Amino-Terminal Charge Affects the Periplasmic Accumulation of Recombinant Heregulin/EGF Hybrids Exported Using theEscherichia coliAlkaline Phosphatase Signal Sequence

AnEscherichia coliexpression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA–hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated byin vitroreplacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein inE. coliperiplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed inE. coliusing the PhoA signal sequence for protein export.     

Cytological and mapping analysis of a novel male sterile type resulting from spontaneous floral organ homeotic conversion in marigold (<Emphasis Type="Italic">Tagetes erecta</Emphasis> L.)

A male sterile line was isolated in marigold (Tagetes erecta L.) and cytological analysis determined this to be a novel genic male sterility trait (Tems). Through the use of amplified fragment length polymorphisms (AFLPs) and bulked segregant analysis (BSA), tightly linked markers of Tems were identified with a view towards a map-based cloning strategy. It was found that spontaneous homeotic conversion of floral organs was the underlying cause of the male sterility in this marigold line. Thus, petals of male sterile plants resembled sepal-like structures and the stamens were partially converted to styles, although without the full characteristics or function of the true style organs. We have constructed a fine marker-based map for the Tems gene. This is intended to provide a tool for marker assisted selection (MAS) strategies in hybrid breeding and map-based cloning strategies for the male sterility locus. We discuss the significance of this spontaneously derived genic male sterility trait relating to the homeotic conversion of floral organs in marigold.     

穿越海南

2001年4月6日 晴 从飞机的舷梯上走下来,一股温湿的风立刻笼罩了我,海南——在我对它有过多次幻想之后的今天,终于又再次踏上了这块葱绿的土地。在接到拍摄任务的几天之内,我甚至请朋友用纸牌占卜——此行安否?答案是有惊无险!唯物主义的我穿着套头衫在北京料峭的春风中登上了飞机,开始了我的穿越海南之行。     

Clinical manifestations of taeniasis in Taiwan aborigines.

From 1974 to 1989, a total of 24,500 aborigines at 67 villages in ten mountainous districts/towns in Taiwan were examined for the Taiwan Taenia infection and 12% were found to be infected. In order to define the clinical manifestations of taeniasis caused by the Taiwan Taenia, 1661 aborigines in ten mountainous districts were surveyed. The overall clinical rate was 76%. The clinical rate was highest among Atayal aborigines (81%), followed by Bunun (66%) and Yami (61%) aborgines and lowest among Ami aborigines (40%). Among 1153 infected people, 10% had passed gravid segments in the faeces for less than 1 year, 24% for 1-3 years, 17% for 4-5 years, 23% for 6-10 years, 16% for 11-20 years, 7% for 21-30 years, and 3% over 30 years. Twenty-six occurrences of gastrointestinal and neurological symptoms were reported by 1258 infected persons. Passing proglottides in the faeces (95%) was the most frequent sign, followed by pruritis ani (77%), nausea (46%), abdominal pain (45%), dizziness (42%), increased appetite (30%), headache (26%), etc.     

Ecotoxicity of soils contaminated with industrial and domestic wastewater in western shenyang,China

Soil samples were collected from 7 sites in the up-, mid-and down-reach along and nearby the wastewater irrigation channel, western Shenyang of China. The concentrations of selected pollutants (mineral oil, PAHs - polycycle aromatic hydrocarbons and Cd) were determined by UV spectrometer, HPLC and AAS (atomic adsorption spectrometer) spectrometer, respectively. Toxicity effects of soils were evaluated by seedling emergence test with root length of wheat as the end-point and by earthworms test with the mortality rate and inhibition rates of body weight as endpoints. Results showed accumulation of pollutants for most soils with concentration of 200.2 mg.kg⁻¹∼1600 mg.kg⁻¹ for mineral oil, 0.33 mg.kg⁻¹∼1.81 mg.kg⁻¹ for Cd and 900.16 mg.kg⁻¹ ∼ 2737.91 mg.kg⁻¹ for PAHs. The inhibition rates of root elongation were from −20% up to 40 %, and mortality rates of earthworms ranged from 0%∼40% from the exposure period of two weeks to eight weeks by sampling interval of two weeks, the inhibition rates of earthworm growth were from −19.36% to 34.53%, showing effects of stimulation at 2 weeks to an increasing effects of inhibition at 4, 6 and 8 weeks, respectively. Mortality rates correlated with the loss of body weight of earthworms.

This study indicated the potential risk of pollutants of environmental low content in soil by the determination of selected chemicals combined with toxicity indexes.

     

Irreversible inhibition of the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase/9-ketoprostaglandin reductase by glutathione thiosulfonate

Oxidation of glutathione disulfide by a mixture of performic and hydrochloric acids leads to the formation of several compounds that are stronger inhibitors than glutathione disulfide of the placental enzyme that possess both NADP-linked 15-hydroxyprostaglandin dehydrogenase and 9-ketoprostaglandin reductase activities. The only one of these inhibitors that has been identified is glutathione thiosulfonate. The others are unstable and may include glutathione sulfinyl sulfone and glutathione disulfone. Since the enzyme appears to have a glutathione binding site in close proximity to its active site and glutathione thiosulfonate reacts with free sulfhydryl groups, the effects of this thiosulfonate on the enzyme were examined in more detail. Glutathione thiosulfonate and methyl methanethiosulfonate cause a time-dependent irreversible inhibition of both the hydroxyprostaglandin dehydrogenase and the ketoprostaglandin reductase activities, presumably by reacting with a free sulfhydryl at the prostaglandin binding site. Experiments with PGA1-glutathione show that this sulfhydryl is not necessary for the catalytic activity of the enzyme as long as the substrate can bind at the glutathione site.     

Ruthenium ammine complexes as electron acceptors for growth stimulation by plasma membrane electron transport

Ammineruthenium(III) complexes have been found to act as electron acceptors for the transplasmalemma electron transport system of animal cells. The active complexes hexaammineruthenium(III), pyridine pentaammineruthenium(III), and chloropentaammineruthenium(III) range in redox potential (E ₀) from 305 to –42 mV. These compounds also act as electron acceptors for the NADH dehydrogenase of isolated plasma membranes. Stimulation of HeLa cell growth, in the absence of calf serum, by these compounds provides evidence that growth stimulation by the transplasma membrane electron transport system is not entirely based on reduction and uptake of iron.     

Deactivation ofCandida rugosa lipase

Summary Deactivation ofCandida rugosa lipase was found to be complex. Hydrophobic interaction induced by iso-octane influenced the initial phase of deactivation, and increased the turn-over rate of the intermediate in the transition phase. After urea-treatment the structure of the last phase was not further influenced by thermal treatment, whereas that of initial phase was more sensitive to temperature change. Charge interaction was important in maintaining the structure during the deactivation, and especially anion charge might be a major factor.