Clinical significance of hmgb1 expression in human gastric cancer.

High mobility group box 1 (HMGB1) has been proved to be implicated in a variety of cell physiological and pathological behaviors including immune response, inflammation and cancer. Accumulating evidence suggests that HMGB1 plays a critical role in the development and progression of multiple malignancies. However, the clinical significance and prognosis of HMGB1 expression in some cancers remain controversial. The present study aimed to investigate whether overexpression of HMGB1 is an independent prognostic factor in patients with gastric cancer. The correlation of HMGB1 expression with clinicopathologic characteristics and prognosis was assessed by immunohistochemical assay through tissue microarray procedure in 50 primary gastric cancer cases. Our results indicated that the positive expression of HMGB1 was significantly increased in the nucleus of gastric cancer tissues compared with the adjacent non-cancerous tissues (ANCT) (64.0% vs 44.0%, P=0.025), but was not linked to the clinicopathologic features, including the TNM stage (P=0.533) and metastatic lymph node (P=0.771), in patients with gastric cancer. Kapalan-Meier and log-rank analysis demonstrated that overexpression of HMGB1 did not exert significant impact on the overall survival of patients with gastric cancer (P=0.805). Furthermore, Cox regression analysis showed that high HMGB1 protein expression did not represent an independent risk factor for patients with gastric cancer (P=0.677). Taken together, our findings suggest that high expression of HMGB1 is not correlated with the clinicopathologic characteristics of gastric cancer, and cannot serve as an independent prognostic biomarker for patients with gastric cancer.

Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency.

An efficient Agrobacterium-mediated durum wheat transformation system has been developed for the production of 121 independent transgenic lines. This improved system used Agrobacterium strain AGL1 containing the superbinary pGreen/pSoup vector system and durum wheat cv Stewart as the recipient plant. Acetosyringone at 400 microM was added to both the inoculation and cultivation medium, and picloram at 10 mg l(-1) and 2 mg l(-1) was used in the cultivation and induction medium, respectively. Compared with 200 microM in the inoculation and cultivation media, the increased acetosyringone concentration led to significantly higher GUS (beta-glucuronidase) transient expression and T-DNA delivery efficiency. However, no evident effects of acetosyringone concentration on regeneration frequency were observed. The higher acetosyringone concentration led to an improvement in average final transformation efficiency from 4.7% to 6.3%. Furthermore, the concentration of picloram in the co-cultivation medium had significant effects on callus induction and regeneration. Compared with 2 mg l(-1) picloram in the co-cultivation medium, increasing the concentration to 10 mg l(-1) picloram resulted in improved final transformation frequency from 2.8% to 6.3%, with the highest frequency of 12.3% reached in one particular experiment, although statistical analysis showed that this difference in final transformation efficiency had a low level of significance. Stable integration of foreign genes, their expression, and inheritance were confirmed by Southern blot analyses, GUS assay, and genetic analysis. Analysis of T(1) progeny showed that, of the 31 transgenic lines randomly selected, nearly one-third had a segregation ratio of 3:1, while the remainder had ratios typical of two or three independently segregating loci.

Expression, purification and antibody preparation of flagellin FlaA from Vibrio alginolyticus strain HY9901.

AIMS: The main aims of this study were to clone and express flagellin flaA gene from Vibrio alginolyticus strain HY9901, also to prepare mouse anti-FlaA polyclonal antibody for future pathogen or vaccine study. METHODS AND RESULTS: The full-length flaA gene was amplified by PCR with designed primers. The open reading frame of flaA gene contains 1131 bp, and its putative protein consists of 376 amino acid residues. Alignment analysis indicated that the FlaA protein was highly conserved. SDS-PAGE indicated that the FlaA protein was successfully expressed in Escherichia coli BL21 (DE3). Then, the recombinant FlaA protein was purified by affinity chromatography, and the mouse anti-FlaA serum was produced. The expression of flaA gene was verified by various immunological methods, including western blotting, enzyme-linked immunosorbent assay (ELISA) and immunogold electron microscopy (IEM). CONCLUSIONS: Flagellin flaA gene was cloned and identified from V. alginolyticus HY9901, the recombinant FlaA protein was expressed and purified, and high-titre FlaA protein-specific antibody was produced. Western blot analysis revealed that the prepared antiserum not only specifically react to FlaA fusion protein, but also to natural FlaA protein of V. alginolyticus. The expressed FlaA protein was demonstrated, for the first time, as the component of flagella from V. alginolyticus by IEM. SIGNIFICANCE AND IMPACT OF THE STUDY: This study may offer important insights into the pathogenesis of V. alginolyticus, provide a base for further studies on the diagnosis and evaluation that whether the FlaA protein could be used as an effective vaccine candidate against infection by V. alginolyticus and other Vibrio species. Additionally, the purified FlaA protein and polyclonal antibody can be used for further functional and structural studies.

[Thirty-nine cases of stage 4 pressure ulcers treated by a two-stage method of debridement and skin flap transfer].

From September 2013 to October 2018, 39 patients (28 males and 11 females, aged 21 to 76 years) with stage 4 pressure ulcers were admitted to the General Hospital of Xinjiang Military Command. The area of pressure ulcers ranged from 2 cm×2 cm to 20 cm×12 cm on admission. The two-stage method of debridement and skin flap transfer was exploited to repair the wounds. In the first stage, a thorough debridement was performed (26 cases underwent debridement once, 10 cases twice, and 3 cases for three times). The skin flap transfer surgery was conducted in the second stage after 6 to 12 days (local skin flap for 16 cases, vascularized island flap for 8 cases, fascial flap for 5 cases, gluteus maximus flap for 5 cases, and biceps femoris flap for 5 cases), with flap area of 4 cm×2 cm to 16 cm×10 cm. Some donor sites were closed by direct suture and the other donor sites which can not be sutured were covered by medium-thickness skin graft from the lateral thigh. All the pressure ulcers of 39 cases were healed with no sinus. During follow-up of 6 months to 5 years, no recurrence of pressure ulcer at the surgical site was observed; the flaps achieved soft texture and good appearance. Thus, the two-stage method of debridement and skin flap transfer achieved good long-term curative effect and could be a preferable option for treating stage 4 pressure ulcers.

Efficient constitutive expression of chitinase in the mother cell of Bacillus thuringiensis and its potential to enhance the toxicity of Cry1Ac protoxin.

Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine-Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry(-)B. The recombinant plasmid was stably maintained over 240 generations in Cry(-)B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies. The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l(-1) Na(2)CO(3)-0.2% beta-mercaptoethanol buffer at a wide range of alkaline pH values, and what's more, the chitinase inclusions potentiated the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera.

Isolation of human sebaceous glands and cultivation of sebaceous gland-derived cells as an in vitro model.

An experimental technique is presented as an in vitro model for the study of human sebaceous gland-derived cells. Intact sebaceous glands were isolated from full-thickness human skin after incubation in dispase (2.4 U/ml) and in deoxyribonuclease (0.02%) by using microsurgical instruments under microscopical observation of the epidermal underface. Subsequently, the ducts of the glands were removed, the isolated gland lobules were seeded on a 3T3-cell feeder layer in Dulbecco's modified Eagle's medium and Ham's F 12 medium (3:1) supplemented with fetal calf serum (10%), L-glutamine, antibiotics, epidermal growth factor (10 ng/ml), hydrocortisone (0.4 microgram/ml), and cholera toxin (10(-9) M), and were then cultivated in a CO2-incubator at 37 degrees C. After 2-3 wk cell outgrowths resulting from the periphery of the gland lobules were obtained and dispersed cells were passaged for three subcultures with or without 3T3-cell feeder layer. The cultured cells preserved in vitro morphologic characteristics and differentiation patterns comparable to those described for normal human sebocytes in vivo, with a high rate of viable cells. Their labeling pattern with MoAb showed close similarities to the pattern reported for sebocytes in vivo but differences to the pattern of keratinocytes in vivo and in vitro. In their cytoplasm oil red and nile red stained droplets were detected, and the observed density and distribution evidenced in vitro lipogenesis. The technique presented here may provide a promising model for further experimental studies on sebaceous gland cell development and function.

Effects of 13-cis-retinoic acid, all-trans-retinoic acid, and acitretin on the proliferation, lipid synthesis and keratin expression of cultured human sebocytes in vitro.

The aim of this study was to determine the effects of 13-cis-RA, all-trans-RA, and acitretin on the proliferation, lipid synthesis, and keratin expression of human sebocytes in vitro and to elucidate possible mechanisms of retinoid action on sebaceous glands at the cellular level. It was found that 13-cis-RA and all-trans-RA decreased sebocyte proliferation in a dose- and time-dependent manner, with a 13-cis-RA-IC50 of 10(-5) M (after 7 d) and 10(-6) M (after 14 d) and an all-trans-RA-IC50 of 10(-7) M (after 14 d; no IC50 after 7 d). Acitretin inhibited sebocyte proliferation only at 10(-5) M. Furthermore, 13-cis-RA was the most potent inhibitor of acetate incorporation into lipids, which indicated lipid synthesis (48.2% reduction), followed by all-trans-RA (-38.6%), and by acitretin (-27.5%). All retinoids tested markedly decreased the synthesis of triglycerides, wax/stearyl esters, and free fatty acids in cultured sebocytes, whereas squalene synthesis remained uninfluenced and cholesterol synthesis slightly increased. On the other hand, keratin 5 was down-regulated, keratin 17 was up-regulated, and the expression of keratin 13 was virtually unaffected by all retinoids tested. Keratins 6 and 16 were down-regulated by 13-cis-RA and by all-trans-RA, keratin 14 was down-regulated by 13-cis-RA only, and keratin 19 was up-regulated by all-trans-RA. These investigations indicate that 13-cis-RA and, to a lesser extent, all-trans-RA are potent inhibitors of both cell proliferation and lipid synthesis in human sebocytes in vitro, whereas acitretin only decreases lipogenesis in this model. In addition, retinoids may modify the differentiation of sebocytes in vitro by modulating keratin expression. Models of cultured human sebocytes are useful tools for further investigations on the sebaceous gland and its activity at the cellular level.

Nucleotide sequence analysis of a transforming gene isolated from nasopharyngeal carcinoma cell line CNE2: an aberrant human immunoglobulin kappa light chain which lacks variable region.

A transforming gene, designated Tx, was isolated from a human nasopharyngeal carcinoma (NPC) cell line CNE2 by transfection and molecular cloning techniques. The Tx gene was analyzed using computer-based bioinformatics and compared with the known sequences in EMBL and GenBank databases. We found that Tx contains human immunoglobulin kappa light chain constant region, five intact joining regions J1-J5, five recombination signal sequences and an N-segment besides classic regulatory sequences such as TATA boxes, CAAT boxes, poly A signals, etc. Interestingly, Tx also contains several binding sites for nuclear transcription factors such as NF-kappaB, NF-IL6, TFIID, etc. In conclusion, there are only several base pairs mutations or deletions compared with normal Ig K JC gDNA fragment. In all, Tx is an aberrant human immunoglobulin kappa light chain that contains the constant region, five joining regions, which lacks the variable regions.

[Air-liquid interface cultivation of human keratinocytes].

For simulation of in vivo conditions, air-liquid interface cultivation of human keratinocytes is reported. After human keratinocytes were grown on a collagen matrix inside plastic rings at the air-liquid interface for 14 days, the culture was cryostat sectioned and labeled with monoclonal antibody against human filaggrin using the APAAP technique. The culture displayed a stratified, multilayered epithelium with a basal cell layer and 9-11 supra-basal layers. Filaggrin was found mainly in the upper cell layers. As the microarchitecture of the culture was essentially similar to in vivo conditions, air-liquid interface cultivation seems to be an appropriate method for the study of epidermal cell growth and differentiation in vitro.

Expression and characterization of a recombinant Cry1Ac crystal protein fused with an insect-specific neurotoxin ω-ACTX-Hv1a in Bacillus thuringiensis.

In order to assess possible enhancement of biopesticide activity, the fusion gene of crystal protein gene cry1Ac with the insect-specific neurotoxin ω-ACTX-Hv1a gene and egfp was expressed in Bacillus thuringiensis acrystalliferous strain Cry-B under the control of the native gene expression system. The fusion recombinant Cry-B(1Ac-ACTX-EGFP) generally produced two or three small crystal-like inclusion bodies in each cell and the GFP signal could be clearly observed. A 166 kDa full-length fusion protein was identified by immunoblot analysis. Virulence of the fusion inclusions was at least fivefold higher toward larvae of Spodoptera exigua. These results demonstrated that a foreign protein could be expressed and accumulate as parasporal inclusions in B. thuringiensis by C-terminal fusion with the native endotoxin while retaining partial insecticidal activity.

Development and validation of a Viviparous-1 STS marker for pre-harvest sprouting tolerance in Chinese wheats.

Pre-harvest sprouting (PHS) of wheat reduces the quality of wheat grain, and improving PHS tolerance is a priority in certain wheat growing regions where conditions favorable for PHS exist. Two new Viviparous-1 allelic variants related to PHS tolerance were investigated on B genome of bread wheat, and designated as Vp-1Bb and Vp-1Bc, respectively. Sequence analysis showed that Vp-1Bb and Vp-1Bc had an insertion of 193-bp and a deletion of 83-bp fragment, respectively, located in the third intron region of the Vp-1B gene. The insertion and deletion affected the expression level of the Vp1 at mature seed stage, more correctly spliced transcripts were observed from the genotypes with either insertion or deletion than that of the wild type. Based on these insertions and deletions, a co-dominant STS marker of Vp-1B gene was developed and designated as Vp1B3, which in most cases could amplify either 845 or 569-bp fragment from the tolerant cultivars, and 652-bp from the susceptible ones. This Vp1B3 marker was mapped to chromosome 3BL using a set of Chinese Spring nulli-tetrasomic and ditelosomic lines. A total of 89 white-grained Chinese wheat cultivars and advanced lines, were used to validate the relationship between the polymorphic fragments of Vp1B3 and PHS tolerance. Statistical analysis indicated that Vp1B3 was strongly associated with PHS tolerance in this set of Chinese germplasm, suggesting that Vp1B3 could be used as an efficient and reliable co-dominant marker in the evaluation of wheat germplasm for PHS tolerance and marker-assisted breeding for PHS tolerant cultivars.

Isolation and characterization of Viviparous-1 genes in wheat cultivars with distinct ABA sensitivity and pre-harvest sprouting tolerance.

Pre-harvest sprouting (PHS) of wheat reduces the quality and economic value of grain, and increasing PHS tolerance is one of the most important traits in wheat breeding. Two new Vp-1B alleles related to PHS tolerance were identified on the 3BL chromosome of bread wheat and were designated Vp-1Bb and Vp-1Bc. Sequence analysis showed that Vp-1Bb has a 193 bp insertion and Vp-1Bc has a 83 bp deletion located in the third intron region of the Vp-1B gene, and that they shared 95.43% and 97.89% similarity, respectively, with the sequence of AJ400713 (Vp-1Ba) at the nucleotide level. Their sequences were deposited in the GenBank under the accession numbers DQ517493 and DQ517494. Semi-quantitative RT-PCR analysis showed that alternatively spliced transcripts of the Vp-1A, Vp-1B, and Vp-1D homologues were present and there were no differences in the splicing patterns or abundances of Vp-1A and Vp-1D from embryos 35 d after pollination between PHS-tolerant and -susceptible cultivars. Although Vp-1Ba, Vp-1Bb, and Vp-1Bc could each produce a set of transcripts, only one was correctly spliced and had the capacity to encode the full-length VP1 protein and was more highly expressed with Vp-1Bb and Vp-1Bc than with Vp-1Ba. Comparison of the expression patterns of Vp-1Ba, Vp-1Bb, and Vp-1Bc on different days after pollination also revealed that the expression of these genes was developmentally regulated. Furthermore, genotypes with different levels of tolerance to PHS respond differently to ABA exposure and differences in transcript levels of Vp-1Ba, Vp-1Bb, and Vp-1Bc were observed after ABA treatment. The results indicated that insertion or deletion in the third intron region might affect the expression of the Vp-1B gene and its sensitivity to ABA, and thus resistance to PHS.

Molecular and biochemical characterization of puroindoline a and b alleles in Chinese landraces and historical cultivars.

Kernel hardness that is conditioned by puroindoline genes has a profound effect on milling, baking and end-use quality of bread wheat. In this study, 219 landraces and 166 historical cultivars from China and 12 introduced wheats were investigated for their kernel hardness and puroindoline alleles, using molecular and biochemical markers. The results indicated that frequencies of soft, mixed and hard genotypes were 42.7, 24.3, and 33.0%, respectively, in Chinese landraces and 45.2, 13.9, and 40.9% in historical cultivars. The frequencies of PINA null, Pinb-D1b and Pinb-D1p genotypes were 43.8, 12.3, and 39.7%, respectively, in hard wheat of landraces, while 48.5, 36.8, and 14.7%, respectively, in historical hard wheats. A new Pinb-D1 allele, designated Pinb-D1t, was identified in two landraces, Guangtouxianmai and Hongmai from the Guizhou province, with the characterization of a glycine to arginine substitution at position 47 in the coding region of Pinb gene. Surprisingly, a new Pina-D1 allele, designated Pina-D1m, was detected in the landrace Hongheshang, from the Jiangsu province, with the characterization of a proline to serine substitution at position 35 in the coding region of Pina gene; it was the first novel mutation found in bread wheat, resulting in a hard endosperm with PINA expression. Among the PINA null genotypes, an allele designed as Pina-D1l, was detected in five landraces with a cytosine deletion at position 265 in Pina locus; while another novel Pina-D1 allele, designed as Pina-D1n, was identified in six landraces, with the characterization of an amino acid change from tryptophan-43 to a 'stop' codon in the coding region of Pina gene. The study of puroindoline polymorphism in Chinese wheat germplasm could provide useful information for the further understanding of the molecular basis of kernel hardness in bread wheat.

Culture of human sebocytes and markers of sebocytic differentiation in vitro.

Human sebocytes obtained as explants after in vitro culture of isolated sebaceous glands were recently shown to maintain in part a sebocytic differentiation. The aim of this study was to further identify markers of sebocytic differentiation in vitro. Therefore, the morphology of cultured human sebocytes, and their differentiation with lipid storing and expression of cellular proteins were investigated by microscopy, electron microscopy, study of cell kinetics, cytochemistry and immunocytochemistry, and were compared to cultured human keratinocytes obtained from the same skin specimens. At first, sebocytes in all stages of sebocytic differentiation were detected in vitro. Abundant cytoplasmic lipids and the absence of desmosomes were identified as their ultrastructural characteristics. Secondly, an increasing number of sebocytes storing lipids was detected during cell proliferation. Sebocytes contained up to 4 times more lipids than keratinocytes in vitro. Squalene and increased quantities of wax/sterol esters could be extracted from secondary sebocyte cultures. Thirdly, the monoclonal antibodies 6B10 (keratin 4), RPN1162 (keratin 7), and OM-1 labeled only sebocytes in vitro. Furthermore, sebocytes presented a marked expression of keratin 19 in comparison to keratinocytes, as detected with CK 4.62, and a lack of RPN1161 (keratins 1 and 2) expression, which was typically found to be expressed in cultured keratinocytes. The culture of human sebocytes possessing several characteristics of sebocytic differentiation in vivo offers unique possibilities in investigating direct effects on sebaceous cell growth, differentiation and their regulation.

[Epstein-Barr virus latent membrane protein 1 induces the telomerase activity in human nasopharyngeal epithelial cells].

Telomerase activation has been linked to cell immortalization in vitro and tumorigenicity in vivo. In this study, for the first, we reported that Epstein-Barr virus activated the telomerase activity of human nasopharyngeal epithelial cells in the early stage of immortalization as tested by the PCR-ELISA. The telomerase activity in nasopharyngeal epithelial cells was only observed in presenescent cells. It was implicated that Epstein-Barr virus induced the escape of nasopharyngeal epithelial cells from senescence via the activation of telomerase. We further showed that telomerase activation in infected cells was dependent on the protein level of latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus using a Tetracycline regulatory cell line expressing LMP1, pTet-on-LMP1-HNE2. The activity of telomerase in nasopharyngeal cells was decreased when the protein level of LMP1 was blocked by antisense LMP1 plasmid DNA. And the activity of telmerase was also related to the carboxyl terminus of LMP1. It was implicated that the ability of Epstein-Barr virus to suppress senescence is associated with telomerase activation by LMP1.