Gene Therapy: The Potential Applicability of Gene Transfer Technology to the Human Germline

The theoretical possibility of applying gene transfer methodologies to the human germline is explored. Transgenic methods for genetically manipulating embryos may in principle be applied to humans. In particular, microinjection of retroviral vector appears to hold the greatest promise, with transgenic primates already obtained from this approach. Sperm-mediated gene transfer offers potentially the easiest route to the human germline, however the requisite methodology is presently underdeveloped. Nuclear transfer (cloning) offers an alternative approach to germline genetic modification, however there are major health concerns associated with current nuclear transfer methods. It is concluded that human germline gene therapy remains for all practical purposes a future possibility that must await significant and important advances in gene transfer technology.     

The exclusion among the Ustilago viruses — A dsRNA restriction modification system?

Summary Specific exclusion relations are know among the three Ustilago maydis viruses that are associated with the cytoplasmically transmitted killer phemomenon. Of the three viruses P1, P4 and P6, only P1, and P4 cancoexist in one host cell. Mutual exclusion occurs between P1 and P6 and P4 unilaterally excludes P6. The exclusion relations were originally defined among the wild-type viruses. Those relations can be modified by two specific segments that are a part of the P4 dsRNA genome and were also found in some sensitive strains that contained part of the viral genome. Also, deletion of the dsRNA segment that is assumed to encode the toxin information permits the formation of hybrid genomes that otherwise cannot be formed. The data is interpreted in terms of a dsRNA restriction modification system in which the killer toxin or a toxin-linked function acts as the restriction factor and segments H3 and H4 or H4 alone contain the necessary information for the modification of certain sites on the M and L segments of the P1 and P4 viruses but not on the P6 segments.     

Functional modification of agonist-antagonist electromyographic activity for rapid movement inhibition

Subjects made a fast elbow extension movement to designated target in response to a go signal. In 45% of trials a stop signal was presented after the go signal, to which subjects were asked to stop the movement as rapidly as possible. The interstimulus interval (ISI), or time interval between the go and stop signals, was randomly varied between 0 and 200 ms. Electromyographic (EMG) activity was recorded from biceps brachii and triceps brachii. Subjects could sometimes completely inhibit initiation of the movements when the ISI was 0 ms, but could rarely do so when the ISI exceeded 100 ms. For responses that were initiated but stopped on the way, the amplitude of the movement decreased linearly as the time interval (=modification time) from the stop signal to EMG onset increased. The peak velocity increased linearly as the movement amplitude increased. This tendency was similar to those previously reported in step-tracking movements with various amplitudes. In spite of the similarity in the kinematics of the movement, the EMG pattern was different from that of step-tracking movement. While the initial agonist burst (AG1) decreased linearly after the modification time exceeded 100 ms, the antagonist burst (ANT) increased compared with the go trial for the modification time from 0 to 200 ms and decreased after the modification time exceeded 300 ms. This change of activation is analogous to functional modification of middle-latency reflex EMG response to load, or cutaneous perturbation. In conclusion, it is suggested that adaptive mechanisms, which would functionally modify the reflex responses, are also continuously working during voluntary movements in response to sudden changes in environmental information. Received: 3 November 1997 / Accepted: 3 February 1998     

Vp130, a chloroviral surface protein that interacts with the host Chlorella cell wall

A protein, Vp130, that interacts with the host cell wall was isolated from Chlorovirus CVK2. From its peptide sequence, the gene for Vp130 was identified on the PBCV-1 genomic sequence as an ORF combining A140R and A145R. In Vp130, the N-terminus was somehow modified and the C-terminus was occupied by 23-26 tandem repeats of a PAPK motif. In the internal region, Vp130 contained seven repeats of 70-73 amino acids, each copy of which was separated by PAPK sequences. This protein was well conserved among NC64A viruses. A recombinant rVp130N protein formed in Escherichia coli was shown not only to bind directly to the host cell wall in vitro but also to specifically bind to the host cells, as demonstrated by fluorescence microscopy. Because externally added rVp130N competed with CVK2 to bind to host cells, Vp130 is most likely to be a host-recognizing protein on the virion.     

Doxorubicin, an RNA synthesis inhibitor, prevents vasoconstriction and inhibits aberrant expression of endothelin-1 in the cerebral vasospasm model of the rat

The present study investigates the effects of bis(7)-tacrine, a novel dimeric acetylcholinesterase inhibitor, on hydrogen peroxide(H₂O₂)-induced cell injury with comparison to the corresponding monomer, tacrine. Exposure of rat pheochromocytoma line PC12 cells to H₂O₂ induced significant cell damage. This reagent also caused redox desequilibrium as indicated by a decrease in activities of intracellular antioxidant enzymes such as glutathione peroxidase as well as catalase and an accumulation of malondialdehyde, a product of lipid peroxidation. Pretreatment of cells with bis(7)-tacrine or tacrine attenuated H₂O₂-induced cell toxicity, and bis(7)-tacrine demonstrated higher potency than tacrine in improving redox desequilibrium. These results suggest that bis(7)-tacrine and tacrine significantly protect against H₂O₂ insult, which might be beneficial for their potential usage in the prevention and treatment of Alzheimer's disease.     

Age-related effects of oxidative metabolism and cyclic AMP signaling on neutrophil apoptosis

Spontaneous as well as Fas-induced polymorphonuclear cell apoptosis is unchanged in the elderly. However, a weak responsiveness to antiapoptotic signals elicited by proinflammatory molecules has been reported in neutrophils isolated from aged humans. To gain insight into this field, here we have evaluated the role of oxidative metabolism and cyclic AMP (cAMP) signaling on age-related neutrophil apoptotic cell death. Results show that although superoxide dismutase (SOD), added exogenously to cell cultures, is able to prolong neutrophil survival in both young and aged individuals, high amounts of the enzyme are further effective in cell cultures of young donors only. Notably, the addition of catalase gives rise to a more striking, yet comparable, inhibition of neutrophil-programmed cell death in both groups of subjects. Furthermore, even low amounts of catalase are enough to restore a normal apoptotic outcome in SOD-treated cell cultures of old donors. Unlike the oxidative metabolism, cAMP signaling activation does not reveal any difference in the apoptotic response of neutrophils isolated from young and aged donors. Thus, supplementation of cell cultures with prostaglandin E2, dibutyryl cAMP or, to a lesser degree, forskolin results in a dose-dependent inhibition of DNA cleavage product appearance in both groups of subjects. The data outline that an impairment of neutrophil antioxidant shield, leading to an augmented cell oxidative load, is likely to occur as a feature of age. This may increase the apoptotic rate of stimulated cells, which may in turn account for the increased susceptibility of elderly individuals to life-threatening infections.     

Fate-Mapping of GM-CSF Expression Identifies a Discrete Subset of Inflammation-Driving T Helper Cells Regulated by Cytokines IL-23 and IL-1β

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Differential sensitivity to Arg side chain modification of IL-1β binding to type I and type II receptors

The central role of interleukin-1 (IL-1) in several disease processes, including fever and inflammation, makes the characterization of ligand-receptor interaction of prime importance.The role of arginine (Arg) side chains of hr-IL-1 in receptor recognition was studied by the modification of Arg residues with the specific reagent 1,2-cyclohexanedione. It was found that chemical modification of Arg residues decreased the binding potential of IL-1 to type I receptor dramatically (by 230-fold) while the affinity to type II receptor was reduced only moderately (by 10-fold), with an insignificant reduction of the dissociation rate.These studies suggest that intact Arg side chains of IL-1 may be necessary for high affinity binding to type I IL-1 receptor, but have less importance for the interaction of IL-1 with type II IL-1 receptor.This observation may be useful in the study of type II IL-1 receptor-mediated biological responses and design of receptor-subtype specific ligands as well.     

慢性乙型肝炎患者肝细胞凋亡的研究

目的 探讨慢性乙型肝炎(CHB)患者肝细胞凋亡与肝组织诱导性一氧化氮合酶(iNOS)、肝脏病理及血清转氮酶水平之间的相关关系.方法 取37例CHB患者和10例正常对照,采用dUTP缺口末端标记技术(TUNEL)检测肝组织中的细胞凋亡情况,免疫组化法检测肝组织iNOS的表达,并常规测定患者肝组织炎症分级和纤维化分期、血清丙氨酸转氨酶(ALT)、HBV DNA水平.结果 所有CHB患者的肝组织中均见到不同程度的TUNEL染色阳性.正常对照肝组织中未见到TUNEL阳性肝细胞.在CHB肝组织中,凋亡指数(A1)与炎症分级(r=0.404,P=0.015)及肝组织iNOS水平(r=0.465,P=0.004)明显相关并有统计学意义,而与血清ALT水平、HBV DNA以及组织学纤维化分期无明显关系.结论 慢性乙型肝炎患者体内氧化应激水平可能是肝细胞凋亡的诱导因素之一,细胞凋亡参与了CHB的肝细胞损伤过程,但并不影响患者的生物化学指标.