Different ionic conditions prompt NHE2 and NHE3 translocation to the plasma membrane

We tested whether NHE3 and NHE2 Na⁺/H⁺ exchanger isoforms were recruited to the plasma membrane (PM) in response to changes in ion homeostasis. NHE2-CFP or NHE3-CFP fusion proteins were functional Na⁺/H⁺ exchangers when transiently expressed in NHE-deficient PS120 fibroblasts. Confocal morphometry of cells whose PM was labeled with FM4-64 measured the fractional amount of fusion protein at the cell surface. In resting cells, 10-20% of CFP fluorescence was at PM and stable over time. A protocol commonly used to activate the Na⁺/H⁺ exchange function (NH₄-prepulse acid load sustained in Na⁺-free medium), increased PM percentages of PM NHE3-CFP and NHE2-CFP. Separation of cellular acidification from Na⁺ removal revealed that only NHE3-CFP translocated when medium Na⁺ was removed, and only NHE2-CFP translocated when the cell was acidified. NHE2/NHE3 chimeric proteins demonstrate that the Na⁺-removal response element resides predominantly in the NHE3 cytoplasmic tail and is distinct from the acidification response sequence of NHE2.     

Multiple full-length NS3 molecules are required for optimal unwinding of oligonucleotide DNA in vitro

NS3 (nonstructural protein 3) from the hepatitis C virus is a 3' --> 5' helicase classified in helicase superfamily 2. The optimally active form of this helicase remains uncertain. We have used unwinding assays in the presence of a protein trap to investigate the first cycle of unwinding by full-length NS3. When the enzyme was in excess of the substrate, NS3 (500 nM) unwound >80% of a DNA substrate containing a 15-nucleotide overhang and a 30-bp duplex (45:30-mer; 1 nM). This result indicated that the active form of NS3 that was bound to the DNA prior to initiation of the reaction was capable of processive DNA unwinding. Unwinding with varying ratios of NS3 to 45:30-mer allowed us to investigate the active form of NS3 during the first unwinding cycle. When the substrate concentration slightly exceeded that of the enzyme, little or no unwinding was observed, indicating that if a monomeric form of the protein is active, then it exhibits very low processivity. Binding of NS3 to the 45:30-mer was measured by electrophoretic mobility shift assays, resulting in K(D) = 2.7 +/- 0.4 nM. Binding to individual regions of the substrate was investigated by measuring the K(D) for a 15-mer oligonucleotide as well as a 30-mer duplex. NS3 bound tightly to the 15-mer (K(D) = 1.3 +/- 0.2 nM) and, surprisingly, fairly tightly to the double-stranded 30-mer (K(D) = 11.3 +/- 1.3 nM). However, NS3 was not able to rapidly unwind a blunt-end duplex. Thus, under conditions of optimal unwinding, the 45:30-mer is initially saturated with the enzyme, including the duplex region. The unwinding data are discussed in terms of a model whereby multiple molecules of NS3 bound to the single-stranded DNA portion of the substrate are required for optimal unwinding.     

Proteomic and genomic characterization of chromatin complexes at a boundary

We have dissected specialized assemblies on the Saccharomyces cerevisiae genome that help define and preserve the boundaries that separate silent and active chromatin. These assemblies contain characteristic stretches of DNA that flank particular regions of silent chromatin, as well as five distinctively modified histones and a set of protein complexes. The complexes consist of at least 15 chromatin-associated proteins, including DNA pol epsilon, the Isw2-Itc1 and Top2 chromatin remodeling proteins, the Sas3-Spt16 chromatin modifying complex, and Yta7, a bromodomain-containing AAA ATPase. We show that these complexes are important for the faithful maintenance of an established boundary, as disruption of the complexes results in specific, anomalous alterations of the silent and active epigenetic states.     

I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions

Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of nonspecifically interacting proteins. We describe a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. I-DIRT will be of broad value for studying protein complexes in biological systems that can be metabolically labeled.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Interaction of propranolol with central serotonergic neurons

Central monoaminergic mechanisms are believed to be involved in cardiovascular regulation. The present study was designed to evaluate the involvement of central serotonergic pathways in the antihypertensive action of propranolol in pentobarbital anesthetized mongrel dogs. Ventriculocisternal perfusion of propranolol (25 ug/kg/min for 30 min) decreased serotonin turnover as indicated by a significant decrease in cerebrospinal fluid levels of 5-hydroxyindoleacetic acid (5-HIAA). This effect was accompanied by a significant reduction in mean arterial pressure and heart rate. These results indicate that propranolol decreases central serotonergic activity and suggests a possible role for central serotonergic pathways in the antihypertensive action of propranolol. Several studies have indicated that central serotonergic pathways participate in the regulation of blood pressure. Brainstem regions including the nucleus tractus solitarius, the raphe nucleus and the anterior hypothalamic preoptic region are involved in cardiovascular control and contain a dense population of serotonergic neurons. A centrally-mediated hypotensive effect of propranolol has been demonstrated. Centrally administered propranolol increases cerebrospinal fluid (CSF) levels of norepinephrine and reduces blood pressure possibly due to decreased peripheral sympathetic nerve activity. Central serotonergic pathways may also be involved in the antihypertensive action of some beta-adrenoceptor antagonists. Destruction of central serotonergic neurons with 5,7-dihydroxytryptamine and desipramine abolished the antihypertensive effect of intracisternal propranolol in sinoaortic denervated dogs. Acute administrations of (-)-propranolol and (-)-pindolol decreased the synthesis rate of serotonin, while acute administration of salbutamol, a beta 2-adrenoceptor agonist, increased 5-HIAA levels in rat brain structures. The present study was designed to evaluate the involvement of central serotonergic pathways in the antihypertensive action of propranolol.     

Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis.     

HUWE1 interacts with PCNA to alleviate replication stress

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S‐phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT‐type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA. We show that HUWE1‐knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono‐ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.     

Series resistance compensation for whole-cell patch-clamp studies using a membrane state estimator

Whole-cell patch-clamp techniques are widely used to measure membrane currents from isolated cells. While suitable for a broad range of ionic currents, the series resistance (R(s)) of the recording pipette limits the bandwidth of the whole-cell configuration, making it difficult to measure rapid ionic currents. To increase bandwidth, it is necessary to compensate for R(s). Most methods of R(s) compensation become unstable at high bandwidth, making them hard to use. We describe a novel method of R(s) compensation that overcomes the stability limitations of standard designs. This method uses a state estimator, implemented with analog computation, to compute the membrane potential, V(m), which is then used in a feedback loop to implement a voltage clamp; we refer to this as state estimator R(s) compensation. To demonstrate the utility of this approach, we built an amplifier incorporating state estimator R(s) compensation. In benchtop tests, our amplifier showed significantly higher bandwidths and improved stability when compared with a commercially available amplifier. We demonstrated that state estimator R(s) compensation works well in practice by recording voltage-gated Na(+) currents under voltage-clamp conditions from dissociated neonatal rat sympathetic neurons. We conclude that state estimator R(s) compensation should make it easier to measure large rapid ionic currents with whole-cell patch-clamp techniques.