The signaling adaptor protein CD3zeta is a negative regulator of dendrite development in young neurons

A novel idea is emergxsing that a large molecular repertoire is common to the nervous and immune systems, which might reflect the existence of novel neuronal functions for immune molecules in the brain. Here, we show that the transmembrane adaptor signaling protein CD3zeta, first described in the immune system, has a previously uncharacterized role in regulating neuronal development. Biochemical and immunohistochemical analyses of the rat brain and cultured neurons showed that CD3zeta is mainly expressed in neurons. Distribution of CD3zeta in developing cultured hippocampal neurons, as determined by immunofluorescence, indicates that CD3zeta is preferentially associated with the somatodendritic compartment as soon as the dendrites initiate their differentiation. At this stage, CD3zeta was selectively concentrated at dendritic filopodia and growth cones, actin-rich structures involved in neurite growth and patterning. siRNA-mediated knockdown of CD3zeta in cultured neurons or overexpression of a loss-of-function CD3zeta mutant lacking the tyrosine phosphorylation sites in the immunoreceptor tyrosine-based activation motifs (ITAMs) increased dendritic arborization. Conversely, activation of endogenous CD3zeta by a CD3zeta antibody reduced the size of the dendritic arbor. Altogether, our findings reveal a novel role for CD3zeta in the nervous system, suggesting its contribution to dendrite development through ITAM-based mechanisms.     

Antibiotic resistance peptides: interaction of peptides conferring macrolide and ketolide resistance with Staphylococcus aureus ribosomes: conformation of bound peptides as determined by transferred NOE experiments

Two antibiotic resistance peptides, the E-peptide (MRLFV) and the K-peptide (MRFFV) conferring macrolide and ketolide resistance, respectively, were studied in the complex state with bacterial Staphylococcus aureus ribosomes. Interactions of antibiotic resistance peptides with ribosomes were investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY), suggesting that the peptide-ribosome interaction was associated with the low-affinity binding level. K-Peptide displayed a significantly better response in TRNOEs NMR experiments, in agreement with a better overall antibiotic activity of ketolides. This difference highlights a mimetic effect displayed by the E- and K-peptides. This study shows that conformation plays an essential role for the affinity binding site and, thus, for the resistance mechanism. Specific conformations were preferred in the bound state; their superimposition exhibited a similar cyclic peptidyl chain, while the side chain region varies. The F4 phenyl moiety in E-peptide has moved out of the turn region compared to its folding in the ketolide resistance peptide. In the K-peptide binding surface, the F4 aromatic chain is maintained by stacking with the guanidyl group of the R2 residue providing a particular hydrophobic and globular fragment, which may be important for the ketolide resistance peptide mode of action. Additionally, T(2) (CPMG) measurements were used to characterize equilibrium binding of antibiotic resistance peptides to bacterial ribosomes. The results bring to the fore E- and K-peptide competition with antibiotics for binding to the ribosomes. Their specific interaction and their competitive effects reveal a novel aspect of interaction of resistance peptides with ribosomes and suggest new insights about their mode of action. The resistance mechanism may imply two steps, a competitive effect of the resistance peptide for the macrolide (or ketolide) binding site followed by a "bottle brush" effect in which the drug and the peptide are driven out their binding site on the ribosome.     

West African Cattle Farmers’ Perception of Tick-Borne Diseases

Worldwide, cattle production is struggling to face the negative impacts caused by ticks and Rhipicephalus (Boophilus) microplus is one of the most harmful ticks for livestock. Most of the people in West Africa depend on cattle farming and subsistence agriculture. The presence of ticks on cattle is a major problem faced by smallholder farmers who fight for their livelihood. National and regional tick control programs could assist these rural communities in protecting their livelihoods against ticks and tick-borne diseases, but only if they take into account the targeted herders and their perception on cattle management and tick control. This paper aims to provide a better insight in the socio-economic characteristics of Beninese cattle farmers, and their perception on tick burden, as well as to document common tick control strategies. Different tick species and their seasonality are well understood by cattle herders. For tick control, many still use manual tick removal, especially in the north of the country. The high cost of acaricides, the lack of financial means of African farmers, and of the local stockbreeders in particular, limits the use of acaricides in livestock breeding in Benin. While aiming to increase the meat or milk production of their animals, stockbreeders who can afford it sometimes turn to an abusive use of acaricides, which might in time lead to an increase in tick resistance. This study remains one of the rare studies to report extensively on the perceptions of West African cattle herders.     

Stable isotope ecology of Miocene bovids from northern Greece and the ape/monkey turnover in the Balkans

Eurasia was home to a great radiation of hominoid primates during the Miocene. All were extinct by the end of the Miocene in Western Eurasia. Here, we investigate the hypothesis of climate and vegetation changes at a local scale when the cercopithecoid Mesopithecus replaced the hominoid Ouranopithecus along the Axios River, Greece. Because they are herbivorous and were much more abundant than primates, bovids are preferred to primates to study climate change in the Balkans as a cause of hominoid extinction. By measuring carbon stable isotope ratios of bovid enamel, we conclude that Ouranopithecus and Mesopithecus both evolved in pure C₃ environments. However, the large range of δ¹³C values of apatite carbonate from bovids combined with their molar microwear and mesowear patterns preclude the presence of dense forested landscapes in northern Greece. Instead, these bovids evolved in rather open landscapes with abundant grasses in the herbaceous layer. Coldest monthly estimated temperatures were below 10 °C and warmest monthly temperatures rose close to or above 20 °C for the two time intervals. Oxygen isotope compositions of phosphate from bulk samples did not show significant differences between sites but did show between-species variation within each site. Different factors influence oxygen isotope composition in this context, including water provenience, feeding ecology, body mass, and rate of amelogenesis. We discuss this latter factor in regard to the high intra-tooth variations in δ¹⁸O_p reflecting important amplitudes of seasonal variations in temperature. These estimations fit with paleobotanical data and differ slightly from estimations based on climate models. This study found no significant change in climate before and after the extinction of Ouranopithecus along the Axios River. However, strong seasonal variations with relatively cold winters were indicated, conditions quite usual for extant monkeys but unusual for great apes distributed today in inter-tropical regions.     

New Pliocene remains of Camelus grattardi (Mammalia,Camelidae) from the Shungura Formation,Lower Omo Valley,Ethiopia, and the evolution of African camels

Abstract

Camels are exceptionally rare in the Plio-Pleistocene fossil record of Africa, hindering attempts to understand the evolution of this family on the continent. Here we describe recently collected camel specimens from the Shungura Formation, Lower Omo Valley, Ethiopia, and attribute these remains to Camelus grattardi. The new specimens date to the late Pliocene (~3 to 2.6 Ma) and consist of three upper molars, one upper premolar, and two proximal metatarsals. The dental specimens confirm this species’ small P4 relative to its molars, a trait that differs significantly from all extant and fossil Old World camels. The metatarsals indicate that C. grattardi was similar in size to the living Bactrian camel, C. bactrianus. Phylogenetically, we find no suitable ancestor, sister, or descendant of the eastern African fossil camel, which suggests greater lineage diversity in Plio-Pleistocene Camelus than previously recognised. Microwear analyses suggest that C. grattardi was likely a mixed-feeder preferring browse, which is consistent with carbon isotopes of enamel from the Turkana Basin. A review of the fossil record of African camels suggests no clear paleoenvironmental association, as fossil camels occur in a range of environments from dry savannas with no permanent water bodies to closed woodlands along the paleo-Omo River.     

Enantiomers of 4-amino-3-fluorobutanoic acid as substrates for gamma-aminobutyric acid aminotransferase. Conformational probes for GABA binding

Gamma-aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5'-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5'-phosphate (PLP) to pyridoxamine 5'-phosphate (PMP). The enzyme then catalyzes the conversion of alpha-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred on the basis of the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches.     

Analytical biochemistry of DNA–protein assemblies from crude cell extracts

Purification of specific DNA–protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA–protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA–protein complexes, showing the benefits to uncouple the DNA–protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA–protein assemblies.     

Analytical biochemistry of DNA--protein assemblies from crude cell extracts

Purification of specific DNA-protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA-protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA-protein complexes, showing the benefits to uncouple the DNA-protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA-protein assemblies.