Evidence for a regulatory action of vanadate on protein phosphorylation in brain microvessels

We investigated the action of vanadate on protein phosphorylation in microvessels isolated from rat brain. We found that a stimulation of protein phosphorylation from 32P-ATP occurs, in the presence of different concentrations of vanadate, 10(-3) M being the most effective dose. This action was time-dependent, and it was more evident after 60 s of treatment. The contribution of ATPase inhibition caused by vanadate appears to be negligible. In addition a stimulation of cAMP-dependent protein kinase activity was observed. The pattern of protein phosphorylation showed that exposure to 10(-3) M vanadate resulted in a nonspecific stimulation of protein phosphorylation concomitantly with a selective inhibition of the 55 KDa protein phosphorylation. The nature of this protein is also discussed.     

Inhibition of substance P degradation in rat brain preparations by peptide hydroxamic acids

A peptidase activity of rat diencephalon membranes, which acts on the C-terminal hexapeptide sequence of substance P, was characterized using the radiolabeled substrate N alpha-[( 125I]iododesaminotyrosyl)-substance P (6-11)-hexapeptide. This activity presents certain characteristics similar to those of the substance-P-degrading enzyme purified from human brain by Lee et al. [Eur. J. Biochem. 114, 315-327 (1981)]. It is inhibited by metal chelators and some thiol reagents, but is insensitive to inhibitors of serine proteases and aminopeptidases. The activity is different from angiotensin-converting enzyme and enkephalinase, since it is not affected by specific inhibitors of these enzymes. Substance P and substance P C-terminal fragments longer than the pentapeptide inhibited the degradation of the radiolabeled substrate with inhibition constants around 200 microM. Short fragments of the substance P sequence, such as Boc-Phe-Phe-OMe and Boc-Phe-Phe-Gly-OEt, were also found to inhibit the degradation of the substrate. When the metal-chelating hydroxamic acid moiety was attached to the carboxyl terminus of these short peptides, potent inhibitors of the substance-P-degrading activity were obtained, with inhibition constants in the micromolar range. The most potent of these compounds, iododesaminotyrosyl-Phe-Phe-Gly-NHOH (IBH-Phe-Phe-Gly-NHOH), is a competitive inhibitor, with a Ki value of 1.9 microM. The degradation of substance P by rat diencephalon slices was inhibited to the same extent (40-50%) by IBH-Phe-Phe-Gly-NHOH (20 microM) and by phosphoramidon (1 microM). A combination of both reagents reduced the degradation rate by 75-80%, suggesting that both enkephalinase and the substance-P-degrading activity are involved in the metabolism of substance P in this preparation. IBH-Phe-Phe-Gly-NHOH seems to be quite specific for the latter enzyme, since at a high concentration (0.1 mM) it did not affect the degradation of the radiolabeled substrate by alpha-chymotrypsin, papain, or thermolysin.     

Detection of a novel immunoreactive endomorphin 2-like peptide in rat brain extracts

To pursue further the possible de novo biosynthetic pathway of endomorphins in rat brain we raised antibodies to endomorphin-2 conjugate in rabbits. Antiserum R1 recognized endomorphin-2 with good selectivity as compared to endomorphin-1 with a median detection value of 65.5+/-7.5 pg/tube (n=7), whereas R4 antiserum recognized both endomorphins with similar sensitivity. Neither antisera recognized YP-related di- or tripeptides or YGGF-related opioid sequences (enkephalins, beta-endorphin, dynorphin). Using the same rat brain extraction-RP-HPLC-gradient separation paradigm as previously, antisera detected 144.6+/-40.0 (n=3) pg/g wet brain weight endomorphin-2-like immunoreactivity in the fraction corresponding to standard endomorphin-2 retention time and also in the fraction matching endomorphin-2-OH standard retention time (179.1+/-30.1 pg/g). Since R1 failed to recognize authentic endomorphin-2-OH, the second immunoreactive species must be different from both endomorphin-2 and endomorphin-2-OH. Possible biosynthetic intermediates to endomorphins, synthetic YPFFG and YPWFG had retention times close to the parent endomorphin standards in RP-HPLC gradient separation profile. The former was a mu-opioid receptor agonist of medium potency in the in vitro assays (rat brain RBA>P gamma S binding and mouse vas deferens), whereas the latter was a weak mu-opioid receptor agonist with a significant delta-opioid receptorial action as well and a definite indication of partial agonism.     

Regional differences in glutaminase activation by phosphate and calcium in rat brain: Impairment in aged rats and implications for regional glutaminase isozymes

Regional regulation of glutaminase by phosphate and calcium was examined in the temporal cortex (TCX), striatum (STR) and hippocampus (HIPP) from adult and aged male F344 rats. Phosphate-dependent glutaminase activity in adult rats was significantly lower (35–43%) in the HIPP (100 and 150 mM) and STR (150 mM) compared to PAG activity in the TCX. Phosphate activation in aged rats was 50–60% lower in the HIPP at concentrations greater than 25 mM compared to the aged TCX or STR. PAG activity in the TCX and STR was unaffected by age, but was significantly reduced (30–50%) in the HIPP from aged rats at phosphate concentrations of 25 mM and greater when compared to adult rats. In adult rats at concentrations of CaCl₂ above 1 mM, PAG activity was significantly lower (60–75%) in the STR and HIPP when compared to the TCX. In aged rats, PAG activity (1 mM CaCl₂) in the HIPP was significantly less (50%) than STR PAG activity in aged rats. Diminished PAG activity was seen only in the TCX (2.5 mM; 32%), and the HIPP (0.5 mM; 25% and 1 mM; 38%) at higher calcium concentrations compared to adult. Phosphate-independent calcium activation of PAG occurred in the HIPP but not in either the TCX or the STR. Addition of phosphate resulted in a synergistic activation of PAG in the STR and TCX, but not in the HIPP. These findings suggest that PAG is regionally regulated by phosphate and calcium, and this regulation is impaired in aged rats. These data also support the hypothesis that isozymes of PAG exist with different regulatory properties.Abbreviation PAG Phosphate-activated glutaminase - L-glutamine amidohydrolase EC 3.5.1.2 - TCX temporal cortex - STR striatum - HIPP hippocampus - F344 Fischer-344 rat     

Ellagic acid in pomegranate suppresses resistin secretion by a novel regulatory mechanism involving the degradation of intracellular resistin protein in adipocytes

Resistin, an adipocytokine, is considered the link between obesity and type 2 diabetes. Pomegranate is a rich source of compounds used to treat metabolic diseases including type 2 diabetes. In this study, we found that consumption of pomegranate fruit extract (PFE) predominantly reduced the serum resistin levels in ovariectomized mice, an animal model with elevated resistin levels in serum and upregulated resistin mRNA expression in white adipose tissue. Moreover, the PFE significantly reduced the secretion and intracellular protein levels of resistin in differentiated murine 3T3-L1 adipocytes, but it did not alter resistin mRNA expression. When de novo protein synthesis was inhibited by the protein synthesis inhibitor cycloheximide, the intracellular resistin protein levels were drastically reduced by the PFE, suggesting that the PFE promoted the degradation of resistin at the protein level. We also found that ellagic acid (EA), a main component of pomegranate, had the same effects on the secretion and intracellular protein level of resistin. These results suggest that EA in pomegranate suppresses resistin secretion by a novel mechanism involving the degradation of intracellular resistin protein in adipocytes.     

Regional and species differences in endogenous prostaglandin biosynthesis by brain homogenates

Endogenously formed prostaglandins (PGs) D₂, E₂ and F_2α were determined in homogenates of brain regions from rat, guinea-pig, rabbit and cat, using gas-chromatography-mass spectrometry. The main PGs formed in the brain regions of the rat were PGD₂, in the guinea-pig PGD₂ and PGF_2α, in the rabbit PGF_2α and in the cat PGE₂. Brain regions from the same animal species showed the same pattern of PG formation. They varied, however, in the amount of total PGs formed, the limbic system and the cerebral cortex being highest and cerebellum lowest.     

A novel peptide modulates alpha7 nicotinic receptor responses: implications for a possible trophic-toxic mechanism within the brain

The alpha7 nicotinic acetylcholine receptor (nAChR) plays a key role in neural development and neurodegeneration. Here, we identify a novel, modulatory receptor ligand, a 14-amino acid peptide (AEFHRWSSYMVHWK) derived from the C-terminus of acetylcholinesterase (AChE). In three different in vitro preparations, this 'AChE-peptide' is bioactive in a ligand-specific and concentration-dependent manner. First, it modulates acutely the effect of acetylcholine (ACh) on Xenopus oocytes transfected with human alpha7, but not alpha4/beta2, nAChR. The action persists when intracellular calcium is chelated with BAPTA or when calcium is substituted with barium ions. This observation suggests that intracellular Ca(2+) signals do not mediate the interaction between the peptide and nAChR, but rather that the interaction is direct: however, the intervention of other mediators cannot be excluded. Secondly, in recordings from the CA1 region in guinea-pig hippocampal slices, AChE-peptide modulates synaptic plasticity in a alpha-bungarotoxin (alpha-BgTx)-sensitive manner. Thirdly, in organotypic cultures of rat hippocampus, long-term exposure to peptide attenuates neurite outgrowth: this chronic, functional effect is selectively blocked by the alpha7 nAChR antagonists, alpha-BgTx and methyllycaconitine, but not by the alpha4/beta2-preferring blocker dihydro-beta-ethroidine. A scrambled peptide variant, and the analogous peptide from butyrylcholinesterase, are ineffective in all three paradigms. The consequences of this novel modulation of the alpha7 nAChR may be activation of a trophic-toxic axis, of relevance to neurodegeneration.     

Red blood cell velocity and oxygen tension measurement in cerebral microvessels by double-wavelength photoexcitation.

Because the regulation of microcirculation in the cerebral cortex cannot be analyzed without measuring the blood flow dynamics and oxygen concentration in cerebral microvessels, we developed a fluorescence and phosphorescence system for estimating red blood cell velocity and oxygen tension in cerebral microcirculation noninvasively and continuously with high spatial resolution. Using red blood cells labeled with fluorescent isothiocyanate to visualize red cell distribution and using the oxygen quenching of Pd-meso-tetra-(4-carboxyphenyl)-porphyrin phosphorescence to measure oxygen tension enabled simultaneous measurement of blood velocity and oxygen tension. We examined how the measurement accuracy was affected by the spatial resolution and by the excitation laser light passing through the targeted microvessel and exciting the oxygen probe dye in the tissue beneath it. Focusing the excitation light into the microvessel stabilized the phosphorescence lifetime at each spatial resolution; moreover, it greatly reduced phosphorescence from the brain tissue. Animal experiments involving acute hemorrhagic shock demonstrated the feasibility of our system by showing that the changes in venular velocity and oxygen tension are synchronized to the change in mean arterial pressure. Our system measures the red cell velocity and oxygen concentration in the cerebral microcirculation by using the differences in luminescence and wavelength between fluorescence and phosphorescence, making it possible to easily acquire information about cerebral microcirculatory distribution and oxygen tension simultaneously.     

Aluminum inhibits proteolytic degradation of amyloid beta peptide by cathepsin D: a potential link between aluminum accumulation and neuritic plaque deposition

Neuritic plaques are the key pathological feature of Alzheimer's disease, and amyloid beta (Abeta) peptides are major component of these plaques. In this study, we demonstrated the influence of aluminum (Al) on the Abeta peptide degradation by cathepsin D. Al did not directly affect the cathepsin D activity using small synthetic substrate. However, when Abeta peptides were used as substrate, the apparent inhibitory effect of Al on cathepsin D activity was observed. This inhibitory effect disappeared by treatment of desferrioxamine. These results indicate that Al has the potential to interact and disrupt Abeta peptide catabolism via the inhibition of proteolytic degradation.     

Regional and species differences in endogenous prostaglandin biosynthesis by brain homogenates.

Endogenously formed prostaglandins (PGs) D2, E2 and F2 alpha were determined in homogenates of brain regions from rat, guinea-pig, rabbit and cat, using gas-chromatography-mass spectrometry. The main PGs formed in the brain regions of the rat were PGD2, in the guinea-pig PGD2 and PGF2 alpha, in the rabbit PGF2 alpha and in the cat PGE2. Brain regions from the same animal species showed the same pattern of PG formation. They varied, however, in the amount of total PGs formed, the limbic system and the cerebral cortex being highest and cerebellum lowest.     

Transport of sugars into microvessels isolated from rat brain: a model for the blood-brain barrier.

Abstract— Microvessels (primarily capillaries) were isolated from the brains of rats 25-35 days of age. This preparation was characterized by light, transmission, and scanning electron microscopy. Transmission electron microscopy revealed that the endothelial cell membranes were intact and were impermeable to horseradish peroxidase. However, scanning electron microscopy revealed that damage to the membrane occurred during isolation. The isolated microvessel preparations were metabolically competent as demonstrated by their ability to metabolize [¹⁴C]glucose. Aliquots of microvessel preparation were incubated with radioactive non-metabolizable analogs of D-glucose at various concentrations. The kinetics of accumulation of radioactivity in the capillaries were analyzed according to a model for carrier-mediated diffusion and affinity constants for 3-O-methyl- D-glucose and 2-deoxyglucose were calculated (about 18 mM at 20°C in each case). These affinity constants are somewhat greater than that expected from whole animal experiments reported by other laboratories. This discrepancy is probably accounted for by the presence of a passive diffusion component. However, despite this complication, the primary mechanism for entry of D-glucose analogues at physiological concentrations is compatible with carrier-mediated transport since: the uptake of sugar analogs was shown to be saturable, to exhibit competition for uptake between structurally similar molecules, and to be non-concentrative. In contrast, the uptake of glycerol, mannitol, and L-glucose by isolated microvessels obeyed the kinetics of simple passive diffusion and was not saturable. Our results are compatible with the concept that the capillary is the anatomic locus of the blood-brain barrier and that this structure contains the carrier-mediated transport system for monosaccharide penetration into brain.     

Depletion of regulatory T cells by anti-GITR mAb as a novel mechanism for cancer immunotherapy

In vitro, engagement of GITR on Treg cells by the agonistic anti-GITR mAb, DTA-1, appears to abrogate their suppressive function. The consequence of in vivo engagement of GITR by DTA-1 is, however, less clear. In this study, we show that Treg cells isolated from DTA-1-treated mice were as potent as those from untreated mice in suppressing conventional CD4 T cells in vitro, indicating that in vivo GITR ligation does not disable Treg cells. Treatment of Foxp3/GFP knock-in mice with DTA-1 led to a selective reduction of circulating Treg cells, suggesting that DTA-1 is a depleting mAb which preferentially targets Treg cells. In tumour-bearing mice, DTA-1-mediated depletion of Treg cells was most marked in tumours but not in tumour-draining lymph node. These features were confirmed in an adoptive transfer model using tumour antigen-specific Treg cells. Interestingly, Treg cells detected in tumour tissues expressed much higher levels of GITR than those in tumour-draining lymph nodes, indicating that the efficiency of depletion might be correlated with the level of GITR expression. Finally, in vivo labelling of GITR in naive or tumour-bearing mice demonstrated that Treg cells constitutively expressed higher levels of GITR than conventional T cells, independent of location and activation state, consistent with the preferential in vivo depletion of Tregs by DTA-1. Thus, depletion of Treg cells represents a previously unrecognised in vivo activity of DTA-1 which has important implications for the application of anti-GITR antibodies in cancer immunotherapy.     

Substrate-stimulated oxygen consumption by isolated rat brain microvessels in the presence and absence of ATP

We report here properties of isolated brain microvessels such as the rate of oxygen consumption with different substrates; the permeabilizing effect of added ATP is studied. With the isolation procedure presented the cerebral endothelium has a metabolic activity comparable to that reported in the literature. The respiratory rate of the microvessels is not affected by the addition of ATP, whereas it is significantly increased by addition of succinate and -chetoglutarate. The exposure of the isolated brain capillaries to ATP, in a Ca²⁺-free medium, increases the uptake of 6-carboxyfluorescein. This may be due to pores opened by ATP in the endothelial cell membrane in the absence of divalent cations.     

Stabilization signals: a novel regulatory mechanism in the ubiquitin/proteasome system

The turnover of cellular proteins is a highly organized process that involves spatially and temporally regulated degradation by the ubiquitin/proteasome system. It is generally acknowledged that the specificity of the process is determined by constitutive or conditional protein domains, the degradation signals, that target the substrate for proteasomal degradation. In this review, we discuss a new type of regulatory domain: the stabilization signal. A model is proposed according to which protein half-lives are determined by the interplay of counteracting degradation and stabilization signals.     

Spatial frequency-based analysis of mean red blood cell speed in single microvessels: investigation of microvascular perfusion in rat cerebral cortex

Background

Our previous study has shown that prenatal exposure to X-ray irradiation causes cerebral hypo-perfusion during the postnatal development of central nervous system (CNS). However, the source of the hypo-perfusion and its impact on the CNS development remains unclear. The present study developed an automatic analysis method to determine the mean red blood cell (RBC) speed through single microvessels imaged with two-photon microscopy in the cerebral cortex of rats prenatally exposed to X-ray irradiation (1.5 Gy).

Methodology/Principal Findings

We obtained a mean RBC speed (0.9±0.6 mm/sec) that ranged from 0.2 to 4.4 mm/sec from 121 vessels in the radiation-exposed rats, which was about 40% lower than that of normal rats that were not exposed. These results were then compared with the conventional method for monitoring microvascular perfusion using the arteriovenous transit time (AVTT) determined by tracking fluorescent markers. A significant increase in the AVTT was observed in the exposed rats (1.9±0.6 sec) as compared to the age-matched non-exposed rats (1.2±0.3 sec). The results indicate that parenchyma capillary blood velocity in the exposed rats was approximately 37% lower than in non-exposed rats.

Conclusions/Significance

The algorithm presented is simple and robust relative to monitoring individual RBC speeds, which is superior in terms of noise tolerance and computation time. The demonstrative results show that the method developed in this study for determining the mean RBC speed in the spatial frequency domain was consistent with the conventional transit time method.     

Integrated response to inducers by communication between a catabolic pathway and its regulatory system

Efficient gene regulation of metabolic pathways implies that the profile of molecules inducing the pathway matches that of the molecules that are metabolized. Gratuitous induction, a well-known phenomenon in catabolic pathways, is the consequence of differences in the substrate and inducer profiles. This phenomenon is particularly evident in pathways for biodegradation of organic contaminants that can be induced by a variety of molecules similar to the real substrates. Analysis of the regulation of tetralin biodegradation genes in mutant strains with mutations that affect each component of the initial dioxygenase enzymatic complex indicated that the response of the regulatory system to potential inducers is altered differently depending on the mutated component. Based on the expression phenotypes of a number of single or double mutants, we propose a model that represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent efficient induction by a molecule that is not a real substrate. This communication allows a better fit of the substrate and inducer profiles, thus minimizing gratuitous induction, without a requirement for optimal coevolution to match the specificity of catabolic enzymes and their regulatory systems. Modulation of the regulatory system in this way not only provides a more appropriate response to potential inducers recognized by the regulatory system but also may properly adjust the levels of gene expression to the substrate availability.     

Regional distribution and in vivo release of tachykinin-like immunoreactivities in rat brain: evidence for regional differences in relative proportions of tachykinins

The regional distribution of various forms of tachykinin-like immunoreactivity (TKLI) was studied in rat brain using radioimmunoassay. TKLI was measured with two different tachykinin-antisera (K12 and E7), which react with neurokinin A (NKA) and neurokinin B (NKB) but not with substance P (SP) and with a specific SP-antiserum. TKLI-K12 and TKLI-E7 were found to have similar regional distributions which were, however, significantly different from that of the substance P-like immunoreactivity (SPLI). Thus, the ratio of the tissue concentrations of TKLI-K12 or TKLI-E7 to that of SPLI was higher in frontal cortex and hippocampus and lower in pons/medulla oblongata than in the other regions studied. Cation-exchange chromatography of neutral water extracts of brain tissue revealed two major immunoreactive components of TKLI-K12 and TKLI-E7, one of which co-eluted with synthetic NKB while the other appeared in the same region as synthetic NKA. The relative quantities of these components varied depending on the brain region studied. No TKLI-K12 or TKLI-E7 co-eluted with synthetic SP. Almost all of the SPLI in acetic acid or water extracts of brain tissue eluted as a single chromatographic component in the same position as synthetic SP. Potassium-stimulated in vivo release of TKLI-K12, TKLI-E7 and SPLI in striatum of rat brain could be demonstrated using intracerebral dialysis. The present results imply that tachykinins, which may serve as neurotransmitters or neuromodulators, are present in different proportions in different regions of rat brain.