Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization

Antibody discovery typically uses hybridoma- or display-based selection approaches, which lack the advantages of directly screening spatially addressed compound libraries as in small-molecule discovery. Here we apply the latter strategy to antibody discovery, using a library of ～10,000 human germline antibody Fabs created by de novo DNA synthesis and automated protein expression and purification. In multiplexed screening assays, we obtained specific hits against seven of nine antigens. Using sequence-activity relationships and iterative mutagenesis, we optimized the binding affinities of two hits to the low nanomolar range. The matured Fabs showed full and partial antagonism activities in cell-based assays. Thus, protein drug leads can be discovered using surprisingly small libraries of proteins with known sequences, questioning the requirement for billions of members in an antibody discovery library. This methodology also provides sequence, expression and specificity information at the first step of the discovery process, and could enable novel antibody discovery in functional screens.     

Evolution of chameleon locomotion,or how to become arboreal as a reptile

High-speed, biplanar X-ray motion analysis, X-ray reconstruction of moving morphology (XROMM) and morphological studies have led to the identification of those traits which are considered to be crucial for the evolution of arboreal locomotion in chameleons. The loss of the extensive lateral undulation typical of reptiles needs to be compensated by high mobility in the shoulder girdle and a clear functional regionalization of the trunk. Large limb excursion angles provide a compliant gait and are made possible by a functional parasagittalization of fore- and hind limbs, at least temporarily. All these evolutionary novelties parallel very similar modifications in the evolution of the locomotor apparatus in therian mammals. We propose that the convergent “invention” of dynamic stability and a compliant gait seem to be responsible for the locomotor similarities between chameleons and mammals.     

Rapid evolution of a novel signalling mechanism by concerted duplication and divergence of a BMP ligand and its extracellular modulators

Gene duplication and divergence is widely considered to be a fundamental mechanism for generating evolutionary novelties. The Bone Morphogenetic Proteins (BMPs) are a diverse family of signalling molecules found in all metazoan genomes that have evolved by duplication and divergence from a small number of ancestral types. In the fruit fly Drosophila, there are three BMPs: Decapentaplegic (Dpp) and Glass bottom boat (Gbb), which are the orthologues of vertebrate BMP2/4 and BMP5/6/7/8, respectively, and Screw (Scw), which, at the sequence level, is equally divergent from Dpp and Gbb. It has recently been shown that Scw has arisen from a duplication of Gbb in the lineage leading to higher Diptera. We show that since this duplication event, Gbb has maintained the ancestral BMP5/6/7/8 functionality while Scw has rapidly diverged. The evolution of Scw was accompanied by duplication and divergence of a suite of extracellular regulators that continue to diverge together in the higher Diptera. In addition, Scw has become restricted in its receptor specificity: Gbb proteins can signal through the Type I receptors Thick veins (Tkv) and Saxophone (Sax), while Scw signals through Sax. Thus, in a relatively short span of evolutionary time, the duplication event that gave rise to Scw produced not only a novel ligand but also a novel signalling mode that is functionally distinct from the ancestral Gbb mode. Our results demonstrate the plasticity of the BMP pathway not only in evolving new family members and new functions but also new signalling modes by redeploying key regulators in the pathway.     

A convenient method for saponin isolation in tumour therapy

Saponinum album (Merck), which is a crude mixture of saponins from Gypsophila paniculata L., was shown to improve the anti cancer therapy when used in vivo in combination with saporin-based targeted toxins. Unfortunately saponinum album cannot be used for further development since Merck has ceased its production in the 1990s. As pure saponins are mandatory for use in medical purposes we developed a convenient method for saponin isolation directly from the roots of Gypsophila paniculata L. The developed method is rapid, cheap and scaling up is also possible. By combining dialysis and HPLC three saponins were isolated in a one-step procedure. Chemical structures of the purified saponins were characterized by extensive one and two-dimensional NMR-spectroscopy and by using ESI-TOF-MS. The biological activities of the purified saponins were also investigated. The method presented herein enabled a rapid and cheap isolation of saponins for tumour therapy.     

Phosphoinositide 3-kinase γ has Multiple Phospholipid Binding Sites

Phosphoinositide 3-kinase γ is a multifunctional enzyme with lipid and protein kinase activities that also acts as a scaffold protein in many diverse signalling processes. The enzyme contains five different domains, but their individual contributions to membrane binding are not fully understood. Here, using in vitro liposome binding assays of individual domains and deletion constructs of human phosphoinositide 3-kinase γ, we show that each domain is capable of binding anionic phospholipids to varying degrees, depending on the charge of the anionic substrate. Moreover, with the exception of the C2-domain, deletion of any single protein domain results in a complete loss of kinase activity toward both lipids and proteins.     

Proteome and radioimmunoassay analyses of pituitary hormones and proteins in response to feed restriction of dairy cows

The hypothalamic-pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2-DE followed by MALDI-TOF-MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non-esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine-rich element-binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co-transcribed homolog, peroxiredoxin III, β-tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood.     

Characterization of rhesus macaque KIR genotypes and haplotypes

Certain combinations of the killer immunoglobulin-like receptors (KIR) and major histocompatibility complex class I ligands in humans predispose carriers to a variety of diseases, requiring sophisticated genotyping of the highly polymorphic and diverse KIR and HLA genes. Particularly, KIR genotyping is challenging due to polymorphisms (allelic substitutions), genomic diversity (presence/absence of genes), and frequent duplications. Rhesus macaques are often used as important animal models of human diseases such as, e.g. AIDS. However, typing of rhesus macaque KIR genes has not been described so far. In this study, we report the identification of additional novel rhesus macaque KIR cDNA sequences and a sequence-specific KIR genotyping assay. From a cohort of four rhesus macaque families with a total of 70 individuals, we identified 25 distinct KIR genotypes. Segregation analyses of KIR genes and of two polymorphic microsatellite markers allowed the identification of 21 distinct KIR haplotypes in these families, with five to 11 segregating KIR genes per haplotype. Our analyses confirmed and extended knowledge on differential gene KIR gene content in macaques and indicate that rhesus macaque and human KIR haplotypes show a comparable level of diversity and complexity.     

Homocysteine and vitamin therapy in stroke prevention and treatment: a review

Homocysteine (Hcy), a sulfur amino acid, is the only direct precursor for L-methionine synthesis through a reaction that requires vitamin B??, representing a connection with "one-carbon" units metabolism. Hcy catabolism requires vitamin B? and as a consequence, alteration in folic acid and B vitamins status impairs Hcy biotransformation. Numerous studies have indicated that Hcy is an independent risk factor for cardio- and cerebrovascular diseases. In the last decade, several clinical trials have investigated the possible correlation between the use of folic acid and vitamins B? and B?? for lowering Hcy plasma concentration and the reduced risk of stroke or its recurrence. This review is aimed to present some aspects of Hcy biochemistry, as well as the mechanisms through which it exerts the toxic effects on the vascular endothelium. We also discuss the results of some of the clinical trials developed to investigate the beneficial effects of vitamin therapy in the prevention and management of stroke.