Polymorphism in the promoter region of the apolipoprotein AI gene associated with differences in apolipoprotein AI levels: The European Atherosclerosis Research Study

The effect associated with the substitution of adenine (A) for guanidine (G) in the promoter region of the apolipoprotein AI gene (?75 bp) with plasma apo AI and high-density lipoprotein (HDL) levels was investigated in the European Atherosclerosis Research Study (EARS). This is a study of healthy offspring (cases) of fathers who had suffered premature myocardial infarction (MI) before age 55 years (n = 565) and age- and sex-matched controls (n = 1,078) from 12 European countries, divided into 5 regions based on geography and language. The frequency of the polymorphism was not significantly different among the regions and the relative frequency of the rare A allele was similar in cases and controls (0.159 vs. 0.142) combining data from all regions. Individuals with one or more A allele had significantly higher plasma apo AI levels (P < 0.05) than individuals homozygous for the G allele. This effect was consistent in all regions. The data were analyzed separately in males and females. In females, those with one or more A allele had significantly higher apo AI levels (P = 0.05) than individuals homozygous for the G allele, and this raising effect of the A allele was greater in cases than controls for both apo AI (5.23% vs. 1.56%) and HDL (4.48% vs. 1.89%). In males, the A allele was associated with higher levels of apo AI and HDL, but the effect was much smaller and the differences did not reach statistical significance. In the females, where the effect of the A allele was strongest, the effect on apo AI associated with genotype was evident in non-smokers, and individuals with one or two A alleles had 3.6% higher apo AI and 3.14% higher HDL levels than individuals homozygous for the G allele. However, in the female smokers the raising effect of the A allele was greatly reduced (0.56%). Thus genetic variation in the promoter region of the apo AI gene is associated with differences in apo AI and HDL levels in healthy individuals throughout Europe, but the effect is modulated by gender, environmental factors such as smoking, and a family history of MI.     

Multiple sclerosis: myelin basic protein induced mRNA expression of proinflammatory cytokines in mononuclear cells is suppressed by interferon-β 1b in vitro

Beta-interferon (IFN-β) is a promising treatment in multiple sclerosis (MS), reducing the exacerbation rate and MRI lesion burden, as well as the disease progression in relapsing-remitting MS. IFN-β was originally defined by its antiviral effects, but the interest has recently been focused on its immunomodulatory properties. Myelin basic protein (MBP) is one of several autoantigens considered to be the target for autoaggressive immune responses, which eventually might lead to the development of MS. To study in-vitro effects of IFN-β1b on MBP induced cytokine expression, mRNA for the Th1 cytokines IFN-γ and TNF-α, the Th2 related IL-4 and IL-6, the cytolytic perforin and the immune response downregulating TGF-β was measured with in situ hybridization after culture of blood mononuclear cells (MNC) in the presence and absence of MBP. Numbers of cells expressing IFN-γ, TNF-α, perforin and IL-4 mRNA were significantly suppressed after culture with 10 U/ml IFN-β1b. No such effect was seen on MBP induced IL-6 or TGF-β mRNA expression. These observations suggest that one of the major effects of IFN-β1b is the induction of a shift in the cytokine mRNA profile towards a more immunosuppressive pattern. In parallel in vitro tests, the control substance dexametasone (40 μg/ml) reduced the numbers of cells expressing mRNA for all cytokines under study with the exception of TGF-β, to an extent equal to or even more pronounced than IFN-β1b.     

Elevated extracellular potassium is associated with a reduced extracellular space in rat neural lobe in vitro

Summary Increased neural activity of neurosecretory cells is accompanied by large increases in extracellular K⁺. The possibility that elevations of this ion might involve fluid redistribution and thus affect the size of the extracellular space and the relationship between pituicytes and axons in the rat neural lobe was explored using rapid freezing and freeze-substitution. Neural lobes were incubated for 15 min before freezing either in a normal medium or one containing a 10 mM increase in KCl (high KCl), a 10 mM increase in KCl balanced by an equimolar reduction in NaCl (high KCl-low NaCl), or only a 10 mM reduction in NaCl (low NaCl). A quantitative assessment of the region of good fixation was made to determine the relative fractions occupied by axons, pituicytes and the extracellular space near the neurohaemal contact zone. In addition, the percentage of basal lamina contacted by pituicytes and axons was calculated, as was the degree of enclosure of axons by pituicytes.In neural lobes incubated in normal medium, the extracellular space accounted for approximately 30% of the cross-sectional area of the neuropil and could be divided into two domains: an extended perivascular space (28–29% of total area); and a narrow (approximately 24 nm; approximately 1% of total) space between closely apposed neurosecretory processes or between these processes and pituicytes. Pituicytes occupied almost 60% of the basal lamina at the neurohaemal contact zone, while axons occupied approximately 20%. Neural lobes incubated in either the high KCl solution, or in the high KCl-low NaCl solution, exhibited a significantly reduced extracellular space, to about 20% of the total area, or a reduction from controls of about one-third. The reduction was complemented by an increased area occupied by axons plus pituicytes. In the high KCl group, the contribution of the narrow spaces (22–24 nm) between apposed processes to the total extracellular space was greatly increased. The group exposed to low NaCl showed high variability in the size of extracellular space, and was thus not significantly different from any other group. No changes in any group were observed in the enclosure of axons by pituicytes, or in the relative amounts of axon and pituicyte apposition to the basal lamina.The subsequent buffering of K⁺ and other ions during periods of increased neuronal activity may be affected by changes in the extracellular space, thereby influencing stimulus-secretion coupling. A shrinkage of the extracellular space and the relative increase in the narrow spaces between processes initiated by elevated K⁺ could also alter the diffusion properties of the neural lobe.     

Norephedrine (phenylpropanolamine) stimulates oxygen consumption and lactate production in the perfused rat hindlimb

d,l-Norephedrine (phenylpropanolamine) which is both a demethylated analogue and a metabolite of d,l-ephedrine, is a reputed anorectic agent. In the present study the proposed most active isomer of this mixture, l-norephedrine has been assessed as a peripherally acting thermogenic agent in the isolated perfused rat hindlimb. l-Norephedrine produced a dose-dependent increase in oxygen uptake and perfusion pressure and increased lactate production. Whereas propranolol potentiated the increase in oxygen uptake and perfusion pressure produced by l-norephedrine, prazosin significantly and nitroprusside totally inhibited both of these changes. Nitroprusside also completely inhibited the increase in lactate production. We conclude that norephedrine has a hitherto unrecognized peripheral thermogenic activity in the perfused rat hindlimb resulting from its interaction with alpha 1 adrenergic receptors that control vasoconstriction in this tissue.     

运动性肥大心肌活细胞胞浆游离Ca2+动态变化的激光共聚焦研究以及局部IGF-Ⅰ和AngⅡ的变化

目的:研究运动与心脏重塑过程中心肌胞浆游离Ca2 ([Ca2 ]c)动态变化的生物学机制.方法:采用激光扫描共聚焦显微技术(LSCM)对STDIn Ca2 荧光试剂负载的运动肥大心肌活细胞[Ca2 ]c动态变化进行研究,采用放射免疫测定运动小鼠心肌局部IGF-Ⅰ和AngⅡ含量.结果:运动肥大心肌[Ca2 ]c变化表现为基值稳态和峰值显著升高,达峰时间延长(P＜0.001).心肌局部IGF-Ⅰ和AngⅡ显著升高(P＜0.001).结论:运动性心肌肥大与[Ca2 ]c变化、机械信号、IGF-Ⅰ和AngⅡ关系密切,机械信号、IGF-Ⅰ和AngⅡ可能是引起[Ca2 ]c显著升高,增强心肌收缩能力的胞外刺激因素.     

Influence of mild hypothermia on vascular endothelial growth factor and infarct volume in brain tissues after cerebral ischemia in rats

BACKGROUND: It has been demonstrated that mild hypothermia has obvious protective effect on both whole and local cerebral ischemia. However, the definite mechanism is still unclear for the brain protection of mild hypothermia on cerebral edema, inhibiting inflammatory reaction, stabilizing blood brain barrier, etc. OBJECTIVE: To investigate the effect of mild hypothermia on the expression of vascular endothelial growth factor and the infarct volume after cerebral ischemia in rats, and analyze the brain protective mechanism of mild hypothermia. DESIGN: A randomized grouping and controlled animal trial. SETTING: Department of Neurology, People's Hospital of Yunyang Medical College. MATERIALS: Twenty adult male SD rats of clean degree, weighing (250±30) g, were provided by the animal experimental center, School of Medicine, Wuhan University. The kits for SP immunohistochemistry were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. METHODS: The experiments were carried out in the laboratory of Department of Neurology, Renmen Hospital of Wuhan University from May to July 2005. ① The 20 rats were divided randomly into normal temperature group (n =10) and mild hypothermia group (n =10). Models of permanent middle cerebral artery occlusion were established with modified nylon suture embolization. The rats were assessed with the Longa standards: 0 point for without nerve dysfunction; 1 for mild neurological deficit (fore claws could no extend completely); 2 for moderate neurological deficit (circling towards the affected side); 3 for severe neurological deficit (tilting towards the affected side); 4 for coma and unconscious; 1-3 points represented that models were successfully established. The rats of the normal temperature group were fed at room temperature, and those in the mild hypothermia group were induced by hypothermia from 2 hours postoperatively, and the rectal temperature was kept at 34-35 ℃ for 72 hours. ② Measurement of infarct volume: All the rats were anesthetized by intraperitoneal injection overdose sodium pentobarbital 7 days postoperatively, and then the heads were cut down to harvest brain. The brain tissues were placed into -20 ℃ refrigerator for 20 minutes, coronal sections of 2 mm were prepared. The infarct sites were not stained, whereas normal brain tissues were stained as red. The infarct volumes were calculated by using MPLAS-500 multimedia color pathological image&&word analytical system. ③ Counting positive cells of vascular endothelial growth factor protein: The brains were harvested by cutting heads, then coronal sections of 2 mm were prepared. Routine dehydration, hyalinization, wax immersion and embedding were performed, then the detected with SP immunohistochemistry, the kits were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. The cells whose cytoplasm was yellow-brown were positive ones, a single sample as a unit, peri-ischemic site and ischemic core were selected, and the corresponding sites in controlateral hemisphere were taken as controls. Five visual fields were selected from each site to be observed under microscope, the cells were counted, and the average number of positive cells was calculated in each group. The numbers of positive cells were determined with the image analytical apparatus. MAIN OUTCOME MEASURES: Number of the positive cells of vascular endothelial growth factor protein; Infarct volume of rat brain tissue. RESULTS: All the 20 rats were involved in the analysis of results. ① Number of positive cells of vascular endothelial growth factor protein in brain tissue: It was obviously lower in the mild hypothermia group than in the normal temperature group [(24.02±5.05), (36.07±2.69) cells/high power visual field, P < 0.01]. ② Comparison of infarct volume of brain tissue: After MCAO, it was obviously smaller in the mild hypothermia group than in the normal temperature group [(153.25±23.14), (253.45±36.21) mm3, P < 0.01]. CONCLUSION: Mild hypothermia can inhibit the expression of vascular endothelial growth factor and decrease the volume of cerebral infarction. The inhibition of mild hypothermia on the expression of vascular endothelial growth factor may be one of the brain protective mechanisms.     

微创手术治疗高血压脑出血

目的 探讨微创手术治疗高血压脑出血的临床疗效。方法 132例高血压脑出血分成微创手术组(68例)和传统开颅手术组(64例)，分析两组手术的特点和手术时机，比较两组手术治疗的疗效。结果 微创组术后GOS良好23例、中残24例、重残9例、植物生存3例、死亡9例；传统组术GOS良好16例、中残15例、重残12例、植物生存6例、死亡15例。两组超早期或早期手术均有良好的预后，而微创组效果更佳。结论 微创手术治疗高血压脑出血能明显提高临床疗效，降低病死率。     

In vitro differentiation of human adipose-derived adult stromal cells into neuron-like cells in hippocampal astrocyte conditioned medium

BACKGROUND: At present, researches on differentiating from human adipose-derived adult stromal cells (hADASC) to neuron-like cells are focus on inducing by artificial-synthetic compound solution; however, hippocampal astrocyte conditioned medium (HCAM) can induce in vitro differentiation from hADASC to neuron-like cells is still unclear. OBJECTIVE: To observe whether HCAM can induce in vitro differentiation from hADASC to neuron-like cells. DESIGN: Randomized control study. SETTING: Department of Neurology, Taixing People's Hospital; Central Laboratory, North China Coal Medical College. MATERIALS: Donor of adipose tissue was donated by female volunteers suffering from caesarean section in the department of obstetrics & gynecology in our hospital and aged 20－35 years. Adipose tissue was collected from subcutaneous tissue of abdomen during the operation. In addition, 8 male newborn Wistar rats within 24 hours with average body mass of 20 g were provided by Animal Institute of Chinese Academy of Medical Sciences. Rabbit-anti-human Nestin polyclonal antibody, rabbit-anti-human glial fibriliary acidic protein (GFAP) polyclonal antibody, rabbit-anti-human neuro-specific enolase polyclonal antibody and mouse-anti-human microtubal associated protein 2 (MAP-2) polyclonal antibody were provided by Wuhan Boster Company. METHODS: The experiment was carried out in the Central Laboratory of North China Coal Medical College from October 2004 to June 2005. hADASC was cultured with HCAM and its growth and morphological changes were observed under inverted phase contrast microscope. Immunocytochemistry, immunofluorescence and Western blotting were used to evaluate the expressions of Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. MAIN OUTCOME MEASURES: Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. RESULTS: On the 3rd day of culture, partial hADASC started deformation from slender shuttle-shape cells to neuron-like cells. It suggested that cells stretched out apophysis, which were mainly double-pole or multiple-pole cells. Five days later, immunohistochemical detection suggested that expression of Nestin (10.5±0.037) was found out in cells; meanwhile, expressions of GFAP (38.4±0.052) and neuro-specific enolase (NSE) (15.7±0.023) were also found out in cells; however, expression of MAP-2 was not observed. Western blot indicated that, 5 days after effect of HCAM, Nestin was found out in hADASC; meanwhile, expressions of GFAP and neuro-specific enolase were also found out; however, expression of MAP-2 was not observed. CONCLUSION: HCAM can induce the differentiation from hADASC to neuron-like cells in vitro.     

Regulation of PTC phenotype and function by TGF-β1: implications for transdifferentiation

Introduction There is now increasing evidence that proximal tubular cells (PTCs) contribute to renal interstitial fibrosis by alteration of matrix turnover and by the generation of pro‐fibrotic cytokines such as TGF‐β1. Recent studies suggest that, through a process of transdifferentiation, the PTCs are one source of the interstitial myofibroblasts that directly drive the fibrotic process. The aim of this work was to examine the role and mechanism by which TGF‐β1 may regulate PTC phenotype and function. Methods Experiments were performed using both primary‐cultures of PTC and the human PTC cell line HK2. All experiments were performed on growth‐arrested cells in the absence of serum. Results TGF‐β1 altered cell phenotype, assessed by light microscopy, with cells appearing elongated and spindle‐shaped. This was associated with loss of cell–cell contact and rearrangement of the actin cytoskeleton, increased formation of stress fibres and focal adhesions. Disruption of the actin cytoskeleton with cytochalasin‐D prevented phenotypic alterations following addition of TGF‐β1. Transient transfection with Smad‐2/‐4 or Smad‐3/‐4 expression vectors did not alter cell phenotype. Previously, we have demonstrated β‐catenin translocation to PTC nuclei and its association with Smad proteins following addition of TGF‐β1, suggesting the possibility that TGF‐β1 may modulate Wnt signalling. Wnt‐responsive Xtwn‐reporter construct was, however, silent in response to TGF‐β1. Similarly, a second Wnt‐/LEF‐1‐regulated element Toplflash, which does not contain Smad‐binding sites, was insensitive to TGF‐β1 signalling. In contrast, phenotypic changes in response to TGF‐β1 were abrogated by inhibitors of the RhoA downstream target ROCK, which also prevented loss of cell–cell contact and adherens junction disassembly. Removal of TGF‐β1 and addition of 1% FCS, however, reverted cell phenotype to a typical cobblestone epitheliod appearance, suggesting that TGF‐β1 did not result in terminal PTC transdifferentiation. Cells grown on tissue culture dishes coated with either type‐I or type‐III collagen also acquired an elongated fibroblastic phenotype; this effect was exaggerated by the addition of TGF‐β1. In contrast to the cells stimulated with TGF‐β1 alone, following stimulation by both TGF‐β1 and exposure to interstitial collagens, cell phenotype was stable in that it was not reversed upon removal of TGF‐β1 and addition of FCS. Addition of TGF‐β1 to cells grown on type‐IV collagen had no greater effect than TGF‐β1 alone. Addition of TGF‐β1 alone had little effect on the expression of α‐SMA. In contrast, cells grown on either type‐I or type‐III collagen, following addition of TGF‐β1, demonstrated marked increased expression of α‐SMA, which appeared to be incorporated into the cell cytoskeleton. Similarly, the combination of interstitial collagen (either type‐I or type‐III) and TGF‐β1 had synergistic effect on the relocation and down‐regulation of the epithelial markers E‐cadherin and cytokeratin. Finally, the results demonstrated synergistic effects of coating with interstitial collagen (either type‐I or type‐III), on cell ‘fibroblastic’ cell function as assessed by cell migration and by the synthesis of type‐III and type‐IV collagen. Conclusion The results of these in vitro experiments suggest that terminal transdifferentiation of proximal tubular epithelial cells is the result of a combination of the effects of the pro‐fibrotic cytokine TGF‐β1 and exposure of the cells to components of the interstitial extra‐cellular matrix to which the cells are not exposed in the absence of damage to the tubular basement membrane.     

Effects of human amniotic fluid on cartilage regeneration from free perichondrial grafts in rabbits.

After the chondrogenic potential of free grafts of perichondrium was shown in several experimental studies, perichondrium has been used to reconstruct cartilage tissue in various clinical situations. This study investigates the effects of human amniotic fluid on neochondrogenesis from free perichondrial grafts in a rabbit model. Since this fluid contains high concentrations of hyaluronic acid, hyaluronic acid-stimulating activator, growth factors, and extracellular matrix precursors during the second trimester, it may have a stimulating effect on neochondrogenesis. Perichondrial grafts, measuring 20 x 20 mm2 were obtained from the ears of 144 New Zealand young rabbits and were sutured over the paravertebral muscles. The rabbits were randomly divided into three groups with 48 rabbits per group. In group 1, 0.3 ml human amniotic fluid, and in group 2, 0.3 ml saline were injected underneath the perichondrial grafts. Group 3 formed the control group in which no treatment was given. Histologically, neochondrogenesis was evaluated in terms of cellular form and graft thickness at 2, 4, 6, and 8 weeks after surgery. In group 1, the mature cartilage was generated quickly and the cartilage plate in this group was significantly thick and extensive when compared with groups 2 and 3 at 8 weeks ( p<0.05 ANOVA). In conclusion, our study shows that human amniotic fluid enhances neochondrogenesis from free perichondrial grafts. The rich content of hyaluronic acid and growth factors possibly participate in this result.