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PubMed2006

Integrative plant biology: role of phloem long-distance macromolecular trafficking.

Recent studies have revealed the operation of a long-distance communication network operating within the vascular system of higher plants. The evolutionary development of this network reflects the need to communicate environmental inputs, sensed by mature organs, to meristematic regions of the plant. One consequence of such a long-distance signaling system is that newly forming organs can develop properties optimized for the environment into which they will emerge, mature, and function. The phloem translocation stream of the angiosperms contains, in addition to photosynthate and other small molecules, a variety of macromolecules, including mRNA, small RNA, and proteins. This review highlights recent progress in the characterization of phloem-mediated transport of macromolecules as components of an integrated long-distance signaling network. Attention is focused on the role played by these proteins and RNA species in coordination of developmental programs and the plant's response to both environmental cues and pathogen challenge. Finally, the importance of developing phloem transcriptome and proteomic databases is discussed within the context of advances in plant systems biology.

Biological Transport↗
PubMed1982

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: role of divalent metals in the dimerization and phosphorylation of enzyme I.

The function of divalent metal ions (Mg2+ and Mn2+) in the dimerization and phosphorylation of enzyme I has been studied. Only a dimeric form of the enzyme can be phosphorylated [Misset, O., Brouwer, M., & Robillard, G. T. (1980) Biochemistry 19, 883--890; Hoving, H., Lolkema, J. S., & Robillard, G. T. (1981) Biochemistry 20, 87--93]. Kinetic studies of phosphoryl-group exchange between phosphoenolpyruvate and pyruvate and measurements of initial enzyme I phosphorylation rates revealed that a divalent metal ion must be bound to the enzyme to render the dimer active. Mn2+ binding experiments by means of electron paramagnetic resonance showed binding of at least one Mn2+ per unphosphorylated dimer with a binding constant comparable to the activation constant found in the kinetic studies and a 10-fold tighter binding of only one Mn2+ per phosphorylated dimer. Gel filtration experiments provided evidence that divalent metals produce about a 10-fold stabilization of the dimers, in addition to their effect on the specific dimer activity. The stability of the dimer was also strongly dependent on salts such as LiCl, NaCl, KCl, and a series of tetraalkylammonium chlorides. The relative effects of these salts suggest that hydrophobic interactions possibly play a significant role in enzyme I dimerization.

Escherichia coli↗
PubMed1993

Red cell antibody screening and identification: a comparison of two column technology methods.

Two commercial column techniques for use in antibody screening and identification procedures were tested in parallel with 1000 random samples sent for ante-natal serological investigation. The DiaMed ID microtyping system uses a sephadex gel contained in microtubes, either neutral or impregnated with anti-human globulin (AHG), for use in two-stage enzyme methods and LISS indirect antiglobulin testing (IAT) respectively. The Ortho Biovue technique consists of a slurry of micro glass spheres which act as the filter to retain haemagglutination reactions within the matrix. Columns containing AHG also possess a macromolecular density barrier to prevent test serum from passing into the column and neutralising the AHG. Both systems offer the advantage of 'no-wash' IAT, which minimises the potential for problems and errors associated with conventional spin-tube techniques. In this comparison of the two column methods, antibody detection rates were found to be similar and the sensitivity of both methods was comparable, although the Biovue technique was prone to exhibit equivocal results, particularly in the IAT.

Antibodies↗
PubMed1986

Metabolism and macromolecular binding of 1-nitronaphthalene in the mouse.

The metabolism and binding of 1-nitronaphthalene (1-NN) to tissue macromolecules was studied in the mouse using tissue microsomes, lung slices, and isolated lung cells. With microsomes, binding was NADPH-dependent. CO inhibited binding in lung microsomes by more than 90% and in liver microsomes by 60-85%. Nitrogen inhibited binding by 70-80% in lung microsomes and 50-75% in liver microsomes. Incubation of the microsomes in pure O2 did not affect the binding of 1-NN. SKF525A inhibited binding in a dose-dependent manner with approximately 50% inhibition of binding obtained at a molar ratio of 1:4, SKF525A/1-NN. The rate of 1-NN binding by liver microsomes was increased by pretreatment of the mice with phenobarbital (1.2 vs. 0.8 nmol/min/mg protein). beta-Naphthoflavone (BNF) pretreatment increased slightly the rate of 1-NN binding by lung microsomes (1.2 vs 0.9 nmol/min/mg protein), but had no effect on binding by liver microsomes. Studies with isolated lung cells indicated that cell cultures enriched in Clara cells were 6-15-fold more active in metabolism and binding of 1-NN than cultures not containing Clara cells. Autoradiography of lung slices incubated in vitro with [14C]1-NN showed the label to be concentrated in the cells of the bronchiolar epithelium. The results indicate that 1-NN is metabolized in vitro by cytochrome P-450 enzymes via an oxidative pathway to bind to tissue macromolecules. Further, this pathway exists in lung cells, and can lead to binding of the compound without the need for extrapulmonary metabolism.

Animals↗
PubMed1999

Acute modulation of albumin microvascular leakage by advanced glycation end products in microcirculation of diabetic rats in vivo.

Advanced glycation end products (AGEs) are nonenzymatic glycosylated adducts of proteins that accumulate in vascular tissue during diabetes and aging. The aim of this work was to study the role of AGEs and of the oxidative mechanisms in diabetes-induced changes in vascular permeability. Intravital videomicroscopy was used to study albumin microvascular leakage in cremaster muscle. The extravasation of a fluorescent macromolecular tracer (fluorescein isothiocyanate-albumin) was measured for 1 h and, after computer-aided image analysis, was expressed as variations of normalized gray levels (arbitrary units). Extravasation of the macromolecular tracer was much higher in diabetic rats than in control rats (slope of extravasation versus time increased by >100%, P < 10(-4)). This increase was significantly inhibited when we blocked AGEs binding to their endothelial receptor by intravenous bolus of soluble recombinant receptor to AGEs (rR-RAGE) (slope of extravasation versus time decreased by 19, 30, and 40%, for 0.5, 2.5, and 5.15 mg/kg rR-RAGE, respectively) or by a 6 mg/kg intravenous bolus of antibody against RAGE (slope decreased by 53%). Systemic injection of probucol (an antioxidant) also significantly inhibited the increase in the extravasation of the macromolecular tracer occurring in experimental diabetes (slope decreased by 51%, P < 10(-4)). These results strongly suggest that in experimental diabetes the interaction of circulating AGEs and endothelial RAGE mediates albumin micro-vascular leakage, possibly via AGE-RAGE-dependent enhanced oxidant stress.

Albumins↗
PubMed2000

Compartmentalization of enzymes and distribution of products in aqueous two-phase systems.

Phase separation is a common phenomenon in water solutions of polymers due to "polymer incompatibility." Polymeric aqueous two-phase systems are much used for separations in biochemistry and cell biology. When macromolecules are included in a phase system, it is often possible to obtain a one-sided distribution to one of the phases, i.e., the macromolecule is compartmentalized within one aqueous phase. This chapter describes the thermodynamic forces which govern the partitioning of molecules in aqueous two-phase systems. For a high molecular weight macromolecule, e.g., an enzyme, both enthalpic and entropic effects contribute to a one-sided partitioning. Molecules of low molecular weight will be more evenly distributed between the phases. These mechanisms are significant in biological systems and can be used for enzyme reactors in bioconversions. Enzymatic reactions can take place with enzyme and substrate compartmentalized in one of the phases. A low-molecular weight product which is evenly partitioned between the phases can be continuously removed from the enzyme-substrate compartment. These principles are described in the enzymatic conversion of cellulose in an aqueous two-phase system.

Animals↗
PubMed1998

Theory of biomolecular recognition.

Specific, noncovalent binding of biomolecules can only be understood by considering structural, thermodynamic, and kinetic issues. The theoretical foundations for such analyses have been clarified in the past year. Computational techniques for both particle-based and continuum models continue to improve and to yield useful insights into an ever wider range of biomolecular systems.

Animals↗
PubMed1994

Testing garlic for possible anti-ageing effects on long-term growth characteristics, morphology and macromolecular synthesis of human fibroblasts in culture.

The beneficial effects claimed for the use of garlic as a nutritional supplement include detoxification, antioxidation, antifungal activity, antibacterial activity, tumour suppression and, possibly, anti-ageing and rejuvenating effects. We have used the Hayflick system of cellular ageing in culture in order to test garlic for its anti-ageing effects on long-term growth characteristics, morphology and macromolecular synthesis of human skin fibroblasts. Our results show that an addition of garlic extract into the normal cell culture medium can support serial subculturing for over more than 55 population doublings in 475 days, and that this treatment has some youth-preserving, anti-ageing and beneficial effects on human fibroblasts in terms of maximum proliferative capacity and morphological characteristics. In comparison, similar or lesser doses of garlic extracts are growth inhibitory for cancerous cells that could not be grown over longer periods in the presence of garlic. To our knowledge, this is the first report of the effects of garlic on the long-term growth characteristics and macromolecular synthesis of normal human skin cells, the results of which have applications for both anti-ageing and anti-cancer research.

Adult↗
PubMed1990

Sugar transport by the bacterial phosphotransferase system. Fluorescence studies of subunit interactions of enzyme I.

Enzyme I of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) exhibits a temperature-dependent monomer/dimer equilibrium. The accompanying paper (Han, M. K., Roseman, S., and Brand, L. (1990) J. Biol. Chem. 265, 1985-1995) shows that the C-terminal -SH residue (Cys-575) can be modified specifically with fluorescent probes such as pyrene maleimide. The derivative retains full enzyme activity, and is capable of forming dimers at room temperature. In the present studies, Enzyme I labeled in this way is found to exhibit a temperature-, concentration-, and pH-dependent monomer/dimer association. The kinetics of dimer formation of Enzyme I is measured in the following way. A derivatized Enzyme I sample is prepared with a pyrene moiety irreversibly attached to the C-terminal -SH residue and 5,5'-dithiobis-2-nitrobenzoic acid reversibly attached to the other 3 -SH residues. This modified enzyme does not form dimers at room temperature. Addition of dithiothreitol results in total release of the thionitrobenzoate anion within 2 min. After the three -SH groups are unblocked, steady-state and nanosecond time-resolved emission anisotropy measurements indicate the dimer is formed over a period of 30 min. In a similar experiment, little dimer formation is observed at 3 degrees C, at temperature at which the native enzyme also does not form dimers. Tryptophan fluorescence is also examined during the release of the thionitrobenzoate. After the completion of thionitrobenzoate release, additional slow steady-state tryptophan fluorescence changes are observed. These results suggest that dimer formation may be preceded by a conformational change following thionitrobenzoate release.

Dithionitrobenzoic Acid↗
PubMed1992

Macromolecular assemblage in the design of a synthetic AIDS vaccine.

We describe a peptide vaccine model based on the mimicry of surface coat protein of a pathogen. This model used a macromolecular assemblage approach to amplify peptide antigens in liposomes or micelles. The key components of the model consisted of an oligomeric lysine scaffolding to amplify peptide antigens covalently 4-fold and a lipophilic membrane-anchoring group to further amplify noncovalently the antigens many-fold in liposomal or micellar form. A peptide antigen derived from the third variable domain of glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1), consisting of neutralizing, T-helper, and T-cytotoxic epitopes, was used in a macromolecular assemblage model (HIV-1 linear peptide amino acid sequence 308-331 in a tetravalent multiple antigen peptide system linked to tripalmitoyl-S-glycerylcysteine). The latter complex, in liposome or micelle, was used to immunize mice and guinea pigs without any adjuvant and found to induce gp120-specific antibodies that neutralize virus infectivity in vitro, elicit cytokine production, and prime CD8+ cytotoxic T lymphocytes in vivo. Our results show that the macromolecular assemblage approach bears immunological mimicry of the gp120 of HIV virus and may lead to useful vaccines against HIV infection.

AIDS Vaccines↗
PubMed1981

The glomerular mesangium: a comparative study of mesangial handling of iron dextran complex and endogenous IgG in mice and rats.

The function and channel system of the glomerular mesangium in mice and rats was investigated by studying the uptake and transport of intravenously injected iron-dextran particles, and the localization of endogenous IgG. Animals were killed at 30 min, 8 h, 1 and 3 days and 1 and 2 weeks after intravenous injection of iron dextran complex. It was found that the tracer was present maximally in the mesangium of the mouse at one day after injection whereas a maximum was not reached until the third day in the rat. Maximal levels of tracer particles in the extra-glomerular lacis area were found at three days in the mouse and at 2 weeks in the rat. Disappearance of the tracer from the blood as indicated by the measured serum iron levels did not seem to differ significantly in the two species. Using an ultrastructural immunoperoxidase technique, considerable amount of endogenous IgG were localized in the mesangial channel system in the stalk region and in the extraglomerular lacis area of mice, whereas in rats only very scanty endogenous IgG was present in these locations. It is suggested that the difference in mesangial handling of macromolecular material in mice and rats is more likely to be due to a different rate of transport through the mesangial channel system than to primary differences in mesangial phagocytotic activity.

Animals↗
PubMed2006

Dendrimers as a scaffold for nitric oxide release.

The preparation and characterization of nitric oxide (NO)-releasing dendrimer conjugates are reported. Generation 3 and 5 polypropylenimine dendrimers (DAB-Am-16 and DAB-Am-64) were modified at the exterior to impart different amine functionalities. The ability to store NO on a dendritic scaffold using N-diazeniumdiolate NO donors was examined via the reaction of primary amine, secondary amine, and amide functionalities with high pressures of NO (5 atm). The secondary amine dendrimer conjugates exhibited a high storage capacity for NO (up to 5.6 micromol NO/mg), greatly increasing the "payload" of released NO over existing macromolecular NO donors. The mechanism of diazeniumdiolate decomposition was proton initiated, generating NO spontaneously under physiological conditions (pH 7.4, 37 degrees C). The NO release durations (>16 h) observed for the secondary amine dendrimers were significantly longer compared to small molecule alkyl secondary amine diazeniumdiolates, thus illustrating a dendritic effect on NO release kinetics. The multivalent exterior of dendrimers allows for the future combination of NO donors and other functionalities on a single molecular scaffold, enabling diverse utility as NO storage/delivery systems.

Azo Compounds↗
PubMed1992

Studies on the mechanism of acetonitrile toxicity. I: Whole body autoradiographic distribution and macromolecular interaction of 2-14C-acetonitrile in mice.

Acetonitrile, a commonly used solvent is known to cause central nervous system dysfunctions. In order to gain an insight onto the mechanism of acetonitrile toxicity, we studied the kinetics of acetonitrile distribution in mice. Male ICR mice were given a tracer dose of 2-14C-acetonitrile intravenously (60 mu mol/kg or 684 mu Ci/kg, spec. act. 11.4 mCi/mmol). At various time intervals (5 min., 0.5, 1, 4, 8, 24 and 48 hr) after treatment, mice were anaesthetized and frozen by immersion in a dry ice/hexane mixture, or they were dissected for collection of organs and tissues. Frozen mice were processed for whole body autoradiography, which allows the detection of non-volatile metabolites of acetonitrile at their sites of accumulation. Covalent binding of acetonitrile metabolites in tissues was determined using trichloroacetic acid followed by ethanol/ether extraction techniques. Whole body autoradiography revealed heavy localization of acetonitrile metabolites in the gastrointestinal tissues and bile. At 5 min., the highest levels of radioactivity occurred in the liver and kidney; levels declined over time. At 24 and 48 hr, acetonitrile derived radioactivity were detected in the gastrointestine, thymus, liver and male reproductive organs. Covalent binding studies at 24 and 48 hr after treatment indicated that 40-50% of the total radioactivity present in the liver was bound to the macromolecular fractions of the tissues. The radioactivity contents of other organs were, in large part (40-50% of total), present in the lipid fraction of the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetonitriles↗
PubMed1992

Utilization of biomacromolecular in vitro assay systems in the prediction of in vivo toxic responses.

Target biomacromolecular assay systems utilize as endpoints quantifiable alterations in key macromolecules elicited by various chemicals and formulations. The EYTEX Assay System, an example of a target biomacromolecular test method, has been used to predict the in vivo ocular irritancy potential of chemicals and formulations. The biomacromolecular reagent is a standardized matrix which can be produced routinely. One hundred test chemicals, representing diverse chemical classes with a wide range of toxic responses in vivo, were evaluated in the EYTEX system. In vitro results were compared to maximum 24-hour Draize scores and Draize classifications. The concordance of the in vitro and in vivo results demonstrated the relevance of the alteration of the EYTEX macromolecular matrix to the prediction of in vivo ocular irritation. The relevance of the in vitro endpoint of turbidity of the EYTEX matrix to in vivo ocular irritation has thus been demonstrated for a large number of test agents in other evaluation studies (1, 2, 3, 4, 5, 6, 7, 8). Integration of macromolecular-based test methods with in vitro cytotoxicity or cellular response methods may provide complementary information about mechanisms of target-induced toxicity. The target biomacromolecular assay systems which provide standardized, reproducible reagents are valuable components of test batteries.

Animal Testing Alternatives↗
PubMed1984

Macromolecular absorption of food antigens in health and disease.

During the neonatal period, the development of the mucosal barrier against penetration of bacteria, toxins and antigens is an important protective mechanism against a variety of pathologic conditions such as inflammatory and allergic reactions. The closure of the gut with regard to the uptake of food antigens in macromolecular form is determined by non-immunologic mechanisms as well as by the immunologic system of the gut. Animal experiments show that various diseases affecting the gut may interfere with intestinal antigen handling. As a result, susceptibility to infection and allergic reactions may ensue. Studies performed in humans show that selective IgA deficiency, preterm delivery, intestinal helminth infection and type of feeding during the neonatal period may influence antigen uptake by the intestinal epithelium. These conditions, as well as various diseases affecting the gut, may cause increased absorption of intraluminal antigens and result in the triggering of allergic type responses.

Animals↗
PubMed1990

Drug delivery systems: 3A. Role of polymers in drug delivery.

At present, polymers represent a class of ubiquitous materials. They are being used for a multitude of purposes and the almost inexhaustible varieties of molecular architecture that macromolecular materials can possess provides the possibility for a myriad of applications. Because of the increased interest being shown in the macromolecules by the pharmaceutical industry for the fabrication of drug delivery systems, numerous polymers have been synthesized and successfully used in drug delivery devices. The necessary conditions for developing the concept of pharmaceutically applicable polymers depend upon delineating a detailed knowledge of the relationship between the structure and properties of polymer networks. A number of polymers have been studied systematically from this point of view and there is every indication that the systems described have the potential to become clinically valuable and therefore marketable drug delivery systems. The potential of these promising polymers is still far from being exhausted and there is a strong possibility that many important developments will be forthcoming in this field in the future. In the current review article, polymers for controlled release have been divided into four major categories: diffusion-controlled systems; chemically controlled systems; solvent-activated systems; and magnetically controlled systems. Polymers as drug carriers also have been divided into various subgroups: soluble, biodegradable, mucoadhesive and other polymeric systems. The latter group includes polymers containing pendant bioactive substituents, matrix systems, heparin-releasing polymers, ionic polymers, oligomers and miscellaneous. At an introductory and fundamental level, an overview of these polymers and the materials science for the design of drug delivery systems will be discussed.

Animals↗
PubMed1992

Investigations on macromolecular precipitation inhibitors of calcium oxalate.

Certain important aspects of the urine oxalate tolerance test (OTT) have been revised. The stirring system has been changed and the test has been adapted to the kinetics of calcium oxalate precipitation. True equilibrium conditions are now ensured during the measurements. Furthermore, the endogenous oxalate concentration is determined and taken into consideration. As a result of these changes, the significance of the test results has greatly improved. The effects of the addition of small amounts of zinc on the precipitation of calcium oxalate have been used in a new variation of the OTT. This new test makes it possible to discriminate much faster and more simply between recurrent stone-formers and other subjects. Tamm-Horsfall protein (THP) has been tested for its effect on the precipitation of calcium oxalate by means of OTT. THP inhibits the precipitation of calcium oxalate, but THP of stone-formers has a diminished inhibitory activity. The inhibitory activity of this protein strongly depends on the method by which it is isolated.

Calcium Oxalate↗
PubMed1980

Placental permeability during early gestation in the hamster. Electron microscopic observations using horseradish peroxidase as a macromolecular tracer.

The permeabilities of the parietal yolk sac placenta and the preplacental region of the hamster conceptus during early postimplantation (day 8) were compared by means of electron microscopy and a macromolecular protein tracer, horseradish peroxidase (HRP). HRP was administered by injection into the maternal venous system; samples of the two placental tissues were obtained for examination at intervals between 4 minutes and 1 hour later. The three layers of the parietal yolk sac wall (from outer to inner: capsular trophoblast, Reichert's membrance, parietal endoderm) appeared to provide little impediment to the passage of HRP from perivitelline maternal blood spaces to the yolk sac cavity. HRP passed through the outer trophoblast layer, both by way of intracellular fenestrae (60-200 nm diameter) and narrower intercellular channels, and completely permeated the meshwork of Reichert's membrane within minutes after injection. The inner parietal endoderm cell layer was widely discontinuous and clearly presented no barrier to HRP movement. HRP reaching the yolk sac cavity was avidly endocytosed by the viceral yolk sac epithelium. In contrast to the parietal yolk sac, the preplacental region of the conceptus was impermeable to HRP. Zonular occluding junctions located between contiguous cells of the chorionic ectoderm layer of the preplacenta were the obvious barrier to the HRP molecules. These results suggest that in this rodent species, during the early postimplantation period of gestation, the pareital yolk sac placenta potentially plays a more important role in the maternal-embryonic transfer of macromolecular substances than does the preplacenta.

Animals↗