维生素D3受体对hsp90β基因表达的调控作用

鉴于热休克蛋白９０β（ｈｓｐ９０β）基因内含子中含有维生素Ｄ３受体（ＶＤＲ）结合位点，为探讨作为核受体家族成员的ＶＤＲ是否对核受体特异分子伴侣的ｈｓｐ９０β基因的表达具有调控作用，我们开展了本项研究。分别将野生型ＶＤＲ、含Ｎ端（１～１３３氨基酸残基）及Ｃ端（２８１～４２７氨基酸残基）片段的ＶＤＲ突变体真核表达质粒与人ｈｓｐ９０β基因调控片段（－１０３９／＋１５３１）介导的氯霉素乙酰基转移酶（ＣＡＴ）报告基因质粒共转染Ｊｕｒｋａｔ细胞，检测正常及经热休克（４２℃，１ｈ）处理后细胞裂解液中ＣＡＴ活性。结果表明ＶＤＲＮ端增强、而Ｃ端抑制ｈｓｐ９０β的组成性表达；在热诱导条件下野生型ＶＤＲ对ｈｓｐ９０β的表达有一定的抑制作用，而其Ｃ端片段的抑制较强。为进一步研究ＶＤＲ对细胞内源性热休克基因表达的影响，我们用ＲＴＰＣＲ方法研究了ＶＤＲ的对细胞内ｈｓｐ９０β基因ｍＲＮＡ水平的影响，发现ＶＤＲ过表达对ｈｓｐ９０β的热诱导表达明显抑制。结果提示ＶＤＲ对ｈｓｐ９０β基因的组成性和热诱导表达的调控机制不同。     

情绪调节量表在青少年人群中的试用

目的:考察情绪调节量表在青少年人群中的信度和效度。方法:对789名中学生施测情绪调节量表,对测试结果进行信、效度分析。结果:情绪调节量表表达抑制维度的α系数为0.77,重新评价维度的α系数为0.83。验证性因素分析的结果显示,标准测量模型与实际数据的拟合程度良好,各项目均在对应的维度上具有0.45以上的完全标准化载荷值。对两个维度的关系进行考察,结果显示独立模型与标准测量模型之间无显著差异。不同年级的学生在两种情绪调节策略的使用上没有显著差异,男生较女生更多使用表达抑制策略。结论:情绪调节量表在青少年人群中具有良好的心理测量学品质,可以在进一步的研究中使用。     

Interaction between pre-landing activities and stiffness regulation of the knee joint musculoskeletal system in the drop jump: implications to performance

The purpose of the present study was to investigate the interaction between the pre-landing activities and the stiffness regulation of the knee joint musculoskeletal system and the takeoff speed during a drop jump (DJ). Nine healthy male subjects performed a DJ test from the height of 50 cm. The surface electromyographic (EMG) activity of the vastus lateralis (VL) muscle was recorded to evaluate both the pre-landing and post-landing muscle activation levels. Simultaneous recording of the jumping motion and ground reaction force was performed by a high-speed video camera (100 frames·s^–1), and a force platform was employed to allow joint moment analysis. Joint stiffness was calculated by a linear regression of the knee joint moment/angle relationship. Elasticity of the knee extensor muscle during DJ was estimated by means of a four-element muscle model consisting of a parallel elastic component, a series elastic component (SEC), a viscous damper, and a contractile element. DJ performance correlated positively with the positive peak power of the knee joint (P<0.01) and with the moment of the knee joint at the end of stretch (P<0.01). However, there was no significant relationship between DJ performance and the positive peak power of the ankle joint. The knee joint moment at the end of stretch correlated with the SEC stiffness during the transmission phase from the end of the initial impact to the onset of the concentric action (P<0.01) and with the maximum rate of isometric force development of the knee extensors (P<0.01). Multiple regression analysis showed that the SEC stiffness during the transmission phase of the knee joint can be explained by a combination of the pre-activity of the VL muscle and the knee joint angular velocity at touchdown (F=5.76, P<0.05). These results seem to emphasize the functional significance of the pre-programmed activity for controlling the subsequent stiffness regulation and then contributing to the performance in DJ. Thus, it can be suggested that the centrally pre-programmed activity and the associated elastic behavior of the SEC in the knee extensor muscle in conjunction with the muscle contractile property play a major role in regulating the performance in DJ. Electronic Publication     

Increased resistance to CD4+CD25hi regulatory T cell‐mediated suppression in patients with type 1 diabetes

Type I diabetes (T1D) is a T cell‐mediated autoimmune disease characterized by loss of tolerance to islet autoantigens, leading to the destruction of insulin‐producing beta cells. Peripheral tolerance to self is maintained in health through several regulatory mechanisms, including a population of CD4⁺CD25^hi naturally occurring regulatory T cells (T_regs), defects in which could contribute to loss of self‐tolerance in patients with T1D. We have reported previously that near to T1D onset, patients demonstrate a reduced level of suppression by CD4⁺CD25^hi T_regs of autologous CD4⁺CD25^‐ responder cells. Here we demonstrate that this defective regulation is also present in subjects with long‐standing T1D (> 3 years duration; P = 0·009). No difference was observed in forkhead box P3 or CD127 expression on CD4⁺CD25^hi T cells in patients with T1D that could account for this loss of suppression. Cross‐over co‐culture assays demonstrate a relative resistance to CD4⁺CD25^hi T_reg‐mediated suppression within the CD4⁺CD25^‐ T cells in all patients tested (P = 0·002), while there appears to be heterogeneity in the functional ability of CD4⁺CD25^hi T_regs from patients. In conclusion, this work demonstrates that defective regulation is a feature of T1D regardless of disease duration and that an impaired ability of responder T cells to be suppressed contributes to this defect.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of β-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities. Received: 9 January / 5 March 1998     

Interferon-α activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-α secretion by immature human natural killer cells

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon- (IFN-) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN- activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN- uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN- resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN- resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-. Unlike IL-2, IFN- did not stimulate the Free cells to proliferate or secrete TNF- and activation of cytotoxicity and modulation of surface antigens by IFN- were independent of TNF-. The failure of IFN- to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN- and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF- and proliferation. These results demonstrate that IFN- serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN- proceeds by TNF--dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.Abbreviations used IFN interferon - IL interleukin - PBL peripheral blood leukocytes - PE phycoerythrin - PE-GAM PE-conjugated Fab2 goat anti-mouse IgG - NK natural killer - NRS normal rabbit serum - TNF tumor necrosis factor - FCS fetal calf serum - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - MACS magnetic cell sorting - ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - PKC protein kinase C - mAb monoclonal antibody - PBMC peripheral blood mononuclear cells - BCLL B-chronic lymphocytic leukemia - E effector - T target     

Thermoregulatory responses to cold during a 7-week acclimatisation process in rabbits

A group of six rabbits reared at +20°C ambient temperature was adapted to moderate cold by housing for seven weeks at +10°C. Rectal and skin temperatures, metabolic heat production and respiratory evaporative heat loss were recorded continuously over 1 h for each animal on 3 days per week in the climatic chamber.There was no significant change either of rectal or of ear skin temperature during the acclimatisation process. On the other hand, metabolic heat production was progressively reduced (20% in the 7th week). Slight changes of mean skin temperature and respiratory evaporative heat loss could not account for compensation. Therefore it must be concluded that both adaptive improvement of peripheral insulation and reduction of heat production were achieved during the acclimatisation process. Both processes together ensure that deviations of core temperature are minimal. The possible origin of the functional adaptive effects is discussed. The results are in full agreement both with former neurophysiological results and with system-theoretical considerations of adaptive processes.Dedicated to the 60th birthday of Professor Dr. Kurt Brück     

Genetics,systems, and alcohol

Under a variety of rubrics (e.g., complexity, self-constructing systems, dissipative structures), interest has recently burgeoned in applying principles of complex systems to a wide variety of scientific issues. A major concern is with emergent properties of systems not derivable from the properties of components of the systems. In this paper, some elementary aspects of systems considerations are applied to phenomena of alcohol pharmacogenetics. It is likely that whole new families of informative phenotypes can be generated by this approach.