Aquaporin belongs to a highly conserved group of membrane proteins called major intrinsic proteins (MIPs) that facilitate water transport across biological membranes. Aquaporins are membrane water channels that play critical roles in controlling the water content of cells and tissues. We focused on GhPIP1;2 which belongs to the PIP subfamily and GhgammaTIP1 which belongs to the gammaTIP group of the TIP subfamily. Northern blot analysis with gene-specific probes and real-time PCR demonstrated that GhPIP1;2 and GhgammaTIP1 are predominantly expressed during cotton fiber elongation, with the highest expression levels at 5 days post anthesis. The high and preferential expression of GhPIP1;2 and GhgammaTIP1 suggests that they may play important roles in supporting the rapid influx of water into vacuoles during cotton fiber cell expansion. Also, the effects of Ca2+ on aquaporins in salinity-stressed plants were studied. Researchers treated the protoplasts and plasma membrane with NaCl or CaCl2, alone or in combination. Under saline conditions, osmotic water permeability (Pf) values decreased in protoplasts and plasma membrane vesicles, and the same reduction was observed in the PIP1 aquaporin abundance, indicating inhibitory effects of NaCl on aquaporin functionality and protein abundance. Two different actions of Ca2+ were observed. Increase in free cytosolic calcium concentrations associated with stress perception may lead to aquaporin closure, however, the extra-calcium would lead to an upregulation of aquaporins. Meanwhile, experiments have demonstrated HvPIP2;1, one of barley aquaporins, has a higher water and CO2 transport activity. The goal of our plant aquaporin research is to determine the key aquaporin species responsible for water and CO2 transport, and to improve plant water relations, stress tolerance, CO2 uptake or assimilation, and plant productivity.
Aquaporins ; physiology ; Cell Enlargement ; Cotton Fiber ; Gossypium ; metabolism ; physiology ; Photosynthesis ; physiology ; Plant Proteins ; physiology ; Sodium Chloride ; pharmacology ; Stress, Physiological ; physiology

OBJECTIVETo study large-scale expansion of SD (Sprague-Dawley) rat's osteoblasts in suspension culture in a rotating wall vessel bioreactor (RWVB).

METHODSThe bioreactor rotation speeds were adjusted in the range of 0 to 20 rpm, which could provide low shear on the microcarriers around 1 dyn/cm2. The cells were isolated via sequential digestions of neonatal (less than 3 days old) SD rat calvaria. After the primary culture and several passages, the cells were seeded onto the microcarriers and cultivated in T-flask, spinner flask and RWVB respectively. During the culture period, the cells were counted and observed under the inverted microscope for morphology every 12 h. After 7 days, the cells were evaluated with scanning electron microscope (SEM) for histological examination of the aggregates. Also, the hematoxylin-eosin (HE) staining and alkaline phosphatase (ALP) staining were performed. Moreover, von-Kossa staining and Alizarin Red S staining were carried out for mineralized nodule formation.

RESULTSThe results showed that in RWVB, the cells could be expanded by more than ten times and they presented better morphology and vitality and stronger ability to form bones.

CONCLUSIONSThe developed RWVB can provide the culture environment with a relatively low shear force and necessary three-dimensional (3D) interactions among cells and is suitable for osteopath expansion in vitro.

Animals ; Bioreactors ; Cell Culture Techniques ; instrumentation ; Cell Enlargement ; Culture Media ; Glucose ; metabolism ; Hydrogen-Ion Concentration ; Lactic Acid ; metabolism ; Osmolar Concentration ; Osteoblasts ; cytology ; metabolism ; ultrastructure ; Rats ; Rats, Sprague-Dawley

Antibiotics were reported to be able to alter bacterial surface properties in subinhibitory concentrations (sub-MICs). The effects of sub-MICs of certain antibiotics on a bacterial surface property such as hemagglutination, as well as on the cell morphology were studied using Streptococcus gordonii and Staphylococcus aureus. The effect of sub-M1Cs of antibiotics on the binding of these bacteria to immobilized fibrinogen were also investigated. The MICs of antibiotics were determined by culturing S. gordonii and S. aureus in media supplemented with serially diluted drug solutions, and one-half the MIC was used as the sub-MIC of the drugs, unless stated otherwise. Sub-MICs of antibiotics did not affect bacterial agglutination of erythrocytes. Microscopic observation of S. gordonii grown at sub-MIC concentration of 0.02 ug/ml of amoxicillin revealed cell enlargement of 1.6 times those grown without the drug. When grown in the sub-MIC amount of 0.08 ug/ml of cefazolin, most S. gordonii cells were enlarged and elongated into rod-shape, resulting in 3 times the size of the cells grown without the antibiotic. The data from the fibrinogen-binding experiments showed that the binding of S. gordonii to immobilized fibrinogen was increased with all the B-lactam drugs tested; the binding of S. aureus to immobilized fibrinogen, on the other hand, was decreased with the same drugs. The results show that low concentrations of certain B-lactam antibiotics are able to cause alterations in cellular morphology of S. gordonii and affect the binding of S. gordonii and S. aureus to immobilized fibrinogen.
Agglutination ; Amoxicillin ; Anti-Bacterial Agents* ; Bacteria ; Cefazolin ; Cell Enlargement ; Erythrocytes ; Fibrinogen ; Hand ; Hemagglutination ; Staphylococcus aureus* ; Staphylococcus* ; Streptococcus gordonii* ; Streptococcus* ; Surface Properties*

The effects of endothelin-1 (ET-1) and other drugs on the reactive oxygen species (ROS) generation and cardiomyocyte hypertrophy were examined in experiments on the cultured neonatal rat cardiomyocytes. The role of ROS on neonatal rat cardiomyocyte hypertrophy induced by ET-1 was studied and the relationship of PKC activation and ROS generation was investigated. The level of intracellular ROS was measured by the ROS-specific probe 2',7'-dichlorofluorescin diacetate (DCF-DA). Cardiomyocyte hypertrophy was determined by the RNA content, the total protein of cells and the cell surface area. The results are as follows. The fluorescence intensity of intracellular DCF-DA increased by 77% in cultured neonatal rat cardiac myocytes treated with ET-1 (10 nmol/L) vs control group. Compared with control group, the fluorescence intensity of intracellular PI, protein content and cell surface area increased by 128%, 87% and 151% respectively (all P<0.01) in cardiac myocytes treated with ET-1 (10 nmol/L). ABT-627, CC, or CAT inhibited the ET-1-induced increase in fluorescence intensity of intracellular DCF-DA by 62%,60% and 51% respectively (all P<0.01), and also attenuated the cardiac hypertrophy. The fluorescence intensity of intracellular DCF-DA increased by 74% (P<0.01) in myocytes treated with PMA (1 micromol/L) vs control group. Therefore, in the course of cardiomyocyte hypertrophy, ET-1 increases intracellular ROS in the cultured neonatal rat cardiac myocytes and inhibits cardiomyocyte hypertrophy induced by ROS. The ET(A) and PKC activation mediate the ROS production and cardiomyocyte hypertrophy induced by ET-1. ROS is necessary in the ET-1-induced cardiomyocyte hypertrophy.
Animals ; Animals, Newborn ; Cell Enlargement ; Cells, Cultured ; Endothelin-1 ; physiology ; Female ; Hypertrophy ; Male ; Myocytes, Cardiac ; cytology ; pathology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism

OBJECTIVETo explore the possible mechanism of lipopolysaccharide (LPS)-induced cardiomyocyte hypertrophy in rats.

METHODSNeonatal rat cardiomyocytes cultured in vitro were stimulated with 100 µg/L LPS for 1, 4 or 8 h and scanned by atomic force microscopy (AFM) for measurement of the two-dimensional area, three-dimensional surface area and volume of each cell. The total proteins and Na(+)-K(+)-ATPase activity in the cardiomyocytes were determined. The same measurements were also carried out in neonatal rat cardiomyocyte cultures stimulated by 0.5 µmol/L ouabain for 8 h and the total protein levels were measured.

RESULTSFollowing a 8-hour stimulation with LPS, the two-dimensional area, three-dimensional surface area and volume of the single cardiomyocyte became enlarged and the total cellular proteins increased significantly as compared with those in the normal control cells (P < 0.05). LPS treatment for 4 and 8 h resulted in significantly decreased activity of Na(+)-K(+)-ATPase in the cardiomyocytes (P < 0.05). In the cells treated with ouabain for 8 h, the two-dimensional area, three-dimensional surface area, volume of the single cardiomyocyte and the total cellular proteins increased significantly in comparison with the normal control group (P < 0.05).

CONCLUSIONLPS can result in cardiomyocyte hypertrophy in rats possibly in relation to lowered Na(+)-K(+)-ATPase activity in the cardiomyocytes after LPS exposure.

Animals ; Animals, Newborn ; Cell Enlargement ; drug effects ; Cells, Cultured ; Hypertrophy ; chemically induced ; Lipopolysaccharides ; Myocytes, Cardiac ; enzymology ; pathology ; Rats ; Rats, Wistar ; Sodium-Potassium-Exchanging ATPase ; metabolism

To investigate the cellular mechanisms of pressure-overload cardiac hypertrophy transition to heart failure, we observed time course of changes in morphology and contractile function of cardiomyocytes in transverse abdominal aortic constriction (TAC) rats. Since TAC rats suffered higher stress, body weight had a slower growth rate compared with that of synchronous control rats. Therefore, the left ventricular to body weight ratio produced experimental bias to evaluate the degree of cardiac hypertrophy. Length and width of collagenase-isolated cardiomyocyte were directly measured. Length, width and calculated surface area of cardiomyocyte showed a progressive increase in 8-, 16-, and 20-week TAC rats. The increasing rate of surface area in cardiomyocytes was higher at the middle stage of TAC (from the eighth to sixteenth week). Due to the constraint of fibrosis formation, the increasing rate of surface area in cardiomyocytes was slower at the late stage of TAC (from the sixteenth to twentieth week). The sarcomere length of cardiomyocytes was unchanged, whereas sarcomere numbers were significantly increased in 8-, 16-, and 20-week TAC rats. Shortening amplitude of unloaded contraction in single cardiomyocyte was significantly enhanced in 1-week TAC rats, but not altered in 8-week TAC rats compared with that in the synchronous control rats. On the contrary, unloaded shortening amplitude of single cardiomyocyte was significantly reduced in 16- and 20-week TAC rats. The above results suggest that the reduced shortening amplitude may be associated with intrinsic molecular alterations in hypertrophied cardiomyocytes.
Animals ; Aorta, Abdominal ; Cardiomegaly ; etiology ; physiopathology ; Cell Enlargement ; Constriction ; Hypertension ; complications ; pathology ; physiopathology ; Male ; Myocardial Contraction ; physiology ; Myocytes, Cardiac ; pathology ; physiology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of microRNA-133a on isoproterenol (ISO)-induced neonatal rat cardiomyocyte hypertrophy and related molecular mechanism focusing on the changes of L-type calcium channel α1C subunit.

METHODSNeonatal rat cardiomyocytes were cultured, cardiomyocyte hypertrophy was induced by isoproterenol (ISO, 10 µmol/L). The cell surface area was measured by phase contrast microscope and Leica image analysis system. The mRNA expressions of atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC), miR-133a and the α1C were detected by qRT-PCR. The protein expression of α1C was evaluated by Western blot. MiR-133a mimic was transfected into cardiomyocytes to investigate the effects of miR-133a on ISO-induced cardiomyocyte hypertrophy. The targets of miR-133a were predicted by online database Targetscan. The 3' untranslated region sequence of α1C was cloned into luciferase reporter vector and then transiently transfected into HEK293 cells. The luciferase activities of samples were measured to verify the expression of luciferase reporter vector. The expression level of α1C was inhibited by RNAi to determine the effects of α1C on cardiomyocyte hypertrophy. Intracellular Ca(2+) content was measured by confocal laser microscope.

RESULTS(1) The expression of miR-133a was significantly reduced in ISO-induced cardiomyocyte hypertrophy (P < 0.01) . Upregulating miR-133a level could suppress the increase of cell surface area, the mRNA expression of ANP and β-MHC (P < 0.01) . (2) α1C was the one of potential target of miR-133a by prediction using online database Targetscan. The luciferase activities of HEK293 cells with the plasmid containing wide type α1C 3'UTR sequence were significantly decreased compared with control group (P < 0.01) . Upregulation of the miR-133a level by miR-133a mimic transfection could suppress the protein expression of α1C (P < 0.05) . (3) The expression of α1C was significantly increased in ISO treated cardiomyocytes (P < 0.05) . Downregulation of α1C by RNAi could markedly inhibit the increase of cell surface area, the mRNA expression of ANP and β-MHC (P < 0.01, P < 0.05, P < 0.05). (4) Downregulation of α1C expression by RNAi or upregulation of miR-133a level by miR-133a mimic transfection significantly inhibited intracellular Ca(2+) content (P < 0.01) .

CONCLUSIONSOur data confirms that α1C is the target of miR-133a. MiR-133a can negatively regulate the expression of L-type calcium α1C subunit, resulting in the decrease of intracellular Ca(2+) content and the attenuation of ISO-induced cardiomyocyte hypertrophy.

Animals ; Calcium Channels, L-Type ; metabolism ; Cell Enlargement ; drug effects ; Cells, Cultured ; Isoproterenol ; pharmacology ; MicroRNAs ; genetics ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Transfection

OBJECTIVETo evaluate the inhibitory effect and related mechanism of bradykinin on mechanical stress induced myocardial hypertrophy.

METHODSNeonatal rat cardiomyocytes were isolated and cultured in silicon plates. All cardiomyocytes were randomly divided into three groups: control group, mechanical stretch group (mechanical stretch of silicon plates to 120% for 30 min) and mechanical stretch plus bradykinin group (1×10(-8) mol/L for 24 h before stretch). The protein synthesis and surface area of cardiomyocytes were detected by [(3)H] leucine incorporation and immunofluorescence of α-MHC, respectively. mRNA expression of atrial natriuretic peptide (ANP) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) was detected by real time-PCR, the phosphorylation of calcineurin (CaN), the expression of Angiotensin II receptor 1 (AT1R) and angiotensin converting enzyme (ACE)by Western blot.

RESULTSThe surface area of cardiomyocytes of mechanical stretch group [(973 ± 103) µm(2)] was significantly enlarged than in control group [(312 ± 29) µm(2)] and this effect could be partly attenuated by bradykinin [(603 ± 74) µm(2), all P < 0.05]. Mechanical stretch also significantly increased the protein synthesis, up-regulated the expression of ANP and decreased the expression of SERCA2, and these effects could be partly reversed by pretreatment with bradykinin. Moreover, bradykinin partly abolished the mechanical stretch-induced increases in CaN phosphorylation, up-regulation of AT1R but preserved the expression of ACE.

CONCLUSIONSBradykinin significantly attenuates mechanical stretch-induced myocardial hypertrophy through inhibition of Ca(2+)/CaN pathway.

Animals ; Bradykinin ; pharmacology ; Calcineurin ; metabolism ; Calcium ; metabolism ; Cell Enlargement ; drug effects ; Cells, Cultured ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Rats ; Stress, Mechanical

OBJECTIVES: Previously, we sought to compile a list of genes expressed during early folliculogenesis by using cDNA microarray to investigate follicular gene expression and changes during primordialprimary follicle transition and development of secondary follicles (Yoon et al., 2005). Among those genes, a group of genes related to the cell size growth was characterized during the ovarian development in the present study. METHODS: We determined ovarian expression pattern of six genes related to the cell size growth (cyr61, emp1, fhl1, socs2, wig1 and wisp1) and extended into CCN family (connective tissue growth factor/cysteine-rich 61/nephroblastoma-overexpressed), ctgf, nov, wisp2, wisp3, including cyr61 and wisp1 genes. Expression of mRNA and protein according to the ovarian developmental stage was evaluated by in situ hybridization, and/or semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry, respectively. RESULTS: Among 6 genes related to the cell size growth, cyr61 and wisp1 mRNA was detected only in oocytes in the postnatal day5 mouse ovaries. cyr61 mRNA expression was limited to the nucleolus of oocytes, while wisp1 was expressed in the cytoplasm and nucleolus of oocytes, except nucleus. cyr61 mRNA expression, however, was found in granulosa cells from secondary follicles. The rest 4 genes in the cell size growth group were detected in oocytes, granulosa and theca cells. Cyr61 and Wisp1 proteins were expressed in the oocyte cytoplasm from primordial follicle stage. Especially, Cyr61 protein was detected in pre-granulosa cells, Wisp1 protein was not. By using RT-PCR, we evaluated and decided that Cyr61 protein is produced by their own mRNA in pre-granulosa cells that was not detected by in situ hybridization. cyr61 and wisp1 genes are happen to be the CCN family members. The other members of CCN family were also studied, but their expression was detected in oocytes, granulose and theca cells. CONCLUSIONS: We firstly characterized the ovarian expression of genes related to the cell size growth and CCN family according to the early folliculogenesis. Cyr61 protein expression in the pre-granulosa cells is profound in meaning. Further functional analysis for cyr61 in early folliculogenesis is under investigation.
Animals ; Cell Enlargement* ; Cell Size* ; Cysteine-Rich Protein 61 ; Cytoplasm ; Female ; Gene Expression ; Genes, vif ; Granulosa Cells ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Mice* ; Oligonucleotide Array Sequence Analysis ; Oocytes ; Ovary ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger ; Theca Cells

Cardiac hypertrophy is an adaptive process to an increased hemodynamic overload. However, the adaption may lead to the fragility of myocardium facing pathological stimuli. In the present study, experiments were designed to explore the susceptibility of hypertrophic myocardiocytes to apoptotic stimuli and the role of protein kinase Cdelta (PKCdelta) during the transition from hypertrophy to apoptosis. Endothelin-1 (ET-1)-treated cardiomyocytes were used as model of cardiac hypertrophy. Angiotensin II (Ang II) was used as an apoptotic stimulus. Cell surface area was measured to determine the extent of hypertrophy. The apoptotic rate in cardiomyocytes was detected by Hoechst 33258. (1) Cell surface area was increased by 42.5% and 67.3% following 1 nmol/L and 10 nmol/L ET-1 treatment, respectively, as compared with serum-free cultured myocytes. So the mildly and moderately hypertrophic myocyte models were set up. (2) Apoptotic rates in serum-free cultured, mildly and moderately hypertrophic myocytes after Ang II treatment were (15.54+/-1.32) %, (20.65+/-1.40) % and (29.33+/-3.52) %, respectively. It is suggested that hypertrophic myocytes are more susceptive to apoptotic stimulus. (3) Rottlerin, a specific inhibitor of PKCdelta depressed apoptotic rates induced by Ang II to (15.88+/-2.25) % in mildly hypertrophic myocytes and to (15.01+/-1.37) % in moderately hypertrophic myocytes; but rottlerin did not affect apoptotic rate induced by Ang II in serum-free cultured myocytes. These results suggest that inhibition of PKCdelta can reduce Ang II-induced apoptosis of hypertrophic cardiomyocytes and that PKCdelta is possibly involved in the apoptotic process of hypertrophic cardiomyocytes.
Angiotensin II ; pharmacology ; Animals ; Animals, Newborn ; Apoptosis ; drug effects ; physiology ; Cardiomegaly ; pathology ; physiopathology ; Cell Enlargement ; drug effects ; Endothelin-1 ; pharmacology ; Heart Failure ; physiopathology ; Myocytes, Cardiac ; cytology ; pathology ; Primary Cell Culture ; Protein Kinase C-delta ; physiology ; Rats ; Rats, Sprague-Dawley