Compositional analysis of the fruiting body of transgenic Flammulina velutipes producing resveratrol.

Two strains of transgenic Flammulina velutipes TF71 and TF7H with 4cl and rs genes with the capability to produce resveratrol were obtained. In the nutrition assessment and analysis of transgenic strain and original strain (F7), the amino acid composition and proximate compositions of fruiting bodies were determined by amino acid automatic instrument and standard methods of the Association of Official Analytical Chemists (AOAC), while 4-coumaric acid, total flavonoids, and resveratrol were extracted by ethyl acetate and quantified by HPLC. Results indicated that significant differences were observed in proximate composition and amino acid between transgenic and original strains, but these detected components were within the normal ranges reported for F. velutipes. Total flavonoids and 4-coumaric acid contents of transgenic strains are lower than F7. Most important of all, resveratrol has been detected from TF71 and TF7H fruiting body but not found in F7, which was firstly produced by a transgenic mushroom.

Bioproduction of baccatin III, an advanced precursor of paclitaxol, with transgenic Flammulina velutipes expressing the 10-deacetylbaccatin III-10-O-acetyl transferase gene.

BACKGROUND: 10-Deacetylbaccatin III (10-DAB) and baccatin III are intermediates in the biosynthesis of Taxol (an anti-cancer drug) and useful precursors for semi-synthesis of the drug. In this study, a bioconversion system was established for the production of baccatin III, an advanced precursor of paclitaxel, in the transgenic mushroom Flammulina velutipes expressing the 10-deacetylbaccatin III-10ß-O-acetyltransferase gene. The expression vector pgFvs-TcDBAT containing the 10-deacetylbaccatin III-10ß-O-acetyltransferase (DBAT) gene was constructed and transformed into the cells of F. velutipes by polyethylene glycol-mediated protoplast transformation. RESULTS: Polymerase chain reaction and Southern blotting analysis verified the successful integration of the exogenous DBAT gene into the genome of F. velutipes. Reverse transcription polymerase chain reaction and enzyme activity analyses confirmed that the DBAT gene was expressed in F. velutipes, and DBAT is able to convert substrate into baccatin III. CONCLUSION: The DBAT gene from the plant Taxus chinensis can be functionally expressed in F. velutipes. Transgenic F. velutipes expressing the DBAT gene is able to produce the target product, baccatin III. This is the first report about the transformation and expression of paclitaxel biosynthetic gene in the edible mushroom F. velutipes. This represents a significant step towards bio-production of paclitaxel and its advanced precursor baccatin III in an edible fungus.

Cloning and characterization of squalene synthase gene from Poria cocos and its up-regulation by methyl jasmonate.

Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate to give presqualene diphosphate and the subsequent rearrangement to form squalene. The gene encoding squalene synthase was cloned from Poria cocos by degenerate PCR and inverse PCR. The open reading frame of the gene is 1,497 bp, which encodes 499 amino acid residues. A phylogenetic analysis revealed that P. cocos SQS belonged to the fungus group, and was more closely related to the SQS of Ganoderma lucidum than other fungi. The treatment of P. cocos with methyl jasmonate (MeJA) significantly enhanced the transcriptional level of P. cocos sqs gene and the content of squalene in P. cocos. The transcriptional level of sqs gene was approximately fourfold higher than the control sample and the squalene content reached 128.62 µg/g, when the concentration of MeJA was 300 µM after 72 h induction.

Evaluation of Burma Reed as Substrate for Production of Pleurotus eryngii.

Burma reed (Neyraudia reynaudiana), a giant C4 grass, was included in substrate at the rates of 0, 20, 40 and 66 % to partially or wholly substitute sawdust and cottonseed hulls to evaluate its suitability for Pleurotus eryngii cultivation. Inclusion of 20 and 40 % Burma reed did not significantly affect linear mycelial growth, dry matter loss, spawn run period and fructification, and achieved high fruiting body yields and biological efficiency of 336.67 g/bag, 67.33 % and 342.15 g/bag, 68.43 %, respectively, which were not significantly different from 350.08 g/bag to 70.02 % obtained from the control substrate. Enzyme assay revealed that on the mixed substrates laccase and manganese peroxidase activity were significantly enhanced, but cellulase was significantly reduced in the middle stage of incubation as compared with the control substrate. Even on Burma reed substrate without sawdust and cottonseed hulls, fruiting body yield (313.56 g/bag) and biological efficiency (62.71 %) were satisfactory, although significantly lower than that on the control substrate. Therefore, Burma reed was a promising potential substrate for P. eryngii production to largely substitute sawdust and cottonseed hulls.

[ATP-dependent chromatin remodeling complex and its function in regulating chromatin structure].

The highly condensed chromatin prevents binding of transcription factors and cofactors to DNA. Therefore, it is crucial to relief the repressive environment for transcription through chromatin remodeling activities. Recently, it is widely accepted that chromatin remodeling is carried out by at least two mechanisms: ATP-dependent chromatin remodeling complex and covalent modifications of histone tails by histone modification complexes. This paper reviews the mechanisms of chromatin remodeling through ATP-dependent chromatin remodeling complex and how the two complexes work together to modify the chromatin structure and regulate the function of transcription mechanism according to recent studies.

Improvement in fruiting body yield by introduction of the Ampullaria crossean multi-functional cellulase gene into Volvariella volvacea.

The multi-functional cellulase gene, mfc, from Ampullaria crossean was transformed into Volvariella volvacea by PEG-mediated protoplast transformation to improve the biological efficiency and fruiting body yield. PCR and Southern blotting indicated that mfc was integrated into the genomes of four transformants. In laboratory and large scale cultivation experiments, the average biological efficiency of the transformants was 18.39+/-1.27% and 27.84+/-3.21%, respectively, considerably higher than the corresponding values for untransformed controls of 12.69+/-1.31% and 20.63+/-2.59%. This is the first report of an improvement in biological efficiency and fruiting body yield of V. volvacea through transgenesis.

Purified polysaccharide from Ginkgo biloba leaves inhibits P-selectin-mediated leucocyte adhesion and inflammation.

AIM: To investigate the anti-inflammatory mechanism of the polysaccharides of Ginkgo biloba leaves (PGBL) by inhibiting leucocyte adhesion. METHODS: The rough PGBL were isolated and purified. The anti-inflammatory effects of purified PGBL (p-PGBL) were assayed by ear edema induced by xylol and the acute peritonitis model in mice. The effect of p-PGBL on inhibiting the interaction between P-selectin and its ligands was investigated by flow cytometry and flow chamber. RESULTS: p-PGBL could effectively inhibit the acute inflammation in mice and interfere with the adhesion of HL-60 cells, a human leukaemia cell line, or neutrophils to P-selectin in static conditions, as well as the adhesion of neutrophils to Chinese hamster ovary cells expressing human P-selectin and human umbilical vein endothelial cells in flow conditions in a dose-dependant manner. CONCLUSIONS: p-PGBL can inhibit the inflammatory process through interfering with the interaction between P-selectin and its ligands.

[The relationship between abnormity of synaptonemal complex and male fertility impairment in human].

It was said that 11% of all men showed their infertility. The genetic origins of male infertility may be classified into three main groups: chromosome abnormalities, microdeletions and gene mutations. Growing literature has shown that spermatogenesis failure or reproductive failure (pregnancy wastages) occurred in male carriers of chromosomal distortion/aberration. But the mechanisms remain largely unsolved. Synaptonemal complex (SC) of human spermatocytes from such carriers offers new information. This review aims to summarize recent development of SC analysis for male infertile diagnosis and sum up our results obtained recently. The relationship between male infertility/sterility and SC abnormity was discussed and reviewed as following five aspects. (1) The association of XY-bivalent and the rearranged autosomes interfere with or affect, by their contact, X chromosome normal functions. (2) Male infertility is related to the incomplete pairing in break regions of rearranged autosomes. (3) SC fragmentation, lateral elements (LEs) swelling and pairing disorder result in spermatogenic failure. (4) This heterosynapsis at early stage of meiosis in rearranged autosomes, results in unbalanced germs and pregnancy wastages. (5) Gene mutations of SC proteins result in male infertility.

Engagement of PSGL-1 upregulates CSF-1 transcription via a mechanism that may involve Syk.

PSGL-1, the optimal selectin ligand demonstrated by in vivo studies to date, is an essential adhesive molecule mediating the rolling of leukocytes on the endothelial cells and the recruitment of leukocytes to the inflamed tissue. Recent studies demonstrated, in addition to its direct role in capture of leukocytes from the bloodstream, PSGL-1 also functions as a signal-transducing receptor and initiates a series of intracellular signal events during the activation of leukocytes. Our present work showed antibody engagement of PSGL-1 upregulated the transcriptional activity of CSF-1 promotor and increased the endogenous expression of CSF-1 mRNA in Jurkat cell. Overexpression of wild-typed non-receptor tyrosine kinase Syk, but not kinase dead mutant of Syk, promoted the upregulation of the transcriptional activity of CSF-1 promoter caused by antibody engagement of PSGL-1. Additionally, special inhibitor of Syk Piceatannol suppressed the increase of CSF-1 mRNA caused by the engagement of PSGL-1. The results suggest that signal transducted by PSGL-1 upregulate the transcriptional activity of CSF-1, and non-receptor tyrosine kinase Syk participates in this pathway.

BAF complex is closely related to and interacts with NF1/CTF and RNA polymerase II in gene transcriptional activation.

Brg- or hBrm-associated factor (BAF) complexes, a chromatin-remodeling complex family of mammalian cells, facilitate transcriptional activity by remodeling nucleosome structure. Brg1 is the core subunit of Brg-associated factor complexes. In the present study, we investigated the spatial relationship between Brg1 and nuclear factor 1 (NF1/CTF) and RNA polymerase II (RNAP II) upon gene transcriptional activation in vivo by employing immuno-gold labeling. The data showed that Brg1 was closely co-localized with NF1/CTF and RNAP II in HeLa cells. Moreover, the co-immunoprecipitation assay further revealed that Brg1 can be isolated together with NF1/CTF and RNAP II in the ConA-stimulated, but not the resting, T lymphocyte. The combined results suggested that BAF complexes can interact with NF1/CTF and RNAP II, and this interaction is closely dependent on the activation of gene transcription.

[Analysis of synaptonemal complex from a carrier with 46,XY,t(11;18) balanced translocation].

Over ten years ago, two male patients of pregnancy wastage, who were the carriers of 4;6 and 4;13 reciprocal translocations, respectively, were analyzed with synaptonemal complexes (SCs) by de Perdigo et al. They suggested that the pregnancy wastage should be caused by heterosynapsis, which reciprocal translocations resulted in. The heterosynapsis might avoid spermatogenesis failure, but easily induced non-disjunction of some chromosomes and led to occurrence of unbalanced gametes. A new male patient of the pregnancy wastage was detected in China three years ago. Karyotyping of the patient showed 11;18 reciprocal translocation. Analysis of synaptonemal complexes (SCs) of the patient was performed using the EM technology of SC surface spreading, SDS treatment, AgNO3 staining. The SCs of 30 spermatocytes at pachytenes were analyzed. The SCs in electron micrographs were measured by the method published by de Perdigo et al. (1991). The results showed that each of the observed spermatocytes displayed 20 autosomal bivalents, a quadrivalent 11;18 and a sexual bivalent. Of the 30 spermatocytes, 21 each exhibited a quadrivalent of adequate quality for measurement and interpretation. The 21 quadrivalents were classified into three types: type I, quadrivalents with fully paired arms with the expected cross-configuration, two quadrivalents were of this type. Type II, quadrivalents with an evident heterosynapsis, forming five SC segments (four fully pairing arms and one middle pairing region), six quadrivalents were of this type. Type III, quadrivalents with four paired arms, but with asynapsed regions around the breakpoints, thirteen quadrivalents were of this type. Based on configuration, quadrivalents were classified into cis-configuration with above three types and trans-configuration with above two types except type I. Each of the analyzed quadrivalents showed four synaptic arms, which respectively paired between chromosome 11 and t18 (the translocated 18;11), 18 and t18, 18 and t11 (the translocated 11;18), and 11 and t11, and the arms were designated as A, B, C, and D correspondingly. The measurements of A and D as well as B and C observed in different cells were taken by calculating the percentages of their lengths in comparison to the length of chromosome 11 and 18, respectively. So were the middle synaptic regions. Measurements of SCs revealed that the location of the putative breakpoints estimated from the fully paired quadrivalents were different from those determined by G-band analysis for mitotic chromosomes. The length of the paired and unpaired segments varied from one quadrivalent to another. For instance, in the case of fully paired A and D arms, the breakpoint was located near to 45.59% and 38.53% of the length of the normal chromosome 11, the breakpoint from the mitotic chromosome was estimated to be near to 45%. The location of the putative breakpoints estimated from the No. 1 quadrivalent was consistent with the breakpoint from the mitotic chromosome. This suggested that only this quadrivalent showed complete homologous pairing. Obviously, the lengths of middle pairing region in type II quadrivalent were different from one quadrivalent to another. The middle pairing region/the length of the normal chromosome 11 is 4.11% - 18%, that/the length of the normal chromosome 18 is 8% - 41.1%. The middle pairing regions should be the heterologous pairing regions in quadrivalents. The quadrivalents with unsynaptic regions also showed partially heterologous synapsis. In 14 of 21 quadrivalents, the paired regions overlapped the mitotic breakpoint position in chromosome 11. In 20 of 21 quadrivalents, the paired regions overlapped the chromosome 18 of mitotic breakpoint position in one or two arms. As synapsis can proceed homologously only up to the breakpoints, a heterosynapsis apparently occurred in 20 quadrivalents. The length of the paired and unpaired segments varied from one quadrivalent to another, and the measurements of the arms of the quadrivalents could not be used to determine breakpoints. The unpaired segment showed a thick and sometimes a split aspect similar to that of the sexual bivalent. Of the 20 quadrivalents with heterosynapsis, four were at early pachytene stages, fourteen at the middle and two at the late, respectively. The heterosynapsis, which occurred on the early pachytene, was without previous homosynapsis. This was different from the classical "synaptic adjustment" during the late pachrtene of spermatocytes. Many researches on reciprocal translocation associated with pregnancy wastage have been reported. It is most possible that the wastage detected in this study was related to the t(11;18). Normal synapsis finishes at early pachytene, while in the observed proband, most of the quadrivalents still left unsynaptic parts during middle and late pachytene stages. Obviously, the synapsis was delayed or incomplete. The unsynaptic regions distributed mainly in the central regions. The central regions perhaps completed the synapsis more late, therefore they had more chances to leave the unsynaptic parts. In addition, the results of SC analysis showed that heterosynapsis occurred wildly and was exhibited from early to late pachytene stages. It has been reported that crossing over could be inhibited by heterosynapsis in mice, man and boar, and normal segregation needs existence of crossing over. Therefore, heterosynapsis may interfere in normal segregation. Both unsynapsis and heterosynapsis could induce incidence of unbalanced gametes. In general, the unbalanced gametes are lethal and could not take part in fertilization, once they fertilize with normal eggs, the risk of pregnancy wastage or other genetic disorders will exist certainly. Our results showed that the heterosynapsis occurred widely, so a poor risk of pregnancy wastages caused by reciprocal translocation probably is not unusual.

[Synaptonemal complexes analysis in male fertility impairment in two men].

Synaptonemal complexes (SCs) of male fertility impairment were analysed in two patients. In case one, his testicular histology was normal and there were 20% abnormal pachytene spermatoeytes, showing unsynaptic and broken SCs. It resulted in spermatogensis with unbalanced chromosomes and pregnancy wastages. In case two, he had no sperms basically. G-banded chromosome analysis of lymphocytes showed normal chromosomal karyotype, but triple fragment, lateral element swelling and fragment breakages of SCs were observed in his spermatocytes. The mechanisms of infertility,impairment of fertility for the observed men were discussed in this paper.

[In situ distribution and configuration of rDNA in the nucleolus of Pisum sativum].

Distribution and configuration of nucleolar DNA in the nucleolus of pea (Pisum sativum) were analyzed using cytochemical NAMA-Ur DNA specific stain method. The results showed that nucleolar DNA distributed in FC and juncture of FC/DFC in the forms of surrounding FC. rDNA in different positions existed in either condensed or uncondensed forms. Nucleolus-associated chromatin outside nucleolus extended in the periphery of FC via channels in the nucleolus.

Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 O-glycans and of tyrosine sulfate residue 51.

Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at N-terminal Thr or Tyr residues. Simultaneous expression of core-2 beta1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptor-counterreceptor pair.

[Degenerate PCR and its application in gene cloning].

Degenerate PCR is introduced in this paper, including what is degenerate PCR, how to design degenerate primers, how to optimize degenerate PCR parameters, how to applying degenerate PCR to obtain full-length gene and which fields can apply degenerate PCR. The limits and recent advances of degenerate PCR are also discussed. Based on this introduction,strategies of gene cloning and applications of degenerate PCR in gene cloning are summarized in brief. Degenerate PCR is a very useful tool for searching and discovering new genes and new members of a protein family.