Hologenomic insights into mammalian adaptations to myrmecophagy.

Highly specialized myrmecophagy (ant- and termite-eating) has independently evolved multiple times in species of various mammalian orders and represents a textbook example of phenotypic evolutionary convergence. We explored the mechanisms involved in this unique dietary adaptation and convergence through multi-omic analyses, including analyses of host genomes and transcriptomes, as well as gut metagenomes, in combination with validating assays of key enzymes' activities, in the species of three mammalian orders (anteaters, echidnas and pangolins of the orders Xenarthra, Monotremata and Pholidota, respectively) and their relatives. We demonstrate the complex and diverse interactions between hosts and their symbiotic microbiota that have provided adaptive solutions for nutritional and detoxification challenges associated with high levels of protein and lipid metabolisms, trehalose degradation, and toxic substance detoxification. Interestingly, we also reveal their spatially complementary cooperation involved in degradation of ants' and termites' chitin exoskeletons. This study contributes new insights into the dietary evolution of mammals and the mechanisms involved in the coordination of physiological functions by animal hosts and their gut commensals.

Recent Advances in Genetics and Genomics of Snub-Nosed Monkeys (Rhinopithecus) and Their Implications for Phylogeny, Conservation, and Adaptation.

The snub-nosed monkey genus Rhinopithecus (Colobinae) comprises five species (Rhinopithecus roxellana, Rhinopithecus brelichi, Rhinopithecus bieti, Rhinopithecus strykeri, and Rhinopithecus avunculus). They are range-restricted species occurring only in small areas in China, Vietnam, and Myanmar. All extant species are listed as endangered or critically endangered by the International Union for Conservation of Nature (IUCN) Red List, all with decreasing populations. With the development of molecular genetics and the improvement and cost reduction in whole-genome sequencing, knowledge about evolutionary processes has improved largely in recent years. Here, we review recent major advances in snub-nosed monkey genetics and genomics and their impact on our understanding of the phylogeny, phylogeography, population genetic structure, landscape genetics, demographic history, and molecular mechanisms of adaptation to folivory and high altitudes in this primate genus. We further discuss future directions in this research field, in particular how genomic information can contribute to the conservation of snub-nosed monkeys.

Mutation Detection and Functional Analysis of MSX1, PAX9, AXIN2, and BMP in Nonsyndromic Congenital Missing Teeth Based on Intelligent Image Detection.

Due to the complexity of clinical manifestations and the lack of standardized diagnostic criteria, it is still difficult to distinguish the etiological types of congenital edentulousness corresponding to genetic defects. This paper studies the application of deep learning image processing and digital image processing in medical images in detail and analyzes the functions of congenital edentulous hotspot genes. The cases in the control group and the study group were collected, and the gene mutations of direct sequence MSX1, PAX9, AXIN2, and BMP were analyzed, and new pathogens were found. The experimental results suggest that PAX9 and MSX1 genes may have a synergistic effect in nonsyndromic congenital edentulous patients. In severely missing teeth, the role of PAX9 may be greater than that of MSX1. The experimental results will help us lay the foundation for further understanding of the disease in the future.

How Good Is Machine Learning in Predicting All-Cause 30-Day Hospital Readmission? Evidence From Administrative Data.

OBJECTIVES: Hospital readmission is a main cost driver for healthcare systems, but existing works often had poor or moderate predictive results. Although the available information differs in different studies, improving prediction is different from the search for important explanatory variables. With large sample size and abundant information, this study explores state-of-the-art machine-learning algorithms and shows their performance in prediction. METHODS: Using administrative data on 1 631 611 hospital stays from Quebec between 1995 and 2012, we predict the probability of 30-day readmission at hospital admission and discharge. We compare the performance between traditional logistic regression, logistic regression with penalization, and more recent machine-learning algorithms such as random forest, deep learning, and extreme gradient boosting. RESULTS: After a 10-fold cross-validation on the training set (80% of the data), machine learning produced very good results on a separate hold-out test set (20% of the data). The importance of explanatory variables is not the same for different algorithms. The area under receiver operating characteristic curve (AUC) reached above 0.79 at hospital admission and above 0.88 at hospital discharge. Diagnostic codes, which include many different categories, are among the most predictive variables. Logistic regression with penalization also produced good results, but a standard logistic regression failed without penalization. The good results are confirmed by calibration curves. CONCLUSION: Although the identification of those at highest risk of readmission is just 1 step to preventing hospital readmissions, 30-day readmission is highly predictable with machine learning.

Transcriptomic profiling of purple broccoli reveals light-induced anthocyanin biosynthetic signaling and structural genes.

Purple Broccoli (Brassica oleracea L. var italica) attracts growing attention as a functional food. Its purple coloration is due to high anthocyanin amounts. Light represents a critical parameter affecting anthocyanins biosynthesis. In this study, 'Purple Broccoli', a light-responding pigmentation cultivar, was assessed for exploring the mechanism underlying light-induced anthocyanin biosynthesis by RNA-Seq. Cyanidin, delphinidin and malvidin derivatives were detected in broccoli head samples. Shading assays and RNA-seq analysis identified the flower head as more critical organ compared with leaves. Anthocyanin levels were assessed at 0, 7 and 11 days, respectively, with further valuation by RNA-seq under head-shading and light conditions. RNA sequences were de novo assembled into 50,329 unigenes, of which 38,701 were annotated against four public protein databases. Cluster analysis demonstrated that anthocyanin/phenylpropanoid biosynthesis, photosynthesis, and flavonoid biosynthesis in cluster 8 were the main metabolic pathways regulated by light and had showed associations with flower head growth. A total of 2,400 unigenes showed differential expression between the light and head-shading groups in cluster 8, including 650 co-expressed, 373 specifically expressed under shading conditions and 1,377 specifically expressed under normal light. Digital gene expression (DGE) analysis demonstrated that light perception and the signal transducers CRY3 and HY5 may control anthocyanin accumulation. Following shading, 15 structural genes involved in anthocyanin biosynthesis were downregulated, including PAL, C4H, 4CL, CHS, CHI, F3H and DFR. Moreover, six BoMYB genes (BoMYB6-1, BoMYB6-2, BoMYB6-3, BoMYB6-4, BoMYBL2-1 and BoMYBL2-2) and three BobHLH genes (BoTT8_5-1, BoTT8_5-2 and BoEGL5-3) were critical transcription factors controlling anthocyanin accumulation under light conditions. Based on these data, a light-associated anthocyanin biosynthesis pathway in Broccoli was proposed. This information could help improve broccoli properties, providing novel insights into the molecular mechanisms underpinning light-associated anthocyanin production in purple vegetables.

Exogenous GM1 Ganglioside Attenuates Ketamine-Induced Neurocognitive Impairment in the Developing Rat Brain.

BACKGROUND: A prolonged exposure to ketamine triggers significant neurodegeneration and long-term neurocognitive deficits in the developing brain. Monosialotetrahexosylganglioside (GM1) can limit the neuronal damage from necrosis and apoptosis in neurodegenerative conditions. We aimed to assess whether GM1 can prevent ketamine-induced developmental neurotoxicity. METHODS: Postnatal day 7 (P7) rat pups received 5 doses of intraperitoneal ketamine (20 mg/kg per dose) at 90-minute intervals for 6 hours. Cognitive functions, determined by using Morris water maze (MWM) including escape latency (at P32-36) and platform crossing (at P37), were compared among the ketamine-exposed pups treated with or without exogenous GM1 (30 mg/kg; n = 12/group). The effect of GM1 on apoptosis in hippocampus was determined by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining and activated caspase 3 measurement. The hippocampal expression of brain-derived neurotrophic factor (BDNF), along with the phosphorylation of protein kinase B (AKT) and extracellular signal-related kinases 1 and 2 (ERK1/2), was detected by western blotting (n = 6/group). Anti-BDNF antibody (2 µg per rat) administered before GM1 treatment was applied to determine the neuroprotective mechanisms of GM1. RESULTS: The rats receiving ketamine exposure experinced cognitive impairment in MWM test compared to the control rats, indicated by prolonged escape latency at P34 (P = .006), P35 (P = .002), and P36 (P = .005). However, in GM1-pretreated rats, ketamine exposure did not induce prolonged escape latency. The exogenous GM1 increased the platform-crossing times at P37 (3.00 ± 2.22 times vs 5.40 ± 1.53 times, mean ± standard deviation; P = .041) and reduced the hippocampal TUNEL-positive cells and cleaved-caspase 3 expression in ketamine-exposed young rats. Ketamine decreased BDNF expression and phosphorylation of AKT and ERK in the hippocampus, whereas exogenous GM1 blocked these ketamine-caused effects. However, for the ketamine-exposed rat pups receiving exogenous GM1, compared to immunoglobulin Y (IgY) isotype control, the BDNF-neutralizing antibody treatment counteracted the exogenous GM1-induced improvement of the escape latency at P36 (41.32 ± 12.37 seconds vs 25.14 ± 8.97 seconds, mean ± standard deviation; P = .036), platform-crossing times at P37 (2.16 ± 1.12 times vs 3.92 ± 1.97 times, mean ± standard deviation; P < .036), apoptotic activity, as well as AKT and ERK1/2 phosphorylation in the hippocampus of ketamine-challenged young rats. CONCLUSIONS: Our data suggest that the exogenous GM1 acts on BDNF signaling pathway to ameliorate the cognitive impairment and hippocampal apoptosis induced by ketamine in young rats. Our study may indicate a potential use of GM1 in preventing the cognitive deficits induced by ketamine in the young per se.

Screening of clubroot-resistant varieties and transfer of clubroot resistance genes to Brassica napus using distant hybridization.

Clubroot is an economically important disease affecting plants in the family Cruciferae worldwide. In this study, a collection of 50 Cruciferae accessions was screened using Plasmodiophora brassicae pathotype 4 in China. Eight of these demonstrated resistance, including three Chinese cabbages, two cabbages, one radish, one kale, and one Brassica juncea. The three clubroot-resistant Chinese cabbages (1003, 1007 and 1008) were then used to transfer the clubroot resistance genes to B. napus by distant hybridization combined with embryo rescue. Three methods including morphological identification, cytology identification, and molecular marker-assisted selection were used to determine hybrid authenticity, and 0, 2, and 4 false hybrids were identified by these three methods, respectively. In total, 297 true hybrids were identified. Clubroot resistance markers and artificial inoculation were utilized to determine the source of clubroot resistance in the true hybrids. As a result, two simple sequence repeat (SSR) and two intron polymorphic (IP) markers linked to clubroot resistance genes were identified, the clubroot resistance genes of 1007 and 1008 were mapped to A03. At last, 159 clubroot-resistant hybrids were obtained by clubroot resistance markers and artificial inoculation. These intermediate varieties will be used as the 'bridge material' of clubroot resistance for further B. napus breeding.

Monosialoganglioside 1 may alleviate neurotoxicity induced by propofol combined with remifentanil in neural stem cells.

Monosialoganglioside 1 (GM1) is the main ganglioside subtype and has neuroprotective properties in the central nervous system. In this study, we aimed to determine whether GM1 alleviates neurotoxicity induced by moderate and high concentrations of propofol combined with remifentanil in the immature central nervous system. Hippocampal neural stem cells were isolated from newborn Sprague-Dawley rats and treated with remifentanil (5, 10, 20 ng/mL) and propofol (1.0, 2.5, 5.0 µg/mL), and/or GM1 (12.5, 25, 50 µg/mL). GM1 reversed combined propofol and remifentanil-induced decreases in the percentage of 5-bromodeoxyuridine(+) cells and also reversed the increase in apoptotic cell percentage during neural stem cell proliferation and differentiation. However, GM1 with combined propofol and remifentanil did not affect ß-tubulin(+) or glial fibrillary acidic protein(+) cell percentage during neural stem cell differentiation. In conclusion, we show that GM1 alleviates the damaging effects of propofol combined with remifentanil at moderate and high exposure concentrations in neural stem cells in vitro, and exerts protective effects on the immature central nervous system.

Caspr interaction with Amyloid Precursor Protein reduces amyloid-ß generation in vitro.

Contactin associated protein (Caspr), an adhesion molecule, plays roles in formation of paranodal junctions in myelinated axons, neurite outgrowth, synaptic plasticity in nervous system. Here we have shown a novel function of Caspr in pathogenesis of Alzheimer's disease (AD). Caspr distributes around amyloid plaques in APP/PS1 mice. Levels of Caspr increase in the cerebral cortex of 7-month-old APP/PS1 mice comparing to wild-type littermates. Caspr decreased protein levels of APP in both HEK-293 cells stably transfected with Indiana mutant APP (V717F; HEK-APP) and CHO cells which express endogenous APP, while it did not alter mRNA levels of APP. Furthermore, Caspr co-localizes and interacts with APP. Amyloid-ß (Aß) 40 and Aß42 generation were also reduced in HEK-APP cells by Caspr overexpression.

Epigenetic reactivation of RANK in glioblastoma cells by curcumin: involvement of STAT3 inhibition.

DNA methylation plays an essential role in carcinogenesis. Promoter hypermethylation can result in transcriptional silencing of specific genes, such as tumor suppressors. Thus far, few reports have investigated the effect of curcumin, an active component of the perennial herb Curcuma longa, on DNA methylation. In the present study, we evaluated the effects of curcumin on receptor activator of NF-κB (RANK) gene expression in human glioblastoma cells. Incubation of cells with therapeutic concentrations of curcumin resulted in a significant elevation of RANK expression at both the mRNA and protein levels in two glioblastoma cell lines. We further confirmed that this elevation was associated with promoter demethylation through methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing PCR. Additionally, we demonstrated that knockdown of STAT3, an oncogenic transcription factor, is sufficient to induce RANK promoter demethylation along with RANK reactivation. These results demonstrated that curcumin induced RANK gene reactivation through epigenetic modification in human glioblastoma cells, and that STAT3 is involved in RANK promoter hypermethylation and epigenetic silencing, thus allowing for further applications of curcumin epigenetic therapy in glioma and therapeutic implications of STAT3 in human glioblastoma.

Neural differentiation ability of mesenchymal stromal cells from bone marrow and adipose tissue: a comparative study.

BACKGROUND AIMS: The characteristics, such as morphologic and phenotypic characteristics and neural transdifferentiation ability, of mesenchymal stromal cells (MSC) derived from different origins have yet to be reported for cases isolated from the same individual. METHODS: The proliferation capacity, secretion ability of neurotrophins (NT) and neural differentiation ability in rat MSC isolated from bone marrow (BMSC) and adipose tissue (ADSC) were compared from the same animal. RESULTS: The ADSC had a significantly higher proliferation capacity than BMSC according to cell cycle and cumulative population doubling analyses. The proportion of cells expressing neural markers was greater in differentiated ADSC than in differentiated BMSC. Furthermore, the single neurosphere derived from ADSC showed stronger expansion and differentiation abilities than that derived from BMSC. The findings from Western blot lent further support to the immunocytochemical data. The mRNA and protein levels of nerve growth factor (NGF) and brain-derived growth factor (BDNF) expressed in ADSC were significantly higher than those in BMSC at different stages before and following induction. CONCLUSIONS: Our study suggests that the proliferation ability of ADSC is superior to that of BMSC. Furthermore, differentiated ADSC expressed higher percentages of neural markers. As one possible alternative source of BMSC, ADSC may have wide potential for treating central nervous system (CNS) diseases.

Potassium channels underlie postsynaptic but not presynaptic GABAB receptor-mediated inhibition on ventrolateral periaqueductal gray neurons.

γ-Aminobutyric acid (GABA) is the principle inhibitory neurotransmitter in adult mammalian brain. GABA receptors B subtype (GABA(B)Rs) are abundantly expressed at presynaptic and postsynaptic neuronal structures in the rat ventrolateral periaqueductal gray (PAG), an area related to pain regulation. Activation of GABA(B)Rs by baclofen, a selective agonist, induces presynaptic inhibition by decreasing presynaptic glutamate release. At the same time, baclofen induces a postsynaptic inhibitory membrane current or potential. We here report that in the ventrolateral PAG, the postsynaptic inhibition is mediated by activation of G protein-coupled inwardly rectifying K(+) (GIRK) channels. Blockade of K(+) channels largely prevents postsynaptic action of baclofen. In contrast, presynaptic inhibition of baclofen is insensitive to K(+) channel blockade. The data indicate that potassium channels play different roles in GABA(B)R-mediated presynaptic and postsynaptic inhibition on PAG neurons.

Production of Nα-acetylated thymosin α1 in Escherichia coli.

BACKGROUND: Thymosin α1 (Tα1), a 28-amino acid Nα-acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining Nα-acetylation. In this study, we describe a novel production process for Nα-acetylated Tα1 in Escherichia coli. RESULTS: To obtain recombinant Nα-acetylated Tα1 efficiently, a fusion protein, Tα1-Intein, was constructed, in which Tα1 was fused to the N-terminus of the smallest mini-intein, Spl DnaX (136 amino acids long, from Spirulina platensis), and a His tag was added at the C-terminus. Because Tα1 was placed at the N-terminus of the Tα1-Intein fusion protein, Tα1 could be fully acetylated when the Tα1-Intein fusion protein was co-expressed with RimJ (a known prokaryotic Nα-acetyltransferase) in Escherichia coli. After purification by Ni-Sepharose affinity chromatography, the Tα1-Intein fusion protein was induced by the thiols ß-mercaptoethanol or d,l-dithiothreitol, or by increasing the temperature, to release Tα1 through intein-mediated N-terminal cleavage. Under the optimal conditions, more than 90% of the Tα1-Intein fusion protein was thiolyzed, and 24.5 mg Tα1 was obtained from 1 L of culture media. The purity was 98% after a series of chromatographic purification steps. The molecular weight of recombinant Tα1 was determined to be 3107.44 Da by mass spectrometry, which was nearly identical to that of the synthetic version (3107.42 Da). The whole sequence of recombinant Tα1 was identified by tandem mass spectrometry and its N-terminal serine residue was shown to be acetylated. CONCLUSIONS: The present data demonstrate that Nα-acetylated Tα1 can be efficiently produced in recombinant E. coli. This bioprocess could be used as an alternative to chemosynthesis for the production of Tα1. The described methodologies may also be helpful for the biosynthesis of similar peptides.

Two duplicate CYP704B1-homologous genes BnMs1 and BnMs2 are required for pollen exine formation and tapetal development in Brassica napus.

S45A, a double recessive mutant at both the BnMs1 and BnMs2 loci in Brassica napus, produces no pollen in mature anthers and no seeds by self-fertilization. The BnMs1 and BnMs2 genes, which have redundant functions in the control of male fertility, are positioned on linkage groups N7 and N16, respectively, and are located at the same locus on Arabidopsis chromosome 1 based on collinearity between Arabidopsis and Brassica. Complementation tests indicated that one candidate gene, BnCYP704B1, a member of the cytochrome P450 family, can rescue male sterility. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of the developing anther showed that pollen-wall formation in the mutant was severely compromised, with a lack of sporopollenin or exine. The phenotype was first evident at the tetrad stage (stage 7) of anther development, coinciding with the maximum BnCYP704B1 mRNA accumulation observed in tapetal cells at stages 7-8 (haploid stage). TEM also suggested that development of the tapetum was seriously defective due to the disturbed lipid metabolism in the S45A mutant. A TUNEL assay indicated that the pattern of programmed cell death in the tapetum of the S45A mutant was defective. Lipid analysis showed that the total fatty acid content was reduced in the S45A mutant, indicating that BnCYP704B1 is involved in lipid metabolism. These data suggest that BnCYP704B1 participates in a vital tapetum-specific metabolic pathway that is not only involved in exine formation but is also required for basic tapetal cell development and function.

Towards map-based cloning: fine mapping of a recessive genic male-sterile gene (BnMs2) in Brassica napus L. and syntenic region identification based on the Arabidopsis thaliana genome sequences.

S45AB, a recessive genic male sterile (RGMS) line, originated as a spontaneous mutant in Brassica napus cv. Oro. The genotypes of sterile (S45A) and fertile plants (S45B) are Bnms1ms1ms2ms2 and BnMs1ms1ms2ms2, respectively. In our previous studies, Yi et al. (Theor Appl Genet 113:643-650, 2006) mapped the BnMs1 locus to a region of 0.4 cM, candidates of which have been identified and genetic transformation is in progress. We describe the fine mapping of BnMs2 exploiting amplified fragment length polymorphism (AFLP) and amplified consensus genetic marker (ACGM) methodologies, and the identification of a collinear region probably containing BnMs2 orthologue in Arabidopsis thaliana. A near isogenic line (NIL) population S4516AB which segregated for BnMs2 locus was generated by crossing, allelism testing and repeated full-sib mating. From the survey of 1,024 AFLP primer combinations, 12 tightly linked AFLP markers were obtained and five of them were successfully converted into co-dominant or dominant sequence characterized amplified region (SCAR) markers. A population of 2,650 sterile plants was screened using these markers and a high-resolution map surrounding BnMs2 was constructed. The closest AFLP markers flanking BnMs2 were 0.038 and 0.075 cM away, respectively. Subsequently, an ACGM marker was developed to delimit the BnMs2 locus at an interval of 0.075 cM. We extended marker sequences to perform BlastN searches against the Arabidopsis genome and identified a collinear region containing 68 Arabidopsis genes, in which the orthologue of BnMs2 might be included. We further integrated BnMs2 linked AFLP or SCAR markers to two doubled-haploid (DH) populations derived from the crosses Tapidor x Ningyou7 (Qiu et al., Theor Appl Genet 114:67-80, 2006) and Quantum x No.2127-17 (available in our laboratory), and BnMs2 was mapped on N16. Molecular markers developed from these investigations will facilitate the marker-assisted selection (MAS) of RGMS lines, and the fine map and syntenic region identified will greatly hasten the process of positional cloning of BnMs2 gene.

Recombinant heat shock protein 65 carrying hepatitis B core antigen induces HBcAg-specific CTL response.

Many studies have provided evidence that heat shock protein 65 (Hsp65) can elicit potent specific cellular adaptive immune responses (e.g. CD8(+) cytotoxic T-cell effectors or classic CTLs) based on their ability to chaperone antigenic peptides. Hsp65 is thus an effective carrier for heterologous peptide epitopes for therapeutic vaccines against cancer or chronic infectious diseases. The core antigen of hepatitis B virus (HBcAg) is extremely immunogenic, and functions as both a T-cell-dependent and a T-cell-independent antigen. Therefore, HBcAg may be a promising candidate target for therapeutic vaccine control of chronic HBV infection. Here, a chimeric protein, Hsp65Bc, was created by fusing the HBcAg sequence to the carboxyl terminus of the Hsp65 sequence in E. coli. Analysis of its antigenicity and immunogenicity revealed that HBc epitopes are surface accessible. Hsp65Bc induced moderate anti-HBc immune responses as well as a strong specific T-cell response in BALB/c mice. These results indicate that Hsp65Bc may have potential as a vaccine for treatment of HBV chronic infection.

Enhanced secretion of heterologous proteins in Pichia pastoris following overexpression of Saccharomyces cerevisiae chaperone proteins.

In Pichia pastoris, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). However, upon introduction of foreign proteins, heterologous proteins are often retained in the cytoplasm or in the ER as a result of suboptimal folding conditions, leading to protein aggregation. The Hsp70 and Hsp40 chaperone families in the cytoplasm or in ER importantly regulate the folding and secretion of heterologous proteins. However, it is not clear which single chaperone is most important or which combination optimally cooperates in this process. In the present study we evaluated the role of the chaperones Kar2p, Sec63, YDJ1p, Ssa1p, and PDI from Saccharomyces cerevisiae. We found that the introduction of Kar2p, Ssa1p, or PDI improves protein secretion 4-7 times. In addition, we found that the combination chaperones of YDJ1p/PDI, YDJ1p/Sec63, and Kar2p/PDI synergistically increase secretion levels 8.7, 7.6, and 6.5 times, respectively. Therefore, additional integration of chaperone genes can improve the secretory expression of the heterologous protein. Western blot experiments revealed that the chaperones partly relieved the secretion bottleneck resulting from foreign protein introduction in P. pastoris. Therefore, the findings from the present study demonstrate the presence of a network of chaperones in vivo, which may act synergistically to increase recombinant protein yields.

Construction and evaluation of anti-gastrin immunogen based on P64K protein.

AIM: To construct two kinds of anti-gastrin immunogen based on P64K protein from Neisseria meningitids and to compare their immunogenic effect. METHODS: G17P64K gene was cloned and ligated into pET28a plasmid, then transformed into BL21(DE3). After inoculation of LB medium and IPTG induction, the recombinant protein was solubly expressed at a high level. The purification of G17P64K fusion protein was similar to that of P64K. An initial step of purification consisting of 30% saturated ammonium sulfate precipitation was done. Additional fine optimizations included phenyl-sepharose, G200 Sephadex gel filtration and Q-sepharose anion exchanger chromatography. Highly purified protein was obtained and sequenced at the N-terminal amino acid residues. Polypeptide was synthesized by Fmoc solid phase chemical method and cross-linked to carrier protein P64K and DT mutant by MBS method and then the rabbit anti-gastrin 17 antibody was prepared by immunizing rabbit with cross-linked and fused protein. The titer and the activity in vitro of antibody were assessed. RESULTS: G17P64K gene and the recombinant bacteria were obtained. After four steps purification, protein sample that has the purity above 90% was achieved. At the 84(th) day after the first immunization, the titer of antibody against cross-linked protein reached 51,200. Evaluation of the antibody in vitro manifested that it had a high inhibitory activity on the growth of tumor cell SW480. CONCLUSION: The P64K-polypeptide cross-linked immunogen immunized rabbit and achieved a higher titer antibody against gastrin 17 than the G17P64K fusion protein immunogen, which could inhibit the growth of the tumor cell SW480.

Secretory expression and characterization of a recombinant-deleted variant of human hepatocyte growth factor in Pichia pastoris.

AIM: To study the secretory expression of human hepatocyte growth factor (hdHGF) gene in Pichia pastoris. METHODS: The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coli-yeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GS115 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Mut(+ ) transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae alpha-factor. RESULTS: The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g (DCW)/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L. CONCLUSION: The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.

Reversal of 5-flouroucial resistance by adenovirus-mediated transfer of wild-type p53 gene in multidrug-resistant human colon carcinoma LoVo/5-FU cells.

AIM: To observe the reversal effects of wide-type p53 gene on multi-drug resistance to 5-FU (LOVO/5-FU). METHODS: After treatment with Ad-p53, LOVO/5-FU sensitivity to 5-Fu was investigated using tetrazolium dye assay. Multidrug resistance gene-1 (MDR1) gene expression was assayed by semi-quantitative reverse transcription-polymerase chain reaction and the expression of p53 protein was examined by Western blotting. RESULTS: The reversal activity after treatment with wide-type p53 gene was increased up to 4.982 fold at 48 h. The expression of MDR1 gene decreased significantly after treatment with wide-type p53 gene, and the expression of p53 protein lasted for about 5 d, with a peak at 48 h, and began to decrease at 72 h. CONCLUSION: Wide-type p53 gene has a remarkable reversal activity for the high expression of MDR1 gene in colorectal cancers. The reversal effects seem to be in a time dependent manner. It might have good prospects in clinical application.