The fertility protein factor and congenital developmental defects

Protein factor of fertility (PFF) has been measured in seminal plasma by immunodiffusion method with sensitivity about 2 mg/L. It has been found that normal level of PFF was from 16 to 256 mg/L, on the average 45.9 mg/L. Family (husband and wife) has been studied in the genetic laboratory by traditional methods. It has been shown that there is the correlation between low PFF levels in seminal plasma (on the average 15.0 mg/L, i.e. from 2 to 64 mg/L) and several developmental defects of fetus. There were anencephalus, hydrocephalus, spina bifida and other morphological defects (coefficient correlation was P less than 0.01). The biological role of PFF in human developmental defects is discussed.     

Occurrence of proteins immunoreactive with anti-coupling factor B in phosphorylating membrane preparations

Coupling factor B has been isolated from beef heart mitochondria, apparently in multiple forms which differ in molecular weight and specific activity. Since it has no known intrinsic catalytic activity, detection and quantitation have been based upon the factor B-dependent stimulation of ATP-linked activities in factor B-deficient submitochondrial particles. This communication reports the development of a reliable and more universally applicable enzyme-linked immunosorbent assay (ELISA) for detection and quantitation of factor B in soluble or membranous preparations. The assay requires nanoliter volumes of rabbit antiserum raised against purified factor B and will detect nanogram amounts of the coupling factor. Analysis of beef heart submitochondrial particles using a competitive binding ELISA indicated a factor B content of 0.27 nmol/mg protein, making factor B stoichiometric with F₁ (0.3–0.6 nmol/mg). Furthermore, application of the factor B ELISA has indicated the presence of material cross-reacting with the beef heart factor B-antiserum in phosphorylating membranes from chloroplasts, Escherichia coli, Paracoccus denitrificans and the thermophilic bacterium, PS3. Negative results were obtained with mitochondria and microsomes from rat liver, purple membranes from Halobium halobacterium and sarcoplasmic reticulum from rabbit skeletal muscle.     

An improved bulk purification method for Escherichia coli elongation factor, Ts

A bulk purification procedure has been designed to maximize the yield of Escherichia coli elongation factor, Ts, with a minimum of effort and time. The enzyme purification is achieved by DEAE-Sepharose and elongation factor Tu-affinity chromatographies. The typical yield is 150 mg/kg of E. coli (B) cells.     

A rapid procedure for production of human basic fibroblast growth factor in Escherichia coli cells.

Human basic fibroblast growth factor has been expressed in Escherichia coli cells at a level of 2-3 mg/l culture, using a rapid procedure which requires only simple DNA manipulative work. The recombinant material has the same potency as natural basic fibroblast growth factor from bovine pituitary glands.     

稀有植物裸果木的组织培养及植株再生

对稀有植物裸果木进行组织培养研究,在MS培养基上裸果木的下胚轴脱分化形成愈伤组织,并进一步分化形成再生植株。激素种类及其浓度是器官脱分化与植株再生的决定因素。诱导下胚轴形成愈伤组织的最适培养基为MS 1mg／L 6-BA 0．5mg／L NAA;芽分化诱导的最适培养基为MS 1 mg／L6-BA;生根的最适培养基为不含任何激素的1／2MS培养基。     

Purification and properties of a low molecular weight protein factor of mitochondrial energy-linked functions.

1. A soluble protein with a molecular weight of 11-12-10(3) has been isolated from bovine-heart mitochondria, which stimulates the following ATP-dependent reactions of submitochondrial particles treated with 0.6 mM EDTA and 1 M NH4OH: reverse electron transfer from succinate to NAD, transhydrogenation from NADH to NADP, and ATP-Pi exchange. The factor has no effect on the NADH oxidase, succinate oxidase and ATPase activities of the particles. 2. The stimulatory effect of the factor in the ATP-dependent reduction of NAD by succinate is 12 mumol-min-1-mg-1 of the factor protein. However, the NH4OH-EDTA treated particles are saturated for maximal activation of the above reaction by very small amounts of the factor (about 20-40 mug factor per mg particle). 3. Electrophoresis of the factor preparation on polyacrylamide gels showed a single protein band plus a nonprotein material which moved at the dye front and was weakly stained with Coomassie Blue. The protein was shown to be required for activation of the particles; whether the fast-moving, nonprotein material is also required is not known. 4. The factor is inhibited by mercurials and N-ethylmaleimide. The former, but not the latter, inhibition is completely reversed by 1,4-dithiothreitol. 5. The NH4OH-EDTA treated particles are also stimulated by rutamycin up to about 0.1 nmol of rutamycin per mg particle; higher rutamycin concentrations inhibit. Depending on the particle preparation, the factor stimulates up to about 3 nmol per mg particle, but does not inhibit at higher concentrations. In addition, under certain conditions in which appropriate concentrations of rutamycin fail to stimulate the particles, the factor still does.     

Preparation of milligrams of pure enantiomers using analytical-scale high-performance liquid chromatography

Pure enantiomers of an agrochemical process intermediate, (RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)-pentan-3-one ( 1 ), have been prepared on the milligram scale under overload chromatographic conditions on an analytical chiral column (250 × 4.6 mm i.d.). The effects of variation of temperature and mobile phase composition on retention factor, separation factor, and peak resolution have been investigated. Effects of flow rate, enantiomer ratio, sample concentration, and column load on productivity are also studied. Seven milligrams of the less retained (+)-enantiomer and 5 mg of the (?)-enantiomer were obtained from a single injection of 21 mg of (RS)- 1 . © 1994 Wiley-Liss, Inc.     

Blood protein technological industrial developments as a mirror of fundamental studies bgy the Institute of Biochemistry of the Ukrainian National Academy of Sciences

We have examined the technology for an industrial chromatographic production highly purified factor VIII concentrate intended for therapy of the hemophilia A and characterized this factor VIII. The final product has been prepared from cryoprecipitate of pooled human plasma using a large-scale procedure combining three conventional chromatographic steps based on AEM and CEM ion exchange and SPG or SHR gel filtration chromatography. The specific activity of the product was 459 +/- 19 IU factor VIII/mg protein (n = 10), corresponding to a purification factor of about 15,000. The concentrate was free of the fibrinogen, alpha-2-macroglobulin, alpha-1-acidglycoprotein, haptoglobin. Only three contaminants could be detected: fibronectin, immunoglobulins A and G (about 0.020, 0.004 and 0.034 microgram/IU factor VIII, respectively). The purity of the final product was confirmed by SDS polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar-Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Another examination was concern to the technology for an industrial chromatographic production highly purified factor IX concentrate intended for therapy of the hemophilia B and characterized this factor IX. The final product has been prepared from pooled human plasma using a large-scale procedure combining four conventional chromatographic steps based on AEM ion exchange, AFM affinity and SGS gel filtration chromatography. The specific activity of the product was 149 +/- 10 IU factor IX/mg protein (n = 10), corresponding to a purification factor of about 9000. The concentrate was free of the vitamin K-dependent clotting factors II, VII and X and of proteins C and S. Most of possible contaminants were absent in this new product. High-molecular-weight kininogen, factor VIII, XI, XII or prekallikrein were not detected. There were no activated factors, such as factors IXa and Xa, no thrombin and no phospholipids. Only two contaminants could be detected: C4 and inter-alpha-trypsin inhibitor (about 0.8 and 1.2 mg/IU factor IX, respectively). The purity of the final product was confirmed by SDS polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar-Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Thrombogenicity tests in rabbits revealed that the high purified factor IX by Institute of Biochemistry technology tested had a lower thrombogenic power than the commercial factors IX tested. The concentrate has been subjected to a special solvent--detergent treatment for definite time and temperature during its production to virus inactivation (it will be describe in following special examination). These data demonstrate that a highly purified therapeutic clotting factor VIII and IX concentrates can be prepared from human plasma by conventional chromatographic methods developed by Institute of Biochemistry of NAS of Ukraine and Combio Ltd.     

Levels of hexachlorocyclohexanes in agricultural environment of Sacco river valley

Aim of this trial was to verify the occurrence and the distribution of hexachlorocyclohexanes (HCHs) in soil, sediment, straw, alfalfa, other animal feed grown in farms with contaminated soil. In the present study two years of monitoring activity in the province of Roma and Frosinone was reported. Experimental trial in two contaminated sites was carried out on uptake and translocation of HCHs in maize and alfalfa. In 19 sites soil, forage and weed has been collected for two years, soil samples consisted in cores of 40 cm to test the presence of HCHs at different deep. The analytical determinations in soil and plant samples were carried out by gas liquid chromatography with electron capture detector and confirmed by mass detector. In the first year (2005- 2006) 68% of soil samples were contaminated (HCHs > LOQ) and 3% of vegetable samples. In the second year (2006- 2007) 42% of soil samples resulted positive and 26% of vegetable matrix. In particular B hexacyclohexane was detected in wheat stem (0.037 mg/kg) with a soil contamination of 0.039 mg/kg and in alfalfa (0.012 mg/kg) with presence in soil of 0.004 mg/kg. Experimental trials on maize evidenced a translocation factor for this isomer stem/soil of 0.006 mg/kg ? and for grain of 0.005 mg/kg. On alfalfa translocation factor root/soil was 0.01 and shot/soil 0.009. A propose to calculate the threshold value of soil contamination to admit crop grown destined to animal feed, would be based on HCHs LOD values weighted with translocation factor.     

Suppressor cell-inducing factor: a new lymphokine secreted by a natural suppressor cell line with natural cytotoxic activity. I. Purification to apparent homogeneity and initial characterization of suppressor cell-inducing factor

The lymphokine suppressor cell-inducing factor (SIF), obtained from 15 liters of serum-free culture supernatants of the natural suppressor cell line, M1-A5, has been purified to apparent homogeneity by a combination of gel filtration, ion exchange chromatography, and reverse-phase-HPLC. Purity of SIF was assessed by the migration of the factor as a single band on SDS-PAGE, and the elution from reverse-phase-HPLC column as a single and sharp peak. SIF activity was retained after both procedures. Two protein factors with SIF activity were isolated from M1-A5 culture supernatants. The first protein factor (SIF alpha) had a Mr of 43 kDa, and the second protein factor (SIF beta) had a Mr of 6 kDa. Final purification of SIF alpha yielded 5 micrograms protein with specific activity of 4 x 10(6) U/mg protein. Final purification of SIF beta yielded 40 micrograms protein with specific activity of 7.5 x 10(7) U/mg protein. The relationship between SIF alpha and SIF beta, as well as the relationship with other suppressor factors, will be addressed.     

Analysis of the relA gene product of Escherichia coli.

The relA gene product, ATP: GTP 3'-pyrophosphotransferase (stringent factor) has been isolated in homogeneous form from an Escherichia coli strain polyploid for this gene at a yield of 1 mg/100 g cells and at a specific activity in a ribosome-activated assay at 37 degrees C of 120 mumol guanosine pentaphosphate formed min-1 mg protein-1. The specific activity in a methanol-activated assay at 25 degrees C was found to be 4 mumol guanosine pentaphosphate formed min-1 mg protein-1. These values are about 100 times higher than reported by others. Our further studies of this enzyme led to the following results. Antibodies raised against this enzyme inhibit the ribosome-activated synthesis of guanosine tetraphosphate and pentaphosphate but have no effect on the much slower synthesis, detected in the absence of ribosomes. The amount of stringent factor in the relA+ strain CP78 is estimated to about 1 copy per 200 ribosomes. The amount of antibody-binding material in CP79 (relA) is at least 5 times lower.     

Detection of a DNA-binding factor associated with mammalian DNA polymerase-alpha.

A DNA-binding factor able to bind to a double-stranded DNA containing no free ends (SV40 DNA I) has been reproducibly detected in highly purified (to 30,000 units/mg) regenrating rat liver DNA polymerase-α. This factor could not be separated from the catalytic unit by any of the separation procedures so far used (ion-exchange chromatographies, gel filtration, sucrose gradients and DNA affinity chromatographies). Dissociation of the DNA binding unit from the catalytic unit may be observed after formation of a nucleoprotein complex between DNA polymerase-α and SV40 DNA I.     

Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region

Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 +/- 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Forty-eight hours after transfection rHFV antigen levels in the conditioned medium were 70 +/- 15 ng/mL. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 +/- 0.012 to 0.124 +/- 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 +/- 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1800 +/- 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1700-2000 units/mg. Immunoprecipitation of [35S]methionine-labeled rHFV revealed a single high molecular mass component (approximately 330 kDa). Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. We have also expressed a mutant, rHFV-des-B811-1441, that lacks a large portion of the highly glycosylated connecting region that is present in factor V. Immunoprecipitation of [35S]methionine-labeled rHFV-des-B811-1441 revealed a single-chain polypeptide with Mr approximately 230 kDa. This mutant constitutively expressed 38 +/- 7% of the activity of the RVV-V-activated protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cd AND Zn ACCUMULATION IN PLANTS FROM THE PADAENG ZINC MINE AREA

Significant cadmium (Cd) contamination In soil and rice has been discovered in Mae Sot, Tak province, Thailand where the rice-based agricultural systems are established in the vicinity of a zinc mine. The prolonged consumption of Cd contaminated rice has potential risks to public health and health impacts of Cd exposed populations in Mae Sot have been demonstrated. The Thai government has prohibited rice cultivation in the area as an effort to prevent further exposure. Phytoextraction, the use of plants to remove contaminants from soil, is a potential option to manage Cd–contaminated areas. However, successful phytoextraction depends on first identifying effective hyperaccumulator plants appropriate for local climatic conditions. Five sampling sites at Padaeng Zinc mine, Tak province were selected to collect plant and soil samples. Total Cd and Zn concentrations in sediments or soils were approximately 596 and 20,673 mg kg^?1 in tailing pond area, 543 and 20,272 mg kg^?1 in open pit area, 894 and 31,319 mg kg^?1 in stockpile area, 1,458 and 57,012 mg kg^?1 in forest area and 64 and 2,733 mg kg^?1 in Cd contaminated rice field. Among a total of 36 plant species from 16 families, four species (Chromolaena odoratum, Gynura pseudochina, Impatiens violaeflora and Justicia procumbens) could be considered as Cd hyperaccumulators since their shoot Cd concentrations exceeded 100 mg Cd kg^?1 dry mass and they showed a translocation factor > 1. Only Justicia procumbens could be considered as a Zn hyperaccumulator (Zn concentration in its shoot more than 10,000 mg Zn kg^?1 dry mass with the translocation factor > 1).     

湖泊富营养化治理: 集中控磷, 或氮磷皆控?

关于湖泊富营养化的治理, 有充足的全生态系统实验和湖泊治理实践表明, 只控磷(P)就可使湖泊贫营养化。但也有不少人认为需要氮(N)和P皆控。由于N和P皆控的成本可达只控P的4—15倍, 故确定富营养化治理是否必须既控P又控N是一个重大而现实的科学问题。针对这个问题, 文章对所有相关观点及其证据的科学性进行了系统辨析。首先, 系统总结了关于富营养化营养驱动与控制的研究历史。其次, 对判定营养控制的主要依据——限制因子的概念发展及判定方法进行了全面回顾与分析, 明确指出该概念的目的是确定促进生物生长的因子。第三, 介绍了新概念——减控因子, 其定义是: 在生态系统管理中, 能够抑制生物个体、种群和群落过度繁盛的必需环境因子, 或直接减灭生物本身的物理(机械)、化学和生物因子, 且成本效益最大。随后, 举例说明了确定减控因子的五个步骤, 即必需性、可控性、可行性、成本分析及实验和应用验证, 证明非限制因子也可成为减控因子, 而限制因子不一定是减控因子。第四, 基于减控因子分析, 指出湖泊富营养化的减控因子是P; 进而, 总结了加拿大和中国的全生态系统实验及大量湖泊治理实践的系统证据。这些充分证明: 仅控P就可控制富营养化, 而减N无助于控制浮游藻类总量, 反而会诱导固氮蓝藻大量生长。第五, 对控N观点的逻辑和实验依据逐一批驳, 指出这些争论或将限制因子混同于减控因子, 或缺乏大尺度的实验证据。第六, 系统辨析了高N的生态效应, 初步确定: 只有总氮和氨氮>5 mg/L时, N才对水生植物等有一定的负面影响且可促进沉积物P的释放。建议先把地表水Ⅰ—Ⅴ类的总氮和氨氮标准限值均放宽至2 mg/L, 后逐步放宽至5 mg/L左右。最后, 指出富营养化治理必须采取系统对策, 以修复物理、化学、水文和生物完整性。在维护湖盆物理完整性的基础上, 最根本的措施是控制外源P负荷总量; 若内源P负荷较大, 则可采取钝化等方法。次之, 应开展水位调控, 以修复水生植被, 实现浊-清稳态转换。综上所述, 湖泊富营养化治理应采取“放宽控N、集中控P的策略”, 以大幅度降低治理成本。     

High-level production of human acidic fibroblast growth factor in E. coli cells: inhibition of DNA synthesis in rat mammary fibroblasts at high concentrations of growth factor.

Recombinant human acidic fibroblast growth factor has been produced in E. coli cells at a level of at least 50 mg/l culture. The recombinant and natural acidic fibroblast growth factors are almost identical to one another when tested on rat mammary fibroblasts for their ability to stimulate DNA synthesis, to bind to the high-affinity surface receptors of the cells and to inhibit DNA synthesis when present in the culture medium at high concentrations. The recombinant acidic fibroblast growth factor binds to two cell-surface polypeptides of molecular masses 160 kDa and 140 kDa, which are the same size as the receptors for basic fibroblast growth factor, and it binds preferentially to the smaller polypeptide.     

The effect of ratibol, retabolil and solasodin on the blood coagulation system

It has been established that ratibol, retabolil and solasodin in doses 5 and 10 mg/kg proved to have a slight anticoagulative effect. The same effect has been observed during 20 days of oral retabolil and solasodin intake. Ratibol was characterized by a two-step action. All three steroids activated factors II, VII, XIII and diminished factor I concentration. These changes were accomplished with the fall of antithrombin activity and decrease in resistance to streptokinase. The fibrinolysis level has been raised under the effect of the steroids studied.     

Purification of a murine leukemia inhibitory factor from Krebs ascites cells

A factor capable of inducing terminal differentiation in the murine myeloid leukemia cell line M1 has been purified to apparent homogeneity from the medium conditioned by Krebs II ascites tumor cells. The factor, termed leukemia inhibitory factor (LIF) is a single chain glycoprotein of apparent Mr 58,000 which induces differentiation and inhibits proliferation of the M1 cell line but not the WEHI-3B D+ murine myeloid leukemic cell line and has no detectable proliferative activity on normal myeloid progenitor cells. It was purified using four successive high-efficiency purification steps--anion-exchange chromatography on DEAE-Sepharose; cation-exchange chromatography on CM-Sepharose; affinity chromatography on lentil lectin-Sepharose; and reverse-phase high-performance liquid chromatography on a phenyl-silica matrix--to a specific biological activity of approximately 1.25 X 10(8) units/mg with an overall purification of 12,000-fold and a yield of 73% for the activity failing to bind to DEAE-Sepharose. Sufficient quantities of the factor (12 micrograms, 200 pmol) have been purified to allow structural and functional analysis of the molecule and comparison with other know differentiation inducers.     

Bioavailability assessment and accumulation by five garden flower species grown in artificially cadmium-contaminated soils

Many studies have been conducted on phytoextraction; however, non-native hyperaccumulator species are not suitable for the natural environment of Taiwan in many cases. Drawing upon previous results, the growth and heavy metal accumulation in artificially cadmium-contaminated soils were compared for five local garden flower species. The treatments included a control (CK), 9.73 +/- 0.05 mg kg(-1) (Cd-10), and 17.6 +/- 0.8 mg kg(-1) (Cd-20). All plants were harvested at 35 days after transplanting and analyzed for Cd content. Cd accumulation in the shoot of French marigold (Tagetes patula L.) and Impatiens (Impatiens walleriana Hook. f.) grown in Cd-20 treatment were 66.3 +/- 6.5 and 100 +/- 11 mg kg(-1), which equated to a removal of 0.80 +/- 0.11 and 0.60 +/- 0.37 mg Cd plant(-1), respectively. The maximum Cd accumulation of Impatiens reached the threshold value (100 mg kg(-1)) characteristic of a Cd hyperaccumulator and its bioconcentration factor (BCF) and translocation factor (TF) were greater than one. Impatiens therefore has the potential to hyperaccumulate Cd from Cd-contaminated soils. With the exception of Garden verbena, significant relationships were found between Cd concentrations in soil extracted by 0.05 M EDTA, 0.005 M DTPA, and 0.01 M CaCl2 and the concentration of Cd in the shoots of the tested garden flowers.