Tryptophan binding to nuclei of rat brain.

Nuclei purified from whole rat brain specifically bind [3H]tryptophan ([3H]Trp) under in vitro conditions. Excess unlabeled Trp (10(-4) M) is an effective inhibitor of in vitro [3H]Trp binding to brain nuclei. Rats tube-fed L-tryptophan (Trp) (30 mg/100 g body wt) 30 min to 4 hr before killing revealed decreased specific binding of [3H]Trp to purified brain nuclei in vitro. By Scatchard analysis, the nuclei from whole brain appear to contain one binding site for [3H]Trp, and the KD is 263 nM. A number of Trp-related compounds, Trp metabolites, or other amino acids and their analogues were observed to compete for in vitro [3H]Trp binding to brain nuclei. The ability of Trp analogues, metabolites, and other cognate compounds to inhibit in vitro [3H]Trp binding to brain nuclei was evaluated and utilized to map the active site of Trp binding.     

Treatment with DNAse I fosters binding to nec PBMC of CRP

CRP is an important inflammatory marker, however, CRP levels are relatively low in patients with SLE. In addition patients with SLE often display low activities and serum levels for DNase I and complement, respectively. Here we show that DNase I treatment of nec PBMC increased their binding of CRP. Consequently, reduced DNase I activity in patients with SLE may contribute to the impaired opsonisation by CRP of dead cells, exacerbating the clearance defect in SLE of apo and nec cells.     

Melatonin binding to the anteroventral and anterodorsal thalamic nuclei in the rat.

Pineal melatonin is a putative humoral coordinator of various circadian body rhythms. Rather few loci in the brain contain such a density of melatonin receptors that specific binding can be demonstrated by autoradiography. In this study we measured, using in vitro receptor autoradiography, displaceable binding of iodo-melatonin to the anteroventral and anterodorsal thalamic nuclei of Wistar rats (0.6-1.4 fmol/mg protein at 100 pM ligand concentration). These nuclei of the limbic system have direct connections with the retina and they participate in learning and memory functions.     

CRP and neutrophils: functional effects and complex uptake.

The uptake of C-reactive protein (CRP)-pneumococcal C-polysaccharide (CPS) complexes by neutrophils was studied. A specific CRP dependent mechanism of uptake was demonstrated. This promoted CPS (complexed to CRP) clearance which was further enhanced by additional complement activation. Physiological concentrations of low density lipoprotein inhibited entry of complexed CPS into neutrophils but had no effect on entry of CRP alone. Pure human CRP was shown to have no effect on neutrophil chemotaxis and oxidative metabolism.     

Platelet inhibitory effects of CRP preparations are due to a co-isolating low molecular weight factor.

We previously reported that C-reactive protein (CRP), an acute phase reactant, inhibits platelet activation through an effect upon a factor(s) critical to ADP-mediated secondary wave platelet aggregation but independent of a direct effect upon platelet contractile elements. However, a role for an accessory factor in this inhibitory effect became of concern because of an inconsistency in the effects of CRP preparations upon the platelet: inhibition was lost upon storage and CRP preparations differed, on a weight basis, in inhibitory capacity and sensitivity to the presence of the CRP ligand C-polysaccharide (CPS(. The studies presented herein were thus intended to assess whether an accessory factor was involved in the inhibition of platelet activation observed with CRP. We report that the activity of the inhibitory CRP preparations resulted from association with a low molecular weight factor (LMF) with an apparent nominal molecular weight of 8300-12,500 and an A280:A260 ratio of approximately 0.4. Purified CRP did not inhibit platelet responsiveness but CRP with associated LMF (CRP-LMF) did. Moreover, the inhibitory capacity of CRP-LMF but not LMF was substantially reversed in the presence of CPS. These studies indicate that the platelet inhibitory properties of CRP preparations result from and are contingent upon the presence of a co-isolating low molecular weight factor.     

Influence of lead acetate and selected metal salts on tryptophan binding to rat hepatic nuclei.

This study evaluated whether lead acetate or other selected metal salts would influence the binding of L-tryptophan to rat hepatic nuclei. Lead salts and other salts of cadmium, zinc, mercury, and molybdenum, when added alone, had only small effects on 3H-tryptophan binding to hepatic nuclei in vitro. However, each of these salts, when added along with unlabeled L-tryptophan (excess, 10(-4) M), caused significantly less inhibition of 3H-tryptophan binding to hepatic nuclei than did unlabeled L-tryptophan alone. Lead acetate (10(-10) to 10(-4) M), when added along with unlabeled L-tryptophan, abrogated the inhibition of binding related to unlabeled L-tryptophan alone. Sodium arsenite (but not potassium arsenate) as well as sodium selenite (at 10(-4) M concentrations) inhibited to a moderate degree the in vitro 3H-tryptophan binding to hepatic nuclei, but addition of 10(-4) dithiothreitol, a protective agent for sulfhydryl groups, diminished this inhibition. Rats receiving a high dose of lead acetate before being tube-fed L-tryptophan displayed a decrease in hepatic protein synthesis compared with the stimulatory response connected with L-tryptophan alone. Thus, the addition of lead acetate and of other metal salts appears to have an inhibitory effect on L-tryptophan binding to hepatic nuclei. Lead acetate was investigated in in vivo experiments and was found to negate the stimulation of hepatic protein synthesis related to L-tryptophan alone.     

Characterization of monoclonal anti-DNA antibodies and visualization of binding to chromosomes and cell nuclei

Monoclonal antibodies against DNA from two hybridoma cell lines were produced and characterized. One had specificity for single stranded (ss) DNA with some cross-reactivity to RNA, while the other was specific for both single (ss) and double stranded (ds) DNA. The latter ds and ss DNA-binding antibody was used as a model for analysing the distribution of the epitope in chromosomes and cell nuclei. A linear correlation between antibody binding and propidium iodide counterstaining was found on flow cytometric analysis of suspended chromosomes. Immunofluorescence of rat myoblast cells showed a speckled distribution of the antibody in the nucleus with a variability between the cells. Using electron microscopy to visualize antibody binding with gold particles, codistribution with uranyl acetate staining of leucocytes was found. These results suggested that the antibody preferentially binds to condensed chromatin in cells and chromosomes.     

Functional assessment and structural basis of antibody binding to human papillomavirus capsid

Persistent high‐risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus‐like particle (VLP)‐based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)‐based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV‐based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational changes in the viral capsid, which can result in concealing the binding site for a cellular receptor like 1A1D‐2 against dengue virus, or inducing premature genome release like E18 against enterovirus 71. Higher‐resolution details on the epitope composition of HPV neutralizing antibodies would shed light on the structural basis of the highly efficacious vaccines and aid the design of next generation vaccines. In‐depth understanding of epitope composition would ensure the development of function‐indicating assays for the comparability exercise to support process improvement or process scale up. Elucidation of the structural elements of the type‐specific epitopes would enable rational design of cross‐type neutralization via epitope re‐engineering or epitope grafting in hybrid VLPs. Copyright © 2015 John Wiley & Sons, Ltd.     

Antiangiogenic and antitumor effects of IMMUNEPOTENT CRP in murine melanoma

Background: Skin cancers are common, and there has recently been a dramatic increase in their incidence, particularly in the occurrence of melanoma. Furthermore, relapse after curative surgical treatment of melanoma remains a significant clinical challenge and accounts for most of the mortality of this disease. Objective: The aim of this study was to determine whether IMMUNEPOTENT CRP affects B16F10 melanoma cells and tumors growth and vascular endothelial growth factor (VEGF) production in vivo and in vitro.

Methods: B16F10 cells and B16F10-inoculated mice were treated with different concentrations of IMMUNEPOTENT CRP. Outcomes were then evaluated using MTT, TUNEL, Caspase-3, senescence, ELISA and colorimetric assays. Parameters related to survival and tumor weight were also assessed.

Results: IMMUNEPOTENT CRP decreased the viability of B16F10 cells by increasing apoptosis of the treated cells, and VEGF production was decreased both in vitro and in vivo. Furthermore, treatment prevented metastasis, delayed the appearance of tumors, decreased tumor weight and improved the survival of tumor-bearing mice.

Discussion: These observations suggest that IMMUNEPOTENT CRP can be used to suppress growth and metastasis by using targeting proteins such as VEGF.     

Functional differences between the magnocellular neurosecretory hypothalamic nuclei in rats

The supraoptic (SON), paraventricultr (PVN), and postoptic (PON) nuclei of the hypothalamus were studied in rats after prolonged adaptation to hypoxia in a pressure chamber (exposure for 6 h daily for 3 months to a simulated altitude of 7600 m). Morphometric measurements showed that the functional activity of all three nuclei rises during the first 5 days of the experiments, but later differences are found in the response of the neurosecretory nuclei. By the 20th day the state of SON is back to normal, but in PVN and PON a reduction in the size of the nucleoli and in the content of neurosecretory substance is evidence of depression of the functional activity of these nuclei. Positive correlation also was found between the volume of the nucleoli in the cells of PON and PVN and the height of the follicular epithelium of the thyroid gland (r=0.82 and 0.81 respectively, P<0.05) whereas no such correlation was found for the cells of SON (r=0.5, P>0.05).I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 7, pp. 3–5, July, 1978.     

Tissue binding of tritiated norepinephrine in pigmented nuclei of human brain

Regional binding of norepinephrine to tissue structures occurs in sections of brain tissue incubated in vitro with tritiated norepinephrine, followed by radioautography. The binding probably represents one of the mechanisms involved in the uptake and storage of catecholamines by nerve cells. Applying this method to a study of human brains showed excessively strong binding of norepinephrine at the surface membranes of pigmented nerve cells in the substantia nigra, nucleus coeruleus, nucleus dorsalis vagi, and others. Such excessive binding was not found in nonpigmented nuclei in the human brain nor in rat brain. The arrangement of sites of binding at the cell membranes strongly suggested synaptic endings. Melanin pigmentation of nerve cells may be related to the amount of catecholamine-containing synapses at the surface of the neurons.     

Functional alignment of feedback effects from visual cortex to thalamus

Following from the classical work of Hubel and Wiesel, it has been recognized that the orientation and the on- and off-zones of receptive fields of layer 4 simple cells in the visual cortex are linked to the spatial alignment and properties of the cells in the visual thalamus that relay the retinal input. Here we present evidence showing that the orientation and the on- and off-zones of receptive fields of layer 6 simple cells in cat visual cortex that provide feedback to the thalamus are similarly linked to the alignment and properties of the receptive fields of the thalamic cells they contact. However, the pattern of influence linked to on- and off-zones is phase-reversed. This has important functional implications.     

Lactoperoxidase binding to streptococci.

There have been conflicting reports regarding the binding of lactoperoxidase to bacterial cell surfaces. We describe here the effects of cell-bound lactoperoxidase on acid production by suspensions of Streptococcus mutans (NCTC 10449) in the presence of hydrogen peroxide and thiocyanate. Saline suspensions of log-phase bacteria were treated with 0.1 mg of lactoperoxidase per ml and were then washed thoroughly. The addition of hydrogen peroxide and thiocyanate markedly reduced the acid production of these lactoperoxidase-treated bacteria but had no effect on the acid production of untreated controls. After a 3-h incubation in saline, the lactoperoxidase-treated bacteria produced acid in the presence of hydrogen peroxide and thiocyanate at the same rate as untreated bacteria. These observations suggest that lactoperoxidase is initially bound to the cell surface in an enzymatically active form at a concentration sufficient to inhibit acid production. The lactoperoxidase is slowly degraded or desorbed as the bacteria stand in saline suspension.     

Functional glutamatergic and glycinergic inputs to several superior olivary nuclei of the rat revealed by optical imaging

Superior olivary complex (SOC) neurons receive excitatory and inhibitory inputs from both ears. We determined the nature of such inputs to the main SOC nuclei with an optical imaging system. To do so, brainstem slices of postnatal (P) rats (P3-13) were treated with the fast voltage-sensitive dye RH795, and ipsilateral and contralateral SOC inputs were activated electrically. Optical signals, equivalent to membrane potential changes, were detected by a 464-photodiode array. The signals consisted mostly of two components which were identified as pre- and postsynaptic potentials in experiments with Ca2+-free solutions. They correlated with morphological structures, i.e. the presynaptic components were prominent in neuropil regions whereas the postsynaptic components dominated in somata regions. Postsynaptic components were distinguished pharmacologically with the glycine receptor blocker strychnine and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Concerning the lateral superior olive, we confirmed the known glutamatergic inputs from the ipsilateral side and the glycinergic inputs from the ipsilateral and contralateral sides. Furthermore, we identified a CNQX-sensitive input from the contralateral side. In the medial superior olive, we corroborated the glutamatergic and glycinergic inputs from the ipsilateral and contralateral sides. Both ipsi- and contralaterally, the glutamatergic input was more pronounced than the glycinergic input. In the superior paraolivary nucleus, we also identified ipsilateral and contralateral inputs. Besides the known glycinergic input from the contralateral side, we found a novel glycinergic input from the ipsilateral side and identified CNQX-sensitive inputs from the contralateral and ipsilateral sides. The latter was very weak and appeared only in 30% of the experiments. The data show the feasibility of identifying functional inputs to the SOC with voltage-sensitive dye recordings.     

Functional activity of CD40 antibodies correlates to the position of binding relative to CD154

In this study we describe the characterization of a panel of 12 anti-mouse CD40 monoclonal antibodies (mAb). Characterization was performed in terms of antibody-binding site relative to the CD154 ligand, and the relationship between position and functional outcome of binding. The antibodies divided into three groups. The first were strong inhibitors of CD154 binding, and induced strong proliferative and activation signals to B cells. Two antibodies gave intermediary inhibition and comparable levels of activation. The remaining antibodies were found to bind outside the CD154 binding site and were poor inducers of B-cell activation. Data presented show a strong correlation between location of mAb binding and the resultant activation signal delivered. This correlation is shown to be independent of the isotype of the antibody involved and of its affinity. Implications of these findings are discussed.