Vast world reserves of methane gas are underutilized as a feedstock for production of liquid fuels and chemicals due to the lack of economical and sustainable strategies for selective oxidation to methanol1. Current processes to activate the strong C–H bond (104 kcal/mol) in methane require high temperatures, are costly and inefficient, and produce waste2. In nature, methanotrophic bacteria perform this reaction under ambient conditions using metalloenzymes called methane monooxygenases (MMOs). MMOs are thus the optimal inspiration for an efficient, green catalyst3. There are two types of MMOs. Soluble MMO (sMMO), which is expressed by several strains of methanotrophs under copper limited conditions, oxidizes methane with a well characterized catalytic diiron center4. Particulate methane monooxygenase (pMMO), an integral membrane metalloenzyme produced by all methanotrophs, is composed of three subunits, pmoA, pmoB, and pmoC, arranged in a trimeric α3β3γ3 complex5. Despite 20 years of research and the availability of two crystal structures, the metal composition and location of the pMMO metal active site are not known. Here we show that pMMO activity is dependent on copper, not iron, and that the copper active site is located in the soluble domains of the pmoB subunit rather than within the membrane. Recombinant soluble fragments of pmoB (spmoB) bind copper and exhibit propylene and methane oxidation activities. Disruption of each copper center in spmoB by mutagenesis indicates that the active site is a dicopper center. These findings resolve the pMMO controversy and provide a promising new approach to developing environmentally friendly C–H oxidation catalysts.

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of CuI and CuII, of which the latter exhibits two distinct type 2 CuII electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu–O/N and Cu–Cu ligand environments with a Cu–Cu interaction at 2.52–2.64 Å. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 Å resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models.

TAR RNA binding protein (TRBP) belongs to an RNA binding protein family that includes the double‐stranded RNA‐activated protein kinase (PKR), Drosophila Staufen and Xenopus xlrbpa. One member of this family, PKR, is a serine/threonine kinase which has anti‐viral and anti‐proliferative effects. In this study we show that TRBP is a cellular down‐regulator of PKR function. Assaying expression from an infectious HIV‐1 molecular clone, we found that PKR inhibited viral protein synthesis and that over‐expression of TRBP effectively countered this inhibition. In intracellular and in cell‐free assays we show that TRBP directly inhibits PKR autophosphorylation through an RNA binding‐independent pathway. Biologically, TRBP serves a growth‐promoting role; cells that over‐express TRBP exhibit transformed phenotypes. Our results demonstrate the oncogenic potential of TRBP and are consistent with the notion that intracellular PKR function contributes physiologically towards regulating cellular proliferation.

MicroRNAs (miRNAs) play diverse roles in regulating cellular and developmental functions. We have profiled the miRNA expression in peripheral blood mononuclear cells from 36 HIV-1 seropositive individuals and 12 normal controls. The HIV-1-positive individuals were categorized operationally into four classes based on their CD4+ T-cell counts and their viral loads. We report that specific miRNA signatures can be observed for each of the four classes.

The authors report 3 experiments on negative mood regulation in which whether mood-congruency or mood-incongruency effects of negative mood on cognition were observed was dependent on an individual's self-esteem (SE). We found that most of our 224 participants tended toward mood-congruent recall under control conditions in which mood was relatively neutral. However, when a negative emotional state was induced, participants low in SE exhibited mood-congruent recall, but high-SE participants did not. In fact, the more negative high-SE participants felt, the more positive were their cognitions (mood-incongruent recall). This pattern was replicated in 3 experiments that included variations in the negative mood inductions and the type of information that was generated or retrieved. Our results suggest a strong link between SE and the regulation of negative emotional states.

Serine/threonine protein phosphatases are central mediators of phosphorylation-dependent signals in eukaryotes and a variety of pathogenic bacteria. Here, we report the crystal structure of the intracellular catalytic domain of Mycobacterium tuberculosis PstPpp, a membrane-anchored phosphatase in the PP2C family. Despite sharing the fold and two-metal center of human PP2Calpha, the PstPpp catalytic domain binds a third Mn(2+) in a site created by a large shift in a previously unrecognized flap subdomain adjacent to the active site. Mutations in this site selectively increased the Michaelis constant for Mn(2+) in the reaction of a noncognate, small-molecule substrate, p-nitrophenyl phosphate. The PstP/Ppp structure reveals core functional motifs that advance the framework for understanding the mechanisms of substrate recognition, catalysis, and regulation of PP2C phosphatases.

The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-A resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO(iso)), chemically reduced (pMMO(red)), and chemically oxidized (pMMO(ox)) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO(iso) contains both Cu(I) and Cu(II) and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 A upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO(iso). Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe(III), is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.

The province of Ontario (Canada) reported more laboratory confirmed rabid animals than any other state or province in Canada or the USA from 1958-91, with the exception of 1960-62. More than 95% of those cases occurred in the southern 10% of Ontario (approximately 100,000 km2), the region with the highest human population density and greatest agricultural activity. Rabies posed an expensive threat to human health and significant costs to the agricultural economy. The rabies variant originated in arctic foxes: the main vector in southern Ontario was the red fox (Vulpes vulpes), with lesser involvement of the striped skunk (Mephitis mephitis). The Ontario Ministry of Natural Resources began a 5 yr experiment in 1989 to eliminate terrestrial rabies from a approximately 30,000 km2 study area in the eastern end of southern Ontario. Baits containing oral rabies vaccine were dropped annually in the study area at a density of 20 baits/km2 from 1989-95. That continued 2 yr beyond the original 5 yr plan. The experiment was successful in eliminating the arctic fox variant of rabies from the whole area. In the 1980's, an average of 235 rabid foxes per year were reported in the study area. None have been reported since 1993. Cases of fox rabies in other species also disappeared. In 1995, the last bovine and companion animal cases were reported and in 1996 the last rabid skunk occurred. Only bat variants of rabies were present until 1999, when the raccoon variant entered from New York (USA). The success of this experiment led to an expansion of the program to all of southern Ontario in 1994. Persistence of terrestrial rabies, and ease of elimination, appeared to vary geographically, and probably over time. Ecological factors which enhance or reduce the long term survival of rabies in wild foxes are poorly understood.