Structure analysis of CCR5 from human and primates

It is well known that the chemokine receptor CCR5 plays very important roles in HIV-1 virus infection. A three-dimensional molecular model of human CCR5 was generated by SYBYL, a distance geometry-based homologous modeling package, using the corresponding transmembrane domain of bacteriorhodopsin as the template. On the basis of human CCR5 model, we also built 18 3D molecular models of CCR5 in primates from Pongo pygmaeus, Pygathrix nemaeus, Macaca assameniss, Trachy-pithecus phayrei, T. francoisi, M. arotoides, Rhinopithecus roxellance, R, bieti, R. avunculus, Hylobates leucogenys, Pan troglodytes, Gorilla gorilla, Cercopithecus aethiops 1, C. aethiops 2, Papio hamadryas M. mulatta, M. fascicularis and M. nemestrina. Structural analyses and statistics results suggested that the main-chains of the primate CCR5 were similar to that of the human CCR5 and that the fit-RMS deviation values of these primate CCR5 were less than 0.1 Angstrom. Moreover, the structures of these CCR5 proteins, except those of the African green monkey 1 (C.aet1), do not have a remarkable difference. It is proved that the 14th residue is possibly very important in the inhibition infections by M-tropic HIV-1, and it is also demonstrated that the 13th residue of human CCR5 was changed from asparagine into aspartic acid in all these primates. It means that the primate CCR5 no longer depend on CD4 for efficient entry, but human CCR5 may have evolved subsequently due to the use of CD4 as a receptor, allowing the high-affinity chemokine receptor-binding site of HIV to be sequestered from host immune surveillance. (C) 2000 Elsevier Science B.V. All rights reserved.

Pharmacokinetics of sifuvirtide, a novel anti-HIV-1 peptide, in monkeys and its inhibitory concentration in vitro

Aim: To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus (HIV) peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion. Methods: Monkeys received 1.2 mg/kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry (SPELC/MS/MS) was established and applied to determine the concentration of sifuvirtide in monkey plasma. A four-I-127 iodinated peptide was used as an internal standard. Fifty percent inhibitory concentration (IC50) of sifuvirtide on cell fusion was determined by co-cultivation assay. Results: The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88-5000 mu g/L, with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10626 +/- 2886 mu g/L and 528 +/- 191 mu g/L, and the terminal elimination half-lives (T,12) were 6.3 +/- 0.9 h and 5.5 +/- 1.0 h, respectively. After sc, T-max was 0.25-2 h, and the absolute bioavailability was 49% +/- 13%. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC50 of 0.33 mu g/L. Conclusion: An on-line SPE-LC/MS/MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T-1/2 of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC50 in vitro.

Changes of urinary steroid conjugates and gonadotropin excretion in the menstrual cycle and pregnancy in the Yunnan snub-nosed monkey (Rhinopithecus bieti)

The Yunnan snub-nosed monkey (Rhinopithecus bieti) is one of the most endangered species in the world, and it is endemic to China. According to our knowledge, there was no information on reproduction for this species. The present study was designed to und

Comparative Expression of Zebrafish Lats1 and Lats2 and Their Implication in Gastrulation Movements

Large tumor suppressor (Lats) is a Ser/Thr kinase, and it presents an important function in tumor suppression. lats was originally identified in Drosophila and recently in mammals. In mammals, it contains two homologues, lats1 and lats2. In the present study, lats1 and lats2 were characterized from zebrafish (Danio rerio), which is the first report of lats in a nonmammalian vertebrate. The primary structure, genomic organization, and phylogenesis of lats from different species were studied, and the results suggest that lats1 is the direct descendant of invertebrate lats, whereas lats2 is formed by genome duplication. In zebrafish, both lats genes are maternally expressed, while they show distinctly different expression profiles during gastrulation. lats1 is almost ubiquitously expressed through development, and lats2 is more prominently expressed in the non-neural ectoderm region of zebrafish gastrula. Most intriguingly, as revealed by cell tracing and gene expression analysis, morpholino-mediated knockdown of either lats1 or lats2 led to obvious defects of cell migration in gastrulation, indicating the functional significance of lats in gastrulation movements. Developmental Dynamics 238:28502859, 2009. (C) 2009 Wiley-Liss, Inc.

Effect of beta-glucan on activity of antioxidant enzymes and Mx gene expression in virus infected grass carp

The effects of beta-glucan, an immunostimulatory agent, on the superoxide dismutase (SOD) and catalase (CAT) activities of erythrocytes and Mx gene expression were studied from grass carp that were challenged with grass carp hemorrhage virus (GCHV). The SOD and CAT activities in erythrocytes and Mx gene expression in spleen from the fish were detected by spectrophotometry and RT-PCR, respectively. Negative control fish were injected with PBS; positive control groups were injected with either P-glucan or GCHV only; and the experimental groups were pre-injected with beta-glucan 15 days prior to injection with GCHV. The results show that the SOD and CAT activities were higher in fish injected with beta-glucan for 15 days than the negative control group injected with PBS. The SOD and CATactivities significantly decreased when the fish were challenged with GCHV, but it was higher in the group pre-treated with beta-glucan than in infected fish not pre-treated, 15 days after GCHV infection. Mx gene expression levels increased during the early stages (at 12 h and 36 h) of GCHV infection, and it remained at higher levels from the 6th till the 10th day in the beta-glucan pre-treated group, but it was failing from the 6th day in the beta-glucan untreated group. The GCHV-infected group pre-treated with P-glucan had a higher survival rate (60%) than the group not pre-treated with P-glucan (20%), suggesting that beta-glucan possesses or enhances anti-viral responses. (C) 2009 Elsevier Ltd. All rights reserved.

Genome-wide identification and characterization of cytochrome P450 monooxygenase genes in the ciliate Tetrahymena thermophila

Background: Cytochrome P450 monooxygenases play key roles in the metabolism of a wide variety of substrates and they are closely associated with endocellular physiological processes or detoxification metabolism under environmental exposure. To date, however, none has been systematically characterized in the phylum Ciliophora. T. thermophila possess many advantages as a eukaryotic model organism and it exhibits rapid and sensitive responses to xenobiotics, making it an ideal model system to study the evolutionary and functional diversity of the P450 monooxygenase gene family. Results: A total of 44 putative functional cytochrome P450 genes were identified and could be classified into 13 families and 21 sub-families according to standard nomenclature. The characteristics of both the conserved intron-exon organization and scaffold localization of tandem repeats within each P450 family clade suggested that the enlargement of T. thermophila P450 families probably resulted from recent separate small duplication events. Gene expression patterns of all T. thermophila P450s during three important cell physiological stages (vegetative growth, starvation and conjugation) were analyzed based on EST and microarray data, and three main categories of expression patterns were postulated. Evolutionary analysis including codon usage preference, sit-especific selection and gene-expression evolution patterns were investigated and the results indicated remarkable divergences among the T. thermophila P450 genes. Conclusion: The characterization, expression and evolutionary analysis of T. thermophila P450 monooxygenase genes in the current study provides useful information for understanding the characteristics and diversities of the P450 genes in the Ciliophora, and provides the baseline for functional analyses of individual P450 isoforms in this model ciliate species.

Effects of sediment anoxia and light on turion germination and early growth of Potamogeton crispus

Potamogeton crispus is a cosmopolitan aquatic species and is widely used as a pioneer species for vegetation restoration of eutrophic lakes. However, many restoration projects applying P. crispus turions have not been successful. Earlier studies focused on effects of light and temperature on turion germination. The purpose of this study was to determine whether sediment anoxia and light interactively affected the turion germination and early growth of P. crispus. Anoxic conditions in the experiment were produced by adding sucrose to the sediment. The germination rate of the turions was 68-73% lower in the highly anoxic condition treatment than in the control. Medium light intensity (10% of natural light at the water surface) was more favorable for germination under slightly anoxic conditions than either low or high light intensity. The growth of newly-formed sprouts was also significantly inhibited by sediment anoxia. Photosynthesis and shoot biomass were reduced under sediment anoxia, whereas total chlorophyll content, root biomass, and soluble protein content were highest in the low anoxic condition treatment. Medium light improved net photosynthesis and biomass production of the sprouts. We conclude that turion germination and sprout growth can be significantly inhibited by sediment anoxia. Medium light intensity may alleviate this inhibition by anoxia, but light has little effect when sediment anoxia is severe. For the purposes of vegetation restoration, more attention should be paid to the role of sediment anoxia, and it is necessary to improve sediment and light conditions for turion germination and early growth of P. crispus in eutrophic lakes. These results will contribute to a more complete understanding of turion germination dynamics of P. crispus and will be useful for future restoration programs.

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA Library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full Length cDNA of carp SLP-76 was 2007 bp, consisting of a T-terminal untranslated region (UTR) of 285 bp, a T-terminal. UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homotogues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2 k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts. (c) 2007 Published by Elsevier Ltd.

Origin of Gymnocypris przewalskii and phylogenetic history of Gymnocypris eckloni (Teleostei : Cyprinidae)

The origins and phylogenetic patterns were assessed for G. przewalskii and G. eckloni by analyzing the complete mtDNA cytochrome b gene sequence (1140bp). Phylogenetic analyses further supported that there were three mtDNA lineages (A-C) identified in G. przewalskii and G. eckloni, demonstrating that outer rakers of the first gill have little significance in the phylogeny of the Gymnocypris fishes. The network established showed that G. eckloni of the Yellow River specific haplotype A1 was a founder and it radiated all haplotypes of G. przewalskii which suggested G. przewalskii might only originate from one of two maternals of G. eckloni from the Yellow River. Fs test and mismatch analysis showed at least two expansion events in the population of G. przewalskii about 0.2734 Ma and 0.0658 Ma, while G. eckloni from Qaidam Basin could have experienced severe bottleneck effect about 0.0693 Ma. The population expansion was detected in subclades A1 and A21 with the most recent common ancestor (TMRCA) about 0.2308 +/- 0.01 Ma and 0.1319 +/- 0.015 Ma, respectively, which were within the geological age range of "Gonghe Movement" event that caused the separation of Lake Qinghai from the upper Yellow River. These results suggested the effect of the fish diversification by rapid uplift of the Qinghai-Tibetan Plateau in the Late Pleistocene.

Relationships between the biomass of algal crusts in fields and their compressive strength

In the desert areas of China investigated by the authors, various biological crusts were predominately associated with three blue-green algal (cyano bacterial) species, Microcoleus vaginatus Gom., Phormidium tenue (Menegh.) Gom. and Seytonema javanicum (Mitz.) Born et Flah. Their biomass and their compressive strength were measured simultaneously in the field in this study. It was also found that the compressive strength of algal crusts was enhanced with the increasing of algal biomass from an undetectable level to a value as high as 9.6mg g(-1) dry soil. However, when the algal biomass decreased, the compressive strength did not descend immediately, but remained relatively steady. The higher the algal biomass became, the thicker were the algal crusts formed. Given the same biomass, the highest compressive strength of man-made algal crusts in fields was found at an algal ratio of 62.5% M. vaginatus, 31.25% P. tenue and 6.25% S. javanicum, and it reached 0.89kgcm(-2). When the biomass of the crusts increased above the value of 8.16 mg chl ag(-1) dry soil, the compressive strength would not ascend easily. It indicated that the compressive strength of man-made algal crusts appeared temporarily saturated in the field. (c) 2006 Elsevier Ltd. All rights reserved.

Changes in the patterns of inorganic nitrogen and TN/TP ratio and the associated mechanism of biological regulation in the shallow lakes of the middle and lower reaches of the Yangtze River

The changes of NH3-N, NO3-N, NO2-N and TN/TP were studied during growth and non-growth season in 33 subtropical shallow lakes in the middle and lower reaches of the Yangtze River. There were significant positive correlations among all nutrient concentrations, and the correlations were better in growth season than in non-growth season. When TP > 0.1 mgL(-1), NH3-N increased sharply in non-growth season with increasing TP, and NO3-N increased in growth season but decreased in non-growth season with TP. These might be attributed to lower dissolved oxygen and low temperature in non-growth season of the hypereutrophic lakes, since nitrification is more sensitive to dissolved oxygen and temperature than anti nitrification. When 0.1 mgL(-1)> TP > 0.035 mgL(-1), TN and all kinds of inorganic nitrogen were lower in growth season than in non-growth season, and phytoplankton might be the vital regulating factor. When TP < 0.035 mgL(-1), inorganic nitrogen concentrations were relatively low and NH3-N, NO2-N had significant correlations with phytoplankton, indicating that NH3-N and NO2-N might be limiting factors to phytoplankton. In addition, TN/TP went down with decline in TIP concentration, and TN and inorganic nitrogen concentrations were obviously lower in growth season than in non-growth season, suggesting that decreasing nitrogen (especially NH3-N and NO3-N) was an important reason for the decreasing TN/TP in growth season. The ranges of TN/TP were closely related to trophic level in both growth and non-growth seasons, and it is apparent that in the eutrophic and hypertrophic state the TN/TP ratio was obviously lower in growth season than in non-growth season. The changes of the TN/TP ratio were closely correlated with trophic levels, and both declines of TN in the water column and TP release from the sediment were important factors for the decline of the TN/TP ratio in growth season.

Effects of solar UV radiation on morphology and photosynthesis of filamentous cyanobacterium Arthrospira platensis

To study the impact of solar UV radiation (UVR) (280 to 400 nm) on the filamentous cyanobacterium Arthrospira (Spirulina) platensis, we examined the morphological changes and photosynthetic performance using an indoor-grown strain (which had not been exposed to sunlight for decades) and an outdoor-grown strain (which had been grown under sunlight for decades) while they were cultured with three solar radiation treatments: PAB (photosynthetically active radiation [PAR] plus UVR; 280 to 700 nm), PA (PAR plus UV-A; 320 to 700 nm), and P (PAR only; 400 to 700 nm). Solar UVR broke the spiral filaments of A. platensis exposed to full solar radiation in short-term low-cell-density cultures. This breakage was observed after 2 h for the indoor strain but after 4 to 6 h for the outdoor strain. Filament breakage also occurred in the cultures exposed to PAR alone; however, the extent of breakage was less than that observed for filaments exposed to full solar radiation. The spiral filaments broke and compressed when high-cell-density cultures were exposed to full solar radiation during long-term experiments. When UV-B was screened off, the filaments initially broke, but they elongated and became loosely arranged later (i.e., there were fewer spirals per unit of filament length). When UVR was filtered out, the spiral structure hardly broke or became looser. Photosynthetic 0, evolution in the presence of UVR was significantly suppressed in the indoor strain compared to the outdoor strain. UVR-induced inhibition increased with exposure time, and it was significantly lower in the outdoor strain. The concentration of UV-absorbing compounds was low in both strains, and there was no significant change in the amount regardless of the radiation treatment, suggesting that these compounds were not effectively used as protection against solar UVR. Self-shading, on the other hand, produced by compression of the spirals over adaptive time scales, seems to play an important role in protecting this species against deleterious UVR. Our findings suggest that the increase in UV-B irradiance due to ozone depletion not only might affect photosynthesis but also might alter the morphological development of filamentous cyanobacteria during acclimation or over adaptive time scales.

Molecular cloning of the viperin gene and its promoter region from the mandarin fish Siniperca chuatsi

A viperin gene has been cloned from the mandarin fish (Siniperca chuatsi). From the first transcription initiation site, the mandarin fish viperin gene extends 3163 nucleotides to the end of the 3' untranslated region, and it contains six exons and five introns. The open reading frame of the viperin transcript has 1062 nucleotides which encode a 354 amino acid peptide. The amino acid sequence of mandarin fish viperin shows high identities with its homologues in teleosts and mammals except for the first 70 amino acids. A characteristic feature in the viperin promoter region was the presence of five putative ICSBP (IRF8) binding sites and one IRFI binding site. The viperin gene expressed mainly in lymphoid tissues before stimulation, but its expression can be examined in almost all the organs investigated after stimulation with virus or Poly I:C. The expression pattern and promoter sequence may be considered as the indirect evidence that the transcription of viperin is regulated by interferons or interferon induced genes. (C) 2004 Elsevier B.V. All rights reserved.

Differential expression of thyroid-stimulating hormone beta subunit in gonads during sex reversal of orange-spotted and red-spotted groupers

We have cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone beta-subunit (TSHbeta) from orange-spotted grouper Epinephelus coioides. It contains 913 nucleotides with an open reading frame encoding 146 amino acids with a 20 amino acid signal peptide. The grouper mature TSHbeta has 75, 70, 61, 59, 41, 42 and 40% identities to that of rainbow trout, Atlantic salmon, zebrafish, European eel, chicken. mouse and human, respectively. RT-PCR analysis indicated that the TSHbeta mRNA was expressed abundantly not only in pituitary but also in gonads. A more interesting finding is to reveal the differential TSHbeta expressions between the ovaries and the transitional gonads or testes in natural individuals of orange-spotted grouper and red-spotted grouper Epinephelus akaara, and in artificial sex reversal individuals of red-spotted grouper induced by MT feeding. In situ hybridization localization provided direct evidence that the TSHbeta was transcribed in the germ cells. In the growing oocytes, the TSHbeta transcripts were concentrated on the ooplasm periphery. In testicular tissues, the intensively expressed TSHbeta cells were found to be spermatogonia and spermatocytes in the spermatogenic cysts. This is the first report of a TSHbeta expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary, and it expresses more transcripts in the gonads during sex reversal or testis than in the ovaries both in E. coioides and E. akaara. Importantly, the TSHbeta identification in germ cells allows us to further investigate the functional roles and the molecular mechanisms in gametogenesis of groupers, especially in sex reversal and in spermatogenesis. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Rapid establishment of pure lines of silver carp (Hypophthalmichthys molirix) and bighead carp (Aristichthys nobilis)

The diversity of gynogenetic, artificial sex reversal and natural silver carp and bighead carp is examined using randomly amplified polymorphic DNA (RAPD) method. All of the 187 bands are obtained and 19 (10.16%) of them are polymorphic in gynogenetic silver carp. Meanwhile 32 (15.61%) out of 205 bands are polymorphic in control group. In gynogenetic bighead carp a total of 232 bands are identified and 11 (4.74%) out of them are polymorphic, while 25 (10.37%) out of 241 bands are polymorphic in control group. The genetic distance of four populations is calculated and it is 0.102 and 0.023 for gynogenetic silver carp and gynogenetic bighead carp respectively. The values of natural silver carp and bighead carp are 0.161 and 0.104. From the UPGMA trees constructed based on genetic distance, the sex reversal individuals that match with the gynogenetic female individuals are picked out. A new breeding process of establishing a pure line is developed.