The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.
Acrosome/*pathology/ultrastructure ; Animals ; Cattle/*physiology ; Cell Membrane/*pathology/ultrastructure ; Cell Separation/veterinary ; Centrifugation, Density Gradient/veterinary ; Cryopreservation/veterinary ; Male ; Microscopy, Electron, Transmission/veterinary ; Povidone/*adverse effects ; Silicon Dioxide/*adverse effects ; Spermatozoa/pathology/ultrastructure

The present study was designed in order to investigate the lectin induced cytoagglutination properties of normal and transformed cells and surface alterations in the early stage of the transformed cells by characterizing the structural changes on the hepatoma surface membrane. Rat and rabbit erythrocytes and Sarcoma 180 ascites tumor cells were used for the lectin-induced cytoagglutination. Plotting % agglutination versus concanavalin A(Con A) concentration, sigmoid curves appeared in all cases. alpha-methyl-D-mannopyranoside(alphaMM) inhibited Con A induced cytoagglutination and the degrees of inhibition depended on the cell types and species. When rats were fed a diet containing 0.06% 3'-methyl-4dimethylaminoazobenzene(3'-Me DAB) for 12 weeks, almost all of the rats had solid liver tumors. Polyacrylamide gel electrophoresis of surface membrane proteins of these rat livers and of transplanted tumor cells showed three distinct protein bands, of which two were absent in normal rat livers. The molecular weights of these proteins were 73,000, 66,000, and 57,000 daltons. Antiserum against primary hepatocarcinoma surface proteins precipitated with three membrane proteins obtained from primary hepatocarcinoma cells as well as transplanted hepatocarcinoma cells, suggesting the presence of specific tumor antigens in these cells.
Animal ; Cell Membrane/pathology* ; Cell Transformation, Neoplastic/ultrastructure* ; Concanavalin A/diagnostic use ; Liver Neoplasms, Experimental/chemically induced ; Liver Neoplasms, Experimental/ultrastructure* ; Methyldimethylaminoazobenzene ; Rabbits ; Rats ; Surface Properties

OBJECTIVETo observe the microstructure of the cell membrane of epileptic neurons using atomic force microscopy (AFM).

METHODSModel of epileptic neurons was established by subjecting the neurons culture for 14 days in vitro to magnesium-free media treatment for 3 h. Patch clamp technique was applied to record the electrophysiological activity of the epileptic neurons. AFM was performed to observe and measure the microstructure of the cell membrane of the epileptic neuron.

RESULTSAfter a 3-hour treatment with magnesium-free media, the epileptic neurons displayed sustained epileptiform discharge, which continued after the neurons were returned to normal medium culture on day 14. Under AFM scanning size of 80 microm x 80 microm and 2 microm x 2 microm, no obvious difference in the morphology of the cell membrane was noted between epileptic and normal neurons; under the scanning size of 500 nm x 500 nm, small pits occurred in the cell membrane in both groups, but no significant difference was found in the dimension of the pits between the two groups (the diameter and depth of the pits was 114.86-/+9.33 nm and 5.71-/+0.69 nm in epileptic neurons, and 116.4-/+9.13 nm and 5.69-/+0.71 nm in the control neurons, respectively, P>0.05).

CONCLUSIONAFM provides a new method for observing neuronal membrane microstructure at nanometer resolutions. No significant alterations occur in the membrane of the neurons after a 3-hour magnesium-free media treatment.

Cell Membrane ; ultrastructure ; Cells, Cultured ; Culture Media ; Epilepsy ; pathology ; Excitatory Postsynaptic Potentials ; Inhibitory Postsynaptic Potentials ; Magnesium ; Microscopy, Atomic Force ; Neurons ; ultrastructure ; Patch-Clamp Techniques

Atomic force microscope (AFM) was used to study biotoxicity of food preservative sodium benzoate (SB) at the single cellular level. Lymphocyte morphology and membrane ultrastructure treated with SB at different concentrations and time were analyzed visually. As compared to the normal lymphocyte, the cell morphology and membrane was significantly changed and its ultrastructure was also complicated. After treated with SB, the Rp-v, Rq, Ra and Z values were changed. The statistical analysis of lymphocytes after treated with SB was studied, and discussed its mechanism.
Animals ; Cell Membrane ; ultrastructure ; Lymphocytes ; drug effects ; pathology ; Mice ; Mice, Inbred BALB C ; Microscopy, Atomic Force ; Sodium Benzoate ; toxicity

OBJECTIVETo investigate the role of mitofusin-2 gene (mfn2) in apoptosis in human breast carcinoma cell line MCF-7 cells after in vitro transfection.

METHODSpEGFP mfn2 was transfected by sofast in vitro. Expression of GFP was observed by Western blot, and the MCF-7 cell proliferation was measured by MTT and cell counting. Apoptosis in MCF-7 cells was observed in annexin-V/PI and chondrosome transmembrane potential of MCF-7 marked in JC-1 by FCM. The Ultrastructure of cells was observed by transmission electron microscopy.

RESULTSThe stable expression of GFP in MCF-7 cells was confirmed by Western blot. Mfn2 significantly inhibited cell proliferation, revealed by MTT, and decrease chondrosome transmembrane potential. Exogenous mfn2 gene significantly induced apoptosis. The apoptotic rate was increased from 3.6% to 16.0% (P < 0.05). Mfn2 gene induced break down and loss of mitochondrial cristae, and rarefaction of mitochondrial ground substance. Swollen mitochondria intensely aggregated around the cell nuclei.

CONCLUSIONMfn2 can strongly induce apoptosis in MCF-7 cells, which may be associated with decrease of mitochondrial transmembrane potential.

Apoptosis ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Female ; GTP Phosphohydrolases ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; Membrane Proteins ; genetics ; metabolism ; Mitochondria ; ultrastructure ; Mitochondrial Proteins ; genetics ; metabolism ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo determine the relationship of thick greasy tongue fur formation and permeability of vascular endothelial cells (ECs) with the protein expression of zonula occludens-1 (ZO-1).

METHODSSprague Dawley rats were randomly divided into a model group of severe acute pancreatitis (SAP) and a sham-operated (SO) group. The SAP rats were further divided into two subgroups on the basis of tongue-coating status: a thick greasy tongue fur group (SAP-TGF) and a normal tongue fur group (SAP-NF). Six rats were chosen randomly from every group mentioned above for an Evans blue assay 5 days after model establishment. For the histomorphology analysis, the expressions of ZO-1 protein and mRNA were studied by hematoxylin-eosin (H&E) staining, transmission electron microscope, Western blot, and Q-PCR using blood and tongue tissues, which were collected from 8 rats randomly chosen from each group.

RESULTSThe papillae density of the rat tongue surface and the caryocinesis frequency of the basal layer were significantly increased in the SAP-TGF group compared with the SO group (P<0.05). Evans blue levels in the tongue tissue of the SAP-TGF group were significantly higher than that of the SO and SAP-NF groups (P<0.05). Vascular ECs were wider and obviously fissured in the SAP-TGF group under transmission electron microscope observation. The protein and mRNA expression of ZO-1 in the SAP-TGF group were lower than those in the SAP-NF (P<0.05).

CONCLUSIONSReproductive activity enhancement of glossal epithelial cells was one of the main characteristics of thick greasy tongue fur formation. An increase in vasopermeability was closely associated with thick greasy tongue fur formation. Tight junction structural variation of vascular ECs might play an important role in the pathological and physiological process of thick greasy tongue fur formation.

Animals ; Blotting, Western ; Cell Membrane Permeability ; Endothelial Cells ; cytology ; Evans Blue ; metabolism ; Gene Expression Regulation ; Male ; Membrane Proteins ; genetics ; metabolism ; Phosphoproteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tongue ; pathology ; ultrastructure ; Zonula Occludens-1 Protein

OBJECTIVETo study the pathological and clinical characteristics of a patient with spontaneous platelet aggregation of his giant and morphologically abnormal platelets.

METHODSPlatelet size and structure were observed under light microscope and electron microscope. Platelet aggregation was measured turbidometrically. Platelet glycoproteins (GP) were analyzed using flow cytometry. PCR and DNA sequencing were performed to identify the gene abnormality.

RESULTSThe patient had spontaneous platelet aggregation of giant platelets with thickened plasma membrane and increased number of granules in various shapes. Aspirin and ticlopidine did not affect the spontaneous aggregation. The expression of GP I b, GP II b, GP III a and P-selectin in the platelet membrane were in normal range. Results of gene analyses for GP I balpha, GP I bbeta and GPIX were also normal.

CONCLUSIONBoth morphological and functional abnormalities of the platelets from the patient were clearly distinguishable from that of other hereditary giant platelet disorders. It would probably represent a novel platelet disorder which had not been reported to date.

Aspirin ; pharmacology ; Bernard-Soulier Syndrome ; metabolism ; pathology ; Blood Platelet Disorders ; metabolism ; pathology ; Cell Size ; physiology ; Child ; Cytoplasmic Granules ; pathology ; ultrastructure ; Female ; Humans ; Platelet Aggregation ; drug effects ; physiology ; Platelet Aggregation Inhibitors ; pharmacology ; Platelet Membrane Glycoproteins ; genetics ; metabolism ; Ticlopidine ; pharmacology

OBJECTIVETo culture rat prostate glandular epithelial cells and study their barrier functions in vitro.

METHODSRat prostate glandular epithelial cells were cultured in vitro. The expression of the tight junction protein claudin-1 was determined by immunohistochemistry, the structure and composition of the epithelial cells observed under the inverted microscope and transmission electron microscope. The transepithelial electrical resistances (TEERs) were monitored with the Millicell system. The permeability of the prostate glandular epithelial cells was assessed by the phenol red leakage test.

RESULTSCompact monolayer cell structures were formed in the prostate glandular epithelial cells cultured in vitro. Immunohistochemistry showed the expression of the tight junction protein claudin-1 and transmission electron microscopy confirmed the formation of tight junctions between the adjacent glandular epithelial cells. The TEERs in the cultured prostate glandular epithelial cells reached the peak of about (201.3 ± 3.5) Ω/cm2 on the 8th day. The phenol red leakage test manifested a decreased permeability of the cell layers with the increase of TEERs.

CONCLUSIONThe structure and function of rat prostate glandular epithelial cells are similar to those of brain capillary endothelial cells, retinal capillary endothelial cells, and intestinal epithelial cells. In vitro cultured prostate glandular epithelial cells have the barrier function and can be used as a model for the study of blood prostate barrier in vitro.

Animals ; Cell Membrane Permeability ; Cells, Cultured ; Claudin-1 ; metabolism ; Electric Impedance ; Epithelial Cells ; pathology ; physiology ; ultrastructure ; In Vitro Techniques ; Male ; Microscopy, Electron, Transmission ; Phenolsulfonphthalein ; pharmacokinetics ; Prostate ; metabolism ; pathology ; Rats ; Tight Junctions

OBJECTIVETo evaluate the effect of bone morphogenic protein 7 (BMP-7) on nephrin expression and distribution in diabetic rat kidneys.

METHODSTwenty rats with diabetes mellitus (DM) induced by streptozotocin (STZ) injection were randomly divided into DM model group and BMP-7 treatment group, with another 10 normal rats serving as the normal control group. The rats in BMP-7 group received intraperitoneal human recombinant BMP-7 injections at 30 microg/kg twice a week for 24 consecutive weeks, while normal saline was administered in rats of the other two groups. Blood glucose and 24 hour urinary protein and creatinine (Ccr) were measured at 8, 16 and 24 weeks, and the rats were sacrificed at 24 weeks to obtain the renal tissues for detecting the expression and distribution of nephrin using immunofluorescence assay and RT-PCR and for examining the expressions of transforming growth factor-beta1 (TGF-beta1) and WT1 using immunohistochemistry.

RESULTSCompared with the normal control group, the DM model group showed significantly increased 24 hour urinary protein, kidney to body weight ratio and TGF-beta1 expression, but had lowered Ccr, glomerular podocyte number and nephrin expression. The linear distribution of nephrin along the capillary loops as found in the normal control group became granular in the kidney of diabetic rats. The rats in BMP-7 group showed less urinary protein excretion, lower TGF-beta1 expression and greater glomerular podocyte number than those in the DM group, and the expression and distribution of nephrin remained normal in the kidney.

CONCLUSIONAdministration of BMP-7 can significantly suppress the down-regulation of nephrin expression and maintain its normal distribution in the podocytes in diabetic rats possibly in association with a direct suppression of TGF-betasignaling.

Animals ; Blood Glucose ; metabolism ; Body Weight ; drug effects ; Bone Morphogenetic Protein 7 ; pharmacology ; Cell Count ; Diabetes Mellitus ; genetics ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Humans ; Kidney ; drug effects ; metabolism ; pathology ; ultrastructure ; Male ; Membrane Proteins ; genetics ; metabolism ; Microscopy, Electron, Transmission ; Organ Size ; drug effects ; Podocytes ; drug effects ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the effect of berbamine on human hepatoma cell line SMMC7721.

METHODSThe effects of 24 h and 48 h incubation with different concentrations (0 to approximately 64 microg/ml) of the berbamine on SMMC7721 cells were evaluated using 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. Hoechst 33258 staining was conducted to distinguish the apoptotic cell, and the appearance of sub-G1 stage was determined by PI (propidium iodide) staining, the percentage of apoptotic cell was determined by flow cytometry following annexin V/PI staining. Flow cytometry was performed to analyze the cell cycle distribution and the mitochondrial membrane potential (psi(m)); the expression of activated caspase3 and caspase9 was analyzed by Western-blot.

RESULTSThe proliferation of SMMC7721 was decreased after treatment with berbamine in a dose- and time-dependent manner. Berbamine could induce apoptosis in SMMC7721 cells and could cause cell cycle arrest in G0/G1 phase, to induce loss of mitochondrial membrane potential (psi(m)) and activate caspase3 and caspase9. Berbamine-induced apoptosis could be blocked by the broad caspase inhibitor z-VAD-fmk.

CONCLUSIONBerbamine exerts antiproliferative effects on human hepatocellular carcinoma SMMC7721 cells. The anticancer activity of berbamine could be attributed partly to its inhibition of cell proliferation and induction of apoptosis in cancer cells through loss in mitochondrial transmembrane potential and caspase activation.

Alkaloids ; pharmacology ; Apoptosis ; drug effects ; Benzylisoquinolines ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Enzyme Activation ; drug effects ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Medicine, Chinese Traditional ; Membrane Potentials ; drug effects ; Mitochondria, Liver ; drug effects ; metabolism ; ultrastructure