细胞壁的制备及其羟脯氨酸含量的测定

细胞壁结构和功能关系的研究越来越被人们所重视。伸展蛋白是植物细胞壁中一类重要的结构蛋白,与细胞壁的精细结构及各种生理功能密切相关。其特点之一是它的羟脯氨酸(Hyp)含量高达30％～40％。因此,细胞壁的制备及其羟脯氨酸含量的分析是细胞壁及其蛋白质研究中最基本又极重要的内容。本文介绍一种细胞壁的制备及其羟脯氨酸含量测定的简单方法。     

颌下腺导管细胞与胶原海绵细胞相容性的实验研究

为探讨胶原海绵对颌下腺 (submandibulargland ,SMG)导管细胞的细胞相容性 ,采用HE染色光镜观察及免疫组化观察SMG导管细胞接种于胶原海绵后 ,细胞的生长情况。光镜下可见接种后第 1d细胞数量较少 ,分散于胶原海绵支架中间 ,第 7d细胞数量明显增加 ,免疫组织化学染色抗IV型胶原抗体染色呈阳性 ,说明细胞与支架材料之间已经有细胞外基质产生。胶原海绵具有良好的细胞相容性 ,是一种理想的支架材料。与胶原海绵复合培养 ,颌下腺导管细胞仍可保持良好的增殖能力。     

微等离子束对豚鼠皮肤超微结构分析和羟脯氨酸测定

目的观察微等离子束对豚鼠皮肤胶原组织作用效应的组织学和超微结构变化及羟脯氨酸含量测定,探讨微等离子束的作用机理。方法选择15只豚鼠,每只豚鼠背部划分为实验侧和空白对照侧2个等分区域,给予60W/10 kJ微等离子束照射,于作用后即刻、1周后和1月后分别切取实验侧及空白对照部位皮肤行组织病理维多利亚-立春红染色,透射电镜分析和羟脯氨酸检测试剂盒进行含量测定。结果 60 W/10 kJ即刻表现为表皮局灶性出现点阵状改变,部分表皮出现汽化缺失或者坏死变性,真皮浅层胶原组织出现点阵化表现和明显均质化;特殊染色显示微等离子束主要影响真皮胶原纤维,形成局灶性胶原纤维凝集和变性。1周后皮肤浅层胶原组织结构逐渐致密,排列有序,有少量组织细胞。1月后皮肤浅层胶原组织明显增厚,胶原纤维增粗并排列致密,弹力纤维呈局灶性增粗。透射电镜显示微等离子束作用后表皮细胞较完整,细胞间结构正常,但真皮胶原丧失正常结构,细胞结构消失,大量细胞凋亡明显,1月后仍可见少量细胞凋亡的表现但胶原结构逐渐恢复,浅层胶原纤维排列明显致密。羟脯氨酸测定显示微等离子束作用1周后羟脯氨酸含量要高于作用之前,但是差异性不具有统计学意义（P〉0.05）;1月后羟脯氨酸含量要明显高于作用前,差异性具有统计学意义（P〈0.05）。结论微等离子束对豚鼠皮肤胶原组织作用有明显的刺激效应,其主要靶组织为真皮胶原组织,可以明显促进皮肤新生胶原的增生。     

牛磺酸、羟脯氨酸、γ－氨基丁酸、鸟氨酸和色氨酸的测定

在日立８３５－５０型氨基酸分析仪上,用７２ｍｉｎ程序进行分析,通常一次只能分析１７种氨基酸。采用本文所述方法则一次能分析２２种氨基酸。在不延长分析时间,不增加成本的情况下,一次多分析了五种氨基酸,提高了仪器工作效率。经过精密度、准确度试验和样品分析证明：该方法测定的数据准确、可靠,是一个又快又省的好方法。     

交联对胶原降解速率的影响

目的：研究交联反应对胶原降解速率的影响。方法：以热交联(DHT)、1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)化学交联以及EDC／DHT交联三种方法对胶原海绵材料进行处理,并测定材料在处理前后的降解速率。结果：各种交联反应均不同程度地提高了胶原的生物稳定性．降低了胶原的降解速率。     

动物脂肪组织中羟脯氨酸测定方法研究

本研究通过试验,提出了盐酸水解—氨基酸自动分析仪快速、准确测定动物脂肪组织中羟脯氨酸的分析方法,该方法适用于动物育种、选种,动物发育期生理生化变化研究和肉质检验等方面。     

同时测定羟脯氨酸、脯氨酸的氨基酸分析仪短程序

本文制定了单独测定样品中羟脯氨酸和脯氨酸的短程序,用本程序测定羟脯氨酸和脯氨酸的峰保留时间的变异系数分别为０．１６％和０．１９％,峰面积的变异系数分别为１．１３％和１．５３％,比标准程序时间减少５０％,节约茚三酮试剂７０％。     

塞隆骨和虎骨的羟脯氨酸含量分析比较

用改良的Woessner第Ⅰ法测定和比较了塞隆骨和虎骨的L 4 羟脯氨酸的含量。结果表明 :原药材中羟脯氨酸含量塞隆骨略低于虎骨 ,但在水煎液干膏部分L 4 羟脯氨酸含量塞隆骨高于虎骨 ,这为塞隆骨与虎骨的药用价值比较提供了成份实验依据     

High-throughput quantification of hydroxyproline for determination of collagen

An accurate and high-throughput assay for collagen is essential for collagen research and development of collagen products. Hydroxyproline is routinely assayed to provide a measurement for collagen quantification. The time required for sample preparation using acid hydrolysis and neutralization prior to assay is what limits the current method for determining hydroxyproline. This work describes the conditions of alkali hydrolysis that, when combined with the colorimetric assay defined by Woessner, provide a high-throughput, accurate method for the measurement of hydroxyproline.     

Fibroblast procollagen production rates in vitro based on [3H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [3H]proline and measuring the subsequent formation of [3H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [3H]proline, and [3H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [14C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45-55]. Procollagen production rates were readily determined by this method using 5 to 20 microCi [3H]proline and approximately 10(6) cells. It was found that 3H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/microgram DNA . h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.     

Fibroblast procollagen production rates in vitro based on [H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [³H]proline and measuring the subsequent formation of [³H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [³H]proline, and [³H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [¹⁴C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45–55]. Procollagen production rates were readily determined by this method using 5 to 20 μCi [³H]proline and approximately 10⁶ cells. It was found that ³H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/μg DNA · h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.     

Effect of silver nanoparticles and hydroxyproline,administered in ovo,on the development of blood vessels and cartilage collagen structure in chicken embryos

It has been considered that concentrations of certain amino acids in the egg are not sufficient to fully support embryonic development of modern broilers. In this study we evaluated embryo growth and development with particular emphasis on one of the major components of connective tissue, collagen. Experiments were performed on Ross 308 chicken embryos from 160 fertilised eggs. Experimental solutions of silver nanoparticles (Ag), hydroxyproline solution (Hyp) and a complex of silver nanoparticles with hydroxyproline (AgHyp) were injected into albumen, and embryos were incubated until day 20. An assessment of the mass of embryo and selected organs was carried out followed by measurements of the expression of the key signalling factors’ fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A). Finally, an evaluation of collagen microstructure using scanning electron microscopy was performed. Our results clearly indicate that Hyp, Ag and AgHyp administered in ovo to chicken embryos did not harm embryos. Comparing to the control group, Hyp, Ag and the AgHyp complex significantly upregulated expression of the FGF-2 at the mRNA and protein levels. Moreover, Hyp, Ag and, in particular, the complex of AgHyp significantly increased blood vessel size, cartilage collagen fibre lattice size and bundle thickness. The general conclusion from this study is that AgHyp treatment may help to build a stronger and longer lasting form of collagen fibres.     

Effect of mercuric chloride on various hydroxyproline fractions in rat serum

Mercuric chloride (HgCl₂) disturbs the collagen metabolism in the body which is reflected by altered hydroxyproline fractions in the serum. The aim of the present investigation was to study the effect of HgCl₂ treatment on various hydroxyproline (Hyp) fractions in rat serum and the effect of 2,3-dimercapto-1-propane sulfonic acid (DMPS) treatment on serum Hyp fractions in HgCl₂ treated rats. Other parameters studied included body weight, food intake, water intake and kidney weight. Doses of HgCl₂ used were 0.1, 0.5, 1.0, 2.0, 3.0 mg/kg body weight and that of DMPS was 100 mg DMPS/kg body weight. All the doses of HgCl₂ used caused significant (p < 0.01) alterations in free, peptide-bound and protein-bound Hyp in the serum when compared with control rats but a dose of 2 mg/kg body weight caused significant (p < 0.001) alteration even in the total serum Hyp when compared to control rats. Administration of DMPS prior HgCl₂ treatment of rats sacrificed 24 h after the treatment caused a significant decrease of 52% (p < 0.01) in free Hyp when compared to similar HgCl₂ treated rats. DMPS treatment with HgCl₂ also caused an increase of 61% (p < 0.001) and 114% (p < 0. 001) in peptide- and protein-bound Hyp respectively, when compared to HgCl₂ treated rats sacrificed 24 h after mercuric chloride and DMPS treatment. Administration of DMPS followed by HgCl₂ to rats which were sacrificed 48 h later caused no significant change in the total and free Hyp when compared to HgCl₂ treated rats which were sacrificed 48 h after the treatment. But there was a significant decrease of 40% (p < 0.001) in peptide-bound Hyp and an increase in of 77% (p < 0.001) in protein-bound Hyp when compared to HgCl₂ treated rats sacrificed 48 h after the treatment. The present study shows that HgCl₂ treatment caused significant alterations in serum Hyp fractions reflecting disturbed composition of connective tissues which were not reversed by DMPS treatment. (Mol Cell Biochem 271: 159–165, 2005)     

Reinforced bioartificial dermis constructed with collagen threads

In this work, a novel type of composite scaffold was designed, which has the suitability of both high biocompatibility and strong mechanical properties, for use in bioartificial dermis applications. The reinforced scaffold consisted of a lyophilized collagen sponge formed around a cross-linked collagen meshwork with an average thread diameter of approximately 55 μm. Fibroblasts were cultured in the reinforced collagen sponge for 7 days, during which time the pores in the sponge became filled with cells that secreted extracellular matrix (ECM) to form a bioartificial dermis. Results of ultimate tensile strength (UTS) measurements and compression tests indicated that the bioartificial dermis formed around the reinforced collagen sponge showed about ten times the strength of the bioartificial dermis formed around a typical collagen sponge (1.5 ± 0.05 vs. 0.15 ± 0.05 and 2.5 ± 0.1 vs. 0.2 ± 0.08 MPa, respectively). As a result, reinforced collagen mesh improved mechanical properties and this technique will be possible to make stronger scaffolds, not only for artificial skin applications but also various artificial tissues, such as synthetic cartilage, bone, and blood vessels.     

Experimental determination of proline hydroxylation and hydroxyproline arabinogalactosylation motifs in secretory proteins

Many secretory and several vacuolar proteins in higher plants contain hydroxylated proline residues. In many cases, hydroxyprolines in proteins are glycosylated with either arabinogalactan or oligoarabinose. We have previously shown that a sporamin precursor is O-glycosylated at the hydroxylated proline 36 residue with an arabinogalactan-type glycan when this protein is expressed in tobacco BY-2 cells (Matsuoka et al., 1995). Taking advantage of the fact that this is the only site of proline hydroxylation and glycosylation in sporamin, we analyzed the amino acid requirement for proline hydroxylation and arabinogalactosylation. We expressed several deletion constructs and many amino acid substitution mutants in tobacco cells and analyzed glycosylation and proline hydroxylation of the expressed sporamins. Hydroxylation of a proline residue requires the five amino acid sequence [AVSTG]-Pro-[AVSTGA]-[GAVPSTC]-[APS or acidic] (where Pro is the modification site) and glycosylation of hydroxyproline (Hyp) requires the seven amino acid sequence [not basic]-[not T]-[neither P, T, nor amide]-Hyp-[neither amide nor P]-[not amide]-[APST], although charged amino acids at the -2 position and basic amide residues at the +1 position relative to the modification site seem to inhibit the elongation of the arabinogalactan side chain. Based on the combination of these two requirements, we concluded that the sequence motif for efficient arabinogalactosylation, including the elongation of the glycan side chain, is [not basic]-[not T]-[AVSG]-Pro-[AVST]-[GAVPSTC]-[APS].     

Effect of strain on human dermal fibroblasts in a three-dimensional collagen sponge

Our aim was to design a simple compression system and investigate the influence of mechanical stress on skin-like structures. Many mechanical compression studies have employed intricate culture systems, so the relationship between extracellular matrix material and the response of skin cells to mechanical stress remains unknown. Our approach uses only glass vials, 6-well plates and standard laboratory equipment. We examined the influence of mechanical stress on human skin fibroblasts embedded within a collagen sponge. The results show that mechanical compression increases MMP-1 and MMP-2 release by the cells into the the cell culture. Our results suggest that pressure on the skin may affect extracellular matrix degradation through some as yet unidentified pathways and that IL-6 mRNA expression may be involved in this effect. Using our approach, the effects of static mechanical stress on protein expression by cells in the culture medium and in sponges can be easily examined, and therefore this system will be useful for further analyses of skin responses to mechanical stress.     

Effect of ascorbate on collagen synthesis by lung embryonic fibroblasts

Summary Total insoluble collagen and hydroxyproline formation were examined in lung embryonic fibroblasts (IMR-90) grown in the presence or absence of added ascorbate. As expected, when the cells from both groups (+ and −ascorbate) are pulsed with [¹⁴C]proline in the presence of ascorbate, the percent hydroxylation in a 24-hr period does not vary significantly. However, there are dramatic differences in the quantity and quality of the insoluble collagen fraction produced by those cells grown for a long period of time with added ascorbate. Those cells deprived of continuous addition of ascorbate to the culture medium do not display large quantities of accumulated collagen in the cell layer fractions as measured by the hydroxyproline content, whereas the cells grown in the presence of ascorbate contain significant amounts of accumulated collagen. A new method for examining the extracellular insoluble collagen produced in cell cultures is described in these studies. With the aid of pancreatic elastase relatively pure insoluble collagen can be obtained from cells grown in culture. In those cells grown in the presence of ascorbate, the purified insoluble collagen yeilds appropriately banded fibrils when examined in the electron microscope and has an amino-acid composition that is compatible with pure collagen. On the other hand, those cells grown in the absence of ascorbate do not yield purified insoluble collagen as determined by these same criteria. The elastase procedure for the purification of insoluble collagen in cell cultures is simple, easy to use and allows one to assess additional aspects of collagen biosynthesis.