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PubMed1982

Demonstration of the Intercellular Compartmentation of l-Menthone Metabolism in Peppermint (Mentha piperita) Leaves.

The metabolism of l-menthone, which is synthesized in the epidermal oil glands of peppermint (Mentha piperita L. cv. Black Mitcham) leaves, is compartmented; on leaf maturity, this ketone is converted to l-menthol and l-menthyl acetate in one compartment, and to d-neomenthol and d-neomenthyl glucoside in a separate compartment. All of the enzymes involved in these reactions are soluble when prepared from whole-leaf homogenates. Mechanical separation of epidermal fragments from the mesophyll, followed by preparation of the soluble enzyme fraction from each tissue, revealed that the neomenthol dehydrogenase and the glucosyl transferase resided specifically in the mesophyll layer, whereas the menthol dehydrogenase and substantial amounts of the acetyl transferase were located in the epidermis, presumably within the epidermal oil glands. These results suggest that the compartmentation of menthone metabolism in peppermint leaves is intercellular, not intracellular.

Journal Article↗
PubMed1983

Paraveinal Mesophyll of Soybean Leaves in Relation to Assimilate Transfer and Compartmentation : III. Immunohistochemical Localization of Specific Glycopeptides in the Vacuole after Depodding.

Immunohistochemical staining was used to determine the cellular distribution of two glycosylated polypeptides (molecular weights of 27 and 29 kilodaltons) which are normally present at low levels in soybean (Glycine max var ;Wye') leaves but which markedly accumulate after depodding. These polypeptides, which comprise a substantial portion of the total leaf soluble protein of depodded plants, were exclusively located in the vacuoles of paraveinal mesophyll and associated bundle sheath cells. These results support the unique role of the soybean leaf paraveinal mesophyll in the transport and spatial compartmentation of nitrogen reserves in relation to seed filling.

Journal Article↗
PubMed1985

Sodium and potassium fluxes and compartmentation in roots of atriplex and oat.

K(+) and Na(+) fluxes and ion content have been studied in roots of Atriplex nummularia Lindl. and Avena sativa L. cv Goodfield grown in 3 millimolar K(+) with or without 3 or 50 millimolar NaCl. Compartmental analysis was carried out with entire root systems under steady-state conditions.Increasing ambient Na(+) concentrations from 0 to 50 millimolar altered K(+), in Atriplex, as follows: slightly decreased the cytoplasmic content (Q(c)), the vacuolar content (Q(v)), and the plasma membrane influx and efflux. Xylem transport for K(+) decreased by 63% in Atriplex. For oat roots, similar increases in Na(+) altered K(+) parameters as follows: plasma membrane influx and efflux decreased by about 80%. Q(c) decreased by 65%, and xylem transport decreased by 91%. No change, however, was observed in Q(v) for K(+). Increasing ambient Na(+) resulted in higher (3 to 5-fold) Na(+) fluxes across the plasma membrane and in Q(c) of both species. In Atriplex, Na(+) fluxes across the tonoplast and Q(v) increased as external Na(+) was increased. In oat, however, no significant change was observed in Na(+) flux across the tonoplast or in Q(v) as external Na(+) was increased. In oat roots, Na(+) reduced K(+) uptake markedly; in Atriplex, this was not as pronounced. However, even at high Na(+) levels, the influx transport system at the plasma membrane of both species preferred K(+) over Na(+).Based upon the Ussing-Teorell equation, it was concluded that active inward transport of K(+) occurred across the plasma membrane, and passive movement of K(+) occurred across the tonoplast in both species. Na(+), in oat roots, was actively pumped out of the cytoplasm to the exterior, whereas, in Atriplex, Na(+) was passively distributed between the free space, cytoplasm, and vacuole.

Journal Article↗
PubMed1986

Compartmental efflux analysis: an evaluation of the technique and its limitations.

Efflux analysis is an established tool for characterizing the exchange properties of multicomponent systems. In this report, we have simulated several three- and four-compartment systems with error-free and imperfect data, the errors being designed to mimic actual, nonbiological variability in isotope efflux studies. The data sets were analyzed using computerized nonlinear regression techniques to identify the important aspects of actual experimental design (uptake times, efflux collection schedules, and total efflux times), and to consider the possibility that a properly designed and executed experiment might fail to resolve compartmentation correctly. The results showed that for any of the systems simulated, including those with error-free four-component data, a reasonable three-component fit was obtainable. Resolution of the additional compartment was not always possible. In correctly resolved systems, failure to estimate the correct decay constants was common, especially when the half-times were separated by less than an order of magnitude. We conclude that efflux analysis, by itself, lacks the power to provide reliable information about multicompartment systems.

Journal Article↗
PubMed1989

P-Nuclear Magnetic Resonance Determination of Phosphate Compartmentation in Leaves of Reproductive Soybeans (Glycine max L.) as Affected by Phosphate Nutrition.

Most leaf phosphorus is remobilized to the seed during reproductive development in soybean. We determined, using (31)P-NMR, the effect phosphorus remobilization has on vacuolar inorganic phosphate pool size in soybean (Glycine max [L.] Merr.) leaves with respect to phosphorus nutrition and plant development. Phosphate compartmentation between cytoplasmic and vacuolar pools was observed and followed in intact tissue grown hydroponically, at the R2, R4, and R6 growth stages. As phosphorus in the nutrient solution decreased from 0.45 to 0.05 millimolar, the vacuolar phosphate peak became less prominent relative to cytoplasmic phosphate and hexose monophosphate peaks. At a nutrient phosphate concentration of 0.05 millimolar, the vacuolar phosphate peak was not detectable. At higher levels of nutrient phosphate, as plants progressed from the R2 to the R6 growth stage, the vacuolar phosphate peak was the first to disappear, suggesting that storage phosphate was remobilized to a greater extent than metabolic phosphate. Under suboptimal phosphate nutrition (</= 0.20 millimolar), the hexose monophosphate and cytoplasmic phosphate peaks declined earlier in reproductive development than when phosphate was present in optimal amounts. Under low phosphate concentrations (0.05 millimolar) cytoplasmic phosphate was greatly reduced. Carbon metabolism was coincidently disrupted under low phosphate nutrition as shown by the appearance of large, prominent starch grains in the leaves. Cytoplasmic phosphate, and leaf carbon metabolism dependent on it, are buffered by vacuolar phosphate until late stages of reproductive growth.

Journal Article↗
PubMed1990

Subcellular compartmentation of uridine nucleotides and nucleoside-5' -diphosphate kinase in leaves.

The subcellular compartmentation of nucleoside diphosphate kinase (EC 2.7.4.6) and the uridine nucleotides has been studied in leaves. Membrane filtration of barley (Hordeum vulgare L.) leaf mesophyll protoplasts and differential centrifugation of spinach (Spinacia oleracea L.) leaf extracts showed that about half the nucleoside diphosphate kinase is present in the cytosol. The activity is adequate to account for the turnover of UTP and UDP during photosynthetic sucrose synthesis. Nonaqueous density gradient centrifugation of freeze-stopped, lyophilized spinach leaf material showed that the uridine nucleotides are predominantly located in the cytosol and that the cytosolic UDP-glucose pool is considerably larger than the UTP or UDP pools.

Journal Article↗
PubMed1991

Compartmentation studies on spinach leaf peroxisomes : evidence for channeling of photorespiratory metabolites in peroxisomes devoid of intact boundary membrane.

In concurrence with earlier results, the following enzymes showed latency in intact spinach (Spinacia oleracea L.) leaf peroxisomes: malate dehydrogenase (89%), hydroxypyruvate reductase (85%), serine glyoxylate aminotransferase (75%), glutamate glyoxylate aminotransferase (41%), and catalase (70%). In contrast, glycolate oxidase was not latent. Aging of peroxisomes for several hours resulted in a reduction in latency accompanied by a partial solubilization of the above mentioned enzymes. The extent of enzyme solubilization was different, being highest with glutamate glyoxylate aminotransferase and lowest with malate dehydrogenase. Osmotic shock resulted in only a partial reduction of enzyme latency. Electron microscopy revealed that the osmotically shocked peroxisomes remained compact, with smaller particle size and pleomorphic morphology but without a continuous boundary membrane. Neither in intact nor in osmotically shocked peroxisomes was a lag phase observed in the formation of glycerate upon the addition of glycolate, serine, malate, and NAD. Apparently, the intermediates, glyoxylate, hydroxypyruvate, and NADH, were confined within the peroxisomal matrix in such a way that they did not readily leak out into the surrounding medium. We conclude that the observed compartmentation of peroxisomal metabolism is not due to the peroxisomal boundary membrane as a permeability barrier, but is a function of the structural arrangement of enzymes in the peroxisomal matrix allowing metabolite channeling.

Journal Article↗
PubMed1992

Functional compartmentation of the Golgi apparatus of plant cells : immunocytochemical analysis of high-pressure frozen- and freeze-substituted sycamore maple suspension culture cells.

The Golgi apparatus of plant cells is engaged in both the processing of glycoproteins and the synthesis of complex polysaccharides. To investigate the compartmentalization of these functions within individual Golgi stacks, we have analyzed the ultrastructure and the immunolabeling patterns of high-pressure frozen and freeze-substituted suspension-cultured sycamore maple (Acer pseudoplatanus L.) cells. As a result of the improved structural preservation, three morphological types of Golgi cisternae, designated cis, medial, and trans, as well as the trans Golgi network, could be identified. The number of cis cisternae per Golgi stack was found to be fairly constant at approximately 1, whereas the number of medial and trans cisternae per stack was variable and accounted for the varying number of cisternae (3-10) among the many Golgi stacks examined. By using a battery of seven antibodies whose specific sugar epitopes on secreted polysaccharides and glycoproteins are known, we have been able to determine in which types of cisternae specific sugars are added to N-linked glycans, and to xyloglucan and polygalacturonic acid/rhamnogalacturonan-I, two complex polysaccharides. The findings are as follows. The beta-1,4-linked d-glucosyl backbone of xyloglucan is synthesized in trans cisternae, and the terminal fucosyl residues on the trisaccharide side chains of xyloglucan are partly added in the trans cisternae, and partly in the trans Golgi network. In contrast, the polygalacturonic/rhamnogalacturonan-I backbone is assembled in cis and medial cisternae, methylesterification of the carboxyl groups of the galacturonic acid residues in the polygalacturonic acid domains occurs mostly in medial cisternae, and arabinose-containing side chains of the polygalacturonic acid domains are added to the nascent polygalacturonic acid/rhamnogalacturonan-I molecules in the trans cisternae. Double labeling experiments demonstrate that xyloglucan and polygalacturonic acid/rhamnogalacturonan-I can be synthesized concomitantly within the same Golgi stack. Finally, we show that the xylosyl residue-linked beta-1,2 to the beta-linked mannose of the core of N-linked glycans is added in medial cisternae. Taken together, our results indicate that in sycamore maple suspension-cultured cells, different types of Golgi cisternae contain different sets of glycosyl transferases, that the functional organization of the biosynthetic pathways of complex polysaccharides is consistent with these molecules being processed in a cis to trans direction like the N-linked glycans, and that the complex polysaccharide xyloglucan is assembled exclusively in trans Golgi cisternae and the trans Golgi network.

Journal Article↗
PubMed1993

A minimal input-output configuration for a priori identifiability of a compartmental model of leucine metabolism.

To develop a model describing the structure and function of a metabolic system using data from an input-output experiment, it is useful to design a pilot tracer study first which contains a predicted maximal amount of information. Having postulated a physiologically reasonable model structure from the pilot data, two questions arise. First, are the model parameters a priori uniquely identifiable? That is, assuming an error-free model structure and data, can the parameters be uniquely identified from the information content of the pilot experiment? Second, if the model parameters are uniquely identifiable, is the pilot experiment a minimal one? That is, is the pilot experiment necessary and sufficient, in the sense of information content, among feasible experiments to guarantee a priori unique identifiability? The purpose of this paper is to determine a minimal input-output configuration for the a priori unique identifiability of a compartmental model describing the metabolism of leucine, an essential amino acid. The original pilot tracer experiment was a two-stage experiment consisting first of a two input-five output experiment followed by a single input-single output experiment. Here we show to guarantee a priori unique identifiability of the leucine model that the single input-single output experiment is not necessary, and that two of the outputs of the multi-input-multi-output experiment are not required.

Bicarbonates↗
PubMed1997

Linear least squares compartmental-model-independent parameter identification in PET.

A simplified approach involving linear-regression straight-line parameter fitting of dynamic scan data is developed for both specific and nonspecific models. Where compartmental-model topologies apply, the measured activity may be expressed in terms of: its integrals, plasma activity and plasma integrals--all in a linear expression with macroparameters as coefficients. Multiple linear regression, as in spreadsheet software, determines parameters for best data fits. Positron emission tomography (PET)-acquired gray-matter images in a dynamic scan are analyzed: both by this method and by traditional iterative nonlinear least squares. Both patient and simulated data were used. Regression and traditional methods are in expected agreement. Monte-Carlo simulations evaluate parameter standard deviations, due to data noise, and much smaller noise-induced biases. Unique straight-line graphical displays permit visualizing data influences on various macroparameters as changes in slopes. Advantages of regression fitting are: simplicity, speed, ease of implementation in spreadsheet software, avoiding risks of convergence failures or false solutions in iterative least squares, and providing various visualizations of the uptake process by straight line graphical displays. Multiparameter model-independent analyses on lesser understood systems is also made possible.

Algorithms↗
PubMed2004

Modulation of cytokine release by differentiated CACO-2 cells in a compartmentalized coculture model with mononuclear leucocytes and nonpathogenic bacteria.

To further investigate the interaction between human mononuclear leucocytes [peripheral blood mononuclear cells (PBMC)] and enterocytes, the effect of a confluent layer of differentiated CACO-2 cells on cytokine kinetics during challenge with bacteria in a compartmentalized coculture model was investigated. Nonpathogenic Escherichia coli were added either to the apical or the basolateral compartment of this transwell cell culture system, the latter of which contained human leucocytes. The synthesis of tumour necrosis factor (TNF-alpha) and interleukin (IL)-12 was significantly suppressed by CACO-2 cells when leucocytes were stimulated directly with bacteria. This suppression was not paralleled by changes in the production of IL-10, IL-6 and transforming growth factor (TGF)-beta. When the bacteria were applied apically to the CACO-2 cell layer, the production of TNF-alpha, IL-12, IL-1beta, IL-8, IL-6, IL-10, TGF-beta and interferon-gamma was pronouncedly lower as compared to the bacterial stimulation of leucocytes beneath the CACO-2 cells. In the latter experiments, IL-6, IL-8 and TNF-alpha were the cytokines being mostly induced by apical addition of E. coli. Quantitative mRNA expression analysis revealed that IL-8 gene expression was equally induced in both CACO-2 and PBMC after apical stimulation with bacteria. Of note, bacteria-stimulated CACO-2 cells produced little or no cytokines in the absence of leucocytes, supporting the concept of leucocyte-epithelial cell cross-talk in modulating cytokine responses in the gut mucosa.

Adult↗
PubMed1975

A compartmental analysis of circulatory lymphocytes in the spleen.

A tentative model describing the passage of circulatory lymphocytes through the spleen is formulated in accord with known anatomical features. In order to preserve isomorphism between the model and the splenic system, the model is formulated in compartmental form and its design allows alternative routes and modes of lymphocyte transit to be considered. The simultaneous differential equations arising from the model are solved using an analogue computer which also provides the means whereby the performance of the model may be compared with suitable dynamic data drawn from literature. This not only allows the selection of a particular configuration of the model in preference to its alternatives, but also allows the numerical determination of certain unknown parameters. In the case of the rat spleen, best agreement between model and experimental data is obtained when between 10 and 25% of the total lymphocyte flux in the model spleen passes through the marginal zone where the average dwell time of the lymphocytes is about 50 min. The white pulp receives a lymphocyte flux from the marginal zone amounting to about 10% of the total splenic flux and the white pulp lymphocytes are sequestered for a period of 4-6 hr before release to the venous circulation. The red pulp receives 90% of the total splenic flux but the majority of lymphocytes find transit through the red pulp in less than 5 min. The remaining flux of lymphocytes, amounting to 10% of the splenic input, is delayed in transit through the red pulp by 2-3 hr before release to the venous circulation.

Animals↗
PubMed2006

Compartmentalization of intracellular proinflammatory cytokines in bronchial intraepithelial T cells of stable lung transplant patients.

Allograft rejection remains a major cause of morbidity and mortality following lung transplantation and is associated with an increased expression of T cell proinflammatory cytokines. We have shown that CD4(+) T cell proinflammatory cytokine production was significantly reduced in peripheral blood and bronchoalveolar lavage (BAL) of stable lung transplant patients, consistent with immunosuppression therapy. However, analysis of inflammatory cytokine profiles of intraepithelial T cells in bronchial brushing (BB) may be more relevant than peripheral blood or BAL T cells for assessing immune graft status. To investigate the immunomodulatory effects of currently used immunosuppressive regimens on bronchial intraepithelial T cell cytokine production, whole blood, BAL and BB from stable lung transplant patients and control volunteers were stimulated in vitro and cytokine production by CD8(+) and CD4(+) T cell subsets determined using multi-parameter flow cytometry. In bronchial intraepithelial T cell subsets in control subjects and transplant patients there was compartmentalization of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha production, a decrease in interleukin (IL)-2 production by CD4(+) T cells and CD4 : CD8 inversion compared with blood and BAL. Although there was a decrease in T cell proinflammatory cytokine production in blood of transplant patients, this was not found in BAL or bronchial intraepithelial CD8 T cell subsets, suggesting that the same level of immunosuppression may not occur in the lung of transplant recipients. Drugs that effectively reduce CD8 T cell proinflammatory cytokine production in the lung compartment may improve current protocols for reducing graft rejection in these patients.

Adult↗
PubMed1989

Compartmentalization of the periplasm at cell division sites in Escherichia coli as shown by fluorescence photobleaching experiments.

Morphological evidence has previously indicated that the periplasmic space of Escherichia coli is compartmentalized at sites corresponding to future sites of cell division. The borders of these morphological compartments are formed by localized zones of adhesion (periseptal annuli). In the present study, the technique of fluorescence recovery after photobleaching was used to determine whether these structures act as barriers to the free movement of proteins within the periplasm. The recovery of fluorescence in the ftsA filaments was found to be uniformly low over at potential sites of cell division and at the cell poles, indicating that these regions are biochemically sequestered from the remainder of the periplasmic space. Our results provide direct evidence for local compartments within the periplasm, primarily located at the sites of past or future cell divisions. The implications of this finding for cell division and other periplasmic processes are discussed.

Biological Transport↗
PubMed2006

Self-compartmentalized bacterial proteases and pathogenesis.

Protein degradation is required for homeostasis of all living organisms. Self-compartmentalized ATP-dependent proteases are required for virulence of several pathogenic bacteria. Among the proteases implicated are ClpP and Lon, as well as the more recently identified bacterial proteasome. It is generally assumed that when a pathogen invades a host, microbial proteins become irreversibly damaged and need to be degraded. However, recent data suggest that proteolysis is also essential for virulence gene regulation. In this review, we will discuss what is known about the relationship between ATP-dependent proteolysis and pathogenesis. In addition, we will propose other potential roles these chambered proteases may have in bacterial virulence. Importantly, these proteases show promise as targets for antimicrobial therapy.

Adenosine Triphosphate↗
PubMed1992

Intranuclear compartmentalization of transcribed and nontranscribed c-myc sequences in Namalva-S cells.

This investigation is centered on the intranuclear localization of transcribed and nontranscribed c-myc sequences in human Namalva-S cells bearing t(8;14) translocation. Southern hybridization showed that the breakpoint in the truncated allele of c-myc lies outside the characteristic 12.8-kbp EcoRI fragment: as Northern analysis indicated, this reorganization induces a high level of c-myc transcription. Following high-salt treatment, EcoRI digestion and centrifugation, isolated nuclei from the same cells are separated into two residual fractions: a heavier P fraction including nuclear matrix structures and a light S fraction representing dehistonized chromatin fibres. Comparative hybridization experiments revealed that the above procedure separates the c-myc sequences between the two fractions. To locate the site of intranuclear c-myc transcription, we performed run-on experiments with two fractions, topologically analogous to the residual P and S fractions but maintaining the original chromatin organization. These experiments indicated that chromatin P fraction harbours actively transcribed c-myc sequences while chromatin S harbours nontranscribed ones. Further experiments have clarified that the transcribed c-myc sequences are firmly bound to the matrix by multiple attachment sites, arranged throughout the entire gene locus. It was found, moreover, that at the site of attachment the interaction between DNA and the matrix components is realized via proteins. Controls with the beta-globin gene, which is constitutively nonexpressed in Namalva-S cells but upon induction is highly expressed in murine erythroleukemia cells, completely confirmed the conclusion we had made for the intranuclear localization of c-myc. Thus the experiments presented here support the more common idea that the transcribed and nontranscribed sequences are precisely compartmentalized.

Animals↗
PubMed2001

Compartmental neurodegeneration and synaptic plasticity in the Wld(s) mutant mouse.

This review focuses on recent developments in our understanding of neurodegeneration at the mammalian neuromuscular junction. We provide evidence to support a hypothesis of compartmental neurodegeneration, whereby synaptic degeneration occurs by a separate, distinct mechanism from cell body and axonal degeneration. Studies of the spontaneous mutant Wld(s) mouse, in which Wallerian degeneration is characteristically slow, provide key evidence in support of this hypothesis. Some features of synaptic degeneration in the absence of Wallerian degeneration resemble synapse elimination in neonatal muscle. This and other forms of synaptic plasticity may be accessible to further investigations, exploiting advantages afforded by the Wld(s) mutant, or transgenic mice that express the Wld(s) gene.

Animals↗
PubMed1981

Continuous determination by a chemiluminescent method of acetylcholine release and compartmentation in Torpedo electric organ synaptosomes.

The detection of acetylcholine (ACh) with a chemiluminescent procedure enables one to follow continuously the release of transmitter from stimulated synaptosomes and to study the compartmentation of ACh in resting and active nerve terminals. A compartment of ACh liberated almost entirely by a single freezing and thawing could be directly measured and compared with a compartment of ACh resistant to several cycles of freezing and thawing but liberated by a detergent (60-70% of the total). It is the compartment liberated by freezing and thawing that is reduced when synaptosomes are stimulated. Up to half the total synaptosomal ACh content is readily releasable provided the calcium entry is maintained, or if a strong releasing agent such as the venom of Glycera convoluta is used. In addition, it is shown that synaptosomes contain only negligible amounts of choline, and that the proportion of the two ACh compartments is not influenced by changing extracellular calcium just before their determination.

Acetylcholine↗