The role of dorsal root ganglia activation and brain-derived neurotrophic factor in multiple sclerosis

Multiple sclerosis (MS) is characterized by focal destruction of the white matter of the brain and spinal cord. The exact mechanisms underlying the pathophysiology of the disease are unknown. Many studies have shown that MS is predominantly an autoimmune disease with an inflammatory phase followed by a demyelinating phase. Recent studies alongside current treatment strategies, including glatiramer acetate, have revealed a potential role for brain-derived neurotrophic factor (BDNF) in MS. However, the exact role of BDNF is not fully understood. We used the experimental autoimmune encephalomyelitis (EAE) model of MS in adolescent female Lewis rats to identify the role of BDNF in disease progression. Dorsal root ganglia (DRG) and spinal cords were harvested for protein and gene expression analysis every 3 days post-disease induction (pdi) up to 15 days. We show significant increases in BDNF protein and gene expression in the DRG of EAE animals at 12 dpi, which correlates with peak neurological disability. BDNF protein expression in the spinal cord was significantly increased at 12 dpi, and maintained at 15 dpi. However, there was no significant change in mRNA levels. We show evidence for the anterograde transport of BDNF protein from the DRG to the dorsal horn of the spinal cord via the dorsal roots. Increased levels of BDNF within the DRG and spinal cord in EAE may facilitate myelin repair and neuroprotection in the CNS. The anterograde transport of DRG-derived BDNF to the spinal cord may have potential implications in facilitating central myelin repair and neuroprotection.     

Representing an ensemble of NMR-derived protein structures by a single structure.

The usefulness of representing an ensemble of NMR-derived protein structures by a single structure has been investigated. Two stereochemical properties have been used to assess how a single structure relates to the ensemble from which it was derived, namely the distribution of phi psi torsion angles and the distribution of chi 1 torsion angles. The results show that the minimized average structure derived from the ensemble (a total of 11 ensembles from the Brookhaven Protein Data Bank were analyzed) does not always correspond well with this ensemble, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue. An alternative method that selects the member of the ensemble which is closest to the "average" of the ensemble has been investigated (a total of 23 ensembles from the Brookhaven Protein Data Bank were analyzed). Although this method selected a structure that on the whole corresponded more closely to the ensemble than did the minimized average structure, this is still not a totally reliable means of selecting a single structure to represent the ensemble. This suggests that it is advisable to study the ensemble as a whole. A study has also been made of the practice of selecting the "best" rather than the most representative member of the ensemble. This too suggests that the ensemble should be studied as a whole. A study of the conformational space occupied by the ensemble also suggests the need to consider the ensemble as a whole, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue.     

Fast sampling,rapid filtration apparatus: Principal characteristics and validation from studies ofd-glucose transport in human jejunal brush-border membrane vesicles

Summary Kinetic data in (brush-border) membrane vesicles which rely on the validity of the initial rate assumption for their interpretation and depend on tracer flux studies using the rapid filtration technique for their experimental measurement have been limited to some extent by the absence of techniques that would allow for real-time data analysis. In this paper, we report on our successful design of a fast sampling, rapid filtration apparatus (FSRFA) which seems to fill up this technical gap since showing the following characteristics: (i) rapid injection (5 msec) and mixing (less than 100 msec) of small amounts of vesicles (10–40 l) with an incubation medium (0.2–1.0 ml); (ii) fast (20 to 80 msec depending on the sample volume) and multiple (up to 18 samples at a maximal rate of 4/sec) sampling of the uptake mixture followed by rapid quenching in the stop solution (approximately 5 msec) according to a predetermined time schedule (any time combination from 0.25 to 9999 sec); and (iii) fast, automated, and sampling-synchronized filtration and washings of the quenched uptake medium (only 15–20 sec are necessary for the first filtration followed by two washings and extra filtrations). As demonstrated using adult human jejunal brush-border membrane vesicles and Na⁺-d-glucose cotransport as models, the FSRFA accurately reproduces the manual aspects of the rapid filtration technique while allowing for very precise initial rate determinations. Moreover, the FSRFA has also been designed to provide as much versatility as possible and, in its present version, allows for a very precise control of the incubation temperature and also permits a few efflux protocols to be performed. Finally, its modular design, which separates the fast sampling unit from the rapid filtration device, should help in extending its use to fields other than transport measurement.     

Energetic approach to the folding of alpha/beta barrels

The folding of a polypeptide into a parallel (alpha/beta)8 barrel (which is also called a circularly permuted beta 8 alpha 8 barrel) has been investigated in terms of energy minimization. According to the arrangement of hydrogen bonds between two neighboring beta-strands of the central barrel therein, such an alpha/beta barrel structure can be folded into six different types: (1) left-tilted, left-handed crossover; (2) left-tilted, right-handed crossover; (3) nontilted, left-handed crossover; (4) nontilted, right-handed crossover; (5) right-tilted, left-handed crossover; and (6) right-tilted, right-handed crossover. Here "tilt" refers to the orientational relation of the beta-strands to the axis of the central beta-barrel, and "crossover" to the beta alpha beta folding connection feature of the parallel beta-barrel. It has been found that the right-tilted, right-handed crossover alpha/beta barrel possesses much lower energy than the other five types of alpha/beta barrels, elucidating why the observed alpha/beta barrels in proteins always assume the form of right tilt and right-handed crossover connection. As observed, the beta-strands in the energy-minimized right-tilted, right-handed crossover (alpha/beta)8-barrel are of strong right-handed twist. The value of root-mean-square fits also indicates that the central barrel contained in the lowest energy (alpha/beta)8 structure thus found coincides very well with the observed 8-stranded parallel beta-barrel in triose phosphate isomerase (TIM). Furthermore, an energetic analysis has been made demonstrating why the right-tilt, right-handed crossover barrel is the most stable structure. Our calculations and analysis support the principle that it is possible to account for the main features of frequently occurring folding patterns in proteins by means of conformational energy calculations even for very complicated structures such as (alpha/beta)8 barrels.     

动脉插管化疗配合膀胱灌注治疗复发性、多发性、表浅性膀胱癌

目的:探讨腹壁下动脉插管化疗(inferior-epigastric artery chemotherapy,IAC)配合吡柔比星(THP)膀胱灌注治疗复发性、多发性和表浅性膀胱癌的疗效。方法:对12例复发性、多发性和表浅性膀胱移行细胞癌患者术后行腹壁下动脉插管化疗3个疗程后,开始行吡柔比星膀胱灌注,每次30 mg,每周1次共12次,以后每2周1次共6次,以后每月1次共6次,第二年重复,第三年每月一次。定期膀胱镜检查,进行随访。结果:12例复发性、多发性、浅表性膀胱移行细胞癌患者,术后随访时间24-48个月,肿瘤复发1例,复发率8.3%。不良反应主要为术后小膀胱,尿路刺激症状和尿常规异常。结论:腹壁下动脉插管化疗配合吡柔比星膀胱灌注治疗复发性、多发性和表浅性膀胱癌的效果明确,疗效满意,患者耐受性好,值得临床推广应用。     

Some aspects of forest soil and litter ecology in the Dawyck Cryptogamic Sanctuary with a particular reference to fungi

Here we report on ecology and biodiversity of fungi in a unique mycological sanctuary in Britain, where data on species composition have been collected since 1994. To complement the biodiversity data by the information on the fungal ecological interactions and their role in the overall ecosystem functioning, soil properties and the composition of forest litter and field layer, bacterial population numbers and fungal biomass (in terms of ergosterol) were measured in 8 plots covered with different vegetations (beech, birch, birch-oak-beech, grass) over a May–Aug. period, and the results were analysed by correlation analysis and stepwise regression modelling together with data on protozoa and nematodes available from parallel research. The results highlighted the complexity of factors influencing temporal dynamics and spatial variability of fungal biomass in soil and forest litter. Most of the registered interactions appeared to be transient, and this should be taken into account while interpreting environmental observations. Interpretation of the specific relationships is given and implications for further research and overall ecosystem functioning are discussed.     

Candidate tumour suppressor Fau regulates apoptosis in human cells: An essential role for Bcl-G

FAU, which encodes a ubiquitin-like protein (termed FUBI) with ribosomal protein S30 as a carboxy-terminal extension, has recently been identified as a pro-apoptotic regulatory gene. This activity may be mediated by Bcl-G (a pro-apoptotic member of the Bcl-2 family) which can be covalently modified by FUBI. FAU gene expression has been shown to be down-regulated in human breast, prostate and ovarian tumours, and this down-regulation is strongly associated with poor prognosis in breast cancer. We demonstrate here that ectopic FAU expression increases basal apoptosis in human T-cell lines and 293T/17 cells, whereas it has only a transient stimulatory effect on ultraviolet-C (UVC)-induced apoptosis. Conversely, siRNA-mediated silencing of FAU gene expression has no effect on basal apoptosis, but attenuates UV-induced apoptosis. Importantly, prior knockdown of Bcl-G expression ablates the stimulation of basal apoptosis by FAU, consistent with an essential downstream role for Bcl-G, itself a candidate tumour suppressor, in mediating the apoptosis regulatory role of FAU. In 293T/17 cells, Bcl-G knockdown also attenuates UV-induced apoptosis, so that Bcl-G may constitute a common factor in the pathways by which both FAU and UV-irradiation induce apoptosis. UV irradiation increases Bcl-G mRNA levels, providing an explanation for the transient nature of the effect of ectopic FAU expression on UV-induced apoptosis. Since failure of apoptosis is fundamental to the development of many cancers, the pro-apoptotic activity of the Fau/Bcl-G pathway offers an attractive explanation for the putative tumour suppressor role of FAU.     

心肌细胞团搏动整数倍节律的非线性动力学机制

利用心肌细胞耦合模型研究心肌整数倍节律的动力学机理。确定性模型仿真揭示了心肌细胞团同步搏动加周期分岔的节律变化规律;随机模型仿真发现在加周期分岔序列中分岔点附近会出现整数倍节律,其中,0-1整数倍节律产生于从静息到周期1的Hopf分岔点附近,1-2整数倍节律产生于周期1和周期2极限环间的加周期分岔点附近;对系统相空间轨道的分析进一步揭示出整数倍节律是由系统运动在相邻的两个轨道之间随机跃迁形成的。上述分析结果不仅阐明了心肌整数倍节律的机理,并且揭示了各种整数倍节律与加周期分岔序列中相邻节律的内在联系,为重新认识心律变化的规律开辟了新的途径。     

pKILLIN: a versatile positive-selection cloning vector based on the toxicity of Killin in Escherichia coli

The invention of DNA cloning over 40 years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes.