

Crevice-forming mutants of bovine pancreatic trypsin inhibitor: stability changes and new hydrophobic surface.

Four mutants of bovine pancreatic trypsin inhibitor (BPTI) with replacements in the rigid core result in the creation of deep crevices on the surface of the protein. Other than crevices at the site of the mutation, few other differences are observed in the crystal structures of wild-type BPTI and the mutants F22A, Y23A, N43G, and F45A. These mutants are highly destabilized relative to wild type (WT). The differences between WT and mutants in the free energy change associated with cooperative folding/unfolding, delta delta G0 (WT-->mut), have been measured by calorimetry, and they are in good agreement with delta delta G0(WT-->mut) values from hydrogen exchange rates. For F22A the change in free energy difference is about 1.7 kcal/mol at 25 degrees C; for the other three mutants it is in the range of 5-7 kcal/mol at 25 degrees C. The experimental delta delta G0(WT-->mut) values of F22A, Y23A, and F45A are reasonably well accounted for as the sum of two terms: the difference in transfer free energy change, and a contribution from exposure to solvent of new surface (Eriksson, A.E., et al., 1992, Science 255, 178-183), if the recently corrected transfer free energies and surface hydrophobicities (De Young, L. & Dill, K., 1990, J. Phys. Chem. 94, 801-809; Sharp, K.A., et al., 1991a, Science 252, 106-109) are used and only nonpolar surface is taken into account. In N43G, three protein-protein hydrogen bonds are replaced by protein-water hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further studies on the effect of aldosterone on electrical resistance of toad bladder

The fall in transepithelial electrical resistance which accompanies aldosterone stimulation of short-circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) in toad urinary bladder has been studied further to evaluate the possible causal role of this response in hormonal stimulation of Na⁺ transport. A steady-state change in tissue conductance was found to depend upon both the simultaneous stimulation of transport by the steroid and the metabolic state of the tissue. Changes in metabolic state alone did not alter resistance. A sustained increase in Na⁺ transport, dependent on pretreatment with aldosterone and elicited by addition of glucose, could be obtained without a sustained decrease in resistance. Amiloride, an inhibitor of Na⁺ uptake, produced changes in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ that were linearly correlated with its effects on tissue conductance. On the basis of the $\text{conductance}\text{-I}{}_{\mathrm{<mtext>sc</mtext>}}$ relationship with amiloride, the $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ response to aldosterone was about two-fold higher than would be predicted from its effects on conductance alone. Despite the apparent lack of a simple quantitative dependence of the change in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ on the change in conductance when the response is fully developed, the results suggest that conductance changes may mediate the initial or early stage of the response.     

Photoaffinity labeling of platelet thrombin-binding proteins

The interaction of thrombin and platelets was studied with a heterobifunctional photoactivable crosslinking agent. Radiolabeled thrombin that was modified with ethyl-N-5-azido-2-nitrobenzoylaminoacetimidate formed two types of complex with platelet proteins; platelet-associated complexes and supernatant complexes. The platelet-associated complexes formed within 20 s. Autoradiography after electrophoresis with sodium dodecyl sulfate indicated that these complexes had apparent masses of 210, 185, 155 and 125 kDa. Formation of the complexes was blocked by hirudin; this is consistent with crosslinking that was a direct consequences of the binding of thrombin to a specific receptor, since hirudin blocks thrombin-induced platelet activation and the saturable binding of thrombin to platelets. The labeled supernatant complex had an apparent mass of about 490 kDa. It also formed in the supernatant solution of platelets after activation with a divalent cation ionophore, suggesting a complex of thrombin with a secreted protein. The supernatant complex did not involve fibrinogen or α₂-macroglobulin, but a similar complex was formed with partially purified secreted glycoprotein G (thrombin-sensitive protein, thrombospondin). Formation of the complex was blocked by hirudin. A similar complex was formed after prolonged (1 h) incubation without photoactivation. It is concluded that thrombin forms high-affinity, hirudin-sensitive complexes with secreted glycoprotein G, as well as with platelet surface proteins.     

DNA cross-linking by intermediates in the mitomycin activation cascade

We have assayed the cross-linking of oligonucleotides containing repeated mitomycin-reactive CpG sites in order to assess the factors that enhance activation of the carbamoyl function at C10, yielding efficient mitomycin cross-linking. Drugs studied include mitomycin C (MC), N-methylmitomycin A (NMA), and the aziridinomitosene of NMA (MS). Drugs were reduced both by catalytic hydrogenation and by diothionite. We find that cross-linking by fully reduced NMA can be increased severalfold by addition of either excess dithionite reductant or the oxidant FeCl3. Enhancement by FeCl3 is not seen with MC or MS, but excess dithionite increases cross-linking by all three compounds. We explain the action of Fe3+ by postulating production of the semiquinone of the monoadduct of mitomycin reacted at the C1-position; according to this mechanism, departure of the carbamate from C10 is more efficient for the semiquinone than for the hydroquinone. However, our results imply that the hydroquinone can also function as a cross-linking agent. Excess dithionite, beyond that required for stoichiometric reduction, activates the carbamate 2-3-fold for cross-linking. We find that the fully reduced leucoaziridinomitosene is highly unstable in solution, yet it produces efficient cross-liking. Hence, this compound is highly reactive in DNA alkylation and a good candidate for the role of primary alkylating agent.     

Differential effects of saturated and unsaturated fatty acid diets on cardiomyocyte apoptosis, adipose distribution, and serum leptin

Fatty acids are the primary fuel for the heart and are ligands for peroxisome proliferator-activated receptors (PPARs), which regulate the expression of genes encoding proteins involved in fatty acid metabolism. Saturated fatty acids, particularly palmitate, can be converted to the proapoptotic lipid intermediate ceramide. This study assessed cardiac function, expression of PPAR-regulated genes, and cardiomyocyte apoptosis in rats after 8 wk on either a low-fat diet [normal chow control (NC); 10% fat calories] or high-fat diets composed mainly of either saturated (Sat) or unsaturated fatty acids (Unsat) (60% fat calories) (n = 10/group). The Sat group had lower plasma insulin and leptin concentrations compared with the NC or Unsat groups. Cardiac function and mass and body mass were not different. Cardiac triglyceride content was increased in the Sat and Unsat groups compared with NC (P < 0.05); however, ceramide content was higher in the Sat group compared with the Unsat group (2.9 +/- 0.2 vs. 1.4 +/- 0.2 nmol/g; P < 0.05), whereas the NC group was intermediate (2.3 +/- 0.3 nmol/g). The number of apoptotic myocytes, assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining, was higher in the Sat group compared with the Unsat group (0.28 +/- 0.05 vs. 0.17 +/- 0.04 apoptotic cells/1,000 nuclei; P < 0.04) and was positively correlated to ceramide content (P < 0.02). Both high-fat diets increased the myocardial mRNA expression of the PPAR-regulated genes encoding uncoupling protein-3 and pyruvate dehydrogenase kinase-4, but only the Sat diet upregulated medium-chain acyl-CoA dehydrogenase. In conclusion, dietary fatty acid composition affects cardiac ceramide accumulation, cardiomyocyte apoptosis, and expression of PPAR-regulated genes independent of cardiac mass or function.     

Metagenomic analysis of two enhanced biological phosphorus removal (EBPR) sludge communities

Enhanced biological phosphorus removal (EBPR) is one of the best-studied microbially mediated industrial processes because of its ecological and economic relevance. Despite this, it is not well understood at the metabolic level. Here we present a metagenomic analysis of two lab-scale EBPR sludges dominated by the uncultured bacterium, "Candidatus Accumulibacter phosphatis." The analysis sheds light on several controversies in EBPR metabolic models and provides hypotheses explaining the dominance of A. phosphatis in this habitat, its lifestyle outside EBPR and probable cultivation requirements. Comparison of the same species from different EBPR sludges highlights recent evolutionary dynamics in the A. phosphatis genome that could be linked to mechanisms for environmental adaptation. In spite of an apparent lack of phylogenetic overlap in the flanking communities of the two sludges studied, common functional themes were found, at least one of them complementary to the inferred metabolism of the dominant organism. The present study provides a much needed blueprint for a systems-level understanding of EBPR and illustrates that metagenomics enables detailed, often novel, insights into even well-studied biological systems.