Molecular evolution of a chordate specific family of G protein-coupled receptors

<p>Abstract</p> <p>Background</p> <p>Chordate evolution is a history of innovations that is marked by physical and behavioral specializations, which led to the development of a variety of forms from a single ancestral group. Among other important characteristics, vertebrates obtained a well developed brain, anterior sensory structures, a closed circulatory system and gills or lungs as blood oxygenation systems. The duplication of pre-existing genes had profound evolutionary implications for the developmental complexity in vertebrates, since mutations modifying the function of a duplicated protein can lead to novel functions, improving the evolutionary success.</p> <p>Results</p> <p>We analyzed here the evolution of the GPRC5 family of G protein-coupled receptors by comprehensive similarity searches and found that the receptors are only present in chordates and that the size of the receptor family expanded, likely due to genome duplication events in the early history of vertebrate evolution. We propose that a single GPRC5 receptor coding gene originated in a stem chordate ancestor and gave rise by duplication events to a gene family comprising three receptor types (GPRC5A-C) in vertebrates, and a fourth homologue present only in mammals (GPRC5D). Additional duplications of GPRC5B and GPRC5C sequences occurred in teleost fishes. The finding that the expression patterns of the receptors are evolutionarily conserved indicates an important biological function of these receptors. Moreover, we found that expression of GPRC5B is regulated by vitamin A <it>in vivo</it>, confirming previous findings that linked receptor expression to retinoic acid levels in tumor cell lines and strengthening the link between the receptor expression and the development of a complex nervous system in chordates, known to be dependent on retinoic acid signaling.</p> <p>Conclusions</p> <p>GPRC5 receptors, a class of G protein-coupled receptors with unique sequence characteristics, may represent a molecular novelty that helped non-chordates to become chordates.</p

Preparations of Meiotic Pachytene Chromosomes and Extended DNA Fibers from Cotton Suitable for Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established

A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes

<p>Abstract</p> <p>Background</p> <p>To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.</p> <p>Results</p> <p>Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an <it>in vivo </it>recombination strategy. Each AMP was then expressed as an Npro fusion protein in <it>Escherichia coli</it>. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On <it>in vitro </it>refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against <it>E. coli</it>, <it>Micrococcus </it>luteus and <it>S. cerevisia</it>.</p> <p>Conclusions</p> <p>The method described in this report allows the fast synthesis of genes that are optimized for over-expression in <it>E. coli </it>and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.</p

Anti-tumor effect of adenovirus-mediated gene transfer of pigment epithelium-derived factor on mouse B16-F10 melanoma

<p>Abstract</p> <p>Background</p> <p>Angiogenesis plays an important role in tumor growth, invasion, and eventually metastasis. Antiangiogenic strategies have been proven to be a promising approach for clinical therapy for a variety of tumors. As a potent inhibitor of tumor angiogenesis, pigment epithelium-derived factor (PEDF) has recently been studied and used as an anticancer agent in several tumor models.</p> <p>Methods</p> <p>A recombined adenovirus carrying PEDF gene (Ad-PEDF) was prepared, and its expression by infected cells and in treated animals was confirmed with Western blotting and ELISA, respectively. Its activity for inhibiting human umbilical vein endothelial cell (HUVEC) proliferation was tested using the MTT assay. C57BL/6 mice bearing B16-F10 melanoma were treated with i.v. administration of 5 × 10⁸IU/mouse Ad-PEDF, or 5 × 10⁸IU/mouse Ad-Null, or normal saline (NS), every 3 days for a total of 4 times. Tumor volume and survival time were recorded. TUNEL, CD31 and H&E stainings of tumor tissue were conducted to examine apoptosis, microvessel density and histological morphology changes. Antiangiogenesis was determined by the alginate-encapsulated tumor cell assay.</p> <p>Results</p> <p>The recombinant PEDF adenovirus is able to transfer the PEDF gene to infected cells and successfully produce secretory PEDF protein, which exhibits potent inhibitory effects on HUVEC proliferation. Through inhibiting angiogenesis, reducing MVD and increasing apoptosis, Ad-PEDF treatment reduced tumor volume and prolonged survival times of mouse bearing B16-F10 melanoma.</p> <p>Conclusion</p> <p>Our data indicate that Ad-PEDF may provide an effective approach to inhibit mouse B16-F10 melanoma growth.</p

Comparative Study of Tumor Targeting and Biodistribution of pH (Low) Insertion Peptides (pHLIP® Peptides) Conjugated with Different Fluorescent Dyes

Purpose Acidification of extracellular space promotes tumor development, progression, and invasiveness. pH (low) insertion peptides (pHLIP® peptides) belong to the class of pH-sensitive membrane peptides, which target acidic tumors and deliver imaging and/or therapeutic agents to cancer cells within tumors. Procedures Ex vivo fluorescent imaging of tissue and organs collected at various time points after administration of different pHLIP® variants conjugated with fluorescent dyes of various polarity was performed. Methods of multivariate statistical analyses were employed to establish classification between fluorescently labeled pHLIP® variants in multidimensional space of spectral parameters. Results The fluorescently labeled pHLIP® variants were classified based on their biodistribution profile and ability of targeting of primary tumors. Also, submillimeter-sized metastatic lesions in lungs were identified by ex vivo imaging after intravenous administration of fluorescent pHLIP® peptide. Conclusions Different cargo molecules conjugated with pHLIP® peptides can alter biodistribution and tumor targeting. The obtained knowledge is essential for the design of novel pHLIP®-based diagnostic and therapeutic agents targeting primary tumors and metastatic lesions

Absence of Ca2+-stimulated adenylyl cyclases leads to reduced synaptic plasticity and impaired experience-dependent fear memory

Ca2+-stimulated adenylyl cyclase (AC) 1 and 8 are two genes that have been shown to play critical roles in fear memory. AC1 and AC8 couple neuronal activity and intracellular Ca2+ increases to the production of cyclic adenosine monophosphate and are localized synaptically, suggesting that Ca2+-stimulated ACs may modulate synaptic plasticity. Here, we first established that Ca2+-stimulated ACs modulate protein markers of synaptic activity at baseline and after learning. Primary hippocampal cell cultures showed that AC1/AC8 double-knockout (DKO) mice have reduced SV2, a synaptic vesicle protein, abundance along their dendritic processes, and this reduction can be rescued through lentivirus delivery of AC8 to the DKO cells. Additionally, phospho-synapsin, a protein implicated in the regulation of neurotransmitter release at the synapse, is decreased in vivo 1 h after conditioned fear (CF) training in DKO mice. Importantly, additional experiments showed that long-term potentiation deficits present in DKO mice are rescued by acutely replacing AC8 in the forebrain, further supporting the idea that Ca2+-stimulated AC activity is a crucial modulator of synaptic plasticity. Previous studies have demonstrated that memory is continually modulated by gene–environment interactions. The last set of experiments evaluated the effects of knocking out AC1 and AC8 genes on experience-dependent changes in CF memory. We showed that the strength of CF memory in wild-type mice is determined by previous environment, minimal or enriched, whereas memory in DKO mice is unaffected. Thus, overall these results show that AC1 and AC8 modulate markers of synaptic activity and help integrate environmental information to modulate fear memory

Association between Alcohol Consumption and Cancers in the Chinese Population—A Systematic Review and Meta-Analysis

Alcohol consumption is increasing worldwide and is associated with numerous cancers. This systematic review examined the role of alcohol in the incidence of cancer in the Chinese population.Medline/PubMed, EMBASE, CNKI and VIP were searched to identify relevant studies. Cohort and case-control studies on the effect of alcohol use on cancers in Chinese were included. Study quality was evaluated using the Newcastle-Ottawa Scale. Data were independently abstracted by two reviewers. Odds ratios (OR) or relative risks (RR) were pooled using RevMan 5.0. Heterogeneity was evaluated using the Q test and I-squared statistic. P<.01 was considered statistically significant.Pooled results from cohort studies indicated that alcohol consumption was not associated with gastric cancer, esophageal cancers (EC) or lung cancer. Meta-analysis of case-control studies showed that alcohol consumption was a significant risk factor for five cancers; the pooled ORs were 1.79 (99% CI, 1.47–2.17) EC, 1.40 (99% CI, 1.19–1.64) gastric cancer, 1.56 (99% CI, 1.16–2.09) hepatocellular carcinoma, 1.21 (99% CI, 1.00–1.46) nasopharyngeal cancer and 1.71 (99% CI, 1.20–2.44) oral cancer. Pooled ORs of the case-control studies showed that alcohol consumption was protective for female breast cancer and gallbladder cancer: OR 0.76 (99% CI, 0.60–0.97) and 0.70 (99% CI, 0.49–1.00) respectively. There was no significant correlation between alcohol consumption and lung cancer, colorectal cancer, pancreatic cancer, cancer of the ampulla of Vater, prostate cancer or extrahepatic cholangiocarcinoma. Combined results of case-control and cohort studies showed that alcohol consumption was associated with 1.78- and 1.40-fold higher risks of EC and gastric cancer but was not significantly associated with lung cancer.Health programs focused on limiting alcohol intake may be important for cancer control in China. Further studies are needed to examine the interaction between alcohol consumption and other risk factors for cancers in Chinese and other populations

Metalloporphyrin intercalation in liposome membranes: ESR study

Liposomes characterized by membranes featuring diverse fluidity (liquid-crystalline and/or gel phase), prepared from egg yolk lecithin (EYL) and dipalmitoylphosphatidylcholine (DPPC), were doped with selected metalloporphyrins and the time-related structural and dynamic changes within the lipid double layer were investigated. Porphyrin complexes of Mg(II), Mn(III), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), and the metal-free base were embedded into the particular liposome systems and tested for 350 h at 24°C using the electron spin resonance (ESR) spin probe technique. 5-DOXYL, 12-DOXYL, and 16-DOXYL stearic acid methyl ester spin labels were applied to explore the interior of the lipid bilayer. Only the 16-DOXYL spin probe detected evident structural changes inside the lipid system due to porphyrin intercalation. Fluidity of the lipid system and the type of the porphyrin complex introduced significantly affected the intermolecular interactions, which in certain cases may result in self-assembly of metalloporphyrin molecules within the liposome membrane, reflected in the presence of new lines in the relevant ESR spectra. The most pronounced time-related effects were demonstrated by the EYL liposomes (liquid-crystalline phase) when doped with Mg and Co porphyrins, whereas practically no spectral changes were revealed for the metal-free base and both the Ni and Zn dopants. ESR spectra of the porphyrin-doped gel phase of DPPC liposomes did not show any extra lines; however, they indicated the formation of a more rigid lipid medium. Electronic configuration of the porphyrin’s metal center appeared crucial to the degree of molecular reorganization within the phospholipid bilayer system