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PubMed2000

Dynamic imaging of the metaphase II spindle and maternal chromosomesin bovine oocytes: implications for enucleation efficiency verification, avoidanceof parthenogenesis, and successful embryogenesis.

Manipulations of DNA and cellular structures are essential for the propagation of genetically identical animals by nuclear transfer. However, none of the steps have been optimized yet. This study reports a protocol that improves live dynamic imaging of the unfertilized bovine oocyte's meiotic spindle microtubules with microinjected polymerization-competent X-rhodamine-tubulin and/or with vital long-wavelength excited DNA fluorochrome Sybr14 so that the maternal chromosomes can be verifiably removed to make enucleated eggs the starting point for cloning. Suitability of the new fluorochromes was compared to the conventional UV excitable Hoechst 33342 fluorochrome. Enucleation removed the smallest amount of cytoplasm (4-7%) and was 100% efficient only when performed under continuous fluorescence, i.e., longer fluorescence exposure. This was in part due to the finding that the second metaphase spindle is frequently displaced (60.7 +/- 10%) from its previously assumed location subjacent to the first polar body. Removal of as much as 24 +/- 3% of the oocyte cytoplasm underneath the polar body, in the absence of fluorochromes, often resulted in enucleation failure (36 +/- 6%). When labeled oocytes were exposed to fluorescence and later activated, development to the blastocyst stage was lowest in the group labeled with Hoechst 33342 (3%), when compared to Sybr14 (19%), rhodamine-tubulin (23%), or unlabeled oocytes (37%). This suggests that longer wavelength fluorochromes can be employed for live visualization of metaphase spindle components, verification of their complete removal during enucleation, and avoidance of the confusion between artifactual parthenogenesis versus "cloning" success, without compromising the oocyte's developmental potential after activation.

Animals↗
PubMed2002

High-resolution scanning patch-clamp: new insights into cell function.

Cell specialization is often governed by the spatial distribution of ion channels and receptors on the cell surface. So far, little is known about functional ion channel localization. This is due to a lack of satisfactory methods for investigating ion channels in an intact cell and simultaneously determining the channels' positions accurately. We have developed a novel high-resolution scanning patch-clamp technique that enables the study of ion channels, not only in small cells, such as sperm, but in submicrometer cellular structures, such as epithelial microvilli, fine neuronal dendrites, and, particularly, T-tubule openings of cardiac myocytes. In cardiac myocytes, as in most excitable cells, action potential propagation depends essentially on the properties of ion channels that are functionally and spatially coupled. We found that the L-type calcium and chloride channels are distributed and colocalized in the region of T-tubule openings, but not in other regions of the myocyte. In addition, chloride channels were found in narrowly defined regions of Z-grooves. This finding suggests a new synergism between these types of channels that may be relevant for action potential propagation along the T-tubule system and excitation-contraction coupling.

Animals↗
PubMed1991

Changes in high-energy phosphate metabolism and cell morphology in four models of acute experimental pancreatitis.

Previous studies using the isolated ex vivo perfused canine pancreatitis preparation showed that during a 4-hour perfusion pancreatitis (edema, weight gain, hyperamylasemia) can be induced by four different stimuli. The stimuli include the intra-arterial infusion of oleic acid (FFA), a 2-hour period of ischemia before perfusion (ISCH), partial obstruction of the pancreatic duct with secretin stimulation (POSS), and the intra-arterial infusion of cerulein at supramaximal doses (CER). In the present study, changes in high-energy phosphate metabolism, as determined by nuclear magnetic resonance spectroscopy, and changes in cellular structure, determined by light and electron microscopy, were documented for all four models of acute pancreatitis. The control preparations remained stable for the 4-hour perfusion period, with no decrease in adenosine triphosphate (ATP) levels. In the FFA preparations, ATP decreased to 36% of baseline levels during the 4-hour perfusion (p less than 0.001). In the ISCH preparations, ATP decreased to undetectable levels during the 2-hour period of ischemia, but recovered rapidly and remained at baseline levels during the perfusion. ATP levels remained stable in the remaining two models of pancreatitis (POSS, CER). Microscopy demonstrated that the initial injury was located chiefly in the capillaries (swollen endothelium, intravascular thrombi) in the FFA and ISCH preparations. In the POSS and CER preparations, capillary changes were minimal and the injury was located chiefly in the acinar cells (swollen endoplasmic reticulum, zymogen granule depletion, vacuolization). The POSS preparations also showed striking dilation of centroacinar lumens reflecting duct obstruction. In additional studies it was shown that the ATP decline in the FFA preparations could be significantly reduced by pretreatment with free radical scavengers. The morphologic changes could be reduced by free radical scavengers in the FFA and ISCH preparations. Any amelioration of morphologic injury in the POSS preparations was obscured by dilatation of centroacinar lumens in both treated and untreated groups. The morphologic changes in the CER preparations were reduced by treatment with a cholecystokinin inhibitor.

Acute Disease↗
PubMed2002

Hepatobiliary pathology.

Technologic advances using cDNA microarray hybridization, liver diseases characterized by mitochondrial DNA depletion, and new work characterizing bile salt transport problems in familial intrahepatic cholestasis syndromes were some of the major highlights of this past year. Analysis of normal livers by cDNA microarrays disclosed 2418 unique gene transcripts encoding a host of cellular structural and functional proteins. This technique was also applied to hepatocellular carcinoma, where enhanced expression of a number of genes involved in antiapoptosis and cell transformation may shed additional light on the process of hepatocarcinogenesis. Mitochondrial DNA depletion seen in Navajo neurohepatopathy and in respiratory chain disorders of infancy was associated with cholestasis and cirrhosis in the former and microvesicular steatosis and oncocytic transformation (mitochondrial hyperplasia) in the latter. Pathologists who routinely examine liver biopsies after liver or bone marrow transplantation should be aware of unusual biopsy features that mimic other diseases, such as the autoimmune hepatitis-like syndrome that may follow liver transplantation and chronic graft-versus-host disease that clinically and pathologically resembles acute hepatitis.

Journal Article↗
PubMed1992

Polyclonal antibodies against the rat NK1 receptor: characterization and localization in the spinal cord.

We have developed antibodies against the NK1 receptor and have investigated its cellular distribution. Rabbit polyclonal antibodies were generated against peptide (19-32) of the rat brain NK1 receptor. They were very specific to the NK1 site as shown by ELISA against various epitopes of NK1, NK2 and NK3 receptors and by immunoblotting of proteins from bacteria transfected with rat brain NK1 receptor cDNA and from rat cortex. Determining how immunostained NK1 receptors are distributed in the rat spinal cord made it possible to identify the cellular structures on which NK1 receptors are located and where they form synapses with SP terminals. In the superficial layers of the dorsal horn, the NK1 receptors appeared mainly of dendritic nature and were, like SP, abundant. In the deep layers of the dorsal horn and in the ventral horn, they were associated mostly with cell bodies.

Amino Acid Sequence↗
PubMed1993

Immunoregulatory cytokine production by tumor-bearing rat spleen cells and its modulation by bleomycin.

The chemotherapeutic agent bleomycin (BLM) increases cytokine production by mitogen-stimulated healthy rat spleen cells without altering the cellular composition of the spleen. In this study, the chronological production of interleukin (IL)-2, IL-6 and tumor necrosis factor (TNF) by untreated and BLM-treated tumor-bearing rat spleen cells is examined. A significant decrease in the production of both IL-2 and TNF could be observed only 5 days after subcutaneous injection of syngeneic KMT-17 tumor cells. Decrease in cytokine production progressed with time with a slight recovery around day 10 after tumor challenge. Administration of BLM, 5 mg/kg, on day 8, restored IL-2 and IL-6 production and significantly increased TNF production by day 14 of tumor burden as compared with the amounts of cytokine produced by the mitogen-stimulated untreated tumor-bearing rat spleen cells. The response of the tumor-bearing rat spleen cells to concanavalin A (ConA), diminished when compared with that of normal rat spleen cells, could be restored to normal levels by treatment with BLM when examined at low concentrations of mitogen but was unaffected at higher concentrations of ConA. Histological examination of the tumor tissue, following continuous intraperitoneal treatment with BLM, 5 mg/kg, from day 8 to 12, shows disruption of cellular structure with significant infiltration of effector cells as compared with undisrupted organization with no visible infiltration of effector cells in the untreated rat tumors.

Animals↗
PubMedDate not supplied

Improvement in the differentiated hepatic phenotype of immortalized human hepatocytes by adenovirus mediated p21 gene transfer.

The p21 molecule, a potent cyclin dependent kinase inhibitor, regulates the transition from the G1 phase to the S phase of the cell cycle and is involved in terminal cellular differentiation. The overexpression of p21 has been shown to induce differentiation in various cell lines. We have made an effort to establish a reliable human hepatocyte cell line as a source of hepatic function in bioartificial liver (BAL) therapy. In this work, we investigated the effect of p21 on the differential phenotype of simian virus 40 large T antigen (SV40Tag) immortalized human hepatocytic NKNT-3 cells. A recombinant adenoviral vector expressing a p21 gene under control of the cytomegalovirus (CMV) promoter (Ad-p21) was used to efficiently transfer genes into NKNT-3 cells. The morphologic alterations, the cell cycle progression, and the expression of p-450 associated enzymes (CYPs) were carefully examined in NKNT-3 cells that had been infected with Ad-p21. Adenovirus mediated gene delivery of p21 was efficiently achieved in NKNT-3 cells without affecting cellular structure. After Ad-p21 infection, NKNT-3 cells were G0/G1 arrested in cell cycle analysis. NKNT-3 cells that had been infected with Ad-p21 showed differentiated hepatic phenotypes in morphology and improvement in protein expression of CYP 3A4 and CYP 2C9. In the present work, we demonstrate that the exogenous expression of p21 enhances the differential phenotype of immortalized hepatocytic NKNT-3 cells.

Adenoviridae↗
PubMed1998

In vivo confocal microscopy of Fuchs' endothelial dystrophy.

PURPOSE: The purpose of this study is to analyze in vivo confocal microscopic findings of corneas with Fuchs' endothelial dystrophy. METHODS: Central corneas of 17 eyes of 11 patients aged 41-86 years were examined using in vivo scanning slit confocal microscopy after being diagnosed with Fuchs' endothelial dystrophy. The cellular structure of the corneas was analyzed morphologically and quantitatively and compared to control results from 22 healthy corneas. RESULTS: Bullae were detected in the basal epithelial layer of one eye. Eight of 17 eyes (47%) exhibited an abnormal Bowman's layer: diffuse bright reflection and absence of nerves. Eleven eyes (65%) exhibited abnormal anterior stroma: lacunae and diffuse increased light reflection due to edema. In 12 eyes (71%), lacunae or dark bands 5-20 microm wide against increased background reflection were noted in the posterior stroma. Descemet's membrane was thickened in all eyes. Dark bands were detected in six eyes (35%). Guttae (137-1,231/mm2) 20-40 microm in diameter were found in every endothelial cell layer. The mean endothelial cell count was 1,202 +/- 850 (cells/mm2 +/- SD; range, 0-2,735). There was a positive correlation between endothelial cell counts obtained by specular microscopy and those obtained by confocal microscopy (r = 0.95). CONCLUSION: In vivo confocal microscopic findings of Fuchs' endothelial dystrophy are described for the first time in a series of cases. Pathological changes in Fuchs' dystrophy were detected in all corneal layers, more frequently in the posterior layers. Endothelial cell counts obtained with confocal microscopy were statistically similar to those obtained with standard specular microscopy.

Adult↗
PubMed1986

Metabolic fate of inhaled Co aerosols in beagle dogs.

Lung retention of 57Co in dogs after the inhalation of physically and chemically uniform particles of Co compounds was similar, indicating little biological variability. The retention of Co oxide particles ranging from 0.3 micron to 2.7 micron geometric diameter, however, depended markedly on their physicochemical parameters. Measuring the retention by a gamma camera, and analyzing excreta and blood samples enabled the distinction of different clearance pathways from the lungs particularly with the use of results of some metabolic studies. Particle dissolution was the predominant clearance pathway. Particle dissolution half-times ranged from 6 to 80 d proportional to the size of the particles. Organ analysis yielded information on the fate of the long-term burden of Co in the lungs and other organs. A fraction less than 10% of the initial lung burden was retained in the lungs of all dogs with a biological half-time of 400 d presumably after being transformed into a nonparticulate state. There were cellular structures in the tracheobronchial tree which accumulated Co significantly. These studies on dogs suggest the dose after human exposure to well-defined Co aerosols can be accurately estimated. Whereas risk assessment after the exposure to undefined aerosols containing radionuclides of Co will mostly be impossible because retention varies widely with the varying physicochemical properties of the aerosol particles.

Administration, Inhalation↗
PubMed2004

Perspectives of chromo and magnifying endoscopy: how, how much, when, and whom should we stain?

The goal of every routine endoscopy in the gut is the early diagnosis of malignant and premalignant changes of the mucosa. Chromo- and magnifying endoscopes are exciting new tools and offer detailed analysis of the colonic mucosal surface and pit pattern architecture. This review summarizes recent advances in endoscopic characterization of colorectal lesions using magnification endoscopy and chromoendoscopy. Surface analysis of the colon using chromoendoscopy allows a prediction between non-neoplastic and neoplastic lesions with high specificity. The precise delineation of the borders and a more detailed macroscopic analysis of the lesions are further advantages. In particular, flat adenomas and early depressed cancers are now more frequently recognized in western countries suggesting that significant lesions were overlooked by conventional endoscopy in the past. Furthermore, chromoendoscopy can be used in a targeted fashion to screen for sporadic adenomas. Finally, in surveillance colonoscopy, patients with long-standing ulcerative colitis have a valuable benefit if targeted biopsies are performed to detect intraepithelial neoplasias after pan-chromoendoscopy with methylene blue. Although there is a long learning curve, chromoendoscopy should thus belong to every endoscopists armamentarium. However, detailed knowledge about the technique, dyes, and specific staining patterns are mandatory before the yield of screening or surveillance colonoscopy can be increased. The new detailed images seen with magnifying chromoendoscopy are unequivocally the beginning of a new era where new optical developments will allow a unique look on cellular structures.

Adenoma↗
PubMed1997

Ultrasonic liposculpturing: extrapolations from the analysis of in vivo sonicated adipose tissue.

The ultrasonic liposculpturing technique is currently gaining increasing popularity. Although ultrasound is an accepted part of our diagnostic medical practice, the way in which it interacts with solid living tissue is still a complex and unsolved biophysical problem. Very few studies, if any, have followed the effects of diffusion of this intriguing technique on the fields of biosafety and interaction mechanisms. We evaluate the results of our standard ultrasound liposculpturing technique in order to recognize the physical mechanism-thermal, cavitational, or "direct"-involved in the damaging process. Our microscopic analysis of sonicated adipose tissue confirms that ultrasound is highly selective in its action, producing disruption of macromolecules and cellular structures probably through microstreaming tissue movement. The results of ultrasonic liposculpturing and standard suction lipoplasty are compared. The main advantages of this new technique are the possibility of a very selective destruction of adipose tissue and the prospective solution to such delicate problems as the irregularity of cellulite.

Adipose Tissue↗
PubMed2001

Influence of different biodegradable carriers on the in vivo behavior of human adipose precursor cells.

The correction of soft-tissue defects presents a challenge in plastic and reconstructive surgery. The implantation of isolated and culture-expanded adipose precursor cells is a solution to this problem because these cells differentiate into adipocytes when implanted in vivo. Appropriate scaffolds are needed in soft-tissue engineering to allow the differentiation of precursor cells. The optimal carrier needs to be defined. In this study, human preadipocytes were isolated and cultured. Three different carrier materials were seeded with 106 preadipocytes each and implanted in 42 nude mice. Sponges and nonwoven carriers based on hyaluronic acid modified by esterification (HYAFF 11) were compared with collagen sponges. Scaffolds without cells served as negative controls in the same animal. After 3 and 8 weeks, the grafts were explanted. Macroscopic appearance, weight, thickness, microscopy, immunohistochemistry, and TEM (scaffold structure, cellularity, penetration depth of the seeded cells, vascularization) were assessed and evaluated for differences in scaffold-cell interactions.Preadipocytes differentiated earlier in vitro when attached to HYAFF 11 scaffolds than to other carrier materials. Macroscopically, all preadipocyte constructs were yellowish and well vascularized, and the controls were white and avascular. Vessel formation was more pronounced around mature adipocytes. Microscopically, HYAFF 11 constructs showed a higher cell density than collagen constructs. The pores of the sponges contained more differentiated adipocytes than the nonwoven carriers, whereas the undifferentiated preadipocytes were more numerous in the nonwoven material. Penetration of adipose precursor cells was deeper and more homogeneous in HYAFF 11 scaffolds. Electron microscopy demonstrated well-differentiated adipocytes and large amounts of extracellular matrix in HYAFF 11 sponges.HYAFF 11 sponges supported the expansion and differentiation of the adipose precursor cells. This carrier is superior to the nonwoven carrier with regard to adipocyte differentiation and superior to the collagen sponge with regard to cellularity. This is a promising method for the reconstruction of soft-tissue defects. Modifications of the scaffold (larger pore size and coating with adipogenic factors) will be examined in further experiments.

Adipocytes↗
PubMed1998

Characterization of apoptosis in a motor neuron cell line.

STUDY DESIGN: Serum withdrawal was introduced to a spinal cord motor neuron cell line to investigate the mode of cell death. OBJECTIVES: To characterize the death of motor neurons in culture, to gain insight into mechanisms that could be important in spinal cord diseases. SUMMARY OF BACKGROUND DATA: Normal reduction of cell number during central nervous system development is brought about by programmed cell death. These same apoptotic processes probably play a role in a variety of central nervous system disorders, including traumatic injury. Although certain proteolytic processes are involved, the molecular details involved in the apoptotic induction have not been fully elucidated. METHODS: To identify apoptosis, several criteria were used, including analysis of chromatin condensation with DNA-specific stains (propidium iodide and Hoechst 33342); in situ end-labeling of DNA fragments in apoptotic nuclei with terminal deoxynucleotidyl transferase; fragmentation of DNA separated on agarose gel electrophoresis; and cleavage of a characteristic substrate for apoptotic proteases, alpha-fodrin, into signature cleavage fragments. RESULTS: The NSC19 cell line exhibited motor neuron characteristics morphologically, with typical cellular structure, and biochemically, by synthesizing choline acetyl transferase. Under various treatments including serum withdrawal (loss of trophic factors), cell loss occurred through an apoptotic cell death pathway. CONCLUSIONS: A murine motor neuron cell line, NSC19, has been used to investigate apoptosis in this in vitro system. Cell death occurs by apoptosis, suggesting that this cell line may provide a useful model for studying apoptotic mechanisms in spinal cord degeneration and injury.

Animals↗
PubMed1995

Cerebral metabolic rate and hypothermia: their relationship with ischemic neurologic injury.

Hypothermia results in a progressive depression of cerebral electrical activity and metabolism. In the setting of cerebral ischemia, large reductions in temperature are associated with a better preservation of high-energy phosphates, a reduced accumulation of toxic metabolites, and an improvement in post-ischemic outcome. With temperature reductions of < or = 6 degrees C, the brain is also partially protected from ischemic neurologic injury; however, this protection does not correlate with measurable alterations in high-energy phosphate depletion or lactate accumulation. Thus, cerebral protection by hypothermia may be due to a variety of factors, including alterations in basal metabolism, ion homeostasis, agonist-specific receptor activity, and cellular structure. Further, the relative influence of these factors may change with progressive reductions in temperature.

Brain↗
PubMed2001

Endothelial function and coronary artery disease.

The endothelium produces a number of vasodilator and vasoconstrictor substances that not only regulate vasomotor tone, but also the recruitment and activity of inflammatory cells and the propensity towards thrombosis. Endothelial vasomotor function is a convenient way to assess these other functions, and is related to the long-term risk of cardiovascular disease. Lipids (particularly low density lipoprotein cholesterol) and oxidant stress play a major role in impairing these functions, by reducing the bioavailability of nitric oxide and activating pro-inflammatory signalling pathways such as nuclear factor kappa B. Biomechanical forces on the endothelium, including low shear stress from disturbed blood flow, also activate the endothelium increasing vasomotor dysfunction and promoting inflammation by upregulating pro-atherogenic genes. In contrast, normal laminar shear stress promotes the expression of genes that may protect against atherosclerosis. The sub-cellular structure of endothelial cells includes caveolae that play an integral part in regulating the activity of endothelial nitric oxide synthase. Low density lipoprotein cholesterol and oxidant stress impair caveolae structure and function and adversely affect endothelial function. Lipid-independent pathways of endothelial cell activation are increasingly recognized, and may provide new therapeutic targets. Endothelial vasoconstrictors, such as endothelin, antagonize endothelium-derived vasodilators and contribute to endothelial dysfunction. Some but not all studies have linked certain genetic polymorphisms of the nitric oxide synthase enzyme to vascular disease and impaired endothelial function. Such genetic heterogeneity may nonetheless offer new insights into the variability of endothelial function.

Animals↗
PubMed2006

Rat brain DNA transcript profile of halothane and isoflurane exposure.

Inhaled anesthetics produce many effects and bind to a large number of brain proteins, but it is not yet clear if this is accompanied by widespread changes in gene expression of the biological targets. Such changes in expression might implicate functionally important targets from the large pool of binding targets. Both rats and isolated primary cortical neurons were exposed to anesthetics and DNA oligonucleotide microarrays were used to detect and quantify transcriptional changes in neural tissue. Using analysis of variance with multiple testing correction, multiple exposures of rats to 0.8 MAC (minimum alveolar concentration) halothane only produced significant changes in a few metabolic genes. No significant in-vivo gene transcriptional response to 0.8 MAC isoflurane was detected. The use of primary cortical neurons allowed exposure to 3 MAC anesthetics without evidence of toxicity. Isoflurane altered several genes involved with neurotransmitter transport, signaling and cellular structure, whereas halothane produced few detectable changes in these cultured cells. Selected genes were confirmed by quantitative reverse transcription-polymerase chain reaction. Although indicating only a small degree of transcriptional regulation, these data implicate several plausible targets, including synaptic vesicle handling, that might contribute to drug action. In addition, the data show different gene expression profiles for the two inhaled anesthetics, suggesting unique pharmacological targets and mechanisms in each case.

Anesthetics, Inhalation↗
PubMed2003

HSP70 interacts with ribosomal subunits of thermotolerant cells.

The expression of heat shock or stress proteins (hsps) is a widespread response to stress that results in the protection of cells from subsequent insults, coined stress tolerance. Stress tolerance is apparently due to the preservation of several cellular structures and processes, such as translation. Protection of protein synthesis has been correlated with the presence of Hsp70. In the present study, Hsp70 was found to interact with translating ribosomes. This interaction is due to the preferential binding of Hsp70 to the 40S ribosomal subunit. Additionally, Hsp70 seems to interact weakly with nascent polypeptides within the 60S subunit. The interaction between Hsp70 and ribosomal subunits could also be observed in vitro conditions. Binding of Hsp70 to ribosomes was salt resistant, suggesting that this protein is not bound to transiently associated translational factors. Moreover, protection of protein synthesis requires new gene expression. We speculate that the binding of Hsp70 to ribosomes is part of a mechanism to guarantee the rapid and abundant protein synthesis during stress, particularly the translation of mRNAs encoding for hsps.

Cell Line↗
PubMed2003

Dynamic observation of oxygenation-induced contraction of and transient fiber-network formation-disassembly in cultured human brain microvascular endothelial cells.

Oxygenation-induced contraction of nonconfluent cultured human brain microvascular endothelial cells (HBECs, n = 30) was examined by video-enhanced contrast-differential interferential contrast microscopy. After administering a continuous gentle blow of pure oxygen gas to the surface of the medium just above the flattened HBEC, the plasma membrane exhibited tensioning and wrinkling, resulting in a strong contraction of the cell body by 14 +/- 7% (P < 0.001). When the cell stopped contracting, transient formation of a fiber network starting from certain spots (possibly adhesion plaques, though these were not visible in the majority of cases) and expanding to the whole cell was observed. The occurrence of fiber network formation was statistically significant (26 of 30 separate cells, P < 0.05). After cessation of oxygen delivery, the observed network of fibers broke up rapidly (in a period of 3.3 +/- 1.2 seconds) into small particles of <0.5 microm in diameter, which subsequently fused into the cellular structure. The HBEC completely recovered the control appearance. The sequential process was completed within 30 seconds and was reproduced in individual cells each time that oxygen gas was supplied. The authors conclude that the HBEC strongly contracts in response to a transient oxygenation stimulus, followed by rapid formation/disassembly of a network structure.

Brain↗