mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Mutations in both sides of the photosystem I reaction center identify the phylloquinone observed by electron paramagnetic resonance spectroscopy

The core of photosystem I (PS1) is composed of the two related integral membrane polypeptides, PsaA and PsaB, which bind two symmetrical branches of cofactors, each consisting of two chlorophylls and a phylloquinone, that potentially link the primary electron donor and the tertiary acceptor. In an effort to identify amino acid residues near the phylloquinone binding sites, all tryptophans and histidines that are conserved between PsaA and PsaB in the region of the 10th and 11th transmembrane alpha-helices were mutated in Chlamydomonas reinhardtii. The mutant PS1 reaction centers appear to assemble normally and possess photochemical activity. An electron paramagnetic resonance (EPR) signal attributed to the phylloquinone anion radical (A(1)(-)) can be observed either transiently or after illumination of reaction centers with pre-reduced iron-sulfur clusters. Mutation of PsaA-Trp(693) to Phe resulted in an inability to photo-accumulate A(1)(-), whereas mutation of the analogous tryptophan in PsaB (PsaB-Trp(673)) did not produce this effect. The PsaA-W693F mutation also produced spectral changes in the time-resolved EPR spectrum of the P(700)(+) A(1)(-) radical pair, whereas the analogous mutation in PsaB had no observable effect. These observations indicate that the A(1)(-) phylloquinone radical observed by EPR occupies the phylloquinone-binding site containing PsaA-Trp(693). However, mutation of either tryptophan accelerated charge recombination from the terminal Fe-S clusters.     

Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.     

Crystal structure of the catalytic domain of UCHL5, a proteasome-associated human deubiquitinating enzyme, reveals an unproductive form of the enzyme

Ubiquitin carboxy-terminal hydrolase L5 (UCHL5) is a proteasome-associated deubiquitinating enzyme, which, along with RPN11 and USP14, is known to carry out deubiquitination on proteasome. As a member of the ubiquitin carboxy-terminal hydrolase (UCH) family, UCHL5 is unusual because, unlike UCHL1 and UCHL3, it can process polyubiquitin chain. However, it does so only when it is bound to the proteasome; in its free form, it is capable of releasing only relatively small leaving groups from the C-terminus of ubiquitin. Such a behavior might suggest at least two catalytically distinct forms of the enzyme, an apo form incapable of chain processing activity, and a proteasome-induced activated form capable of cleaving polyubiquitin chain. Through the crystal structure analysis of two truncated constructs representing the catalytic domain (UCH domain) of this enzyme, we were able to visualize a state of this enzyme that we interpret as its inactive form, because the catalytic cysteine appears to be in an unproductive orientation. While this work was in progress, the structure of a different construct representing the UCH domain was reported; however, in that work the structure reported was that of an inactive mutant [catalytic Cys to Ala; Nishio K et al. (2009) Biochem Biophys Res Commun 390, 855-860], which precluded the observation that we are reporting here. Additionally, our structures reveal conformationally dynamic parts of the enzyme that may play a role in the structural transition to the more active form.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

A new brace treatment similar for adolescent scoliosis and kyphosis based on restoration of thoracolumbar lordosis. Radiological and subjective clinical results after at least one year of treatment

Study design

A prospective treatment study with a new brace was conducted Objective. To evaluate radiological and subjective clinical results after one year conservative brace treatment with pressure onto lordosis at the thoracolumbar joint in children with scoliosis and kyphosis.

Summary of background data

Conservative brace treatment of adolescent scoliosis is not proven to be effective in terms of lasting correction. Conservative treatment in kyphotic deformities may lead to satisfactory correction. None of the brace or casting techniques is based on sagittal forces only applied at the thoracolumbar spine (TLI= thoracolumbar lordotic intervention). Previously we showed in patients with scoliosis after forced lordosis at the thoracolumbar spine a radiological instantaneous reduction in both coronal curves of double major scoliosis.

Methods

A consecutive series of 91 children with adolescent scoliosis and kyphosis were treated with a modified symmetric 30 degrees Boston brace to ensure only forced lordosis at the thoracolumbar spine. Scoliosis was defined with a Cobb angle of at least one of the curves [greater than or equal to] 25 degrees and kyphosis with or without a curve <25 degrees in the coronal plane. Standing radiographs were made i) at start, ii) in brace at beginning and iii) after one year treatment without brace.

Results

Before treatment start ??in brace?? radiographs showed a strong reduction of the Cobb angles in different curves in kyphosis and scoliosis groups (sagittal n = 5 all p < 0.001, pelvic obliquity p < 0.001). After one year of brace treatment in scoliosis and kyphosis group the measurements on radiographs made without brace revealed an improvement in 3 Cobb angles each.

Conclusion

Conservative treatment using thoracolumbar lordotic intervention in scoliotic and kyphotic deformities in adolescence demonstrates a marked improvement after one year also in clinical and postural criteria. An effect not obtained with current brace techniques.     

NMR structure of the N-terminal coiled coil domain of the Andes hantavirus nucleocapsid protein

The hantaviruses are emerging infectious viruses that in humans can cause a cardiopulmonary syndrome or a hemorrhagic fever with renal syndrome. The nucleocapsid (N) is the most abundant viral protein, and during viral assembly, the N protein forms trimers and packages the viral RNA genome. Here, we report the NMR structure of the N-terminal domain (residues 1-74, called N1-74) of the Andes hantavirus N protein. N1-74 forms two long helices (alpha1 and alpha2) that intertwine into a coiled coil domain. The conserved hydrophobic residues at the helix alpha1-alpha2 interface stabilize the coiled coil; however, there are many conserved surface residues whose function is not known. Site-directed mutagenesis, CD spectroscopy, and immunocytochemistry reveal that a point mutation in the conserved basic surface formed by Arg22 or Lys26 lead to antibody recognition based on the subcellular localization of the N protein. Thus, Arg22 and Lys26 are likely involved in a conformational change or molecular recognition when the N protein is trafficked from the cytoplasm to the Golgi, the site of viral assembly and maturation.