Site-directed mutation of ß-galactosidase from Streptococcus thermophilus for galactooligosaccharide-enriched yogurt making.

ß-Galactosidase is one of the most important enzymes used in dairy processing. It converts lactose into glucose and galactose, and also catalyzes galactose to form galactooligosaccharides (GOS), so-called prebiotics. However, most of the ß-galactosidases from the starter cultures have low transgalactosylation activities, the process that results in galactose accumulation in yogurt. Here, a site-directed mutation strategy was attempted, to genetically modify ß-galactosidase from Streptococcus thermophilus. Out of 28 Strep. thermophilus strains, a ß-galactosidase gene named bgaQ, encoded for high ß-galactosidase hydrolysis activity (BgaQ), was cloned from the strain Strep. thermophilus SDMCC050237. It was 3,081 bp in size, with 1,027 deduced amino acid residuals, which belonged to the GH2 family. After replacing the Tyr801 and Pro802 around the active sites of BgaQ with His801 and Gly802, the GOS synthesis of the generated mutant protein BgaQ-8012 increased from 20.5% to 26.7% at 5% lactose, and no hydrolysis activity altered obviously. Subsequently, the purified BgaQ or BgaQ-8012 was added to sterilized milk inoculated with 2 starters from Strep. thermophilus SDMCC050237 and Lactobacillus delbrueckii ssp. bulgaricus ATCC11842. The GOS yields with added BgaQ or BgaQ-8012 increased to 5.8 and 8.3 g/L, respectively, compared with a yield of 3.7 g/L without enzymes added. Meanwhile, the addition of the BgaQ or BgaQ-8012 reduced the lactose content by 49.3% and 54.4% in the fermented yogurt and shortened the curd time. Therefore, this study provided a site-directed mutation strategy for improvement of the transgalactosylation activity of ß-galactosidase from Strep. thermophilus for GOS-enriched yogurt making.

[Study on the oral mucosal diseases in patients with cerebrovascular diseases].

Objective: To investigate the prevalence of oral mucosal diseases (OMD) in patients with cerebrovascular disease. Methods: A total of 182 patients with cerebrovascular disease and 166 controls were examined for OMD to compare the differences of prevalence rates. Results: The prevalence of OMD in patients with cerebrovascular disease appeared higher than that in the control group. Oral candidiasis was most commonly seen (11.1%, 20/182), followed by fissured tongue (5.0%, 9/182), traumatic ulcer (2.8%, 5/182), herpes labialis (2.2%, 4/182), recurrent oral ulcer (1.6%, 3/182), chronic cheilitis (1.6%, 3/182) and oral leukokeratosis (1.6%, 3/182). Conclusion: Patients with cerebrovascular diseases were susceptible to OMDs, especially to oral candidiasis that called for more attention.

[Alterations of mTOR pathway and autophagy in early type 2 diabetic cardiomyopathy in rats].

Objective: To investigate the alterations of mTOR signaling pathway and autophagy in the development of type 2 diabetes and early diabetic cardiomyopathy and to study their roles in pathogenesis of diabetic myocardium. Methods: A type 2 diabetes rat model was established by injection of streptozocin after five-week of high fat diet. The rats were randomly divided into control group, experiment group of 2 weeks and experiment group of 4 weeks. Alterations of mTOR, p-mTOR, S6K1, Beclin-1 and LC3-Ⅱ expression in myocardium were determined by Western blot and immunohistochemistry. Results: Compared with the control group, the expression of mTOR, p-mTOR, S6K1, Beclin-1 and LC3-Ⅱ level increased significantly in the experiment group of 2 weeks. The expression of p-mTOR and S6K1 increased significantly in the experiment group of 4 weeks compared with those of the experiment group of 2 weeks. Conclusions: mTOR signaling pathway is activated in early diabetic myocardial injury via autophagy. The findings may provide a new therapeutic target for diabetic cardiomyopathy.

Nimotuzumab with cisplatin or fluorouracil on human esophageal squamous cell carcinoma EC1 cells.

OBJECTIVE: To investigate the effects of nimotuzumab (h-R3) with cisplatin (DDP) or fluorouracil (5-FU) on human esophageal squamous cell carcinoma (ESCC) EC1 cells. MATERIALS AND METHODS: The assignment included blank control, h-R3 alone, DDP alone, 5-FU alone, h-R3 combined with DDP, and h-R3 combined with 5-FU. The cell proliferation in each group was measured by MMT method 48 h post dose. The effect on the cell cycle was determined by flow cytometry, and the effect on cell apoptosis was determined by flow cytometry and TUNEL test 48 h post dose. RESULTS: The inhibitory effect of h-R3 on the proliferation of EC1 cells was weak. The maximum inhibition rate was 10.10 ± 0.58% 48 h post dose, and the difference in the inhibition rate between the h-R3 with chemotherapeutic agents and the chemotherapeutic agent alone was not statistically significant (p > 0.05). Flow cytometry demonstrated no obvious change in the EC1 cells after h-R3 treatment (p > 0.05). Flow cytometry and TUNEL test demonstrated that the difference in the apoptosis rate between h-R3 combined with chemotherapeutic agents and blank control was not statistically significant (p > 0.05). CONCLUSIONS: h-R3 had no significant effect on human ESCC EC1 cells in vitro, with or without the combination of chemotherapeutic agents.

Expression and significance of glucosylceramide synthase in colorectal carcinoma tissues.

OBJECTIVE: To investigate the impression of glucosylceramide synthase (GCS) in colorectal carcinoma tissues. MATERIALS AND METHODS: Immunohistochemical method was used to detect the expression of GCS in 188 cases of colorectal carcinoma tissues and 80 cases of non-cancerous colorectal tissues. The expression of GCS and the relation with age, gender, tumor location, Tumor stage, differentiation, lymph node metastasis were analyzed. Thus, we further discussed whether to accept neoadjuvant chemotherapy can affect the expression of GCS. RESULTS: GCS was overexpressed in colorectal carcinoma tissues compared with non-cancerous colorectal tissues; the expression of GCS was associated with lymph node metastasis, but not associated with age, gender, tumor location, differentiation and Tumor stage. What's more, the positive expression rate of GCS in patients who receiving neoadjuvant chemotherapy was higher than that in untreated patients. CONCLUSIONS: GCS was upregulated in colorectal carcinoma tissues. It might not only play an important role in controlling the generation and progression of colorectal carcinoma, but also be a factor of MDR.

Sequence characterization and promoter identification of porcine APC10 gene.

APC10 protein, a subunit of the anaphase-promoting complex (APC), plays an essential role in the progression of cells from mitosis to G1. In this study, we cloned and sequenced partial cDNA, intron 1 and 5'-flanking sequences of porcine APC10. The partial cDNA is 595 bp long and has an open reading frame of 558 bp which encodes 185 putative amino acids. Real-time PCR analysis revealed that the porcine APC10 mRNA expression shows a wide distribution and expression levels varies within a small different range in detected tissues. The deduced protein has a high identity with other eight species and comprises a conserved DOC domain. The phylogenetic tree indicated that porcine APC10 has the closest genetic relationship with human, monkey and dog. Promoter activity was demonstrated by transient transfection of 5'-deletion promoter/luciferase constructs into PK15 cells, and indicated that the proximal region from -2,052 to -1,764 is necessary for basal promoter activity. Positive cis-regulatory elements are present from -2,544 to -2,052 and from -3,114 to -2,774, while negative cis-regulatory elements may be present from -2,774 to -2,544. Yeast one-hybrid assay revealed Sp1 can interact with proximal promoter region of porcine APC10.

Molecular characterization and association analysis of FBXO40 with partial hematological indexes in pig.

F-box proteins are quite significant ubiquitin-proteasome pathway regulators in eukaryotic cells. FBXO40, a member of this large family, alters its expression pattern in muscle atrophy. Here we isolated most of the verified porcine FBXO40 coding sequence (CDS) (2258 bp) and assigned it to the porcine chromosome 13q4.1-4.6 by using the INRA-Minnesota porcine radiation hybrid panel, and we also explored the tissue expression distributions, which is relatively high in longissimus dorsi muscle, heart, low in kidney, small intestine, brain, hypophysis, lymphonode, thymus, spleen, large intestine, ovary, stomach, and undetectable in testis, liver, uterus and thyroid gland. Inferring phylogenetic tree was constructed to study the evolutionary implications. Moreover, a HindII (HincII)-RFLP (A/C) polymorphism in 3'-untranslated region (3'-UTR) of porcine FBXO40 gene was demonstrated by sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. Statistical analysis result of this polymorphism showed that the allele A was predominant in all detected indigenous breeds, but C in western introduced commercial breeds. The SNP was further analyzed in our experimental pig population including Tongcheng, Landrace, Large White, and crossbreds of Large White × (Landrace × Tongcheng) and Landrace × (Large White × Tongcheng). The association analysis results indicated that the A/C base substitution was associate with some hematological indexes, the hemoglobin concentration (P < 0.0001), mean corpuscular volume hemoglobin concentration (P = 0.0002) and mean corpuscular volume (P = 0.0138).

Expression and SNP association analysis of porcine FBXL4 gene.

As a kind of E3 ligase, the product of FBXL4 gene belongs to a member of FBLs which is the biggest eukaryotic subfamily of F-BOX proteins, it can recognize some substrate through particular protein-protein interaction domains. To investigate its functions, the polymorphism and association analysis was analyzed. The partial cDNA of porcine FBXL4 with 2384 bp long was first cloned; the deduced protein comprises a conserved F-BOX domain at position from the 277th to 332nd amino acid. The phylogenetic tree indicated porcine FBXL4 has the closest genetic relationship with bovine FBXL4 than other selected animal species. Ten tissue expression level of porcine FBXL4 mRNA fluctuated remarkably in a large range by quantitative RT-PCR analysis. For two identified SNPs, the genotyping analysis of Tail showed TT genotype owned dominance in introduced Landrace pig and miniature Guizhou and Wuzhishan breeds, but CC genotype was more than two other genotypes in miniature Laiwu breed. While in another genotyping analysis of BsaJI, CC genotype was obviously more than other genotypes in two kinds of Chinese miniature pig breeds and introduced Landrace pig breeds. Furthermore, the association analysis with immune traits and blood parameters revealed that SNP Tail was significantly associated with the lymphocyte percentage (P = 0.0166) and the antibody levels for pseudorabies virus vaccination (P = 0.0001) of neonate piglets at 0 day. Meanwhile, SNP BsaJI was significantly associated with lymphocyte percentage of individuals at 32 days (P = 0.0351), neutrophil percentage (P = 0.0005), the absolute lymphocyte count (P = 0.0458), and the mixed cells (P = 0.0010) of neonate piglets at 0 day.

The chromosomal localization, expression pattern and polymorphism analysis of porcine FSCN1 gene differently expressed from LongSAGE library.

Fascin homologue 1 (FSCN1) has established roles in cell adhesion, motility, and cell-cell interactions. Our LongSAGE analysis suggested that FSCN1 was potentially differentially expressed in prenatal pig skeletal muscle. We have cloned the genomic DNA and mRNA sequence of FSCN1 gene and mapped it to SSC3p16-p17. The FSCN1 gene was differently expressed during prenatal skeletal muscle development and exhibited different expression pattern between Tongcheng and Landrace pigs. In Tongcheng pigs, FSCN1 expression was similar at 33 and 65 days post conception (dpc), and then sharply increased to a peak at 90 dpc. In Landrace pigs, however, expression increased between 33 and 65 dpc, peaked at 65 dpc, and was down-regulated thereafter. Significantly different expression levels between Tongcheng and Landrace were observed at 65 and 90 dpc. In postnatal pigs, it was strongly expressed only in the brain, but weakly in skeletal muscle and other tissues. We initially identified 32 SNPs through genomic DNA of FSCN1 gene. Association analysis suggested that the 6840(C/T) mutation was significantly associated with the age at market weight (AGE) (p = 0.0004), average day gain from birth to market (ADG1) (p = 0.0002), and average day gain at testing period (ADG2) (p < 0.0001). Our study suggested that FSCN1 gene plays an in prenatal skeletal muscle development and was a candidate gene for meat production trait.

Partial molecular characterization, expression pattern, polymorphism and association analysis of porcine SKP2 gene.

As a component of E3 ubiquitin protein ligases called SCFs, SKP2 protein belongs to a member of FBLs protein which is the biggest eukaryotic subfamily of F-BOX proteins with 12 members. In this study, we cloned and sequenced partial cDNA, intron 1 and intron 6 of porcine SKP2 gene. The partial cDNA is 1,402 bp long and has an open reading frame of 1,272 bp which encodes 424 putative amino acids. The deduced protein comprises a conserved F-BOX domain at position from the 90th to 140th amino acid. The phylogenetic tree indicated that porcine SKP2 has the closest genetic relationship with bovine SKP2 than other selected animal species. Quantitative RT-PCR analysis displayed that the tissue expression level of porcine SKP2 fluctuated remarkably in a large range, and it expressed in thymus with the highest level and in longissimus dorsi muscle with the lowest level. Two SNPs were identified, meanwhile, further polymorphism analysis with Cfr42I showed that AA genotype was in dominance absolutely among four kinds of unrelated Chinese indigenous miniature and one introduced Landrace pig breeds. In addition, association analysis with immune traits and blood parameters revealed that the SNP Cfr42I in intron 1 was significantly associated with red cell distribution width of neonate piglets at 0 day (P = 0.027).

Partial molecular cloning, characterization, and analysis of the subcellular localization and expression patterns of the porcine OTUB1 gene.

The product of the OTUB1 gene is a member of the OTU superfamily of predicted cysteine proteases and inhibits cytokine gene transcription via its interaction with a ubiquitin protease and E3 ubiquitin ligase. To further understand the functions of the porcine OTUB1 gene, the subcellular localization of porcine OTUB1 protein was analyzed. We first cloned a partial DNA sequence of porcine OTUB1 which contained an 816 bp ORF encoding 271 amino acids. The deduced protein product was found to contain an OTU domain. The corresponding porcine OTUB1 protein was subsequently demonstrated to localize predominantly in the nucleus by confocal fluorescence microscopy. By spatial expression analysis, we further found that OTUB1 is highly expressed in the brain, liver, spleen, lung, kidney, large intestine, small intestine, stomach, ovary, uterus and thymus. In contrast, only low levels of this gene were evident in the heart, dorsal muscles and leg muscle of the pig. This is the first report to show the subcellular localization of porcine OTUB1, and our current data provides us with an important basis for conducting further studies on the functions and regulatory mechanisms underlying the role of OTUB1 gene in the immune system.

Prolactin receptor as a candidate gene for prolificacy of small tail han sheep.

DNA polymorphism of the ovine prolactin receptor gene (PRLR) was investigated and used to study its effect on litter size in sheep. By means of PRLR gene sequence homology between sheep and human, three primer pairs were designed for polymerase chain reaction (PCR) amplification within intron 1 and exon 10 of the PRLR gene in sheep. In these parts of the gene the single nucleotide polymorphisms were detected by PCR-single strand conformation polymorphism (SSCP) in 314 Small Tail Han ewes. These poly-morphisms were used to study the associations with litter size. The results indicated that there were three genotypes (AA, AB and BB) detected by three primer pairs. For three primer pairs the frequency of allele A was 0.96, 0.79, 0.68; and the frequency of allele B was 0.04, 0.21, 0.32, respectively. The frequency of genotype AA was 0.93, 0.62, 0.51; the frequency of genotype AB was 0.06, 0.34, 0.34; the frequency of genotype BB was 0.01, 0.04, 0.15, respectively. The Small Tail Han ewes with genotype BB or AB had 0.64-0.76 or 0.44-0.54 more lambs than those with genotype AA, respectively. These results preliminarily showed that the prolactin receptor locus is either a major gene that influences the prolificacy in Small Tail Han sheep or is in close linkage with such a gene.