[Emphasis on the diagnosis of chronic kidney disease in diabetes mellitus].

Chronic kidney disease (CKD) in diabetes mellitus includes diabetic kidney disease (DKD), non-diabetic kidney disease (NDKD) or a combination of NDKD and DKD. The clinical and renal pathological manifestations of DKD in type 1 diabetes are different from those in type 2 diabetes. Renal biopsy histopathology is the gold standard for distinguishing DKD from NDKD. However, based on the same pathological diagnosis, DKD patients may still have different disease progression and prognosis due to individual differences in molecular biological mechanisms. Metabonomics, proteomics, transcriptomics and artificial intelligence offer hope for biomarkers to diagnose and predict the progress of DKD.

Screening potential SSR markers of the anadromous fish Coilia nasus by de novo transcriptome analysis using Illumina sequencing.

RNA-Seq technology has been widely applied to transcriptomics, genomics, molecular marker development, and functional gene studies. In the genome, microsatellites are simple sequence repeats (SSR) with a high degree of polymorphism that are used as DNA markers in many molecular genetic studies. Using traditional methods such as magnetic bead enrichment, only a few microsatellite markers have been isolated. Coilia nasus is an anadromous, small-to-moderately sized fish species that is famous as an important fishery resource. Here, we have identified a large number of microsatellites from the fish brains by using Illumina sequencing. About 20 million Illumina reads were assembled into 148,845 unigenes. A total of 13,038 SSR motifs were identified via analysis of 3,958,293,117 (3.96 Gb) nucleotides to produce a comprehensive transcript dataset for the C. nasus brain, including mono-, di-, tri-, tetra-, and penta-repeat motifs. The most abundant type of repeat motif was di-nucleotide (42.97%), followed by mono-nucleotide (38.86%), tri-nucleotide (16.21%), tetra-nucleotide (1.83%), and penta-nucleotide (0.05%) repeat units, which is similar to the results obtained in studies in other species. These data provide a base of sequence information to improve molecular-assisted markers to study C. nasus genetic diversity.

Decreased expression of ING2 gene and its clinicopathological significance in Chinese NSCLC patients.

The inhibitor of growth 2 (ING2) is a member of lNG family, involved in cell cycle regulation, DNA repair, apoptosis and senescence, and participating in chromatin remodeling and transcriptional regulation by histone modification. Recent researches suggest ING2 plays roles in carcinogenesis both as tumor suppressor gene and ongocene depending on tumor types and cell status. Here, we investigated the status of ING2 in a series of 64 Chinese non-small cell lung cancer (NSCLC)patients using immunohistochemistry (IHC) and confirmed the results with Western blotting. RT-PCR results revealed the expression level of ING2 was consistent with mRNA level. The IHC results showed that ING2 protein expression was significantly decreased in NSCLC samples compared with normal lung tissues (P

Alteration of HLA-F and HLA I antigen expression in the tumor is associated with survival in patients with esophageal squamous cell carcinoma.

Alteration of human leukocyte antigen (HLA) expression, such as decreased HLA I (HLA-A, -B and -C) antigens and elevated nonclassical HLA I antigens (HLA-E, -F and -G), was reported to have an unfavorable prognosis in various cancers. In our study, HLA-F expression in 105 primary esophageal squamous cell carcinoma (ESCC) lesions and 62 case-matched adjacent normal tissues, and HLA I antigens among 68 cases were analyzed by immunohistochemistry. Data revealed that HLA-F expression was observed in 58.1% (61/105) of the ESCC lesions and in 54.8% (34/62) of the normal esophageal tissues. Among the 62 case-matched samples, HLA-F expression (lesion vs. normal tissue) was upregulated, unchanged and downregulated in 13 (21.0%), 6 (9.6%) and 43 (69.4%) cases, respectively. Patients with HLA-F positive had a worse survival than those with HLA-F negative (p = 0.040). Patients with upregulated HLA-F expression (lesion vs. normal tissue) had significantly worse survival than those with HLA-F unchanged and downregulated (p = 0.010). Furthermore, decreased HLA I expression was observed in 41.2% (28/68) patients and was with worse prognosis in comparison to those with preserved HLA I expression (p = 0.001). Multivariate analysis using Cox's proportional hazards model revealed that upregulated HLA-F expression (p = 0.026) and downregulated HLA I expression (p = 0.013) could be an independent unfavorable prognostic factor. In conclusion, our study provided the evidence that alteration of HLA I and HLA-F antigen expression was associated with survival in patients with ESCC.

VNTR polymorphism of human IL1RN in Chinese Han and She ethnic populations.

Interleukin 1 receptor antagonist (IL-1Ra) is an important anti-inflammatory molecule encoded by the IL1RN gene. The polymorphism of IL1RN characterized by variable numbers of an 86 bp tandem repeat (VNTR) sequence in intron 2 has been described. Moreover, frequencies of the IL1RN alleles vary among different ethnics. In the present study, we analysed the IL1RN polymorphism in intron 2 in 256 Chinese Han and 252 Chinese She individuals. Four alleles including IL1RN*1, *2, *3 and IL1RN*4 were identified in this study. Data revealed that the distribution of the IL-1RN genotypes and allele was significantly different between the two Chinese populations (P < 0.001). Among them, 66.8% (171/256) and 86.5% (218/252) were homozygous for the allele IL-1RN*1 in Chinese Han and She individuals respectively. Homozygosity for allele IL-1RN*2 was only observed in Chinese Han with the percentage of 0.8% (2/256). Heterozygosity for IL-1RN*1/2, IL1RN*1/3 and IL1RN*1/4 was 30.9% (79/256), 0.4% (1/256) and 1.2% (3/256) in Chinese Han, whereas only heterozygosity for IL-1RN*1/2 was found in Chinese She (13.5%, 34/252). Frequencies of the most common allele IL-1RN*1 and IL-1RN*2 were 83.0% and 16.2% for Chinese Han and 93.3% and 6.7% for Chinese She respectively. The rare allele IL-1RN*3 and IL-1RN*4 was only observed in the Chinese Han population with the frequency of 0.2% and 0.6% respectively. Our findings suggested that the ethnic background plays an important role in IL-1Ra gene variation in different populations.

Polymorphism of human CD1a, CD1d, and CD1e in exon 2 in Chinese Han and She ethnic populations.

CD1 molecules are the major histocompatibility complex (MHC)-like glycoproteins specialized in capturing and presenting a variety of glycolipid to antigen-specific T-cells. There are five closely linked CD1 genes termed as CD1a, CD1b, CD1c, CD1d, and CD1e. CD1 gene features limited the polymorphism in exon 2 which encodes for the alpha1 domain. Few investigations on the allele frequencies of the CD1 genes have been reported to date; however, variation of CD1 allele frequency in different ethnics has been observed. In the current study, the CD1a, CD1d, and CD1e gene polymorphisms in exon 2 (alleles 01 and 02) in a group of normal Chinese Han and She individuals were analyzed. Similar allele prevalence was observed between the two populations. The CD1e allele frequency was 37.1% (allele 01); 62.9% (allele 02) and 39.3% (allele 01); 60.7% (allele 02) for Han and She populations, respectively. CD1e was the only polymorphic gene with a genotype frequency for Chinese Han (01/01, 11.0%; 01/02, 52.2%; 02/02, 36.8%) and She (01/01, 13.2%; 01/02, 52.1%; 02/02, 34.7%) individuals, respectively. No CD1a allele 01 and CD1d allele 02 were observed in either population. Our findings indicate that the polymorphism of CD1a, CD1d, and CD1e genes in exon 2 is very limited in the Chinese Han and She ethnics.

IL-1 receptor antagonist gene polymorphism in idiopathic recurrent spontaneous abortion in a Chinese Han population.

Interleukin-1 receptor antagonist (IL-1Ra) has been supposed to play important roles in pregnancy. The purpose of this study was to evaluate the association between the polymorphisms of IL-1Ra gene (IL1RN) variable number tandem repeat (VNTR) in intron 2 with idiopathic recurrent spontaneous abortion (RSA). Ninety-two RSA patients and hundred normal women with at least one live birth and no history of miscarriage were included in the study. Frequencies of the IL1RN alleles and genotypes were determined. Data revealed that the prevalence of IL1RN allele and genotype was not significant between the RSA and control group (all P > 0.05). Our finding indicated that the polymorphism VNTR of IL1RN gene in intron 2 may not be a risk factor for RSA in the Chinese Han population.

Ethnic variation of the HLA-G*0105N allele in two Chinese populations.

Unlike high polymorphic classical human leukocyte antigen (HLA) class I molecules, the genetic polymorphism of HLA-G is very limited. However, the prevalence of HLA-G alleles among different ethnic populations varied dramatically. The HLA-G null allele (HLA-G*0105N) is defined by a cytosine deletion (Delta C) at position 1597 in exon 3, which disrupts the reading frame and alters the expression of HLA-G proteins. The HLA-G*0105N allelic frequency was investigated in previous studies and possible roles were addressed. In the current study, a total of 310 Chinese Han and 260 Chinese She ethnic minority population had been genotyped for the G*0105N polymorphism. Marked difference was observed that the G*0105N allelic frequency in Chinese Han was 1.61%, while no copy of the null allele was observed in the Chinese She minority population (P(c) = 0.0073). Data also revealed that no homozygote of HLA-G*0105N allele exists in this Chinese Han population. Furthermore, significant difference was found for the frequencies of HLA-G*0105N both in Chinese Han and in Chinese She populations when compared with other ethnic populations. Taken together, our results indicated that ethnic variation of the HLA-G*0105N polymorphism among different ethnic populations is possibly the result of evolution. However, the advantages of the selection of this allele are necessary to be further investigated.

Ascorbic acid and cell survival of adriamycin resistant and sensitive MCF-7 breast tumor cells.

The ability of human cells to regenerate ascorbic acid from dehydroascorbate is partially dependent on the glutathione redox status of the cell and the relative activity of dehydroascorbate reductases. Mammalian dehydroascorbate reductase activity is associated with two proteins known as thioltransferase (glutaredoxin) and protein disulfide isomerase. We compared the specific activity of thioltransferase, protein disulfide isomerase, and other GSH-related enzymes in Adriamycin-resistant human breast tumor cells, MCF-7 ADRR, and Adriamycin-sensitive, MCF-7 WT, tumor cells. MCF-7 ADRR cells had higher activities of glutathione peroxidase (34.7 fold), nonseleno-glutathione peroxidase (glutathione S-transferases; 5.3 fold), thioredoxin (2.3 fold), and thioltransferase (4.0 fold) compared with the WT Adriamycin-sensitive cell line. Thioltransferase was detected in Western blots in extracts of ADRR MCF-7 cells but not in WT MCF-7 cells. alpha-Tocopherol in the membrane and cytosolic fractions was 2.8 and 3.0 fold higher, respectively, in Adriamycin-resistant compared with Adriamycin-sensitive cells. Supplementation of MCF-7 cells with L-ascorbic acid 2-phosphate (2 and 10 mM) had no effect on WT cell viability after 5 days incubation with up to 0.33 microM Adriamycin. In contrast, supplementation of ADRR MCF-7 cells with L-ascorbic acid 2-phosphate resulted in enhanced resistance up to 3.4 microM Adriamycin over a 5-day incubation. Both lines of MCF-7 cells demonstrated the ability to utilize ascorbic acid as the 2-phosphate derivative. After 48 h incubation with 8.6 microM Adriamycin, the resistant cells maintained normal viability and ascorbate-dehydroascorbate levels, whereas drug-sensitive cells had significantly lower ascorbate with a higher percent dehydroascorbate and increased cell death as judged by cell protein levels (52% of controls).

Host-species-dependent physiological characteristics of hemiparasite Santalum album in association with N2-fixing and non-N2-fixing hosts native to southern China.

Understanding the interactions between the hemiparasite Santalum album L. and its hosts has theoretical and practical significance in sandalwood plantations. In a pot study, we tested the effects of two non-N2-fixing (Bischofia polycarpa (Levl.) Airy Shaw and Dracontomelon duperreranum Pierre) and two N2-fixing hosts (Acacia confusa Merr. and Dalbergia odorifera T. Chen) on the growth characteristics and nitrogen (N) nutrition of S. album. Biomass production of shoot, root and haustoria, N and total amino acid were significantly greater in S. album grown with the two N2-fixing hosts. Foliage and root δ(15)N values of S. album were significantly lower when grown with N2-fixing than with non-N2-fixing hosts. Significantly higher photosynthetic rates and ABA (abscisic acid) concentrations were seen in S. album grown with D. odorifera. Similarity in the proportional amounts of amino acid of root xylem sap between S. album and its host D. odorifera was also evident, suggesting major access to nitrogenous solutes from D. odorifera to S. album. Irrespective of host species, S. album clearly appeared to optimize xylem sap extraction from its hosts by higher transpiration and lower water-use efficiency than its host. The growth of two non-N2-fixing hosts parasitized by S. album was significantly greater than the equivalent values for unparasitized treatments, and lower growth and photosynthesis were observed for parasitized A. confusa, and significant decreases in root N, photosynthesis and transpiration for parasitized D. odorifera compared with unparasitized treatments. Furthermore, foliage ABA concentrations were significantly higher in all hosts parasitized by S. album than in their unparasitized counterparts. Our study is probably the first to report on host dependence and preference in the hemiparasite S. album, and the generated results may have important implications for understanding of the physiological interactions between host species and parasitic plants, and for successfully mixing plantations of S. album with D. odorifera.

Two-way transfer of nitrogen between Dalbergia odorifera and its hemiparasite Santalum album is enhanced when the host is effectively nodulated and fixing nitrogen.

Nutrient translocation from a host plant is vital to the growth and survival of its root parasitic plant, but few studies have investigated whether a parasitic plant is also able to transfer nutrients to its host. The role of N2-fixation in nitrogen (N) transfer between 7-month-old Dalbergia odorifera T. Chen nodulated with Bradyrhizobium elkanii DG and its hemiparasite Santalum album Linn. was examined by external (15)N labeling in a pot study. Four paired treatments were used, with (15)N given to either host or hemiparasite and the host either nodulated or grown on combined N. N2-fixation supplied 41-44% of total N in D. odorifera. Biomass, N and (15)N contents were significantly greater in both nodulated D. odorifera and S. album grown with paired nodulated D. odorifera. Significantly higher total plant (15)N recovery was in N donor D. odorifera (68-72%) than in N donor S. album (42-44%), regardless of the nodulation status in D. odorifera. Nitrogen transfer to S. album was significantly greater (27.8-67.8 mg plant(-1)) than to D. odorifera (2.0-8.9 mg plant(-1)) and 2.4-4.5 times greater in the nodulated pair than in the non-nodulated pair. Irrespective of the nodulation status, S. album was always the N-sink plant. The amount of two-way N transfer was increased by the presence of effective nodules, resulting in a greater net N transfer (22.6 mg plant(-1)) from host D. odorifera to hemiparasite S. album. Our results may provide N management strategies for D. odorifera/S. album mixed plantations in the field.

Characterization of a pathogenic H9N2 influenza A virus isolated from central China in 2007.

The entire genome of the A/Chicken/Hubei/C1/2007 (H9N2) virus, isolated from central China in 2007, was completely sequenced and phylogenetically analyzed. Phylogenetic analysis demonstrated that A/Chicken/Hubei/C1/2007 (H9N2) virus represents multiple reassortant lineages, with genes coming from the early mainland China strain (Ck/Beijing/1/94), an H9N2 virus with special genotype (Ck/shanghai/F/98) and other lineages from poultry in Asia. Infection studies indicated that A/Chicken/Hubei/C1/2007 (H9N2) virus replicated efficiently in MDCK cells and in BALB/c mice. The H9N2 virus also replicated to high titers in chicken respiratory tracts and caused overt clinical signs in chickens. Our results suggest that attention should be paid to the natural evolution of H9N2 influenza viruses and to the control of H9N2 influenza viruses in animals.

[Screening and cloning of the down-regulation gene by recombinant interferon-B using suppression subtractive hybridization technique.].

BACKGROUND: To construct a subtractive cDNA library of target genes down-regulated in human hepatocarcinoma cell line HepG2 cells treated with IFNB, and clone genes of the down-regulation by IFNB using suppression subtractive hybridization (SSH) technology and bioinformatics techniques. METHODS: The mRNA was isolated from HepG2 cells induced by recombinant interferon-B and 0.9 percent sodium chloride, respectively, then cDNA was synthesized. After restriction enzyme Rsa I digestion, small sizes cDNAs were obtained. Then tester cDNA was divided into two portions and each was ligated to the specific cDNA adaptor 1 and adaptor 2 respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the DNA fragment was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5a. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS: The subtractive library of genes down-regulation in HepG2 cells treated with recombination interferon-B was constructed successfully. The amplified library contained 58 positive clones. Colony PCR and sequence analysis was performed in 35 clones randomly, and the full length sequences were obtained with bioinformatics method. Altogether 12 coding sequences were obtained. CONCLUSION: A subtractive cDNA library of genes down-regulation in HepG2 cells treated with IFNB using SSH technique was constructed successfully, which brings some new clues for studying the regulation mechenism of IFNB in liver cells.

The systemic administration of Ig-4-1BB ligand in combination with IL-12 gene transfer eradicates hepatic colon carcinoma.

We have previously shown that the local-membrane bound 4-1BB ligand and IL-12 gene transfer induced a significant antitumor response in a mouse colon carcinoma model. However, a high viral dose was required in order to achieve the best efficacy. In this study, we hypothesize that the systemic administration of soluble Ig-4-1BB ligand can give rise to better T-cell immune activation than local gene delivery. With potential clinical applications in mind, we further compare whether the natural 4-1BB ligand fused to mouse IgG2a (Ig-4-1BBL) would be as effective as the agonistic anti-4-1BB antibody. The dimeric form of Ig-4-1BBL was purified from HeLa cells transduced with a recombinant adenovirus (ADV/Ig-4-1BBL) expressing Ig-4-1BBL. Functional activity was confirmed by the ligand's ability to bind to activated splenic T cells or bone marrow (BM)-derived dendritic cells (DCs) that express 4-1BB receptor. The soluble Ig-4-1BBL efficiently costimulated CD3-activated T-cell proliferation in vitro. More importantly, it induced tumor-specific CTLs as effectively as the agonistic anti-4-1BB antibody. When combined with IL-12 gene transfer, systemic administration of the Ig-4-1BBL proved to be more potent than local gene delivery. In addition, the Ig-4-1BBL is as potent as the agonistic anti-4-1BB antibody for the treatment of hepatic MCA26 colon carcinoma, resulting in 50% complete tumor regression and long-term survival. In long-term surviving mice, both treatment modalities induced persistent tumor-specific CTL activity. In summary, these results suggest that the systemic delivery of Ig-4-1BBL can generate a better antitumor response than local gene delivery. Ig-4-1BBL had equivalent biological functions when compared to the agonistic anti-4-1BB antibody. Thus, soluble 4-1BBL dimmer can be developed as a promising agent for cancer therapy in humans.

Dehydroascorbate reduction.

Dehydroascorbic acid is generated in plants and animal cells by oxidation of ascorbic acid. The reaction is believed to occur by the one-electron oxidation of ascorbic acid to semidehydroascorbate radical followed by disproportionation to dehydroascorbic acid and ascorbic acid. Semidehydroascorbic acid may recycle to ascorbic acid catalyzed by membrane-bound NADH-semidehydroscorbate reductase. However, disproportionation of the free radical occurs at a rapid rate, 10(5) M-1 s-1, accounting for measurable cellular levels of dehydroascorbate. Dehydroascorbate reductase, studied earlier and more extensively in plants, is now recognized as the intrinsic activity of thioltransferases (glutaredoxins) and protein disulfide isomerase in animal cells. These enzymes catalyze the glutathione-dependent two-electron regeneration of ascorbic acid. The importance of the latter route of ascorbic acid renewal was seen in studies of GSH-deficient rodents (Meister, A. (1992) Biochem. Pharmacol. 44, 1905-1915). GSH deficiency in newborn animals resulted in decreased tissue ascorbic acid and increased dehydroascorbate-to-ascorbate ratios. Administration of ascorbic acid daily to GSH-deficient animals decreased animal mortality and cell damage from oxygen stress. A cellular role is proposed for dehydroascorbate in the oxidation of nascent protein dithiols to disulfides catalyzed in the endoplasmic reticulum compartment by protein disulfide isomerase.

alpha-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria.

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an alpha-lipoic acid dependent or independent manner. The alpha-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of alpha-lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, alpha-lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an alpha-lipoic acid, coenzyme A, and pyruvate or alpha-ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an alpha-lipoic acid (K0.5 = 1.4 +/- 0.8 mM) and lipoamide (K0.5 = 0.9 +/- 0.3 mM) dependent manner.

Sucrose metabolism in lima bean seeds.

Developing and germinating lima bean (Phaseolus lunatus var Cangreen) seeds were used for testing the sucrose synthase pathway, to examine the competition for uridine diphosphate (UDP) and pyrophosphate (PPi), and to identify adaptive and maintenance-type enzymes in glycolysis and gluconeogenesis. In developing seeds, sucrose breakdown was dominated by the sucrose synthase pathway; but in the seedling embryos, both the sucrose synthase pathway and acid invertase were active. UDPase activity was low and seemingly insufficient to compete for UDP during sucrose metabolism in seed development or germination. In contrast, both an acid and alkaline pyrophosphatase were active in seed development and germination. The set of adaptive enzymes identified in developing seeds were sucrose synthase, PPi-dependent phosphofructokinase, plus acid and alkaline pyrophosphatase; and, the adaptive enzymes identified in germinating seeds included the same set of enzymes plus acid invertase. The set of maintenance enzymes identified during development, in the dry seed, and during germination were UDP-glucopyrophosphorylase, neutral invertase, ATP and UTP-dependent fructokinase, glucokinase, phosphoglucomutase, ATP and UTP-dependent phosphofructokinase and sucrose-P synthase.

Identification of actively filling sucrose sinks.

Certain actively filling plant sucrose sinks such as a seed, a tuber, or a root can be identified by measuring the uridine diphosphate and pyrophosphate-dependent metabolism of sucrose. Sucrolysis in both active and quiescent sucrose sinks was tested and sucrose synthase was found to be the predominant sucrose breakdown activity. Sucrolysis via invertases was low and secondary in both types of sinks. Sucrose synthase activity dropped markedly, greater than fivefold, in quiescent sinks. The tests are consistent with the hypothesis that the sucrose filling activity, i.e. the sink strength, of these plant sinks can be measured by testing the uridine diphosphate and pyrophosphate-dependent breakdown of sucrose. Measuring the initial reactions of sucrolysis shows much promise for use in agriculture crop and tree improvement research as a biochemical test for sink strength.