Episodic evolution of growth hormone in primates and emergence of the species specificity of human growth hormone receptor

Growth hormone (GH) evolution is very conservative among mammals, except for primates and ruminant artiodactyls. In fact, most known mammalian GH sequences differ from the inferred ancestral mammalian sequence by only a few amino acids. In contrast, the human GH sequence differs from the inferred ancestral sequence by 59 amino acids. However, it is not known when this rapid evolution of GH occurred during primate evolution or whether it was due to positive selection. Also, human growth hormone receptor (GHR) displays species specificity; i.e., it can interact only with human (or rhesus monkey) GH, not with nonprimate GHS: The species specificity of human GHR is largely due to the Leu-->Arg change at position 43, and it has been hypothesized that this change must have been preceded by the His-->Asp change at position 171 of GH. Is this hypothesis true? And when did these changes occur? To address the above issues, we sequenced GH and GHR genes in prosimians and simians. Our data supported the above hypothesis and revealed that the species specificity of human GHR actually emerged in the common ancestor of Old World primates, but the transitional phase still persists in New World monkeys. Our data showed that the rapid evolution of primate GH occurred during a relatively short period (in the common ancestor of higher primates) and that the rate of change was especially high at functionally important sites, suggesting positive selection. However, the nonsynonymous rate/synonymous rate ratio at these sites was <1, so relaxation of purifying selection might have played a role in the rapid evolution of the GH gene in simians, possibly as a result of multiple gene duplications. Similar to GH, GHR displayed an accelerated rate of evolution in primates. Our data revealed proportionally more amino acid replacements at the functionally important sites in both GH and GHR in simians but, surprisingly, showed few coincidental replacements of amino acids forming the same intermolecular contacts between the two proteins.     

Liver engraftment potential of hepatic cells derived from patient-specific induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) are potential renewable sources of hepatocytes for drug development and cell therapy. Differentiation of human iPSCs into different developmental stages of hepatic cells has been achieved and improved during the last several years. We have recently demonstrated the liver engraftment and regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo. Here we describe the in vitro and in vivo activities of hepatic cells derived from patient specific iPSCs, including multiple lines established from either inherited or acquired liver diseases, and discuss basic and clinical applications of these cells for disease modeling, drug screening and discovery, gene therapy and cell replacement therapy.     

Efficiency of different PCR-based marker systems for assessment of Iris pumila genetic diversity

We investigated informativeness and effectiveness of different marker types (ISSR, IRAP, REMAP, RGAP and LP-PCR that employ primers based on the conservative sequences of abiotic stress response genes) to study genetic diversity of Iris pumila L. By the number of amplicons per primer, number of polymorphic amplicons per primer and resolving power index (Rp), ISSR-markers were the most efficient followed by LP-PCR-markers. In order of decreasing value of indicators of genetic diversity “the percentage of polymorphic bands”, and “the average Jaccard? genetic distance between plants”, marker systems may be arranged as follows: ISSR > RAPD > LP-PC > RGAP ≈ IRAP. For ISSR-markers, the percentage of polymorphic bands was 1.3–1.7 times higher than for the others, and the average genetic distance was 1.2–1.3 times higher. Different marker systems were ranked by the value of Nei? gene diversity and the Shannon? index as follows: ISSR > RAPD ≈ LP-PCR > RGAP ≈ IRAP, with the highest and the lowest values differing 1.4 times. Genetic population structure was investigated with program Structure 2.3. The data of all marker systems suggest that all genomes under study belonged to one population. The PCoA and cluster analyses based on genetic distances showed distinctions in clustering generated from different markers data and summarized data, as well as the lack of strong clusters. Mantel test revealed significant positive correlation between the matrices of genetic distances generated by the data of almost all marker systems. The strongest correlation was found between RGAP- and IRAP-markers (r = 0.452, p = 0.01) and between RGAP and ISSR (r = 0.430, p = 0.01). ISSR, RAPD and LP-PCR proved to be more effective for the study of I. pumila genetic diversity, nevertheless, joint use of different marker systems will provide a more comprehensive assessment of variation in different genomic regions.     

Modulations of behavioral consequences of minor cortical ischemic lesion by application of free radicals scavengers

Functional and morphological consequences of ischemic lesions are partially related to the production of reactive oxygen species (ROS). The aim of the study was to create a unilateral photothrombic lesion with minimal morphological changes and minor sensorimotor and cognitive deficits and also to test whether the application of ROS scavengers after the end of induction of ischemia had improved the functional outcome. Adult Wistar male rats were randomly divided into five groups: naive control, sham operated animals, animals with induced ischemia, and two groups of animals with induced ischemia and subsequent ROS scavenger application -melatonin or tempol. The group subjected to ischemia showed a significant decline in performance in sensorimotor tests and the Morris water maze (MWM) test, compared to control animals. Tempol (50 mg/kg, i.p.) did not improve sensorimotor function and did not change spatial learning. Melatonin (100 mg/kg, i.p.), on the contrary, resulted in a significant improvement in animals' performances. All the ischemia subjected animals had increased speed of swimming in the MWM test, compared to the control group. Our findings showed that subsequent application of ROS scavengers improve ischemia outcomes, with melatonin being more potent. Conversely, neither melatonin, nor tempol decreased swimming speed cased by ischemia.     

GAP-43 regulates NCAM-180-mediated neurite outgrowth

The neural cell adhesion molecule (NCAM), and the growth-associated protein (GAP-43), play pivotal roles in neuronal development and plasticity and possess interdependent functions. However, the mechanisms underlying the functional association of GAP-43 and NCAM have not been elucidated. In this study we show that (over)expression of GAP-43 in PC12E2 cells and hippocampal neurons strongly potentiates neurite extension, both in the absence and in the presence of homophilic NCAM binding. This potentiation is crucially dependent on the membrane association of GAP-43. We demonstrate that phosphorylation of GAP-43 by protein kinase C (PKC) as well as by casein kinase II (CKII) is important for the NCAM-induced neurite outgrowth. Moreover, our results indicate that in the presence of GAP-43, NCAM-induced neurite outgrowth requires functional association of NCAM-180/spectrin/GAP-43, whereas in the absence of GAP-43, the NCAM-140/non-receptor tyrosine kinase (Fyn)-associated signaling pathway is pivotal. Thus, expression of GAP-43 presumably acts as a functional switch for NCAM-180-induced signaling. This suggests that under physiological conditions, spatial and/or temporal changes of the localization of GAP-43 and NCAM on the cell membrane may determine the predominant signaling mechanism triggered by homophilic NCAM binding: NCAM-180/spectrin-mediated modulation of the actin cytoskeleton, NCAM-140-mediated activation of Fyn, or both.     

Balancing Light Absorption and Charge Transport in Vertical SnS2 Nanoflake Photoanodes with Stepped Layers and Large Intrinsic Mobility

Significant optical absorption in the blue–green spectral range, high intralayer carrier mobility, and band alignment suitable for water splitting suggest tin disulfide (SnS₂) as a candidate material for photo‐electrochemical applications. In this work, vertically aligned SnS₂ nanoflakes are synthesized directly on transparent conductive substrates using a scalable close space sublimation (CSS) method. Detailed characterization by time‐resolved terahertz and time‐resolved photoluminescence spectroscopies reveals a high intrinsic carrier mobility of 330 cm² V^?1 s^?1 and photoexcited carrier lifetimes of 1.3 ns in these nanoflakes, resulting in a long vertical diffusion length of ≈1 µm. The highest photo‐electrochemical performance is achieved by growing SnS₂ nanoflakes with heights that are between this diffusion length and the optical absorption depth of ≈2 µm, which balances the competing requirements of charge transport and light absorption. Moreover, the unique stepped morphology of these CSS‐grown nanoflakes improves photocurrent by exposing multiple edge sites in every nanoflake. The optimized vertical SnS₂ nanoflake photoanodes produce record photocurrents of 4.5 mA cm^?2 for oxidation of a sulfite hole scavenger and 2.6 mA cm^?2 for water oxidation without any hole scavenger, both at 1.23 V_RHE in neutral electrolyte under simulated AM1.5G sunlight, and stable photocurrents for iodide oxidation in acidic electrolyte.     

Male-driven evolution

The strength of male-driven evolution - that is, the magnitude of the sex ratio of mutation rate - has been a controversial issue, particularly in primates. While earlier studies estimated the male-to-female ratio (alpha) of mutation rate to be about 4-6 in higher primates, two recent studies claimed that alpha is only about 2 in humans. However, a more recent comparison of mutation rates between a noncoding fragment on Y and a homologous region on chromosome 3 gave an estimate of alpha = 5.3, reinstating strong male-driven evolution in hominoids. Several studies investigated variation in mutation rates among genomic regions that may not be related to sex differences and found strong evidence for such variation. The causes for regional variation in mutation rate are not clear but GC content and recombination are two possible causes. Thus, while the strong male-driven evolution in higher primates suggests that errors during DNA replication in the germ cells are the major source of mutation, the contribution of some replication-independent factors such as recombination may also be important.     

Global analysis of protein phosphorylation networks in insulin signaling by sequential enrichment of phosphoproteins and phosphopeptides

Although the important role of protein phosphorylation in insulin signaling networks is well recognized, its analysis in vivo has not been pursued in a systematic fashion through proteome-wide studies. Here we undertake a global analysis of insulin-induced changes in the rat liver cytoplasmic and endosomal phosphoproteome by sequential enrichment of phosphoproteins and phosphopeptides. After subcellular fractionation proteins were denatured and loaded onto iminodiacetic acid-modified Sepharose with immobilized Al3? ions (IMAC-Al resin). Retained phosphoproteins were eluted with 50 mM phosphate and proteolytically digested. The digest was then loaded onto an IMAC-Al resin and phosphopeptides were eluted with 50 mM phosphate, and resolved by 2-dimensional liquid chromatography, which combined offline weak anion exchange and online reverse phase separations. The peptides were identified by tandem mass spectrometry, which also detected the phosphorylation sites. Non-phosphorylated peptides found in the flow-through of the IMAC-Al columns were also analyzed providing complementary information for protein identification. In this study we enriched phosphopeptides to ~85% purity and identified 1456 phosphopeptides from 604 liver phosphoproteins. Eighty-nine phosphosites including 45 novel ones in 83 proteins involved in vesicular transport, metabolism, cell motility and structure, gene expression and various signaling pathways were changed in response to insulin treatment. Together these findings could provide potential new markers for evaluating insulin action and resistance in obesity and diabetes.     

Synthesis of beta-hydroxy-alpha-amino acids with a reengineered alanine racemase

The Y265A mutant of alanine racemase (alrY265A) was evaluated as a catalyst for the synthesis of beta-hydroxy-alpha-amino acids. It promotes the PLP-dependent aldol condensation of glycine with a range of aromatic aldehydes. The desired products were obtained with complete stereocontrol at C(alpha) (ee>99%, D) and moderate to high selectivity at C(beta) (up to 97% de). The designed enzyme is thus similar to natural d-threonine aldolases in its substrate specificity and stereoselectivity. Moreover, its ability to utilize alanine as an alternative donor suggests an expanded scope of potential utility for the production of biologically active compounds.