Proteomic identification of proteins specifically oxidized in Caenorhabditis elegans expressing human Abeta(1-42): implications for Alzheimer's disease

Protein oxidation has been shown to lead to loss of protein function, increased protein aggregation, decreased protein turnover, decreased membrane fluidity, altered cellular redox poteintial, loss of Ca2+ homeostaisis, and cell death. There is increasing evidence that protein oxidation is involved in the pathogenesis of Alzheimer's disease and amyloid beta-peptide (1-42) has been implicated as a mediator of oxidative stress in AD. However, the specific implications of the oxidation induced by Abeta(1-42) on the neurodegeneration evident in AD are unknown. In this study, we used proteomic techniques to identify specific targets of oxidation in transgenic Caenorhabditis elegans (C. elegans) expressing human Abeta(1-42). We identified 16 oxidized proteins involved in energy metabolism, proteasome function, and scavenging of oxidants that are more oxidized compared to control lines. These results are discussed with reference to Alzheimer's disease.     

In vitro and in vivo oxidative stress associated with Alzheimer's amyloid beta-peptide (1-42)

The amyloid beta-peptide (A beta)-associated free radical oxidative stress model for neuronal death in Alzheimer's disease (AD) brain predicts that neuronal protein oxidation is a consequence of A beta-associated free radicals [8]. In this study we have used both in vitro and in vivo models of beta-amyloid (A beta) toxicity to detect free radical induced oxidative stress by the measure of protein carbonyl levels. These model systems employed cultured hippocampal neurons exposed to exogenous synthetic A beta(1-42) and transgenic Caenorhabditis elegans (C. elegans) animals expressing A beta(1-42). We also investigated the importance of the A beta(1-42) Met35 residue for free radical formation in peptide solution and for peptide-induced protein oxidation and neuronal toxicity in these model systems. A beta(1-42) in solution yielded an EPR spectrum, suggesting that free radicals are associated with this peptide; however, neither the reverse [A beta(42-1)] nor methionine-substituted peptide [A beta(1-42)Met35Nlc] gave significant EPR spectra, suggesting the importance of the methionine residue in free radical formation. A beta(1-42) addition to cultured hippocampal neurons led to both neurotoxicity (30.1% cell death, p < 0.001) and increased protein oxidation (158% of controls, p < 0.001). and both of those effects were not observed with reverse or Met35Nle substituted peptides. C. elegans transgenic animals expressing human A beta(1-42) also had significantly increased in vivo protein carbonyls (176% of control animals, p < 0.001), consistent with our model. In contrast, transgenic animals with a Met35cys substitution in A beta(1-42) showed no increased protein carbonyls in vivo, in support of the hypothesis that methionine is important in A beta-associated free radical oxidative stress. These results are discussed with reference to the A beta-associated free radical oxidative stress model of neurotoxicity in AD brain.     

Oxidative stress precedes fibrillar deposition of Alzheimer's disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model

Alzheimer's disease is a progressive, neurodegenerative disorder characterized by senile plaques and neurofibrillary components. Abeta(1-42) is a principal component of senile plaques and is thought to be central to the pathogenesis of the disease. The Alzheimer's disease brain is under significant oxidative stress, and the Abeta(1-42) peptide is known to cause oxidative stress in vitro. One controversy in the amyloid hypothesis is whether or not Abeta plaques are required for toxicity. We have employed a temperature-inducible Abeta expression system in Caenorhabditis elegans to create a strain of worms, CL4176, in which Abeta(1-42) is expressed with a non-permissive temperature of 23 degrees C. The CL4176 strain allows examination of the temporal relationship between Abeta expression, oxidative stress, and Abeta fibril formation. CL4176 were under increased oxidative stress, evidenced by increased protein oxidation indexed by increased carbonyl levels, 24 and 32 h after temperature upshift as compared to the control strain, CL1175. The increased oxidative stress in CL4176 occurred in the absence of Abeta fibril formation, consistent with the notion that the toxic species in Abeta toxicity is pre-fibrillar Abeta and not the Abeta fibril. These results are discussed with reference to Alzheimer's disease.     

Alzheimer's amyloid beta-peptide (1-42): involvement of methionine residue 35 in the oxidative stress and neurotoxicity properties of this peptide

In the interesting debate entitled "Challenging Views of Alzheimer's Disease II," we defended the position that factors such as oxygen, the single methionine residue of amyloid beta-peptide(1-42) [Abeta(1-42)], and redox metal ions were important for the oxidative stress and neurotoxic properties of this peptide that is critically involved in the pathogenesis of Alzheimer's disease. This brief review summarizes some of our findings relevant to the role of the single methionine residue of Abeta(1-42) in the oxidative stress and neurotoxic properties of this peptide.     

Efflux of human and mouse amyloid beta proteins 1-40 and 1-42 from brain: impairment in a mouse model of Alzheimer's disease

Brain to blood transport is believed to be a major determinant of the amount of amyloid beta protein (AbetaP) found in brain. Impaired efflux has been suggested as a mechanism by which AbetaP can accumulate in the CNS and so lead to Alzheimer's disease (AD). To date, however, no study of the efflux of the form of AbetaP most relevant to AD, AbetaP1-42, has been conducted, even though a single amino acid substitution in AbetaP can greatly alter efflux. Here, we examined the efflux of AbetaP mouse1-42, mouse1-40, human1-42, and human1-40 in young CD-1, young senesence accelerated mouse (SAM) P8, and aged SAMP8 mice. The SAMP8 mouse with aging spontaneously overproduces AbetaP and develops cognitive impairments reversed by AbetaP-directed antibody or phosphorothioate antisense oligonucleotide. CD-1 mice transported all forms of AbetaP, although mouse1-42 and human1-40 were transported faster than the other forms. There was a decrease in the saturable transport of mouse1-42 in SAMP8 mice regardless of age. Efflux of mouse1-40 and human1-42 was only by a non-saturable mechanism in young SAMP8 mice and their efflux was totally absent in aged SAMP8 mice. These differences in the efflux of the various forms of AbetaP among the three groups of mice supports the hypothesis that impaired efflux is an important factor in the accumulation of AbetaP in the CNS.     

Increased amyloid beta-peptide (1-40) level in brain of streptozotocin-induced diabetic rats

The aims of the study were to investigate whether the level of amyloid β-peptide (Aβ) (1–40) was increased in brain of diabetic rats and whether the increase was associated with dysfunction of P-glycoprotein at the blood–brain barrier. A diabetes-like condition was induced by single administration of 65 mg/kg streptozotocin via i.p. injection. Aβ (1–40) levels in brain of the diabetic rats were measured using an enzyme linked immunosorbent assay (ELISA) kit. The in vivo brain-to-blood efflux and blood-to-brain influx transport of [¹²⁵I]-labeled human amyloid-β-peptide (hAβ) (1–40) were measured using the brain efflux index and brain permeability coefficient-surface area product, respectively. [¹⁴C]inulin served as a reference compound. The results showed that Aβ (1–40) levels significantly increased in temporal cortex and hippocampus of the diabetic rats. The brain remaining percentage of [¹²⁵I]hAβ (1–40) in diabetic rats significantly increased at 30 min after intracerebral microinjection, accompanied by decrease of the brain efflux index. Pretreatment of P-glycoprotein inhibitors verapamil or cyclosporin A significantly increased the brain remaining percentage of [¹²⁵I]hAβ (1–40). The brain permeability coefficient-surface area product of [¹²⁵I]hAβ (1–40) was increased in diabetic rats, accompanied by increased Aβ (1–40) levels in plasma. The present study demonstrated that a diabetic state could increase Aβ (1–40) levels in brain, which might be explained, at least in part, by the decline in brain-to-blood efflux of Aβ (1–40) due to deficient cerebral P-glycoprotein function in diabetic rats.     

Amyloid beta-peptide(1-42) contributes to the oxidative stress and neurodegeneration found in Alzheimer disease brain

Oxidative stress is extensive in Alzheimer disease (AD) brain. Amyloid beta-peptide (1-42) has been shown to induce oxidative stress and neurotoxicity in vitro and in vivo. Genetic mutations that result in increased production of Abeta1-42 from amyloid precursor protein are associated with an early onset and accelerated pathology of AD. Consequently, Abeta1-42 has been proposed to play a central role in the pathogenesis of AD as a mediator of oxidative stress. In this review, we discuss the role of Abeta1-42 in the lipid peroxidation and protein oxidation evident in AD brain and the implications of such oxidative stress for the function of various proteins that we have identified as specifically oxidized in AD brain compared to control, using proteomics methods. Additionally, we discuss the critical role of methionine 35 in the oxidative stress and neurotoxic properties exhibited by Abeta1-42.     

In vivo protection by the xanthate tricyclodecan-9-yl-xanthogenate against amyloid beta-peptide (1-42)-induced oxidative stress

Considerable evidence supports the role of oxidative stress in the pathogenesis of Alzheimer's disease. One hallmark of Alzheimer's disease is the accumulation of amyloid beta-peptide, which invokes a cascade of oxidative damage to neurons that can eventually result in neuronal death. Amyloid beta-peptide is the main component of senile plaques and generates free radicals ultimately leading to neuronal damage of membrane lipids, proteins and nucleic acids. Therefore, interest in the protective role of different antioxidant compounds has been growing for treatment of Alzheimer's disease and other oxidative stress-related disorders. Among different antioxidant drugs, much interest has been devoted to "thiol-delivering" compounds. Tricyclodecan-9-yl-xanthogenate is an inhibitor of phosphatidylcholine specific phospholipase C, and recent studies reported its ability to act as a glutathione-mimetic compound. In the present study, we investigate the in vivo ability of tricyclodecan-9-yl-xanthogenate to protect synaptosomes against amyloid beta-peptide-induced oxidative stress. Gerbils were injected i.p. with tricyclodecan-9-yl-xanthogenate or with saline solution, and synaptosomes were isolated from the brain. Synaptosomal preparations isolated from tricyclodecan-9-yl-xanthogenate injected gerbils and treated ex vivo with amyloid beta-peptide (1-42) showed a significant decrease of oxidative stress parameters: reactive oxygen species levels, protein oxidation (protein carbonyl and 3-nitrotyrosine levels) and lipid peroxidation (4-hydroxy-2-nonenal levels). Our results are consistent with the hypothesis that modulation of free radicals generated by amyloid beta-peptide might represent an efficient therapeutic strategy for treatment of Alzheimer's disease and other oxidative-stress related disorders. Based on the above data, we suggest that tricyclodecan-9-yl-xanthogenate is a potent antioxidant and could be of importance for the treatment of Alzheimer's disease and other oxidative stress-related disorders.     

Characterization of neuronal dystrophy induced by fibrillar amyloid beta: implications for Alzheimer's disease

Amyloid deposition, neuronal dystrophy and synaptic loss are characteristic pathological features of Alzheimer's disease (AD). We have used cortical neuronal cultures to assess the dystrophic effect of fibrillar amyloid beta (Abeta) and its relationship with neurotoxicity and synaptic loss. Treatment with fibrillar Abeta led to the development of neuritic dystrophy in the majority of the neurons present in the culture. Morphometric analysis and viability assays showed that neuronal dystrophy appeared significantly earlier and at lower Abeta concentrations than neurotoxicity, suggesting that both effects are generated independently by different cellular mechanisms. The development of dystrophic features required Abeta fibril formation and did not depend on the presence of the RHDS adhesive domain in the sequence of Abeta. Finally, a dramatic reduction in the density of synaptophysin immunoreactivity was closely associated with dystrophic changes in viable neurons. These results suggest that aberrant plastic changes and loss of synaptic integrity induced by fibrillar Abeta may play a significant role in the development of AD pathology.     

Cerebrospinal fluid levels of amyloid beta-peptide1-42, but not tau have positive correlation with brain glucose metabolism in humans.

To address the question of whether assay for cerebrospinal fluid (CSF) levels of amyloid beta-peptide 1-42 (A(beta)1-42) and tau allow us to monitor the neurodegenerative processes that lead to a progressive and massive death of neurons in Alzheimer's disease (AD) and non-AD patients, cerebral glucose metabolism using 2-[18F] fluoro-2-deoxy-glucose was quantified by positron emission tomography in fifteen AD patients and nine non-AD patients with defined levels of CSF-A(beta)1-42 and CSF-tau. The CSF-A(beta)1-42 levels, but not the CSF-tau levels, in both AD and non-AD patients consistently and significantly correlated with global and, in particular, temporal lobe glucose metabolism. Results from our study suggest that the CSF-A(beta)1-42 levels may reflect residual brain function and help monitoring progression of dementing disorders.     

Plasma levels of beta-amyloid (1-42) in Alzheimer's disease and mild cognitive impairment

We compared plasma levels of beta-amyloid 1-42 (pg/ml) found for 146 sporadic Alzheimer (AD) patients, 89 subjects with mild cognitive impairment (MCI) and 89 age-matched controls (CT). AD patients had significantly lower levels (38, 54, 52; p<0.01), unrelated to severity of the disease as assessed by MMSE score, age, sex or APOE4 status. Twenty cases investigated at two time points 18 months apart did not demonstrate further decreases. Thus, the reduction in beta-amyloid 1-42 may be a marker for AD status, specifically, a transition from normal status or MCI to AD, rather than a marker for neurodegenerative processes occurring in the disease.     

Enhancement of amyloid beta 42 secretion by 28 different presenilin 1 mutations of familial Alzheimer's disease.

Families bearing mutations in the presenilin 1 (PS1) gene develop early onset familial Alzheimer's disease (FAD). Further, some PS1 mutants enhance secretion of the longer form of amyloid beta protein (Abeta42). We constructed cDNAs encoding human PS1 harboring 28 FAD-linked mutations, and examined the effects of the expressed PS1 mutants on Abeta42 secretion in beta amyloid precursor producing COS-1 cells. All the mutants significantly enhanced the ratio of Abeta42 to total Abeta compared with wild-type PS1. However, the increase in Abeta42 ratio in cells with each PS1 mutation did not correlate with the reported age of onset of FAD caused by that mutation. These results suggest that increased Abeta42 secretion is important for the development of Alzheimer's disease (AD), but may not be the only factor contributing to the onset of AD.     

Abeta as a bioflocculant: implications for the amyloid hypothesis of Alzheimer's disease

Research into Alzheimer's disease (AD) has been guided by the view that deposits of fibrillar amyloid-beta peptide (Abeta) are neurotoxic and are largely responsible for the neurodegeneration that accompanies the disease. This 'amyloid hypothesis' has claimed support from a wide range of molecular, genetic and animal studies. We critically review these observations and highlight inconsistencies between the predictions of the amyloid hypothesis and the published data. We show that the data provide equal support for a 'bioflocculant hypothesis', which posits that Abeta is normally produced to bind neurotoxic solutes (such as metal ions), while the precipitation of Abeta into plaques may be an efficient means of presenting these toxins to phagocytes. We conclude that if the deposition of Abeta represents a physiological response to injury then therapeutic treatments aimed at reducing the availability of Abeta may hasten the disease process and associated cognitive decline in AD.     

Alterations in brain antioxidant enzymes and redox proteomic identification of oxidized brain proteins induced by the anti-cancer drug adriamycin: implications for oxidative stress-mediated chemobrain

Adriamycin (ADR) is a chemotherapeutic for the treatment of solid tumors. This quinone-containing anthracycline is well known to produce large amounts of reactive oxygen species (ROS) in vivo. A common complaint of patients undergoing long-term treatment with ADR is somnolence, often referred to as “chemobrain.” While ADR itself does not cross the blood brain barrier (BBB), we recently showed that ADR administration causes a peripheral increase in tumor necrosis factor α (TNF-α), which migrates across the BBB and leads to inflammation and oxidative stress in brain, most likely contributing to the observed decline in cognition. In the current study, we measured levels of the antioxidant glutathione (GSH) in brains of mice injected intraparitoneally (i.p.) with ADR, as well as the levels and activities of several enzymes involved in brain GSH metabolism. We observed significantly decreased GSH levels, as well as altered GSH/GSSG ratio in brains of ADR treated mice relative to saline-treated controls. Also observed in brains of ADR treated mice were increased levels of glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR). We also observed increased activity of GPx, but a significant reduction in GST and GR activity in mice brain, 72 h post i.p. injection of ADR (20 mg/kg body weight). Furthermore, we used redox proteomics to identify specific proteins that are oxidized and/or have differential levels in mice brains as a result of a single i.p. injection of ADR. Visinin like protein 1 (VLP1), peptidyl prolyl isomerase 1 (Pin1), and syntaxin 1 (SYNT1) showed differential levels in ADR treated mice relative to saline-treated controls. Triose phosphate isomerase (TPI), enolase, and peroxiredoxin 1 (PRX-1) showed significantly increased specific carbonylation in ADR treated mice brain. These results further support the notion ADR induces oxidative stress in brain despite not crossing the BBB, and that antioxidant intervention may prevent ADR-induced cognitive dysfunction.     

Inflammation of the brain in Alzheimer's disease: implications for therapy

We briefly describe the similarities and differences between a systemic and a local immune reaction and review the evidence that the latter occurs in Alzheimer's disease (AD) brains. The evidence comes mainly from studies on the complement system, microglia, and cytokines, all of which are important actors in the inflammatory process. The evidence is now overwhelming that the complement proteins and many of the mediators of inflammation are produced locally by brain cells. We will mention briefly the many epidemiological studies indicating that the use of anti-inflammatory drugs reduces the incidence and slows the progress of AD. Mention will also be made of some recent work on animal models of possible relevance to AD and inflammation.     

β-淀粉样蛋白(1-42)对PC12细胞的氧化损伤

目的：探讨Aβ（1-42）对PC12细胞的氧化损伤作用。 方法: 利用不同浓度的Aβ(1-42)处理PC12细胞，采用MTT法、抗氧化酶活性测定以及RT-PCR分析观察Aβ(1-42)对PC12细胞的影响，分析Aβ(1-42)与SeGPx、MnSOD基因表达的关系。 结果: 随着Aβ(1-42)浓度的增加，SeGPx的表达不断下降；0.1 μmol/L Aβ(1-42)能诱导MnSOD的活性增加，Aβ(1-42) 对PC12的损伤作用能被acetovanilon（一种抑制内源性自由基产生的化合物）所抑制。 结论: Aβ(1-42)能介导PC12细胞内源性自由基的产生增多。     

Nonfibrillar diffuse amyloid deposition due to a gamma(42)-secretase site mutation points to an essential role for N-truncated A beta(42) in Alzheimer's disease

Amyloidogenic processing of the amyloid precursor protein (APP) with deposition in brain of the 42 amino acid long amyloid beta-peptide (A beta(42)) is considered central to Alzheimer's disease (AD) pathology. However, it is generally believed that nonfibrillar pre-amyloid A beta(42) deposits have to mature in the presence of A beta(40) into fibrillar amyloid plaques to cause neurodegeneration. Here, we describe an aggressive form of AD caused by a novel missense mutation in APP (T714I) directly involving gamma-secretase cleavages of APP. The mutation had the most drastic effect on A beta(42)/A beta(40) ratio in vitro of approximately 11-fold, simultaneously increasing A beta(42) and decreasing A beta(40) secretion, as measured by matrix-assisted laser disorption ionization time-of-flight mass spectrometry. This coincided in brain with deposition of abundant and predominant nonfibrillar pre-amyloid plaques composed primarily of N-truncated A beta(42) in complete absence of A beta(40). These data indicate that N-truncated A beta(42) as diffuse nonfibrillar plaques has an essential but undermined role in AD pathology. Importantly, inhibiting secretion of full-length A beta(42 )by therapeutic targeting of APP processing should not result in secretion of an equally toxic N-truncated A beta(42).     

Expression of somatostatin receptor subtypes (SSTR1-5) in Alzheimer's disease brain: an immunohistochemical analysis

Somatostatin, widely distributed in human cortical brain regions, acts through specific high affinity somatostatin receptors (SSTR1-5) to exert profound effects on motor, sensory, behavioral, cognitive and autonomic functions. Somatostatin levels are consistently decreased in the cortex of Alzheimer's disease (AD) brain and in cerebrospinal fluid, and have become reproducible markers of this disease. In the present study, the distributional pattern of SSTR1-5 antigens in the frontal cortex of AD and age-matched control brains was studied using antipeptide polyclonal rabbit antibodies directed against the five human somatostatin receptor subtypes. All five SSTRs were differentially expressed as membrane and cytoplasmic proteins in cortical neurons with significant variations in control vs. AD brain. In AD cortical brain region, somatostatin and neuropeptide-Y-positive neurons decreased (>70%), and glial fibrillary acidic protein-positive astrocytes significantly increased (>130%) in comparison to control brain. SSTR2 and 4 were the predominant subtypes followed by SSTR1, 3 and 5. AD cortex showed a marked reduction in neuronal expression of SSTR4 and 5 and a modest decrease in SSTR2-like immunoreactivity without any changes in SSTR1 immunoreactive neurons. In contrast, SSTR3 was the only receptor subtype that increased in AD cortex. In AD cortex, SSTR1-, 3- and 4-like immunoreactivities were strongly expressed in glial cells but not SSTR2 and 5. These findings suggest the differential loss of immunoreactivity of SSTR2, 4 and 5 but not SSTR1, and increased SSTR3 in frontal cortex of AD brain as well as subtype-selective glial expression in AD brain. In summary, subtype-selective changes in the expression of SSTRs at protein levels in AD cortical regions suggest that somatostatin and SSTR-containing neurons are pathologically involved in AD and could possibly be used as markers of this disease.     

Intracellular accumulation of beta-amyloid(1-42) in neurons is facilitated by the alpha 7 nicotinic acetylcholine receptor in Alzheimer's disease

Amyloid beta(1-42), a major component of amyloid plaques, binds with exceptionally high affinity to the alpha 7 nicotinic acetylcholine receptor and accumulates intracellularly in neurons of Alzheimer's disease brains. In this study, we investigated the possibility that this binding plays a key role in facilitating intraneuronal accumulation of amyloid beta(1-42). Consecutive section immunohistochemistry and digital imaging were used to reveal the spatial relationship between amyloid beta(1-42) and the alpha 7 receptor in affected neurons of Alzheimer's disease brains. Results showed that neurons containing substantial intracellular accumulations of amyloid beta(1-42) invariably express relatively high levels of the alpha 7 receptor. Furthermore, this receptor is highly co-localized with amyloid beta(1-42) within neurons of Alzheimer's disease brains. To experimentally test the possibility that the binding interaction between exogenous amyloid beta(1-42) and the alpha 7 receptor facilitates internalization and intracellular accumulation of amyloid beta(1-42) in Alzheimer's disease brains, we studied the fate of exogenous amyloid beta(1-42) and its interaction with the alpha 7 receptor in vitro using cultured, transfected neuroblastoma cells that express elevated levels of this receptor. Transfected cells exhibited rapid binding, internalization and accumulation of exogenous amyloid beta(1-42), but not amyloid beta(1-40). Furthermore, the rate and extent of amyloid beta(1-42) internalization was related directly to the alpha 7 receptor protein level, since (1) the rate of amyloid beta(1-42) accumulation was much lower in untransfected cells that express much lower levels of this receptor and (2) internalization was effectively blocked by alpha-bungarotoxin, an alpha 7 receptor antagonist. As in neurons of Alzheimer's disease brains, the alpha 7 receptor in transfected cells was precisely co-localized with amyloid beta(1-42) in prominent intracellular aggregates. Internalization of amyloid beta(1-42) in transfected cells was blocked by phenylarsine oxide, an inhibitor of endocytosis.We suggest that the intraneuronal accumulation of amyloid beta(1-42) in Alzheimer's disease brains occurs predominantly in neurons that express the alpha 7 receptor. In addition, internalization of amyloid beta(1-42) may be facilitated by the high-affinity binding of amyloid beta(1-42) to the alpha 7 receptor on neuronal cell surfaces, followed by endocytosis of the resulting complex. This provides a plausible explanation for the selective vulnerability of neurons expressing the alpha 7 receptor in Alzheimer's disease brains and for the fact that amyloid beta(1-42) is the dominant amyloid beta peptide species in intracellular accumulations and amyloid plaques.     

Entry of the synthetic ACTH(4-10) analogue into the rat brain following intravenous injection.

In view of known central effects of N-terminal ACTH fragments, a possibility of their entry into the brain was studied. Rat blood and brain extracts after intravenous injection of the tritiated synthetic ACTH(4-10) analogue, Met-Glu-His-Phe-Pro-Gly-Pro, were subjected to a high-performance liquid chromatographic analysis. At two time points the labelled peptide was detected in brain extracts. The brain to blood ratios of peptide content in brain and blood were found to be significantly higher than those calculated for a distribution of labelled bovine serum albumin in rat brain capillaries and blood. This strongly suggests that this peptide penetrates into the brain tissues, its quantity not exceeding 0.01% of dose injected. Peptide diffusion through the vascular epithelium of brain capillaries could account for the data obtained.