Recent advances in production of 5-aminolevulinic acid using biological strategies

5-Aminolevulinic acid (5-ALA) is the precursor for the biosynthesis of tetrapyrrole compounds and has broad applications in the medical and agricultural fields. Because of the disadvantages of chemical synthesis methods, microbial production of 5-ALA has drawn intensive attention and has been regarded as an alternative in the last years, especially with the rapid development of metabolic engineering and synthetic biology. In this mini-review, recent advances on the application and microbial production of 5-ALA using novel biological approaches (such as whole-cell enzymatic-transformation, metabolic pathway engineering and cell-free process) are described and discussed in detail. In addition, the challenges and prospects of synthetic biology are discussed.     

Quantitative phosphoproteome analysis of lysophosphatidic acid induced chemotaxis applying dual-step (18)O labeling coupled with immobilized metal-ion affinity chromatography

Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O labeling plus (16)O/ (18)O-methanol esterification for quantitation, a macro-immobilized metal-ion affinity chromatography trap for phosphopeptide enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis.     

Advanced glycation end products serve as ligands for lectin-like oxidized low-density lipoprotein receptor-1(LOX-1): biochemical and binding characterizations assay

Advanced glycation end products (AGEs) are a class of complex heterogeneous compounds which accumulate with age and is known to be involved in the pathogenesis of several diseases from diabetes to atherosclerosis. AGEs serve as ligands for multiple receptors including scavenger receptor (SR-A), CD36, and SR-BIota. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays an important role in both atherosclerosis and is found to be an endothelial cell receptor for AGEs. To explore the binding characterization of AGEs to LOX-1, AGEs were prepared by three different reducing sugars (d-glucose, d-fructose, and d-ribose) and the biochemical characterization including, free amino groups, free amine content, fructosamine residues, carbonyl content, fluorescence, and absorbance were determined. The binding activity was determined by FITC labeled AGEs using Chinese hamster ovary-K1 cells stably transfected with human LOX-1 gene. The obtained AGEs showed significant differences in the extent of side chain modifications, carbonyl content, fluorescence, and absorption models. All of the AGEs showed specific and saturable binding to hLOX-1-CHO-K1 cells. Furthermore, dose-dependent binding processes were observed. However, the maximal cellular binding of AGEs differs between the sugars (glucose > ribose > fructose). In addition, oxidized low-density lipoprotein (ox-LDL) could significantly inhibit the binding of AGEs to LOX-1 with different inhibitory efficiency. LOX-1 serves as receptor for AGEs which may give some insight into the role of LOX-1 in the pathogenesis of diabetes and related disorders.     

CYTOGENETIC STUDIES IN THE GENUS TRIBOLIUM (POACEAE: ARUNDINEAE)

The genus Tribolium Desv. consists of nine species, i.e., T. utriculosum (Nees) Renv., T. ciliare (Stapf) Renv., T. echinatum (Thunb.) Desv., T. hispidum (Thunb.) Desv., T. acutiflorum (Nees) Renv., T. obliterum sensu Davidse, T. glomeratum sensu Davidse, T. uniolae (L.f.) Renv., and T. brachystachyum (Nees) Renv. The genus has a basic chromosome number of 6, and from diploid to hexaploid specimens have been examined. Precocious segregation of metaphase I bivalents were observed in four species. Multivalent formation results in unequal chromosome segregation during anaphase I, and several cells with an 11–13 chromosome distribution have been observed. The presence of univalents and anaphase I bridges in all T. brachystachyum specimens suggests a hybrid origin for the species. B-chromosomes were present in specimens from four species. The B-chromosomes are similar to the euchromosomes with the exception that they do not participate in meiosis. The B-chromosomes have a possible isochromosome origin. The cytogenetic evidence presented supports the combination of Plagiochloa and Lasiochloa into Tribolium and indicates that the genus is closely related to Urochlaena, whereas it is not closely related to Prionanthium.     

Changes of Photosystem Ⅱ Electron Transport in the Chlorophyll-deficient Oilseed Rape Mutant Studied by Chlorophyll Fluorescence and Thermoluminescence

The photosystem Ⅱ (PSII) complex of photosynthetic membranes comprises a number of chlorophyll-binding proteins that are important to the electron flow. Here we report that the chlorophyll b-deficient mutant has de creased the amount of light-harvesting complexes with an increased amount of some core polypeptides of PSII,including CP43 and CP47. By means of chlorophyll fluorescence and thermoluminescence, we found that the ratio of Fv/Fm, qP and electron transport rate in the chlorophyll b-deficient mutant was higher compared to the wild type.In the chlorophyll b-deficient mutant, the decay of the primary electron acceptor quinones (QA-) reoxidation was decreased, measured by the fluorescence. Furthermore, the thermolumlnescence studies in the chlorophyll b deficient mutant showed that the B band (S2/S3QB-) decreased slightly and shifted up towards higher temperatures.In the presence of dichlorophenyl-dimethylurea, which is inhibited in the electron flow to the second electron acceptor quinines (QB) at the PSII acceptor side, the maximum of the Q band (S2QA-) was decreased slightly and shifted down to lower temperatures, compared to the wild type. Thus, the electron flow within PSll of the chlorophyll b-deficient mutant was down-regulated and characterized by faster oxidation of the primary electron acceptor quinine QA- via forward electron flow and slower reduction of the oxidation S states.     

Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4°C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 10⁹ CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.     

Adenosine diphosphate ribosylation factor-GTPase-activating protein stimulates the transport of AUX1 endosome, which relies on actin cytoskeletal organization in rice root development

Polar auxin transport, which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers, mediates various processes of plant growth and development. Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein, GNOM. However, the mediation of auxin influx carrier recycling is poorly understood. Here, we report that overexpression of OsAGAP, an ARF-GTPase-activating protein in rice, stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1. AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption. Furthermore, OsAGAP is involved in actin cytoskeletal organization, and its overexpression tends to reduce the thickness and bundling of actin filaments. Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells, and was only slightly promoted when the actin filaments were completely disrupted by Lat B. Thus, we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.     

Cloning of the flap endonuclease-1 gene in Bombyx mori and identification of an antiapoptotic function

The flap endonuclease-1 (FEN-1) gene is involved in DNA replication and repair, and it maintains genomic stability as well as the accuracy of DNA replication under normal growth conditions. However, FEN-1 also plays an important role in apoptosis and cancer development. We cloned the BmFEN-1 gene from Bombyx mori, which was 1343?bp in length and possessed an 1143?bp ORF (123-1266). It consists of seven introns and eight exons that encode a protein with 380 amino acids that has the typical XPG domain. The N-terminal motif is located at amino acids 95-105, and the proliferating cell nuclear antigen interaction motif is located at amino acids 337-344. RNA interference-mediated reduction of BmFEN-1 expression induced cell cycle arrest in S phase in BmE-SWU1?cells. These results suggest that BmFEN-1 can inhibit apoptosis and promote cell proliferation.     

Changes in plasma inbulin related to the type of dietary carbohydrate in overweight hyperlipidemic male patients

Five patients with mild diabetes mellitus or angina pectoris were studied, each of whom consumed two sequential diets containing 40 to 50% carbohydrate. When 75% of the dietary carbohydrate was derived from food containing polysaccharides, the mean plasma insulin response to oral glucose was decreased relative to that seen following complementary diets providing carbohydrate mainly as simple sugars. Under both dietary conditions, glucose tolerance was improved compared to that seen prior to the study. These results suggest that with sugar restriction an improvement in the efficiency of the pancreatic beta-cell mechanism occurs. This effect may have been mediated by changes in the rate of secretion of pancreatic glucagon, or by an enteric secretogogue of insulin.     

Phylogenetic analysis of chinese rhesus macaques (Macaca mulatta) based on mitochondrial control region sequences

Between one and six subspecies of Chinese rhesus macaques (Macaca mulatta) have been proposed based on morphological differences and/or their geographic distribution. In this study, a 489 base pair fragment of the mitochondrial control region was amplified from 230 DNA samples collected from rhesus macaques in the Sichuan province in Western China. The fragment was then sequenced and aligned with 208 sequences from wild rhesus macaques, sampled throughout the species' geographic range in China downloaded from GenBank. Phylogenetic analysis of the 182 unique sequences identified among these samples divided Chinese rhesus macaques into two western haplogroups (haplogroups A and B) and three older eastern haplogroups (haplogroups C, D, and E), whose differentiation probably occurred during the penultimate glacial event. During the warming after the penultimate glacial event, haplogroups A, B, and E rapidly expanded and a relatively young subhaplogroup of haplogroup E, E', limited to Southern China but shared with Vietnamese rhesus macaques, was reintroduced from Indochina during the last glacial event. One haplotype most closely related to subhaplogroup E' probably represents the isolation of Hainan Island, to where it is restricted, from the mainland by the formation of the Qiongzhou Strait approximately 8,500 years ago. The distribution of haplogroups both informs the phylogeographic history of dispersal of Chinese rhesus macaques and has implications for their suitability as animal models in biomedical research.