Enrichment and efficient screening of ES cells containing a targeted mutation: the use of DT‐A gene with the polyadenylation signal as a negative selection maker

Gene targeting in embryonic stem (ES) cells via homologous recombination can occur at very low frequency. In order to enrich homologous recombinants before screening, a negative selection marker, such as thymidine kinase (TK) and diphtheria toxin A fragment (DTA), has been commonly used. In this study, we developed a negative selection marker using DTA gene with polyadenylation signal and it was designated DTApA. To determine the difference in targeting efficiency of the negative selections, we constructed three different targeting vectors for each negative selection (first, TK at the 3 end, second, TK at both the 5 and 3 ends <2 X TK>, and third, DTApA at the 5 end of the homologous sequences). Gene targeting experiments using these constructs clearly showed that negative selection using DTApA was more efficient than that using TK for homologous recombination and that negative selection using DTApA was as efficient as that using 2 X TK. Considering the fact that the use of DTApA is more convenient for construction of targeting vectors than that of 2 X TK, DTApA is an efficient negative selection marker.In addition, we examined long and accurate PCR (LAPCR) for screening gene targeted clones. The use of LAPCR with genomic DNAs from ES cell clones facilitated simple detection of homologous recombinants, suggesting that the screening with LAPCR is compatible with the use of longer homologous sequences of both arms in vector design. Our results indicate that the use of DTApA for negative selection together with the application of LAPCR for screening ensures efficient and timesaving screening for homologous recombinants.     

Feedback regulation of RNA polymerase subunit synthesis after the conditional overproduction of RNA polymerase in Escherichia coli

Summary The and subunit of RNA polymerase are thought to be controlled by a translational feedback mechanism regulated by the concentration of RNA polymerase holoenzyme. To study this regulation in vivo, an inducible RNA polymerase overproduction system was developed. This system utilizes plasmids from two incompatibility groups that carry RNA polymerase subunit genes under lac promoter/operator control. When the structural genes encoding the components of core RNA polymerase (, and ) or holoenzyme (, , and ⁷⁰) are present on the plasmids, induction of the lac promoter results in a two fold increase in the concentration of functional RNA polymerase. The induction of RNA polymerase overproduction is characterized by an initial large burst of synthesis followed by a gradual decrease as the concentration of RNA polymerase increases. Overproduction of RNA polymerase in a strain carrying an electrophoretic mobility mutation in the rpoB gene results in the specific repression of synthesis off the chromosome. These results indicate that RNA polymerase feedback regulation controls synthesis in vivo.     

Biochemical and immunological responses of hairy cell leukemia patients to interferonβ

Summary Ten hairy-cell leukemia patients were treated with interferon (IFN-) at a dose rate of 2 × 10⁶ IU/m² × 5 days for 4 weeks (induction therapy) and, thereafter, at the same dose three times a week for 11 months (maintenance therapy). The effect of this treatment on serum neopterin, 2-microglobulin, (2–5)oligoadenylate [(2–5)A _n ] levels, intracellular (2–5)A _n values and human Mx protein synthesis was analysed. There were significant rises in serum neopterin and (2–5)A _n levels during both induction and maintenance, whereas 2-microglobulin levels rose only during induction. Rises in intracellular (2–5)A _n were documented mainly during induction, but they were not significantly higher than pretherapy values. IFN provoked an increase in human Mx protein synthesis over the entire induction — maintenance period, but was only significantly higher than baseline during induction. All markers proved useful for monitoring the effects of IFN dose schedules, but were not predictive of clinical outcome. Natural killer activity and IFN production, which were initially defective, followed a different trend from that of the other factors studied, in that increases were documented only late in the course of therapy when the disease was already in remission.     

Variable Deposition of Amyloid β-Protein (Aβ) with the Carboxy-Terminus that Ends at Residue Valine40 (Aβ40) in the Cerebral Cortex of Patients with Alzheimer's Disease: A Double-Labeling Immunohistochemical Study with Antibodies Specific for Aβ40 and the Aβ that Ends at Residues Alanine42/Threonine43 (Aβ42)

Amyloid -protein (A) deposits in the cerebral cortices of patients with Alzheimer's disease (AD) were investigated immunohistochemically to determine their carboxy terminal sequences. Antibodies specific for A terminating at residue valine₄₀ (A40) and at residues alanine₄₂/threonine₄₃ (A42) were used. Virtually all parenchymal A deposits were positive for A42. Many of these deposits were also partially or completely labeled for A40. The degree of A40 labeling varied from area to area within a given brain and from AD case to AD case. In contrast to parenchymal deposits, A40 labeled essentially all the vascular deposits which constitute amyloid angiopathy (AA), with A42 occurring variably in some of these deposits. Occasional AA was found, however, in which A42 predominated or was exclusively deposited. Such a diversity of A species, both in brain parenchyma and in AA, suggests that multiple C-terminal processing mechanisms occur in the cell types responsible for these deposits.     

ATTRACTION D‘ACROLEPIOPSIS ASSECTELLA,EN OLFACTOMÈTRE,PAR DES SUBSTANCES ALLÉLOCHIMIQUES VOLATILES D’ALLIUM PORRUM

Résumé Pour rechercher le poireau, Acrolepiopsis assectella doit utiliser des stimulus olfactifs issus du végétal-hôte et notamment les substances soufrées spécifiques. Le pouvoir attractif de l'«odeur» du poireau et celui de différents thiosulfinates et disulfures trouvés dans cette plante sont donc étudiés et comparés par olfactométrie. Le comportement de mâles et de femelles vierges et fécondées est observé, en fonction de l'âge des imagos, pendant la scotophase. Alors qu'un courant d'air sans «odeur» significative déclenche essentiellement l'orientation et l'immobilisation des insectes face au flux, la plus part des mâles et des femelles quel que soit leur état sexuel sont attirés par le courant d'air lorsque celui-ci transporte l'«odeur» du poireau ou les substances volatiles étudiées. La sensibilité à l'«odeur» de la plante-hôte semble se modifier avec l'âge des insectes, les mâles âgés et les jeunes femelles étant les plus attirés. Cependant, ces dernières conservent une forte sensibilité une fois fécondées. Aux âges où l'«odeur» du poireau est la plus efficace, les thiosulfinates déclenchent chez les mâles et les femelles vierges une attraction plus importante que les disulfures, le radical propyle étant plus actif que le méthyle. Le thiosulfinate de dipropyle, labile, très abondant dans le poireau, semble donc avoir un rôle informationnel prépondérant.

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Summary To find the leek, Acrolepiopsis assectella uses olfactory stimulants from the hostplant, and specially the specific sulphur compounds. The attractiveness of the leek smell and of different thiosulfinates and disulfides found in this plant were studied and compared in an olfactometer. The behaviour of , and virgin or mated was observed during the scotophase according to to the age of the adults. In a clean air current the insects orient and remain stationary. The majority of and virgin or mated move upwind when the air current bears the leek smell or the voltile sulphur compounds. The sensitivity to the hostplant smell seems to change with the age of the insect, old and young being most attracted. Once mated, the retain a high sensitivity. When the responsiveness to the leek smell is highest (at 1 day and 5 days) the thiosulfinates have a greater effect than the disulfides in both and virgin . The propyl moiety is more active than the methyl one. The labile dipropyl thiosulfinate (propyl propanethiosulfinate), very profuse in the leek, seems to have the major role.

     

A comparison of male and female recombination frequency in wheat using RFLP maps of homoeologous group 6 and 7 chromosomes

A novel approach was used to compare male and female recombination rates in wheat. Doubled haploid lines were developed from an F₁ using two distinct approaches: the anther-culture technique and the Hordeum bulbosum system, from which sets of lines were developed from male and female meioses, respectively. The genotype of the lines was established at RFLP and isozyme markers polymorphic on chromosomes of homoeologous groups 6 and 7, and male and female linkage maps were calculated using this information. The markers in one segment of chromosome 6B exhibited disturbed segregation frequencies in the anther-culture population. The male and female maps differed significantly in recombination frequency between some markers on two chromosomes, and these were consistent in direction within chromosomes and inconsistent in direction between chromosomes. In two of the four chromosomes studied the male map was much longer than the female map. These results suggest that significant differences may exist in male and female recombination frequencies in bread wheat which are specific to certain chromosomal segments but are inconsistent in direction between chromosomes. Other factors, such as environmental influences, may also be important in creating differences.     

Synthesis of New 15-Ketosterol Analogues from Ergosterol

Ergosteryl acetate was converted through three stages into 3-acetoxy-24-methyl-5-cholesta-8(14),22-diene-15-one in 32% overall yield. The product was transformed to 3-hydroxy-24-methyl-5-cholesta-8(14),22-diene-15-one, 3-hydroxy-24-methyl-5-cholesta-8(14),22-diene-15-one, and 24-methyl-5-cholesta-8(14),22-diene-3,15-dione. The compounds were characterized by ¹H and ¹³C NMR spectra.     

DNA variations in regenerated plants of pea (Pisum sativum L.)

Summary The aim of this study was to determine whether DNA variations could be detected in regenerated pea plants. Two different genotypes were analyzed by cytogenetic and molecular techniques: the Dolce Provenza cultivar and the 5075 experimental line. Dolce Provenza regenerated plants showed a reduction in DNA content, particularly at the level of unique sequences and ribosomal genes. Moreover, regeneration was associated with an increase in DNA methylation of both internal and external cytosines of the CCG sequence. On the other hand, the DNA content of the 5075 line remained stable after regeneration. DNA reduction was found only in 5075 plants regenerated from callus cultures maintained for long incubation periods (about a year). The DNA variations observed are discussed both in relation to the genotype source and the role of tissue-culture stress.     

Zur Frage des Zeipunktes einer Pr?gung auf die Heimatregion beim Halsbandschn?pper(Ficedula albicollis)

Zusammenfassung Junge Halsbandschnäpper wurden handaufgezogen, flogen im Flugkäfig aus und wurden dort selbständig. Darauf wurden sie 90 km nach Süden verfrachtet und in einem von dieser Art unbewohnten Gebiet freigelassen. Im nächsten Frühjahr siedelten sich mindestens 9 dort an, was 19% Rückkehrern entspricht, wenn die Hälfte der Vögel waren. kehrten in geringerer Zahl zurück und wurden nicht restlos erfaßt.Eine weitere Gruppe wurde erst vor Ende der Jugendmauser verfrachtet. Auch davon kehrten 18-19% der zurück. Ein Zeitraum von rund 2 Wochen vor dem Wegzug reichte also zur Prägung auf ein Gebiet als Heimat aus.Von einer dritten Gruppe von insgesamt 68 Schnäppern (= ca. 34 ), die erst nach Ende der Jugendmauser zur Wegzugzeit aufgelassen wurde, konnte später keiner nachgewiesen werden, auch nicht am Aufzuchtsort. Letzteres könnte an der Ungunst der örtlichen Verhältnisse liegen.Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Interactions of protein kinase CK2β subunit within the holoenzyme and with other proteins

Protein kinase CK2 is a ubiquitous, highly conserved protein kinase with a tetrameric 22 structure. For the formation of this tetrameric complex a - dimer seems to be a prerequisite. Using the two-hybrid system and a series of CK2 deletion mutants, we mapped domains involved in - and - interactions. We also detected an intramolecular b interaction within the amino acid stretch 132-165.Using CK2 as a bait in a two-hybrid library screening several new putative cellular partners have been identified, among them the S6 kinase p90rsk, the putative tumor suppressor protein Doc-1, the Fas-associated protein FAF1, the mitochondrial translational initiation factor 2 and propionyl CoA carboxylase subunit.     

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (_m), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of _m may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of _m may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of _m has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of _m early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of _m may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect _m. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in _m.     

Considerations on the astroglial architecture and the columnar organization of the cerebral cortex

Present knowledge on astroglial roles in brain organization and function indicates that these cells can regulate the extracellular ionic composition and modulate neuronal activity. In this regard, the panglial synctium formed by electrically and chemically coupled stellate astrocytes (general mammalian architecture) is believed to provide an intracellular pathway for the redistribution of ions and molecules within the cerebral cortex. Long astroglial interlaminar processes (primate-specific architecture) that run parallel to apical dendrites in the cerebral cortex of primates, may provide additional properties to the glial participation in cortical physiology and function. Since these processes are exclusively present within the primate order, functional models of cortical computations for these species should incorporate the astroglial interlaminar architecture in addition to the panglial synctium. This study analyzes possible implications of interlaminar astroglial processes, for the regulation of the extracellular ionic composition and segregation of functional columns in the cerebral cortex.     

Evolutionary origin of the class A and class C β-lactamases

Summary The protein sequences of 18 class A -lactamases and 2 class C -lactamases were analyzed to produce a rooted phylogenetic tree using the DD peptidase of Streptomyces R61 as an outgroup. This tree supports the penicillin-binding proteins as the most likely candidate for the ancestoral origin of the class A and class C -lactamases, these proteins diverging from a common evolutionary origin close to the DD peptidase. The actinomycetes are clearly shown as the origin of the class A -lactamases found in other non-actinomycete species. The tree also divides the -lactamases from the Streptomyces into two subgroups. One subgroup is closer to the DD peptidase root. The other Streptomyces subgroup shares a common branch point with the rest of the class A -lactamases, showing this subgroup as the origin of the non-actinomycete class A -lactamases. The non-actinomycete class A -lactamase phylogenetic tree suggests a spread of these -lactamases by horizontal transfer from the Streptomyces into the non-actinomycete gram-positive bacteria and thence into the gram-negative bacteria. The phylogenetic tree of the Streptomyces class A -lactamases supports the possibility that horizontal transfer of class A -lactamases occurred within the Streptomyces.     

Synthesis of selectively radiolabeled hexasaccharides for the determination of enzymatic regioselectivity

Poly-N-acetyllactosamines provide backbone structures for functional modifications such as sialyl Lewis X. To understand how the biosynthesis of poly-N-acetyllactosamines is regulated, two branched oligosaccharides of the structure Gal1,4GlcNAc1, 6(Gal1,4GlcNAc1,2)-Man1,6Man-octyl 1 and 2 were synthesized in which one of the terminal galactose units was selectively radiolabeled. Hexasaccharides 1 and 2 were assembled from the chemically synthesized pentasaccharide precursors GlcNAc1,6(Gal1,4GlcNAc1,2)-Man1,6Man-octyl3 and Gal1,4GlcNAc1,6(GlcNAc1, 2) - Man1,6 Man-octyl 4 respectively, through treatment with UDP-1-[³H]-Gal and 1,4 galactosyltransferase. Compounds 1 and 2 were subsequently incubated with UDP-GlcNAc and the UDP-GlcNAc: Gal1-4Glc(NAc)1,3-N-acetylglucosaminyltransferase (i-GlcNAc transferase) resulting in a partial conversion to a mixture of heptasaccharides which were purified by HPLC. The branch selectivity of the addition of N-acetylglucosamine to compounds 1 and 2 was then characterized by endo--galactosidase digestion of the heptasaccharides, followed by isolation of the resultant pentasaccharides on C18 reverse-phase silica cartridges. Comparison of the amount of radiolabel to a control reaction lacking endo--galactosidase indicated the favored site of GlcNAc addition to be the lower 1,2-branch over the 1,6-branch by a 3:1 ratio.     

Molecular switch of F0F1-ATP synthase,G-protein,and other ATP-driven enzymes

Exchange-out of amide tritium from labeled -subunit of ₃₃ complex of F₀F₁-ATP synthase was not accelerated by ATP, suggesting that hemagglutinin-type transition of coiled-coil structure did not occur in -subunit. Local topology of nucleotide binding site and switch II region of G-protein resemble those of F₁- subunit and other proteins which catalyze ATP-triggered reactions. Probably, binding of nucleotide to F₀F₁-ATP synthase induces conformational change of the switch II-like region with transforming subunit structure from open to closed form and this transformation results in loss of hydrogen bonds with the subunit, thus enabling the subunit to move.     

Complex sex-determining mechanisms in three species of South American grasshoppers (Orthoptera,Acridoidea)

The karyotype of three species of South American grasshoppers are studied in this paper. Leiotettix sanguineus has two chromosome races, one of them with 2n=23 and an XO sex mechanism and the other, as far as we know limited to the Cerro Chato population, with 2n=22 and an XY sex mechanism. Leiotettix politus has two kinds of individuals, one with 2n=14 and XY sex chromosomes and the other 2n=13 and an X₁X₂Y mechanism. Dichroplus dubius presents 2n=21 and an X₁X₂Y sex chromosomes. One of the three specimens studied shows aberrant behaviour in the meiotic process.     

Enzyme-catalyzed synthesis of alkyl β-D-glycosides with crude homogenate ofSulfolobus solfataricus

Summary Crude homogenate of thermophilic archaebacteriumSulfolobus solfataricus, possessing a -glycosidase, has been used to synthesize different alkyl -D-glycosides starting from phenyl -D-glucoside, phenyl -D-galactoside and lactose as carbohydrate donors. High product yield (95% with respect to the carbohydrate donor) of octyl -D-glucoside has been obtained in a two-phase system containing 5% of water. The enantioselection for the galactosyl transfer to the secondary hydroxyl group of propane-1,2-diol is higher than that found using -galactosidase fromE. coli.     

Purification and immunocytochemical localization of kafirins inSorghum bicolor (L. Moench) endosperm

Summary Kafirins are the storage proteins of sorghum and are found in protein bodies in the seed endosperm. They have been classified as -, -, and -kafirins according to differences in molecular weight, solubility, and structure. The kafirins were purified, amino acid composition was determined, and immunolocalization methods were used to determine the organization of the protein bodies and distribution of kafirins throughout the endosperm. All three groups of kafirins were low in lysine. -Kafirins and -kafirins were relatively high in cysteine, and -kafirins were relatively high in methionine. Transmission electron microscopy showed that protein bodies in the peripheral endosperm were spheroid with concentric rings and few darkly stained inclusions. In contrast, protein bodies of the central endosperm were irregularly shaped with a higher proportion of darkly stained material. The light staining regions of the protein bodies are composed primarily of -kafirins with minor portions of - and -kafirins. The dark staining regions, however, are composed primarily of - and -kafirins. Immunoelectron microscopy showed that protein bodies in the peripheral endosperm contain predominantly a-kafirin with minor amounts of - and -kafirin. Central endosperm protein bodies are also predominantly -kafirin, but have a higher proportion of -kafirin and -kafirin than the peripheral endosperm protein bodies.Abbreviations GAR-HRP Goat anti-rabbit horseradish peroxidase - IgG immunoglobulin G - 2-ME 2-mercaptoethanol - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffer saline - TBS-T Tris buffer saline with Tween - TBS-T-B Tris buffer saline with Tween and bovine serum albumin - TCA trichloroacetic acid - UV ultraviolet     

Analysis of responses to human synthetic adrenomedullin and calcitonin gene-related peptides in the hindlimb vascular bed of the cat

Vasodilator responses to human adrenomedullin (hADM), a newly discovered hypotensive peptide, human calcitonin gene-related peptide- (hCGRP-) and hCGRP-, which share structural homology with hADM, were compared in the hindlimb vascular bed of the cat under constant flow conditions. Injections of hADM (0.003-1 nmol), hCGRP-, and hCGRP- (0.003-0.3 nmol) into the perfusion circuit caused dose-related decreases in hindlimb perfusion pressure. Vasodilator responses to hCGRP- and hCGRP- were similar in potency and duration, and the doses of hCGRP- and hCGRP- required to reduce hindlimb perfusion pressure 40 mm Hg (ED40 mm Hg) were significantly lower than the ED40 mm Hg for hADM. The duration of the hindlimb vasodilator responses to hCGRP- and hCGRP- were significantly longer than the duration of the response to hADM. Amylin, a peptide that shares structural homology with ADM and with CGRP, had no significant effect on hindlimb perfusion pressure when injected in doses up to 1 nmol. Decreases in hindlimb perfusion pressure in response to hADM, hCGRP-, and hCGRP- were not altered by L-N5-(1-iminoethyl)-ornithine (L-NIO) in a dose of the nitric oxide synthase inhibitor that decreased the vasodilator response to acetylcholine or by the cyclooxygenase inhibitor, meclofenamate, in a dose that decreased the vasodilator response to archidonic acid. The present data demonstrate that hADM, hCGRP-, and hCGRP- have potent, but relatively short-lasting, vasodilator activity, and that vasodilator responses are not dependent on the release of nitric oxide or vasodilator prostaglandins in the hindlimb vascular bed of the cat.