eNOS gene deletion restores blood-brain barrier integrity and attenuates neurodegeneration in the thiamine-deficient mouse brain

Wernicke's encephalopathy is a cerebral disorder caused by thiamine (vitamin B₁) deficiency (TD). Neuropathologic consequences of TD include region-selective neuronal cell loss and blood-brain barrier (BBB) breakdown. Early increased expression of the endothelial isoform of nitric oxide synthase (eNOS) occurs selectively in vulnerable brain regions in TD. We hypothesize that region-selective eNOS induction in TD leads to altered expression of tight junction proteins and BBB breakdown. In order to address this issue, TD was induced in C57BL/6 wild-type (WT) and eNOS^−/− mice by feeding a thiamine-deficient diet and treatment with the thiamine antagonist pyrithiamine. Pair-fed control mice were fed the same diet with additional thiamine. In medial thalamus of TD-WT mice (vulnerable area), increased heme oxygenase-1 and S -nitrosocysteine immunostaining was observed in vessel walls, compared to pair-fed control-WT mice. Concomitant increases in IgG extravasation, decreases in expression of the tight junction proteins occludin, zona occludens-1 and zona occludens-2, and up-regulation of matrix metalloproteinase-9 in endothelial cells were observed in the medial thalamus of TD-WT mice. eNOS gene deletion restored these BBB alterations, suggesting that eNOS-derived nitric oxide is a major factor leading to cerebrovascular alterations in TD. However, eNOS gene deletion only partially attenuated TD-related neuronal cell loss, suggesting the presence of mechanisms additional to BBB disruption in the pathogenesis of these changes.     

A novel solid phase approach to Aia‐containing peptides

A strategy was developed to directly assemble 4‐amino‐1,2,4,5‐tetrahydro‐indolo[2,3‐c]‐azepin‐3‐ones on solid‐phase‐supported peptide sequences. Fmoc‐ and Boc‐based strategies were investigated. The Fmoc‐strategy approach strongly depends on the peptide sequence being synthesized while the Boc‐based synthesis leads to excellent results for all the selected peptide analogs. The method was applied to prepare Aia‐analogs of several bioactive peptides containing one or more Trp‐residues which were shown to be important for biological recognition. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Concentration of Disease-Associated Prion Protein with Silicon Dioxide

Reagents that can precipitate the disease-associated prion protein (PrP^Sc) are vital for the development of high sensitivity tests to detect low levels of this disease marker in biological material. Here, a range of minerals are shown to precipitate both ovine cellular prion protein (PrP^C) and ovine scrapie PrP^Sc. The precipitation of prion protein with silicon dioxide is unaffected by PrP^Sc strain or host species and the method can be used to precipitate bovine BSE. This method can reliably concentrate protease-resistant ovine PrP^Sc (PrP^res) derived from 1.69 μg of brain protein from a clinically infected animal diluted into either 50 ml of buffer or 15 ml of plasma. The introduction of a SiO₂ precipitation step into the immunological detection of PrP^res increased detection sensitivity by over 1,500-fold. Minerals such as SiO₂ are readily available, low cost reagents with generic application to the concentration of diseases-associated prion proteins.     

Pathogenetic mechanisms in hereditary dysfunctions of complex I of the respiratory chain in neurological diseases

This paper covers genetic and biochemical aspects of mitochondrial bioenergetics dysfunction in hereditary neurological disorders associated with complex I defects. Three types of hereditary complex I dysfunction are dealt with: (i) homozygous mutations in the nuclear genes NDUFS1 and NDUFS4 of complex I, associated with mitochondrial encephalopathy; (ii) a recessive hereditary epileptic neurological disorder associated with enhanced proteolytic degradation of complex I; (iii) homoplasmic mutations in the ND5 and ND6 mitochondrial genes of the complex, cohexistent with mutation in the nuclear PINK1 gene in familial Parkinsonism. The genetic and biochemical data examined highlight different mechanisms by which mitochondrial bioenergetics is altered in these hereditary defects of complex I. This knowledge, besides clarifying molecular aspects of the pathogenesis of hereditary diseases, can also provide hints for understanding the involvement of complex I in sporadic neurological disorders and aging, as well as for developing therapeutical strategies.     

Prion Protein NMR Structure from Tammar Wallaby (Macropus eugenii) Shows that the β2-α2 Loop Is Modulated by Long-Range Sequence Effects

NMR structures are presented for the recombinant construct of residues 121-230 from the tammar wallaby (Macropus eugenii) prion protein (PrP) twPrP(121-230) and for the variant mouse PrPs mPrP[Y225A,Y226A](121-231) and mPrP[V166A](121-231) at 20 °C and pH 4.5. All three proteins exhibit the same global architecture as seen in other recombinant PrP^Cs (cellular isoforms of PrP) and shown to prevail in natural bovine PrP^C. Special interest was focused on a loop that connects the β2-strand with helix α2 in the PrP^C fold, since there are indications from in vivo experiments that this local structural feature affects the susceptibility of transgenic mice to transmissible spongiform encephalopathies. This β2-α2 loop and helix α3 form a solvent-accessible contiguous epitope, which has been proposed to be the recognition area for a hypothetical chaperone, the “protein X”. This hypothetical chaperone would affect the conversion of PrP^C into the disease-related scrapie form (PrP^Sc) by moderating intermolecular interactions related to the transmission barrier of transmissible spongiform encephalopathies between different species. In contrast to mPrP(121-231) and most other mammalian PrP^Cs, the β2-α2 loop is well defined at 20 °C in tammar wallaby PrP and in the two aforementioned variants of mPrP, showing that long-range interactions with helix α3 can have an overriding influence on the structural definition of the β2-α2 loop. Further NMR studies with two variant mPrPs, mPrP[Y225A](121-231) and mPrP[Y226A](121-231), showed that these interactions are dominantly mediated by close contacts between residues 166 and 225. The results of the present study then lead to the intriguing indication that well-defined long-range intramolecular interactions could act as regulators of the functional specificity of PrP^C.     

鼻咽癌放疗后致迟发性放射性脑病34例临床分析

目的：探讨鼻咽癌放疗后致迟发性放射性脑病的临床特征。方法：对中南大学湘雅医院神经内科所收治的34例鼻咽癌放疗后致迟发性放射性脑病患者临床资料、治疗方法及预后进行回顾性研究。结果：34例迟发性放射性脑病患者的临床特征包括头痛、头晕、记忆力下降、精神异常、抽搐、复视、面瘫或面部麻木、听力下降、言语增多、肢体无力、走路不稳、肢端麻木等。头部MRIT1WI呈低信号,T2WI呈高信号,增强后呈不规则强化。34例均随访,其中死亡11例,占32.35%;现存活23例,17例伴有严重症状,生存质量差,占50%。结论：鼻咽癌放疗后致迟发性放射性脑病临床表现各异,患者预后较差,临床上需尽早诊断,积极治疗,定期随诊。     

Transgenic analysis of the physiological functions of Mahogunin ring finger‐1 isoforms

Mahogunin Ring Finger‐1 (Mgrn1) null mutant mice have a pleiotropic phenotype that includes the absence of yellow hair pigment, abnormal head shape, reduced viability, and adult‐onset spongiform neurodegeneration. Mgrn1 encodes a highly conserved E3 ubiquitin ligase with four different isoforms which are differentially expressed and predicted to localize to different subcellular compartments. To test whether loss of specific isoforms causes different aspects of the mutant phenotype, we generated transgenes for each isoform and bred them onto the null mutant background. Mice expressing only isoform I or III appeared completely normal. Isoform II rescued or partially rescued the mutant phenotypes, whereas isoform IV had little or no effect. Our data show that different Mgrn1 isoforms are not functionally equivalent in vivo and that the presence of only isoform I or III is sufficient for normal development, pigmentation, and neuronal integrity. genesis 47:524–534, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons

It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5 h with [U-¹³C]glucose to monitor de novo synthesis of alanine and glutamine in the absence and presence of 5.0 mM NH₄Cl and 10 mM MSO. Ammonia exposure led to increased incorporation of label but not to a significant increase in the amount of these amino acids. However, in the presence of MSO, glutamine synthesis was blocked and synthesis of alanine increased leading to an elevated content intra- as well as extracellularly of this amino acid. Treatment with MSO led to a dramatic decrease in glutamine content and increased the intracellular contents of glutamate and aspartate. The large increase in alanine during exposure to MSO underlines the importance of the GDH and ALAT biosynthetic pathway for ammonia fixation, and it points to the use of a GS inhibitor to ameliorate the brain toxicity and edema induced by hyperammonemia, events likely related to glutamine synthesis.     

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.