Crystal structure of a glycosylated Fab from an IgM cryoglobulin with properties of a natural proteolytic antibody

An increase in circulating levels of specific NEFAs (non-esterified fatty acids) has been implicated in the pathogenesis of insulin resistance and impaired glucose disposal in skeletal muscle. In particular, elevation of SFAs (saturated fatty acids), such as palmitate, has been correlated with reduced insulin sensitivity, whereas an increase in certain MUFAs and PUFAs (mono- and poly-unsaturated fatty acids respectively) has been suggested to improve glycaemic control, although the underlying mechanisms remain unclear. In the present study, we compare the effects of palmitoleate (a MUFA) and palmitate (a SFA) on insulin action and glucose utilization in rat L6 skeletal muscle cells. Basal glucose uptake was enhanced approx. 2-fold following treatment of cells with palmitoleate. The MUFA-induced increase in glucose transport led to an associated rise in glucose oxidation and glycogen synthesis, which could not be attributed to activation of signalling proteins normally modulated by stimuli such as insulin, nutrients or cell stress. Moreover, although the MUFA-induced increase in glucose uptake was slow in onset, it was not dependent upon protein synthesis, but did, nevertheless, involve an increase in the plasma membrane abundance of GLUT1 and GLUT4. In contrast, palmitate caused a substantial reduction in insulin signalling and insulin-stimulated glucose transport, but was unable to antagonize the increase in transport elicited by palmitoleate. Our findings indicate that SFAs and MUFAs exert distinct effects upon insulin signalling and glucose uptake in L6 muscle cells and suggest that a diet enriched with MUFAs may facilitate uptake and utilization of glucose in normal and insulin-resistant skeletal muscle.     

Interaction of the N-terminus of chicken skeletal essential light chain 1 with F-actin

Skeletal myosin has two isoforms of the essential light chain (ELC), called LC1 and LC3, which differ only in their N-terminal amino acid sequence. The LC1 has 41 additional residues containing seven pairs of Ala-Pro, which form an elongated structure, and two pairs of lysines located near the N-terminus. When myosin subfragment-1 (S1) binds to actin, these lysines may interact with the C-terminus of actin and be responsible for the isoform specific properties of myosin. Here we employ cross-linking to identify the LC1 residues that are in contact with actin. S1 was reconstituted with various LC1 mutants and reacted with the zero-length cross-linker 1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide (EDC). Cross-linking occurred only when actin was in molar excess over S1. Wild-type LC1 could be cross-linked through the terminal alpha-NH2 group, as well as via the two pairs of lysines. In a mutant ELC, where the lysines were deleted but two arginines were introduced near the N-terminus, the light chain could still be cross-linked via the terminal alpha-NH2 group. When the charge was reduced in the N-terminal region while retaining the Ala-Pro rich region, the mutant could not be cross-linked. These results suggest that as long as the N-terminus contains charged residues and an Ala-Pro rich extension, the binding between LC1 and actin can occur.     

Substitution of a single residue in Stichodactyla helianthus peptide, ShK-Dap22, reveals a novel pharmacological profile

ShK, a peptide isolated from Stichodactyla helianthus venom, blocks the voltage-gated potassium channels, K(v)1.1 and K(v)1.3, with similar high affinity. ShK-Dap(22), a synthetic derivative in which a diaminopropionic acid residue has been substituted at position Lys(22), has been reported to be a selective K(v)1.3 inhibitor and to block this channel with equivalent potency as ShK [Kalman et al. (1998) J. Biol. Chem. 273, 32697-32707]. In this study, a large body of evidence is presented which indicates that the potencies of wild-type ShK peptide for both K(v)1.3 and K(v)1.1 channels have been previously underestimated. Therefore, the affinity of ShK-Dap(22) for both channels appears to be ca. 10(2)-10(4)-fold weaker than ShK. ShK-Dap(22) does display ca. 20-fold selectivity for human K(v)1.3 vs K(v)1.1 when measured by the whole-cell voltage clamp method but not in equilibrium binding assays. ShK-Dap(22) has low affinity for K(v)1.2 channels, but heteromultimeric K(v)1.1-K(v)1.2 channels form a receptor with ca. 200-fold higher affinity for ShK-Dap(22) than K(v)1.1 homomultimers. In fact, K(v)1.1-K(v)1.2 channels bind ShK-Dap(22) with only ca. 10-fold less potency than ShK and reveal a novel pharmacology not predicted from the homomultimers of K(v)1.1 or K(v)1.2. The concentrations of ShK-Dap(22) needed to inhibit human T cell activation were ca. 10(3)-fold higher than those of ShK, in good correlation with the relative affinities of these peptides for inhibiting K(v)1.3 channels. All of these data, taken together, suggest that ShK-Dap(22) will not have the same in vivo immunosuppressant efficacy of other K(v)1.3 blockers, such as margatoxin or ShK. Moreover, ShK-Dap(22) may have undesired side effects due to its interaction with heteromultimeric K(v)1.1-K(v)1.2 channels, such as those present in brain and/or peripheral tissues.     

Intersection tests for single marker QTL analysis can be more powerful than two marker QTL analysis

Background

It has been reported in the quantitative trait locus (QTL) literature that when testing for QTL location and effect, the statistical power supporting methodologies based on two markers and their estimated genetic map is higher than for the genetic map independent methodologies known as single marker analyses. Close examination of these reports reveals that the two marker approaches are more powerful than single marker analyses only in certain cases.Simulation studies are a commonly used tool to determine the behavior of test statistics under known conditions. We conducted a simulation study to assess the general behavior of an intersection test and a two marker test under a variety of conditions. The study was designed to reveal whether two marker tests are always more powerful than intersection tests, or whether there are cases when an intersection test may outperform the two marker approach.We present a reanalysis of a data set from a QTL study of ovariole number in Drosophila melanogaster.

Results

Our simulation study results show that there are situations where the single marker intersection test equals or outperforms the two marker test. The intersection test and the two marker test identify overlapping regions in the reanalysis of the Drosophila melanogaster data. The region identified is consistent with a regression based interval mapping analysis.

Conclusion

We find that the intersection test is appropriate for analysis of QTL data. This approach has the advantage of simplicity and for certain situations supplies equivalent or more powerful results than a comparable two marker test.

     

Identification of a multifunctional binding site on Ubc9p required for Smt3p conjugation

Ubiquitin-like proteins (ub-lps) are conjugated by a conserved enzymatic pathway, involving ATP-dependent activation at the C terminus by an activating enzyme (E1) and formation of a thiolester intermediate with a conjugating enzyme (E2) prior to ligation to the target. Ubc9, the E2 for SUMO, synthesizes polymeric chains in the presence of its E1 and MgATP. To better understand conjugation of ub-lps, we have performed mutational analysis of Saccharomyces cerevisiae Ubc9p, which conjugates the SUMO family member Smt3p. We have identified Ubc9p surfaces involved in thiolester bond and Smt3p-Smt3p chain formation. The residues involved in thiolester bond formation map to a surface we show is the E1 binding site, and E2s for other ub-lps are likely to bind to their E1s at a homologous site. We also find that this same surface binds Smt3p. A mutation that impairs binding to E1 but not Smt3p impairs thiolester bond formation, suggesting that it is the E1 interaction at this site that is crucial. Interestingly, other E2s and their relatives also use this same surface for binding to ubiquitin, E3s, and other proteins, revealing this to be a multipurpose binding site and suggesting that the entire E1-E2-E3 pathway has coevolved for a given ub-lp.     

Mechanism of generation of spontaneous miniature outward currents (SMOCs) in retinal amacrine cells

A subtype of retinal amacrine cells displayed a distinctive array of K(+) currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of -60 to -40 mV. Depolarizations above approximately -40 mV were associated with the disappearance of SMOCs and the appearance of transient (I(to)) and sustained (I(so)) outward K(+) currents. I(to) appeared at about -40 mV and its apparent magnitude was biphasic with voltage, whereas I(so) appeared near -30 mV and increased linearly. SMOCs, I(to), and a component of I(so) were Ca(2+) dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of -20 mV and above. Their mean amplitude was Nernstian with respect to [K(+)](ext) and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca(2+)-free solutions, indicative of BK-type Ca(2+)-activated K(+) currents. Dihydropyridine Ca(2+) channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca(2+) permeation through the kainic acid receptor had no effect. Blockade of organelle Ca(2+) channels by ryanodine, or intracellular Ca(2+) store depletion with caffeine, eradicated SMOCs. Internal Ca(2+) chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca(2+) influx through L-type Ca(2+) channels and its subsequent amplification by Ca(2+)-induced Ca(2+) release via the ryanodine receptor leads to a localized elevation of internal Ca(2+). This amplified Ca(2+) signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs.     

Multiple nuclear-gene phylogenies: application to pinnipeds and comparison with a mitochondrial DNA gene phylogeny

Phylogenetic analyses of closely related species should use information from multiple, independent genes with relatively high rates of sequence evolution. To investigate species for which there are few prior sequence data for single-copy nuclear (scnDNA) genes, primers for gene amplification can be designed to highly conserved regions of exons in order to amplify both coding (exons) and noncoding (introns) sequences. We have explored this approach in a phylogenetic analysis of six species of pinnipeds that, together with terrestrial carnivore outgroups, encompass divergence times < or = 40-50 Mya. We sequenced one intron from each of the aldolase A (ALD-A), aldolase C (ALD-C), and histone H2AF genes; one exon from the major-histocompatibility-complex DQA gene; a H2AF processed pseudogene (psi H2AF); and, for comparison with the nuclear genes, the 5' portion of the mitochondrial DNA (mtDNA) control region. The pinniped psi H2AF genes were found to be of limited use because they were paralogous with the gene in the outgroup. The rate of silent substitution in scnDNA (primarily introns) was 5-10-fold lower than that for mtDNA control region I, and scnDNA sequence divergence increased linearly with time < or = 40-50 Mya. Alleles at three polymorphic scnDNA loci (ALD-A, H2AF, and DQA) in the southern elephant seal were paraphyletic with respect to the allele from the closely related northern elephant seal, while the more numerous mtDNA alleles were monophyletic. This we attribute to the consequences of a higher mutation rate rather than to a lower effective population size of mtDNA compared with scnDNA. Within the short (i.e., < 500-bp) sequences of individual scnDNA sequences, phylogenetically informative variation was insufficient to obtain robust phylogenies. However, the combined scnDNA sequences produced a well-supported phylogeny congruent with that derived from mtDNA. This analysis illustrates the high resolution of mtDNA sequences compared with a similar length of scnDNA sequence, but it also demonstrates the utility of combining information from multiple short scnDNA sequences obtained using broadly applicable primers.