A cell polarity protein aPKCλ is required for eye lens formation and growth

The organisation of individual cells into a functional three-dimensional tissue is still a major question in developmental biology. Modulation of epithelial cell shape is a critical driving force in forming tissues. This is well illustrated in the eye lens where epithelial cells elongate extensively during their differentiation into fibre cells. It is at the lens equator that epithelial cells elongate along their apical-basal axis. During this process the elongating epithelial cells and their earliest fibre cell derivatives remain anchored at their apical tips, forming a discrete region or modiolus, which we term the lens fulcrum. How this is achieved has received scant attention and is little understood. Here, we show that conditional depletion of aPKCλ, a central effector of the PAR polarity complex, disrupts the apical junctions in elongating epithelial cells so that the lens fulcrum fails to form. This results in disorganised fibre cell alignment that then causes cataract. Interestingly, aPKCλ depletion also promotes epithelial-mesenchymal transition of the lens epithelial cells, reducing their proliferation, leading ultimately to a small lens and microphthalmia. These observations indicate that aPKCλ, a regulator of polarity and apical junctions, is required for development of a lens that is the correct size and shape.     

A membrane-bound vertebrate globin

The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2)-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O(2) binding with a variable affinity (P(50)～1.3-12.5 torr), depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.     

Synthesis of sulfonamide-containing N-hydroxyindole-2-carboxylates as inhibitors of human lactate dehydrogenase-isoform 5

N-Hydroxyindole-2-carboxylates possessing sulfonamide-substituents at either position 5 or 6 were designed and synthesized. The inhibitory activities of these compounds against isoforms 1 and 5 of human lactate dehydrogenase were analysed, and K_i values of the most efficient inhibitors were determined by standard enzyme kinetic studies. Some of these compounds displayed state-of-the-art inhibitory potencies against isoform 5 (K_i values as low as 5.6 μM) and behaved as competitive inhibitors versus both the substrate and the cofactor.     

Development of a new class of aromatase inhibitors: design, synthesis and inhibitory activity of 3-phenylchroman-4-one (isoflavanone) derivatives

Aromatase (CYP19) catalyzes the aromatization reaction of androgen substrates to estrogens, the last and rate-limiting step in estrogen biosynthesis. Inhibition of aromatase is a new and promising approach to treat hormone-dependent breast cancer. We present here the design and development of isoflavanone derivatives as potential aromatase inhibitors. Structural modifications were performed on the A and B rings of isoflavanones via microwave-assisted, gold-catalyzed annulation reactions of hydroxyaldehydes and alkynes. The in vitro aromatase inhibition of these compounds was determined by fluorescence-based assays utilizing recombinant human aromatase (baculovirus/insect cell-expressed). The compounds 3-(4-phenoxyphenyl)chroman-4-one (1h), 6-methoxy-3-phenylchroman-4-one (2a) and 3-(pyridin-3-yl)chroman-4-one (3b) exhibited potent inhibitory effects against aromatase with IC(50) values of 2.4 μM, 0.26 μM and 5.8 μM, respectively. Docking simulations were employed to investigate crucial enzyme/inhibitor interactions such as hydrophobic interactions, hydrogen bonding and heme iron coordination. This report provides useful information on aromatase inhibition and serves as a starting point for the development of new flavonoid aromatase inhibitors.     

The preclinical pharmacology of the high affinity anti-IL-6R Nanobody? ALX-0061 supports its clinical development in rheumatoid arthritis

IntroductionThe pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology.MethodsALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control.ResultsALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration.ConclusionsALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0651-0) contains supplementary material, which is available to authorized users.     

Proton transfer in the photosynthetic reaction center of Blastochloris viridis

Photosynthetic reaction centers of Blastochloris viridis require two quanta of light to catalyse a two-step reduction of their secondary ubiquinone Q(B) to ubiquinol. We employed capacitive potentiometry to follow the voltage changes that were caused by the accompanying transmembrane proton displacements. At pH 7.5 and 20 degrees C, the Q(B)-related voltage generation after the first flash was contributed by a fast, temperature-independent component with a time constant of approximately 30 micros and a slower component of approximately 200 micros with activation energy (E(a)) of 50 kJ/mol. The kinetics after the second flash featured temperature-independent components of 5 micros and 200 micros followed by a component of 600 micros with E(a) approximately 60 kJ/mol.     

Phylogenetic overview of the Boletineae

The generic and sub-generic relationships in the Boletineae (Boletales) were studied using nuclear large subunit (nuc-lsu), translation elongation factor 1-alpha (tef1), and DNA directed RNA polymerase largest subunit (RPB1). The Boletineae, with the exclusion of Hydnomerulius pinastri, was strongly supported and the status of the families Boletaceae and Paxillaceae is discussed. Members of the genus Boletus are found throughout the phylogeny, with the majority not closely related to the type species, Boletus edulis. Many of the traditional, morphologically defined genera are not supported as monophyletic and additional sampling and taxonomic revisions are needed. The majority of the Boletineae are confirmed or putatively ectomycorrhizal (ECM), but two putatively mycoparasitic lineages (one lineage of Buchwaldoboletus lignicola and Chalciporus piperatus and the second Pseudoboletus parasiticus) are strongly supported.     

Multidrug resistance gene deficient (mdr1a–/–) mice have an altered caecal microbiota that precedes the onset of intestinal inflammation

Aim:  To compare caecal microbiota from mdr1a ^–/– and wild type (FVB) mice to identify differences in the bacterial community that could influence the intestinal inflammation.
Methods and Results:  Caecal microbiota of mdr1a ^–/– and FVB mice were evaluated at 12 and 25 weeks of age using denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR. DGGE fingerprints of FVB and mdr1a ^–/– mice (with no intestinal inflammation) at 12 weeks revealed differences in the presence of DNA fragments identified as Bacteroides fragilis , B. thetaiotaomicron , B. vulgatus and an uncultured alphaproteobacterium. Escherichia coli and Acinetobacter sp. were only identified in DGGE profiles of mdr1a ^–/– mice at 25 weeks (with severe intestinal inflammation), which also had a lower number of total bacteria in the caecum compared with FVB mice at same age.
Conclusions:  Differences found in the caecal microbiota of FVB and mdr1a ^–/– mice (12 weeks) suggest that the lack of Abcb1 transporters in intestinal cells due to the disruption of the mdr1a gene might lead to changes in the caecal microbiota. The altered microbiota along with the genetic defect could contribute to the development of intestinal inflammation in mdr1a ^–/– mice.
Significance and Impact of the Study:  Differences in caecal microbiota of mdr1a ^–/– and FVB mice (12 weeks) suggest genotype specific colonization. The results provide evidence that Abcb1 transporters may regulate host interactions with commensal bacteria. Future work is needed to identify the mechanisms involved in this possible cross-talk between the host intestinal cells and microbiota.     

Effects of exogenous FGF-1 treatment on regeneration of the lens and the neural retina in the newt, Notophthalmus viridescens

Experiments were designed to compare the effects of recombinant newt fibroblast growth factor-1 (rnFGF-1) and recombinant human glial growth factor (rhGGF) on lens and retina regeneration in the eyes of adult newts. Both eyes were retinectomized and lentectomized. Beginning 3 days after the operation, one eye was given either 0.1 microg of rnFGF-1 or 0.1 microg of rhGGF in 1 microl of phosphate-buffered saline (PBS) per injection, three per week. Contralateral operated eyes served as controls and were treated with PBS alone or were not injected. In eyes that were not injected, injected with PBS alone, or with PBS containing rhGGF, regeneration of both the retina and the lens proceeded normally as described in the literature. In these control eyes, the entire retinal pigmented epithelium (RPE) depigmented/dedifferentiated and a retina rudiment formed from which a new retina regenerated by the end of the experiment at day 41 post-operation. Likewise, only a small area of dorsal iris depigmented/dedifferentiated and formed a lens vesicle from which a lens subsequently regenerated. The vitreous remained relatively free of loose cells.In eyes given rnFGF-1, the RPE depigmented/dedifferentiated and formed what appeared to be a retina rudiment but a new retina did not regenerate. Instead, vesicles were seen associated with the retina rudiment. In eyes given rnFGF-1, both the dorsal iris and ventral iris depigmented/dedifferentiated and lens regeneration occurred but the new lenses had abnormal fiber cells and the lens epithelium was very thin or absent. In addition, ectopic lenses usually regenerated in rnFGF-1-treated eyes. An abundance of loose cells were present in the vitreous of rnFGF-1-treated eyes associated largely with the RPE and the dorsal and ventral irises.The results are consistent with the view that the timely expression of FGFs is involved in the depigmentation/dedifferentiation of the RPE and dorsal iris and is necessary for proper regeneration of the lens and neural retina. Continued presence of FGF results in continued and excessive dedifferentiation, resulting in the lack of retina regeneration and abnormal lens regeneration.