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PubMed2006

A ramble through the cell: how can we clear such a complicated trail?

The arrangement of course information in a logical sequence for molecular life science (MLS) courses remains a matter of some controversy, even within a single subdiscipline such as biochemistry. This is due to the explosion of knowledge, the latest bioinformatic revelations, and the observation that new discoveries sometimes reveal specific connections between previously disparate topics. However, the general outlines of biomedical information are in place, at least the knowledge that should be conveyed to undergraduates taking cell and molecular biology and biochemistry. Despite the increasing amount and complexity of the information to be presented, integration and unification are possible because the molecular reactions and interactions that underlie all life processes are coming into view: they are common to all cellular structural rearrangements, nucleic acid functions, and biochemical reactions, whether of plant or animal origin. Also, it is no longer possible to draw clear boundaries between cell biology, biochemistry, and molecular biology that would not violate the fundamental unity of our understanding. Therefore, an arrangement of content is proposed for a two-semester course that aims to present a unified portrait of upper-division undergraduate MLS.

Cells↗
PubMed1996

Cholesterol and mental disorder.

BACKGROUND: Cholesterol plays an important part in cellular structure and function and changes in serum levels may affect neurotransmission in the central nervous system. METHOD: A MEDLINE literature search was made covering the period 1990-95 with systematic searching of citations from the articles identified. Representative articles were selected, focusing on those aspects which had not been thoroughly reviewed elsewhere, namely suicidal ideation, depression, personality and schizophrenia. RESULTS: Lowering cholesterol levels have been associated with an increase in violent deaths in cardiovascular primary prevention studies. However, altered cholesterol levels have also been reported in relation to other psychiatric disorders. CONCLUSION: There is substantial evidence that serum cholesterol levels may be associated with variations in mental state or personality. Further work is needed to clarify this and to elucidate the mechanisms involved.

Brain↗
PubMed1980

Choline incorporation into lecithin in response to insulin or dexamethasone in homogeneous cell cultures of rat lung epithelial cells and fibroblasts.

Labeled choline incorporation into adult rat lung alveolar epithelial cells and adult rat lung fibroblasts in monolayer culture was determined after incubation with insulin (Ins) 10 micrograms/ml, Dexamethasone (Dex) 10(-6)M, or no drug (ND). Incubation periods were 1, 3, 4, and 5 hours. The lecithin (phosphatidyl choline - PC) recovered was separated inot disaturated phosphatidyl choline (DSPC) and unsaturated phosphatidyl choline (USPC). Results expressed as specific activity per hour (see Table) indicate that the incorporation of choline into PC and USPC was greater in fibroblasts (F) than in epithelial cells (E) whether ND, Dex or Ins was present. For incorporation into DSPC, there was no difference between E and F whether ND, Dex or Ins was present. There was significant increase in choline incorporation into PC or USPC for both cell types when Ins was present, whereas there was no difference for either cell type when Dex was present. Insulin significantly increased choline incorporation into DSPC in E cells only. Dex was no different from ND in DSPC incorporation in either cell type. We attribute the greater lecithin synthesis of the F cells to a more rapid increase in cellular structural lipids in the fibroblast cell. Dex had no effect on either cell type possibly from the short-term exposure or possibly because the effect of dexamethasone on alveolar epithelial cells is mediated by product(s) from other lung cells, and thus requires a mixed cell culture to have its effect. We suggest that further study of isolated homogeneous cell lines will not be fruitful in the evaluation of mechanisms of acceleration of lung maturation.

Animals↗
PubMed2000

Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis.

The adult skeleton regenerates by temporary cellular structures that comprise teams of juxtaposed osteoclasts and osteoblasts and replace periodically old bone with new. A considerable body of evidence accumulated during the last decade has shown that the rate of genesis of these two highly specialized cell types, as well as the prevalence of their apoptosis, is essential for the maintenance of bone homeostasis; and that common metabolic bone disorders such as osteoporosis result largely from a derangement in the birth or death of these cells. The purpose of this article is 3-fold: 1) to review the role and the molecular mechanism of action of regulatory molecules, such as cytokines and hormones, in osteoclast and osteoblast birth and apoptosis; 2) to review the evidence for the contribution of changes in bone cell birth or death to the pathogenesis of the most common forms of osteoporosis; and 3) to highlight the implications of bone cell birth and death for a better understanding of the mechanism of action and efficacy of present and future pharmacotherapeutic agents for osteoporosis.

Animals↗
PubMed1983

Role of disulfide bonds in human growth hormone binding and dissociation in isolated rat hepatocytes and liver plasma membranes.

The role of disulfide bonds and sulfhydryl groups in rat hepatocytes and rat liver plasma membranes in the binding of human GH (hGH) has been studied. Since hGH binding involves uptake and irreversible binding, the effect of disulfide reducing agents [dithiothreitol (DTT) and 2-mercaptoethanol (ME)] and an alkylating agent [N-ethylmaleimide (NEM)] on the time course of binding and displacement of [125I]hGH was determined in the hepatocytes and membranes. The time course of binding and displacement of [125I]hGH was similar in membranes and cells, indicating that the irreversible nature of hGH binding is not dependent upon an intact cellular structure. Both 1% ME and 10 mM DTT prevented further binding of [125I]hGH when added at 60 min of the 300-min binding incubation for both hepatocytes and plasma membranes. The ME caused some initial dissociation of bound [125I]hGH, with subsequent binding to levels that were present when the ME was added. Only ME caused an increase in nonspecific binding with the plasma membranes. Both ME and DTT caused an increase in displacement of [125I]hGH in the presence of excess unlabeled hGH. The amount of [125I]hGH remaining bound in the presence of DTT and unlabeled hGH approached nonspecific levels by 240 min of incubation. NEM caused an increase in the total [125I]hGH bound, but this was apparently due to increased nonspecific binding in the presence of NEM. The effect of the reducers on binding was not secondary to an effect on hGH, since the disulfides of [125I]hGH were not reduced under the conditions of binding with either ME or DTT. The effect of the ME or DTT on binding could be reversed or prevented by subsequent or simultaneous addition of an oxidizer such as NAD or oxidized glutathione. The data indicate that disulfide bonds in the membranes are intimately involved in the maintenance of a receptor structure necessary for hGH binding. The disruption of the disulfides also results in increased dissociation and displacement of the bound [125I]hGH, indicating a possible role in the irreversible nature of hGH binding. This represents a partial delineation of the hGH binding process.

Animals↗
PubMed1991

Analysis of the thyroglobulin internalization process using in vitro reconstituted thyroid follicles: evidence for a coated vesicle-dependent endocytic pathway.

We have designed a new experimental system based on in vitro reconstituted thyroid follicles (RTF) to study the relative implication of macropinocytosis and micropinocytosis processes in the internalization of thyroglobulin (Tg). Thyrocytes cultured in the presence of TSH reorganize in histiotypic and functional follicles. Tg, which accumulates into the newly formed intrafollicular lumen (IL), was pulse labeled with [125I]iodide. Basal or TSH-activated Tg internalization, i.e. transfer from IL to cells, was assessed by measuring [125I]Tg in the cells and the IL; the IL fraction was collected after selective opening of lumina by a short treatment of RTF in a calcium-free medium. We used the ratio between cellular and IL labeled Tg contents as an endocytic index. TSH caused a very rapid increase in the cellular uptake of labeled Tg; the endocytic index increased by a factor of 4-8. The TSH effect was maximum after 15-20 min. TSH had no effect when the chase-incubation was performed at 4 C, but exhibited the same stimulatory action in terms of both time course and amplitude of action at 20 and 37 C. The macropinocytosis-related cellular structures, the pseudopods, were never observed in RTF maintained at 20 C; they were rare at 37 C and only found after 30 min of TSH treatment. At 20 as well as 37 C, the action of TSH on Tg endocytosis was concentration dependent in the range of 0.05-10 mU/ml. A fraction of Tg internalized by thyrocytes was found in coated vesicles. The labeled Tg content of purified coated vesicles varied with the temperature of the chase-incubation and was increased in TSH-treated RTF. Taken together, these data show that endocytosis of Tg by thyroid follicular cells in resting or moderately activated states does not proceed via the pseudopod formation-dependent mechanism, also termed macropinocytosis. Tg internalization would be related to what is referred as micropinocytosis and would involve a coated vesicle-dependent endocytic pathway.

Animals↗
PubMed2005

Distinct mechanisms govern the localisation of Drosophila CLIP-190 to unattached kinetochores and microtubule plus-ends.

CLIP-170 was the first microtubule plus-end-tracking protein to be described, and is implicated in the regulation of microtubule plus-ends and their interaction with other cellular structures. Here, we have studied the cell-cycle-dependent mechanisms which localise the sole Drosophila melanogaster homologue CLIP-190. During mitosis, CLIP-190 localises to unattached kinetochores independently of spindle-checkpoint activation. This localisation depends on the dynein-dynactin complex and Lis1 which also localise to unattached kinetochores. Further analysis revealed a hierarchical dependency between the proteins with respect to their kinetochore localisation. An inhibitor study also suggested that the motor activity of dynein is required for the removal of CLIP-190 from attached kinetochores. In addition, we found that CLIP-190 association to microtubule plus-ends is regulated during the cell cycle. Microtubule plus-end association is strong in interphase and greatly attenuated during mitosis. Another microtubule plus-end tracking protein, EB1, directly interacts with the CAP-Gly domain of CLIP-190 and is required to localise CLIP-190 at microtubule plus-ends. These results indicate distinct molecular requirements for CLIP-190 localisation to unattached kinetochores in mitosis and microtubule ends in interphase.

Animals↗
PubMed1991

Human immunodeficiency virus type 1 protease microinjected into cultured human skin fibroblasts cleaves vimentin and affects cytoskeletal and nuclear architecture.

In human skin fibroblasts microinjected with purified human immunodeficiency virus type 1 protease (HIV-1 PR), stress fibers were lost and alterations in nuclear morphology and condensation of nuclear chromatin were observed. Thereafter, the vimentin intermediate filament (IF) network collapsed. No effect was seen on the microtubules. While complicated by loss of affected cells from the substratum, a minimum estimate of the proportion of cells demonstrating these effects is 50%. Observation of single cells demonstrated that these effects were largely irreversible and were steps leading to the death of the HIV-1 PR-injected cells. After microinjection of various dilutions of the HIV-1 PR, it was observed that the changes in nuclear morphology and chromatin condensation were detectable under conditions where little or no effect was observed on both stress fibers and the IF network. Proteins of cells labelled with [35S]methionine and microinjected with either HIV-1 PR or BSA were subjected to two-dimensional gel electrophoresis. The major differences in the gel patterns were a diminution in the amount of vimentin and the appearance of novel products comigrating with cleavage products obtained after treatment of vimentin with HIV-1 PR in vitro. Thus, the HIV-1 PR is capable not only of cleaving IF subunit proteins in vivo, but also can catalyze alterations in other cellular structures.

Bisbenzimidazole↗
PubMed1998

Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells.

Using a computerized fluorescence microscope system to observe fluorescently stained cellular structures in vivo, we have examined the dynamics of chromosomes and microtubules during the process of meiosis in the fission yeast Schizosaccharomyces pombe. Fission yeast meiotic prophase is characterized by a distinctive type of nuclear movement that is led by telomeres clustered at the spindle-pole body (the centrosome-equivalent structure in fungi): the nucleus oscillates back and forth along the cell axis, moving continuously between the two ends of the cell for some hours prior to the meiotic divisions. To obtain a dynamic view of this oscillatory nuclear movement in meiotic prophase, we visualized microtubules and chromosomes in living cells using jellyfish green fluorescent protein fused with alpha-tubulin and a DNA-specific fluorescent dye, Hoechst 33342, respectively. Continuous observation of chromosomes and microtubules in these cells demonstrated that the oscillatory nuclear movement is mediated by dynamic reorganization of astral microtubules originating from the spindle-pole body. During each half-oscillatory period, the microtubules extending rearward from the leading edge of the nucleus elongate to drive the nucleus to one end of the cell. When the nucleus reversed direction, its motion during the second half of the oscillation was not driven by the same microtubules that drove its motion during the first half, but rather by newly assembled microtubules. Reversible inhibition of nuclear movement by an inhibitor of microtubule polymerization, thiabendazole, confirmed the involvement of astral microtubules in oscillatory nuclear movement. The speed of the movement fluctuated within a range 0 to 15 micron/minute, with an average of about 5 microm/minute. We propose a model in which the oscillatory nuclear movement is mediated by dynamic instability and selective stabilization of astral microtubules.

Cell Division↗
PubMed2002

Three-dimensional reconstruction of cytoplasmic membrane networks in parietal cells.

There is general agreement that stimulation and consequent secretion of gastric parietal cells result in a great expansion of the apical canalicular membrane at the expense of an extensive intracellular network of membranes rich in the gastric proton pump (H,K-ATPase). However, there is ongoing controversy as to the precise nature of the intracellular membrane network, conventionally called tubulovesicles. At the heart of this controversy lies the question of whether tubulovesicles are a distinct membrane compartment or whether they are continuous with the apical plasma membrane. To address this controversy we used high-pressure, rapid freezing techniques to fix non-stimulated (resting) rabbit gastric glands for electron microscopy. Ultra-thin (60-70 nm) serial sections were used for conventional TEM; 400-500 nm sections were used for tomography. Images were digitized and models constructed using Midas and Imod software (http://bio3d.colorado.edu ). Images were aligned and contours drawn on specific cellular structures. The contours from a stack of serial sections were arranged into objects and meshed into 3D structures. For resting parietal cells our findings are as follows: (1) The apical canaliculus is a microvilli-decorated, branching membrane network that extends into and throughout the parietal cell. This agrees well with a host of previous studies. (2) The plentiful mitochondria form an extensive reticular network throughout the cytoplasm. This has not previously been reported for the parietal cell, and the significance of this observation and the dynamics of the mitochondrial network remain unknown. (3) H,K-ATPase-rich membranes do include membrane tubules and vesicles; however, the tubulovesicular compartment is chiefly comprised of small stacks of cisternae. Thus a designation of tubulocisternae seems appropriate; however, in the resting cell there are no continuities between the apical canaliculus and the tubulocisternae or between tubulocisternae. These data support the recruitment-recycling model of parietal cell stimulation.

Animals↗
PubMed1988

Video-enhanced light microscopy and its applications in cell biology.

The combination of novel optical microscopic techniques with advanced video and digital image-processing technology now permits dramatic improvements in the quality of light-microscope images. Such video-enhanced light microscopy has lead to a renaissance in the applications of the light microscope for the study of living cells in two important areas: the intensification of faint fluorescence images, permitting observation of fluorescently labelled cells under conditions of very low illuminating intensity; and the enhancement of extremely low contrast images generated by minute cellular structures, so that these may be clearly seen and their normal intracellular movements recorded. Application of both these aspects of video-enhanced light microscopy have recently led to major discoveries concerning the functioning of the living cell. In this review I discuss the equipment, procedures and image-processing principles employed in these applications, and describe and illustrate some of the spectacular results that have recently been obtained.

Animals↗
PubMed2004

The role of muscarinic receptors and intracellular Ca2+ in the spectral reflectivity changes of squid iridophores.

In this paper we describe changes in spectral reflectivity of the light reflectors (iridophores) of the squid Alloteuthis subulata. The spectral changes that can be seen in living squid, can also be brought about by superfusing whole skin preparations with acetylcholine (ACh) (20 micro mol l(-1)) and muscarine (30 micro mol l(-1)) but not nicotine (up to 50 mmol l(-1)), suggesting that cholinergic muscarinic receptors are involved. Changing the osmolarity of the external solution had no effect on spectral reflectivity. To study the iridophores at the cellular level, iridophores were isolated enzymatically. Lucifer Yellow filled the iridophores uniformly, showing cellular individuality. Isolated iridophore cells were loaded with Fura-2 AM and cytoplasmic Ca(2+) was recorded ratiometrically. Intracellular Ca(2+) (resting concentration at 66.16 nmol l(-1)) increased transiently after addition of ACh (50 micro mol l(-1)), muscarine (25 micro mol l(-1)), but not nicotine (up to 5 mmol l(-1)). Ca(2+) also increased when superfused with potassium chloride (10 mmol l(-1)) and caffeine (2.5 mmol l(-1)). Hypo- and hyperosmotic solutions had no effects on the cytoplasmic Ca(2+). By presenting direct evidence that iridophores are polarised cellular structures containing Ca(2+) stores and that they are activated via cholinergic muscarinic receptors, we demonstrate that Ca(2+) is involved in the reflectivity changes of the iridophores of A. subulata. Specimens were prepared for transmission electron microscopy. It was found that the orientations of the plates with respect to the skin surface are in good agreement with the expected orientations based on the prediction that the iridophores act as multilayer reflectors.

Acetylcholine↗
PubMed1985

Excitation-contraction coupling: the link between the surface and the interior of a muscle cell.

The control of the contractile state of a muscle cell, a process called excitation-contraction coupling, involves a sequence of steps and is passed along through a series of cellular structures. The excitation of the fibre, initiated at the motor end-plate, spreads rapidly over the surface of the fibre and into the fibre along the T-system networks as an action potential. This excitation is coupled to the sarcoplasmic reticulum (SR) by a mechanism not yet identified with certainty. The SR then responds by releasing stored calcium from its interior compartment into the myoplasm. The diffusion and binding of this calcium to regulatory proteins initiates the mechanical events of contraction, which subsequently are turned off when calcium is removed from these regulatory binding sites. Some of this calcium may temporarily bind to proteins in the myoplasm, but eventually it gets pumped back into the internal compartment of the SR by a calcium-ATPase in the SR membrane. The system then is poised for a repeat performance.

Animals↗
PubMed1997

Dynamics of actin in cardiac myofibrils and fibroblast stress fibers.

The exchangeability of actin in cardiac myofibrils and fibroblast stress fibers was investigated using fluorescent analogue cytochemistry in combination with fluorescence recovery (FR) after photobleaching. Living embryonic chicken cardiac myocytes and fibroblasts were microinjected with rhodamine (rh)-labeled muscle and nonmuscle actins. After incorporation of the fluorescent actin analogue into cellular structures, small areas of labeled structures were photobleached with a laser pulse. In cardiac myofibrils, FR in their proximal striated portions occurred at a slower rate than that in their proximal nonstriated and distal terminal portions with each rh-isoactin injected. Thus, nascent myofibrils at different developmental stages display different actin exchangeabilities. Further, in all portions of myofibrils, FR of rh-muscle actin was faster than that of rh-nonmuscle actin. This indicates that actin molecules in cardiac myofibrils cannot be readily exchanged by heterotypic nonmuscle actin. In fibroblasts, photobleaching of stress fibers yielded similar results in both their proximal mid-points and distal terminal portions, and the FR rate was consistently faster than that observed in any part of the myofibrils. This result seems to be related to the dynamic properties of actin filaments in stress fibers at all portions. Further, the fact that stress fibers possessed a similar exchange rate with muscle and nonmuscle actins appears to be related to a more primitive nature of stress fibers than myofibrils.

Actins↗
PubMed2005

Live cell imaging to study signaling molecules in allergic reactions.

Mast cells are widely distributed throughout the body, predominantly near blood vessels and nerves, and express effector functions in allergic reactions, inflammatory diseases, and host defense. The activation of mast cells results in secretion of the preformed chemical mediators in their granules by a regulated process of exocytosis and leads to synthesis and secretion of lipid mediators and cytokines. Their soluble factors contribute to allergic inflammation. Mast cells are associated with hypersensitivity reactions, not only in the classical immunoglobulin E (IgE)-dependent mechanism but also in an IgE-independent manner. In particular, investigations of potential anatomical and functional interactions between mast cells and the nervous system have recently attracted great interest. To understand these molecular mechanisms in mast cell activation, the ability to visualize, track, and quantify molecules and events in living mast cells is an essential and powerful tool. Recent dramatic advances in imaging technology and labeling techniques have enabled us to carry out these tasks with high spatiotemporal resolution using confocal laser scanning microscopes, green fluorescent protein and its derivatives, and image analysis systems. Here we review our investigations of the dynamic processes of intracellular signaling molecules, cellular structure, and interactions with neurons in mast cells to provide basic and valuable information for allergy and clinical immunology using these new imaging methods.

Animals↗
PubMed2005

Use of three-dimensional collagen gels to study mechanotransduction in T47D breast epithelial cells.

Several pathological and disease conditions can alter the mechanical properties of the extracellular matrix (ECM). Conversely, some diseases may arise from changes in the density or rigidity of the ECM. This necessitates the use and development of in vitro models to understand how both biophysical and biochemical signals regulate complex cellular behaviors. T47D breast epithelial cells will differentiate into duct-like tubules when cultured in a floating three-dimensional (3D) collagen gel, but not a 3D collagen gel that is left attached to the culture dish. This paper details several protocols we have developed for analyzing breast cell biology in 3D matrices, including culturing cells in 3D collagen gels, immunostaining cellular structures, and performing biochemical procedures directly from cells embedded in collagen gels.

Journal Article↗
PubMed1979

Effect of allopurinol (zyloric) on patients undergoing open heart surgery.

We observed previously that allopurinol, which was used for the treatment of gout, had a life saving effect after experiment on traumatic shock rats and hemorrhagic shock rabbits. To evaluate the effect of allopurinol fifteen patients undergoing open heart surgery who were considered to have similar metabolic derangement in shock patient were examined. Allopurinol was given orally 2 mg per kg body weight twice before the start of nitrous oxide, oxygen and halothane anesthesia. In the control group of ten patients who were not treated with allopurinol, serum uric acid increased, the lactate/pyruvate ratio rose and beta-glucuronidase activity increased respectively after open heart surgery as in shock. But the metabolic changes of the fifteen patients pretreated with allopurinol were less significant, although same tendency was observed. The heart beat of all patients except one case in the allopurinol group, started spontaneously after extracorporeal circulation without using DC counter shock. In the control group all patients needed DC counter shock. We concluded that allopurinol was effective in preventing damage of cellular structures and derangements of metabolism of patients undergoing open heart surgery.

Allopurinol↗
PubMed1996

Effects of microwave tissue coagulation on the livers of normal rabbits: a comparison of findings of image analysis and histopathological examination.

The livers of normal rabbits were subjected to microwave tissue coagulation (MTC), and comparison was made of the subsequent time-course changes in tissue observed on MRI, CT and histopathological examination. 16 rabbits were used. MTC was performed with a 21 gauge needle electrode inserted into the liver at laparotomy. 1-2 h after thermal coagulation, a region with slightly lower attenuation than that of surrounding normal liver parenchyma was observed on CT, and no enhancement was detected. With MRI, change from high signal intensity to iso-signal intensity from the inner zone to the margin was found on T1 weighted images (T1WI), and heterogeneous high signal intensity was observed on T2 weighted images (T2WI). On Gd-DTPA enhanced MRI, no enhancement occurred. 1-4 weeks after coagulation, the cellular structure at the site of coagulation was lost on histological examination, and the tissue became necrotic. On CT, homogeneous water density was observed, and no enhancement was detected. With MRI, regions of iso- or slightly low signal intensity were observed on T1WI, and regions of heterogeneous high to low signal intensity were seen on T2WI. After 1 week, a granulation layer consisting mainly of fibrous tissue developed, and a ring-shaped enhancement was observed in the low signal intensity region on T1WI and in the high signal intensity region on T2WI. The ring-shaped enhancement was also noted on CT. MRI appears to be useful for observation of time-course changes following MTC therapy because of its sensitivity in the detection of tissue changes.

Animals↗