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.

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.

[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.

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.

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.

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.

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.

Dipolar filtered 1H-13C heteronuclear correlation spectroscopy for resonance assignment of proteins.

Resonance assignment is necessary for the comprehensive structure determination of insoluble proteins by solid-state NMR spectroscopy. While various 2D and 3D correlation techniques involving 13C and 15N spins have been developed for this purpose, H chemical shift has not been exploited sufficiently. We demonstrate the combination of the regular 1H-13C heteronuclear correlation (HETCOR) experiment and a dipolar filtered HETCOR technique to obtain better resolved 1H chemical shift spectra. The dipolar filtered experiment, MELODI-HETCOR. simplifies the 1H spectra by suppressing the directly bonded C-H correlation peaks and retaining only the medium- and long-range cross peaks. We apply this MELODI-HETCOR technique to several amino acids and proteins with various isotopic labeling patterns. The enhanced 1H chemical shift resolution allows the assignment of overlapping H alpha and H beta resonances in Ser, identifies the 1H chemical shift differences between neutral and cationic imidazole rings of His, and permits the assignment of residues with side chain nitrogen atoms in ubiquitin. The potential utility of this dipolar filtered HETCOR technique to resonance assignment of extensively labeled proteins is discussed.

Vasopressin-activated calcium-mobilizing (VACM-1) receptor mRNA is present in peripheral organs and the central nervous system of the laboratory rat.

The distribution and physiological role of the neuropeptide, arginine vasopressin (AVP), and its three receptor subtypes, V1a, V1b and V2, has been well described. A fourth AVP receptor, VACM-1, was recently discovered and appears to be a member of the cullin gene family. The objective of this research is to characterize VACM-1 receptor mRNA expression in the CNS as well as in various tissues and organs of the laboratory rat. Northern blotting of poly(A) + RNA from various tissues demonstrated the size of VACM-1 MRNA in the rat is approximately 6.3 kb. RT-PCR indicated the transcript is present in all twelve tissues examined: brainstem, cerebral cortex, cerebellum, hypothalamus, aorta, gastrointestinal tract, heart, kidney medulla, liver, lung, skeletal muscle, and spleen. Quantitative realtime PCR confirmed RT-PCR results that VACM-1 mRNA is in all organs and tissues and expression levels are similar in all tissues examined. The transcript encoding VACM-1, a novel AVP receptor, appears to be ubiquitously expressed in various tissues of the laboratory rat. VACM-1 shares some similarities with both V1 and V2 receptors, as it binds AVP analogues that independently recognized either of these receptors. Therefore, many functions ascribed to activation of the previously known AVP receptors could at least in part be mediated by VACM-1.

Retinoic acid reduces p11 protein levels in bronchial epithelial cells by a posttranslational mechanism.

p11 is a member of the S100 family of proteins, is the cellular ligand of annexin II, and interacts with the carboxyl region of 85-kDa cytosolic phospholipase A(2) (cPLA(2)), inhibiting cPLA(2) activity and arachidonic acid (AA) release. We studied the effect of retinoic acid (RA) on PLA(2) activity in human bronchial epithelial cells and whether p11 contributes to these effects. The addition of 10(-6) M RA resulted in reduced p11 protein levels at 4 days, with the greatest effect observed on days 6 and 7. This effect was dose related (10(-6) to 10(-9) M). RA treatment (10(-6) M) had no effect on cPLA(2) protein levels. p11 mRNA levels were unchanged at 6 and 8 days of treatment (correlating with maximum p11 protein reduction). Treatment with RA reduced p11 levels in control cells and in cells transfected with a p11 expression vector, suggesting a posttranslational mechanism. Lactacystin (10(-6) M), an inhibitor of the human 26S proteasome, blocked the decrease in p11 observed with RA treatment. Compared with control cells (n = 3), RA-treated cells (n = 3) had significantly increased AA release after treatment with the calcium ionophore A-23187 (P = 0.006). Therefore, RA reduces p11 protein expression and increases PLA(2) activity and AA release.

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.

Dexamethasone alters arachidonate release from human epithelial cells by induction of p11 protein synthesis and inhibition of phospholipase A2 activity.

The effect of the glucocorticosteroid, dexamethasone, on arachidonic acid (AA) release and on protein levels of p11 and cytosolic phospholipase A2 (cPLA2) was studied in two epithelial cell lines, HeLa cells and BEAS-2B cells. Dexamethasone treatment of HeLa cells and BEAS-2B cells increased cellular p11 protein and mRNA levels in a time- and dose-dependent manner. It had little effect on levels of cPLA2 protein. In order to determine if increased p11 protein expression resulted in increased interaction between p11 and cPLA2, anti-cPLA2 antibodies were used to immunoprecipitate p11.cPLA2 complexes and Western blots of the immunoprecipitate were used to detect p11. In cells treated with dexamethasone, more p11 was detected in the anti-cPLA2 immunoprecipitate compared with control cells. Dexamethasone treatment of HeLa cells prelabeled with [3H]AA decreased the release of [3H]AA under basal conditions and after stimulation with the calcium ionophore A23187 (10(-6) M). In order to determine if altering the p11 protein levels in HeLa cells independent of glucocorticosteroid treatment could also produce an effect on [3H]AA release, cells were stably transfected with plasmids expressing either p11 antisense mRNA or p11 mRNA. Cloned HeLa cells expressing p11 antisense mRNA exhibited less cellular p11 protein compared with control cells and greater [3H]AA release compared with cells transfected with a control vector. Cloned HeLa cells stably transfected with a p11 expression vector exhibited increased p11 cellular protein and diminished [3H]AA release under basal conditions and in response to A23187. Therefore, dexamethasone alteration of epithelial cell AA release may be due in part to induction of p11 protein expression.

Tumor necrosis factor-alpha stimulates human Clara cell secretory protein production by human airway epithelial cells.

Clara cell secretory protein (CCSP), or CC10, is an inhibitor of secretory phospholipase A2 which may be produced by phagocytic cells and by a variety of other cells in the airway. Tumor necrosis factor-alpha (TNF-alpha) is capable of activating phospholipases and inducing the expression of a variety of genes in the airway epithelium which may modulate the airway inflammatory response. Therefore, it was of interest to determine whether this proinflammatory cytokine could induce the production of a counterregulatory protein such as CCSP which might modulate the production of eicosanoid mediators in the airway. Using a human bronchial epithelial cell line (BEAS-2B), CCSP messenger RNA (mRNA) levels were detected by ribonuclease protection assay. TNF treatment of these cells increased CCSP mRNA levels in a time- and dose-dependent manner. The CCSP mRNA level increased in response to TNF-alpha (20 ng/ml) stimulation after 8 to 36 h with the peak increase at 18 h. Immunoblotting of CCSP protein released into the culture media demonstrated that TNF-alpha induced the synthesis and secretion of CCSP protein in a time-dependent manner over 8 to 18 h. The results of a CCSP reporter gene activity assay, nuclear run-on assay, and CCSP mRNA half-life assay indicated that the TNF-alpha-induced increases in CCSP gene expression are regulated at the post-transcriptional level. We conclude that TNF-alpha induces airway epithelial cell expression of human CCSP protein and may modulate airway inflammatory responses in this manner.

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.

Interferon-gamma stimulates human Clara cell secretory protein production by human airway epithelial cells.

Clara cell secretory protein (CCSP) is an inhibitor of secretory phospholipase A2. It is produced by airway epithelial cells and is present in airway secretions. Because interferon (IFN)-gamma can induce gene expression in airway epithelial cells and may modulate the inflammatory response in the airway, it was of interest to study the effect of this cytokine on epithelial cell CCSP mRNA expression and CCSP protein synthesis. A human bronchial epithelial cell line (BEAS-2B) was used for this study. CCSP mRNA was detected by ribonuclease protection assay. IFN-gamma was found to increase CCSP mRNA expression in a time- and dose-dependent manner. The CCSP mRNA level increased after IFN-gamma (300 U/ml) treatment for 8-36 h, with the peak increase at 18 h. Immunobloting of CCSP protein also demonstrated that IFN-gamma induced the synthesis and secretion of CCSP protein in a time-dependent manner. Nuclear run-on, CCSP reporter gene activity assay, and CCSP mRNA half-life assay demonstrated that IFN-gamma-induced increases in CCSP gene expression were mediated, at least in part, at the posttranscriptional level. The present study demonstrates that IFN-gamma can induce increases in steady-state mRNA levels and protein synthesis of human CCSP protein in airway epithelial cells and may modulate airway inflammatory responses in this manner.

P11, a unique member of the S100 family of calcium-binding proteins, interacts with and inhibits the activity of the 85-kDa cytosolic phospholipase A2.

Using a two hybrid system screen of a human cDNA library, we have found that p11, a unique member of the S100 family of calcium-binding proteins, interacts with the carboxyl region of the 85-kDa cytosolic phospholipase A2 (cPLA2). p11 synthesized in a cell-free system interacts with cPLA2 in vitro. The p11-cPLA2 complex is detectable from a human bronchial epithelial cell line (BEAS 2B). Furthermore, p11 inhibits cPLA2 activity in vitro. Selective inhibition of p11 expression in the BEAS 2B cells by antisense RNA results in an increased PLA2 activity as well as an increased release of prelabeled arachidonic acid. This study demonstrates a novel mechanism for the regulation of cPLA2 by an S100 protein.