bluuebunny/crossref_metadata_2025 huggingface dataset:
Post-Harvest Loses and Socioeconomic Sustenance of Yam Farmers in Southern Agricultural Zone of Nasarawa State
Authors: Iorzua D. A. , Ikwuba A. A. , Aan J. T. , Nwafor S. C.
Published: November 2020

Aims: The study determined the effect of post-harvest losses of yam on the socio-economic sustenance of farmers in Nasarawa State Nigeria. It also described the socio-economic status of farmers; determined the average quantities of yam lost for the years in review (2014-2018).

Study Design: The study employed the use of cross sectional design.

Place and Duration of Study: The study was conducted in Nasarawa state, Nigeria between October 2019 to June 2020.

Methodology: Using the multistage sampling technique, data for the study were collected from a sample of three hundred and eighty seven (387) respondents.

Results: That majority of the respondents (322) 83%, were male, and within the age range of 31-40 years 158(41%) followed by the age bracket of 41-50: 97(25%); 323(84%) of the respondents were married with average household size of 8.0 persons per household while 87% of the respondents have had one form of formal education or the other.

The Ordinary Least Square result of the effect of postharvest losses on the socioeconomic sustenance of the respondents revealed that the coefficient of simple determination (r2) was 0.850which implies that 85.00% of the variations in the socioeconomic sustenance of the respondents was explained by the independent variable included in the model (post-harvest losses) while 15.00% unexplained was due to error. F-prob. value of 0.0000 was observed from the analysis which is less than 0.05, indicating that the variables included in the estimated regression model were correct and significant at 1% significant level. The coefficient of post-harvest losses with the value of (-4.327) was statistically significant and negatively related to the socioeconomic sustenance of the respondents at 1 percent.

Conclusion: The study concluded that there is a relationship between the post-harvest losses and socioeconomic sustenance of farmers. Therefore, the relevant government and agricultural agencies, Non- Governmental Organizations (NGOs), Community Based Organizations (CBOs) as well as individuals need to strengthen their effort toward reducing post-harvest losses of yam by educating through demonstration and encouraging farmer to use modern technologies and materials in yam storage.
Conformational change of mastoparan from wasp venom on binding with phospholipid membrane
Authors: Higashijima Tsutomu , Wakamatsu Kaori , Takemitsu Mari , Fujino Masahiko , Nakajima Terumi , Miyazawa Tatsuo
Published: July 2002

The conformational change upon binding with phospholipid membrane has been studied of mastoparan from wasp venom, a tetradecapeptide causing the degranulation of mast cells. The 270‐MHz 1H‐NMR spectra and CD spectra indicate that the mastoparan molecule takes the α‐helical conformation in methanol solution, but a much less ordered form in aqueous solution. On binding with phospholipid membrane, the α‐helical conformation is formed even in aqueous medium. Such a conformational change is primarily due to the interaction between the aliphatic side chains of mastoparan and the hydrophobic interior of phospholipid membrane, in contrast to the case of melittin from bee venom.
Glycosylation of yeast aspartyl—tRNA synthetase
Authors: Colas Bernard , Boulanger Yves
Published: July 2002

Several lines of evidence establish that the crystallizable aspartyl—tRNA synthetase from Baker's yeast contains some covalently bound glucose: (i) a positive staining of the enzyme was obtained after polyacrylamide gel electrophoresis followed by the concanavalin A‐peroxidase test which is specific for glucose and mannose containing proteins; (ii) thin‐layer chromatography and gas‐liquid chromatography revealed the presence of glucose in enzyme hydrolysates; (iii) immunoaffinoelectrophoresis in agarose gels containing concanavalin A and antibodies raised against aspartyl—tRNA synthetase showed that the enzyme was able to precipitate entirely in the lectin. Finally incubation of the enzyme with [14C]glucose or [14C]glucose 6‐phosphate led to the incorporation of radioactivity into trichloroacetic acid‐precipitable protein. Indeed immunoprecipitation of [14C]glucose‐labelled aspartyl‐tRNA synthetase with specific antibodies using the rocket method followed by autoradiography gave a radioactive peak. This last result also demonstrates the possibility of in vitro glycosylation of yeast aspartyl—tRNA synthetase.
Effect of glucose‐6‐P on the catalytic and structural properties of glycogen phosphorylase a
Authors: Melpidou Angeliki E. , Oikonomakos Nikolaos G.
Published: July 2002

Kinetic studies of muscle phosphorylase a in cationic buffer (pH 6.8) demonstrate that glucose‐6‐P competitively inhibits the binding of the substrate, glucose‐1‐P, to the enzyme. The inhibitory effect of glucose‐6‐P is largely overcome by glycerol‐2‐P. AMP counteracts inhibition of the enzyme by glucose‐6‐P, while glucose and glucose‐6‐P can interact to produce a synergistic inhibition of phosphorylase a activity. Preincubation of phosphorylase a with glucose‐6‐P at 20°C results in ∼3‐fold increase in activity, while ultracentrifugation experiments carried out under the same conditions showed that phosphorylase a can be converted to dimers by glucose‐6‐P.
Ferredoxin—ferredoxin NADP reductase interaction
Authors: Forti Giorgio , Bracale Marcella
Published: July 2002

Ferredoxin‐NADP reductase (FNR) and ferredoxin form a complex when the former is membrane‐bound as they do when both components are in solution, with the same dissociation constant. The rate constant of NADP photoreduction, first order with respect to the complex, is more than 20‐times higher when FNR is membrane‐bound than when the enzyme is in solution. The Arrhenius activation energy is identical in both conditions. These observations are interpreted in terms of ‘entropic catalysis’ of NADP reduction by the thylakoid‐bound FNR.
Effects of ciglitazone on insulin resistance and thermogenic responsiveness to acute cold in brown adipose tissue of genetically obese (ob/ob) mice
Authors: Mercer Stewart W. , Trayhurn Paul
Published: July 2002

Genetically obese (ob/ob) mice develop a marked insulin resistance in brown adipose tissue soon after weaning, and this is paralleled by a fall in the acute activation of the mitochondrial proton conductance pathway in the tissue on cold exposure. Treatment of ob/ob mice with ciglitazone, a new oral hypoglycaemic, led to a restoration of insulin sensitivity in brown adipose tissue. The amelioration of insulin resistance was accompanied by a normalization of the acute, cold‐induced increase in mitochondrial GDP binding. These results support the hypothesis that the development of insulin resistance in brown adipose tissue is an important factor in the impaired thermogenic responsiveness of obese mice.
Ruthenium red inhibits the mitochondrial Ca2+ uptake in intact bovine spermatozoa and increases the cytosolic Ca2+ concentration
Authors: Rigoni Fernanda , Deana Renzo
Published: July 2002

The uptake and cycling of Ca2+ by ejaculated bovine spermatozoa are almost completely abolished by ruthenium red, antimycin A or FCCP. The inhibitory effect of ruthenium red is also observed after washing of the dye‐pretreated cells followed by addition of digitonin or filipin. In contrast, the inhibition is overcome by A23187 treatment. It is concluded that ruthenium red penetrates into intact spermatozoa, inhibits the mitochondrial Ca2+ uptake ‘in situ’, and causes the observed increase of the cytosolic free Ca2+ concentration.
The N‐terminal half of the heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayers
Authors: Blaustein Robert O. , Germann William J. , Finkelstein Alan , DasGupta Bibhuti R.
Published: July 2002

The heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayer membranes. Channel activity is confined to the N‐terminal half of this chain; the C‐terminal half is inactive. Channel activity is stimulated by low pH (4.5–5.5) on the cis side (the side to which protein is added), neutral pH on the opposite (trans) side, and cis positive voltages. These findings are strikingly similar to those previously reported for analogous fragments of diphtheria and tetanus toxins.
A sequence variant in the N‐terminal region of the catalytic subunit of the cAMP‐dependent protein kinase
Authors: Hotz Agnes , König Norbert , Kretschmer Jürgen , Maier Gernot , Ponstingl Herwig , Kinzel Volker
Published: July 2002

Protein kinase; Cyclic AMP; (Bovine)
Synthetic phosphopeptides are substrates for casein kinase II
Authors: W. Litchfield David , Arendt Anatol , Lozeman Fred J. , Krebs Edwin G. , Hargrave Paul A. , Palczewski Krzysztof
Published: July 2002

Casein kinase II is a protein serine/threonine kinase that exhibits a preference for acidic substrates. Previous studies have demonstrated that a glutamic acid 3 amino acids C‐terminal (+3) to a serine or threonine is required for phosphorylation. To examine the ability of phosphoserine and phosphothreonine residues to serve as specificity determinants for casein kinase II, phosphopeptides containing either of these phosphoamino acids in the +3 position were synthesized and tested as substrates. Phosphopeptides containing phosphoserine in the +3 position were readily phosphorylated. In contrast, corresponding phosphothreonine‐containing peptides were very poorly phosphorylated. These results imply that prior phosphorylation of substrate proteins on serine, but not threonine residues, may be important in regulating their phosphorylation by casein kinase II.
Site‐directed mutagenesis of the N‐terminal region of IGF binding protein 1; analysis of IGF binding capability
Authors: Brinkman A. , Kortleve D.J. , Schuller A.G.P. , Zwarthoff E.C. , Drop S.L.S.
Published: July 2002

To define domains involved in IGF binding 60 N‐terminal amino acid residues of IGFBP‐1 were deleted. This deletion resulted in loss of IGF binding suggesting that the N‐terminus may enclose an IGF binding domain. However, most point mutations introduced in this region did not affect IGF binding. In contrast to Cys‐34, only substitution of Cys‐38 for a tyrosine residue abolished IGF binding. With the determination that all 18 cysteine residues are involved in disulphide bond formation our data suggest that, although not all cysteines contribute to the same extent, the ligand binding site may be spatially organized.
Cell‐free translation in reversed micelles
Authors: Nametkin Sergey N. , Kolosov Michael I. , Ovodov Sergey Yu. , Alexandrov Alexander N. , Levashov Andrey V. , Alakhov Valery Yu. , Kabanov Alexander V.
Published: July 2002

Cell‐free translation in reversed micelles (RM) of surfactants in organic solvents is demonstrated using as an example the synthesis of human interleukin‐2 by the wheat germ translation system solubilized in Brij 96 (oleyl‐poly(10)oxyethylene ether) RM in cyclohexane. The translation system components and the product were recovered from the RM system by acetone precipitation. The recovery and translation reaction yields depended on the degree of surfactant hydration. The translation yields in Brij 96 RM were close to that observed in regular aqueous solution. The Brij 96 RM system is regarded as a promising media for the cell‐free synthesis of hydrophobic proteins. Meanwhile, no translation reaction was observed in Aerosol OT (sodium bis(2‐ethylhexyl) sulfosuccinate) RM in octane, which presumably is due to the ability of Aerosol OT to bind Mg2− ions necessary for the functioning of the translation apparatus.
Enhanced acetylcholine secretion in neuroblastoma x glioma hybrid NG108‐15 cells transfected with rat choline acetyltransferase cDNA
Authors: Kimura Yasuhiro , Oda Yoshio , Deguchi Takeo , Higashida Haruhiro
Published: July 2002

Neuroblastoma x glioma hybrid NG108‐15 cells and mouse neuroblastoma N18TG‐2 and NIE‐115 celIs were transiently transfected with the sense cDNA coding for rat choline acetyltransferase (ChAT). All transfected cell lines showed a high level of ChAT activity. ACh secretion was monitored by recording miniature end‐plate potentials (MEPPs) in striated muscle cells that had been co‐cultured with transfected cells. The number of muscle cells with synaptic responses and the MEPP frequency were higher in co‐culture with transfected NG108‐15 cells than with control or mock cells. No tsynaptic response was detected in muscle cells co‐cultured with transfected N18TG‐2 or N1E‐115 cells. The results show that ACh secretion into the synaptic cleft was enhanced due to ChAT overexpression in NG108‐15 hybrid cells but not in neuroblastoma cells.
Reduction in the amount of 8‐hydroxy‐2'‐deoxyguanosine in the DNA of SV40‐transformed human fibroblasts as compared with normal cells in culture
Authors: Barciszewski Jan , Rattan Suresh I.S. , Siboska Gunhild E. , Otzen Daniel E. , Clark Brian F.C.
Published: July 2002

DNA damage due to oxidative free radicals is considered to be a major cause of ageing and age‐related diseases including cancer. Of more than 20 modifications formed in DNA by the action of hydroxyl radicals, 8‐hydroxy‐2'‐deoxyguanosine (oh8dG) is potentially highly mutagenic and is known to occur most frequently. Using HPLC combined with electrochemical (HPLC/EC) detection of oh8dG, fivefold higher levels of oh8dG are detected in the DNA of cultured normal human skin fibroblasts as compared with SV40‐transformed human fibroblasts MRC‐5V2. In comparison, the levels of oh8dG were similar in the growth medium of both types of cells. Applications of this method range from studies on the genomic stability and instability of normal and cancerous cells to the clinical and laboratory testing of toxic substances and drugs.
Study of phosphorylation of translation elongation factor 2 (EF‐2) from wheat germ
Authors: Smailov Salim K. , Lee Anatoly V. , Iskakov Bulat K.
Published: July 2002

Phosphorylation of elongation factor 2 (EF‐2) by specific Ca2+/calmodulin‐dependent kinase is considered as a possible mechanism of regulation of protein biosynthesis in animal cells at the level of polypeptide chain elongation. In this report we show that wheat germ EF‐2 can be intensively phosphorylated by the rabbit reticulocyte EF‐2 kinase. Phosphorylation results in inhibition of the activity of plant EF‐2 in poly(U)‐dependent cell‐free translation system. Thus, the activity of EF‐2 in plant cells can be potentially regulated by phosphorylation. However, we could not detect endogenous EF‐2 kinase activity in wheat germ either in vitro or in vivo. Furthermore, EF‐2 kinase activity is not displayed in different organs of wheat and other higher plants.
Identification of a novel protein kinase C inhibitor in microsomes from phytohaemagglutinin activated human peripheral blood mononuclear cells
Authors: Gandini Enrico , Orlando Patrizia , Selvatici Rita , Balboni Alessandra , Boninsegna Sara , Rubini Michele
Published: July 2002

A peptide inhibiting either corpuscolate or purified PKC has been identified from microsomes of PHA‐activated human PBMC but it is not detectable in microsomes of resting PBMC. The peptide was obtained from a microsomal preparation in an oligomeric form that could be transformed into a monomeric form by β‐MSH. The active peptide (IN) was retained on a PC‐11 chromatographic column and could be eluted with Nad. IN is ineffective on PKC‐dependent protamine phosphorylation of protamine and on Ca2+ and phospholipid‐independent activity generated by mild hydrolysis with trypsin of PK‐C. Ca2+ binding is permissive for IN activity. IN inhibits particulate PK.C in PHA‐activated PBMC, but is ineffective after TPA activation. All these data indicate that IN acts at the regulatory domain of PKC.
Cytochrome P‐450: hexameric structure of the purified LM4 form
Authors: Myasoedova K.N. , Tsuprun V.L.
Published: July 2002

Purified cytochrome P‐450LM4 was found to be monodisperse in 20% glycerol by analytical ultracentrifugation. Its S
20,w value was quite similar to that of hexameric P‐450LM2. At lower glycerol concentrations the P‐450LM4 oligomers showed a tendency to aggregate. The P‐450LM4 oligomers were immobilized on Ultrogel A4 under conditions allowing only one covalent link to the matrix per oligomer. In the presence of SDS, the oligomers dissociated leaving only 1/6th of the initial amount of bound protein on the matrix, suggesting that the purified P‐450LM4 is a hexamer. This was confirmed by electron microscopy. The quaternary structure of the P‐450LM4 was similar to that demonstrated earlier for P‐450LM2.
Role of Lys‐110 of human NADH‐cytochrome b5 reductase in NADH binding as probed by site‐directed mutagenesis
Authors: Fujimoto Yoshio , Shirabe Komei , Nagai Takushi , Yubisui Toshitsugu , Takeshita Masazumi
Published: July 2002

Lys‐110 of human NADH‐cytochrome b
5 reductase was replaced by Ala, Met, or Arg by site‐directed mutagenesis to evaluate the role of the residue. K
m values of purified Lys‐110 → Ala and Lys‐110 → Met mutants for NADH were approximately 200‐fold and 1,100‐fold higher than that of the wild‐type, respectively, while the value of the Arg mutant was almost the same as that of the wild‐type. These results indicate that the positive charge at position 110 is important for NADH binding. The k
cat value of Lys‐110 → Ala was not affected, indicating that the residue only participates in the binding process in the reaction by forming an ionic interaction with phosphoryl group of NADH.
Kinetics and energetics of redox regulation of ATP synthase from chloroplasts
Authors: Strelow Frank , Rumberg Bernd
Published: July 2002

The rate of ATP hydrolysis catalyzed by the membrane‐bound CF0F1 ATP synthase from chloroplasts served as a probe for the determination of the reduction grade of the enzyme treated with dithiothreitol (DTT) or thioredoxin. Rate constants for reduction were obtained. It turns out that reduction by thioredoxin is about a factor of 6,000 more effective than DTT reduction. The activation profiles with respect to ΔpH were obtained for reduced and oxidized ATPases. The activation curve of reduced enzyme turns out to have its half‐maximum degree of activation at ΔPH = 1.65, which is considerably lower than reported hitherto. The corresponding value of the oxidized enzyme has been obtained from the rate of ATP hydrolysis in the case of incomplete reduced ATPases, taking into account the aforementioned rate constants, and comes to ΔpH = 3.35.
A point mutation found in the WT1 gene in a sporadic Wilms' tumor without genitourinary abnormalities is identical with the most frequent point mutation in Denys‐Drash syndrome
Authors: Akasaka Yoshikiyo , Kikuchi Haruhito , Nagai Toshihiro , Hiraoka Nobuyoshi , Kato Shingo , Hata Jun-ichi
Published: July 2002

We have analyzed exon 9 of the WT1 gene of 18 non‐familial/sporadic unilateral Wilms' tumors (WTs) from Japanese patients, by the polymerase chain reaction single‐strand conformation polymorphism (PCR‐SSCP) method. After screening these WTs, a nucleotide alternation, which was present on both alleles, was found in only one case. Furthermore, PCR‐SSCP analysis of the constitutional DNA revealed that this patient carried the mutation on only one allele in the germline. Sequence analysis showed that the tumor carried a point mutation (C‐1180 to T‐1180) in WT1 exon 9 of both alleles, resulting in an Arg‐394 to Trp‐394 amino acid substitution within the third zinc finger domain of the WT1 product. Interestingly, this mutation is identical with the most frequent point mutation associated with the Denys‐Drash syndrome. However, the classical triad of Denys‐Drash syndrome does not apply to this patient. This is in the first report of the point mutation in the zinc finger domain of both WT1 alleles in a sporadic unilateral WT without genitourinary abnormalities, and the mutation suggests that some sporadic WTs carry the Denys‐Drash WT1 mutations.
Rate limitation within a single enzyme is directly related to enzyme intermediate levels
Authors: Kholodenko Boris N. , Westerhoff Hans V. , Brown Guy C.
Published: July 2002

The extents to which different rate constants limit the steady‐state rate of an isolated enzyme can be quantified as the control coefficients of those constants and elemental steps. We have found that the sum of the control coefficients of rate constants characterising unidirectional rates depleting a particular enzyme intermediate is equal to the concentration of that enzyme intermediate as a fraction of the total enzyme concentration. Together with simple measurements this powerful relation may be used (i) to estimate certain enzyme intermediate levels, in particular the free enzyme concentration, and (ii) to estimate the control coefficients of rate constants and steps.
Immunological significances of invariant chain from the aspect of its structural homology with the cystatin family
Authors: Katunuma Nobuhiko , Kakegawa Hisao , Matsunaga Youichi , Saibara Toshiji
Published: July 2002

The primary structure of p31 of invariant chain (Ii‐chain) shows about 50% homology with those of the cystatin family which are endogenous cysteine protease inhibitors. The binding domains between Ii‐chain and HLA‐DR‐7 were estimated from the structural homology between cystatin and Ii‐chain and also between cathepsins and DR‐7, respectively. The QL64–71 and GS76–88 of Ii‐Chain were estimated to be the binding domains with GG45–51, and VS57–63 of HLA‐DR7, respectively. The purified human Ii‐chain from spleen is capable of forming four molecular forms from monomer to tetramer by redox‐potential dependent disulfide bond formation. The Ii‐chain inhibits cathepsin L and H competitively as a dimer and the Ki value for cathepsin L was 4.1 × 10−8 M, but cathepsin B was not inhibited at all. The Ii‐chain showed mainly a dimer (60 kDa) under the assay condition of cathepsins with cysteine and was not degraded by these cathepsins. The Ii‐chain may play an important role in the regulation of antigenic peptide presentation to MHC class II.
α‐Dystroglycan deficiency correlates with elevated serum creatine kinase and decreased muscle contraction tension in golden retriever muscular dystrophy
Authors: Ervasti James M. , Roberds Steven L. , Anderson Richard D. , Sharp Nicholas J.H. , Kornegay Joe N. , Campbell Kevin P.
Published: July 2002

The dystrophin—glycoprotein complex was examined in dystrophin‐deficient dogs with golden retriever muscular dystrophy (GRMD) using immunoblot and immunofluorescence analysis. The dystrophin‐associated proteins were substantially reduced in muscle from dogs with GRMD. Interestingly, regression analysis revealed a strong correlation between the amount of α‐dystroglycan and serum creatine kinase levels and the contraction tension measured for a given peroneus longus muscle.
Isolation of a cDNA clone encoding a novel membrane protein expressed in lymphocytes
Authors: Shimizu Yoshifumi , Ogawa Hiroyasu , Oka Yoshihiro , Mizuno Ryuzo , Sakoda Saburo , Kishimoto Tadamitsu , Sugiyama Haruo
Published: July 2002

By subtractive hybridization using single‐stranded phagemids with directional inserts, we isolated a mouse cDNA clone, LSM‐1, from temperature‐sensitive Abelson virus‐tranformed immature B cells whose differentiation was being induced after the shift from the permissive (35°C) to the non‐permissive temperature (39°C). LSM‐1, which encodes an as yet unknown peptide of 197 amino acids, has a putative signal sequence and a trans‐membrane region, and is expressed in B‐ and T‐cell lines, in spleen, thymus, and bone marrow of adult mice, and in embryos.
DNA fragmentation into 200–250 and/or 30–50 kilobase pair fragments in rat liver nuclei is stimulated by Mg2+ alone and Ca2+/Mg2+ but not by Ca2+ alone
Authors: Cain Kelvin , Inayat-Hussain Salmaan H. , Wolfe James T. , Cohen Gerald M.
Published: July 2002

Internucleosomal cleavage of DNA has often been regarded as the biochemical hallmark of apoptosis. We now demonstrate in isolated rat liver nuclei that DNA is initially cleaved into ⩾700, 200–250 kbp and 30–50 kbp fragments via a multi‐step process, which is activated by Mg+ and Mg2++Ca2+ but not by Ca2+ alone. The subsequent internucleosomal cleavage requires both cations. These findings demonstrate that a key event in the apoptotic process is the fragmentation of DNA into large kbp fragments by either a Mg2+ ‐dependent process (which can be potentiated by Ca2+) and/or by a Ca2+/Mg2+ activated endonuclease(s).
Enterocytic differentiation of the human Caco‐2 cell line is correlated with down‐regulation of fibronectin and laminin
Authors: Levy Peggy , Loreal Olivier , Munier Annie , Yamada Yoshihiko , Picard Jacques , Cherqui Gisèle , Clement Bruno , Capeau Jacqueline
Published: July 2002

Human intestinal Caco‐2 cells were used to examine the expression of fibronectin (FN) and laminin (LN) during enterocytic differentiation. Combination of immunoprecitation. Western and Northern blotting revealed that Caco‐2 cells expressed a classical FN and a variant form of LN: besides B1 and B2 chains, LN contained a 350‐kDa heavy chain instead of the 400‐kDa A chain. Throughout Caco‐2 cell differentiation, FN and LN synthesis decreased at both mRNA and protein levels. These data indicate that enterocytic differentiation involves both transcriptional and/or post‐transcriptional down‐regulation of FN and LN gene expression.
Polychlorinated biphenyls modulate protooncogene expression in Chang liver cells
Authors: Hornhardt Sabine , Jenke Hans-Stephan , Michel Günter
Published: July 2002

In Chang liver cells we studied the influence of polychlorinated biphenyls (PCB) on the expression of different protooncogenes. In cells incubated with medium supplemented with PCB we observed an early effect after 3 h on c‐erbA and c‐erbH RNA level. This reduction of RNA was due to a delayed transcriptional activation of the genes. The PCB congener 3,3',4,4',5‐pentachlorobiphenyl (5‐CB) in a 1,000‐fold lower concentration influenced protooncogene expression in the same manner. In contrast c‐raf RNA level increased transiently after 24 h. The fact that persistent chemicals like PCB interfere with protooncogene expression is particularly interesting in view of their tumor promoting activity.
Achievement of renaturation of subtilisin BPN' by a novel procedure using organic salts and a digestible mutant of Streptomyces subtilisin inhibitor
Authors: Matsubara Mamoru , Kurimoto Eiji , Kojima Shuichi , Miura Kin-ichiro , Sakai Tomoya
Published: July 2002

The pro‐sequences of proteases have been considered to be required for the refolding of denatured proteases. However, here we report achievement of almost complete restoration of enzymatic activity of subtilisin BPN' in the absence of its pro‐sequence. The presence of 2 M potassium acetate in the folding medium enhanced the refolding efficiency of guanidine hydrochloride (GdnHCl)‐denatured subtilisin BPN' by up to 28%, and other organic salts were also found to be useful, suggesting that general contribution of the bulky hydrophobic moieties of the salts to the formation of a favorable environment required for folding. This finding will provide new insights into the folding mechanisms not only of proteases but also of various other proteins. Almost complete restoration of enzymatic activity of denatured subtilisin in the organic salt solution was accomplished by further addition of mutated Streptomyces subtilisin inhibitor (SSI), which had been converted to a digestible temporary inhibitor by removal of the disulfide bridge near the reactive site.
Surface display on staphylococci: a comparative study
Authors: Robert Alain , Samuelson Patrik , Andréoni Christine , Bächi Thomas , Uhlén Mathias , Binz Hans , Thien Ngoc Nguyen  , Ståhl Stefan
Published: July 2002

Two different host‐vector expression systems, designed for cell surface display of heterologous receptors on Staphylococcus xylosus and Staphylococcus carnosus, respectively, were compared for the surface display of four variants of a 101 amino acid region derived from the G glycoprotein of human respiratory syncytial virus (RSV). Surface localization of the different chimeric receptors was evaluated by a colorimetric assay and by fluorescence‐activated cell sorting. It was concluded that the S. carnosus system was better both in the ability to translocate inefficiently secreted peptides and in the number of exposed hybrid receptors. The potential use of the described staphylococci as live bacterial vaccine vehicles or alternatives to filamentous phages for surface display of protein libraries is discussed.
Accumulation of xylitol in the mammalian lens is related to glucuronate metabolism
Authors: Goode Derek , Lewis Mary E , Crabbe M.James C
Published: July 2002

Cataract remains the major cause of blindness worldwide and a common complication of diabetes. Polyol accumulation in the lens is associated with cataract formation. Here we present evidence for a novel pathway for xylitol production in the lens involving glucuronate metabolism. Xylitol can be produced in rat and bovine lens from glucose, via the enzymes myo‐inositol‐oxygen oxidoreductase, d‐glucuronate reductase, l‐gulonate NAD+‐3‐oxidoreductase and l‐iditol‐NAD+‐5‐oxidoreductase, which have been found in the mammalian lens for the first time. Glucuronate reductase has been purified and was inhibited by thiol quenching reagents. UDP‐glucuronyl transferase is also present in mammalian lenses; this enzyme may be an anti‐toxic defense mechanism in the lens.
Therapeutic approach to ovarian cysts in tamoxifen‐treated women with breast cancer
Authors: Shushan A. , Peretz T. , Mor‐Yosef S.
Published: July 2002

Objectives: To present our clinical experience with gonadotropin‐releasing hormone agonist (GnRHa) treatment of tamoxifen‐treated women who had developed ovarian cysts, and to discuss the diagnostic and therapeutic options in such cases. Methods: The study included six tamoxifen‐treated premenopausal women with breast cancer who developed ovarian cysts and were followed up by our outpatient clinic. Tamoxifen was administered orally (20 mg/day). All patients underwent a pelvic examination and vaginal ultrasound using a 5‐MHz vaginal probe. Blood samples for serum gonadotropins, estradiol (E2) and CA 125 levels were collected at the time of the ovarian cyst detection and every month thereafter. After detection of an ovarian cyst, the women were treated by monthly injections of GnRHa. Results: The patients' mean age was 44 years (range 37–51 years) and all had stage‐II or ‐III breast cancer. The mean duration of tamoxifen administration at the time of ovarian cyst detection was 15 months (range 3–40 months). All six ovarian cysts were found to be simple cysts, measuring more than 30 × 30 mm. Serum E2 levels of the six patients at the time of ovarian cyst detection were between 939 and 1796 pg/ml and were suppressed to less than 25 pg/ml after GnRHa therapy. After between three and six monthly injections of GnRHa all six ovarian cysts disappeared. On follow‐up 6 months later, all patients had a normal pelvic examination. Conclusion: The findings of this preliminary report suggest that GnRHa might be an appropriate treatment for tamoxifen‐treated women who develop ovarian cysts. It was demonstrated that the GnRHa caused regression of the ovarian cysts and enabled continuation with the adjuvant tamoxifen treatment.
Comparative outcome of first-admission patients with delusional beliefs
Authors: Jørgensen P
Published: July 2002

A study sample of 88 patients suffering from delusional beliefs was examined from first admission to follow-up. From the time of index admission, global functioning differs when distributed in accordance with ICD-10 categories. Patients with the discharge diagnoses affective disorder and acute and transient psychotic disorder have similar and optimistic perspective. The most pessimistic perspectives apply to patients with schizophrenia and an intermedium, but a rather pessimistic position also applies to patients with persistent delusional disorder. According to the discharge diagnosis following first admission, the score of global assessment functioning (GAF) at follow-up (8 years) is: schizophrenia 37; affective disorder 73; persistent delusional disorder 47; and acute and transient psychotic disorder 70. Generally, short-term outcome bears close resemblance to that of middle-term observation. During the observation period, 13 patients died. Amongst the surviving 75 patients, diagnosis at follow-up differed from that given at first-admission discharge, in eight cases using ICD-10 and in ten cases using DSM-III-R. Therefore, the use of ICD-10 as well as DSM-III-R enables clinicians to estimate a high stability (89%) diagnostic category with minimal delay.
Three‐dimensional NMR structure of the sixth ligand‐binding module of the human LDL receptor: comparison of two adjacent modules with different ligand binding specificities
Authors: Clayton Daniel , Brereton Ian M. , Kroon Paulus A. , Smith Ross
Published: July 2002

The sixth ligand‐binding module of the low‐density lipoprotein receptor contributes to the binding of apolipoprotein B100‐containing lipoproteins. 1H NMR spectroscopy, DYANA and X‐PLOR structure calculations were used to determine that this module has a well defined structure with a backbone conformation similar to other modules. Structures from calculations that simulated the presence of a calcium ion showed increased resolution without large increases in energy, increased deviations from idealised geometry or violations of experimental constraints. Investigation of the surface properties of this module indicates there are significant differences from the fifth module, which binds apolipoprotein E‐containing lipoproteins in addition to apolipoprotein B100‐containing lipoproteins.
A δ opioid receptor lacking the third cytoplasmic loop is generated by atypical mRNA processing in human malignomas
Authors: Mayer Peter , Tischmeyer Helga , Jayasinghe Melany , Bonnekoh Bernd , Gollnick Harald , Teschemacher Hansjörg , Höllt Volker
Published: July 2002

δ Opioid receptors were identified in human melanomas by RT‐PCR and radioligand binding. In all tumors an additional PCR amplificate was detected in which 144 bp within the third exon were deleted. This fragment corresponded to the third cytoplasmic domain of the receptor protein. The short variant resulted from atypical mRNA processing. There were no common splice recognition sequences around the deleted fragment; instead its excision resembled the removal of a transposon. The deletion was not detected in normal human melanocytes nor in human or rat brain. However, it was present in a human neuroblastoma cell line (SH‐SY5Y). Thus, it appears that the occurrence of the short δ opioid receptor is correlated to malignancy.
cDNA micro array identification of a gene differentially expressed in adenovirus type 5‐ versus type 12‐transformed cells
Authors: Vertegaal Alfred C.O. , Kuiperij H.Bea , van Laar Theo , Scharnhorst Volkher , van der Eb Alex J. , Zantema Alt
Published: July 2002

Proteins encoded by non‐oncogenic adenovirus type 5 and oncogenic adenovirus type 12 differentially affect expression of a number of cellular genes. We have used cDNA micro array analysis to identify a cellular gene that is expressed in Ad12‐ but not in Ad5‐transformed cells. This cellular gene was found to be the gene encoding follistatin‐related protein, a TGF‐β inducible gene. Consistently, a constitutive factor binding to Smad binding elements was found in adenovirus type 12‐transformed cells.
Mechanisms for the transport of unconjugated bilirubin in human trophoblastic BeWo cells
Authors: Pascolo Lorella , Fernetti Cristina , Garcia-Mediavilla Maria Victoria , Ostrow J.Donald , Tiribelli Claudio
Published: July 2002

To evaluate mechanisms that mediate passage of unconjugated bilirubin (UCB) across placenta, the transport of [3H]UCB was studied in the human trophoblastic, BeWo cell line. When plotted against the unbound UCB concentration [Bf], uptake exhibited saturative kinetics with a similar apparent K
m (∼30 nM) for BeWo cells grown either in polarized (Transwell) or non‐polarized fashion (dish). UCB release from cells, but not uptake, was inhibited by sulfobromophthalein but not by taurocholate, and almost abolished by MK571, a specific inhibitor of the activity of multidrug resistance‐associated proteins (MRPs). MRP1 and MRP5 were both present in BeWo cells and the expression of MRP1, but not MRP5, was markedly higher in polarized cells. These data indicate that UCB is taken up from the fetal circulation by a still undefined, saturative process not shared by other organic anions and is then excreted to maternal circulation by proteins of the MRP family.
Induction of phosphotyrosine in the gap junction protein, connexin431
Authors: Mikalsen Svein-Ole , Husøy Trine , Vikhamar Gunnhild , Sanner Tore
Published: July 2002

The protein‐tyrosine phosphatase inhibitors pervanadate, permolybdate, H2O2, and to a much lesser extent vanadate, increased the amount of cellular phosphotyrosine and induced tyrosine phosphorylation of connexin43 (Cx43) in early passage hamster embryo fibroblasts. The presence of phosphotyrosine in Cx43 immunoprecipitates from pervanadate‐treated cells was shown by a phosphotyrosine‐specific antibody and a phosphotyrosine‐specific phosphatase. Pervanadate‐induced Cx43 tyrosine phosphorylation was further verified by phosphoamino acid analysis, while no phosphotyrosine was present in control cells. This is the first observation of tyrosine phosphorylation of connexins in normal cells.
Detection of glycosyl‐phosphatidylinositol‐anchored proteins on the surface of Nicotiana tabacum protoplasts
Authors: Takos Adam M , Dry Ian B , Soole Kathleen L
Published: July 2002

Glycosyl‐phosphatidylinositol (GPI)‐anchored plasma membrane proteins have been found to be widespread in eukaryotes and protozoa but have not been reported in higher terrestrial plants. A sensitive biotin‐based assay has been used to detect the presence of GPI‐anchored proteins on the outer surface of cultured Nicotiana tabacum cells. Six proteins with molecular weights of 92, 84, 60.5, 54.5, 39.5 and 37 kDa were found to move from a Triton X‐114 detergent‐rich phase to an aqueous phase following incubation with phosphatidylinositol‐specific phospholipase C (PtdIns‐PLC). The behaviour of these proteins is consistent with the presence of a GPI‐anchor. Seven GPI‐anchored proteins were also detected on the surface of tobacco leaf protoplasts with molecular weights of 67.5, 62, 39, 33.5, 27, 23 and 15.6 kDa. These data demonstrate the presence of multiple GPI‐anchored proteins on the plasma membrane of higher plant cells. © 1997 Federation of European Biochemical Societies.
Effect of various glycosidase treatments on the resistance of the HIV‐1 envelope to degradation
Authors: Papandreou Marie-Jeanne , Fenouillet Emmanuel
Published: July 2002

Using a CD4‐binding assay to assess the conformation of the human immunodeficiency virus envelope glycoprotein (CHO+
Env), we studied the effect of treatment with various glycosidases on the stability of Env in denaturing environments and in biological media: cleavage from Env of either high‐mannose‐type glycans (HMT−
Env) by endoglycosidase H or sialic acid residues (Sial−
Env) by sialidase did not alter Env stability whereas its complete deglycosylation (CHO−
Env) by N‐glycanase had a large effect. The influence of glycan removal on Env sensitivity to proteases was also studied. Thrombin cleavage within V3 was affected by N‐glycanase treatment; both HMT−
Env and CHO−
Env displayed an increased sensitivity to other endoproteases. Thus, partial deglycosylation increases Env sensitivity to proteases but only its total deglycosylation alters its stability.
A novel model for the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator
Authors: Annereau Jean-Philippe , Wulbrand Ulrich , Vankeerberghen Anne , Cuppens Harry , Bontems François , Tümmler Burkhard , Cassiman Jean-Jacques , Stoven Véronique
Published: July 2002

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most frequent mutation is the deletion of F508 in the first nucleotide binding fold (NBF1). It induces a perturbation in the folding of NBF1, which impedes posttranslational maturation of CFTR. Determination of the three‐dimensional structure of NBF1 would help to understand this defect. We present a novel model for NBF1 built from the crystal structure of bovine mitochondrial F1‐ATPase protein. This model gives a reasonable interpretation of the effect of mutations on the maturation of the protein and, in agreement with the CD data, leads to reconsideration of the limits of NBF1 within CFTR.
A disulfide bond between conserved cysteines in the extracellular loops of the human VIP receptor is required for binding and activation
Authors: Knudsen Sanne Møller , Tams Jeppe Wegener , Wulff Birgitte S , Fahrenkrug Jan
Published: July 2002

The importance of two highly conserved cysteines in the human vasoactive intestinal peptide receptor 1 (hVIPR 1) was examined. By site‐directed mutagenesis each Cys residue was converted into Ala or Ser. The mutant and wild‐type genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215‐Ala/Ser and Cys285‐Ala/Ser showed at least a 10‐fold decrease in binding affinity and receptor potency when compared to the wild type. In contradiction to the wild‐type receptor, both mutations were insensitive to dithiothreitol (DTT). The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilising the receptor in the correct conformation for ligand binding and activation.
Intrinsic nucleoside diphosphate kinase‐like activity as a novel function of 14‐3‐3 proteins
Authors: Yano Mihiro , Mori Sachie , Niwa Yasuharu , Inoue Masahiro , Kido Hiroshi
Published: July 2002

14‐3‐3 proteins play a role in many cellular functions as molecular chaperone and adapter proteins: they bind to and modulate several proteins involved in cell proliferation and differentiation, and also function ATP‐dependently in targeting of precursors to mitochondria. We show here that 14‐3‐3 purified from a human lymphoblastoma and also its recombinant τ isoform exhibited intrinsic nucleoside diphosphate (NDP) kinase‐like activity. 14‐3‐3 proteins preferentially catalyzed the transfer of the γ‐phosphate group from ATP, dATP or dGTP to all nucleoside diphosphates and this transfer involved acid‐labile phosphoenzyme intermediates. They also simultaneously catalyzed the reverse reaction of ATP hydrolysis. These properties of 14‐3‐3 are similar to those of NDP kinase, but not to those of adenylate kinase.
The effect of activin and FSH on the differentiation of rat granulosa cells
Authors: Kishi Hiroshi , Minegishi Takashi , Tano Mari , Kameda Takashi , Ibuki Yoshito , Miyamoto Kaoru
Published: July 2002

The differentiation of granulosa cells is regulated by follicle‐stimulating hormone (FSH) and local ovarian factors. To further analyze the role of FSH and activin in this process, we have examined the effect of FSH and activin on FSH and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor induction in granulosa cells. Granulosa cells from diethylstilbestrol (DES)‐primed immature rats produce activin and maintain FSH receptor without LH/hCG receptor expression in the absence of FSH. On the other hand, FSH induced granulosa cells to differentiate into more mature granulosa cells in which higher LH/hCG receptor expression and diminished activin production were observed.
Individual promoter and intron p53‐binding motifs from the rat Cyclin G1 promoter region support transcriptional activation by p53 but do not show co‐operative activation
Authors: Yardley Gina , Zauberman Arie , Oren Moshe , Jackson Paul
Published: July 2002

The rat Cyclin G1 gene promoter contains one p53‐binding motif upstream of the transcription start site, and a second motif downstream in the first intron. We have investigated the possibility that these motifs co‐operate to permit high level promoter activation by p53. Although individual motifs supported p53‐dependent, orientation‐independent transcriptional activation, using reporter plasmids containing both motifs, we found no evidence for co‐operative promoter activation either after co‐transfection with human p53 expression plasmids, or after exposure of transfected cells to cisplatin and UV‐radiation.
The LIM homeobox protein mLIM3/Lhx3 induces expression of the prolactin gene by a Pit‐1/GHF‐1‐independent pathway in corticotroph AtT20 cells
Authors: Girardin Stephen E , Benjannet Suzanne , Barale Jean-Christophe , Chrétien Michel , Seidah Nabil G
Published: July 2002

mLIM3, a member of the LIM homeobox family, was recently demonstrated to be critical for proliferation and differentiation of the pituitary cell lineage. Using a pool of degenerate oligonucleotides we determined the DNA sequence ANNAGGAAA(T/C)GA(C/G)AA as the set preferentially recognized by mLIM3. A nearly identical sequence is found in the prolactin (PRL) promoter, within a 15‐mer stretch from nucleotides (nts) −218 to −204 which is highly conserved between human, rat, and bovine. In order to test the hypothesis of a transcriptional effect of mLIM3 on the prolactin promoter, stable transfectants of mLIM3 cDNA in AtT20 tumor cells revealed that PRL mRNA expression was induced in 3 separate stable clones. Gel retardation experiments performed using nuclear extracts isolated from one of the AtT20/mLIM3 stable transfectants revealed affinity towards the 15‐mer element of the PRL promoter. From these results, we propose that the PRL promoter element (nts −218 to −204) could be functional in vivo. Finally, we demonstrate that in AtT20 cells prolactin mRNA expression is not induced by the Pit‐1/GHF‐1 pathway and that growth hormone mRNA is not detected concomitantly with prolactin. We conclude that mLIM3 may play a key role in inducing PRL gene expression in lactotrophs by binding to a conserved motif close to a Pit‐1/GHF‐1 site within the proximal PRL promoter.
The murine nuclear orphan receptor GCNF is expressed in the XY body of primary spermatocytes
Authors: Bauer Uta-Maria , Schneider-Hirsch Stefan , Reinhardt Sigrid , Benavente Ricardo , Maelicke Alfred
Published: July 2002

We have studied the expression of the nuclear orphan receptor GCNF (germ cell nuclear factor) on the mRNA and protein level in pubertal and adult mouse testes. We show by Northern and Western blot analyses and by in situ hybridization that GCNF is expressed in spermatocytes and round spermatids of adult mouse testis suggesting that GCNF may be a transcriptional regulator of spermatogenesis. Since the GCNF protein is accumulated in the XY body of late pachytene spermatocytes, it may be involved in transcriptional inactivation of sex chromosomes.
Immunoglobulin‐derived polypeptides enter the regulated secretory pathway in AtT‐20 cells
Authors: Castle Anna M. , Huang Amy Y. , Castle J.David
Published: July 2002

Constitutively secreted proteins have traditionally been believed to be excluded from the regulated secretory pathway. In this work we show that kappa light chain and Fc fragment, two markers of the constitutive pathway, are present in the regulated pathway in AtT‐20 cells. They colocalize with the endogenous hormone ACTH and they exhibit stimulus‐dependent secretion. The Fc fragment, which undergoes intracellular transport at the same rate as the ACTH precursor POMC, enters the forming secretory granules, however, it is partially lost during granule maturation. These observations show that classic constitutive secretory markers are not excluded from the regulated secretory pathway and that efficient sorting for regulated secretion occurs above a background of proteins which enter the granules without sorting.