抗寒锻炼中不同抗寒性小麦细胞膜糖蛋白的细胞化学研究

本研究根据植物细胞的特点,修改了在动物和人体细胞方面立的酶标Con A的电镜细胞化学方法,成功地展现了2个不同抗寒性冬小麦品种幼苗在抗寒锻炼和脱锻炼过程中细胞膜系统上糖蛋白的分布动态,显示与Con A连接的标志酶-辣根过氧化物酶活性的反应产物呈颗粒状分散分布在质膜、内质网、核膜及液泡膜的一些部位上,揭示糖蛋白在冬小麦细胞膜系统上的分布似有其特定的位点。经抗寒锻炼后,强抗寒性品种燕大1817细胞内的糖蛋白在内质网和核膜上的分布量明显地增加;同时,几乎所有的胞间连丝通道中都有糖蛋白的分布。脱锻炼后,内质网和核膜上的糖蛋白分布量又减少,胞间连丝通道中的糖蛋白也消失,基本上回复到抗寒锻炼前的分布状态。抗寒性弱的冬小麦品种郑州39-1幼苗在同样的抗寒锻炼和脱锻炼过程中不产生这些明显的变化。这些结果说明,抗寒锻炼中内质网和核膜上糖蛋白分布量的增加,以及糖蛋白输入胞间连丝的动态变化是与植物抗寒力的提高和保持稳定密切相关的。     

POLARIZATION STUDIES IN COLLODION MEMBRANES AND IN SYNTHETIC PROTEIN-LIPOID MEMBRANES

1. Collodion membranes of high polarizability and low resistance can be obtained either by addition of certain ether-soluble substances such as phosphatides, olive oil, mastix, and gum benzoin, to the collodion or by drying collodion membranes for a limited time under pressure. 2. The permeability of membranes of different polarization has been measured by means of conductivity methods. 3. Sintered glass filter plates of Jena glass crucibles on which proteins and lipoids have been adsorbed show polarization. It could be shown that some narcotics which react with lecithin cause an increase in polarization of the protein-lipoid-glass system. Substitutions of the protein but not of the lipoid were possible, without causing a decrease in the polarizability of the membranes.     

CARBOHYDRATE-PEPTIDE LINKAGES IN GLYCOPROTEINS AND MUCOPOLYSACCHARIDES FROM BRAIN

Abstract— Treatment of glycopeptides, prepared from glycoproteins of rat and rabbit brain, with NaOH-NaBH₄ leads to the destruction of a portion of the serine, threonine and galactosamine present, and the appearance in acid hydrolysates of alanine, α-aminobutyric acid and galactosaminitol. These results indicate that N-acetylgalactosamine at the reducing end of oligosaccharide chains in brain glycoproteins is linked O-glycosidically to the hydroxyl groups of serine and threonine residues. 2-acetamido-1-(L-β-aspartamido)-l,2-dideoxy-β-D-glucose was also detected after partial acid hydrolysis of the alkali-stable glycopeptides, and most of the carbohydrate in brain glycoproteins appears to be linked by N-acetylglucosaminylasparagine linkages. The results of the treatment of the sulphated mucopolysaccharides from bovine brain with alkaline-borohydride indicate that the polysaccharide chains in chondroitin sulphate and heparan sulphate are linked exclusively to serine.     

INVOLVEMENT OF MANNOSYL-PHOSPHORYL-DOLICHOLS IN MANNOSE TRANSFER TO BRAIN GLYCOPROTEINS

Abstract— Mannose was transferred from GDP-[¹⁴C]mannose by homogenates of embryonic chick and adult rat brain to mannolipids with properties identical to manriosyl-phosphoryl-dihydropolyisoprenols. Embryonic chick brain formed six-fold larger quantities of mannolipid than adult rat brain. The reaction was stimulated by Mn²⁺ ions and Triton X-100 but inhibited by EDTA. Phosphoenolpyruvic acid had no effect on the reaction. A crude mitochondrial fraction was two to three times more active than the microsomal fraction. All radioactivity in the mannolipid could be displaced by the addition of non-radioactive GDP-mannose. The endogenous lipid acceptor in brain was readily labelled in vivo by injection of [³H]mevalonate into the amniotic sac of 7-day-old embryos. The mannolipid formed had the properties of an acidic phospholipid on column and TLC, was stable to dilute alkali but readily cleaved by dilute acid. Synthesis was markedly stimulated by the addition of pig liver or calf brain dolichol phosphate in the presence of Triton X-100 and Mn²⁺. The mannolipid so formed displayed chemical characteristics identical to the endogenous lipid acceptor. Incubation of the purified radioactive mannolipid with the 'post-nuclear' fraction from 14-day-old embryonic chick brain in the presence of EDTA and Triton X-100 resulted in the transfer of 40-50 per cent of the radioactive mannose to protein and 40-45 per cent to water soluble compounds. The efficiency of transfer of radioactivity from endogenously formed mannolipid with or without the addition of dolichol phosphate was similar to exogenously added highly purified mannolipid. These results are compatible with the hypothesis that synthesis of the mannose core of brain glycoproteins involves the synthesis first of mannosyl-phosphoryl-dolichols followed by transfer of the mannose to glycoprotein.     

SYNTHESIS OF GLYCOPROTEINS AND GANGLIO-SIDES IN DEVELOPING RAT BRAIN

Abstract— Intracerebral injections of radioactive fucose into developing rats resulted in specific labelling of the brain glycoproteins in their fucose moieties. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the radioactive glycoproteins were very heterogeneous with regard to molecular weight. A procedure utilizing [³H]fucose and [¹⁴C]fucose together with double-label counting techniques was developed for comparing the electrophoretic patterns of newly synthesized glycoproteins from different samples of tissue. By the use of this procedure we showed that the incorporation of radioactive fucose into the glycoproteins of high mol. wt. was relatively greater in the brains of 5-day-old rats than in those of 25-day-old rats. Intracerebral injection of N -[ Ac -³H]acetyl- d -mannosamine resulted in a high degree of specificity for the labelling of sialic acid moieties in glycoproteins and gangliosides. The ratio of the d.p.m. of N -[³H]acetylmannosamine incorporated into glycoproteins to the d.p.m. incorporated into gangliosides was higher in 5-day-old rats than in 15- or 25-day-old rats. Experiments in which 15-day-old rats were injected with a mixture of [¹⁴C]fucose and N -[³H]acetylmannosamine showed that there were differences in the relative degrees of incorporation of the two radioactive precursors into the various glycoproteins. The greatest incorporation of [¹⁴C]fucose relative to that of N- [³H]acetylmannosamine occurred in some of the glycoproteins of smaller mol. wt.     

RAPID INTRACELLULAR TRANSPORT OF FUCOSE-CONTAINING GLYCOPROTEINS IN RETINAL GANGLION CELLS

[3H]Fucose was incorporated into glycoproteins in the rabbit retinal ganglion cells and subsequently transported at a rapid rate along the optic pathway to the nerve terminals of the lateral geniculate body and the superior colliculus. Radioautographic results indicated a preferential labelling of the terminal part of the axon. Cell fractionation showed that the major part of the transported fucose-containing glycoproteins were associated with membranes. Sodium dodecy 1 sulphate electrophoresis of rapidly transported glycoproteins showed that most of the polypeptides had a mol. wt. of more than 40,000.     

IONIC TRANSFERENCE NUMBERS IN CELLOPHANE MEMBRANES

The "apparent" cation transference number within cellophane is determined for HCl, KCl, NH₄Cl, NaCl, and LiCl. The method consists in measuring the E.M.F. in a concentration chain employing Ag:AgCl electrodes or calomel electrodes and calculating from formulas derived for cases of simple, unconstrained diffusion. The transference numbers and the cation mobilities relative to the chloride ion were found to be higher in the cellophane (relative cation mobilities increased about 40 per cent). The effect of the membrane is discussed. It is emphasized that with the introduction of a membrane as a liquid junction new factors are introduced, which are not considered in the formulas ordinarily used. Such factors may be activity changes due to dimensional or other reasons and particularly electrical effects exhibited by the membrane upon the ionic diffusion. Accordingly the transference number, as determined, may lack well defined physical significance.     

RAPID TRANSPORT OF FUCOSYL GLYCOPROTEINS TO NERVE ENDINGS IN MOUSE BRAIN

Abstract— Mice were injected intracerebrally with mixtures of [³H]fucose and [¹⁴C]gluco-samine, and incorporation into macromolecules in various subcellular fractions of brain was studied at 1, 2, 3 and 4 h after administration of the precursors. There was a lag of several hours between the incorporation of [³H]fucose into the glycoproteins of the whole brain fractions and of that into the soluble and particulate glycoproteins of the nerve ending fractions. In contrast, no lag was observed between the incorporation of [¹⁴C]glucosamine into the macromolecules of the whole brain fractions and of that into the soluble macro-molecules of the nerve ending fraction. We conclude that fucosyl glycoproteins of the nerve ending fraction were synthesized in the nerve cell bodies and transported to nerve endings by rapid axoplasmic transport, whereas a substantial proportion of the glucosamine in the soluble macromolecules of the nerve ending fraction was incorporated by the nerve endings themselves. In addition, our evidence indicates that cyclobeximide inhibited fucose incorporation into brain glycoproteins by inhibiting the synthesis of acceptor proteins rather than fucosyl transferase.     

DECREASED AXONAL FLUX OF RETROGRADELY TRANSPORTED GLYCOPROTEINS IN EARLY EXPERIMENTAL DIABETES

Abstract— Anterograde and retrograde flux of axonal transported glycoproteins were examined in streptozotocin diabetic rats with 4 weeks'duration of the metabolic derangement.
[³H]Fucose and [¹⁴C]NeuNAc were injected into the fifth lumbar root ganglion and the accumulation of TCA-PTA insoluble activity proximal and distal to a sciatic nerve ligature was measured.
Accumulation of glycoproteins during 2 h collection periods was decreased distal to a ligature in diabetic animals whereas no abnormality of proximal accumulation was observed. These findings demonstrate an abnormality of the retrograde transport of glycoproteins in early experimental diabetes.     

AGE VARIATIONS IN CORTICAL MEMBRANES OF ROTIFERS

Electron micrographs were made of rotifers of known ages embedded in Vestopal and stained with lead hydroxide. In epithelial cells lining the gut of the rotifer, anatomical continuity is seen between the cell membrane, the cortical membranes, and the rough endoplasmic reticulum at the basal region of the cells. At their luminal surface, these cells possess a terminal web, and colloidal gold is taken up via food vacuoles at this surface. Such pinocytosis does not occur on the peripheral or distal surface (pseudocoelomic) of these cells. Exposure of rotifers to adenine inhibits the formation of cortical infoldings of the cell membrane and the formation of the rough reticulum, whereas the ribosomes and the Golgi apparatus appear to be unaffected. The suggestion is made that the rough reticulum may be derived from modifications of the cell membrane and that the rate of formation of the rough reticulum in adult and elderly rotifers is lower than in the actively growing animal.     

HISTOCHEMICAL STAINING AND CHARACTERIZATION OF GLYCOPROTEINS IN ACID-SECRETING CELLS OF FROG STOMACH

Glycoproteins were histochemically localized in oxyntic cells of the frog stomach by staining with periodic acid-silver methenamine. Reduction of silver was most intense on (a) the outer aspect of the apical plasmalemma, (b) within the tubular smooth membrane system characteristic of oxyntic cells, and (c) within cisternae and vesicles of the Golgi complex. Other membrane components such as those from the mitochondria, nucleus, junctional complex, lateral and basal cell membranes showed little or no stainability. Gastric mucosal homogenates were fractionated by centrifugation for further morphological and chemical analysis. The staining reaction of the microsomal fraction (40,000 g x 60 min) was similar to that of the tubular membranous components of intact oxyntic cells. Carbohydrate analyses showed that all cell fractions are extremely low in acidic sugars, uronic and sialic acids, while neutral sugars and hexosamines are relatively abundant. The microsomal fraction contains the largest proportion of carbohydrates, ca. 9% of the fat-free dry weight. Another distinguishing feature is that glucosamine is the only detectable hexosamine in the microsomal fraction. These histochemical and chemical data indicate that neutral glycoproteins are associated with membranous components which have been implicated in the process of HCl secretion by oxyntic cells. The staining pattern within the cells supports the hypothesis of interrelationships between the Golgi membranes, tubular smooth membranes, and apical surface membrane.     

汉滩病毒糖蛋白G1G2和核蛋白NP在痘苗病毒天坛株中的联合表达

以汉滩病毒７６／１１８株Ｍ、Ｓ片段分别插入转南粒ＰＪＳＢ１１７５的Ｐ１１和Ｐ７．５启动子下游,构建成重组质粒ＰＪＳＢ１１７．５Ｓ。采用Ｌｉｐｏｆｅｃｔｉｎ转染针重组质粒分别与痘苗病毒天坛株ＴＫｖｖ和表达Ｓ片的重线痘苗病毒ＶＪＳＡ１１７５Ｓ进行共转染,免疫酶斑和蓝白筛法筛选并纯化,得到了重组病毒ＶＪＳＢ１１Ｍ５Ｓ。Ｂｕｄｒ的选择压力试验表明,外源基因确定插入在ＴＫ基因区;ＰＣＲ及打点杂交证实了两个片     

THE SIZE OF PORES IN COLLODION MEMBRANES

By the application of Poiseuille''s law to the rate of flow of water through collodion membranes, it is calculated that the membranes used had pore radii of the order of 0.3 to 2 x 10^–6 cm. On the same basis the number of pores per sq. cm. appears to vary from 270 x 10¹⁰ to 7 x 10¹⁰, decreasing with increase in pore size. Reasons are given for preferring these figures for the radii to figures, 100 times as large, which were calculated by others. Microscopic examination of the membranes, with dark-field illumination, indicates that they are made up of solid granules or filaments of collodion much less than 1 x 10^–4 cm. in thickness.     

AXONAL TRANSPORT OF SULFATED GLYCOPROTEINS AND MUCOPOLYSACCHARIDES IN THE GARFISH OLFACTORY NERVE

—Application of ³⁵SO₄ to the olfactory mucosa of the long-nosed garfish is found to label sulfated macromolecules which are transported down the olfactory nerve. The transported molecules pass along the nerve as a discrete peak whose leading edge has a transport velocity of 206 ± 6 mm/day. A large portion of the radioactivity from the peak is deposited along the axon. At 2 days after isotope application 83% of the total nerve radioactivity is in the axons and the remaining 17% has accumulated at the terminals in the olfactory bulb. Characterization of sulfated material in the migrating peak indicates that both sulfated glycoproteins (isolated as glycopeptides) and mucopolysaccharides, including chondroitin sulfate and heparan sulfate, are undergoing transport.     

OBSERVATIONS ON THE BASEMENT MEMBRANES IN RAT KIDNEY

Basement membranes in the kidney are made up of a homogeneous matrix. In argyria, silver passes from the blood in the ionic form and diffuses into the kidney basement membranes in which it is precipitated. X-ray diffraction studies of "silver-stained" rat kidneys show that most of the silver in the kidneys is combined with some form of sulfur. Histochemical staining for sulfhydryls and disulfides demonstrates the presence of these groups in basement membranes. It appears that silver ions combine with either or both the sulfhydryl or disulfide groups in the basement membranes and also in mitochondria (when the silver diffuses into a cell).     

粘液糖蛋白去糖基化作用的研究进展

由于粘液糖蛋白中的寡糖侧链对其蛋白质多肽链的水解具有保护作用,为了研究粘液糖蛋白多肽链的结构与功能,必须进行去糖基化作用的处理。处理的方法主要有酶法和化学法。到目前为止,以碘酸三氯甲烷(TFMSA)预处理,再以高碘酸盐氧化,然后在碱性条件下进行β-消去的方法最为理想。