[Clinical and genetic analysis of pulmonary embolism induced by a novel gene mutation of the antithrombin â ¢ gene].

Objective: To improve the clinical management of acute pulmonary embolism caused by antithrombin Ⅲ (AT Ⅲ) deficiency through gene sequence analysis of the SERPINC1 gene. Methods: The diagnosis and treatment of a 33-year-old male patient with chest pain was reviewed. All exon sequences and flanking regions of 7 related genes of thrombophilia were subjected to detection by high-throughput next generation sequencing technology. The gene mutation was inquired in the gene database and the pathogenic probability of the mutant gene was predicted by Mutation Taster software. Results: The patient was diagnosed with acute pulmonary embolism (intermediate-low risk), with the ATⅢ activity less than 50%. Anticoagulation with nadroparin calcium combined with warfarin was administrated, but hemoptysis was aggravated, and then the medication was replaced by anticoagulant of rivaroxaban. In the end, the embolus was gradually absorbed. A heterozygous missense mutation of c.1148T>A (p.L383H) in the SERPINC1 gene was detected. The gene database and Mutation Taster confirmed the mutation as a new pathogenic mutation with the pathogenic probability of 0.999 999 851 200 991. Conclusions: C.1148T>A (p.L383H) is a novel pathogenic mutation in SERPINC1 gene that complements and updates the gene mutation spectrum of hereditary AT Ⅲ deficiency. The new oral anticoagulant rivaroxaban may be used as the first-line treatment for these patients.

Spin-valley locking and bulk quantum Hall effect in a noncentrosymmetric Dirac semimetal BaMnSb2.

Spin-valley locking in monolayer transition metal dichalcogenides has attracted enormous interest, since it offers potential for valleytronic and optoelectronic applications. Such an exotic electronic state has sparsely been seen in bulk materials. Here, we report spin-valley locking in a Dirac semimetal BaMnSb2. This is revealed by comprehensive studies using first principles calculations, tight-binding and effective model analyses, angle-resolved photoemission spectroscopy measurements. Moreover, this material also exhibits a stacked quantum Hall effect (QHE). The spin-valley degeneracy extracted from the QHE is close to 2. This result, together with the Landau level spin splitting, further confirms the spin-valley locking picture. In the extreme quantum limit, we also observed a plateau in the z-axis resistance, suggestive of a two-dimensional chiral surface state present in the quantum Hall state. These findings establish BaMnSb2 as a rare platform for exploring coupled spin and valley physics in bulk single crystals and accessing 3D interacting topological states.

[Comparison of quality of life of hypopharyngeal squamous cell carcinoma patients after laryngeal preservation surgery and total laryngectomy].

Objective: To evaluate and compare the quality of life (QOL) in patients with hypopharyngeal squamous cell carcinoma after laryngeal preservation surgery and total laryngectomy. Methods: We selected parts of the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 and the Head and Neck Module (EORTC QLQ-C30 and EORTC QLQ-H&N35) and designed the QOL questionnaire. We investigated 42 patients with hypopharyngeal squamous cell carcinoma underwent laryngeal preservation surgery and 38 patients underwent total laryngectomy by QOL questionnaire and followed up their survival. Results: The somatic function dimension, psychological function dimension, and social function dimension of patients underwent laryngeal preservation surgery were (92.46±15.71), (80.56±22.67) and (90.08±19.50), respectively, which were higher than (79.39±32.75), (68.42±25.05) and (61.84±29.55) of the total laryngectomy group (P<0.05), while the economic dimension was not significantly different between the two groups (P>0.05). The social function dimension (including social support and socialization, family relationship) of laryngeal preservation surgery group were (89.04±25.47) for postoperative time < 70 months and (90.94±13.28) for postoperative time ≥70 months, which were higher than (65.48±29.14) and (57.35±30.32) of the total laryngectomy group (P<0.01). Conclusions: The somatic function dimension, psychological function and social function of patients with hypopharyngeal squamous cell carcinoma underwent laryngeal preservation surgery obtain a better QOL than patients underwent total laryngectomy. Therefore, we should improve the laryngeal function and QOL of patients under the premise of ensuring the survival rate.

Effect of Crystal Symmetry on the Spin States of Fe3+ and Vibration Modes in Lead-free Double-Perovskite Cs2AgBi(Fe)Br6.

We show by electron spin resonance (ESR) and Raman spectroscopies that the crystal phase transition of the lead-free double-perovskite Cs2AgBiBr6 has a profound symmetry-breaking effect on the high spin states of, for example, a transition-metal ion Fe3+ and the vibrational modes. It lifts their degeneracy when the crystal undergoes the cubic-tetragonal phase transition, splitting the six-fold degenerate S = 5/2 state of Fe3+ to three Kramer doublets and the enharmonic breathing mode Tg of the MBr6 octahedra (M = Ag, Bi, Fe) into Eg + Ag. The magnitudes of both spin and Raman line splitting are shown to directly correlate with the strength of the tetragonal strain field. This work, in turn, demonstrates the power of the ESR and Raman spectroscopies in probing structural phase transitions and in providing in-depth information on the interplay between the structural, spin, and vibrational properties of lead-free double perovskites, a newly emerging and promising class of materials for low-cost and high-efficiency photovoltaics and optoelectronics.

[Application of preventive flap-placement of terminal ileostomy in laparoscopic radical resection of low rectal cancer].

Objective: To evaluate the value of preventive flap placement of terminal ileostomy in laparoscopic radical resection of low rectal cancer. Methods: A retrospective analysis was conducted in the patients (n=63) who received preventive terminal ileostomy in laparoscopic radical resection of low rectal cancer in our institution from April 2016 to March 2018, including 33 patients who underwent ileostomy with flap-placement (flap group), and 30 patients who underwent ileostomy with stent (stent group). Clinical data were collected from both groups and statistically analyzed. Results: All patients were successfully completed laparoscopic radical resection with preventive ileostomy. All patients of stent group received stoma-closure surgery one month later after rectal resection. There were significantly statistical differences in operating time of ileostomy (28.9±4.3 vs 36.3±2.3, t=11.73, P<0.001) and overall stoma-related complications (1 vs 7, χ(2)=4.155, P=0.042), but no difference in anastomosis leakage, operating time of stoma-reversal, parastomal infection, parastomal hernia and parastomal prolapse. Conclusions: Preventive flap placement of terminal ileostomy represents a secure and feasible approach to laparoscopic low rectal cancer resection. Patients can be released from the discomfort of removing the stent and may suffer fewer stoma-related complications.

Characterization, culture medium optimization and antioxidant activity of an endophytic vitexin-producing fungus Dichotomopilus funicola Y3 from pigeon pea [Cajanus cajan (L.) Millsp.].

AIMS: The aim of this study was to characterize a fungal endophyte Y3 from pigeon pea (Cajanus cajan [L.] Millsp), as a novel producer of vitexin, and its culture medium optimization and antioxidant activity. METHODS AND RESULTS: The endophyte from the leaves of pigeon pea was identified as Dichotomopilus funicola by the morphological and molecular characteristics. The most important medium variables affecting vitexin production in liquid culture of D. funicola Y3 were screened by Plackett-Burman design, and three culture medium constituents (i.e. l-phenylalanine, salicylic acid and CuSO4 ·5H2 O) were identified to play significant roles in vitexin production. The most significant factors were further optimized using by central composite design with response surface methodology. The DPPH radical-scavenging assay indicated that fungal vitexin exhibited notable antioxidant activity with an EC50 value of 164 µg l-1 . CONCLUSIONS: First, a novel endophyte vitexin-producing Dichotomopilus funicola Y3 was isolated from pigeon pea (Cajanus cajan[L.] Millsp.). The maximum vitexin yield was obtained as 78·86 mg l-1 under the optimum culture medium constituents: 0·06 g l-1  l-phenylalanine, 0·21 g l-1 salicylic acid, and 0·19 g l-1 CuSO4 ·5H2 O in medium, which is 4·59-fold higher than that in the unoptimized medium. Also, fungal vitexin clearly demonstrated its antioxidant potential. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings provide an alternative source for large-scale production of vitexin by endophytic fungal fermentation and have a promising prospect in food and pharmaceutical industry.

The safety profile of subcutaneous allergen immunotherapy in children with asthma in Hangzhou, East China.

BACKGROUND: The aim of the current study is to evaluate the prevalence, severity and possible risk factors of systemic reactions (SRs) to subcutaneous allergen immunotherapy (SCIT) in children and adolescents with asthma in Hangzhou, east China's Zhejiang province. METHODS: From January 2011 to December 2016, this survey analysed the SCIT-related SRs involving 429 patients (265 children and 134 adolescents) affected by allergic asthma. Recorded data included demographics, diagnosis, patient statuses, pulmonary function testing results before and after each injection, allergen dosage, and details of SRs. RESULTS: All patients finished the initial phase and six patients withdrew during the maintenance phase. There were 2.59% (328/12,655) SRs in all injections (3.28% in children and 1.47% in adolescents); 15.62% (67/429) patients experienced SRs (18.49% children and 10.98% adolescents). There were 54.57% SRs of grade 1; 42.37% SRs of grade 2; 3.05% SRs of grade 3; and no grades 4 or grade 5 SRs occurred in patients. Most reactions were mild, and were readily controlled by immediate emergency treatment. There was no need for hospitalisation. The occurrence of SRs was significantly higher in children than that in adolescents (p<0.01). A higher ratio of SRs was found among patients with moderate asthma. CONCLUSION: This retrospective survey showed that properly-conducted SCIT was a safe treatment for children and adolescents with asthma in Hangzhou, East China. Children and patients with moderate asthma may be prone to develop SRs.

[Clinical effect of preservation of the left colonic artery in laparoscopic anterior resection for rectal cancer].

Objective: To evaluate the value and feasibility of preservation of the left colonic artery (LCA) in laparoscopic anterior resection for rectal cancer. Methods: The clinical data of 97 patiens who received laparoscopic anterior resection of rectal cancer from 2009.3 to 2015.3 were randomly divided into two groups, including 52 cases with preservation of LCA and 45 cases without preservation of LCA. The operation time, quantity of bleeding, number of lymph nodes removed around the root of inferior mesenteric artery (IMA), the rate of lymph node metastasis around the root of IMA, the incidence of transverse colostomy and anastomotic leak were compared between the two groups. Results: All 97 operations were successfully completed by laparoscopic operation. There were significantly statistical differences in operation time, quantity of bleeding and transverse colon stoma between two groups(P<0.05), but no difference in the number of lymph nodes removed and the rate of lymph node metastasis. Conclusions: The preservation of the left colonic artery in laparoscopic anterior resection of rectal cancer can preserve more supplying vessels for anastomosis and prevent anastomotic leak.

[Role of cytokine-matrix metalloproteinase axis on promoting vascular neointima hyperplasia in mice].

Objective: To observe the effects of tumor necrosis factor-α (TNF-α) and platelet derived growth factor (PDGF) on vascular neointimal hyperplasia on matrix metalloproteinase 9/2 gene knockout (MMP9/2-/-) mice and explore related mechanisms. Methods: Mice of control group, MMP9-/- group, MMP2-/- group and MMP9/2-/- group were studied. Femoral artery was injured by transluminal wire, the mRNA expression levels of TNF-α and PDGF on femoral artery were detected by RT-PCR; the protein expression of MMP9 and MMP2 were assessed by Western blot on day 0, 1, 3, 7, 14 and 28 post injury. Mice in control group received TNF-α(5 ng/ml, 0.10 ml), TNF-α(0.05 ml)+ MMP inhibitor SB-3CT(0.50 ng/ml, 0.05 ml) injection, or PDGF-bb (10 ng/ml, 0.10 ml)and PDGF-bb(0.05 ml)+ SB-3CT(0.05 ml)injection around injured artery, intimal hyperplasia at 2 and 4 weeks after injury was observed. Intimal hyperplasia at 2 and 4 weeks after injury was also observed in MMP9/2-/- mice. TNF-α(5 ng/ml, 0.10 ml)was injected to MMP2-/- mice, PDGF-bb (0.1 ml) was injected to MMP9-/- mice around injured artery, intimal hyperplasia at 2 and 4 weeks after injury was observed. The degree of neointimal hyperplasia were observed by the Elastica-van Gieson staining and the area of neointima and media of the arteries were measured by SigmaPlot and intima ratio was calculated. Vascular smooth muscle cell (VSMC) mediums of MMP9-/- and MMP2-/- mice were stimulated by TNF-α and PDGF-bb, respectively, and migration assay, and proliferation assay were performed, relative migration and proliferation cells numbers were counted. Results: (1) mRNA expression of TNF-α (235.33±23.68) and PDGF-bb (3.30±0.56) in femoral arteries peaked at 1 day after injury, while MMP9 or MMP2 protein expression peaked at 7 or 28 days after injury. (2)In control mice, TNF-α intervention significantly enhanced intimal hyperplasia at 2 weeks after injury (2.21±0.05 vs. 1.55±0.03 in blank control group, P<0.05), while PDGF-bb intervention significantly enhanced intimal hyperplasia at 4 weeks after injury (2.60±0.07 vs. 1.89±0.04, P=0.03). (3) Intima hyperplasia was significantly higher in control group than in MMP9/2-/- group at 2 weeks (1.63±0.05 vs. 0.46±0.01, P=0.008) and 4 weeks (2.24±0.06 vs. 0.51±0.01) after injury(P=0.005). (4) TNF-α intervention stimulated intimal hyperplasia in MMP2-/-mice (intimal ratio at 2 weeks after injury: 1.73±0.05 vs.1.23±0.03, P=0.02)and PDGF-bb intervention stimulated intimal hyperplasia in MMP9-/-mice(intimal ratio at 4 weeks after injury: 2.32±0.06 vs.1.35±0.03, P=0.03). (5) Reduced VSMC migration was evidenced in MMP9-/- mice post TNF-α stimulation (1.45±0.03 vs. 2.16±0.04 in control group, P=0.03), while reduced VSMC proliferation post PDGF was seen in MMP2-/- group (1.15±0.02 vs.1.82±0.04 in control group, P=0.03). Conclusions: TNF-α induced MMP9 activation plays a major role on promoting VSMC migration at the first 2 weeks after vascular injury, while PDGF induced MMP2 activation plays a crucial role on VSMC proliferation on the following 2 weeks after vascular injury in this mice model. Thus, the axis of TNF-α-MMP9-VSMC migration axis and PDGF-MMP2-VSMC proliferation axis are the two major working mechanisms responsible for intimal hyperplasia post vascular injury.

Potent effects of peracetylated (-)-epigallocatechin-3-gallate against hydrogen peroxide-induced damage in human epidermal melanocytes via attenuation of oxidative stress and apoptosis.

BACKGROUND: Damage to melanocytes induced by oxidative stress plays an important role in the pathogenesis of vitiligo. A polyphenol found in green tea, (-)-epigallocatechin-3-gallate (EGCG), exhibits certain antioxidative effects in the treatment of various diseases. The major problem that limits the clinical application of this polyphenol is its low bioavailability and stability. Peracetylated EGCG (AcEGCG), a fully acetylated derivative of EGCG, is more stable and bioavailable than EGCG, but the effects of its action on human epidermal melanocytes have not been elucidated. AIM: To compare the protective effects of AcEGCG and EGCG on hydrogen peroxide (H2 O2 )-induced damage to human melanocytes. METHODS: Effects of AcEGCG and EGCG on human melanocytes were examined by measuring cell viability, levels of reactive oxygen species (ROS), the mitochondrial membrane potential (ΔΨm)and protein levels of caspase-9, caspase-3 and p38 mitogen-activated protein kinase. RESULTS: Both AcEGCG and EGCG decreased ROS generation, restored lost mitochondrial membrane potential and reduced H2 O2 -induced apoptosis in melanocytes. All of these effects were more pronounced with AcEGCG than with EGCG. Furthermore, AcEGCG effectively suppressed H2 O2 -induced p38 mitogen-activated protein kinase phosphorylation, which has been suggested to contribute to melanocyte damage. CONCLUSIONS: AcEGCG is a more potent agent than EGCG for protection of melanocytes from oxidative damage.

[Effect of radiation dose and dose rate on pulmonary fibrosis in mice].

OBJECTIVE: To study the effect of radiation dose and dose rate on radiation-induced pulmonary fibrosis in mice. METHODS: Twenty-four C57BL/6 mice were randomly divided into a control group (n=6) and an irradiation group(n=18). The irradiation group was further assigned to 3 subgroups according to the whole lung radiation with 15 Gy at 400 cGy/min, 20 Gy at 400 cGy/min and 20 Gy at 100 cGy/min, while the control group received sham-irradiation. All mice were scanned with computed tomograph (CT) 20 weeks post-irradiation, and then they were sacrificed and lung tissues were collected. H&E staining, sirius red staining, lung fibrosis scored and hydroxyproline content analysis were used to assess lung fibrosis and collagen deposition. Real time PCR was used to measure the mRNA expression of type Ⅰ collagen. Immunohistochemical staining was used to detect the activatin and distribution of a-SMA(+) -myofibroblasts. RESULTS: Compared to the control group, mice from irradiation groups exhibited significant pulmonary consolidation and collagen deposition.At the same dose rate, the higher irradiated dose used, the more severe pulmonary fibrosis was.On the other hand, with the same dose, the dose rate had less effect on pulmonary fibrosis. CONCLUSION: The effect of radiation dose on the degree of pulmonary fibrosis in mice is more than effect of the dose rate.

The Brassica rapa FLC homologue FLC2 is a key regulator of flowering time, identified through transcriptional co-expression networks.

The role of many genes and interactions among genes involved in flowering time have been studied extensively in Arabidopsis, and the purpose of this study was to investigate how effectively results obtained with the model species Arabidopsis can be applied to the Brassicacea with often larger and more complex genomes. Brassica rapa represents a very close relative, with its triplicated genome, with subgenomes having evolved by genome fractionation. The question of whether this genome fractionation is a random process, or whether specific genes are preferentially retained, such as flowering time (Ft) genes that play a role in the extreme morphological variation within the B. rapa species (displayed by the diverse morphotypes), is addressed. Data are presented showing that indeed Ft genes are preferentially retained, so the next intriguing question is whether these different orthologues of Arabidopsis Ft genes play similar roles compared with Arabidopsis, and what is the role of these different orthologues in B. rapa. Using a genetical-genomics approach, co-location of flowering quantitative trait loci (QTLs) and expression QTLs (eQTLs) resulted in identification of candidate genes for flowering QTLs and visualization of co-expression networks of Ft genes and flowering time. A major flowering QTL on A02 at the BrFLC2 locus co-localized with cis eQTLs for BrFLC2, BrSSR1, and BrTCP11, and trans eQTLs for the photoperiod gene BrCO and two paralogues of the floral integrator genes BrSOC1 and BrFT. It is concluded that the BrFLC2 Ft gene is a major regulator of flowering time in the studied doubled haploid population.

Peroxisome proliferator-activated receptor δ confers resistance to peroxisome proliferator-activated receptor γ-induced apoptosis in colorectal cancer cells.

Peroxisome proliferator-activated receptor γ (PPARγ) may serve as a useful target for drug development in non-diabetic diseases. However, some colorectal cancer cells are resistant to PPARγ agonists by mechanisms that are poorly understood. Here, we provide the first evidence that elevated PPARδ expression and/or activation of PPARδ antagonize the ability of PPARγ to induce colorectal carcinoma cell death. More importantly, the opposing effects of PPARδ and PPARγ in regulating programmed cell death are mediated by survivin and caspase-3. We found that activation of PPARγ results in decreased survivin expression and increased caspase-3 activity, whereas activation of PPARδ counteracts these effects. Our findings suggest that PPARδ and PPARγ coordinately regulate cancer cell fate by controlling the balance between the cell death and survival and demonstrate that inhibition of PPARδ can reprogram PPARγ ligand-resistant cells to respond to PPARγ agonists.

Analysis of the CDR3 region of alpha/beta T-cell receptors (TCRs) and TCR BD gene double-stranded recombination signal sequence breaks end in peripheral blood mononuclear cells of T-lineage acute lymphoblastic leukemia.

Recently, numerous reports have highlighted the restriction of the CDR3 length of T-cell receptor (TCR) beta chain in T-cells infiltrating solid tumors and hematological malignancies. However, these studies ignored the restriction of CDR3 length of TCR alpha chain and few of them attempted to reveal the mechanisms of the oligo-clonal expansion of T cells in the tumors. The primary aims of this study were twofold to: (i) analyze the CDR3 length of TCR alpha and beta chain in peripheral blood mononuclear cells of T-lineage acute lymphoblastic leukemia (T-ALL); and (ii) discover the relationship between the clonality of T cells and the process of TCR rearrangement in peripheral T cells. To this end, we investigated the TCR BV and TCR AV family spectratypes of two T-ALL patients and healthy controls using the immunoscope spectratyping technique. We found that the spectratypes exhibited a Gaussian distribution in healthy controls. However, the TCR repertoires of the two patients were highly restricted in the number of different TCR BV and TCR AV family members present. Furthermore, we found that the peripheral blood mononuclear cells (PBMC) of two T-ALL patients had the recombination signal sequence (RSS) 5'- and 3'-breaks end in the TCR BD2 gene using a specialized ligation-mediated polymerase chain reaction, implying the ongoing recombination of the TCR beta gene. Analysis of the particular CDR3 length of TCR alpha/beta T cells might be helpful for further study of the individualized therapy of T-ALL. This information will also be helpful in exploring new immunological pathogenesis and facilitating the design of a T-ALL vaccine, as well as in improving our understanding of healthy human T-cell development.

Comparative SAGE analysis of the response to hypoxia in human pulmonary and aortic endothelial cells.

We utilized serial analysis of gene expression (SAGE) to analyze the temporal response of human pulmonary artery endothelial cells (HPAECs) to short-term chronic hypoxia at the level of transcription. Primary cultures of HPAECs were exposed to 1% O2 hypoxia for 8 and 24 h and compared with identical same-passage cells cultured under standard (5% CO2-95% air) conditions. Hierarchical clustering of significant hypoxia-responsive genes identified temporal changes in the expressions of a number of well-described gene families including those encoding proteins involved in thrombosis, stress response, apoptosis, angiogenesis, and cell proliferation. These experiments build on previously published data describing the transcriptomic response of human aortic endothelial cells (HAECs) obtained from the same donor and cultured under identical conditions, and we have thus taken advantage of the immortality of SAGE data to make direct comparisons between these two data sets. This approach revealed comprehensive information relating to the similarities and differences at the level of mRNA expression between HAECs and HPAECs. For example, we found differences in the cell type-specific response to hypoxia among genes encoding cytoskeletal factors, including paxillin, and proteins involved in metabolic energy production, the response to oxidative stress, and vasoreactivity (e.g., endothelin-1). These efforts contribute to the expanding collection of publicly available SAGE data and provide a foundation on which to base further efforts to understand the characteristics of the vascular response to hypoxia in the pulmonary circulation relative to systemic vasculature.

Phosphatidylinositol 3-kinase/Akt pathway mediates heme oxygenase-1 regulation by lipopolysaccharide.

The stress-inducible protein heme oxygenase-1 exerts potent antiinflammatory, antiapoptotic and cytoprotective effects in vitro and in vivo. Another important mediator of cytoprotection, the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway activates many proteins involved in the maintenance of cellular homeostasis. Since activation of heme oxygenase-1 and PI3K/Akt both protect the cellular environment, we postulated that PI3K/Akt can regulate the induction of heme oxygenase-1 by proinflammatory stress. The treatment of primary murine macrophage cells (RAW 264.7) with lipopolysaccharide induced heme oxygenase-1 protein and mRNA expression, and increased the phosphorylation of Akt and p38 mitogen activated protein kinase (p38 MAPK). These cellular effects of lipopolysaccharide were markedly diminished by pre-treatment with wortmannin, a specific inhibitor of PI3K. Furthermore, lipopolysaccharide-inducible heme oxygenase expression was blocked by SB203580, a specific inhibitor of p38 MAPK. Both wortmannin and SB203580 decreased lipopolysaccharide-inducible NF-E2-related factor (Nrf2) DNA binding activity. Transfection of macrophages with dominant negative mutants of PI3K, Akt and Nrf2, as well as wortmannin treatment, significantly reduced the transcriptional activity of a minimal heme oxygenase-1 promoter luciferase construct (D33HO-1luc). We demonstrate, to our knowledge for the first time, that upon proinflammatory stimulation heme oxygenase-1 gene expression in macrophages depends on PI3K/Akt and p38 MAPK acting upstream of Nrf2-dependent promoter activation.

Genome-wide analysis of the endothelial transcriptome under short-term chronic hypoxia.

We have utilized serial analysis of gene expression (SAGE) to analyze the temporal response of human aortic endothelial cells (HAECs) to short-term chronic hypoxia at the level of transcription. Primary cultures of HAECs were exposed to 1% O2 hypoxia for 8 and 24 h and compared with identical same passage cells cultured under standard (5% CO2-95% air) conditions. A total of 121,446 tags representing 37,096 unique tags were sequenced and genes whose expression levels were modulated by hypoxia identified by novel statistical analyses. Hierarchical clustering of genes displaying statistically significant hypoxia-responsive alterations in expression revealed temporal modulation of a number of major functional gene families including those encoding heat shock factors, glycolytic enzymes, extracellular matrix factors, cytoskeletal factors, apoptotic factors, cell cycle regulators and angiogenic factors. Within these families we documented the coordinated modulation of both previously known hypoxia-responsive genes, numerous genes whose expressions have not been previously shown to be altered by hypoxia, tags matching uncharacterized UniGene entries and entirely novel tags with no UniGene match. These preliminary data, which indicate a reduction in cell cycle progression, elevated metabolic stress and increased cytoskeletal remodeling under acute hypoxic stress, provide a foundation for further analyses of the molecular mechanisms underlying the endothelial response to short-term chronic hypoxia.

C-Terminus of desmoyokin/AHNAK protein is responsible for its translocation between the nucleus and cytoplasm.

We previously demonstrated that desmoyokin gene is identical to AHNAK gene, which is downregulated in neuroblastomas. Whereas desmoyokin/AHNAK protein is distributed in the nucleus and cytoplasm in nonepithelial tissues, it is distributed in the cell membrane in epithelial tissues. It is present diffusely in the cytoplasm and nucleus of epithelial cell lines cultured in low calcium condition. Low to normal calcium shift translocates it to the cell boundary. In this study, we investigated which domain(s) of desmoyokin/AHNAK protein are responsible for its different distribution. We constructed three different eukaryotic expression plasmids, pN-DY, pM-DY, and pC-DY, which expressed N-terminus, central domain, and C-terminus of this molecule, respectively, when transfected into COS-7 cells, normal human keratinocytes, and HeLa cells. In normal calcium medium, whereas N-terminus and central domain of desmoyokin/AHNAK protein were present mainly in the cytoplasm, C-terminus was present in the nucleus, cytoplasm, and weakly in the cell membrane. In low calcium medium, C-terminus was present exclusively in the nucleus, and a part of the molecules translocated from the nucleus to the cytoplasm, 3 h after the shift to normal calcium medium or 3 h after addition of protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate in low calcium medium. Calcium shift showed no effects on the distribution of N-terminus and central domain. These results suggested that C-terminus, but neither N-terminus nor central domain, is responsible for the translocation of this protein into the nucleus. This study may also suggest that C-terminus play a role in the translocation to the cell membrane, although further evidence is necessary.

[Phacoemulsification through a small pupil].

OBJECTIVE: To discuss the surgical techniques and attention points in phacoemulsification through a small pupil. METHOD: Eighty three cataracts of 77 patients were emulsified by using non-cut pupil dilation, cystotome or diathermic high-frequency capsulorrhexis. RESULTS: The 66 cataracts of 83 eyes were emulsified by means of the above method. After the surgery, all the pupils were recovered to normal size (2 to 3 mm), and none of them were damaged. In 17 eyes, the procedure was performed after separation of synechia and excision of the organized membranes; after the surgery, a round pupil was obtained in 15 cases and an irregular pupil in 2 cases. The visual acuity at postoperative 1 month was >or= 0.5 in 46 eyes (55.4%) and corrected >or= 0.5 in 71 eyes (85.5%). CONCLUSION: After phacoemulsification through a small pupil by non-cut pupil dilation method, the pupil can be recovered to normal and no unfavorable reaction is seen.