Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the ‘immobilization antigen’, constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

第四双小核草履虫减数分裂产物的退化特征分析

通过吖啶橙和Hoechst 33342两种活体荧光染料双染的方法对第四双小核草履虫(Paramecium tetraurelia)接合生殖过程中小核减数分裂产物进行观察,结果发现位于口旁锥外的小核分裂产物呈蓝绿色或黄绿色,表明它们以凋亡的方式发生退化.     

The effect of temperature on candidate gene expression in the brain of honey bee Apis mellifera (Hymenoptera: Apidae) workers exposed to neonicotinoid imidacloprid

Neonicotinoid insecticides are potent agonists of nicotinic acetylcholine receptors and are a major factor in the decline of pollinators worldwide. Several studies show that low doses of this neurotoxin influence honey bee physiology, however, little is known about how insecticides interact with other environmental variables. We studied the effects of two neonicotinoid Imidacloprid doses (IMD, 0, 2.5, and 10 ppb), and three temperatures (20, 28, and 36°C) on gene expression in the brains of worker honey bees (Apis mellifera). Using qRT-PCR we quantified the expression of eight key genes related to the nervous system, stress response, and motor and olfactory capacities. Gene expression tended to increase with the low IMD dose, which was further intensified in individuals maintained in the cold treatment (20°C). At 20°C the octopamine receptor gene (oa1) was underexpressed in bees that were not exposed to IMD, but overexpressed in individuals exposed to 2.5 ppb IMD. Also, heat shock proteins (hsp70 and hsp90) increased their expression at high temperatures (36°C), but not with IMD doses. These results suggest that despite the low insecticide concentrations used in this study (a field-realistic dose), changes in gene expression associated with honey bee physiological responses could be induced. This study contributes to the understanding of how neonicotinoid residual doses may alter honey bee physiology.     

The effect of temperature on veratridine action in squid giant axons

Resting membrane potential and intracellular sodium and potassium concentrations were determined at 5 and 21°C in normal and veratridine-treated axons of the squid Doryteuthis plei. 300 μM veratridine produced an increase in the intracellular sodium concentration, which changed from 52 to 284 mM in 10 min of exposure at 21°C, and from 76 to 260 mM at 5°C. Under the same treatment the intracellular potassium concentration changed from 357 to 221 mM (21°C) and from 334 to 194 mM (5°C). All the changes could be prevented by adding 1 μM tetrodotoxin. Veratridine (30, 100 and 300 μM) increased the resting sodium permeability of the giant axon, and the effect was greater at 21°C. The affinity of the membrane for veratridine increases when the nerves are cooled, the three concentrations tested produce maximum activation of the sodium channels at 5°C. But only the higher two concentrations are saturating at 21°C.     

Molecular expression of xylanase gene in Cryptococcus albidus

In the yeast Cryptococcus albidus, the utilization of xylan as compared to xylose requires at least an inducible endoxylanase enzyme, secreted in the culture medium. The endoxylanase induction was monitored by immunoprecipitation of in vivo and in vitro synthesized products. The mature endoxylanase is a highly glycosylated enzyme with an apparent molecular weight of 48000. Upon chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight was reduced to 40000. Addition of tunicamycin to the culture medium resulted in the synthesis of a modified polypeptide having a molecular weight of 40000. Poly(A)-containing RNA isolated from the yeast was translated in the rabbit reticulocyte protein-synthesizing system. The appearance of a translatable xylanase mRNA was observed in xylan-grown cells but not in xylose-grown cells. The polypeptide identified as xylanase had a molecular weight of 44000. This suggests that the xylanase is synthesized as a precursor, containing a peptide signal sequence of 35 residues.     

The effect of temperature change on gene expression in Paramecium primaurelia

When paramecia grown at 24 degrees C are transferred rapidly to 32 degrees C, DNA and protein synthesis continue uninterrupted but at higher rates. Electron microscopic observations indicate that more of the macronuclear chromatin is transcribed at the elevated temperature. This interpretation is supported by hybridization experiments which show that the percentage of the macronuclear genome transcribed into poly(A)+RNA is 24 degrees C and 35% at 32 degrees C. Kinetic analysis of cDNA-poly(A)+RNA hybridizations reveals three abundance classes of poly(A)+RNA and indicates that the number of genes expressing low abundance sequences is about 9000 at 24 degrees C and 13000 at 32 degrees C. The intermediately abundant and highly abundant classes are represented by 100-200 and 1-3 different kinds of RNA sequence, respectively. Cross hybridization shows that changes occur throughout the distribution of abundance classes of poly(A)+RNA with increase in temperature.     

Differential gene expression in lymphocytes from malnourished children

Malnutrition, which is widespread in developing countries, may be particularly devastating during childhood, when tissue development is occurring and nutrient requirements are great. Since protein-energy malnutrition potentially involves many cellular alterations, we have evaluated gene expression changes in lymphocytes from malnourished children using differential hybridization cloning. A cDNA library was generated from well-nourished children and differential screenings were performed with cDNAs obtained from well-nourished and malnourished children who presented with bacterial gastrointestinal infections. Differential expression was detected for genes involved in cell development and differentiation, and for genes involved in lymphocyte and mitochondrial functions. The genes detected in the present study suggest mechanisms for the changes in cell growth and immune function that are associated with protein-energy malnutrition. Two down-regulated genes in malnourished children may represent mechanisms of protection against immunosuppression. This finding clearly merits further investigation.     

Differential mRNA expression levels and gene sequences of carboxylesterase in both deltamethrin resistant and susceptible strains of the cotton aphid, Aphis gossypii

Extensive use of insecticides on cotton has prompted resistance development in the cotton aphid, Aphis gossypii (Glover) in China. A deltamethrin‐selected population of cotton aphids from Xinjiang Uygur Autonomous Region, China with 228.59‐fold higher resistance to deltamethrin was used to examine how carboxylesterase conferred resistance to this pyrethroid insecticide. The carboxylesterase activity in the deltamethrin‐resistant strain was 3.67‐, 2.02‐ and 1.16‐fold of the susceptible strain when using α‐naphthyl acetate (α‐NA), β‐naphthyl acetate (β‐NA) and α‐naphthyl butyrate (α‐NB) as substrates, respectively. Carboxylesterase cDNA was cloned and sequenced from both deltamethrin‐resistant and susceptible strains. The cDNA contained 1581 bp open reading frames (ORFs) coding a 526 amino acid protein. Only one amino acid substitution (Val⁸⁷‐Ala) was observed between deltamethrin‐resistant and susceptible strains but it is not genetically linked to resistance by the catalytic triad and signature motif analysis. The real‐time polymerase chain reaction analysis indicated that the resistant strain had a 6.61‐fold higher level of carboxylesterase mRNA than the susceptible strain. The results revealed that up‐regulation of the carboxylesterase gene, not modified gene structure, may be responsible for the development of resistance in cotton aphids to deltamethrin.     

温度对香蕉花蓟马发育和存活的影响

通过在人工气候箱内饲养香蕉花蓟马,测定了14、18、22、26℃和30℃5个温度下香蕉花蓟马不同虫态的发育历期,分析了温度与香蕉花蓟马发育速率的关系,计算了其发育起点温度、有效积温及存活率。结果表明:香蕉花蓟马各虫态的发育历期随温度的升高而缩短,发育速率和温度之间有很大的相关性;香蕉花蓟马卵、一龄若虫、二龄若虫、预蛹、蛹及世代的发育起点温度分别为8.26、10.56、7.92、9.17、9.53、8.69℃,世代的有效积温为153.81d·℃。26℃下香蕉花蓟马的世代存活率最高,为63.16%;30℃下的存活率最低,为46.34%。     

基因表达差异显示分析技术在创伤研究中的应用

基因是细胞增殖,分化,成熟等各项生命活动的调控中心,也是许多痢疾发生,发展和转归的决定性因素。基因表达的变化必然导致细胞,组织,器官乃至整个机体的各种异常。包括创伤在内的各种内外刺激,都可不同程度地引起基因表达的变化,最终妨碍机体健康。随着生物信息学的逐渐兴起和分子生物学的不断发展并向其他学科的逐渐渗透,业已建立起一系列研究基因表达变化的切实可行的技术手段（即“基因表达差异分析技术”,如DNA微阵列）,对捕获基因表达的种种变化具有重要价值。这些技术已经在肿瘤及其他疾病的研究中得到广泛应用,近几年也逐渐进入创伤研究领域,在一定程度上推动了创伤研究的发展。