Some considerations on the ion transport properties of the rod disc membrane

The ion transport properties of the disc membranes in rod outer segments are discussed on the basis of available data. The properties of an air-water interface film of spectroscopically intact and chemically regenerable rhodopsin are presented, and results of studies of ion binding to these films are reported.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Chitin biosynthesis in imaginal discs cultured in vitro

Summary We have investigated the stimulation of cuticle production by imaginal discs ofPlodia interpunctella in tissue culture. We turned to biochemical methods to assess the quantitative effects of beta-ecdysone on chitin biosynthesis in wing discs incubated with 0.5 C of C¹⁴-glucosamine for the final 24 h of culture.We demonstrated that imaginal discs ofP. interpunctella respond to increasing concentrations of -ecdysone with increased synthesis. The threshold is between 0.01 and 0.1 g/ml of hormone (2×10^–8 M to 2×10^–7 M). These data represent the first demonstration of quantitative biosynthesis of chitin by a developing tissue in vitro in relation to varying amounts of hormone. Additionally, protein synthesis during the -ecdysone-dependent period was necessary for chitin synthesis. This system thus lends itself to a detailed investigation of the control of chitin biosynthesis.We wish to dedicate this paper to the memory of a fine colleague and friend, Dr. Andrzej Dutkowski     

A new non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells

Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, “tissue incubation”, for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7–10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3–4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α6 and laminin α5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.     

Importance of mechanics and kinematics in determining the stiffness contribution of the vertebral column during body-caudal-fin swimming in fishes

Whole-body stiffness in fishes has important consequences for swimming mode, speed and efficiency, but the contribution of vertebral column stiffness to whole-body stiffness is unclear. In our opinion, this lack of clarity is due in part to the lack of studies that have measured both in vitro mechanical properties of the vertebral column as well as in vivo vertebral kinematics in the same species. Some lack of clarity may also come from real variation in the mechanical role of the vertebral column across species. Previous studies, based on either mechanics or kinematics alone, suggest species-specific variation in vertebral column locomotor function that ranges from highly stiff regimes that contribute greatly to whole-body stiffness, and potentially act as a spring, to highly compliant regimes that only prohibit excessive flexion of the intervertebral joints. We review data collected in combined investigations of both mechanics and kinematics of three species, Myxine glutinosa, Acipenser transmontanus, and Morone saxatilis, to illustrate how mechanical testing within the context of the in vivo kinematics more clearly distinguishes the role of the vertebral column in each species. In addition, we identify species for which kinematic data are available, but mechanical data are lacking. We encourage further investigation of these species to fill these mechanical data gaps. Finally, we hope these future combined analyses will identify certain morphological, mechanical, or kinematic parameters that might be associated with certain vertebral column functional regimes with respect to body stiffness.     

MSC response to pH levels found in degenerating intervertebral discs

Painful degenerative disc disease is a major health problem and for successful tissue regeneration, MSCs must endure and thrive in a harsh disc microenvironment that includes matrix acidity as a critical factor. MSCs were isolated from bone marrow of Sprague-Dawley rats from two different age groups (<1 month, n = 6 and 4-5 months, n = 6) and cultured under four different pH conditions representative of the healthy, mildly or severely degenerated intervertebral disc (pH 7.4, 7.1, 6.8, and 6.5) for 5 days. Acidity caused an inhibition of aggrecan, collagen-1, and TIMP-3 expression, as well as a decrease in proliferation and viability and was associated with a change in cell morphology. Ageing had generally minor effects but young MSCs maintained greater mRNA expression levels. As acidic pH levels are typical of increasingly degenerated discs, our findings demonstrate the importance of early interventions and predifferentiation when planning to use MSCs for reparative treatments.     

Effects of phospholipid hydrolysis on the aggregate structure in DPPC/DSPE-PEG2000 liposome preparations after gel to liquid crystalline phase transition

Upon storage of phospholipid liposome samples, lysolipids, fatty acids, and glycerol-3-phosphatidylcholine are generated as a result of acid- or base-catalyzed hydrolysis. Accumulation of hydrolysis products in the liposome membrane can induce fusion, leakage, and structural transformations of the liposomes, which may be detrimental or beneficial to their performance depending on their applications as, e.g., drug delivery devices. We investigated in the present study the influence of phospholipid hydrolysis on the aggregate morphology of DPPC/DSPE-PEG₂₀₀₀ liposomes after transition of the phospholipid membrane from the gel phase to liquid crystalline phase using high performance liquid chromatography (HPLC) in combination with static light scattering, dynamic light scattering, and cryo-transmission electron microscopy (cryo-TEM). The rates of DPPC hydrolysis in DPPC/DSPE-PEG₂₀₀₀ liposomes were investigated at a pH of 2, 4, or 6.5 and temperatures of 22 °C or 4 °C. Results indicate that following phase transition, severe structural reorganizations occurred in liposome samples that were partially hydrolyzed in the gel phase. The most prominent effect was an increasing tendency of liposomes to disintegrate into membrane discs in accordance with an increasing degree of phospholipid hydrolysis. Complete disintegration occurred when DPPC concentrations had decreased by, in some cases, as little as 3.6%. After extensive phospholipid hydrolysis, liposomes and discs fused to form large bilayer sheets as well as other more complex bilayer structures apparently due to a decreased ratio of lysolipid to palmitic acid levels in the liposome membrane.     

外源性uPA对兔椎间盘作用的初步研究

目的：通过不同浓度外源性尿激酶型纤溶酶原激活剂（uPA）兔椎间盘内注射,探讨uPA对椎间盘作用。方法：健康大白兔72只,随机分为实验组48只,阴性对照组16只,空白对照组8只。实验组：分为三个亚组,L4／5椎间盘内分别注射浓度为10、20、40ng／μl的uPA各1μl。阴性对照组：椎间盘内注射1μl的生理盐水。空白对照组：不作任何处理。分别于第3、6周取相应椎间盘,大体观察、HE染色、SABC法测基质金属蛋白酶-3（MMP-3）的表达及蛋白多糖含量测定。结果：实验组MMP．3表达显著增强,蛋白多糖含量明显降低,与同期对照组比较（P〈0．05）,有显著统计学意义。实验组在不同时间及不同浓度比较也有统计学意义。结论：外源性uPA能够导致兔椎间盘内蛋白多糖含量降低,MMP-3表达显著增强,可能在椎间盘退变过程中其重要作用。     

正常与退变椎间盘髓核和纤维环中生化成份的研究

本工作测定了椎间盘的髓核和纤维环中的生化成份,结果提示在退变的椎间盘中,糖醛酸,总己糖胺、半乳糖胺均明显下降,而羟脯氨酸的含量明显增加。在退变的纤维环中尚有组成硫酸角质素的主要成份葡萄糖胺量的明显下降。在退变的髓核和纤维环中,总蛋白质含量呈增加趋势,水的含量呈下降趋势。提示退变椎间盘中蛋白多糖含量降低,组成改变及胶原蛋白的增加可能是诱发椎间盘突出症的物质基础。