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PubMed2000

Mapping of surrogate markers of cellular components and structures using laser desorption/ionization mass spectrometry.

Laser desorption/ionization mass spectrometry (LDI-MS) has been used to assess the potential of using surrogate markers, bound to cellular structures containing nucleic acids, to image or map the position of these structures within biological samples. In this study, organic dyes were used as markers because of their established use in the histochemical marking of nucleic acids, and also because they are amenable to LDI-MS. Eight cationic dyes were tested and all could be desorbed from nucleic acid samples without additional matrix after specifically binding to these molecules. Methylene Blue was the best of these based on its sensitivity to detection by LDI-MS and the fact that it can be washed from the tissue in areas where it was not specifically bound to provide low-intensity background signals. Experiments are reported which characterize the M(+) ion signal obtained from Methylene Blue with regard to sensitivity, reproducibility and possible use for quantitation. This dye was used to map (with a lateral resolution of 25 microm) several nucleic acid-containing samples spotted on prepared surfaces, and to image the location of nucleic acids in two model tissues, retinal vertical sections and thyroid whole mount sections.

Animals↗

PubMed2005

Development of aortic valve sclerosis in a rabbit model of atherosclerosis: an immunohistochemical and histological study.

BACKGROUND AND AIM OF THE STUDY: It has been suggested that aortic valve sclerosis (AVS) is an atherosclerotic disease process that can proceed to aortic stenosis. The absence of reports studying an animal model of the early stages of this disease has precluded the development of preventive therapeutic strategies. A cholesterol-fed (0.25% cholesterol in chow) rabbit model of atherosclerosis that is characterized by a moderate level of hypercholesterolemia was studied to determine its efficacy as a model of early AVS. Cellular, structural and morphological changes in the aortic valves of these rabbits were studied. METHODS: Twenty rabbits were assigned randomly to four experimental groups: Group 1 received normal chow for 40 weeks; group 2 received 0.25% cholesterol-supplemented chow for 20 weeks; group 3 received 0.25% cholesterol-supplemented chow for 40 weeks; and group 4 received 0.25% cholesterol-supplemented chow for 20 weeks followed by normal chow for an additional 20 weeks. The aortas and aortic valves were analyzed using immunohistochemical and histological methods to detect cellular and structural components of the developing lesions. RESULTS: All rabbits in groups 2, 3 and 4 developed atherosclerotic lesions in their aortas. Aortic valves from these animals demonstrated thickening, lipid deposition, a change in collagen content and organization, a reorganization of elastin, and the presence of both macrophage infiltrate and osteopontin. CONCLUSION: These findings were consistent with the suggestion of a link between atherosclerosis and AVS. Results were also similar to changes reported in human sclerotic aortic valves, suggesting the suitability of this rabbit model of atherosclerosis as a model for AVS.

Animals↗

PubMed1995

Vitamin E deficiency as a model of precocious brain aging: assessment by X-ray microanalysis and morphometry.

Vitamin E (alpha-tocopherol) is a known biological antioxidant able to quench the lipid peroxidation chain and to protect the cellular structures (e.g., plasma membranes) from the attack of free radicals which are reported to play a primary role in aging. To assess whether the absence of alpha-tocopherol from the diet of young laboratory animals may be considered a reliable model of precocious brain aging, intracellular ionic content of brain cortex pyramidal cells, ultrastructural features of synaptic contact zones, synaptic mitochondria and perykarial mitochondria positive to the succinic dehydrogenase (SDH) histochemical reaction with copper ferrocyanide have been investigated by X-ray microanalysis and computer-assisted morphometry in young, adult, old and 11-month-old vitamin E deficient rats. Our data document significant alterations of intracellular ionic content, synaptic contact areas and synaptic and perykarial mitochondria in aging. Vitamin E deficiency caused similar alterations in adult animals. Taking into account the known role of alpha-tocopherol in protecting the cellular membrane structure, we support that the common process underlying the changes found in aging and vitamin E deficiency is an excessive deterioration of the neuronal membrane.

Aging↗

PubMed2002

Learning cell biology as a team: a project-based approach to upper-division cell biology.

To help students develop successful strategies for learning how to learn and communicate complex information in cell biology, we developed a quarter-long cell biology class based on team projects. Each team researches a particular human disease and presents information about the cellular structure or process affected by the disease, the cellular and molecular biology of the disease, and recent research focused on understanding the cellular mechanisms of the disease process. To support effective teamwork and to help students develop collaboration skills useful for their future careers, we provide training in working in small groups. A final poster presentation, held in a public forum, summarizes what students have learned throughout the quarter. Although student satisfaction with the course is similar to that of standard lecture-based classes, a project-based class offers unique benefits to both the student and the instructor.

Biology↗

PubMed2001

Morphological investigation of Toxoplasma gondii in vivo by a multiple beam interference microscope.

A recently developed technique, namely multiple beam interference microscopy, has been applied to investigate the morphology of the parasite Toxoplasma gondii for the first time. The interference pattern obtained from the multiple internal reflection of a T. gondii, sandwiched between a glass plate and a cover plate, was focused on the objective of a conventional microscope. Because of the enhance contrast, several details of sub cellular structure and separating compartments are clearly visible. Details reveal the presence of a nucleus, lipid body, dense granule, rhoptry and amylopectin. The wall thickness of the membrane of the lipid body and the amylopectin is of the order of 0.02 microm and can be clearly distinguished with the help of the present technique. The same parasite has also been examined with the help of atomic force microscopy, and because of its thick membrane, the inner structural details were not observed at all. Sub cellular details of T. gondii observed with the present technique have been reported earlier only by low amplification transmission electron microscopy and not by any optical microscopic technique.

Animals↗

PubMed1999

Direct visualization of a protein nuclear architecture.

Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus.

Animals↗

PubMed1983

High-resolution autoradiography in mouse brain 24 h after radiolabelled CDP-choline administration.

A group of mice was given 10 microCi/animal of cytidine diphosphate choline (CDP-choline, citicoline, Somazina) (methyl 14C). Animals were sacrificed 24 h after administration and their brains isolated for electron microscopy and autoradiography. The main purpose of the present study was to examine the intracellular distribution of the drug and its possible specificity site in specific organelles and cellular structures. While a large quantity of radioactivity appeared in cellular membranes, no selectivity was in its distribution thus being detected in several cellular organelles and free hyaloplasm.

Animals↗

PubMed2006

Human development VIII: a theory of "deep" quantum chemistry and cell consciousness: quantum chemistry controls genes and biochemistry to give cells and higher organisms consciousness and complex behavior.

Deep quantum chemistry is a theory of deeply structured quantum fields carrying the biological information of the cell, making it able to remember, intend, represent the inner and outer world for comparison, understand what it "sees", and make choices on its structure, form, behavior and division. We suggest that deep quantum chemistry gives the cell consciousness and all the qualities and abilities related to consciousness. We use geometric symbolism, which is a pre-mathematical and philosophical approach to problems that cannot yet be handled mathematically. Using Occam's razor we have started with the simplest model that works; we presume this to be a many-dimensional, spiral fractal. We suggest that all the electrons of the large biological molecules' orbitals make one huge "cell-orbital", which is structured according to the spiral fractal nature of quantum fields. Consciousness of single cells, multi cellular structures as e.g. organs, multi-cellular organisms and multi-individual colonies (like ants) and human societies can thus be explained by deep quantum chemistry. When biochemical activity is strictly controlled by the quantum-mechanical super-orbital of the cell, this orbital can deliver energetic quanta as biological information, distributed through many fractal levels of the cell to guide form and behavior of an individual single or a multi-cellular organism. The top level of information is the consciousness of the cell or organism, which controls all the biochemical processes. By this speculative work inspired by Penrose and Hameroff we hope to inspire other researchers to formulate more strict and mathematically correct hypothesis on the complex and coherence nature of matter, life and consciousness.

Behavior↗

PubMed2003

Decellularization of rat aortic valve allografts reduces leaflet destruction and extracellular matrix remodeling.

OBJECTIVES: Decellularization of aortic valve allografts in advance of transplantation is a promising approach to overcome immune-induced early graft failure. In this study the effects of in vitro cell extraction on extracellular matrix molecules and in vivo remodeling of decellularized aortic valves were investigated in a heterotopic aortic valve rat implantation model. METHODS: Rat aortic valve conduits were decellularized by a 2-step detergent-enzymatic extraction method involving sodium dodecyl sulfate in combination with RNase and DNase. Cellular and acellular allogeneic (2x, n = 4) and syngeneic valve grafts (2x, n = 3) were grafted infrarenally into the descending aorta for 21 days. Immunohistochemical techniques were used to study extracellular matrix constitution (elastin, collagen, fibronectin, and chondroitin sulfate) and cellular infiltration. RESULTS: The decellularization procedure resulted in a complete loss of all cellular structures from the entire valve conduit with minimal damage to the extracellular matrix. All transplanted cellular allografts became deformed, swollen, and acellular with major changes in extracellular matrix structure. The transplanted decellularized allografts, however, retained normal preserved valve leaflets comparable to transplanted cellular and acellular syngeneic grafts. With the exception of cellular syngeneic grafts, all other grafts showed retrovalvular thrombi. CONCLUSIONS: Damage to the valves caused by decellularization technique is much less than the damage caused by the recipient's immune response. In vitro removal of viable cells in (cryopreserved) homografts may decrease graft failure. Seeding with autologous or major histocompatibility complex-matched donor endothelial cells will be necessary to diminish damage induced by an absent blood-tissue barrier.

Animals↗

PubMed2006

Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye.

PURPOSE: The purpose of this work was to demonstrate the use of the combined imaging modality of multiphoton autofluorescence and second-harmonic generation (SHG) microscopy in obtaining spectrally resolved morphologic features of the cornea, limbus, conjunctiva, and sclera in whole, ex vivo porcine eyes. METHODS: The 780-nm output of a femtosecond, titanium-sapphire laser was used to induce broadband autofluorescence (435-700 nm) and SHG (390 nm) from various regions of the surface of ex vivo porcine eyes. A water-immersion objective was used for convenient imaging of the curved surface of the eye. RESULTS: Multiphoton autofluorescence was useful in identifying cellular structures of the different domains of the ocular surface, and the SHG signal can be used to resolve collagen organization within the cornea stroma and sclera of ex vivo porcine eyes. CONCLUSIONS: Multiphoton autofluorescence and SHG microscopy have been demonstrated to be an effective technique for resolving, respectively, the cellular and collagen structures within the ocular surface of ex vivo porcine eyes. SHG imaging resolved the difference in structural orientations between corneal and sclera collagen fibers. Specifically, the corneal collagen is organized in a depth-dependent fashion, whereas the scleral collagen is randomly packed. Because this technique does not require histologic preparation procedures, it has the potential to be applied for in vivo studies with minimal disturbance to the eye.

Animals↗

PubMed2004

Effect of ischemia-reperfusion on Na+, K+-ATPase expression in human liver tissue allograft: image analysis by confocal laser scanning microscopy.

We have analyzed the effect of ischemia-reperfusion on expression of hepatic Na+,K+-ATPase on bile canalicular (BCM) and basolateral membranes (BLM) in human liver allografts using confocal laser scanning microscopy imaging. Na+, K+-ATPase, an integral membrane enzyme, plays a key role in the physiology and structure of hepatocytes, where it maintains the electrochemical gradients for Na+ and K+ across the cell membrane. The concentrations of these ions as well as their gradients regulate the active transport across the plasma membrane for bile acid and water from sinusoidal to canalicular membranes. In addition, Na+,K+-ATPase is also involved in cellular structure because of its close relationship with submembrane microfilaments and its implication in tight junction assembly. Therefore, Na+,K+-ATPase appears as an indicator of tissue viability and hepatic functionality during liver transplantation. Its localization and its function in BCM are still controversial. As in previous studies, we found an enzyme expression in both BLM and BCM. We show that ischemia induced a decrease in Na+,K+-ATPase expression only in BCM. This result could be explained by the differences in biochemical membrane environment between basolateral and bile canalicular Na+,K+-ATPase. Membrane lipid fluidity, which is more elevated in BLM than in BCM, could protect the enzyme during ischemia. After reperfusion, Na+,K+-ATPase expression was strongly decreased in both BCM and BLM. This alteration following reperfusion is probably due to multiple factors: direct alteration of the enzyme catalytic subunit and modification of its environment and membrane lipid fluidity by free radicals and changes in ATP levels and ionic distribution. This important decrease in Na+,K+-ATPase expression of both BLM and BCM could disturb not only hepatic secretory function but also cellular volume and structure during the postoperative period.

Adult↗

PubMed2006

Coupling the distribution of RNA polymerase to global gene regulation and the dynamic structure of the bacterial nucleoid in Escherichia coli.

Prokaryotic genomes are contained in a cellular structure termed the nucleoid. However, despite a complete genome sequence and years of intensive study of Escherichia coli, our knowledge of nucleoid structure remains quite rudimentary. Moreover, little is known about the in vivo relationship between nucleoid structure and global gene regulation. Recent studies have shown that the structure of the nucleoid responds dynamically to changing environmental conditions and that this metastable nature of the nucleoid is mediated to a large extent by the distribution and activity of RNA polymerase (RNAP). For example, during rapid growth, the nucleoid is highly condensed with RNAP concentrated into transcription foci or factories, structures analogous to the eukaryotic nucleolus, where active transcription of rRNA genes occurs. However, during nutrient starvation and/or limitation, RNAP is redistributed throughout the genome and this is accompanied by a decondensation of the nucleoid. Thus, the distribution of RNAP, global gene regulation and the dynamic structure of the nucleoid are coupled in the bacterial cell.

Cell Proliferation↗

PubMed1990

Role of microfilaments in maintenance of proximal tubule structural and functional integrity.

To determine the selective effect of microfilament disruption on both cellular structure and function, microfilament-specific doses of cytochalasin D (10 microM) were used in an isolated perfused kidney system. Structurally, cytochalasin D resulted in extensive disruption of the apical surface with blebbing, vacuolization, and patchy loss and fusion of microvilli. Functionally, cytochalasin D resulted in an initial decrease in glomerular filtration rate (300.8 +/- 29.9 vs. 541.6 +/- 51 microliters.min-1.g-1, P less than 0.05) with subsequent stabilization throughout the duration of the perfusion. In contrast, the tubular reabsorption of sodium decreased significantly in a linear fashion from 97.1 +/- 0.7 to 64.3 +/- 7.0% over the duration of the perfusion. Similarly, the tubular reabsorption of lithium decreased linearly from 74.8 +/- 2.6%, before the addition of cytochalasin, to 33.6 +/- 6.8% by the end of the perfusion. Correlation of the decrements in percent tubular reabsorption of sodium and lithium for individual kidneys was 0.87 (P less than 0.01), suggesting the effect of microfilament disruption on tubular reabsorption of sodium was localized primarily to the proximal tubule. Because ischemic injury is characterized by time-dependent structural alterations in the apical membrane of proximal tubule cells, we set out to determine whether microfilament disruption occurs during ischemic acute renal failure. Utilizing indirect immunofluorescence with an anti-actin antibody, control kidneys demonstrated intact circumferential apical immunofluorescence representing brush-border and terminal web actin staining. Fifteen minutes of ischemia resulted in multiple large gaps in the terminal web, and 50 min of ischemia caused diffuse redistribution of actin immunofluorescence throughout the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗

PubMed1991

Calcium-binding sites in the inner ear after pure-tone stimulation.

Five guinea pigs were exposed to an interrupted 90 dB SPL pure tone of 3.2 kHz for a total application time of 5 min. Following sound application all animals were decapitated and the cochleae were removed. After that, calcium-binding sites were located by the potassium pyroantimonate precipitation method. Another three animals served as control animals and did not receive the sound treatment. Findings confirmed previous studies showing the spatial arrangements of precipitate rich regions in the inner ear's two acellular structures (the basilar membrane and tectorial membrane) and the two cellular structures (the inner hair cells and Huschke's teeth). By using semiquantitative evaluation we found a relative diminution of precipitable calcium in inner hair cells and in Huschke's teeth of sound-exposed animals when compared to untreated animals.

Acoustic Stimulation↗

PubMed2005

Branched-chain alpha-keto acids accumulating in maple syrup urine disease induce reorganization of phosphorylated GFAP in C6-glioma cells.

In this study we investigate the effects of the branched-chain keto acids (BCKA) alpha-ketoisocaproic (KIC), alpha-ketoisovaleric (KIV), and alpha-keto-beta-methylvaleric (KMV) acids, metabolites accumulating in maple syrup urine disease (MSUD), on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and cytoskeletal reorganization in C6-glioma cells. We observed that after 3 h treatment with KIC, KIV, or KMV cells showed retracted cytoplasm with bipolar processes containing packed GFAP filaments as revealed by immunocytochemistry. Western Blot analysis by anti-GFAP monoclonal antibody demonstrated that BCKA were not able to alter GFAP immunocontent in total cell homogenate, but the immunocontent as well as the in vitro (32)P incorporation into GFAP recovered into the high salt Triton-insoluble cytoskeletal fraction were significantly increased. Western Blot using monoclonal antiphosphoserine antibody showed that BCKA induced increased immunocontent of phosphoserine-containing amino acids in several proteins in total cell homogenate. In addition, the immunocontent of phosphoserine-containing amino acids was also greatly increased in GFAP recovered in the high-salt Triton insoluble cytoskeletal fraction, corresponding to the polymerized intermedite filament (IF) proteins present in the cell. In conclusion, our results indicate that KIC, KIV, or KMV increased the serine/threonine in vitro phosphorylation of GFAP leading to increased Triton-insoluble GFAP immunocontent and cytoskeletal reorganization. Considering IF networks can be regulated by phosphorylation of polypeptide subunits leading to reorganization of the IF filamentous structure, we could suppose that GFAP hyperphosphorylation and disorganization of cellular structure could be involved in the brain damage characteristic of MSUD patients.

Animals↗

PubMed1994

Application of microwave fixation techniques in pathology to neuroscience studies: a review.

The introduction of microwave energy into the scientist's repertoire of fixation modalities offers for the first time in relatively large specimens the potential for 'instantaneous' preservation of cellular structure for light and electron microscopy with minimal alteration of cellular biochemistry and antigenicity. Because of the rapid evolution of this new technology, we provide a classification system of newly generated microwave methods as applied to specimen preservation for microscopic analysis. With emphasis on neuronal tissue, we review qualitative and quantitative microscopy data of specimens fixed by two microwave methods in common use: (1) microwave stabilization and (2) fast and ultrafast, primary microwave-chemical fixation. In addition, we provide a table of neuropeptides or proteins in neuronal tissues that are preserved by various microwave fixation methods for histochemistry, immunohistochemistry, and immuno-electron microscopy studies. Commercial microwave ovens have limitations which can result in irreproducible fixation results. Therefore, we present a calibration protocol that is used to identify the best locations for fixation within large cavity (i.e., household) microwave ovens. We also provide a standardization protocol to improve the reproducibility of microwave fixation in calibrated, large-cavity microwave ovens.

Animals↗

PubMed2003

Simultaneous imaging and functional assessment of cytoskeletal protein connections in passively loaded single muscle cells.

We describe a novel system that permits simultaneous confocal imaging of protein interactions and measurement of cell mechanical properties during passive loading. A mechanical apparatus was designed to replace the stage of a confocal microscope, enabling cell manipulation, force transduction, and imaging. In addition, image processing algorithms were developed to quantify the degree of connectivity between subcellular structures. Using this system, we examined the interactions among three cellular structures thought to be linked by the muscle's intermediate filament system: Z-disks, nuclei, and the costamere protein complexes located at the muscle cell surface. Fast Fourier transforms (FFTs) and autocorrelations (ACs) were implemented to quantify image periodicity and relative phase shifts among structures. We demonstrated in sample wild-type muscle cells that there was significant connectivity among Z-disks in the same fiber at various sarcomere lengths, as well as between Z-disks and the costamere complexes. This approach can be applied to any cell system in which structural periodicity and mechanical connectivity are of interest.

Actinin↗

PubMedDate not supplied

Mobilisation of structural proteins during exercise.

In general, the mobilisation of structural proteins is necessary for enzyme synthesis and for renewing cellular structures with amino acids and precursors of nucleotic acids. However, during exercise the adaptive synthesis of proteins occurs only in the liver to some extent. In muscle tissue most protein synthesis is suppressed, although the synthesis of certain proteins in muscle remains unchanged or even increases. The general suppression of protein synthesis in muscle leaves much of the free amino acid pool unused. The breakdown of tissue proteins may also increase in various tissues, but there is no convincing evidence for proteolysis of contractile proteins in active muscle. As a result of these processes, an increased pool of available free amino acids is created. The main use of free amino acids is connected with the energy requirement of muscular activity, through the oxidation of branched-chain amino acids and the use of alanine in gluconeogenesis. In active muscles the output of alanine is increased. It is based on usage of pyruvate, which is produced in increased amounts due to intensified glycogenolysis and glycolysis, and of amino groups, which are liberated in oxidation of branched-chain amino acids. In the liver, alanine is consumed. The carbon skeleton of alanine is required for gluconeogenesis and the liberated amino groups are used in ureagenesis. The branched-chain amino acids are transported from the liver to active muscle for their oxidation. The increases in the free amino acid pool, in the rate of the glucose-alanine cycle, and in the use of amino acids in the liver are stimulated by an increased level of glucocorticoids and a decreased level of insulin during exercise. During recovery after exercise the use of amino acids for adaptive protein synthesis is intensified. This coincides with a persistently high rate of protein breakdown, constituting an increased rate of protein turnover. During recovery, the production of 3-methylhistidine by previously active muscles increases. It results in an increase in urinary output of 3-methylhistidine after exercise. Immediately after exercise the level of free 3-methylhistidine is elevated in the intestine for only a short time and the fact that it does not contribute significantly to the delayed increase in the excretion of 3-methylhistidine excretion after exercise must be considered as a sign of increased turnover of contractile proteins, helping to restore a good contractile function.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗