Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over‐expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step‐wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys‐rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Eye size variation reflects habitat and daily activity patterns in colubrid snakes

The functioning of the vertebrate eye depends on its absolute size, which is presumably adapted to specific needs. Eye size variation in lidless and spectacled colubrid snakes was investigated, including 839 specimens belonging to 49 genera, 66 species and subspecies. Variations of adult eye diameters (EDs) in both absolute and relative terms between species were correlated with parameters reflecting behavioral ecology. In absolute terms, eye of arboreal species was larger than in terrestrial and semiaquatic species. For diurnal species, EDs of terrestrial species do not differ from semiaquatic species; for nocturnal species the ED of terrestrial species is larger than fossorial species but not different from semiaquatic species. In relative terms, ED did not differ significantly by habitat for diurnal species. Although the ED of terrestrial species is larger than fossorial species there were no differences for nocturnal species between semiaquatic and fossorial snakes. In contrast to other vertebrates studied to date, colubrid EDs in absolute and relative terms are larger in diurnal than in nocturnal species. These observations suggest that among colubrid snakes, eye size variation reflects adaptation to specific habitats, foraging strategies and daily activities, independently of phylogeny. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Akt Activation Emulates Chk1 Inhibition and Bcl2 Overexpression and Abrogates G2 Cell Cycle Checkpoint by Inhibiting BRCA1 Foci

Akt is perhaps the most frequently activated oncoprotein in human cancers. Overriding cell cycle checkpoint in combination with the inhibition of apoptosis are two principal requirements for predisposition to cancer. Here we show that the activation of Akt is sufficient to promote these two principal processes, by inhibiting Chk1 activation with concomitant inhibition of apoptosis. These activities of Akt cannot be recapitulated by the knockdown of Chk1 alone or by overexpression of Bcl2. Rather the combination of Chk1 knockdown and Bcl2 overexpression is required to recapitulate Akt activities. Akt was shown to directly phosphorylate Chk1. However, we found that Chk1 mutants in the Akt phosphorylation sites behave like wild-type Chk1 in mediating G2 arrest, suggesting that the phosphorylation of Chk1 by Akt is either dispensable for Chk1 activity or insufficient by itself to exert an effect on Chk1 activity. Here we report a new mechanism by which Akt affects G2 cell cycle arrest. We show that Akt inhibits BRCA1 function that induces G2 cell cycle arrest. Akt prevents the translocation of BRCA1 to DNA damage foci and, thereby, inhibiting the activation of Chk1 following DNA damage.     

Hydrolysis of fish oil by hyperactivated rhizomucor miehei lipase immobilized by multipoint anion exchange

Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20‐ to 25‐fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide‐activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl‐Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Association between FABP2 Ala54Thr polymorphisms and type 2 diabetes mellitus risk: a HuGE Review and Meta‐Analysis

Many studies have examined the association between the FABP2 (rs1799883) Ala54Thr gene polymorphism and type 2 diabetes mellitus risk (T2DM) in various populations, but their results have been inconsistent. To assess this relationship more precisely, A HuGE review and meta‐analysis were performed. The PubMed and CNKI database was searched for case‐control studies published up to April 2014. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. Ultimately, 13 studies, comprising 2020 T2DM cases and 2910 controls were included. Overall, for the Thr carriers (Ala/Thr and Thr/Thr) versus the wild‐type homozygotes (Ala/Ala), the pooled OR was 1.18 (95% CI = 1.04–1.34, P = 0.062 for heterogeneity), for Thr/Thr versus Ala/Ala the pooled OR was 1.17 (95% CI = 1.05–1.41 P = 0.087 for heterogeneity). In the stratified analysis by ethnicity, the significantly risks were found among Asians but not Caucasians. This meta‐analysis suggests that the FABP2 (rs1799883) Ala54Thr polymorphisms are associated with increased susceptibility to T2DM risk among Asians but not Caucasians.     

Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing neurons. Ultrastructure, adrenergic innervation and putative transmitter action

The neuropeptide thyrotropin releasing hormone (TRH) is capable of influencing both neuronal mechanisms in the brain and the activity of the pituitary-thyroid endocrine axis. By the use of immunocytochemical techniques, first the ultrastructural features of TRH-immunoreactive (IR) perikarya and neuronal processes were studied, and then the relationship between TRH-IR neuronal elements and dopamine-beta-hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT)-IR catecholaminergic axons was analyzed in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN). In control animals, only TRH-IR axons were detected and some of them seemed to follow the contour of immunonegative neurons. Colchicine treatment resulted in the appearance of TRH-IR material in parvocellular neurons of the PVN. At the ultrastructural level, immunolabel was associated with rough endoplasmic reticulum, free ribosomes and neurosecretory granules. Non-labelled axons formed synaptic specializations with both dendrites and perikarya of the TRH-synthesizing neurons. TRH-IR axons located in the parvocellular units of the PVN exhibited numerous intensely labelled dense-core and fewer small electron lucent vesicles. These axons were frequently observed to terminate on parvocellular neurons, forming both bouton- and en passant-type connections. The simultaneous light microscopic localization of DBH or PNMT-IR axons and TRH-synthesizing neurons demonstrated that catecholaminergic fibers established contacts with the dendrites and cell bodies of TRH-IR neurons. Ultrastructural analysis revealed the formation of asymmetric axo-somatic and axo-dendritic synaptic specializations between PNMT-immunopositive, adrenergic axons and TRH-IR neurons in the periventricular and medial parvocellular subnuclei of the PVN. These morphological data indicate that the hypophysiotrophic, thyrotropin releasing hormone synthesizing neurons of the PVN are directly influenced by the central epinephrine system and that TRH may act as a neurotransmitter or neuromodulator upon other paraventricular neurons.