In Vitro and In Vivo Binding of S-Adenosyl-L-Homocysteine to Membranes from Rat Cerebral Cortex

Membranes from rat cerebral cortex are able to bind S-adenosyl-L-homocysteine (SAH) with a KD of 5 . 10(-7) M and n of 170 pmol/g fresh tissue (i.e. 20 mg protein). The binding is enhanced by Mg2+ and Ca2+ but not K+ and Na+. gamma-Aminobutyric acid, diazepine, noradrenaline and alpha antagonists are without any effect; S-adenosyl-L-methionine, adenosine and adenosine triphosphate inhibit SAH binding. Linkage with an adenosine receptor has not been expressly demonstrated by our method. SAH binding proteins are more abundant in the crude synaptosomal pellet (P2). A similar fixation seems to occur on brain membranes after [3H]SAH administration to rat. The binding might be linked to a methylase activity or an adenosine receptor.     

New Insight into Erythrocyte through In Vivo Surface-Enhanced Raman Spectroscopy

The article presents a noninvasive approach to the study of erythrocyte properties by means of a comparative analysis of signals obtained by surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RS). We report step-by-step the procedure for preparing experimental samples containing erythrocytes in their normal physiological environment in a mixture of colloid solution with silver nanoparticles and the procedure for the optimization of SERS conditions to achieve high signal enhancement without affecting the properties of living erythrocytes. By means of three independent techniques, we demonstrate that under the proposed conditions a colloid solution of silver nanoparticles does not affect the properties of erythrocytes. For the first time to our knowledge, we describe how to use the SERS-RS approach to study two populations of hemoglobin molecules inside an intact living erythrocyte: submembrane and cytosolic hemoglobin (Hb_sm and Hb_c). We show that the conformation of Hb_sm differs from the conformation of Hb_c. This finding has an important application, as the comparative study of Hb_sm and Hb_c could be successfully used in biomedical research and diagnostic tests.     

In Vivo Dynamics of Nuclear Pore Complexes in Yeast

While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP– Nup49p into existing recipient NPCs. Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures. This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.     

Novel Iron Complexes Behave Like Superoxide Dismutase In Vivo

Novel iron and copper complexes having tris[N-(5-methyl-2-pyridylmethyl)-2-aminoethyl]amine (5MeT-PAA), tris[N-(3-methyl-2-pyridylmethyl)-2-aminoethyl]amine(3MeTPAA),rris[N-(5-methoxycarbonyl-2-pyridylmethyl)-2-aminoethyl]amine (TNAA), tris[(2-thienylmethyI)-2-aminoethyl]amine (TTAA), tris[(2-furylniethyl)-2-aminoethyl]amine (TFAA) or tris[(2-imidazolyl)-2-aminoethyl]amine (TIAA) as ligand. were synthesized to examine the superoxide dismutase (SOD) activity. The concentrations of Fe-3MeTPAA and Fe-TIAA equivalent to 1 unit of SOD (IC₅₀) were 0.5 μM and I.O μM. respectively. Fe-3MeTPAA and Fe-TIAA had higher SOD activity than other Fe and Cu complexes and protected Escherichiu coli cells from paraquat toxicity. In case of using tris[N-(Cmethyl-2-pyridylrnethyl)-2-aminoethyl]amine (6MeTPAA) as ligand, the Fe complex could not be obtained, which may be due to the steric hindrance of Cmethyl substituent. Generally, Cu complexes had low SOD activity, compared with Fe complexes, and could not suppress paraquat toxicity.     

In Vivo and In Vitro Binding of Vip3Aa to Spodoptera frugiperda Midgut and Characterization of Binding Sites by 125I Radiolabeling

Bacillus thuringiensis vegetative insecticidal proteins (Vip3A) have been recently introduced in important crops as a strategy to delay the emerging resistance to the existing Cry toxins. The mode of action of Vip3A proteins has been studied in Spodoptera frugiperda with the aim of characterizing their binding to the insect midgut. Immunofluorescence histological localization of Vip3Aa in the midgut of intoxicated larvae showed that Vip3Aa bound to the brush border membrane along the entire apical surface. The presence of fluorescence in the cytoplasm of epithelial cells seems to suggest internalization of Vip3Aa or a fragment of it. Successful radiolabeling and optimization of the binding protocol for the ¹²⁵I-Vip3Aa to S. frugiperda brush border membrane vesicles (BBMV) allowed the determination of binding parameters of Vip3A proteins for the first time. Heterologous competition using Vip3Ad, Vip3Ae, and Vip3Af as competitor proteins showed that they share the same binding site with Vip3Aa. In contrast, when using Cry1Ab and Cry1Ac as competitors, no competitive binding was observed, which makes them appropriate candidates to be used in combination with Vip3A proteins in transgenic crops.     

Zolpidem Displays Heterogeneity in Its Binding to the Nonhuman Primate Benzodiazepine Receptor In Vivo

Abstract: The distinctive pharmacological activity of zolpidem in rats compared with classical benzodiazepines has been related to its differential affinity for benzodiazepine receptor (BZR) subtypes. By contrast, in nonhuman primates the pharmacological activity of zolpidem was found to be quite similar to that of classical BZR agonists. In an attempt to explain this discrepancy, we examined the ability of zolpidem to differentiate BZR subtypes in vivo in primate brain using positron emission tomography. The BZRs were specifically labeled with [¹¹C]flumazenil. Radiotracer displacement by zolpidem was monophasic in cerebellum and neocortex, with in vivo Hill coefficients close to 1. Conversely, displacement of [¹¹C]flumazenil was biphasic in hippocampus, amygdala, septum, insula, striatum, and pons, with Hill coefficients significantly smaller than 1, suggesting two different binding sites for zolpidem. In these cerebral regions, the half-maximal inhibitory doses for the high-affinity binding site were similar to those found in cerebellum and neocortex and ～100-fold higher for the low-affinity binding site. The low-affinity binding site accounted for <32% of the specific [¹¹C]-flumazenil binding. Such zolpidem binding characteristics contrast with those reported for rodents, where three different binding sites were found. Species differences in binding characteristics may explain why zolpidem has a distinctive pharmacological activity in rodents, whereas its pharmacological activity in primates is quite similar to that of classical BZR agonists, except for the absence of severe effects on memory functions, which may be due to the lack of substantial zolpidem affinity for a distinct BZR subtype in cerebral structures belonging to the limbic system.     

Immune Complexes Indirectly Suppress the Generation of Th17 Responses In Vivo

The precise context in which the innate immune system is activated plays a pivotal role in the subsequent instruction of CD4⁺ T helper (Th) cell responses. Th1 responses are downregulated when antigen is encountered in the presence of antigen-IgG immune complexes. To assess if Th17 responses to antigen are subject to similar influences in the presence of immune complexes we utilized an inflammatory airway disease model in which immunization of mice with Complete Freund’s Adjuvant (CFA) and ovalbumin (Ova) induces a powerful Ova-specific Th1 and Th17 response. Here we show that modification of that immunization with CFA to include IgG-Ova immune complexes results in the suppression of CFA-induced Th17 responses and a concurrent enhancement of Ova-specific Th2 responses. Furthermore, we show the mechanism by which these immune complexes suppress Th17 responses is through the enhancement of IL-10 production. In addition, the generation of Th17 responses following immunization with CFA and Ova were dependent on IL-1α but independent of NLRP3 inflammasome activation. Together these data represent a novel mechanism by which the generation of Th17 responses is regulated.     

Slipped Misalignment Mechanisms of Deletion Formation: In Vivo Susceptibility to Nucleases

Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3′ exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3′ ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.     

Gelsolin Modulates Phospholipase C Activity In Vivo through Phospholipid Binding

Gelsolin and CapG are actin regulatory proteins that remodel the cytoskeleton in response to phosphatidylinositol 4,5-bisphosphate (PIP₂) and Ca²⁺ during agonist stimulation. A physiologically relevant rise in Ca²⁺ increases their affinity for PIP₂ and can promote significant interactions with PIP₂ in activated cells. This may impact divergent PIP₂- dependent signaling processes at the level of substrate availability. We found that CapG overexpression enhances PDGF-stimulated phospholipase C_γ (PLC_γ) activity (Sun, H.-q., K. Kwiatkowska, D.C. Wooten, and H.L. Yin. 1995. J. Cell Biol. 129:147–156). In this paper, we examined the ability of gelsolin and CapG to compete with another PLC for PIP₂ in live cells, in semiintact cells, and in vitro. We found that CapG and gelsolin overexpression profoundly inhibited bradykinin-stimulated PLC_β. Inhibition occurred at or after the G protein activation step because overexpression also reduced the response to direct G protein activation with NaF. Bradykinin responsiveness was restored after cytosolic proteins, including gelsolin, leaked out of the overexpressing cells. Conversely, exogenous gelsolin added to permeabilized cells inhibited response in a dose-dependent manner. The washout and addback experiments clearly establish that excess gelsolin is the primary cause of PLC inhibition in cells. In vitro experiments showed that gelsolin and CapG stimulated as well as inhibited PLC_β, and only gelsolin domains containing PIP₂-binding sites were effective. Inhibition was mitigated by increasing PIP₂ concentration in a manner consistent with competition between gelsolin and PLC_β for PIP₂. Gelsolin and CapG also had biphasic effects on tyrosine kinase– phosphorylated PLC_γ, although they inhibited PLC_γ less than PLC_β. Our findings indicate that as PIP₂ level and availability change during signaling, cross talk between PIP₂-regulated proteins provides a selective mechanism for positive as well as negative regulation of the signal transduction cascade.     

EGF Downregulates Presynaptic Maturation and Suppresses Synapse Formation In Vitro and In Vivo

Neuronal differentiation, maturation, and synapse formation are regulated by various growth factors. Here we show that epidermal growth factor (EGF) negatively regulates presynaptic maturation and synapse formation. In cortical neurons, EGF maintained axon elongation and reduced the sizes of growth cones in culture. Furthermore, EGF decreased the levels of presynaptic molecules and number of presynaptic puncta, suggesting that EGF inhibits neuronal maturation. The reduction of synaptic sites is confirmed by the decreased frequencies of miniature EPSCs. In vivo analysis revealed that while peripherally administrated EGF decreased the levels of presynaptic molecules and numbers of synaptophysin-positive puncta in the prefrontal cortices of neonatal rats, EGF receptor inhibitors upregulated these indexes, suggesting that endogenous EGF receptor ligands suppress presynaptic maturation. Electron microscopy further revealed that EGF decreased the numbers, but not the sizes, of synaptic structures in vivo. These findings suggest that endogenous EGF and/or other EGF receptor ligands negatively modulates presynaptic maturation and synapse formation.

     

Radical Formation and Accumulation In Vivo, In Desiccation Tolerant and Intolerant Mosses

Water loss in a desiccation-sensitive moss resulted in destruction of chlorophyll, loss of carotenoids and increased lipid peroxidation, indicating the presence of damaging forms of activated oxygen. These effects were exaggerated when the plants were desiccated at high light intensities. During water-deprivation there was a build up of a free radical, detected in vivo, with a close correlation between molecular damage and radical accumulation. In contrast, in a desiccation-tolerant moss there was almost no indication of molecular (oxidative) damage. However a stable radical similar in type and concentration to that found in the desiccation-sensitive species accumulated, particularly under high irradiances. The stable radical appears to be one of the end-products of a process initiated by environmental stress, desiccation and high irradiance: its association with molecular damage depending on the degree to which the species is tolerant of desiccation. Identification of the radical in intact tissue from EPR and ENDOR studies, suggests that this is not a short-lived proxy-radical but instead is relatively stable and carbon-centred.     

Radical Formation and Accumulation In Vivo,In Desiccation Tolerant and Intolerant Mosses

Water loss in a desiccation-sensitive moss resulted in destruction of chlorophyll, loss of carotenoids and increased lipid peroxidation, indicating the presence of damaging forms of activated oxygen. These effects were exaggerated when the plants were desiccated at high light intensities. During water-deprivation there was a build up of a free radical, detected in vivo, with a close correlation between molecular damage and radical accumulation. In contrast, in a desiccation-tolerant moss there was almost no indication of molecular (oxidative) damage. However a stable radical similar in type and concentration to that found in the desiccation-sensitive species accumulated, particularly under high irradiances. The stable radical appears to be one of the end-products of a process initiated by environmental stress, desiccation and high irradiance: its association with molecular damage depending on the degree to which the species is tolerant of desiccation. Identification of the radical in intact tissue from EPR and ENDOR studies, suggests that this is not a short-lived proxy-radical but instead is relatively stable and carbon-centred.     

In Vivo Kinetics of Nitrogenase Formation in Clostridium pasteurianum

Clostridium pasteurianum exhibits diauxic growth when grown in the presence of both NH(3) and N(2); no nitrogenase activity or formation was detected either serologically or by activity during growth on NH(3). During the 60-min lag that ensued after NH(3) was consumed and before growth resumed, molybdoferredoxin and azoferredoxin were first detected by activity measurements and serologically at 25 and 40 min, respectively. With the use of rifampin and dactinomycin, it was found that azoferredoxin messenger ribonucleic acid was initiated between 25 and 30 min after the inception of the lag and was completed by 38 min. An explanation of these results and their relation to possible models for the regulation of nitrogenase is given.     

Prevalence of Heterotypic Tumor/Immune Cell-In-Cell Structure In Vitro and In Vivo Leading to Formation of Aneuploidy

Cell-in-cell structures refer to a unique phenomenon that one living cell enters into another living cell intactly, occurring between homotypic tumor cells or tumor (or other tissue cells) and immune cells (named as heterotypic cell-in-cell structure). In the present study, through a large scale of survey we observed that heterotypic cell-in-cell structure formation occurred commonly in vitro with host cells derived from different human carcinomas as well as xenotypic mouse tumor cell lines. Most of the lineages of human immune cells, including T, B, NK cells, monocytes as well as in vitro activated LAK cells, were able to invade tumor cell lines. Poorly differentiated stem cells were capable of internalizing immune cells as well. More significantly, heterotypic tumor/immune cell-in-cell structures were observed in a higher frequency in tumor-derived tissues than those in adjacent tissues. In mouse hepatitis models, heterotypic immune cell/hepatocyte cell-in-cell structures were also formed in a higher frequency than in normal controls. After in vitro culture, different forms of internalized immune cells in heterotypic cell-in-cell structures were observed, with one or multiple immune cells inside host cells undergoing resting, degradation or mitosis. More strikingly, some internalized immune cells penetrated directly into the nucleus of target cells. Multinuclear cells with aneuploid nucleus were formed in target tumor cells after internalizing immune cells as well as in situ tumor regions. Therefore, with the prevalence of heterotypic cell-in-cell structures observed, we suggest that shielding of immune cells inside tumor or inflammatory tissue cells implies the formation of aneuploidy with the increased multinucleation as well as fine-tuning of microenvironment under pathological status, which may define distinct mechanisms to influence the etiology and progress of tumors.     

Potent Neutralization of Staphylococcal Enterotoxin B In Vivo by Antibodies that Block Binding to the T-Cell Receptor

To develop an antibody (Ab) therapeutic against staphylococcal enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a “class T" anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed “class M" Abs. A systematic engineering approach was applied to affinity-mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen–deuterium exchange coupled to mass spectrometry revealed that the class T epitope on SEB overlapped with the T-cell receptor binding site, whereas other evidence suggested that the class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed ∼ 50-fold more potent toxin-neutralizing efficacy than the best class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two class M binding sites and two class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bsAb engineering.