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PubMed1977

Absorption of horseradish peroxidase by the principal cells of the large intestines of postnatal developing rats.

The absorption of horseradish peroxidase in the rat large intestine during the postnatal developing period was investigated by cytochemical and ultrastructural methods. In the ceca and ascending colons from 4-, and 5-day-old rats, intraluminaly injected peroxidase became localised on microvilli, in the intracytoplasmic network of the apical tubules and small vacuoles (tubulo-vacuolar system), in the large supranuclear vacuole, in the Golgi apparatus, on the lateral and basal cell membranes of the surface principal cells, and also in the lumen of blood capillaries in the subjacent propria. On the other hand, the protein tracer was not seen in the large intestinal epithelial cells that lack tubulo-vacuolar systems and large supranuclear vacuoles within their cytoplasm. In the descending colons of rats of any postnatal age, the epithelial principal cells are devoid of these membrane systems, and exogenous peroxidase was not detected in the cytoplasm of the principal cells. It is assumed that at least the proximal portion of the large intestine in neonatal animals has the capacity to absorb macromolecular protein through those surface principal cells that have specialized intracellular membrane systems.

Animals↗
PubMed2003

Safety and utilization of blood components as therapeutic delivery systems.

In recent years, natural blood components have been extensively studied as the advanced therapeutic delivery systems. The blood components which can potentially be used as the therapeutic delivery systems include different types of cells, such as erythrocytes and lymphocytes, macromolecular complexes such as lipoproteins and antibody or albumin conjugates and other molecules. This review article covers the progress in this topic, specifically, including the safety issues and the utilization of these component. It can be seen through the literature that the blood components as the therapeutic delivery systems have a number of advantages over traditional pharmaceutical products. The efficacy and practice of the applications, however, require significant amount of development work in the near future.

Animals↗
PubMed2006

Counter-selectable marker for bacterial-based interaction trap systems.

Counter-selectable markers can be used in two-hybrid systems to search libraries for a protein or compound that interferes with a macromolecular interaction or to identify macromolecules from a population that cannot mediate a particular interaction. In this report, we describe the adaptation of the yeast URA3/5-FOA counter-selection system for use in bacterial interaction trap experiments. Two different URA3 reporter systems were developed that allow robust counter-selection: (i) a single copy F' episome reporter and (ii) a co-cistronic HIS3-URA3 reporter vector. The HIS3-URA3 reporter can be used for either positive or negative selections in appropriate bacterial strains. These reagents extend the utility of the bacterial two-hybrid system as an alternative to its yeast-based counterpart.

Escherichia coli↗
PubMed1997

Calibration and application of an X-ray image intensifier/ charge-coupled device detector for monochromatic macromolecular crystallography.

Charge-coupled device (CCD)-based X-ray detectors allow data to be collected much more quickly (approximately 10 times) than with current on-line imaging-plate systems. At the ESRF, X-ray image intensifier/CCD detector systems have been developed. These have great potential as fast read-out detectors for macromolecular and other forms of crystallography. They are relatively large sensitive X-ray detectors but have two inherent weaknesses: convex detection surfaces leading to spatial distortion and non-uniformity of intensity response, and susceptibility to small changes in magnetic fields. A large improvement has been made to the accuracy obtained by non-uniformity of response calibration and correction, using fluorescence from doped lithium borate glasses. Monochromatic macromolecular crystallography demonstration experiments with external user groups have shown that high-quality results may be obtained under real experimental conditions.

Journal Article↗
PubMed1999

Inhibition of apoptosis after ischemia-reperfusion in rat myocardium by cycloheximide.

Macromolecular synthesis inhibitors protect cells from apoptosis in many systems. To determine whether the protein synthesis inhibitor cycloheximide (CHX) might inhibit apoptosis and protect the myocardium during ischemia-reperfusion, we subjected isovolumic isolated perfused rat hearts to 25 min of normothermic global ischemia followed by reperfusion. We monitored coronary flow, end-diastolic pressure and rate-pressure product (RPP) throughout and assessed apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). Regardless of the treatment regimen (only before ischemia; only during reperfusion; or both before ischemia and during reperfusion), CHX significantly improved functional recovery during reperfusion. These effects were most pronounced when CHX was present during reperfusion. When hearts were treated with CHX only during reperfusion the recovery of sinus rhythm was more frequent in the CHX-treated hearts than control hearts (80% v 53%) and earlier for CHX-treated than control hearts: 6.4 +/- 2 v 19.4 +/- 4.7 min of reperfusion. The maximal RPP recoveries for the CHX-treated hearts were 45 +/- 4.0% (P=0.005) of pre-ischemic values, compared to 26 +/- 3% for controls. In control hearts reperfused for 2 h, TUNEL identified 49.5 +/- 10 intact nuclei and 7.5 +/- 2 fragmented nuclei per 1000 nuclei counted. A significantly lower incidence of labeled nuclei with or without fragmentation was observed in CHX treated hearts: 7.6 +/- 3.4 (P=0.009) intact labeled nuclei and 1.8 +/- 0.7/10(3)fragmented labeled nuclei. Our results suggest that CHX-induced inhibition of apoptosis in reperfused myocardium is cardioprotective and promotes functional recovery in vitro.

Animals↗
PubMed1992

The sequence homologies of cytochromes P-450 and active-site geometries.

The amino acid sequence alignment of 16 cytochrome P-450 proteins representative of the major families is reported. The sequence matching process has been carried out on the basis of maximum homology by residue type, retention of secondary structure and minimization of deletions/insertions except where additional loop regions exist. From the starting point of known reported sequence homology matching from the literature, a realignment on the basis of conserved residues involved in both structure and function gives rise to a self-consistent set of sequences which correlates with known mechanistic and structural data. Once fitted, these archetypal sequences form a straightforward template for the alignment of all P-450 subfamilies. Computer modelling of the active-site regions constructed from homology with the bacterial form of the enzyme (P-450CAM) evinces the correct substrate specificity. Furthermore, the construction of the macromolecular assembly of components of the cytochrome P-450 system on the microsomal endoplasmic reticular membrane is presented from the evidence of site-directed mutagenesis, analysis by molecular probes, X-ray crystallography and molecular modelling.

Amino Acid Sequence↗
PubMed1975

An ultrastructural study of the microfilaments in rat brain by means of E-PTA staining and heavy meromyosin labeling. II. The synapses.

To identify structures involved in the translocation of the synaptic vesicles towards the presynaptic membrane, an ultrastructural study has been undertaken by means of (1) the E-PTA stain and (2) the HMM-labeling procedure. Using serial sections of E-PTA stained nervous tissue, especially those made in transversal and tangential planes, the geometric order of the presynaptic grid and of its constituents has been described in detail. It consisted of dense projections having the shape of small truncated pyramids cut parallel to their hexagonal bases which rested on the electron-lucent presynaptic membrane. The dense projections were arranged at the points of equilateral triangles. Around each dense projection, six asymmetric hexagonal holes were seen to be arrayed in an hexagonal pattern, forming thus the presynaptic sieve. From the spiny tops of the dense projections, which appeared as specialized structures of the dense material coating the inner surface of the plasma membrane at the level of the synaptic cleft, fine filaments, 40--60 A in diameter, radiated and formed a three-dimensional meshwork pervading the presynaptic bag. The dense cytoplasmic coating delineating the plasma membrane served as anchor points for these microfilaments. Upon incubation with rabbit skeletal muscle HMM the microfilaments underwent specific structural changes, consisting of: (1) a striking increase in diameter; (2) the association of periodic and polarized substructures with their surfaces. The synaptic vesicles and mitochondria were seen to be attached to the numerous HMM-decorated filaments or enmeshed in the network formed by these filaments. The actin-like filaments were anchored to the plasma membrane at many points and to the presynaptic dense projections. Following incubation in the buffer alone or in buffer HMM solutions containing Na+ pyrophosphate or ATP, no arrowheaded structures were observed. Thus, a network consisting of actin-like filaments was demonstrated in the presynaptic bag. Of particular interest was the structural relation of the actin-like filaments with the occasional, tapered myosin-like filaments. The role of the presynaptic actin-like network in the transport of synaptic vesicles towards the presynaptic membrane by a mechanism of chemomechanical transduction is discussed. In the postsynaptic dendrite or dendritic spine, a filamentous network was observed to be attached to the subsynaptic web by means of the E-PTA stain and of the HMM-labeling procedure. The occurrence of an actin-like meshwork in the postsynaptic region is suggested to produce changes in the macromolecular configuration of the postsynaptic membrane by a "mechanoenzyme" system similar to that described in the mitochondrial membrane.

Actins↗
PubMed2002

Theoretical and computational advances in biomolecular NMR spectroscopy.

Recent developments in experimental and computational aspects of NMR spectroscopy have had a significant impact on the accuracy and speed of macromolecular structure determination in solution, particularly with regard to systems of high complexity (such as protein complexes). These include experiments designed to provide long-range orientational and translational restraints, improvements in internal coordinate dynamics used for simulated annealing, and the development of database potentials of mean force to improve the description of the non-bonded contacts.

Databases, Factual↗
PubMed2002

Modeling supramolecular assemblages.

There has been some progress (but not much) in simulating supramolecular assemblages in the past year. The two main technical advances have been, firstly, the establishment of a protocol for extracting equilibrium thermodynamic data from forced (i.e. nonequilibrium) simulations and experiments, and, secondly, the development of a method for accurately calculating the electrostatics of enormous systems. Some recent applications have demonstrated the increasing feasibility of performing meaningful simulations of very large systems.

Computer Simulation↗
PubMed1999

Molecular order versus vitrification in high-sugar blends of gelatin and kappa-carrageenan.

The understanding of synthetic polymer viscoelasticity was applied to the small deformation properties of high-sugar gelatin and kappa-carrageenan mixtures. The glass transition zone in sugar/gelatin mixtures exhibited a dominant liquid-like response, which was followed by the method of reduced variables. The glass transition temperature predicted by the WLF/free volume approach coincided with the crossover of storage and loss modulus at the onset of the glassy state. The viscoelastic spectrum was resolved into a basic function of temperature and a basic function of time, thus rationalizing their effect on the vitrification of the mixture. Vitrification was only one in a plethora of viscoelastic properties. Manipulation of the composition of the mixture generated a continuous gelatin matrix or a viscous sugar phase suspending a dispersion of ice crystals. Besides the small-molecule crystallinity, vitrification can be halted by ordered macromolecular helices observed in the kappa-carrageenan/potassium system. Thus, the solid-like component of the sugar/kappa-carrageenan network remained prevalent over the predicted frequency window. The WLF equation was unable to follow the progress of viscoelastic functions, which were better described by an energetic barrier of rotation from one state to another.

Carbohydrates↗
PubMed1987

The immunohistochemical localisation of microfibril-associated glycoprotein (MAGP) in elastic and non-elastic tissues.

We have previously identified the major antigen of elastin-associated microfibrils as a 31kD glycoprotein which we named microfibril-associated glycoprotein or MAGP. Affinity-purified antibodies to MAGP were shown to localise specifically to elastin-associated microfibrils in sections of bovine foetal nuchal ligament. In the present paper we compare the localisation of anti-MAGP antibodies and anti-tropoelastin antibodies in a range of bovine elastic and non-elastic tissues. The results show that anti-MAGP antibodies invariably localised to immuno-reactive elastic fibres, wherever they occurred. Extensive additional localisation was observed in a number of tissues. This extra distribution of anti-MAGP antibodies was found to correspond to those structures exhibiting the oxytalan histochemical staining reaction in tissues such as skin, periodontal ligament and ocular zonule. Since these oxytalan fibres have been shown to consist of 12 nm microfibrils which are morphologically similar to those of elastic fibres (and unpublished data from this laboratory confirm this conclusion), the results suggest that MAGP is a component of 12 nm microfibrils in both elastic and non-elastic tissues. Anti-tropoelastin antibodies did not localise to these oxytalan fibres, suggesting that tropoelastin is not a component of 12 nm microfibrils. MAGP was also detected in extracellular matrix regions of tissues such as skeletal muscle, Achilles tendon and spleen, suggesting that 12 nm microfibrils, containing one or more macromolecular constituents in common, make up an important structural system within the extracellular matrix in a wide range of elastic and non-elastic tissues.

Animals↗
PubMed2005

Maturation of phage T7 involves structural modification of both shell and inner core components.

The double-stranded DNA bacteriophages are good model systems to understand basic biological processes such as the macromolecular interactions that take place during the virus assembly and maturation, or the behavior of molecular motors that function during the DNA packaging process. Using cryoelectron microscopy and single-particle methodology, we have determined the structures of two phage T7 assemblies produced during its morphogenetic process, the DNA-free prohead and the mature virion. The first structure reveals a complex assembly in the interior of the capsid, which involves the scaffolding, and the core complex, which plays an important role in DNA packaging and is located in one of the phage vertices. The reconstruction of the mature virion reveals important changes in the shell, now much larger and thinner, the disappearance of the scaffolding structure, and important rearrangements of the core complex, which now protrudes the shell and interacts with the tail. Some of these changes must originate by the pressure exerted by the DNA in the interior of the head.

Bacteriophage T7↗
PubMed1990

A nervous system-specific isotype of the beta subunit of Na+,K(+)-ATPase expressed during early development of Xenopus laevis.

We have previously described the isolation of several genes expressed exclusively in the nervous system of adult Xenopus laevis and activated in the embryo shortly after neural induction. The sequence of one of these cDNAs, 24-15, identifies the corresponding protein as an isotype of the beta subunit of Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting); EC 3.6.1.37]. This form is distinct from the previously described beta 1 subunit of Xenopus, and the protein sequence comparison suggests that it is not the frog homolog of the mammalian beta 2 subunit; therefore, we refer to the 24-15 protein as the beta 3 subunit of Na+,K(+)-ATPase of Xenopus. Antisera directed against beta 3-subunit fusion protein detected a protein in adult brain extracts with the size and properties expected for a Na+,K(+)-ATPase beta subunit. In Xenopus the beta 1 and beta 3 subunits are expressed as maternal mRNAs at similar levels; during embryogenesis rapid accumulation of beta 3 mRNA begins at stage 14 (early neurula), and the rapid accumulation of beta 1 mRNA begins at stage 23/24. In situ hybridization of antisense RNA probes to tadpole brain sections indicates that beta 3 subunit is expressed throughout the developing brain. We suggest that beta 3 is a major Na+,K(+)-ATPase beta subunit present during early nervous system development in the frog.

Amino Acid Sequence↗
PubMed1983

Pathogenesis of Shigella diarrhea. VIII. Evidence for a translocation step in the cytotoxic action of Shiga toxin.

Shigella toxin interacts with HeLa cells by binding to a glycoprotein cell surface receptor and, after a lag period, inactivates the 60S ribosomal subunit and inhibits protein synthesis. The lag period may be due to energy-dependent translocation of the toxin to the interior of the cells. This step was investigated with agents known to inhibit glucose metabolism, mitochondrial energy production, or macromolecular synthesis and with drugs that alter the cytoskeletal system or lysosomes. Metabolic inhibitors reduced both cytotoxicity and binding of the toxin. All agents tested except actinomycin D also diminished endocytosis. Effects on cytotoxicity were partially reversed in the presence of the membrane permeabilizer dimethylsulfoxide. Various cytochalasins, colchicine, vinca alkaloids, chloroquine, and steroids also reduced the activity of the toxin. These diverse agents are probably acting on a common pathway affecting the internalization and/or intracellular processing of shigella toxin. Translocation of this toxin apparently occurs by endocytosis from the cell surface.

Antitoxins↗
PubMed1983

Detection of ornithine decarboxylase antizyme in mouse brain.

Ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, is known to be regulated by a macromolecular inhibitor, termed antizyme, in a number of cellular systems. The present results show that the antizyme is also a functional component of polyamine metabolism in the brain. It could be demonstrated both in normal randomly selected mice and in animals which had been subjected either to intracerebroventricular injection of saline, which is known to cause a transient activation of ornithine decarboxylase, or to 1,3-diamino-2-propanol, an antizyme-inducing agent. When compared to tissues or cell systems studied so far, the cytosol fraction from mouse brain homogenate appeared to contain an exceptionally high amount of antizyme, that was bound to some material other than active ornithine decarboxylase. This feature was seen in all the animal groups studied, being most prominent after saline injection, when the amount of dissociable antizyme exceeded 14-fold the corresponding released ornithine decarboxylase activity. In untreated animals the excess was about eightfold and after 1,3-diamino-2-propanol about fivefold.

Animals↗
PubMed1989

[Interaction of component (E) with tumor cellular DNA].

Some antitumor activities of component (E), extracted from the root of Fagopynum Cymosum (TreV) meisn (FCTM), have recently been discovered in vitro and in vivo. The component (E)'s pattern of action with tumor cellular DNA at the molecular pharmacological level was investigated by macromolecular synthesis experiment (MSE) and human DNA interaction HDI system established in our laboratory. The experiments demonstrated that, in vitro, the agent could markedly inhibit incorporation of H3-TdR into the cellular DNA, and the IC50 in P388 and SGC-7901 was 17.86 micrograms/ml and 110.4 micrograms/ml, respectively. The agent, at level of mg/ml, could produce an intercalation reversion pattern with DNA within a short time (2 hours). But when the interval was prolonged for over 4 hours, the pattern of action changed to intercalation irreversible pattern. According to these observations, the authors infer that component (E) interacts with DNA in two ways-directly and indirectly. The indirect action, especially in low concentrations, probably plays the major role. The authors have also compared the interaction of component (E) with those of components (CD1) and (CB3) extracted from FCTM by the same method and found that component (E) is the most active agent against the DNA of cancer cells among the extracts from FCTM.

Adenocarcinoma↗
PubMed2005

Pressure-induced protein unfolding in the ternary system AOT-octane-water is different from that in bulk water.

In a cellular environment, the presence of macromolecular cosolutes and membrane interfaces can influence the folding-unfolding behavior of proteins. Here we report on the pressure stability of alpha-chymotrypsin in the ternary system bis(2-ethylhexyl)sodium sulfosuccinate-octane-water using FTIR spectroscopy. The ternary system forms anionic reverse micelles which mimic cellular conditions. We find that inclusion of a single protein molecule in a reverse micelle does not alter its conformation. When pressurized in bulk water, alpha-chymotrypsin unfolds at 750 MPa into a partially unfolded structure. In contrast, in the ternary system, the same pressure increase induces a random coil-like unfolded state, which collapses into an amorphous aggregate during the decompression phase. It is suggested that the unfolding pathway is different in a cell-mimicking environment due to the combined effect of multiple factors, including confinement. A phase transition of the reverse micellar to the lamellar phase is thought to be essential to provide the conditions required for unfolding and aggregation, though the unfolding is not a direct result of the phase transition. Our observations therefore suggest that membranes may cause the formation of alternative conformations that are more susceptible to aggregation.

Animals↗
PubMed1991

Viral and cellular gene expression in CD4+ human lymphoid cell lines infected by the simian immunodeficiency virus, SIV/Mne.

Our laboratory has undertaken an analysis of cellular and viral gene expression in CD4+ human lymphoid cell lines infected by the human and simian immunodeficiency viruses, HIV-1 and SIV/Mne, respectively. The purpose of the current study was to: (i) examine the effects of SIV/Mne infection on host macromolecular synthesis and compare the results to those in the HIV-1 system; and (ii) investigate the mechanisms responsible for the restriction of SIV/Mne infection in CD4 positive lymphoid cells which are readily infected by HIV-1. First we determined that SIV does not impose selective blocks on host macromolecular synthesis, unlike HIV-1, which induces both the selective inhibition of cellular protein synthesis and the degradation of cellular mRNAs (Agy, M., Wambach, M., Foy, K., and Katze, M. G., 1990, Virology 177, 251-258). No such selective reduction in cellular mRNA stability or protein synthesis was observed in cells infected by SIV/Mne. Additional differences between SIV and HIV-1 were observed using a panel of CD4+ human cell lines. While HIV-1-infected all cell lines. SIV/Mne efficiently infected only the MT-4, C8166, and 174 x CEM cell lines. Repeated efforts to infect CEM or Jurkat cells were unsuccessful as determined by PCR analysis of viral DNA. HUT 78 cells supported a limited infection detectable only by PCR analysis. These data suggest the block in viral replication in the nonsusceptible cell lines is at an early step. Interestingly, all the SIV susceptible cells were virally transformed, C8166 and MT-4 by HTLV-1, and 174 x CEM by Epstein-Barr virus. Furthermore FACS analysis revealed that all susceptible cells expressed two B cell associated markers, B7/BB1 and CD40. These observations taken together highlight differences between the HIV and SIV viruses, and suggest that for efficient replication, SIV/Mne may require an additional cell surface molecule, cofactors provided by transforming viruses, or a complex interplay between the two.

Animals↗