Horseradish peroxidase: a reliable or a misleading tool for the investigations on the origin of the proteins of the aqueous humor?

The concept of the blood-aqueous barrier is largely based on the use of horseradish peroxidase (HRP). The present investigation was designed to check its reliability as a macromolecular tracer, especially with regard to the transport of plasma proteins. Rabbits were killed 5 min to 24 h after being intravenously injected with HRP. The tracer diffused rapidly, reaching the aqueous humor of the eye in 3 min or less and was detected at high concentration in the narrow space between the outer epithelial layer of the ciliary epithelium and the wall of the pervious capillaries in the stroma of the processes. HRP appeared to migrate from the blood to the posterior chamber, permeating the tight junctions, viz., the anatomical basis of the blood-aqueous barrier. It was detected at higher concentration at the anterior surface of the iris, at short time intervals; this was interpreted as penetration of the tracer from the aqueous humor of the anterior chamber. The choroid was also labeled in continuation with the reaction in the stroma of the pars plana of the ciliary body which, in turn, sometimes reached the iris root. Therefore, the pervious blood vessels of the choroid could be a source of macromolecules for the iris root. HRP also induced the formation of lysosomes in the ciliary epithelium. This can hardly be accepted as the way in which plasma proteins are physiologically transported to the aqueous humor. However, the pathway of HRP migration over short time intervals seems to be in agreement with previous research indicating that the entrance of serum albumin into the posterior chamber is the first step of its incorporation into the aqueous humor. Received: 7 June 1996 / Accepted: 15 January 1997     

Formation of zeatin allylic phosphate by the microsomal fractions of bulb disks of Iris × hollandica Tub. and tubers of Helianthus tuberosus L.

It is shown that in bulbous Iris zeatin originates from a nucleotide. This nucleotide is probably zeatin-allylic-phosphate, in which a phosphate group is attached to the isoprenoid side-chain of zeatin. The formation of zeatin-allylic-phosphate from t-zeatin and 8-[¹⁴C]-zeatin by the microsomal fractions of Iris bulb disks and Helianthus tubers was demonstrated. The responsible enzyme was partially purified. 5-AMP was found to be a phosphate group delivering substrate. Adenosine and adenine inhibited the enzyme reaction. The significance of the results is discussed in relation to cytokinin biosynthesis and the occurrence of bud blast in Iris.     

Effects of Corticotropin-(1–24)-Tetracosapeptide on Polyphosphoinositide Metabolism and Protein Phosphorylation in Rabbit Iris Subcellular Fractions

Abstract: Effects of the neuropeptide corticotropin-(1–24) -tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 ×g supernatant fraction [30–50% (NH₄)₂SO₄ precipitate; ASP_30–50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The ³²P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [γ-³²P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP_30–50 fractions were resolved into six and nine labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30–60 s), and it was dependent on the presence of Mg²⁺ in the incubation medium; in general it was inhibited by high concentrations (>0.2 mM) of Ca²⁺. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose-dependent manner, with maximal effect observed at 50–100 μ of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 μM) inhibited significantly the endogenous phosphorylation of six microsomal phosphoproteins (100K, 84K, 65K, 53K, 48K, and 17K). In the ASP_30–50 fraction, ACTH inhibited the phosphorylation of three phosphoproteins (53K, 48K, and 17K) and stimulated the labeling of six phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K). The effects of ACTH on lipid and protein phosphorylation are probably Ca²⁺-independent; thus the neuropeptide effects were not influenced by either 1 μM EGTA or low concentrations of Ca²⁺ (50 μ.M). We conclude that a relationship may exist between polyphosphoinositide metabolism and protein phosphorylation in the rabbit iris smooth muscle.     

不同产地苍术药材化学成分的比较

江苏、安徽及湖北５个产地苍术（Ａｔｒａｃｔｙｌｏｄｅｓｌａｎｃｅａ（Ｔｈｕｎｂ．）ＤＣ．）商品药材的化学分析结果表明：它们在某些药材性状、一些常规化学成分、挥发油成分和含量上存在显著差异。江苏茅苍术根茎挥发油的化学成分复杂,由此探讨了它作为地道药材的特殊性。同时对苍术的“断面暴露稍久可析出结晶”的性状和《药典》（１９９０年版）规定的“层析谱上应显有苍术素斑点”的鉴别实验进行讨论。     

白及块茎铜,锌超氧物歧化酶的纯化及其性质

白及（Ｂｌｅｉｌｌａｓｔｒｉａｔａ（Ｔｈｕｎｂ．）Ｒｅｉｃｈｂ．ｆ．）的ＳＯＤ同工酶只有一条较宽的谱带,确认为Ｃｕ·Ｚｎ－ＳＯＤ。其块茎ＳＯＤ总活性和比活性都高,且含有丰富的白及胶;经丙酮分级沉淀,ＳｅｐｈａｄｅｘＧ１００凝胶过滤和ＤＥＡＥ－纤维素柱层析分离纯化,获得对ＣＮ￣－敏感的淡兰色Ｃｕ·ＺｎＳｏＤ粉末。在凝胶电泳染色图谱上,纯化后的酶与粗酶液的ＳＯＤ区带相对应,且其酶活性染色带与蛋白染色带位置对应,表明已纯化到均一程度。该酶分子量约３３ＫＤ,亚基分子量约为１６．４ＫＤ;紫外光区的吸收峰在２６４．６ｎｍ,等电聚焦电泳呈现一条蛋白区带,ｐＨ值在４．３５左右;该酶在ｐＨ６．０～１０．０,温度在５０℃范围内具稳定性。纯化后的酶为４５６３．２ｕ／ｍｇ·蛋白,纯化了５１倍,活力回收为２２．３％。上述酶没有过氧化氢酶活性。提取过程中还得到高质量的副产品白及胶。     

金银花花形基因的发掘及生物信息学分析

金银花作为我国重要的中药材,具有消炎、抗菌、抗病毒、抗氧化、防癌等多种功效。随着金银花市场供需矛盾日益加剧,通过分子标记辅助选择育种方法来培育高产优质品种势在必行。通过NCBI的Blast工具扫描金银花蛋白组数据发掘花形候选基因,并执行候选基因的亲缘关系分析、结构域分析、表达模式分析、理化性质分析、蛋白质结构预测等一系列生物信息学分析。依据拟南芥调控花形的ABE类基因,通过NCBI-Blast工具扫描金银花氨基酸序列,筛选出包含MADS结构域的8个调控花形的金银花候选基因。经LjaFGD表达模式分析发现,金银花的花中GWHGAAZE016592和GWHGAAZE014905表达量显著高于其他部位,可能正向调控金银花花形。GWHGAAZE014905是一个包含MADS结构域的调控花器官发育的B类基因;GWHGAAZE016592是AP3同源基因。生物信息学分析发现,GWHGAAZE016592和GWHGAAZE014905均是稳定的亲水蛋白,属于非分泌蛋白,包括Motif1、Motif3、Motif4、Motif2、Motif6和Motif5,蛋白质三级结构模板为6byy.2.A和4ox0.2.C。GWHGAAZE014905被定位到细胞核上,而GWHGAAZE016592被定位到叶绿体上,且包含1个位于151~173 bp的跨膜螺旋区域,属于膜蛋白。研究结果为分子标记辅助选择方式培育道地高产优质金银花品种提供了基因资源和分子标记。     

鸢尾柄锈菌一新变种及柄锈属国内一新记录

本文报道采自东北地区的鸢尾柄锈菌一新变种和柄锈属一国内新记录种。新变种命名为鸢尾柄锈菌多孔变种[Puccinia iridis(DC.)Wallr.var.polyporis W.C.Liu var.nov.],区别于原变种的主要特征是其夏孢子小,芽孔5—7个,散生;而后者夏孢子较大,芽孔2—4个,赤道生。新记录种是短果茴芹柄锈菌(Puccinia pimpinellae-brachycarpae Tranzsch.et Ere-meeva),寄生在短果茴芹(Pimpinella brachycarpa Nakai)上。标本保存于沈阳农业大学真菌标本室和中国科学院微生物研究所真菌标本室。     

Comparison of carbohydrate utilization and energy charge in the yellow flag iris (Iris pseudacorus) and garden iris (Iris germanica) under anoxia

Carbohydrate and energy metabolism of the flooding- and anoxia-tolerant Iris pseudacorus and the intolerant Iris germanica rhizomes were investigated under experimental anoxic conditions. Rhizomes of I. pseudacorus and I. Germanica were incubated in the absence of oxygen from 0 to 60 and 16 days, respectively. Amounts of glucose, total reducing sugars and non-reducing sugars (starch, fructan and oligosaccharides) in the rhizomes were measured. Ethanol concentration and adenylate energy charge were determined enzymatically. Glucose content of I. pseudacorus rhizomes decreased gradually during the first 30 days under anoxia and then increased at the same time as adenylate energy charge values started to decline. In I. germanica rhizomes the changes were more dramatic and the time scale was much shorter than in I. pseudacorus but the changes were similar. Non-reducing sugar content of I. pseudacorus rhizomes decreased rapidly during the first 15 days under oxygen deprivation and then increased again, to near starting levels at 35 days. In I. germanica the amount of non-reducing sugars decreased gradually during the anoxic incubation. Under aerobic control conditions, adenylate energy charge (AEC) of I. pseudacorus and I. germanica rhizome tissue was 0.87±0.01 and 0.81±0.01, respectively. In I. pseudacorus AEC remained high until 30 days under anoxia. In contrast, the energy charge of I. germanica rhizome tissue remained above 0.6 for 4 days only. Large amounts of ethanol were found in anoxic rhizome tissues of I. pseudacorus (up to 0.21 M ) and I. germanica (0.06 M ) after 45 days and 8 days, respectively. The results are discussed in relation to flooding tolerance of these species.     

Reflective organelles in the anterior pigment epithelium of the iris of the European starling Sturnus vulgaris

The ultrastructure and chemical composition of reflective organelles in the anterior pigment epithelium of the iris of the European starling Sturnus vulgaris were examined. The reflective organelles produced a diffuse white reflectance at the iris mid-section which was visible only when the stroma was removed. The pigment granules were clear, angular, and birefringent under the light microscope. In electron micrographs the granules were irregular in shape and density, sometimes crystalline in appearance, but more often they were lost during sectioning or staining. Guanine was abundant in the modified pigment epithelium of the starling, but not in the pigment epithelia of other birds that lacked birefringent granules. Pteridines, such as xanthopterin and leucopterin, were present in small amounts. Pteridines were also present in the iris stroma which had no reflective organelles. The reflective organelles in the starling pigment epithelium resemble both the reflecting platelets of lower vertebrate chromatophores and the reflective granules in the tapeta of various vertebrates. Possible derivation of the organelles from these sources is discussed.     

Improved detection of iris severe mosaic virus in secondarily-infected iris bulbs

The influence of wounding and high-temperature treatment on the detection of iris severe mosaic virus (ISMV) in secondarily ISMV-infected iris bulbs was studied. Wounding of the bulbs just after lifting, followed by storage for 3 wk at 17°C or 20°C, increased the detectability of ISMV to 100% reliability. High-temperature treatment and consecutive storage at 17°C induced a similar improvement of detection. It is concluded that a certain degree of stress, such as wounding or high-temperature treatment, ultimately leads to an increase in viral antigens and thus to improvement of detection. It is hypothesised that the virus titre increases by the altered metabolism during the repair reactions as a response to stress applied to the bulbs.