bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains.
Authors: Crawford N M , Smith M , Bellissimo D , Davis R W
Published: May 2006

The sequence of nitrate reductase (EC 1.6.6.1) mRNA from the plant Arabidopsis thaliana has been determined. A 3.0-kilobase-long cDNA was isolated from a lambda gt10 cDNA library of Arabidopsis leaf poly(A)+ RNA. The cDNA hybridized to a 3.2-kilobase mRNA whose level increased 15-fold in response to treatment of the plant with nitrate. An open reading frame encoding a 917 amino acid protein was found in the sequence. This protein is very similar to tobacco nitrate reductase, being greater than 80% identical within a section of 450 amino acids. By comparing the Arabidopsis protein sequence with other protein sequences, three functional domains were deduced: (i) a molybdenum-pterin-binding domain that is similar to the molybdenum-pterin-binding domain of rat liver sulfite oxidase, (ii) a heme-binding domain that is similar to proteins in the cytochrome b5 superfamily, and (iii) an FAD-binding domain that is similar to NADH-cytochrome b5 reductase.
Down-regulation of beta-adrenergic receptors: agonist-induced reduction in receptor mRNA levels.
Authors: Hadcock J R , Malbon C C
Published: May 2006

Incubation of DDT1 MF-2 hamster vas deferens cells with beta-adrenergic agonists results in a time- and concentration-dependent decreases in both beta-adrenergic receptor (beta AR) responsiveness and receptor number. Receptor mRNA levels were quantified by DNA-excess solution hybridization by using a 170-nucleotide single-stranded probe derived from the hamster beta 2AR cDNA. RNA blot analysis of poly(A)+-selected RNA with the solution probe revealed a 2.2-kilobase species. Digestion of the RNA/solution probe mixture with S1 endonuclease revealed a single species of RNA (170 bases) that was protected by the solution probe. DDT1 MF-2 cells were found to contain 0.38 pg of beta AR mRNA per microgram of total cellular RNA. Incubation (16 hr) with isoproterenol decreased beta AR mRNA levels in cells by 40%. This agonist-induced decrease in receptor mRNA levels was found to be dependent on the time of incubation and the dose of agonist. The decrease in beta AR mRNA was half-maximal at 0.1-0.5 microM isoproterenol. The beta-adrenergic antagonists CGP 20712A (beta 1-selective) and ICI 118,551 (beta 2-selective) blocked in a dose-dependent fashion the ability of isoproterenol to effect receptor mRNA levels. The beta 2-adrenergic antagonist displayed a potency 25-fold greater than that of the beta 1-adrenergic antagonist, in agreement with the subtype of receptor (beta 2) expressed by these cells. For down-regulated cells in which receptor mRNA levels declined in response to agonist, the addition of the antagonist ligand (-)-propranolol (1 microM) was able to restore receptor mRNA levels to 90% of the control value within 12 hr. Full recovery of steady-state beta AR mRNA was achieved within 60 hr. These studies provide a molecular explanation for the down-regulation of GTP-binding regulatory protein (G protein)-linked cell-surface receptors that accompanies desensitization.
Secondary structure of complement component C3a anaphylatoxin in solution as determined by NMR spectroscopy: differences between crystal and solution conformations.
Authors: Nettesheim D G , Edalji R P , Mollison K W , Greer J , Zuiderweg E R
Published: May 2006

Two-dimensional 1H NMR investigations were used to locate elements of regular secondary structure in the human complement protein C3a (the des-Arg77 derivative) in solution. The results were compared to a refined crystal structure based on the 3.2-A resolution structure of des-Arg77-C3a [Huber, R., Scholze, H., Paques, E. P. & Deisenhofer, J. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1389-1399]. In excellent agreement with the x-ray data, helices occur in the regions of residues 17-28 and 36-43 in solution. In contrast to the x-ray data, where a third long helix was found from residue 47 to residue 73, the solution data show a shorter helix in the region from residue 47 to residue 66, followed by a transition range at positions 67-70, leading into a six-residue carboxyl-terminal peptide in dynamic random coil conformation. At the amino terminus, a well-defined helix is observed in solution for the residues 8-15 region, which, like the carboxyl terminus, gradually changes to dynamic random coil toward the end of the polypeptide chain. This is at variance with the x-ray data as well, in which residues 13-15 are nonhelical and no electron density could be assigned to the first 12 residues due to disorder.
Canonical formulation of linkage thermodynamics.
Authors: Di Cera E , Gill S J , Wyman J
Published: May 2006

The canonical structure of the group of thermodynamic potentials obtained from the energy of a physico-chemical system removes any possible distinction between physical and chemical binding phenomena. Transformations of these potentials by means of Jacobians give linkage relations for equilibrium thermodynamics. Linkage matrices are introduced to explore the functional properties of a biological macromolecule. They provide a quantitative basis for a broader approach and understanding of generalized binding phenomena.
Identification of two integral membrane proteins of Plasmodium falciparum.
Authors: Smythe J A , Coppel R L , Brown G V , Ramasamy R , Kemp D J , Anders R F
Published: May 2006

We describe the isolation and cloning of two integral membrane protein antigens of Plasmodium falciparum. The antigens were isolated by Triton X-114 temperature-dependent phase separation, electrophoretically transferred to nitrocellulose, and used to affinity-purify monospecific human antibodies. These antibodies were used to isolate the corresponding cDNA clones from a phage lambda gt11-Amp3 cDNA expression library. Clone Ag512 corresponds to a Mr 55,000 merozoite rhoptry antigen, and clone Ag513 corresponds to a Mr 45,000 merozoite surface antigen. Both proteins can be biosynthetically labeled with [3H]glucosamine and [3H]myristic acid, suggesting that they may be anchored in membranes via a glycosylphosphatidylinositol moiety. Similarities in the C-terminal sequences of the Mr 45,000 merozoite surface antigen and the Trypanosoma brucei variant surface glycoproteins provides further evidence that this antigen has a glycosylphosphatidylinositol anchor.
Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats.
Authors: Di Chiara G , Imperato A
Published: May 2006

The effect of various drugs on the extracellular concentration of dopamine in two terminal dopaminergic areas, the nucleus accumbens septi (a limbic area) and the dorsal caudate nucleus (a subcortical motor area), was studied in freely moving rats by using brain dialysis. Drugs abused by humans (e.g., opiates, ethanol, nicotine, amphetamine, and cocaine) increased extracellular dopamine concentrations in both areas, but especially in the accumbens, and elicited hypermotility at low doses. On the other hand, drugs with aversive properties (e.g., agonists of kappa opioid receptors, U-50,488, tifluadom, and bremazocine) reduced dopamine release in the accumbens and in the caudate and elicited hypomotility. Haloperidol, a neuroleptic drug, increased extracellular dopamine concentrations, but this effect was not preferential for the accumbens and was associated with hypomotility and sedation. Drugs not abused by humans [e.g., imipramine (an antidepressant), atropine (an antimuscarinic drug), and diphenhydramine (an antihistamine)] failed to modify synaptic dopamine concentrations. These results provide biochemical evidence for the hypothesis that stimulation of dopamine transmission in the limbic system might be a fundamental property of drugs that are abused.
Clone-forming ability and differentiation potential of migratory neural crest cells.
Authors: Baroffio A , Dupin E , Le Douarin N M
Published: May 2006

The neural crest of vertebrate embryos gives rise to a variety of differentiated cell types, including neuronal and non-neuronal cells of peripheral ganglia (sensory and autonomic), pigment cells, and mesectodermal derivatives. Neural crest cells were taken from quail embryos at the level encompassing mesencephalon and metencephalon and the developmental potentials were evaluated by culturing them as single cells on 3T3 feeder layers. Such conditions proved to be particularly favorable for survival, proliferation, and differentiation of quail neural crest cells. Two hundred and forty-three clones that contained from 1 to greater than 20,000 cells were analyzed after 7-10 days of culture. Phenotype analysis provided evidence for the existence of cells with extremely diverse developmental potencies. A few committed neuron progenitors were observed as well as some pluripotent cells, able to differentiate into several types of neurons, non-neuronal cells, and melanocytes, and many cells with intermediate developmental potencies. These cloning experiments revealed the striking heterogeneity of migrating neural crest cells in terms of their capacity for differentiation and their potential for proliferation.
Climate and red spruce growth and decline in the northern Appalachians
Authors: Johnson A. H. , Cook E. R. , Siccama T. G.
Published: May 2006

Between the mid-1960s and mid-1980s, red spruce (
Picea rubens
Sarg.) died at unusual rates on the mountains of New York and western New England. We determined the relationship between standardized tree ring widths and monthly climate data for calibration and verification periods from 1856 to 1981 and found that after about 1960, there was a distinct shift in the temperature variables related to standardized ring widths in vigorous spruce. The beginning of widespread spruce mortality, regionwide growth decreases, and the shift in response to climate in the early 1960s corresponds to the onset of a decade of unusually cold winters and several consecutive years when severe winter damage was noted across the Northeast in this species. We suggest that the episodes of winter damage are an important initiating and synchronizing factor in the red spruce decline.
Sequence analysis of cDNA and genomic DNA for a putative pertussis toxin-insensitive guanine nucleotide-binding regulatory protein alpha subunit.
Authors: Matsuoka M , Itoh H , Kozasa T , Kaziro Y
Published: May 2006

We have isolated cDNA clones from rat C6 glioma cells coding for several guanine nucleotide-binding regulatory protein (G protein) alpha subunits (G alpha). The cDNA clones were then used to isolate human chromosomal genes. Among human genomic clones isolated by cross-hybridization with the rat cDNA for the alpha subunit of the inhibitory G protein Gi2, termed Gi2 alpha, a clone designated lambda HGi62 was found to contain a sequence that is highly homologous but distinct from any of the known G alpha sequences, and we have tentatively designated this sequence Gx alpha. We have searched a rat brain cDNA library with the Gx alpha sequence and isolated a cDNA clone containing a rat sequence similar to human Gx alpha. The cDNA contained a single open reading frame of 1065 nucleotides coding for a protein of 355 amino acids with a calculated molecular weight of 40,879. The amino acid sequence of rat Gx alpha shows 66% and 40% similarity with rat Gi2 alpha and rat Gs alpha (the alpha subunit of the stimulatory G protein), respectively. By RNA blot hybridization analysis, mRNA of approximately 3.2 kilobases was detected mainly in brain. Interestingly, the deduced amino acid sequence of Gx alpha predicts that the Gx alpha protein may be refractory to modification by pertussis toxin since the cysteine residue in the fourth position from the C terminus of pertussis toxin-sensitive G alpha is replaced by isoleucine.
Insulin stimulates the dephosphorylation and activation of acetyl-CoA carboxylase.
Authors: Witters L A , Watts T D , Daniels D L , Evans J L
Published: May 2006

The mechanism underlying the ability of insulin to acutely activate acetyl-CoA carboxylase [acetyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.2; AcCoA-Case] has been examined in Fao Reuber hepatoma cells. Insulin promotes the rapid activation of AcCoACase, as measured in cell lysates, and this stimulation persists to the same degree after isolation of AcCoACase by avidin-Sepharose chromatography. The insulin-stimulated enzyme, as compared with control enzyme, exhibits an increase in both citrate-independent and -dependent activity and a decrease in the Ka for citrate. Direct examination of the phosphorylation state of isolated 32P-labeled AcCoACase after insulin exposure reveals a marked decrease in total enzyme phosphorylation coincident with activation. The dephosphorylation due to insulin appears to be restricted to the phosphorylation sites previously shown to regulate AcCoACase activity. All of these effects of insulin are mimicked by a low molecular weight autocrine factor, tentatively identified as an oligosaccharide, present in conditioned medium of hepatoma cells. These data suggest that insulin may activate AcCoACase by inhibiting the activity of protein kinase(s) or stimulating the activity of protein phosphatase(s) that control the phosphorylation state of the enzyme.
Type 1 plasminogen activator inhibitor gene: functional analysis and glucocorticoid regulation of its promoter.
Authors: van Zonneveld A J , Curriden S A , Loskutoff D J
Published: May 2006

Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, we have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5' flanking region of the gene revealed a perfect "TATA box" at position -28 to position -23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5' flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk- fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides -305 and +75 of the plasminogen activator inhibitor type 1 gene.
Immunological function of HLA-C antigens in HLA-Cw3 transgenic mice.
Authors: Dill O , Kievits F , Koch S , Ivanyi P , Hämmerling G J
Published: May 2006

The human major histocompatibility complex encodes three classical class I antigens, HLA-A, -B, and -C. Of these HLA-A and -B act as strong transplantation antigens and as restriction molecules for recognition of foreign antigen by cytotoxic T lymphocytes. In contrast, little is known about HLA-C and it is not clear whether HLA-C has the same functional properties as HLA-A and -B. Transgenic C57BL/6 mice expressing the HLA-Cw3 gene were established. Functional studies demonstrated that transgenic skin was rapidly rejected by normal C57BL/6 mice and that cytotoxic T lymphocytes generated by immunization of the Cw3 transgenic mice with influenza and Sendai virus were restricted by the Cw3 molecule. These data suggest that HLA-Cw3 has immunological functions comparable to those of HLA-A and -B.
Electron paramagnetic resonance measurements of free radicals in the intact beating heart: a technique for detection and characterization of free radicals in whole biological tissues.
Authors: Zweier J L , Kuppusamy P
Published: May 2006

Free radicals have been hypothesized to be important mediators of disease in a variety of organs and tissues. Electron paramagnetic resonance (EPR) spectroscopy can be applied to directly measure free radicals; however, it has not been possible to measure important biological radicals in situ because conventional spectrometer designs are not suitable for the performance of measurements on whole organs or tissues. We report the development of an EPR spectrometer designed for optimum performance in measuring free radicals in intact biological organs or tissues. This spectrometer consists of a 1- to 2-GHz microwave bridge with the source locked to the resonant frequency of a recessed gap loop-gap resonator. With this spectrometer, radical concentrations as low as 0.4 microM can be measured. Isolated beating hearts were studied in which simultaneous real time measurements of free radicals and cardiac contractile function were performed. This in vivo EPR technique was applied to study the kinetics of free radical uptake and metabolism in normally perfused and globally ischemic hearts. In addition, we show that this technique can be used to noninvasively measure tissue oxygen consumption. Thus, it is demonstrated that EPR spectroscopy can be applied to directly measure in vivo free radical metabolism and tissue oxygen consumption. This technique offers great promise in the study of in vivo free radical generation and the effects of this radical generation on whole biological tissues.
Paramecium calcium channels are blocked by a family of calmodulin antagonists.
Authors: Ehrlich B E , Jacobson A R , Hinrichsen R , Sayre L M , Forte M A
Published: May 2006

Although the voltage-sensitive Ca channel present in Paramecium has been subjected to detailed physiological and genetic analysis, no organic ligands have been described that block this channel with high affinity and that ultimately can be used to identify channel components. Based on a previous observation that the naphthalene sulfonamide calmodulin antagonist W-7 can block Paramecium Ca channels at high concentration, we have synthesized analogs of W-7 that block these channels at concentrations of less than 1 microM. The effectiveness of these compounds was tested both by a sensitive behavioral assay and on Ca channels that had been incorporated into planar lipid bilayers. Despite the fact that these compounds are effective Paramecium calmodulin antagonists, two independent lines of evidence suggest that W-7 and its analogs block the Ca channel by a mechanism that is independent of their action on calmodulin. In addition, the sensitivity to W-7 or dihydropyridines of Ca channels present in a number of eukaryotic phyla has been used to identify similarities in Ca channels from widely diverse organisms. It appears that the pharmacological specificity provides a means to group Ca channels.
Effects of ras-encoded proteins and platelet-derived growth factor on inositol phospholipid turnover in NRK cells.
Authors: Kamata T , Kung H F
Published: May 2006

The role of ras-encoded proteins and platelet-derived growth factor (PDGF) in inositol phospholipid metabolism has been studied. PDGF stimulates inositol phospholipid turnover in confluent normal rat kidney (NRK) cells and enhances hydrolysis of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate in NRK cell membranes in the presence of guanosine 5'-[gamma-thio]triphosphate. The stimulatory effect of PDGF on phosphatidylinositol bisphosphate hydrolysis is not inhibited by pretreatment of NRK cells with pertussis toxin, implying that PDGF-stimulated phospholipase C activity of NRK cells is regulated by a pertussis toxin-insensitive guanine nucleotide-binding protein (G protein) that is different from Gi (inhibitory G protein) or Go (G protein of unknown function). When bacterially made human normal or oncogenic T24 ras protein is added to 32P-labeled NRK cell membranes in the presence of guanosine 5'-[gamma-thio]triphosphate, normal ras protein increases by 3-fold the formation of inositol trisphosphate, whereas T24 ras protein has no significant effect. In addition, normal ras protein and PDGF have additive effects on inositol trisphosphate production. Taken together, these data suggest that normal ras protein stimulates inositol phospholipid turnover in NRK cells by means of a pathway different from the PDGF-regulated one and that oncogenic ras protein is without significant stimulatory effect in this action.
Specificity of action of guanine nucleotide-binding regulatory protein subunits on the cardiac muscarinic K+ channel.
Authors: Logothetis D E , Kim D H , Northup J K , Neer E J , Clapham D E
Published: May 2006

The cardiac muscarinic receptor stimulates a potassium-selective ionic current (IK.ACh) through activation of a guanine nucleotide-binding regulatory protein. Purified alpha and beta gamma subunits of the guanine nucleotide-binding regulatory protein have each been reported to open the K+ channel. We have reported that nanomolar concentrations of purified brain beta gamma subunits activated IK.ACh in chicken embryonic atrial patches. In contrast, J. Codina, A. Yatani, D. Grenet, A.M. Brown, and L. Birnbaumer [(1987) Science 236, 442-445] subsequently reported that picomolar concentrations of activated erythrocyte alpha subunits (i.e., the 40-kDa alpha subunit that the authors call alpha K) opened K+ channels in guinea pig atrial patches. In this paper, we further explore the specificity of various beta gamma and alpha subunits in embryonic chicken and neonatal rat atrial patches. Beta gamma subunits from either human placenta (beta 35 gamma) or bovine brain (beta 35,36 gamma) activated IK.ACh whereas transducin beta gamma (beta 36 gamma) did not. The beta gamma activation was consistent in rat and chicken patches [118 of 123 patches (97%)]. Beta gamma subunits opened K+ channels at concentrations greater than or equal to 200 pM and maximally activated the channel at 10 nM. Beta gamma or guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) channel activation could be reversed by alpha 41-GDP. The purified brain beta gamma preparation was contaminated with less than 0.01% unactivated alpha. The detergent (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPS), used to suspend the hydrophobic beta gamma, did not activate IK.ACh alone, with buffer, with heat-inactivated beta gamma, or with transducin beta gamma. Unactivated alpha subunits did not open K+ channels. Activated, alpha subunits purified from human erythrocytes (alpha 40-GTP[gamma-S]) or bovine brain (alpha 39-GTP[gamma-S]) at concentrations of 10 pM or higher (up to 1 nM) opened K+ channels less frequently in chicken atrial patches [5 of 27 patches (19%) and 9 of 35 patches (26%), respectively] than in rat atrial patches [5 of 11 patches (45%) and 11 of 19 patches (58%), respectively]. Negative results were not due to patch vesicle formation. Other experiments indicated that alpha and beta gamma activated the same population of channels. Activation of the channel by both beta gamma and alpha subunits implies a more complicated scheme for guanine nucleotide-binding regulatory protein action than previously proposed.
A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice.
Authors: Pittius C W , Hennighausen L , Lee E , Westphal H , Nicols E , Vitale J , Gordon K
Published: May 2006

Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation.
Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes.
Authors: Kim Y K , Wells S , Lau Y F , Lee A S
Published: May 2006

Recent evidence on the transcriptional regulation of the human thymidine kinase (TK) gene raises the possibility that cell-cycle regulatory sequences may be localized within its promoter. A hybrid gene that combines the TK 5' flanking sequence and the coding region of the bacterial neomycin-resistance gene (neo) has been constructed. Upon transfection into a hamster fibroblast cell line K12, the hybrid gene exhibits cell-cycle-dependent expression. Deletion analysis reveals that the region important for cell-cycle regulation is within -441 to -63 nucleotides from the transcriptional initiation site. This region (-441 to -63) also confers cell-cycle regulation to the herpes simplex virus thymidine kinase (HSVtk) promoter, which is not expressed in a cell-cycle manner. We conclude that the -441 to -63 sequence within the human TK promoter is important for cell-cycle-dependent expression.
Protein dynamics and reaction rates: mode-specific chemistry in large molecules?
Authors: Bialek W , Onuchic J N
Published: May 2006

Reactive events in proteins may be strongly coupled to a few specific modes of protein motion or they may couple nonspecifically to the dense continuum of protein and solvent modes. We summarize the evidence that at least some biologically important reactions can be described in terms of a few specific modes, and we propose experiments to quantify the strength of coupling to the continuum. We also show that large entropic effects--solvent ordering, for example--can be rigorously incorporated in few-mode models without losing mode specificity. Within our description, the dynamics that determine chemical reaction rates can be summarized by a small number of parameters directly related to spectroscopic and thermodynamic data. Mode specificity allows protein dynamics to contribute directly to the control and specificity of biochemical reaction rates.
Statistical geometry in sequence space: a method of quantitative comparative sequence analysis.
Authors: Eigen M , Winkler-Oswatitsch R , Dress A
Published: May 2006

A statistical method of comparative sequence analysis that combines horizontal and vertical correlations among aligned sequences is introduced. It is based on the analysis mainly of quartet combinations of sequences considered as geometric configurations in sequence space. Numerical invariants related to relative internal segment lengths are assigned to each such configuration and statistical averages of these invariants are established. They are used for internal calibration of the topology of divergence and for quantitative determination of the noise level. Comparison of computer simulations with experimental data reveals the high sensitivity of assignment of basic topologies even if much randomized. In addition, these procedures are checked by vertical analysis of the aligned sequences to allow the study of divergences with positionally varying substitution probabilities.
Characterization of a benzyladenine binding-site peptide isolated from a wheat cytokinin-binding protein: sequence analysis and identification of a single affinity-labeled histidine residue by mass spectrometry.
Authors: Brinegar A C , Cooper G , Stevens A , Hauer C R , Shabanowitz J , Hunt D F , Fox J E
Published: May 2006

A wheat embryo cytokinin-binding protein was covalently modified with the radiolabeled photoaffinity ligand 2-azido-N6-[14C]benzyladenine. A single labeled peptide was obtained after proteolytic digestion and isolation by reversed-phase and anion-exchange HPLC. Sequencing by classical Edman degradation identified 11 of the 12 residues but failed to identify the labeled amino acid. Analysis by laser photodissociation Fourier-transform mass spectrometry of 10 pmol of the peptide independently confirmed the Edman data and also demonstrated that the histidine residue nearest the C terminus (underlined) was modified by the reagent in the sequence Ala-Phe-Leu-Gln-Pro-Ser-His-His-Asp-Ala-Asp-Glu.
Biparental defensive endowment of eggs with acquired plant alkaloid in the moth Utetheisa ornatrix.
Authors: Dussourd D E , Ubik K , Harvis C , Resch J , Meinwald J , Eisner T
Published: May 2006

The eggs of Utetheisa ornatrix contain pyrrolizidine alkaloids. These compounds are contributed by both parents, who sequester them as larvae from their food plants. Females receive alkaloid from the males at mating, apparently by seminal infusion, and transmit this alkaloid together with alkaloid of their own to the eggs. Field and laboratory tests showed that the alkaloids protect eggs from predators. The alkaloidal contribution of the male, although smaller than that of the female, itself provides significant egg protection. A previously identified pheromone, derived by the male from the alkaloid and emitted during precopulatory behavior, may announce the male alkaloidal worth to the female.
Reconstruction of human evolution: bringing together genetic, archaeological, and linguistic data.
Authors: Cavalli-Sforza L L , Piazza A , Menozzi P , Mountain J
Published: May 2006

The genetic information for this work came from a very large collection of gene frequencies for "classical" (non-DNA) polymorphisms of the world aborigines. The data were grouped in 42 populations studied for 120 alleles. The reconstruction of human evolutionary history thus generated was checked with statistical techniques such as "boot-strapping". It changes some earlier conclusions and is in agreement with more recent ones, including published and unpublished DNA-marker results. The first split in the phylogenetic tree separates Africans from non-Africans, and the second separates two major clusters, one corresponding to Caucasoids, East Asians, Arctic populations, and American natives, and the other to Southeast Asians (mainland and insular), Pacific islanders, and New Guineans and Australians. Average genetic distances between the most important clusters are proportional to archaeological separation times. Linguistic families correspond to groups of populations with very few, easily understood overlaps, and their origin can be given a time frame. Linguistic superfamilies show remarkable correspondence with the two major clusters, indicating considerable parallelism between genetic and linguistic evolution. The latest step in language development may have been an important factor determining the rapid expansion that followed the appearance of modern humans and the demise of Neanderthals.
Physical mapping of large DNA by chromosome fragmentation.
Authors: Vollrath D , Davis R W , Connelly C , Hieter P
Published: May 2006

A technique is described for physically positioning any cloned DNA on a native or artificial Saccharomyces cerevisiae chromosome. The technique involves splitting a chromosome at a specific site by transformation with short linear molecules containing the cloned DNA at one end and telomeric sequences at the other. Recombination between the end of the linear molecules and homologous chromosomal sequences gives rise to chromosome fragments comprising all sequences distal or proximal to the mapping site depending on the orientation of the cloned DNA. The recombinant products are recovered by screening for stabilization of a suppressor tRNA on the linear molecules using a colony color assay. The cloned DNA is positioned relative to the chromosome ends by sizing the chromosomal fragments using alternating contour-clamped homogeneous electric field gel electrophoresis. Application of this technique to organisms other than S. cerevisiae and to the analysis of exogenous DNA cloned in yeast is discussed.
Genetic analysis of indefinite division in human cells: identification of four complementation groups.
Authors: Pereira-Smith O M , Smith J R
Published: May 2006

Hybrids obtained following fusion of normal human diploid fibroblasts with different immortal human cell lines exhibited limited division potential. This led to the conclusion that the phenotype of cellular senescence is dominant and that immortal cells arise as a result of recessive changes in the growth control mechanisms of the normal cell. We have exploited the fact that immortality is recessive and, by fusing immortal human cell lines with each other, assigned 21 cell lines to at least four complementation groups for indefinite division. A wide variety of cell lines was included in the study to determine what parameters, if any, would affect complementation group assignment. The results indicate that cell type, embryonal layer of origin, and type of tumor do not affect group assignment. There does not appear to be any correlation between expression of an activated oncogene and group assignment. However, all of the immortal simian virus 40-transformed cell lines studied (with the exception of one xerodermapigmentosum fibroblast-derived line) assign to the same group, indicating that this virus immortalizes various human cells by the same processes. The assignment of immortal human cells to distinct groups provides the basis for a focused approach to determine the genes important in normal growth regulation that have been modified in immortal cells.
Molecular characterization of T-cell antigen receptor expression by subsets of CD4- CD8- murine thymocytes.
Authors: Pearse M , Gallagher P , Wilson A , Wu L , Fisicaro N , Miller J F , Scollay R , Shortman K
Published: May 2006

Precursors of all T-lineage cells are found in a population of thymocytes that lack the CD4 and CD8 surface glycoproteins. These "double-negative" thymocytes are markedly heterogeneous in their expression of other surface markers and include cells at various stages of development. In this study, CD4- CD8- adult murine thymocytes were separated into subsets based on the expression of the "heat stable antigen" (HSA) and of Ly 1 (CD5). The sorted subsets were analyzed directly (without prior expansion in culture) for T-cell antigen receptor (TcR) gene rearrangement and mRNA expression and for TcR and CD3 cell-surface protein expression. Very little surface CD3 or TcR expression was detected on the major HSA+ Ly 1low subset. However, the HSA+ Ly 1high, HSA- Ly 1high, and HSA- Ly 1low subsets all contained cells with surface expression of CD3 and TcR. In contrast to previous studies, we found no subset that exclusively expressed either the alpha beta or gamma delta heterodimer, although the ratio of alpha beta+ to gamma delta+ varied widely. Two of these three subsets (HSA- Ly 1low and HSA- Ly 1high) showed very high usage of V beta 8 gene products in the alpha beta heterodimer, but nevertheless included approximately equal to 15% non-V beta 8 alpha beta forms. All CD4- CD8- subsets were found to have extensively rearranged their TcR gamma genes and to express gamma mRNA. Expression of a high ratio of mature [1.3 kilobases (kb)] to truncated (1.0 kb) beta message and presence of alpha message was largely restricted to subsets with TcR alpha beta surface expression.
Sequence and organization of the diversity, joining, and constant region genes of the human T-cell delta-chain locus.
Authors: Takihara Y , Tkachuk D , Michalopoulos E , Champagne E , Reimann J , Minden M , Mak T W
Published: May 2006

In this paper we describe the genomic organization and sequence of the human T-cell receptor delta-chain diversity, joining, and constant genes. There is one delta-chain constant region gene (C delta) located approximately equal to 85 kilobases (kb) upstream of the alpha-chain constant region. The delta-chain constant region consists of four exons, whose organization is very similar to that of the C alpha exons, suggesting that C alpha and C delta may have arisen from a gene duplication event. The first exon encodes most of the extracellular constant domain, the second encodes a hinge-like region, and the third encodes the entire transmembrane segment and intracytoplasmic portion, whereas the last exon contains exclusively 3' untranslated sequences. Three joining segments, J delta 1, J delta 2, and J delta 3, are found approximately equal to 12, approximately equal to 5.7, and approximately equal to 3.4 kb upstream of the first exon of C delta. Two functional diversity gene segments, D delta 1 and D delta 2, which can be productively translated in all three reading frames, are found 1 and 9.6 kb upstream of J delta 1. The presence of two D delta with such potential for diversity may offset the limited repertoire of the J delta and V delta genes. The spacer distribution in the recombinational signals flanking D delta and J delta segments allows recombination with V alpha gene segments; however, examination of delta-chain messages does not indicate that this is the case, suggesting that the delta chain uses unique variable gene segments and raising the question as to the reasons for this phenomenon.
Induction of macrophage antitumor activity by acetylated low density lipoprotein containing lipophilic muramyl tripeptide.
Authors: Shaw J M , Futch W S , Schook L B
Published: May 2006

A method has been developed for the selective delivery of lipophilic immunomodulators to macrophages, which results in the induction of antitumor activity. This method utilizes exhaustively acetylated low density lipoprotein (acetyl-LDL) to deliver the lipophilic immunomodulator, muramyl tripeptide phosphatidylethanolamine (MTP-PtdEtn; amide composed of N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine and dipalmitoyl phosphatidylethanolamine) to macrophages (M phi) by means of a scavenger lipoprotein receptor pathway. The binding of acetyl-LDL:MTP-PtdEtn to M phi showed specificity since minimal competition was observed in the presence of excess native LDL or phosphatidylcholine/cholesterol liposomes. Additional binding studies showed that acetyl-LDL may serve as a suitable delivery vehicle to a wide variety of M phi in different stages of activation. Cytostatic and tumoricidal activities by thioglycolate-elicited M phi against two tumor cell lines were examined in vitro following incubation with the acetyl-LDL:MTP-PtdEtn complex. Cytostatic activity against B16F10 melanoma cells was induced after the incubation of thioglycolate-elicited M phi with a minimum of 25 micrograms of acetyl-LDL protein containing 2.5 micrograms of bound MTP-PtdEtn (approximately equal to 40 molecules per particle of acetyl-LDL). The induction of cytostasis was not affected by liposome bilayers, which were also endocytosed by the M phi. In addition, tumoricidal activity against P815 mastocytoma cells was demonstrated at a 40:1 effector-to-target ratio using 18 micrograms of the acetyl-LDL:MTP-PtdEtn complex containing 3.6 micrograms of MTP-PtdEtn (approximately equal to 80 molecules per particle). These studies describe a method for the induction of antitumor activity by use of a chemically modified serum component, acetyl-LDL, to direct lipophilic immunomodulators to M phi.
A simple "neural induction" model with two interacting cleavage-arrested ascidian blastomeres.
Authors: Okado H , Takahashi K
Published: May 2006

A single anterior-animal blastomere, which includes the presumptive neural region in the eight-cell embryo of the Halocynthia, a protochordate, when dissociated, cleavage-arrested with cytochalasin B, and cultured in isolation, differentiated exclusively to epidermal type judging from membrane excitability and immunoreactivity. However, when the same blastomere was cultured in contact with a single anterior-vegetal blastomere, which includes the presumptive notochordal region, it displayed Na spikes and showed no expression of the epidermal antigen, suggesting that "neural induction" resulted in a single cell during the interaction with a single neighboring cell. This simple two-cell system can be used for further studies on the induction mechanism.
"Ultraviolet footprinting" accurately maps sequence-specific contacts and DNA kinking in the EcoRI endonuclease-DNA complex.
Authors: Becker M M , Lesser D , Kurpiewski M , Baranger A , Jen-Jacobson L
Published: May 2006

The "UV footprinting" technique has been used to detect contacts between EcoRI endonuclease and its recognition sequence at single nucleotide resolution. Comparison of the UV-footprinting results to the published crystal structure of the EcoRI endonuclease-DNA complex allows us to determine how UV light detects protein-DNA contacts. We find that kinking of the DNA helix in the complex greatly enhances the UV photoreactivity of DNA at the site of the kink. In contrast to kinking, contacts between the endonuclease and the DNA bases inhibit the UV photoreactivity of DNA. Similar analysis of a proteolytically modified endonuclease that exhibits the same sequence specificity as wild-type enzyme but that does not cleave DNA supports these conclusions. Furthermore, detection of enhanced photoreactivity at the same kink in the modified enzyme-DNA complex allows us to conclude that the loss of cleavage activity by the modified endonuclease is not due to its failure to kink DNA.
Detection of DNA looping due to simultaneous interaction of a DNA-binding protein with two spatially separated binding sites on DNA.
Authors: Mukherjee S , Erickson H , Bastia D
Published: May 2006

We describe different and relatively rapid biochemical techniques to detect protein-mediated DNA looping. These techniques, based on enhancement of DNA knotting and that of ligase-catalyzed cyclization, were used to show that the replication initiator protein of plasmid R6K can bring together two intramolecular gamma origin of replication sequences located as far apart as 2 kilobases. The site-site interaction causes looping out of the intervening DNA sequence as visualized by electron microscopy. Because the autoregulatory sequence of the initiator cistron also binds initiator protein, we investigated whether the gamma origin-bound protein can participate in autoregulation by interaction of the two sites through a protein bridge. We discovered that the two sites do not interact in vitro at their natural locations when on opposite faces of the double helix. Moving the two sites to the same face of the double helix by introducing a half turn into the intervening sequence allows protein-mediated site-site interaction to occur.
Developmental regulation of embryonic genes in plants
Authors: Borkird Chumpol , Choi Jung H. , Jin Zheng-Hua , Franz Gerald , Hatzopoulos Polydefkis , Chorneau Robert , Bonas Ulla , Pelegri Francisco , Sung Z. Renee
Published: May 2006

Somatic embryogenesis from cultured carrot cells progresses through successive morphogenetic stages termed globular, heart, and torpedo. To understand the molecular mechanisms underlying plant embryogenesis, we isolated two genes differentially expressed during embryo development. The expression of these two genes is associated with heart-stage embryogenesis. By altering the culture conditions and examining their expressions in a developmental variant cell line, we found that these genes were controlled by the developmental program of embryogenesis and were not directly regulated by 2,4-dichlorophenoxyacetic acid, the growth regulator that promotes unorganized growth of cultured cells and suppresses embryo morphogenesis. These genes are also expressed in carrot zygotic embryos but not in seedlings or mature plants.
Conservation of gene repertoire but not gene order in pepper and tomato
Authors: Tanksley Steven D. , Bernatzky Robert , Lapitan Nora L. , Prince James P.
Published: May 2006

Homologies of tomato and pepper genes have been compared, and genetic linkage maps have been constructed based on a common set of cDNA clones and selected single-copy genomic clones. We report here that the gene repertoire of these two species is highly conserved, yet the linear order of the genes on the chromosomes has been greatly modified. Although the two species share the same number of centromeres, the chromosomal regions around those centromeres have undergone extensive rearrangements. Accompanying the extensive chromosome rearrangement has been a change in locus number for approximately 12% of the loci detected by random cDNA clones. Duplicated loci within each genome are normally found on different chromosomes and are not confined to one species, thus ruling out gene duplication as an explanation for the 4-fold higher DNA content of pepper. At least one of the duplications occurred since the divergence of tomato and pepper from their last common ancestor.
The oldest fossil bee: Apoid history, evolutionary stasis, and antiquity of social behavior
Authors: Michener Charles D. , Grimaldi David A.
Published: May 2006

Trigona prisca
, a stingless honey bee (Apidae; Meliponinae), is reported from Cretaceous New Jersey amber (96-74 million years before present). This is about twice the age of the oldest previously known fossil bee, although
Trigona
is one of the most derived bee genera.
T. prisca
is closely similar to modern neotropical species. Most of bee evolution probably occurred during the ≈50 million years between the beginning of the Cretaceous when flowering plants (on which bees depend) appeared and the time of
T. prisca
. Since then, in this phyletic line of Meliponinae, there has been almost no morphological evolution. Since the fossil is a worker, social organization had arisen by its time.
Homologous recombination can restore normal immunoglobulin production in a mutant hybridoma cell line.
Authors: Baker M D , Pennell N , Bosnoyan L , Shulman M J
Published: May 2006

We report here the occurrence of homologous recombination between transferred and chromosomal immunoglobulin genes. Specifically, we have corrected a chromosomal immunoglobulin gene mutation by transferring pSV2neo vectors encoding the constant region of the immunoglobulin mu heavy chain to mutant hybridoma cells that bear a 2-base-pair deletion in the third constant region exon of their chromosomal mu gene. After DNA transfer, we detected G418-resistant transformants that produce normal IgM. Analysis of the DNA structure of the mu gene in these transformants indicates that in four of five cases the mu gene has been restored as a result of the integration of a single copy of the transfer vector by a reciprocal homologous recombination event; the fifth case seems to have resulted from gene conversion or double crossover. These results suggest that this technology might be adapted for mapping immunoglobulin gene mutations by marker rescue and for more convenient engineering of specifically altered immunoglobulin.
Molecular and cytogenetic evidence for the location of Tdy and Hya on the mouse Y chromosome short arm.
Authors: Roberts C , Weith A , Passage E , Michot J L , Mattei M G , Bishop C E
Published: May 2006

Using a combination of in situ mapping and DNA analysis with recombinant DNA probes specific for the Sxr region of the mouse Y chromosome, we show that both the gene(s) controlling sex determination and the expression of the male-specific antigen H-Y (Tdy and Hya, respectively) are located on the minute short arm of the mouse Y chromosome. We demonstrate that the H-Y- variant of Sxr (Sxr') arose by a partial deletion within the Sxr region. Also, we show that intrachromosomal recombination between the Y short arm and Sxr' can sometimes occur during male meiosis, restoring the deleted DNA sequences and resulting in an H-Y+ mouse (male 719 in this paper). Based on these results, we propose a model for the generation of the original Sxr region and the Sxr' and Sxr719 variants.