Cathepsin D displays in vitro β-secretase-like specificity

The formation of Aβ and Aβ-containing fragments is likely a key event in the process of neural degeneration in Alzheimer's disease. The N-terminal residue (Asp-1) of Aβ and its C-terminally extended sequences is liberated from the β-amyloid precursor protein (βAPP) by β-secretase(s). This activity appears highly increased by the presence (N-terminally to Asp-1) of a double-mutation (KM→NL) found in several Swedish families affected by early onset Alzheimer's disease. By means of synthetic peptides encompassing the ‘normal' (N peptide) and mutated (ΔNL peptide) sequences targeted by β-secretase(s), we have detected a human brain protease displaying preferred efficiency for the ΔNL peptide than for the non-mutated analog. This activity is sensitive to pepstatin, maximally active at acidic pH and hydrolyses the two peptides at the expected M/D or L/D cleavage sites. Such acidic activity is also detected in rat brain, PC12 cells and primary cultured astrocytes. The pepstatin sensitivity and pH maximum of the brain activity that appeared reminiscent of those displayed by the acidic protease cathepsin D led us to examine this enzyme as a putative β-secretase-like candidate. Purified cathepsin D displays higher catalytic parameters for the ΔNL peptide than for the non-mutated peptide, cleaves these two substrates at the expected M/D or L/D sites, and is maximally active at acidic pH. However, cathepsin D does not cleave peptides bearing mutations that were previously shown to drastically lower or fully block Aβ secretion by transfected cells. Furthermore, cathepsin D hydrolyses recombinant baculoviral ΔNLβAPP₇₅₁ at a 6-fold higher rate than βAPP₇₅₁ and gives rise to a 12-kDa C-terminal product that is recognized by antibodies fully specific of the N-terminus of Aβ. Altogether, our study indicates that cathepsin D displays several in vitro β-secretase-like properties that suggests that this protease could fulfill such a role, at least in the Swedish genetic form of Alzheimer's disease. © 1997 Elsevier Science B.V. All rigths reserved.     

Coexistence of S100β and putative transmitter agents in vagal and glossopharyngeal sensory neurons of the rat

The coexistence of S100β with calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), nicotinamide adenosine dinucleotide phosphate-diaphorase (NADPH-d), and tyrosine hydroxylase (TH) was examined in the glossopharyngeal and vagal sensory ganglia. S100β immunoreactive (-ir) neurons in the jugular and petrosal ganglia frequently colocalized CGRP- or SP-ir, whereas S100β-ir neurons in the nodose ganglion infrequently contained CGRP- or SP-ir. No S100β-ir neurons in the jugular and petrosal ganglia showed SOM-ir while the small number of SOM-ir neurons in the nodose ganglion colocalized S100β-ir. Many neurons in the nodose ganglion colocalized S100β-ir and NADPH-d activity, whereas S100β-ir neurons in the jugular and nodose ganglia infrequently contained NADPH-d activity. S100β- and TH-ir were frequently colocalized in nodose ganglion but not in petrosal or jugular ganglion neurons. These findings suggest relationships between S100β and specific putative transmitters in functions of subpopulations of vagal and glossopharyngeal sensory neurons.     

β-Amyloid(Phe(SO3H))25–35 in rat nucleus basalis induces behavioral dysfunctions, impairs learning and memory and disrupts cortical cholinergic innervation

Long-term behavioral effects, changes in learning and memory functions and aberrations of cholinergic fibers projecting to the parietal cortex were investigated after bilateral injections of β-amyloid(Phe(SO₃H)²⁴)25–35 peptide in rat nucleus basalis magnocellularis (nbm). The β-amyloid peptide used in these experiments contained the original β-amyloid 25–35 sequence which was coupled to a phenylalanine-sulphonate group at position 24. This additional residue serves as a protective cap on the molecule without influencing its neurotoxic properties and results in water-solubility, stability and low rates of peptide metabolism. In this paper, home cage, locomotor and open-field activities, passive shock-avoidance and ‘Morris' water maze learning abilities were assessed throughout a 35-day survival period. Subsequently, acetylcholinesterase (AChE) histochemistry was used to visualize alterations of parietal cortical cholinergic innervation. In response to the neurotoxic action of β-amyloid(Phe(SO₃H)²⁴)25–35, a progressive hyperactivity developed in the rats in their home cages which were maintained throughout the 5-week post-injection period. This was accompanied by a significant hypoactivity in the novel environment of a locomotor arena. β-Amyloid(Phe(SO₃H)²⁴)25–35-treated animals showed greatly impaired cortical memory functions in the step-through passive shock-avoidance paradigm, while spatial learning processes remained unaffected. Moreover, β-amyloid(Phe(SO₃H)²⁴)25–35 injections in the nucleus basalis suppressed explorative behavior in rats and inhibited conditioned stress responses 28 days after surgery. Reductions of cortical cholinergic (AChE-positive) projections provided anatomical substrate for the behavioral changes. This indicated extensive, long-lasting neurodegenerative processes as a result of β-amyloid(Phe(SO₃H)²⁴)25–35 infusion.     

Factors produced by activated macrophages reduce accumulation of Alzheimer''s β-amyloid protein in vascular smooth muscle cells

Smooth muscle cells (SMCs) isolated from amyloid-angiopathy affected brain vessels accumulate intracellularly amyloid-β peptide (Aβ). Now we demonstrate that accumulation of Aβ in SMCs can be reduced by factors secreted by macrophages – IL-1, IL-6, TNF-, TGF-β1 or PGE₂ – probably by stimulating the non-amyloidogenic processing of Aβ precursor protein (PP). It is suggested that brain macrophages may regulate AβPP/Aβ metabolism under physiological conditions and prevent β-amyloidosis. The disturbance of this regulatory function of brain macrophages may result in excessive production and accumulation of Aβ.     

The neurochemical and behavioral effects of β-amyloid peptide(25–35)

Beta-amyloid protein (A β) fragments have been shown to be neurotoxic and/or enhance neuronal vulnerability when injected into the hippocampus. We investigated alterations in monoamine contents, including norepinephrine (NE), 5-HT and dopamine (DA) in the rat locus coeruleus (LC) one week following the injection of beta-amyloid peptide fragment 25–35 (β(25–35)) into the left dorsal hippocampal areas CA1–3. A single treatment of β(25–35) had no effect on any monoamine levels. Rats that received two treatments (separated by 7 days) revealed significant elevations in NE, 5-HT, and 5-HIAA as compared with the control group injected with ddH₂O. However, these changes were observed in the LC on the contralateral side, whereas the injected side exhibited no significant change. These effects may result from an enhanced synthesis of NE by the contralateral LC neurons to compensate for the loss of tyrosine hydroxylase and accompanying recurrent inhibition in a small number of their population. In a second experiment, the influence of β(25–35) on spatial learning was evaluated using a Morris water maze task. Rats received bilateral injections of β(25–35) into hippocampal areas CA1–3. The results indicate that β(25–35)-treated rats exhibited significantly longer latencies and swim distances to locate the submerged platform than did members of the control group.     

GABAA-receptor subunit composition in the circadian timing system

In the present study, the distribution of GABA_A-receptor ₁-, ₂-, ₃-, ₅-, β_{2, 3}- and γ₂-subunits were localized immunohistochemically with subunit specific antibodies in the rat circadian timing system (CTS). The areas examined include the principal circadian pacemaker, the suprachiasmatic nucleus (SCN), and areas that receive important SCN input including the intergeniculate leaflet (IGL), subparaventricular zone (SPVZ), paraventricular hypothalamic nucleus (PVH), the retrochiasmatic area (RCh) and the paraventricular nucleus of the thalamus (PVT). The SCN has an unusual pattern with immunoreactivity for the ₂-, ₃-, ₅- and γ₂-subunits but not for the commonly expressed ₁- and β_{2, 3}-subunits. In all of the areas receiving SCN efferent input (SPVZ, PVH, RCh, PVT and IGL), staining is present either for all six subunits or for the three common subunits, ₁-, β_{2, 3}- and γ₂. There is some evidence for a differential distribution of subunits at the cellular level. The ₂- and β_{2, 3}-subunits are predominantly expressed in neuropil the ₃-, ₅- and γ₂-subunits are predominantly expressed over perikarya and the ₁-subunit is expressed over both neuropil and perikarya in the areas in which subunit immunoreactivity is found. The demonstration of this regional and cellular expression of GABA_A-receptor subunits should contribute to our understanding of GABAergic neurotransmission in the CTS.     

Bax expression in mammalian neurons undergoing apoptosis, and in Alzheimer's disease hippocampus

Recent studies indicate that the proto-oncogene Bax, and other related proteins (eg Bcl-2) may play a major role in determining whether cells will undergo apoptosis under conditions which promote cell death. Increased expression of Bax has been found to promote apoptosis, while over-expression of Bcl-2 can inhibit apoptosis. To investigate the role of Bax in nerve cell death in the rat brain we examined the level of Bax expression in cells undergoing apoptosis, using a hypoxic-ischemic stroke model. We found that Bax was expressed at high levels in the nuclei of neurons in the hippocampus, cortex, cerebellum, and striatum on the control side, and that Bax levels increased in hippocampal neurons undergoing apoptosis on the stroke side, and then declined (correlating with cell loss). In the Alzheimer's disease hippocampi we found a concentrated localisation of Bax in senile plaques, which correlated with the localisation of β-amyloid protein in adjacent sections from the same brains. β-Amyloid positive plaques are thought to contribute to the Alzheimer's disease process, possibly via an apoptotic mechanism, and this may occur via an increase in Bax in these areas. Bax was also strongly stained in τ-positive tangles in Alzheimer's disease hippocampi, suggesting Bax may play a role in tangle formation. In addition, we observed a loss of Bax expression in the dentate granule cells of Alzheimer's disease hippocampi compared with moderate Bax expression in control hippocampi, and this loss may be related to the survival of these neurons in Alzheimer's disease. Finally, we observed substantially different staining patterns of Bax using three different commercially available antisera to Bax, indicating the need for caution when interpreting results in this area.     

Opposing effects of different steroid sulfates on GABAA receptor-mediated chloride uptake

Steroids with the 3-hydroxy-5- or 5β-reduced configurations of the A ring interact with the γ-aminobutyric acid (GABA) type A receptor chloride channel complex and potentiate the stimulation of Cl⁻ uptake by GABA agonists. Conversely, the sulfate esters of 3β-hydroxy-5-ene neurosteroids pregnenolone and dehydroepiandrosterone behave as inhibitory modulators. In the present work, steroid sulfates were tested for their ability to modulate muscimol-induced chloride ion uptake into cortical synaptoneurosomes. 3-Hydroxy-5-pregnan-20-one sulfate and several other 3-hydroxy-steroid sulfates potentiated, whereas 3β-hydroxy-steroid sulfates inhibited muscimol effect. It is concluded that GABA-agonistic or antagonistic properties of steroid sulfates depend on the or β orientation of the sulfate moiety linked to the A ring.     

Effects of amyloid precursor protein derivatives andoxidative stress on basal forebrain cholinergic systems inALZHEIMERS disease

The dysfunction and degeneration of cholinergic neuronal circuits in the brain is aprominent feature of Alzheimers disease. Increasing data suggest that age-related oxidative stresscontributes to degenerative changes in basal forebrain cholinergic systems. Experimental studieshave shown that oxidative stress, and membrane lipid peroxidation in particular, can disruptmuscarinic cholinergic signaling by impairing coupling of receptors to GTP-binding proteins.Altered proteolytic processing of the β-amyloid precursor protein (APP) may contributeto impaired cholinergic signaling and neuronal degeneration in at least two ways. First, levels ofcytotoxic forms of amyloid β-peptide (Aβ) are increased ; Aβdamages and kills neurons by inducing membrane lipid peroxidation resulting in impairment ofion-motive ATPases, and glucose and glutamate transporters, thereby rendering neuronsvulnerable to excitotoxicity. The latter actions of Aβ may be mediated by4-hydroxynonenal, an aldehydic product of membrane lipid peroxidation that covalently modifiesand inactivates the various transporter proteins. Subtoxic levels of Aβ can also suppresscholine acetyltransferase levels, and may thereby promote dysfunction of intact cholinergiccircuits. A second way in which altered APP processing may endanger cholinergic neurons is byreducing levels of a secreted form of APP which has been shown to modulate neuronalexcitability, and to protect neurons against excitotoxic, metabolic and oxidative insults. Mutationsin presenilin genes, which are causally linked to many cases of early-onset inherited Alzheimersdisease, may increase vulnerability of cholinergic neurons to apoptosis. The underlying mechanismappears to involve perturbed calcium regulation in the endoplasmic reticulum, which promotesloss of cellular calcium homeostasis, mitochondrial dysfunction and oxyradical production.Knowledge of the cellular and molecular underpinnings of dysfunction and degeneration ofcholinergic circuits is leading to the development of novel preventative and therapeuticapproaches for Alzheimers disease and related disorders.     

Several GABAA receptor subunits are expressed in LHRH neurons of juvenile female rats

Gamma aminobutyric acid (GABA), the dominant inhibitory neurotransmitter in brain, is involved in the developmental regulation of LHRH secretion. Morphological studies in rodents have demonstrated that LHRH neurons are innervated by GABA-containing processes, suggesting that LHRH secretion is under direct transsynaptic GABAergic control. While GABA acts through two different receptors, GABA_A and GABA_B, to exert its effects, it appears that GABA_A receptors are able to mediate both inhibitory and stimulatory effects of GABA on LHRH neurons. GABA_A receptors are heterooligomeric ligand-gated anion channels that exhibit a diverse array of functional and pharmacological properties. This diversity is determined by the structural heterogeneity of the receptors, which are assembled from the combination of different classes of subunits with multiple isoforms. Although several studies have described the effect of GABA_A receptor stimulation on LHRH and/or gonadotropin release in prepubertal animals, nothing is known about the receptor subunits that may be expressed in LHRH neurons at this phase in development. Double immunohistofluorescence followed by confocal laser microscopy revealed that subsets of prepubertal LHRH neurons are endowed with ₁, ₂, β_2/3, and γ₂ GABA_A receptor subunits. Combined immunohistochemistry for LHRH neurons and in situ hybridization for GABA_A subunit mRNAs confirmed that the genes encoding the ₁, ₂, β₃ and γ₂ subunits, but not the γ₁ subunit, are expressed in LHRH neurons. Notwithstanding the relative insensitivity of these methods, both the immunohistochemical and hybridization histochemical approaches employed indicate that only a fraction of LHRH neurons are endowed with GABA_A receptors. This arrangement suggests that those LHRH neurons bearing the appropriate GABA_A receptors are responsible for either the entire secretory response to direct GABAergic inputs or for its initiation.     

Nasal administration of myelin basic protein prevents relapsing experimental autoimmune encephalomyelitis in DA rats by activating regulatory cells expressing IL-4 and TGF-β mRNA

This study explores nasal administration of myelin basic protein (MBP) as a potential means of inducing tolerance to relapsing experimental autoimmune encephalomyelitis (PR-EAE), an experimental multiple sclerosis (MS) model that was induced in DA rats by immunization with rat spinal cord homogenate and incomplete Freund's adjuvant. DA rats received a total dosage of 0, 6, 60, 600 μg/rat of bovine MBP on ten consecutive days prior to immunization. EAE with typical course was observed in control rats receiving only PBS nasally, and in rats receiving 6 μg/rat of MBP. Rats receiving 60 μg/rat of MBP developed acute EAE but no relapse during 60 days of observation post immunization (p.i.). Only one of eight rats receiving 600 μg/rat of MBP developed slight, transient EAE. This protection was confirmed at the histology level and was associated with decreased levels of MBP-reactive IFN-γ secreting Th1-like spleen cells on day 13 and 60 p.i. Rats receiving 60 and 600 μg/rat of MBP showed decreased serum anti-MBP IgG2b antibody levels on day 60 p.i., and rats receiving 600 μg/rat of MBP had marginally increased anti-MBP IgG1 antibody levels in serum compared to control EAE rats. Cytokine mRNA profiles in central nervous system (CNS) and spleen mononuclear cells were evaluated. Dose-dependent reduction of TNF- mRNA expression were observed both in CNS and in splenocytes. Increased IL-4 and TGF-β mRNA expression were observed in CNS of low (6 μg/rat) and median (60 μg/rat) dose of MBP tolerized rats and in splenocytes of rats tolerized with 600 μg/rat of MBP. We conclude that nasal administration of MBP in DA rat prevents EAE induced by immunization with whole rat spinal cord homogenate that, besides MBP, contains multiple antigenic myelin proteins. A mechanism involving MBP-reactive regulatory cells expressing IL-4 and TGF-β mRNA acts as part in the induction of this tolerance.     

Synchronization analysis using joint peri-stimulus time histograms for human motor units

The concurrence of action potentials from two motor units (MUs) happens more frequently in human than what one would expect by chance. It can be visualized as the central peak of the Cross-Correlogram of spike trains from two MUs. It is widely believed that a common pre-synaptic input to motoneurons from the cortical tract generates the concurrence. A number of synchronization indices have been proposed in order to evaluate the quantity of the common input, however, these indices are dependent on the firing rates of the MUs. To resolve this issue, we introduce a new synchronization index β using a joint peri-stimulus time histograms (JPSTH). In this paper, we discuss the dependence of synchronization indices on firing rates using a spike model, and the application of JPSTH and the index β to experimental data for the quantitative analysis of MU synchronization. We confirm that the novel index β represents the synchronization state independently from firing rates, and that it also accurately traces event-related temporal modulation of synchronization. In conclusion, we consider the index β to be a valuable index for the analysis of MU synchronization.     

Chronic neuroinflammation in rats reproduces components of the neurobiology of Alzheimer''s disease

Inflammatory processes may play a critical role in the pathogenesis of the degenerative changes and cognitive impairments associated with Alzheimer's disease (AD). In the present study, lipopolysaccharide (LPS) from the cell wall of gram-negative bacteria was used to produce chronic, global inflammation within the brain of young rats. Chronic infusion of LPS (0.25 μg/h) into the 4th ventricle for four weeks produced (1) an increase in the number of glial fibrillary acidic protein-positive activated astrocytes and OX-6-positive reactive microglia distributed throughout the brain, with the greatest increase occurring within the temporal lobe, particularly the hippocampus, (2) an induction in interleukin-1β, tumor necrosis factor- and β-amyloid precursor protein mRNA levels within the basal forebrain region and hippocampus, (3) the degeneration of hippocampal CA3 pyramidal neurons, and (4) a significant impairment in spatial memory as determined by decreased spontaneous alternation behavior on a T-maze.     

Presynaptic inhibition by noradrenaline of the EPSC evoked in neonatal rat sympathetic preganglionic neurons

Visually identified and electrophysiologically characterized sympathetic preganglionic neurons (SPNs) were recorded using the whole-cell voltage clamp technique in slices of neonatal rat spinal cord. Monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the nucleus intercalatus in the presence of strychnine (5 μM) and bicuculline (10 μM). These EPSCs were abolished by the antagonist of AMPA-type glutamate receptors, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 10 μM). Bath applied noradrenaline (NA; 0.5–50 μM) dose-dependently and reversibly decreased by up to around 60% the amplitude of the EPSC, without affecting the holding current. The EPSC depression by NA was not accompanied by a change in EPSC reversal potential (around +5 mV), nor were inward currents generated by pressure application of glutamate affected by NA application. A comparable degree of EPSC depression was also seen with the ₂-adrenoceptor agonist clonidine (5 μM), and the _2A-agonist oxymetazoline (5 μM), while the ₁-agonist phenylephrine (100 μM) caused only a 22% depression. The EPSC depression caused by NA (10 μM) was completely antagonized by either the -antagonist phentolamine (10 μM) or the ₂-antagonist idazoxan (2 μM). Conversely, the β-adrenoceptor antagonist popranolol (5 μM), and the ₁-, _2B- and _2C-antagonist prazosin (2 μM) were without effect. These results indicate that activation of presynaptic _2A-adrenoceptors on inputs to SPNs decreases glutamate release.     

δ1-OPIOID receptor-mediated controlofacetylcholine (ACh) release in human neocortex slices

In slices of human neocortex, prelabelled with [³H]-choline, the release of [³H]-acetylcholine reflects the evoked release of endogenous acetylcholine whichwaselicited by the same electrical stimulation paradigm. [³H]-Acetylcholine releasewasdepressed by the δ-opioid receptor agonist -Pen₂- -Pen₅-enkephalin. When the nerve endings were depolarized byelevatingextracellular potassium the evoked [³H]-acetylcholine release was similarlydepressed by -Pen₂- -Pen₅-enkephalin in theabsence, but notin the presence, of tetrodotoxin which blocks action potential propagation.Therefore, the δ-opioid receptor inhibiting [³H]-acetylcholine release should notbe located tocholinergic nerve terminals, but rather to interneurons. The somatostatin₂receptorpartial agonist octreotide per se did not influence action potential-evoked [³H]-acetylcholine release, but prevented the inhibition of release of [³H]-acetylcholine by -Pen₂- -Pen₅-enkephalin.Similarly, the δ₁-opioid receptor antagonist 7-benzylidenenaltrexon perse did notinfluence [³H]-acetylcholine release, but prevented of the inhibition ofrelease by -Pen₂- -Pen₅-enkephalin.

From the presentfindings we conclude : (1) The evoked release of [³H]-acetylcholine from humanneocortex slices reflects the release of endogenous acetylcholine.(2) It is inhibited in an indirectmanner by opioid receptors of the δ₁-subtype,which (3) are not localized oncholinergic axon terminals but on soma and dendrites ofsomatostatin-containing interneurons,where they inhibit somatostatin release. (4) Theseinterneurons innervate cholinergic nerveendings in the human neocortex and appear to facilitateacetylcholine release via somatostatin₂ receptors.     

Apolipoprotein E genotype analysis in Chinese Han ethnic children with Wilson's disease, with a concentration on those homozygous for R778L

Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by a large number of different mutations in the ATP7B gene. R778L mutation is mostly observed in Chinese, Japanese and Korean patients, whereas the H1069Q point mutation in the ATP7B gene is the most frequent mutation in European patients with WD. In our previous study we did not find a significant correlation between genotype and phenotype (age of onset and clinical presentation) in patients homozygous (37 patients) or heterozygous (52 patients) for R778L. It was reported that European patients homozygous for H1069Q who were also homozygous for the ApoE genotype ε3/3 developed clinical symptoms 5–11 years later than did patients with genotypes other than ApoE ε3/3. In the present study (i) we firstly observed that ApoE ε3/3 did not delay the onset of WD; (ii) no association between ApoE genotype and WD clinical presentation in Chinese Han children, including those patients homozygous for R778L. Thus we conclude that the onset of WD in Chinese children is not related to ApoE ε3/3, although the high frequency of ApoE ε3/3 in Chinese Han children with WD was not significantly different from that in controls.     

Reproducible and efficient murine CNS gene delivery using a microprocessor-controlled injector

To develop a reproducible gene transfer method for the murine CNS we evaluated delivery of various gene vehicles using mechanical or manual stereotaxic intracranial inoculation. A microprocessor controlled microsyringe pump (The World Precision Instruments/UltraMicroPump) programmable for volume, rate and syringe size and designed to dispense nanoliter and picoliter volumes was compared to a standard manual deliver method. Gene transfer efficiency of two viral vectors, two synthetic cationic lipid molecules, and naked DNA were evaluated in mice injected unilaterally in two brain regions. Animals received 1 μl over 10 min. of either HSVlac (1×10⁵ b.f.u), AdLac (1×10⁵ p.f.u), Tfx-10 or Tfx-20 (2.6 μg DNA in 2.0 μl Tfx; 1:1 charge ratio of DNA to liposome), or naked DNA (HSVlac plasmid, 10 μg/μl). After 4 days, animals from each group were perfused and tissue prepared for X-gal histochemical detection of β-galactosidase expression. Blue cells were observed in the HSV, Adenovirus, and Tfx-20 groups only at the injection site in animals injected using the UMP. Animals injected manually exhibited fewer blue cells and positive cells were not restricted to the injection site. To quantify expression, tissue punches harvested from the injection sites as well as other brain regions were analyzed using a chemiluminescent reporter assay to detect β-galactosidase (Galacto-Light). These data indicated increased activity in all animals injected with a lacZ containing vector via the UMP as compared to manual delivery: A 41% increase in the expression levels of β-gal in HSVlac infected animals (p=0.0029); a 29% increase in Adlac infected animals (p=0.01); a 56% increase in Tfx-10 transduced animals (p=0.04); a 24% increase in Tfx-20 transduced animals (p=0.01); and a 69% increase in naked DNA gene transfer (p=0.05). Total β-galactosidase activity was greatest in HSVlac infected mice followed by Adlac>Tfx-20™>Tfx-10™=naked DNA.     

Long-term postnatal effect of prenatal irradiation on the astrocyte proliferative response to brain injury

Pregnant Wistar rats were exposed to a single 1.0 Gy dose of γ-irradiation on gestational day 13, 15, 17 or 19. Thirty-day-old male offspring received a mechanical lesion in the left cerebral hemisphere. One, 2 or 4 days after the injury the rats were injected with [³H]thymidine and sacrificed 4 h after the injection. Thereafter, brain sections were immunostained for GFAP or S100β protein, subjected to autoradiography and examined microscopically to record immunopositive astrocytes labelled with [³H]thymidine. Statistically significant elevation of the reactive astrocyte proliferation was revealed on the 2nd day following injury in brains irradiated on gestational day 15. The results represent the first in vivo evidence that a low-dose prenatal γ-irradiation can induce a long-term increase in the ability of astroglia to proliferate in response to injury.     

多胺类似物DENSPM对人胶质母细胞瘤SNB19细胞生长的影响

目的探讨多胺类似物DENSPM对人胶质母细胞瘤sNB19细胞生长的影响。方法采用MTS法、流式细胞术、高效液相色谱法和逆转录-聚合酶链反应,分别检测经DENSPM处理后的SNB19细胞存活率、细胞周期变化和细胞内过氧化氢水平,以及细胞内多胺水平和鸟氨酸脱羧酶、亚精胺/精胺-N1-乙酰基转移酶、多胺氧化酶mRNA表达变化。结果经DENSPM处理后,SNB19细胞存活率随着药物浓度的增加逐渐降低(F=81.915,P=0.001),并于体外培养72h在细胞增殖周期中出现典型的亚凋亡峰;细胞内腐胺、亚精胺和精胺水平显著下降,而乙酰亚精胺和乙酰精胺水平略有上升(均P<0.01);鸟氨酸脱羧酶表达水平下降(P<0.01),而亚精胺/精胺-N1-乙酰基转移酶和多胺氧化酶水平上升(P<0.05或P<0.01);但细胞内过氧化氢水平与DENSPM处理前差异无统计学意义(P>0.05)。结论 DENSPM可抑制人胶质母细胞瘤SNB19细胞的生长并诱导其发生凋亡,而诱导细胞凋亡的可能机制是肿瘤细胞内多胺表达水平下降。DENSPM可能具有治疗胶质母细胞瘤的潜在临床应用价值。