Acute nutrient treatment causes alterations in intra-follicular antioxidation and AKT signaling.

This study aimed to determine if short-term nutrient alteration affects (1) ovarian morphology, (2) plasma and ovarian antioxidant capability and (3) cell apoptosis and AKT signaling within the ovary. After estrus synchronization, 24 Hu sheep were assigned to three groups based on the nutrient requirement recommended for maintenance (M): 1 × M (Control), 1.5 × M (S) and 0.5 × M (R) during days 7-14 of their estrous cycle. The results indicated that undernourishment significantly increased the counts and volume of follicles <2.5 mm and decreased the counts and volume of follicles ≥2.5 mm (P < 0.05). Feed restriction altered the plasma and follicular redox balance within follicles ≥2.5 mm by inhibiting total antioxidant capacity, increasing malondialdehyde concentration (P < 0.05) and reducing the mRNA expression levels of superoxide dismutase 2 (SOD2) and glutathione peroxidase (GSH-PX), as well as the activities of total SOD and GSH-PX. Feed restriction also attenuated B-cell lymphoma-2 (BCL2) but enhanced Bcl-2-associated X protein (BAX) and BAX/BCL2 transcription and translation levels in granulosa cells (P < 0.05). Uniform staining intensities of AKT and P-AKT-Ser473 were observed in each follicle stage, whereas weaker P-AKT-Thr308 staining in the antral follicle than in the pre-antral follicle suggested possible involvement of P-AKT-Thr308 during the beginning of follicle development. P-AKT-Ser473 levels in follicles ≥2.5 mm was significantly reduced in the R group (P < 0.05). The results presented in this study demonstrate that suppressed folliculogenesis caused by feed restriction might be associated with attenuated AKT signaling, reduced follicular antioxidant capacity and enhanced granulosa cells apoptosis.

Study on physiological and psychological comprehensive nursing of elderly tumor patients after surgery.

The aim of this study is to formulate nursing schemes for elderly tumor patients after surgery according to their clinical characteristics, and give effective guidance for alleviating the patients’ psychological anxiety. One hundred elderly tumor patients admitted to the oncology department of the Affiliated Cancer Hospital of Harbin Medical University were included and divided into an intervention group (50) and a control group (50). Nursing intervention was performed on the intervention group, and routine nursing was performed in the control group. One day before surgery, all the patients were asked to fill in a self-rating anxiety scale (SAS) and a self-rating depression scale (SDS), and their blood pressure and heart rate data were measured. After surgery, the patients were asked to fill in a form which investigated their pain degree, recovery situation and satisfaction degree. The heart rate and blood pressure of the patients in the intervention group recovered faster than those of the control group, with lower SAS and SDS scores and shorter recovery time. In conclusion, effective nursing intervention played a crucial role in the postoperative recovery of elderly tumor patients by reducing pain and anxiety degrees, which improved the patients’ satisfaction with the nursing.

[Clinicopathological characteristics and prognosis of different molecular types of breast cancer].

OBJECTIVE: To investigate the clinicopathological characteristics of 4 subtypes of breast cancer, Luminal A, Luminal B, human epidermal growth factor receptor 2(HER2), triple-negative and their associated prognostic factors. METHODS: The clinical characteristics and prognosis of 102 patients with breast cancer who treated in Wuhan University Renmin Hospital from January 2011 to November 2012 were retrospectively analyzed. RESULTS: The proportion of each subtype was Luminal A 27.5%, Luminal B 35.3%, HER2 14.7%, and triple-negative 22.5%. The age range was from 22 to 75 years old, with median age of 48.0 years old. All patients underwent surgery and 98 cases were also treated with chemotherapy/endocrine+ Herceptin therapy (96.1%)after surgery. The tumor size, histological grade, and lymph node metastasis had significant difference in different subtypes of breast cancer (P<0.05). Further analysis showed the proportion of HER2 tumor≤2 cm (4, 26.7%)had a tendency of smaller than that in luminal A(20, 71.4%) or triple negative(16, 69.6%) (P=0.009>0.008, P=0.019>0.008). Histological Ⅲ grade proportion in patients with HER2 subtype (8, 53.3%) was in greater tendency than that with Luminal A subtype(23, 82.1%) (P=0.039>0.008). The proportion of patients with no metastatic lymph nodes in triple negative subtype(7, 30.4%) had a tendency of smaller than that in HER2 subtype(7, 46.7%) or Luminal B subtype(17, 47.2%)(P=0.019>0.008, P=0.016>0.008). There were no significant differences in age of onset, menstruation status, operation, family cancer history, and the risk of recurrence and metastasis in different subtypes (P>0.05). In Luminal A subtype, three-years overall survival rate was 100%, and one-, two-, three-year event-free survival rate were all 92.9%. In Luminal B subtype, three-year overall survival rate was 100%, event-free survival rate at one-, two- and three-year were all 97.2%. In HER2 subtype, three-year overall survival rate was 100%, one-, two- and three-year event-free survival rate were 100%, 100%, and 93.3%, respectively. The 1, 2, 3 years overall survival rate of triple-negative subtype were 100%, 95.7%, and 95.7%, respectively. One-, two- and three-year event-free survival rate of triple-negative subtype were 95.7%, 91.3%, and 90.0% respectively. Whether triple negative subtype, recurrence risk, age of onset, menstrual status, tumor size, histological grade, lymph node metastasis, surgery, breast cancer or other family history of cancer were not significantly related with three-year event-free survival rate (P>0.05). CONCLUSIONS: Luminal subtype had the largest proportion and better prognosis. The proportion of tumor >2 cm and histological grade Ⅲ in patients with HER2 subtype was larger, and three-year event-free survival rate of this patients was lower. Triple-negative subtype had a greater tendency of lymph node metastasis and the lowest three-year event-free survival. Tumor size, histological grade and lymph node metastasis may be the important factors for the prognosis of breast cancer.

Role of mechanical strain and estrogen in modulating osteogenic differentiation of mesenchymal stem cells (MSCs) from normal and ovariectomized rats.

Bone's adaptability to loading depends upon the process of bone remodeling. This adaptive mechanism is restricted in postmenopausal osteoporosis. Differentiation of mesenchymal stem cells (MSCs) is crucial to bone remodeling and regeneration. It is well accepted that mechanical loading influences the fate of MSC differentiation. The aim of this study was to explore the possible restricted mechanism in osteoporotic condition, through investigating response of MSCs from both sham-operated and ovariectomized rats. MSCs were exposed to estrogen and mechanical strain (2%, 1Hz, 6h/day) for 3 days. Osteogenic differentiation and ß-catenin protein in MSCs were examined. Exposure to estrogen and mechanical strain alone enhanced expression of Runx2 (Cbfα1), type I collagen (ColI) and activated ß-catenin protein in MSCs from both sham-operated and OVX rats. MSCs from both sham-operated and OVX rats stimulated with both mechanical strain and estrogen had higher expression of osteogenic genes and activated ß-catenin protein than these cells exposed to estrogen and mechanical strain alone. Osteoporotic MSCs had lower expression of osteogenic genes and protein in the absence and presence of stimulation than did MSCs from sham-operated rats. Cumulatively, our results indicate that mechanical strain and estrogen in vitro enhance osteogenic potential and activation of ß-catenin in MSCs from both sham-operated and OVX rats. Estrogen augments strain-induced osteogenic potential and activity of ß-catenin in MSCs.

Functional role of NKX2-5 and Smad6 expression in developing rheumatic heart disease.

OBJECTIVE: Rheumatic heart disease (RHD) results due to the cross reaction of the host immune system when it develops immunity against group A streptococcal infection. This autoimmune disease progress with different pathological conditions and the genes associated with it are still less understood. MATERIALS AND METHODS: To understand the role of NKX2-5 and Smad-6 in developing an RHD, we successfully developed RHD model using BALB/c mice and we evaluate the expression of NKX2-5 and Smad-6 in different conditions. RESULTS: The disease conditions are confirmed through histological sectioning of RHD heart tissue with its associated Aschoff bodies. The histological of control heart tissue in the absence of NKX2-5 looks abnormal with an enlarged nucleus and in the absence of Smad-6 the solid nature of heart tissue loosens. The mice developed a complex form of acute RHD with tissue hardening in the absence of either NKX2-5 or Smad-6 which are confirmed in NKX2-5 or Smad-6 null mice. Immunohistochemical studies reveal that the NKX2-5 and Smad-6 expression get down regulated on developing with RHD. Through experiments, we detected that both Nkx2-5 and Smad-6 are both inter-dependable and it negatively regulated each other by inhibiting them. In the absence of NKX2-5 or Smad-6, a severe form of RHD is observed together with down-regulation of either NKX2-5 or Smad-6. CONCLUSIONS: The present investigation of NKX2-5 and Smad-6 in RHD provides a new insight of data that helps to understand the disease pathogenesis.

Intrafollicular expression and potential regulatory role of cocaine- and amphetamine-regulated transcript in the ovine ovary.

Follicular growth is regulated by a complex interaction of pituitary gonadotropins with local regulatory molecules. Previous studies demonstrated an important role for cocaine- and amphetamine-regulated transcript (CART) in regulation of granulosa cell estradiol production associated with dominant follicle selection in cattle. However, intraovarian expression and actions of CART in other species, including sheep, are not known. The objective of this study was to investigate the expression of CART in sheep follicles and determine the effects of CART on indices of ovine granulosa cell function linked to follicular development. Results demonstrated the expression of CART messenger RNA and prominent intraovarian localization of CART peptide in granulosa cells of sheep follicles. Granulosa cell CART messenger RNA was lower, but follicular fluid estradiol concentrations were higher in large (>5 mm) follicles vs smaller 3- to 5-mm follicles harvested from sheep ovaries of abattoir origin. CART treatment inhibited follicle stimulating hormone-induced estradiol production by cultured ovine granulosal cells and also blocked the follicle stimulating hormone-induced increase in granulosa cell numbers. Results demonstrate expression of CART in sheep follicular tissues and suggest potential biological actions of CART, which are inhibitory to ovine follicular growth and development.

Leukemia inhibitory factor induces the 85-kDa cytosolic phospholipase A2 gene expression in cultured human bronchial epithelial cells.

Leukemia inhibitory factor (LIF) has become increasingly recognized as an important regulator of inflammation. This study is designed to determine whether LIF has an effect on arachidonate metabolism in human airway epithelial cells. LIF (100 ng/ml) induced a significantly increased release of prelabeled [3H] arachidonic acid (AA) from the human bronchial epithelial cell line (BEAS 2B cell) as well as from the primary cultures of human bronchial epithelial cells. Exposure of the LIF stimulated BEAS 2B cells to calcium ionophore A23187 (10(-5) M, 15 min) caused a further increase of [3H]AA release. To identify the role of cytosolic phospholipase A2 (cPLA2) in this upregulation of AA release, further experiments were performed to determine the expression of cPLA2 in the BEAS 2B cells. Immunoblot analysis indicated that LIF increased cPLA2 protein expression. Ribonuclease protection assay showed that LIF induced an increase of cPLA2 mRNA levels following 3 h to 24 h treatment. Nuclear run-on experiments suggested that LIF upregulated cPLA2 gene expression through post-translational regulation. These results demonstrate that LIF induces cPLA2 gene expression and modulates arachidonate metabolism in airway epithelial cells.

An Arabidopsis embryonic lethal mutant with reduced expression of alanyl-tRNA synthetase gene.

In present paper, one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant. The embryo development of this mutant is arrested in globular stage. The cell division pattern is abnormal during early embryogenesis and results in disturbed cellular differentiation. Most of mutant embryos are finally degenerated and aborted in globular stage. However, a few of them still can germinate in agar plate and produce seedlings with shorter hypocotyl and distorted shoot meristem. To understand the molecular basis of the phenotype of this mutant, the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening. According to the sequence analysis and similarity searching, a 936 bp cDNA sequence (EMBL accession #: Y12555) from selected positive clone shows a 99.8% (923/925bp) sequence homology with Alanyl-tRNA Synthetase (AlaRS) gene of Arabidopsis thaliana. Furthermore, the data of in situ hybridization experiment indicate that the expression of AlaRS gene is weak in early embryogenesis and declines along with globular embryo 'development' in this mutant. Accordingly, the reduced expression of AlaRS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.

Cytogenetic and molecular identification of a de novo direct duplication of the long arm of chromosome 4(q21.3-->q31.3).

We report on a 3-year-old boy with moderate developmental retardation, microcephaly, and malformations of ears, lids, mouth, and thumbs. Cytogenetic analysis demonstrated a direct duplication of chromosome subregion 4(q21.3-->q31.3). Confirmation of this specific rearrangement was performed by fluorescent in situ hybridization (FISH) with a chromosome painting probe and by means of quantitative Southern hybridization with DNA probes localized within the chromosome 4 region presumed to be duplicated.

Transmission electron microscopy of chromosomes by longitudinal section preparation: application to fragile X chromosome analysis.

We developed a method for the preparation of ultrathin longitudinal sections of chromosomes enabling TEM studies of whole chromosomes. By using a novel "repeat chill" method of exposing the glass slide and plastic block interface to liquid nitrogen, it was possible to separate consistently hardened epoxy resin-embedded chromosome spreads from glass slides for ultrathin longitudinal sectioning of entire spreads and of individual chromosomes. The method was applied to analyze the fragile X chromosome. The ultrastructure of centromeres, telomeres, fragile sites and other chromosomal areas can now be studied in detail.

Tumor necrosis factor alpha stimulation of human Clara cell secretory protein production by human airway epithelial cells.

Clara cell secretory protein (CCSP) or uteroglobin/CC10 is a product of epithelial cells in a variety of organs including the lung. CCSP has anti-inflammatory properties and may act as an inhibitor of secretory phospholipase A2's. Tumor necrosis factor alpha (TNF-alpha) is capable of inducing the expression of gene products including a variety of cytokines and chemokines in the airway epithelium that may upregulate the airway inflammatory response. Therefore, it was of interest to determine whether this proinflammatory cytokine might also induce the production of a counterregulatory protein such as CCSP, which might modulate the inflammatory response in the airway. Normal human tracheobronchial epithelial cells in primary culture and a human bronchial epithelial cell line (BEAS-2B) were studied. CCSP mRNA levels in BEAS-2B cells were detected by ribonuclease protection assay. CCSP mRNA levels increased in response to TNF-alpha (20 ng/mL) stimulation after 8-36 h, with the peak increase at 18 h. Immunoblotting of CCSP released from BEAS-2B cells into the culture media demonstrated that TNF-alpha induced the synthesis and secretion of CCSP over 8 to 18 h. Similarly, TNF stimulated the release of CCSP from human tracheobronchial epithelial cells in primary culture at 8 and 18 h. The CCSP reporter gene including 801 bases 5' of the transcription start site did not increase transcriptional activity in response to TNF-alpha stimulation. A CCSP mRNA half-life assay indicated that TNF-alpha induced increases in CCSP mRNA at least in part at a posttranscriptional level. Therefore, TNF-alpha induces airway epithelial cell expression of human CCSP and may modulate airway inflammatory responses in this manner.

Characterization of a strain of cerebral endothelial cells derived from goat brain which retain their differentiated traits after long-term passage.

A strain of cerebral endothelial cells was established from isolated cortical microvessels of caprine brain. These cells, which are referred to as EC1 cells, can be routinely subcultured to 32 passages without the loss of differentiated morphologic and immunologic traits. The ability to routinely subculture EC1 cells is an important asset, given that isolated cerebral endothelial cells in mammals generally lose their differentiated traits after only 2 to 3 passages. EC1 cells were shown to contain Factor VIII-related antigen, which is a specific marker for cells of endothelial origin. EC1 cells morphologically demonstrated a scarcity of pinocytotic vesicles on their apical surfaces, a lack of trans-cytoplasmic vesicles, and the ability to form in culture confluent monolayers with tight junctional complexes. Therefore, EC1 cells possess specific antigenic and ultrastructural features which classify them as being small vessel endothelial cells of the blood-brain barrier type. Cytogenetic evaluation of EC1 cells demonstrated a normal female goat 60,XX karyotype and confirmed the apparent non-transformed nature of EC1 cells due to the lack of chromosome abnormalities or rearrangements. Using scanning electron microscopy, EC1 cells were also shown to form confluent monolayers on mixed nitrocellulose filters, a feature that will enable the development of an in vitro system to study trans-endothelial transport. Given that EC1 cells are readily subcultured and grow well on nitrocellulose filters, and that they resemble cerebral endothelium in vivo, it seems evident that EC1 cells can be used as a versatile model for the study of blood-brain barrier function, regulation, and pathology.

Surgical treatment of atherosclerotic infrarenal abdominal aortic aneurysms. A review of 30 years' experience.

During the 30 years from 1960 to 1989, 100 sequential patients with atherosclerotic infrarenal abdominal aortic aneurysm (AAA) were admitted to Zhongshan Hospital. Twenty-eight were not treated surgically and 72 underwent resection of the AAA with prosthesis replacement. Nineteen non-surgical patient were followed up, and 8 died from ruptures with a five-year survival rate of 41.3 +/- 13%. In 72 surgically treated patients, the operative mortality was 2.8%. The five-year survival rate is 77.5 +/- 6.2%, which is higher in comparison with the non-surgical group (P < 0.01). The data show that the operation for AAA is safe and the postoperative long-term results are satisfactory.

Activation of ß-catenin stimulated by mechanical strain and estrogen requires estrogen receptor in mesenchymal stem cells (MSCs).

BACKGROUND AND OBJECTIVES: Mechanical stimulation and hormones act via interconnected signaling pathways to influence the function of bone cells. Estrogen receptor (ER) and ß-catenin play important role in bone formation and have implicated in mechanotransduction in bone cells. To investigate the interaction between mechanotransduction and estrogenic signaling in mesenchymal stem cells (MSCs), this study examined the effect of mechanical strain and estrogen on activation of ß-catenin in MSCs, and the role of ER in response to mechanical strain and estrogen in MSCs. MATERIALS AND METHODS: MSCs were exposed to mechanical strain (2%, 1 Hz) and estrogen (100 nM). The ER inhibitor, ICI182,780 was used to assess the role of ER in activation of ß-catenin stimulated by mechanical strain and estrogen. Changes of activated ß-catenin in the nuclei were determined by immunoflourescent test. The expression of ß-catenin was detected by western blotting. RESULTS: Mechanical strain and estrogen augment, respectively, activation of ß-catenin and accumulation of activated ß-catenin in the nuclei of MSCs. Combined treatment with estrogen and mechanical strain had higher levels of activated ß-catenin than the cells exposed to mechanical strain or estrogen. After MSCs were pre-treated by ICI182,780, the level of activated ß-catenin expression induced by mechanical strain or estrogen was depressed. Meanwhile, ICI182,780 blocked effect of combined stimulation on activation of ß-catenin in MSCs. CONCLUSIONS: Our study demonstrates that mechanical strain and estrogen both promote the levels of activated ß-catenin in MSCs. Estrogen receptor implicates in activation of ß-catenin stimulation by mechanical strain and estrogen in MSCs.

Current targeting strategies for adenovirus vectors in cancer gene therapy.

Adenovirus vectors (Adv) are the most frequently used vectors in gene therapy research, especially in cancer gene therapy. However, despite encouraging preclinical and early clinical results, the successful clinical utility of gene therapy has not yet been fully realized. Challenges to clinical trial success for targeted Adv include inefficient Adv-mediated gene transfer (because many tumor cells lack Adv receptors), poor transduction in tumor tissues after systemic administration, accumulation and undesirable transgene expression in the liver. This review summarizes current targeting strategies for Adv to overcome these obstacles. Strategies include transductional selectivity through genetic modification of viral coat proteins, transcriptional selectivity by means of tumor-specific promoters, and selective biodistribution from conjugation with targeting ligands or polymers such as polyethylene glycol (PEG). Furthermore, combining selective biodistribution and active targeting ligands such as proteins, antibodies and peptides is an intriguing and promising approach that will also be covered in this review. These studies have provided new insights into our understanding of the utility of Adv in cancer gene therapy.

Effects of anionic lipid and ion concentrations on the topology and segmental mobility of colicin Ia channel domain from solid-state NMR.

Channel-forming colicins are bacterial toxins that spontaneously insert into the inner cell membrane of sensitive bacteria to form voltage-gated ion channels. It has been shown that the channel current and the conformational flexibility of colicin E1 channel domain depend on the membrane surface potential, which is regulated by the anionic lipid content and the ion concentration. To better understand the dependence of colicin structure and dynamics on the membrane surface potential, we have used solid-state NMR to investigate the topology and segmental motion of the closed state of colicin Ia channel-forming domain in membranes of different anionic lipid contents and ion concentrations. Colicin Ia channel domain was reconstituted into membranes with different POPG and KCl concentrations. 1H spin diffusion experiments indicate that the protein contains a small domain that inserts into the hydrophobic center of the 70% anionic membrane, similar to when it binds to the 25% anionic membrane. Measurements of C-H and N-H dipolar couplings indicate that, on the sub-microsecond time scale, the protein has the least segmental mobility under the high-salt and low-anionic lipid condition, which has the most physiological membrane surface potential. Measurement of millisecond time scale motions yielded similar results. These suggest that optimal channel activity requires the protein to have sufficient segmental rigidity so that entire helices can undergo cooperative conformational motions that are required for translocating the channel-forming helices across the lipid bilayer upon voltage activation.

Structure distribution in an elastin-mimetic peptide (VPGVG)3 investigated by solid-state NMR.

Elastin is an extracellular-matrix protein that imparts elasticity to tissues. We have used solid-state NMR to determine a number of distances and torsion angles in an elastin-mimetic peptide, (VPGVG)3, to understand the structural basis of elasticity. C-H and C-N distances between the V6 carbonyl and the V9 amide segment were measured using 13C-15N and 13C-1H rotational-echo double-resonance experiments. The results indicate the coexistence of two types of intramolecular distances: a third of the molecules have short C-H and C-N distances of 3.3 +/- 0.2 and 4.3 +/- 0.2 A, respectively, while the rest have longer distances of about 7 A. Complementing the distance constraints, we measured the (phi, psi ) torsion angles of the central pentameric unit using dipolar correlation NMR. The -angles of P7 and G8 are predominantly ~150, thus restricting the majority of the peptide to be extended. Combining all torsion angles measured for the five residues, the G8 C chemical shift, and the V6-V9 distances, we obtained a bimodal structure distribution for the PG residues in VPGVG. The minor form is a compact structure with a V6-V9 C=O-HN hydrogen bond and can be either a type II -turn or a previously unidentified turn with Pro (phi = -70, psi= 20 +/- 20) and Gly ( phi= -100 +/- 20, psi = -20 +/- 20). The major form is an extended and distorted beta-strand without a V6-V9 hydrogen bond and differs from the ideal parallel and antiparallel beta-strands. The other three residues in the VPGVG unit mainly adopt antiparallel beta-sheet torsion angles. Since (VPGVG)3 has the same 13C and 15N isotropic and anisotropic chemical shifts as the elastin-mimetic protein (VPGXG)n (X = V and K, n = 195), the observed conformational distribution around Pro and Gly sheds light on the molecular mechanism of elastin elasticity.