Cell surface-expression of the phosphate-binding protein PstS: System development, characterization, and evaluation for phosphorus removal and recovery

Simultaneous overabundance and scarcity of inorganic phosphate(P_i) is a critical issue driving the development of innovative water/wastewater treatment technologies that not only facilitate Pi removal to prevent eutrophication,but also recover P_i for agricultural reuse.Here,a cell-surface expressed high-affinity phosphate binding protein(PstS) system was developed,and its P_i capture and release potential was evaluated.E.coli was genetically modified to express PstS on its outer membrane using the ice nucleation protein(INP)as an anchoring motif.Verification of protein expression and localization were performed utilizing SDS-polyacrylamide gel electrophoresis(SDS-PAGE),western blot,and outer membrane separation analyses.Cell surface characterization was investigated through acid-base titration,X-ray photoelectron spectroscopy(XPS),and Fourier transform infrared spectroscopy(FTIR).These tests provided information on the macromolecular structure and composition of the bacteria surface as well as the proton-exchange properties of the surface functional groups(i.e.,pKa values).Phosphate desorption and adsorption batch experiments were conducted to evaluate the effects of temperature,pH,and ionic strength on phosphate capture and release.The PstS surface-displayed cells demonstrated greater potential to release and capture phosphate compared to non-modified cells.Higher temperatures up to 40℃,basic pH conditions(pH=10.5),and higher ionic strength up to 1.0 mol/L KCl promoted 20%-50% higher phosphate release.     

Molecular analysis of genomic DNA allows rapid,and accurate,prenatal diagnosis of peroxisomal D-bifunctional protein deficiency

Prenatal diagnosis was requested for a couple with a previous child affected by the peroxisomal disorder D-bifunctional protein deficiency. Prior analysis of the D-bifunctional protein cDNA sequence from the propositus had shown that it was missing 22 bp. This was subsequently attributed to a point mutation in the intron 5 donor site (IVS5+1G>C) of the D-bifunctional protein gene. Consistent with parental consanguinity, the patient was shown to be homozygous for this mutation, which is associated with loss of a Hph 1 restriction site in the genomic sequence. Prenatal testing of the fetus using genomic DNA isolated from uncultured amniocytes indicated that both alleles of the D-bifunctional protein had the IVS5+1G>C substitution. The peroxisomal defect was later confirmed biochemically using cultured amniocytes, which were found to have elevated levels of very long chain fatty acids (VLCFA). This is the first report of prenatal diagnosis of D-bifunctional protein deficiency using molecular analysis of genomic DNA. Copyright © 2002 John Wiley & Sons, Ltd.     

Procurement of exogenous ammonia by the swallowtail butterfly, Papilio polytes, for protein biosynthesis and sperm production

How to acquire sufficient quantity of nitrogen is a pivotal issue for herbivores, particularly for lepidopterans (butterflies and moths) of which diet quality greatly differs among their life stages. Male Lepidoptera often feed from mud puddles, dung, and carrion, a behavior known as puddling, which is thought to be supplementary feeding targeted chiefly at sodium. During copulation, males transfer a spermatophore to females that contains, besides sperm, nutrients (nuptial gifts) rich in sodium, proteins, and amino acids. However, it is still poorly understood how adults, mostly nectarivores, extract nitrogen from the environment. We examined the availability of two ubiquitous inorganic nitrogenous ions in nature, viz. ammonium (or ammonia) and nitrate ions, as nutrients in a butterfly, and show that exogenous ammonia ingested by adult males of the swallowtail, Papilio polytes, can serve as a resource for protein biosynthesis. Feeding experiments with ¹⁵N-labeled ammonium chloride revealed that nitrogen was incorporated into eupyrene spermatozoa, seminal protein, and thoracic muscle. Ammonia uptake by males significantly increased the number of eupyrene sperms in the reproductive tract tissues. The females also had the capacity to assimilate ammonia into egg protein. Consequently, it is evident that acquired ammonia is utilized for the replenishment of proteins allocable for reproduction and somatic maintenance. The active exploitation of exogenous ammonia as a nutrient by a butterfly would foster better understanding of the foraging and reproductive strategies in insects.     

Phytotoxicity of cadmium on protein, proline and antioxidant enzyme activities in growing Arachis hypogaea L. seedlings

Phytotoxicity of cadmium on growing Arachis hypogaea L. seedlings was studied. Seeds were exposed to 25, 50, and 100 μmol/L CdCl2 concentrations, for a period of 10, 15, 20 and 25 d. The extent of damage to chlorophyll, protein, proline, nitrate and nitrite reductase, antioxidant enzyme activity in leaves and roots were evaluated after 10 d of cadmium stress. The higher concentration of cadmium （100 μmol/L） resulted （leaves and roots） total chlorophyll 91.01%, protein 79.51%, 83.61%, nitrate reductase 79.39%, 80.72% and nitrite reductase 77.07%, 75.88% activity decreased with increase in cadmium concentrations and exposure periods. Cadmium caused significant changes in the activity of antioxidative enzymes. Contrastingly Cd treated plant tissues showed an increase in proline 159.87%, 239.6%, gluthion reductase （GR） 337.72%, 306.14%, superoxide disumutase （SOD） 688.56%, 381.72%, ascorbate peroxidase （APX） 226.47%, 252.14%, peroxidase （POD） 72.19%, 60.29% and catalase （CAT） 228.96%, 214.74% as compared to control. Cadmium stress caused a significant increase in the rate of SOD activity in leaves and roots of plant species. Results show the crop A. hypogaea is highly sensitive even at very low cadmium concentrations.     

Maternal serum human chorionic gonadotrophin and pregnancy-associated plasma protein a,markers for fetal down syndrome at 8–14 weeks

Maternal serum levels of human chorionic gonadotrophin and its subunits (intact, α, and free βhCG) and pregnancy-associated plasma protein A (PAPP-A) were measured in 279 women between 8 and 14 weeks' gestation. This group included 23 pregnancies in which the fetus had Down syndrome (DS), diagnosed either at birth or during the second trimester (n=17) or from chorionic villus sampling (CVS) (n=6). Normal medians were determined from the 258 apparently normal pregnancies. The median levels of intact hCG (1·4 MOM) and free βhCG (2·1 MOM) were significantly raised, whereas the median level of PAPP-A (0·39 MOM) was significantly lower in the DS pregnancies when compared with the control group. Levels of αhCG were similar in both the control and the DS pregnancies. Analysis of samples taken prior to 14 weeks' gestation demonstrated that only PAPP-A (0·34 MOM) was significantly altered in DS pregnancies. However, after the exclusion of DS cases diagnosed at CVS, the median intact hCG (1·56 MOM), free βhCG (2·27 MOM), and αhCG (1·8 MOM) were all raised in DS pregnancies. This emphasizes the problem of the interpretation of biochemical markers when DS cases are diagnosed at CVS.     

鲢鱼、草鱼和尼罗罗非鱼热休克蛋白70基因cDNA全序列的克隆与分析

采用RT-PCR技术和RACE技术分别从淡水食毒藻鱼类鲢鱼（Hypophthalmichthys molitrix）、尼罗罗非鱼（Oreochromis nilotica）及草食性鱼类草鱼（Ctenopharyngodon idella）肝脏扩增出热休克蛋白70（HSP70）cDNA全序列,并与其它鱼类、两栖类和哺乳类动物的HSP70氨基酸进行了同源性比较. 结果表明,鲢鱼、草鱼和尼罗罗非鱼肝脏HSP70基因cDNA全长分别为2356 bp、2348 bp和2242 bp,分别编码649、649和638个氨基酸.鲢鱼、草鱼和尼罗罗非鱼HSP70与其它鱼类、两栖类和哺乳类动物HSP70氨基酸同源性均较高,表明其在进化上高度保守,且承担着重要的生理功能.构建系统进化树发现,鲢鱼、草鱼与其它鲤科鱼类斑马鱼、银鲫（Carassius auratus gibelio）的HSP70氨基酸同源性较高,处于同一进化树分枝;而尼罗罗非鱼HSP70未与其它鲈形目鱼类,如鲷科鱼类聚为一枝,而是占据一个独立的分枝,这与克隆得到的鲢鱼、草鱼肝脏HSP70基因可能为结构型HSP70,而尼罗罗非鱼肝脏HSP70基因可能为诱导型HSP70的结果相一致.     

Nickel accumulation and its effect on biomass, protein content and antioxidative enzymes in roots and leaves of watercress (Nasturtium officinale R. Br.)

In order to understand its response towards nickel stress,watercress(Nasturtium officinale R.Br.) was exposed to nickel(1-25 mg/L) for 1,3,5 and 7 days.The accumulation and translocation of nickel were determined and the influence of nickel on biomass,protein content and enzymatic antioxidants was examined for both roots and leaves.It was determined that N.officinale could accumulate appreciable amounts of Ni in both roots and leaves.Nickel accumulated particularly in the roots of plants.Biomass increased at low nickel concentrations but certain measurable change was not found at high concentrations.Under stress conditions the antioxidant enzymes were up-regulated compared to control.An increase in protein content and enzyme activities was observed at moderate exposure conditions followed by a decline at both roots and leaves.The maximum enzyme activities were observed at different exposure conditions.Our results showed that N.officinale had the capacity to overcome nickel-induced stress especially at moderate nickel exposure.Therefore,N.officinale may be used as a phytoremediator in moderately polluted aquatic ecosystems.     

Drosophila glue protein gene expression

The primary targets of steroid hormones are genes. For the ecdysone-controlled genes of Drosophila larval glue proteins proximal and distal control elements were identified by mutagenesis and sequence comparison. Their presence is required for the correct stage- and tissue-specific expression of these genes. The supposed function of these elements is described in a working model.     

应用应激蛋白HSP70作为生物标志物研究锌、铜及其联合毒性对鲫鱼肝脏的影响

将应激蛋白 (HSP70 )作为生物标志物 ,以银鲫 (Carassiusauratusgibelio)作为实验生物 ,经过 4 0d不同浓度铜 (Cu2 )、锌(Zn2 )及其混合物暴露后 ,运用SDS PAGE和WesternBlotting方法研究了鲫鱼肝脏组织内应激蛋白HSP70的诱导表达情况 .结果表明 :在实验浓度下 ,Cu2 ,Zn2 及其混合物对鱼肝脏内HSP70有显著的诱导 (P <0 0 5 ) ,而将Cu2 和Zn2 实验组与其混合物实验组相比 ,Cu2 ,Zn2 表现为一定的协同作用 .实验还发现 ,在国家渔业水质标准Zn2 浓度下 ,鱼肝脏组织内HSP70已经有明显的表达 ,说明分子生物学指标要比传统的环境检测指标敏感 ,对污染物具有早期预警的作用 ,将其作为生物标志物来监测环境中的重金属污染具有重要意义     

Nickel accumulation and its effect on biomass, protein content and antioxidative enzymes in roots and leaves of watercress （Nasturtium officinale R. Br.）

In order to understand its response towards nickel stress, watercress (Nasturtium o cinale R. Br.) was exposed to nickel (1–25 mg/L) for 1, 3, 5 and 7 days. The accumulation and translocation of nickel were determined and the influence of nickel on biomass, protein content and enzymatic antioxidants was examined for both roots and leaves. It was determined that N. o cinale could accumulate appreciable amounts of Ni in both roots and leaves. Nickel accumulated particularly in the roots of plants. Biomass increased at low nickel concentrations but certain measurable change was not found at high concentrations. Under stress conditions the antioxidant enzymes were up-regulated compared to control. An increase in protein content and enzyme activities was observed at moderate exposure conditions followed by a decline at both roots and leaves. The maximum enzyme activities were observed at di erent exposure conditions. Our results showed that N. o cinale had the capacity to overcome nickel-induced stress especially at moderate nickel exposure. Therefore, N. o cinale may be used as a phytoremediator in moderately polluted aquatic ecosystems.