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.      

Differential Effects of Motor Efference Copies and Proprioceptive Information on Response Evaluation Processes

It is well-kown that sensory information influences the way we execute motor responses. However, less is known about if and how sensory and motor information are integrated in the subsequent process of response evaluation. We used a modified Simon Task to investigate how these streams of information are integrated in response evaluation processes, applying an in-depth neurophysiological analysis of event-related potentials (ERPs), time-frequency decomposition and sLORETA. The results show that response evaluation processes are differentially modulated by afferent proprioceptive information and efference copies. While the influence of proprioceptive information is mediated via oscillations in different frequency bands, efference copy based information about the motor execution is specifically mediated via oscillations in the theta frequency band. Stages of visual perception and attention were not modulated by the interaction of proprioception and motor efference copies. Brain areas modulated by the interactive effects of proprioceptive and efference copy based information included the middle frontal gyrus and the supplementary motor area (SMA), suggesting that these areas integrate sensory information for the purpose of response evaluation. The results show how motor response evaluation processes are modulated by information about both the execution and the location of a response.     

Synergistic effects of hormone therapy drugs and usnic acid on hormone receptor‐positive breast and prostate cancer cells

The aim of this study was to investigate the combined effects of usnic acid (UA) and Tamoxifen (Tam) or Enzalutamide (Enz) on hormone receptor‐positive breast and prostate cancer (BC and PC), respectively. The antiproliferative and apoptotic effects of Tam or Enz alone and in combination with UA on MCF7 and LNCaP cancer cells were detected. The results of the WST‐1 assay indicated that UA was a promising anticancer compound that significantly enhanced the effectiveness of hormone therapy drugs compared with each drug alone (combination index < 1). In addition, the combination of UA with Tam or Enz remarkably induced more cell cycle arrest at the G0/G1 phase and apoptosis than only drug‐treated cells (P < 0.01). Consequently, our findings suggest that the combination of UA with Tam or Enz may be a potential therapeutic approach for the treatment of BC and PC and further studies are required to exploit the potential mechanisms of synergistic effects.     

2'Halo-ATP and -GTP analogues: rational phasing tools for protein crystallography

The solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into protein crystals. Several 2'-halogenated nucleotides have been reported as potential universal phasing tools for nucleotide binding proteins. However, only limited data are available dealing with the effect of 2'-substitution on recognition by the protein. We have determined equilibrium dissociation constants of 2'-halogenated ATP analogues for the ATP binding proteins UMP/CMP kinase and the molecular chaperone DnaK. Whereas the affinities to UMP/CMP kinase are of the same order of magnitude as for unsubstituted ATP, the affinities to DnaK are drastically decreased to undetectable levels. For 2'-halogenated GTP analogues, the kinetics of interaction were determined for the small GTPases p21ras(Y32W) (fluorescent mutant) and RabS. The rates of association were found to be within about one order of magnitude of those for the nonsubstituted nucleotides, whereas the rates of dissociation were accelerated by factors of approximately 100 (p21ras) or approximately 10(5) (Rab5), and the resulting equilibrium dissociation constants are in the nm or microM range, respectively. The data demonstrate that 2'halo-ATP and -GTP are substrates or ligands for all proteins tested except the chaperone DnaK. Due to the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 10(5)-fold decrease in affinity as observed for Rab5 places the equilibrium dissociation constant in the microM range, so that they are still well suited for crystallization of the G-protein:nucleotide complex.     

Double chip protein arrays using recombinant single-chain Fv antibody fragments

Protein arrays permit the parallel analysis of many different markers in a small sample volume. However, the problem of cross-reactivity limits the degree of multiplexing in parallel sandwich immunoassays (using monoclonal antibodies (mAbs)), meaning antibodies must be prescreened in order to reduce false positives. In contrast, we use a second chip surface for the local application of detection antibodies, thereby efficiently eliminating antibody cross-reactions. Here, we illustrate the potential advantages of using single-chain Fv fragments rather than mAbs as capture and detection molecules with this double chip technology.     

Melatonin ameliorates ionizing radiation-induced oxidative organ damage in rats

This study was designed to study the effects of the potential radioprotective properties of pharmacological doses of melatonin against organ damage induced by whole-body irradiation (IR) in rats. A total of 32 male Sprague-Dawley rats were exposed to irradiation performed with a LINAC producing 6 MV photons at a focus 100 cm distant from the skin. Under ketamine anaesthesia, each rat received a single whole-body dose of 800 cGy. Immediately before and after IR, rats were treated with either saline or melatonin (20 mg/kg and 10 mg/kg, i.p.) and decapitated at 12-h after exposure to irradiation. Another group of rats was followed for 72-h after IR, where melatonin (10 mg/kg, i.p.) injections were repeated once daily. Tissue levels of malondialdehyde (MDA)--an index of lipid peroxidation--, glutathione (GSH)--a key to antioxidant--and myeloperoxidase (MPO) activity--an index of neutrophil infiltration--were estimated in liver, lung, colon and intestinal tissues. The results demonstrate that both 12-h and 72-h following IR, tissue levels of MDA were elevated (p<0.05-0.001), while GSH levels were reduced (p<0.05-0.001) in all organs. On the other hand, melatonin, reduced the levels of MDA and increased the GSH levels significantly, (p<0.05-0.001). MPO activity was increased significantly in the colonic tissue at the both 12-h and 72-h, and in the hepatic tissue at the 72-h following IR, which were reduced by melatonin (p<0.01-0.001). In the lung tissue enzyme activity was decreased at 72nd h of post-irradiation. In conclusion, the increase in MDA levels and MPO activity and the concomitant decrease in GSH levels demonstrate the role of oxidative mechanisms in irradiation-induced tissue damage, and melatonin, by its free radical scavenging and antioxidant properties, ameliorates irradiation-induced organ injury. Thus, supplementing cancer patients with adjuvant therapy of melatonin may have some benefit for successful radiotherapy.     

Ultrafast electron transfer in the complex between fluorescein and a cognate engineered lipocalin protein, a so-called anticalin

Anticalins are a novel class of engineered ligand-binding proteins with tailored specificities derived from the lipocalin scaffold. The anticalin FluA complexes fluorescein as ligand with high affinity, and it effects almost complete quenching of its steady-state fluorescence. To study the underlying mechanism, we have applied femtosecond absorption spectroscopy, which revealed excited-state electron transfer within the FluA*Fl complex to be responsible for the strong fluorescence quenching. On the basis of a comparison of redox potentials, either tryptophan or tyrosine may serve as electron donor to the bound fluorescein group in its excited singlet state, thus forming the fluorescein trianion radical within 400 fs. The almost monoexponential rate points to a single, well-defined binding site, and its temperature independence suggests an (almost) activationless process. Applying conventional electron transfer theory to the ultrafast forward and slower back-rates, the resulting electronic interaction is rather large, with approximately 140 cm(-1) for tyrosine, which would be consistent with a coplanar arrangement of both aromatic moieties within van der Waals distance. The weak residual steady-state fluorescence originates from a small (approximately 10%) component with a time constant in the 40-60 ps range. These results demonstrate the power of time-resolved absorption spectroscopy as a diagnostic tool for the elucidation of a fluorescence quenching mechanism and the temporal profiles of the processes involved. The high structural and dynamic definition of the complexation site suggests the anticalin FluA to be a promising model in order to tailor and probe electronic interactions and energetics in proteins.     

A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin

We demonstrate that the bilin-binding protein, a member of the lipocalin family of proteins, can be structurally reshaped in order to specifically complex digoxigenin, a steroid ligand commonly used for the non-radioactive labelling of biomolecules. 16 amino acid residues, distributed across the four loops which form the binding site of the bilin-binding protein, were subjected to targeted random mutagenesis. From the resulting library the variant DigA16 was obtained by combined use of phage display and a filter-sandwich colony screening assay, followed by in vitro affinity maturation. DigA16 possesses strong binding activity and high specificity for the digoxigenin group, with a K(D) of 30.2(+/-3.6) nM. The derivative compound digitoxigenin is bound even more tightly, with a K(D) of 2.0(+/-0.52) nM, whereas the steroid glycoside ouabain is not recognized at all. Fusion proteins between DigA16 and alkaline phosphatase were constructed and shown to retain both the digoxigenin-binding function and enzymatic activity, irrespective of whether the enzyme was fused to the N or the C terminus of the bilin-binding protein variant. Our findings suggest that the lipocalin scaffold can be generally employed for the construction of specific receptor proteins, so-called "anticalins", which provide a promising alternative to recombinant antibody fragments.     

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.