Sic1 is phosphorylated by CK2 on Ser201 in budding yeast cells

We have previously identified Ser201 of Sic1, a yeast cyclin-dependent kinase inhibitor, as an in vitro target of protein kinase CK2. Here we present new evidence, by using specific anti-P-Ser201 antibodies and 2-D gel electrophoresis coupled to MALDI mass spectrometry analysis, that Sic1 is phosphorylated in vivo on Ser201 shortly after its de novo synthesis, during late anaphase in glucose-grown cells. This phosphorylation is also detected in Sic1 immunopurified from G1 cells. In agreement with these data we also show that the catalytic alpha' subunit of CK2, whose function is required for cell cycle progression, is detected in Sic1 immunopurified complexes, and that phosphorylation on Ser201 is reduced after CK2 inactivation at the non-permissive temperature in a cka1delta cka2(ts) yeast strain. These data strongly support the notion that CK2 phosphorylates Sic1 in vivo.     

Somatic embryogenesis in <Emphasis Type="Italic">Pinus nigra</Emphasis>: embryogenic tissue initiation,maturation and regeneration ability of established cell lines

The effect of plant growth regulators (PGR), 6-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and sugars (sucrose, maltose, glucose, fructose) on the initiation of somatic embryogenesis of Pinus nigra Arn. was investigated. Megagametophytes containing immature zygotic embryos have been used as explants. The experiments were done in the years 2000 and 2001. Higher initiation frequencies were obtained in 2001 when the zygotic embryos showed uniformity, being in the precotyledonary stage of development. Embryogenic tissue initiation occurred on all the media tested, including PGR-free medium. Relatively high initiation frequencies were obtained on media containing either NAA (9.09 %) or 2,4-D (7.14 %) alone. Somatic embryos were present as bipolar structures and showed differences in morphological features among cell lines. Plantlet regeneration occurred in cell lines containing bipolar somatic embryos composed of compact meristematic embryo “head” and suspensor organized into bundles.We highly appreciate the financial support from VEGA, Slovak Grant Agency, project No. 2/2089/22.     

A high standard metabolic rate constrains juvenile growth

The allocation of energy to various components of an individual's energy budget is often viewed as a competitive process. As such, a tradeoff may exist between production (growth) and maintenance metabolism. One view of a potential tradeoff, termed “the principle of allocation”, suggests that individuals with lower maintenance metabolic expenditures may have higher growth rates. To determine whether such a tradeoff exists, I analyzed the relationship between growth rate and maintenance metabolism of 225 juvenile snapping turtles housed in the laboratory. I measured growth from hatching to 6 months of age, and then measured oxygen consumption and calculated standard metabolic rate. Mean growth rate was 0.19 g d⁻ and mean standard metabolic rate (SMR) was 1.41 kJ d⁻. Maintenance metabolism and growth were negatively correlated after both were adjusted for body mass. The results support the “principle of allocation” theory: individuals with higher standard metabolic rates tended to have low growth rates.     

Actin: From Cell Biology to Atomic Detail

Over the past 2 decades our knowledge about actin filaments has evolved from a rigid “pearls on a string” model to that of a complex, highly dynamic protein polymer which can now be analyzed at atomic detail. To achieve this, exploring actin's oligomerization, polymerization, polymorphism, and dynamic behavior has been crucial to understanding in detail how this abundant and ubiquitous protein can fulfill its various functions within living cells. In this review, a correlative view of a number of distinct aspects of actin is presented, and the functional implications of recent structural, biochemical, and mechanical data are critically evaluated. Rational analysis of these various experimental data is achieved using an integrated structural approach which combines intermediate-resolution electron microscopy-based 3-D reconstructions of entire actin filaments with atomic resolution X-ray data of monomeric and polymeric actin.     

Methodology for the development of a drug library based upon collision-induced fragmentation for the identification of toxicologically relevant drugs in plasma samples

The possibility of creating a robust mass spectral library with use of high-performance liquid chromatography–atmospheric pressure–electrospray ionization (HPLC–AP–ESI) for the identification of drugs misused in cases of clinical toxicology has been examined. Factors reported as influencing the fragmentation induced by “source transport region collision induced dissociation” (CID) have been tested in this study (i.e. solvent, pH, different acids or buffer salts and their concentration, different organic modifiers and the modifier concentration). The tests performed on a few “model drugs” were analysed with use of two different single quadrupole instruments. The large number of mass spectra obtained appears to be affected by the mobile phase conditions to only a minor extent. This also holds for the mass spectra obtained at two different instruments (laboratories). Subsequently breakdown curves have been measured for about 20 randomly chosen drugs by variation of the kinetic energy of their ions in the CID zone through changing the fragmenter voltage. These breakdown curves were used to optimize the fragmenter voltage for each drug. The optimized fragmenter voltages were then applied by use of a variably ramped fragmenter voltage to acquire mass spectra for the library. The chromatographic separations were run on a Zorbax Stable bond column using a 10-mM ammonium formate–acetonitrile gradient method. Spiked blank serum and patient samples with a total of 40 different drugs were extracted with use of a standard basic liquid–liquid extraction (LLE) method. A search of significant peaks in the chromatogram by application of the developed mass spectral library is shown to result in a more than 95% positive identification.     

The synthesis of P-(2-acetamido-2-deoxy-α-D-glucopyranosyl) P-ficaprenyl pyrophosphate

2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α,β-D-glucopyranosylammonium phosphate was prepared by the action of crystalline phosphoric acid on 2-acetamido-1,3,4,6-tetra-O-acetyl-β-D-glucopyranose. The α-D anomer (3) was the main product, and was isolated pure by preparative thin-layer chromatography or by removal of the β-D anomer (6) by selective acid hydrolysis. Ficaprenyl phosphate was prepared from ficaprenol, obtained as an isomeric mixture (mainly C₅₅) from an extract of Ficus elastica. Compound 3 was converted into the free acid and then into the tributyl-ammonium salt, which was treated with P¹-diphenyl P²-ficaprenyl pyrophosphate to give the acetylated pyrophosphate diester 8, characterized by analytical, spectral, and hydrogenolytic studies. Deacetylation of 8 gave the synthetic “lipid intermediate”, P¹-(2-acetamido-2-deoxy-D-glucopyranosyl) P²-ficaprenyl pyrophosphate (9), the properties of which were compared with those of natural substances considered to be active in the biosynthesis of teichoic acids.     

Use of mass spectrometry to probe the nucleophilicity of cysteinyl residues of prolyl hydroxylase domain 2

Prolyl hydroxylase domain 2 (PHD2) plays an important role in hypoxic sensing in humans. Here we report studies on the reactivity of cysteinyl residues of the catalytic domain of PHD2 using an approach in which nondenaturing electrospray ionization–mass spectrometry (ESI–MS) analyses were combined with the use of a thiol library and residue substitution. Among the seven cysteinyl residues of the PHD2 catalytic domain, Cys201 was found to be predominantly modified by thiols or N-ethylmaleimide. Selective modification of Cys201 was further demonstrated with methanethiosulfonate, a spin-labeled probe. The modified PHD2 will be useful in electron paramagnetic resonance studies on PHD2. The results demonstrate the use of a combined library/residue substitution/ESI–MS approach for analyzing residue reactivity.     

The Second Catalytic Domain of Protein Tyrosine Phosphatase δ (PTPδ) Binds to and Inhibits the First Catalytic Domain of PTPς

The LAR family protein tyrosine phosphatases (PTPs), including LAR, PTPδ, and PTPς, are transmembrane proteins composed of a cell adhesion molecule-like ectodomain and two cytoplasmic catalytic domains: active D1 and inactive D2. We performed a yeast two-hybrid screen with the first catalytic domain of PTPς (PTPς-D1) as bait to identify interacting regulatory proteins. Using this screen, we identified the second catalytic domain of PTPδ (PTPδ-D2) as an interactor of PTPς-D1. Both yeast two-hybrid binding assays and coprecipitation from mammalian cells revealed strong binding between PTPς-D1 and PTPδ-D2, an association which required the presence of the wedge sequence in PTPς-D1, a sequence recently shown to mediate D1-D1 homodimerization in the phosphatase RPTPα. This interaction was not reciprocal, as PTPδ-D1 did not bind PTPς-D2. Addition of a glutathione S-transferase (GST)–PTPδ-D2 fusion protein (but not GST alone) to GST–PTPς-D1 led to ~50% inhibition of the catalytic activity of PTPς-D1, as determined by an in vitro phosphatase assay against p-nitrophenylphosphate. A similar inhibition of PTPς-D1 activity was obtained with coimmunoprecipitated PTPδ-D2. Interestingly, the second catalytic domains of LAR (LAR-D2) and PTPς (PTPς-D2), very similar in sequence to PTPδ-D2, bound poorly to PTPς-D1. PTPδ-D1 and LAR-D1 were also able to bind PTPδ-D2, but more weakly than PTPς-D1, with a binding hierarchy of PTPς-D1>>PTPδ-D1>LAR-D1. These results suggest that association between PTPς-D1 and PTPδ-D2, possibly via receptor heterodimerization, provides a negative regulatory function and that the second catalytic domains of this and likely other receptor PTPs, which are often inactive, may function instead to regulate the activity of the first catalytic domains.     

Testing German shepherd puppies to assess their chances of certification

Behavioral activity of 7-week-old German shepherd puppies was tested and the activities analyzed if they could be used for predicting police efficiency of the individual. In total 206 individuals sired by 42 sires and 44 dams were used. The activities were divided into 10 tasks in which reactions and behavior of pups were scored from 0 to 5 points. All pups were tested separately from other conspecifics. Probability that the puppy will pass the certification was tested by a logistic regression. Of the 206 puppies, 148 passed the certification while 58 failed. Some tested behavioral variables were moderately to highly correlated with one another. Therefore we applied a factor analysis. Three factors were retained accounting for 100% of the shared variance. After inspection of the rotated factor pattern matrix and its confidence intervals, it appeared that variables “Independent movement and interactions with the tester”, “Negotiating obstacles”, “Entering a room”, “Behavior toward a person”, and “Behavior in new environments” loaded on Factor 1 (“Factor for movement”), while variables “Response to distracting stimuli caused by a shovel”, “Response to a distracting noise while left alone in a room”, and “Response to loud distracting stimuli” on Factor 2 (“Factor for responding to noise”) and variables “Retrieval” and “Tug of war” on Factor 3 (“Factor for attitude to predation”). In the final logistic regression model, the probability that the puppy will pass the certification depended on the higher weight at the time of testing (, P = 0.0005), on the “Factor for attitude to predation” (, P = 0.0007), on the “Factor for responding to noise”, where the higher the score, the weaker was the response (, P = 0.0232), and on the “Factor for movement” showing an increasing probability with decreasing score (, P = 0.0219). The tests in our study seem to be a good base which might enable selection for suitable dogs as early as 7 weeks of age. The puppies having high probability to pass certification in adulthood were heavy individuals willing to chase, catch, and fetch a tennis ball, and follow a rag drawn away from them, while weakly responding to a distracting noise in various situations and showing low activity while negotiating obstacles and moving and interacting with the tester. To conclude, contrary to skeptical assumptions expressed by various authors, the specific puppy tests for police dogs provide a reliable tool for predicting future service ability of a puppy. Differences in methodology are likely to contribute to a lack of consensus among various studies.     

Structural Analysis of F18 Fimbriae Expressed by Porcine Toxigenic Escherichia coli

The F18 fimbriae expressed by porcine toxigenic Escherichia coli strains are 1- to 2-mm-long filaments that mediate the adhesion of the bacteria to enterocytes. The backbone of these fimbriae is built from a major structural 15.1-kDa protein, FedA. The structure of isolated negatively stained F18 fimbriae imaged by dark-field scanning transmission electron microscopy (STEM) was resolved to approximately 2 nm. Analyzing their helical symmetry showed the axially repeating units to alternate in a “zigzag” manner around the helical axis with an axial rise of 2.2 nm. Two repeating units give rise to the observed 4.3-nm helical repeat, which is practically identical to the pitch of the one-start helix formed. Additionally, an axially repeating pattern with a 27-nm spacing was found on rotary-shadowed fimbriae. Mass-per-length determination of unstained F18 fimbriae by STEM revealed the axially repeating unit to have a molecular mass of 25.4 kDa, indicating that it is a FedA monomer, with the difference in mass arising from the minor subunits, FedE and FedF. The presence of the latter two proteins might cause the observed 27-nm axial pattern.     

Electrochemistry of some substituted pteridines

Some substituted 4(3H)-pteridones have been investigated by classical polarography, cyclic voltammetry, and controlled potential electrolysis; based on these results, the following reaction path for substituted 2-amino-4(3H)-pteridones seems likely. In neutral and slightly alkaline solution the first step is a reversible two-electron reduction to the unreducible 5,8-dihydro derivative, which tautomerizes into a reducible 7,8-dihydropteridone at a rate depending on the substituents. At a more negative potential the 7,8-dihydro derivative is reduced in a two-electron reaction to the unreducible 5,6,7,8-tetrahydropteridone. This compound can be oxidized in a reversible reaction to a 6,7-dihydropteridone, a “quinonoid” form, which tautomerizes into the 7,8-dihydropteridone. The data from cyclic voltammetry favour the formulation of the “quinonoid” form as the 6,7-dihydropteridone, an “o-quinonoid” structure.     

Different responses of two contrasting wheat genotypes to abscisic acid application

Purpose of this study was to investigate different responses of two wheat genotypes (Triticum aestivum L.) from the wet and dry climate regions to exogenous abscisic acid (ABA) application under well-watered and water-stressed conditions. Exogenous ABA was applied to the leaves by spraying and changes in dry matter accumulation and allocation, endogenous ABA content and carbon isotope ratio (δ¹³C) were monitored. The ABA application significantly decreased stem height, total biomass, total leaf area, total grain mass and leaf area/mass ratio, and significantly increased root/aboveground biomass ratio, endogenous ABA content and δ¹³C under well-watered and water-stressed conditions. Compared with the wet climate genotype, the dry climate genotype was more responsive to exogenous ABA application, resulting in lower stem height, total biomass, total leaf area, total grain mass and leaf area/mass ratio, and higher root/aboveground biomass ratio, endogenous ABA content and δ¹³C under all experimental treatments.The research was supported by the Program of “100 Distinguished Young Scientists” and “ Knowledge Innovation Engineering” of the Chinese Academy of Sciences (No. KSCX2-SW-115).     

Protein analysis of dwarfed transgenic rice plants overexpressing GA2-oxidase gene

Using 2-D electrophoresis, we analyzed proteins from transgenic rice overexpressing gibberellin acid (GA) catabolic enzyme, GA2-oxidase. These results indicate eight specific proteins differentially expressed in the transformed rice stems of T₁ generation, but non in case of T₂ generation. Proteins isolated from different stages of leaves of T₁ generation showed no significant differences, except one-month-old leaf, where five differentially expressed proteins are visible.This work was supported in part by a research project of an Identification and Analysis of Proteins for Gene Discovery and Elucidation of Functions of Useful Genes in Rice Genome from Ministry of Agriculture, Forestry and Fisheries, and also supported by a part of grant from the Program of Basic Research Activities for Innovative Biosciences. Martin Hajduch was supported by European Commission’s specific research and technological development program “Confirming the International Role of Community Research” 1998-2002 (EU Fellowship to Japan). The authors are solely responsible for the content of this paper and it does not represent the opinion of the Community, and the Community is not responsible for any of use that might be made of date appearing herein.     

Perinatal adaptation in mammals: the impact of metabolic rate

Mammalian birth is accompanied by profound changes in metabolic rate that can be described in terms of body size relationship (Kleiber's rule). Whereas the fetus, probably as an adaptation to the low intrauterine pO₂, exhibits an “inappropriately” low, adult-like specific metabolic rate, the term neonate undergoes a rapid metabolic increase up to the level to be expected from body size. A similar, albeit slowed, “switching-on” of metabolic size allometry is found in human preterm neonates whereas animals that are normally born in a very immature state are able to retard or even suppress the postnatal metabolic increase in favor of weight gain and O₂ supply. Moreover, small immature mammalian neonates exhibit a temporary oxyconforming behavior which enhances their hypoxia tolerance, yet is lost to the extent by which the size-adjusted metabolic rate is “locked” by increasing mitochondrial density. Beyond the perinatal period, there are no other deviations from metabolic size allometry among mammals except in hibernation where the temporary “switching-off” of Kleiber's rule is accompanied by a deep reduction in tissue pO₂. This gives support to the hypothesis that the postnatal metabolic increase represents an “escape from oxygen” similar to the evolutionary roots of mitochondrial respiration, and that the overall increase in specific metabolic rate with decreasing size might contribute to prevent tissues from O₂ toxicity.     

Some connections between physiognomic traits and intelligence

A sample of 84 female subjects (students) was taken and split up on the basis of the IST-ratings (“Intelligence Structure Test” by Amthauer) into two extreme groups of “more intelligent” (N = 14) and “less intelligent” (N = 11) subjects with an aim to testing Kozeny's findings. Using his photographic-statistical method, Kozeny had observed that “more intelligent” people have a more open eye, a smaller and straighter mouth, a stronger chin, and—above all in its side dimensions—a better developed forehead. These physiognomic indicators of differences in intelligence were confirmed to a large extent by the measurements taken from the portraits of the subjects.     

Stimulation by DIF Causes an Increase of Intracellular CainDictyostelium discoideum

Using fluorescence-activated cell sorting (FACS), we have studied the effect of the differentiation-inducing factor (DIF) on cellular Ca²⁺inDictyostelium discoideum.We have shown previously that freshly starved or postaggregation amoebae are heterogenous with respect to the amounts of cellular Ca²⁺that they contain; the L or “low Ca²⁺” class exhibits a prespore tendency and the H or “high Ca²⁺” class exhibits a prestalk tendency. Upon adding DIF, within 2 min there is an approximately twofold increase in the relative fraction of amoebae falling in the H class. A major part of the increase is caused by Ca²⁺influx from the extracellular medium. Therefore a rise in the level of cellular Ca²⁺is an early step in the signal transduction pathway following stimulation by DIF. Also, in parallel with the cellular heterogeneity in respect of Ca²⁺content, there is a heterogeneity in the response to DIF, which appears to be restricted to L cells.     

Glucuronic acid conjugates

The methods of assay in body fluids of 1-β-alkyl, 1-β-phenyl and 1-β-acyl glucuronic acids (“glucuronide conjugates”) have been reviewed. Most of the 78 references cited (from the literature of the period 1990–1997) concern the glucuronide conjugates of drug metabolites, and these have been considered, for reasons of accessibility, within sections of individual drug classes such as analgesics, anti-cancer agents and opioids. Other glucuronide conjugates are considered under “miscellaneous compounds”. A few gas chromatography and capillary electrophoresis methods are described, but the major technique of assay (62 citations) is reversed-phase high-performance liquid chromatography.     

Phosphorus-containing purines and pyrimidines: A new class of transition state analogs

Synthetic routes to the [1,5,2]-diazaphosphorine (“4-phosphapyrimidine”), imidazo[4,5-e][1,5,2]-diazaphosphorine (“6-phosphapurine”), and imidazo[4,5-d][1,3,2]-diazaphosphorine (“2-phosphapurine”) ring systems have been developed. Appropriately functionalized derivatives of these heterocycles are desired as possible transition state analogs of the nucleoside deaminases.     

A label-free mass spectrometry method for the quantification of protein isotypes

Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as “built-in” internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from βI-, βII-, βIII-, βIVb-, and βV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the β-tubulin isotypes. To show the utility of this method, we determined that βI-tubulin represented 77% and βIII-tubulin represented 3.2% of the total HeLa β-tubulin.