A constitutively open potassium channel formed by KCNQ1 and KCNE3

Mutations in all four known KCNQ potassium channel alpha-subunit genes lead to human diseases. KCNQ1 (KvLQT1) interacts with the beta-subunit KCNE1 (IsK, minK) to form the slow, depolarization-activated potassium current I(Ks) that is affected in some forms of cardiac arrhythmia. Here we show that the novel beta-subunit KCNE3 markedly changes KCNQ1 properties to yield currents that are nearly instantaneous and depend linearly on voltage. It also suppresses the currents of KCNQ4 and HERG potassium channels. In the intestine, KCNQ1 and KCNE3 messenger RNAs colocalized in crypt cells. This localization and the pharmacology, voltage-dependence and stimulation by cyclic AMP of KCNQ1/KCNE3 currents indicate that these proteins may assemble to form the potassium channel that is important for cyclic AMP-stimulated intestinal chloride secretion and that is involved in secretory diarrhoea and cystic fibrosis.     

Natural methyl bromide and methyl chloride emissions from coastal salt marshes

Atmospheric methyl bromide (CH3Br) and methyl chloride (CH3Cl), compounds that are involved in stratospheric ozone depletion, originate from both natural and anthropogenic sources. Current estimates of CH3Br and CH3Cl emissions from oceanic sources, terrestrial plants and fungi, biomass burning and anthropogenic inputs do not balance their losses owing to oxidation by hydroxyl radicals, oceanic degradation, and consumption in soils, suggesting that additional natural terrestrial sources may be important. Here we show that CH3Br and CH3Cl are released to the atmosphere from all vegetation zones of two coastal salt marshes. We see very large fluxes of CH3Br and CH3Cl per unit area: up to 42 and 570 micromol m(-2) d(-1), respectively. The fluxes show large diurnal, seasonal and spatial variabilities, but there is a strong correlation between the fluxes of CH3Br and those of CH3Cl, with an average molar flux ratio of roughly 1:20. If our measurements are typical of salt marshes globally, they suggest that such ecosystems, even though they constitute less than 0.1% of the global surface area, may produce roughly 10% of the total fluxes of atmospheric CH3Br and CH3Cl.     

Voice-selective areas in human auditory cortex

The human voice contains in its acoustic structure a wealth of information on the speaker's identity and emotional state which we perceive with remarkable ease and accuracy. Although the perception of speaker-related features of voice plays a major role in human communication, little is known about its neural basis. Here we show, using functional magnetic resonance imaging in human volunteers, that voice-selective regions can be found bilaterally along the upper bank of the superior temporal sulcus (STS). These regions showed greater neuronal activity when subjects listened passively to vocal sounds, whether speech or non-speech, than to non-vocal environmental sounds. Central STS regions also displayed a high degree of selectivity by responding significantly more to vocal sounds than to matched control stimuli, including scrambled voices and amplitude-modulated noise. Moreover, their response to stimuli degraded by frequency filtering paralleled the subjects' behavioural performance in voice-perception tasks that used these stimuli. The voice-selective areas in the STS may represent the counterpart of the face-selective areas in human visual cortex; their existence sheds new light on the functional architecture of the human auditory cortex.     

KIR expression on self-reactive CD8+ T cells is controlled by T-cell receptor engagement

Natural killer cell tolerance is maintained by the interaction of killer inhibitory receptors (KIRs) with self-major histocompatibility complex class I gene products. A subset of T cells also expresses inhibitory receptors, but the functional significance of these receptors on T cells is unclear. Here we show that, in the absence of T-cell receptor (TCR) engagement, KIRs expressed on CD8+ T cells are slowly downregulated by KIR ligands expressed on antigen-presenting cells. The resulting expression levels of KIR are no longer able to inhibit T-cell function. In contrast, TCR engagement sustains KIR expression, and re-induces functional levels of KIR expression after ligand-induced downregulation of KIR. Our data indicate that KIR expression on CD8+ T cells in vivo may be maintained through continuous encounters with antigen. As KIR-mediated inhibition of T-cell activation can be bypassed at high antigen concentrations, dynamic KIR expression may mediate T-cell tolerance to self-antigens by sparing self-reactive T cells, thus enabling them to mediate potentially useful immune functions to quantitatively or qualitatively different antigens.     

Single-molecule studies of the effect of template tension on T7 DNA polymerase activity

T7 DNA polymerase catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3'-->5' exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a 'right hand' shape which is common to most polymerases with fingers, palm and thumb domains. The rate-limiting step for replication is thought to involve a conformational change between an 'open fingers' state in which the active site samples nucleotides, and a 'closed' state in which nucleotide incorporation occurs. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of approximately 34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.     

Structure of a ligand-binding intermediate in wild-type carbonmonoxy myoglobin

Small molecules such as NO, O2, CO or H2 are important biological ligands that bind to metalloproteins to function crucially in processes such as signal transduction, respiration and catalysis. A key issue for understanding the regulation of reaction mechanisms in these systems is whether ligands gain access to the binding sites through specific channels and docking sites, or by random diffusion through the protein matrix. A model system for studying this issue is myoglobin, a simple haem protein. Myoglobin has been studied extensively by spectroscopy, crystallography, computation and theory. It serves as an aid to oxygen diffusion but also binds carbon monoxide, a byproduct of endogenous haem catabolism. Molecular dynamics simulations, random mutagenesis and flash photolysis studies indicate that ligand migration occurs through a limited number of pathways involving docking sites. Here we report the 1.4 A resolution crystal structure of a ligand-binding intermediate in carbonmonoxy myoglobin that may have far-reaching implications for understanding the dynamics of ligand binding and catalysis.     

A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae

Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.     

Evidence that humans evolved from a knuckle-walking ancestor

Bipedalism has traditionally been regarded as the fundamental adaptation that sets hominids apart from other primates. Fossil evidence demonstrates that by 4.1 million years ago, and perhaps earlier, hominids exhibited adaptations to bipedal walking. At present, however, the fossil record offers little information about the origin of bipedalism, and despite nearly a century of research on existing fossils and comparative anatomy, there is still no consensus concerning the mode of locomotion that preceded bipedalism. Here we present evidence that fossils attributed to Australopithecus anamensis (KNM-ER 20419) and A. afarensis (AL 288-1) retain specialized wrist morphology associated with knuckle-walking. This distal radial morphology differs from that of later hominids and non-knuckle-walking anthropoid primates, suggesting that knuckle-walking is a derived feature of the African ape and human clade. This removes key morphological evidence for a Pan-Gorilla clade, and suggests that bipedal hominids evolved from a knuckle-walking ancestor that was already partly terrestrial.