Endopeptidase Isoenzyme Characteristics in Cucumis sativus Leaves During Dark-induced Senescence

The changes and characteristics of endopeptidase （EP） isoenzymes in cucumber （Cucumis sativus L.） leaves during dark-induced senescence were investigated by activity staining after gradient-polyacrylamide gel electrophoresis （G-PAGE） containing co-polymerized gelatin as substrate. The results showed that both the chlorophyll and the protein contents of leaves were decreased, and the protein degradation was correlated with the increase of proteolytic activity during the course of leaf senescence. Meanwhile, nine cucumber endopeptidases isoenzymes （CEP） with 140, 120, 106, 94, 76, 55, 46, 39 and 35 kDa molecular weights were detected. Four of these, CEP2, 3, 4 and CEP9 appeared all the time, but the changes of the activity were different during incubation. Another four CEPs （CEP5, 6, 7 and CEP8） whose activities increased with dark-induced time were only detected in senescent leaves. Furthermore, the biochemical properties of these nine CEP were also characterized. All the CEPs had high activities from 35 ℃ to 45 ℃, and the optimum temperature was found to be 40 ℃. However, the activities of CEPs were not detected below 25 ℃ or over 60 ℃. The activity bands appeared at a wide range of pH from 5.0 to 9.0, but the optimum pH was found at 7.0. No CEPs were detected at pH 4 or pH 10. By inhibition analysis we concluded that CEP2, 3, 4 and CEP9 were serine endopeptidases and CEP6 was a kind of cysteine protease. It is suggested that serine endopeptidases might play a major role in cucumber leaf senescence, and for the first time, six senescencerelated endopeptidases （CEP1, 5, 6, 7, 8 and 9） were found in cucumber leaves.     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase／Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Characters of Cysteine Endopeptidases in Wheat Endosperm during Seed Germination and Subsequent Seedling Growth

The endopeptidases （EPs） in wheat endosperm during seed germination and subsequent seedling growth were characterized by gradient-polyacrylamide gel electrophoresis with gelatin copolymerized into the gel. Four cysteine EPs （EP1, EP2, EP3 and EP4） were detected in wheat endosperm during the 7 d growth after seed imbibition. The results also showed that the activities of all of these EPs increased continuously, and EP2 first appeared and had the highest proteolytic activity among the four EPs in this experimental process. The optimum pH and temperature of all four EPs were 4.0 and 40.0 ~C. All EPs were completely inhibited by 25 μmol/L E-64 and had no good thermal stabilities, especially EP1. In addition, these EPs had different substrate specificities to albumins, globulins, gliadins and glutenins; the main storage proteins of mature wheat endosperm. Among them, EP2 had the highest proteolytic activities on globulins, gliadins and glutenins, and might be the most important and specific EP with potential to be tightly correlated with seedling development.     

Degradation of Rubulose-1．5-Bisphosphate Carboxylase／Oxygenase in Wheat Leaves During Dark-induced Senescence

The degradation of Ribulose-1.5-bisphosphate carboxylase／oxygenase(Rubisco,EC4.1.1.39)in wheat(TnticumaestivumL．CV．Yan6mai 158)leaves dunng dark—induced senescence was studied．An／in vivo degradation product of Rubisco large subunit(LSU)with molecular weiht of 50kD was detected by SDS—PAGE and immunobloted with antibody against tobacco Rubisco．This fragment could also be detected in natural senescence．The result also suggested that the Rubisco holoenzyme had not dissociated when LSU hydrolyzed from 53 kD to 50kD．And．LSUcould be fragmented to 50kD at 30-35℃and at DH7．5 in crude enzyme extracts of wheat leaves dark—induced for 48h．which suggested that maybe LSU was degraded to 50 kD by anunknown protease in chloroplast．     

Effects of salicylic acid on protein kinase activity and chloroplast D1 protein degradation in wheat leaves subjected to heat and high light stress

In the north of China, wheat plants are often stressed by heat and high light during grain-filling stage, which leads to injury in photosynthetic apparatus and decline in photosynthetic rate. In order to develop a method to protect photosynthetic apparatus in wheat leaves subjected to heat and high light stress, the effects of SA (salicylic acid) and FSBA (5′-p-fluorosulfonylbenzoyl adenosine) on PK (protein kinase) activity, D1 protein degradation and the performance of PSII were investigated in present work. Our results showed that PK activity enhanced under heat and high light stress and declined when stress was removed. FSBA pretreatment resulted in marked decreases in PK activity and D1 protein level, suggesting a correlationship between degradation of D1 protein and phosphorylation. After 2 h of stress, D1 protein level in water-pretreated leaves decreased to 79% of control and then recovered to 81% after 3 h of recovery. This clearly indicated that the damage of D1 protein induced by heat and high light stress was reversible. Compared to the control, SA pretreatment could not only increase PK activity, retard the degradation of D1 protein during heat and high light stress, but also accelerate the recovery of D1 protein level when the stress was removed. Correspondingly, F_v/F_m (maximum photochemical efficiency of PSII), Φ_PSII (actual photochemical efficiency of PSII), ETR (electron transfer rate) and Pn (net photosynthetic rate) in SA-treated leaves were higher than that in leaves of control under both stress and non-stress conditions. Taken together, our results revealed that SA pretreatment could significantly alleviate damages of heat and high light stress on D1 protein and PSII of wheat leaves, and accelerate restoration of photosynthetic function.     

Effects of salicylic acid on protein kinase activity and chloroplast D1 protein degradation in wheat leaves subjected to heat and high light stress

In the north of China, wheat plants are often stressed by heat and high light during grain-filling stage, which leads to injury in photosynthetic apparatus and decline in photosynthetic rate. In order to develop a method to protect photosynthetic apparatus in wheat leaves subjected to heat and high light stress, the effects of SA (salicylic acid) and FSBA (5′-p-fluorosulfonylbenzoyl adenosine) on PK (protein kinase) activity, D1 protein degradation and the performance of PSII were investigated in present work. Our results showed that PK activity enhanced under heat and high light stress and declined when stress was removed. FSBA pretreatment resulted in marked decreases in PK activity and D1 protein level, suggesting a correlationship between degradation of D1 protein and phosphorylation. After 2 h of stress, D1 protein level in water-pretreated leaves decreased to 79% of control and then recovered to 81% after 3 h of recovery. This clearly indicated that the damage of D1 protein induced by heat and high light stress was reversible. Compared to the control, SA pretreatment could not only increase PK activity, retard the degradation of D1 protein during heat and high light stress, but also accelerate the recovery of D1 protein level when the stress was removed. Correspondingly, F_v/F_m (maximum photochemical efficiency of PSII), Φ_PSII (actual photochemical efficiency of PSII), ETR (electron transfer rate) and Pn (net photosynthetic rate) in SA-treated leaves were higher than that in leaves of control under both stress and non-stress conditions. Taken together, our results revealed that SA pretreatment could significantly alleviate damages of heat and high light stress on D1 protein and PSII of wheat leaves, and accelerate restoration of photosynthetic function.     

Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50℃ and 60 ℃; for EG45 it was 50 ℃. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 ℃, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 ℃ for 24 h. However, less than 10% residual activity of EG45 was detected at 50 ℃. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan （from oat spelt） and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.     

菠菜叶片中乙醇酸氧化酶3种同工酶的生化特性

By DEAE cellulose and Sepharose 6B chromatography, the proteins containing glycolate oxidase isozymes GOⅡ and GOⅢ were extracted from spinach green leaves. The protein containing GOⅡ showed two bands of 67±2 kD and 40±2 kD in SDS PAGE whose specific activity of glycolate oxidase was 33.4 U·mg -1 ·min -1 .It migrated towards cathode in Native PAGE in pH 8.3 buffer system. pI of GOⅡwas about 9.4 detected by IEF. The protein containing GOⅢ showed three bands of 67±2 kD, 40±2 kD and 38±2 kD in SDS PAGE whose specific activity of glycolate oxidase 14.4 U·mg -1 ·min -1 and could not migrate anywhere in the same Native PAGE. pI of GOⅢ was about 8.3 detected by IEF. The 40±2 kD might be the subunits of GOⅡ and GOⅢ. Antibodies of the protein containing GOⅡ and GOⅢ were prepared respectively. GOⅡ was very unstable and could change into GOⅢ artifact; GOⅢ was also unstable and could change into GOⅠartifact whose Mr ≈470 kD and pI ≈7.4 . This GOⅠ(specific activity: 9.8 U·mg -1 ·min -1 ), showing one 40±2 kD band in SDS PAGE, could be purified on another Sepharose 6B chromatography. The specific activity of GOⅡ decreased rapidly to about half of its original value and then was relatively stable when stored in 50% glycerol at -20℃. The results above explained why GOⅡ was extracted difficultly, and GOⅢ were easily confused with GOⅠ and GOⅡ.     

Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants （Camellia sinensis （L.） O. Kuntze）

β-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity,with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50℃ and was stable at temperatures lower than 40℃. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.     

Structural and Functional Damage Caused by Boron Deficiency in Sunflower Leaves

Boron deficiency induced a dramatic inhibition in sunflower plant growth, shown by a reduction in dry mass of roots and shoots of plants grown for 10 d in nutrient solution supplied with 0.02 μM B. This low B supply facilitated the appearance of brown purple pigmentation on the plant leaves over the entire growth period. Compared to B-sufficient (BS) leaves, leakage from B-deficient (BD) leaves was 20 fold higher for potassium, 38 fold for sucrose, and 6 fold for phenolic compounds. High level of membrane peroxidation was detected by measuring peroxidase activities as well as peroxidative products in BD sunflower plants. Soluble and bound peroxidase activities measured in BD thylakoid membranes were accelerated two fold compared to those detected in BS-membranes. No detectable change in soluble peroxidase activity in roots whereas a 4 fold stimulation in bound peroxidase activity was detected. Thylakoid membranes subjected to low B supply showed enhancement in lipoxygenase activity and malondialdehyde (MDA) content in parallel with 40 and 30 % decrease of linoleic and linolenic acid contents (related to total unsaturated fatty acids). A slower rate of Hill reaction activity (40 %) and a suppressed flow of electron transfer of the whole chain (30 %) were detected in BD thylakoid membranes. This reduction was accompanied with a decline in the activity of photosystem 2 shown by a diminished rate of oxygen evolution (42 %) coupled with a quenching (27.5 %) in chlorophyll a fluorescence emission spectra at 685 nm (F₆₈₅). Thus B is an important element for membrane maintenance, protection, and function by minimizing or limiting production of free oxygen radicals in thylakoid membranes of sunflower leaves. This revised version was published online in June 2006 with corrections to the Cover Date.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.