bluuebunny/crossref_metadata_2025 huggingface dataset:
Paneth cell differentiation in the developing intestine of normal and transgenic mice.
Authors: Bry L , Falk P , Huttner K , Ouellette A , Midtvedt T , Gordon J I
Published: May 2006

Paneth cells represent one of the four major epithelial lineages in the mouse small intestine. It is the only lineage that migrates downward from the stem-cell zone located in the lower portion of the crypt of Lieberkühn to the crypt base. Mature Paneth cells release growth factors, digestive enzymes, and antimicrobial peptides from their apical secretory granules. Some of these factors may affect the crypt stem cell, its transit-cell descendants, differentiating villus-associated epithelial lineages, and/or the gut microflora. We used single and multilabel immunocytochemical methods to study Paneth cell differentiation during and after completion of gut morphogenesis in normal, gnotobiotic, and transgenic mice as well as in intestinal isografts. This lineage emerges coincident with cytodifferentiation of the fetal small intestinal endoderm, formation of crypts from an intervillus epithelium, and establishment of a stem-cell hierarchy. The initial differentiation program involves sequential expression of cryptdins, a phospholipase A2 (enhancing factor), and lysozyme. A dramatic increase in Paneth cell number per crypt occurs during postnatal days 14-28, when crypts proliferate by fission. Accumulation of fucosylated and sialylated glycoconjugates during this period represents the final evolution of the lineage's differentiation program. Establishment of this lineage is not dependent upon instructive interactions from the microflora. Transgenic mice containing nucleotides -6500 to +34 of the Paneth cell-specific mouse cryptdin 2 gene linked to the human growth hormone gene beginning at its nucleotide +3 inappropriately express human growth hormone in a large population of proliferating and nonproliferating cells in the intervillus epithelium up to postnatal day 5. Transgene expression subsequently becomes restricted to the Paneth cell lineage in the developing crypt. Cryptdin 2 nucleotides -6500 to +34 should be a useful marker of crypt morphogenesis and a valuable tool for conducting gain-of-function or loss-of-function experiments in Paneth cells.
A cysteine residue in the third membrane-spanning segment of the human D2 dopamine receptor is exposed in the binding-site crevice.
Authors: Javitch J A , Li X , Kaback J , Karlin A
Published: May 2006

The binding site in G-protein-linked neurotransmitter receptors is formed among their membrane-spanning segments. Because the binding site is in the plane of the bilayer and is accessible to charged, water-soluble agonists, it must lie in a crevice open to the extracellular, aqueous medium. Information about the structure of these receptors can be obtained by identifying the residues in the membrane-spanning segments which face this water-filled crevice. Human D2 dopamine receptor was expressed in human embryonic kidney 293 cells. Small, charged, sulfhydryl-specific methanethiosulfonate (MTS) derivatives irreversibly inhibited the binding of the D2-specific antagonist [3H]YM-09151-2 to these cells. The highly polar MTS derivatives should react with cysteine sulfhydryl groups only at the water-accessible surface of the receptor, which includes the surface of the binding-site crevice. In contrast, these reagents will have little access to sulfhydryls facing the lipid bilayer or buried in the protein interior. Positively charged MTS reagents irreversibly inhibited binding several hundredfold faster than a negatively charged MTS reagent, consistent with the affinity of the binding site for positively charged dopamine agonists and antagonists. Furthermore, both agonists and antagonists of the D2 receptor protected against irreversible inhibition by the MTS reagents. To identify the susceptible cysteine, we mutated, one at a time, five transmembrane and two extracellular cysteine residues to serine. Only the mutation of Cys118 to serine decreased the susceptibility of antagonist binding to irreversible inhibition by the MTS reagents. Thus, Cys118, a residue in the middle of the third membrane-spanning segment, is exposed in the D2 receptor binding-site crevice.
Another way of being anisogamous in Drosophila subgenus species: giant sperm, one-to-one gamete ratio, and high zygote provisioning.
Authors: Bressac C , Fleury A , Lachaise D
Published: May 2006

It is generally assumed that sexes in animals have arisen from a productivity versus provisioning conflict; males are those individuals producing gametes necessarily small, in excess, and individually bereft of all paternity assurance. A 1- to 2-cm sperm, 5-10 times as long as the male body, might therefore appear an evolutionary paradox. As a matter of fact, species of Drosophila of the Drosophila subgenus differ from those of other subgenera by producing exclusively sperm of that sort. We report counts of such giant costly sperm in Drosophila littoralis and Drosophila hydei females, indicating that they are offered in exceedingly small amounts, tending to a one-to-one gamete ratio after a single mating. As a result, most of them are successfully involved in a fertilization. Hence, the concept of "paternity assurance of individual sperm" arises. Evidence is further provided here that almost the entire sperm is incorporated into the egg during fertilization. Labeling with specific antibodies in fertilized eggs reveals intact axonemes up to late gastrulation. The question, then, is why selection has favored such an unusual strategy. Explanations related to some prefertilization functions are ruled out. It is therefore tentatively proposed that virtually every giant sperm constitutes a "direct paternal legacy to the embryo," which, in contrast to any male-derived nuptial gift, cannot be minimized by female remating. We suggest that dramatic shortage of giant sperm with a high prospect of fusion and increased zygote provisioning is merely another way of being anisogamous.
Native-like in vivo folding of a circularly permuted jellyroll protein shown by crystal structure analysis.
Authors: Hahn M , Piotukh K , Borriss R , Heinemann U
Published: May 2006

A jellyroll beta-sandwich protein, the Bacillus beta-glucanase H(A16-M), is used to probe the role of N-terminal peptide regions in protein folding in vivo. A gene encoding H(A16-M) is rearranged to place residues 1-58 of the protein behind a signal peptide and residues 59-214. The rearranged gene is expressed in Escherichia coli. The resultant circularly permuted protein, cpA16M-59, is secreted into the periplasm, correctly processed, and folded into a stable and active enzyme. Crystal structure analysis at 2.0-A resolution, R = 15.3%, shows cpA16M-59 to have a three-dimensional structure nearly identical with that of the parent beta-glucanase. An analogous experiment based on the wild-type Bacillus macerans beta-glucanase, giving rise to the circularly permuted variant cpMAC-57, yields the same results. Folding of these proteins, therefore, is not a vectorial process depending on the conformation adopted by their native N-terminal oligopeptides after ribosomal synthesis and translocation through the cytoplasmic membrane.
Developmentally regulated cell death on expression of the fungal avirulence gene Avr9 in tomato seedlings carrying the disease-resistance gene Cf-9.
Authors: Hammond-Kosack K E , Harrison K , Jones J D
Published: May 2006

Plant defense responses are induced when the products of disease-resistance genes and pathogen avirulence genes interact. We report here the effects of expressing the Cladosporium fulvum avirulence Avr9 gene product in a tomato line containing the Cf-9 disease-resistance gene. A synthetic Avr9 gene was constructed to produce constitutive high-level expression of AVR9 peptide in the plant apoplast. Avr9 expression in Cf-9-containing tomato lines is lethal, but cell death is developmentally regulated, in that necrosis is not visible until 10 days after planting seed. Plant lines lacking Cf-9 and expressing Avr9 remain healthy. The synthetic Avr9 gene exhibited the same specificity of action as the authentic C. fulvum Avr9 gene. Our results have significant implications for strategies using the gene combination Avr9/Cf-9 to engineer plants with enhanced disease resistance.
Cloning of a cDNA for the FAD-linked glycerol-3-phosphate dehydrogenase from rat liver and its regulation by thyroid hormones.
Authors: Müller S , Seitz H J
Published: May 2006

A full-length 2.4-kb cDNA for the FAD-linked glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) was cloned from rat liver using PCR techniques. The cloned gene encodes a protein of 727 amino acids. The calculated molecular mass of 80,898 Da is higher than the apparent molecular mass observed by SDS/PAGE (74,000 Da) of the purified enzyme. This result indicates that the enzyme is synthesized as a precursor with a putative mitochondrial signal sequence. mRNA for this gene was detected in liver, heart, muscle, brain, testes, and pancreas. With the exception of testes, basal expression levels were very low in all tissues examined. However, application of thyroid hormones led to a 10- to 15-fold increase in liver glycerol-3-phosphate dehydrogenase mRNA, whereas hypothyroidism further decreased the mRNA level.
Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I.
Authors: Carver T E , Hochstrasser R A , Millar D P
Published: May 2006

Fluorescence depolarization decays were measured for 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) probes attached internally to 17-mer.27-mer oligonucleotides bound to Klenow fragment of DNA polymerase I. The time-resolved motions of the dansyl probes were sensitive indicators of DNA-protein contacts, showing that the protein binds to DNA with two footprints, corresponding to primer termini at either the polymerase or 3'-5' exonuclease sites. We examined complexes of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus caused a 3- to 4-fold increase in the equilibrium partitioning of DNA into the exonuclease site; the largest effects were observed for purine-purine mismatches. Two or more consecutive G.G mismatches caused the DNA to bind exclusively at the exonuclease site, with a partitioning constant at least 250-fold greater than that of the corresponding matched DNA sequence. Internal single mismatches produced larger effects than the same mismatch at the primer terminus, with a delta delta G relative to the matched sequence of -1.1 to -1.3 kcal/mol for mismatches located 2, 3, or 4 bases from the primer terminus. Although part of the observed effects may be attributed to the increased melting capacity of the DNA, it appears that the polymerase site also promotes movement of DNA into the exonuclease site by rejecting aberrant primer termini. These effects suggest that the polymerase and exonuclease sites act together to recognize specific errors that distort the primer terminus, such as frameshifts, in addition to proofreading misincorporated bases.
Purification of the Golgi adenosine 3'-phosphate 5'-phosphosulfate transporter, a homodimer within the membrane.
Authors: Mandon E C , Milla M E , Kempner E , Hirschberg C B
Published: May 2006

Sulfation of proteoglycans, secretory and membrane proteins, and glycolipids occurs in the lumen of the Golgi apparatus. Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the sulfate donor in these reactions, must be transported from the cytosol, its site of synthesis, into the lumen of the Golgi apparatus. We have identified and purified to apparent homogeneity the rat liver Golgi membrane PAPS transporter by a combination of conventional and affinity chromatography as well as photoaffinity radiolabeling with adenosine 3',5'-bisphosphate, a competitive inhibitor of PAPS transport. The transporter, a 75-kDa protein, was purified 70,000-fold over homogenate (6% yield) and transported PAPS into phosphatidylcholine liposomes selectively and in a saturable manner (apparent Km of 1.7 microM). Radiation target-inactivation analyses of the transport activity in rat liver Golgi vesicles, together with the above described biochemical approaches, demonstrate that the PAPS transporter within the Golgi membrane is a homodimer.
Engineered biosynthesis of novel polyketides: influence of a downstream enzyme on the catalytic specificity of a minimal aromatic polyketide synthase.
Authors: McDaniel R , Ebert-Khosla S , Fu H , Hopwood D A , Khosla C
Published: May 2006

To identify the minimum set of polyketide synthase (PKS) components required for in vivo biosynthesis of aromatic polyketides, combinations of genes encoding subunits of three different aromatic PKSs--act from Streptomyces coelicolor A3(2) (an actinorhodin producer), fren from Streptomyces roseofulvus (a frenolicin and nanaomycin producer), and tcm from Streptomyces glaucescens (a tetracenomycin producer)--were expressed in a recently developed Streptomyces host-vector system. The "minimal" components (ketosynthase/putative acyltransferase, chain length-determining factor, and acyl carrier protein) were produced with and without a functional polyketide ketoreductase and/or cyclase, and the polyketide products of these recombinant strains were structurally characterized. Several previously identified polyketides were isolated in addition to two previously unidentified polyketides, dehydromutactin and SEK 15b, described here. The results proved that the act cyclase is not required for the biosynthesis of several aberrantly cyclized products that have been previously reported. They are also consistent with earlier conclusions that the minimal PKS controls chain length as well as the regiospecificity of the first cyclization and that it can do so in the absence of both a ketoreductase and a cyclase. However, the ability of the minimal tcm PKS to synthesize two different singly cyclized intermediates suggests that it is unable to accurately control the course of this reaction by itself. In the presence of a downstream enzyme, the flux through one branch of the cyclization pathway increases relative to the other. We propose that these alternative specificities may be due to the ability of downstream enzymes to associate with the minimal PKS and to selectively inhibit a particular branch of the cyclization pathway.
PapD chaperone function in pilus biogenesis depends on oxidant and chaperone-like activities of DsbA.
Authors: Jacob-Dubuisson F , Pinkner J , Xu Z , Striker R , Padmanhaban A , Hultgren S J
Published: May 2006

Adhesive P pili of uropathogenic Escherichia coli were not assembled by a strain that lacks the periplasmic disulfide isomerase DsbA. This defect was mostly attributed to the immunoglobulin-like pilus chaperone PapD, which possesses an unusual intrasheet disulfide bond between the last two beta-strands of its CD4-like carboxyl-terminal domain. The DsbA-dependent formation of this disulfide bond was critical for PapD's proper folding in vivo. Interestingly, the absence of the disulfide bond did not prevent PapD from folding in vitro or from forming a complex with the pilus adhesin in vitro. We suggest that DsbA maintains nascently translocated PapD in a folding-competent conformation prior to catalyzing disulfide bond formation, acting both as an oxidant and in a chaperone-like fashion. Disulfide bond formation in pilus subunits was also mediated by DsbA even in the absence of PapD. However, the ability of pilus subunits to achieve native-like conformations in vivo depended on PapD. These results suggest that a productive folding pathway for subunits requires sequential interactions with DsbA and the PapD chaperone.
Sequential imputation for multilocus linkage analysis.
Authors: Irwin M , Cox N , Kong A
Published: May 2006

A Monte Carlo method called sequential imputation is proposed for multilocus likelihood computations. This method is most useful in mapping situations where the data consist of large pedigrees with substantial missing information and it is desirable to perform linkage analysis utilizing data from many polymorphic markers simultaneously. A pedigree example with 155 individuals, 9 loci, and 155,520 haplotypes is used for illustration.
Latent capacities for gametogenic cycling in the semelparous invertebrate Nereis.
Authors: Golding D W , Yuwono E
Published: May 2006

Most nereid polychaetes are strictly semelparous, a single episode of reproduction being invariably followed by death. Endocrine manipulation in Nereis diversicolor by the regular implantation of cerebral ganglia from immature donors unveils characteristics associated with a capacity to engage in repeated gametogenic cycling. Such manipulation permits full maturation of the gametes but blocks spawning. Gamete resorption then leads on to another bout of gametogenesis and a new cohort of gametes is formed. The neurosecretory system adopts a cyclical pattern of activity, which parallels that of gametogenesis. Repair and maintenance of the soma continue throughout sexual maturation, as shown by the persistence of feeding and the capacity for regenerative segment proliferation. In consequence, life is extended apparently indefinitely. These latent capacities are reminiscent of features of iteroparous life histories, characterized by repeated breeding, and are postulated to be vestiges of an iteroparous ancestry. They also constitute a preadaptation for iteroparity and reveal how readily a reversal to this condition could occur. The study suggests that reproductive strategies may be unexpectedly labile in even their most fundamental aspects.
Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors.
Authors: Finco T S , Beg A A , Baldwin A S
Published: May 2006

The ubiquitous transcription factor NF-kappa B is regulated by its cytoplasmic inhibitor I kappa B. A variety of cellular stimuli cause the dissociation of NF-kappa B from I kappa B, allowing NF-kappa B to translocate to the nucleus and regulate gene expression. Although the activation of NF-kappa B in vivo is associated with the phosphorylation and degradation of I kappa B alpha, it has remained unclear how each of these events contributes to this process. Recently, studies utilizing protease inhibitors have suggested that the proteolysis of I kappa B alpha is a necessary event in the activation of NF-kappa B. We demonstrate in this study that these and an additional protease inhibitor also completely repress inducible phosphorylation of I kappa B alpha. This surprising result suggests a more complex role of proteases in NF-kappa B activation. In addition, data presented here indicate that many of these inhibitors also directly modify NF-kappa B and inhibit its DNA binding activity. Due to the pleiotropic effects of these protease inhibitors, it is difficult to conclude from their use how I kappa B alpha phosphorylation and degradation contribute to NF-kappa B activation. In the present study, a more direct approach demonstrates that phosphorylation of I kappa B alpha alone is not sufficient for NF-kappa B activation.
Rapid detection of homologous recombinants in nontransformed human cells.
Authors: Williams S R , Ousley F C , Vitez L J , DuBridge R B
Published: May 2006

Gene targeting is a technique by which a preselected site in the genome of a living cell can be modified by inserting, deleting, or exchanging DNA sequences. The application of this technology to cells with a limited life-span, such as nontransformed human somatic cells, requires the development of simplified and efficient procedures to allow the isolation of correctly modified cells from the much larger pool of random integrants. The current study describes the development of a widely applicable strategy for detecting homologous recombinants in human cells by using an ELISA-based screen. When this system is used accurately targeted clones can be detected with high efficiency as soon as 14 days following transfection. Data are presented demonstrating the utility of this detection system in isolating targeted recombinants at the beta 2-microglobulin locus in both human retinal pigmented epithelial cells and human keratinocytes.
Asymmetrical blockade of the Ca2+ release channel (ryanodine receptor) by 12-kDa FK506 binding protein.
Authors: Chen S R , Zhang L , MacLennan D H
Published: May 2006

A soluble 12-kDa FK506 binding protein (FKBP12), the cellular receptor of the immunosuppressive drug FK506, is tightly associated with the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum [Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument-Bromage, H., Tempst, P. & Marks, A. (1992) J. Biol. Chem. 267, 9474-9477]. We have assessed the role of excess free FKBP12 in the function of single Ca2+ release channels incorporated into planar lipid bilayers. The addition of human recombinant FKBP12 (hFKBP12) to the cytoplasmic face of the Ca2+ release channel blocked the flow of cytoplasmic to luminal current (outward current) in a concentration-dependent manner but had no significant effect on the flow of luminal to cytoplasmic current (inward current). The luminal to cytoplasmic flow of current was modulated by Ca2+, Mg2+, ATP, caffeine, and ryanodine in the presence and absence of FKBP12. An immunosuppressive drug, L-683,590, an analog of FK506, did not block or reverse the asymmetrical hFKBP12 blockade of single Ca2+ release channels in planar lipid bilayers. FKBP12 may play a role in regulation of the flow of ions into the lumen of the sarcoplasmic reticulum through the Ca2+ release channel.
Angiogenin promotes invasiveness of cultured endothelial cells by stimulation of cell-associated proteolytic activities.
Authors: Hu G , Riordan J F , Vallee B L
Published: May 2006

Angiogenin, a potent inducer of neovascularization in the chicken chorioallantoic membrane and rabbit cornea, promotes endothelial cell invasion of Matrigel basement membrane. A transformed bovine aortic endothelial cell line, GM 7373, is 5 times more invasive when cultured in the presence of 1 microgram of bovine angiogenin per ml than in its absence. A polyclonal anti-angiogenin antibody and alpha 2-antiplasmin neutralize the effect of angiogenin, but an angiogenin-binding protein (actin) does not. Further, this concentration of angiogenin induces a 14-fold increase in the cell-associated proteolytic activity of cultured endothelial cells, determined with a tissue-type plasminogen activator-specific peptide as the substrate. In addition, cells cultured on a three-dimensional fibrin gel in the presence of angiogenin are 3 times more capable of dissolving the gel and forming focal defects in the underlying matrix. The results indicate that angiogenin can enhance the ability of endothelial cells to digest extracellular matrix components and degrade basement membrane, thereby facilitating cell invasion and migration. Binding of angiogenin to its cell-surface binding protein (actin) followed by dissociation of the angiogenin-actin complex from the cell surface and subsequent activation of tissue-type plasminogen activator/plasmin are likely steps involved in the processes of endothelial cell invasion and angiogenesis.
AFC1, a LAMMER kinase from Arabidopsis thaliana, activates STE12-dependent processes in yeast.
Authors: Bender J , Fink G R
Published: May 2006

In the yeast Saccharomyces cerevisiae a kinase cascade activates the transcription factor STE12 leading to mating in haploid cells and pseudohyphal growth in diploid cells. To investigate related signal transduction pathways in higher plants, we have isolated a putative protein kinase gene from Arabidopsis thaliana that restores STE12-dependent functions to yeast with mutations in this signal transduction pathway. This Arabidopsis gene, AFC1, induces three STE12-dependent processes even in signal transduction-defective yeast strains: mating-specific gene expression in haploid yeast, mating of haploid yeast to yield diploids, and pseudohyphal growth in diploid yeast. AFC1 has no effect on transcription of the STE12 gene and, instead, is likely to activate the STE12 protein. However, AFC1 has only limited homology to FUS3 and KSS1, the endogenous yeast kinase regulators of STE12. AFC1 is a member of a recently described CDC2-related kinase subfamily, the LAMMER kinases. A close AFC1 homolog, AFC2, lacks STE12 activation phenotypes, indicating the specificity of AFC1. The phenotypes of AFC1 in yeast provide us with tools to elucidate the role of this kinase in Arabidopsis.
DNA oxidative damage and life expectancy in houseflies.
Authors: Agarwal S , Sohal R S
Published: May 2006

The objective of this study was to explore the relationship between oxidative molecular damage and the aging process by determining whether such damage is associated with the rate of aging, using the adult housefly as the experimental organism. Because the somatic tissues in the housefly consist of long-lived postmitotic cells, it provides an excellent model system for studying cumulative age-related cellular alterations. Rate of aging in the housefly was manipulated by varying the rate of metabolism (physical activity). The concentration of 8-hydroxydeoxyguanosine (80HdG) was used as an indicator of DNA oxidation. Exposure of live flies to x-rays and hyperoxia elevated the level of 8OHdG. The level of 8OHdG in mitochondrial as well as total DNA increased with the age of flies. Mitochondrial DNA was 3 times more susceptible to age-related oxidative damage than nuclear DNA. A decrease in the level of physical activity of the flies was found to prolong the life-span and corresponding reduce the level of 8OHdG in both mitochondrial and total DNA. Under all conditions examined, mitochondrial DNA exhibited a higher level of oxidative damage than total DNA. The 8OHdG levels were found to be inversely associated with the life expectancy of houseflies. The pattern of age-associated accrural of 8OHdG was virtually identical to that of protein carbonyl content. Altoghether, results of this study support the hypothesis that oxidative molecular damage is a causal factor in senescence.
Peroxynitrite induces both vasodilatation and impaired vascular relaxation in the isolated perfused rat heart.
Authors: Villa L M , Salas E , Darley-Usmar V M , Radomski M W , Moncada S
Published: May 2006

The effects of the oxidant species peroxynitrite (ONOO-) on coronary perfusion pressure and vasodilatation elicited by acetylcholine, isoproterenol, and S-nitroso-N-acetyl-DL-penicillamine were investigated in the isolated perfused rat heart. ONOO- (0.3-1000 microM) caused a concentration-dependent vasodilatation of the coronary vasculature. This dilator response was inhibited by oxyhemoglobin, indicating that it was due to the generation of nitric oxide. Tachyphylaxis to ONOO- developed rapidly, so that the response disappeared after three or four applications of this compound. ONOO- not only induced tachyphylaxis but also inhibited the vasodilatation induced by the three vasodilators studied. This latter effect of ONOO- was critically dependent on its concentration, since it occurred at 3 microM, which was subthreshold as a dilator, and at 1000 microM, which was supramaximal, but not at 30 and 100 microM. These latter concentrations inhibited the responses to vasodilators only in the presence of oxyhemoglobin. Thus, a wide range of concentrations of ONOO- induce a vascular dysfunction, as evidenced by the tachyphylaxis to its own vasodilator actions and the long-lasting impairment of the responses to other vasodilators. However, at the same time ONOO- generates nitric oxide, which at certain concentrations of ONOO- is sufficient to counteract its deleterious action. Coinfusion of S-nitroso-N-acetyl-DL-penicillamine or prostacyclin at low concentrations that did not produce vasodilatation also protected against ONOO(-)-induced vascular dysfunction: these compounds may be protective through a common mechanism, as yet undefined.
Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells.
Authors: Jacobsson G , Bean A J , Scheller R H , Juntti-Berggren L , Deeney J T , Berggren P O , Meister B
Published: May 2006

Several proteins that are of importance for membrane trafficking in the nerve terminal have recently been characterized. We have used Western blot and immunohistochemistry to show that synaptotagmin, synaptobrevin/VAMP (vesicle-associated membrane protein), SNAP-25 (synaptosomal-associated protein of 25 kDa), and syntaxin proteins are present in cells of the islets of Langerhans in the endocrine pancreas. Synaptotagmin-like immunoreactivity (-LI) was localized to granules within the cytoplasm of a few endocrine cells located in the periphery of the islets, identified as somatostatin-containing cells, and in many nerve fibers within the islets. VAMP-LI was seen in granules of virtually all pancreatic islet cells and also in nerve fibers. SNAP-25-LI and syntaxin-LI were predominantly present in the plasma membrane of the endocrine cells, including insulin-producing beta cells. In situ hybridization, using isoform-specific oligonucleotide probes, detected VAMP-2, cellubrevin, SNAP-25, syntaxin 1A, 4, and 5, and munc-18 mRNAs in isolated pancreatic islets and in insulin-producing cells. The results show the presence of several synaptic proteins at protein and mRNA levels in pancreatic islet cells, suggesting that they may have specific roles in the molecular regulation of exocytosis also in insulin-secreting cells.
Phytochrome assembly in living cells of the yeast Saccharomyces cerevisiae.
Authors: Li L , Lagarias J C
Published: May 2006

The biological activity of the plant photoreceptor phytochrome requires the specific association of a linear tetrapyrrole prosthetic group with a large apoprotein. As an initial step to develop an in vivo assay system for structure-function analysis of the phytochrome photoreceptor, we undertook experiments to reconstitute holophytochrome in the yeast Saccharomyces cerevisiae. Here we show that yeast cells expressing recombinant oat apophytochrome A can take up exogenous linear tetrapyrroles, and, in a time-dependent manner, these pigments combine with the apoprotein to form photoactive holophytochrome in situ. Cell viability measurements indicate that holophytochrome assembly occurs in living cells. Unlike phytochrome A in higher plant tissue, which is rapidly degraded upon photoactivation, the reconstituted photoreceptor appears to be light stable in yeast. Reconstitution of photoactive phytochrome in yeast cells should enable us to exploit the power of yeast genetics for structure-function dissection of this important plant photoreceptor.
Increased internal Ca2+ mediates neural induction in the amphibian embryo.
Authors: Moreau M , Leclerc C , Gualandris-Parisot L , Duprat A M
Published: May 2006

The molecular mechanism of neural induction is still unknown and the identity of the natural inducer remains elusive. It has been suggested that both the protein kinase C and cAMP signal transduction pathways may be involved in mediating its action. Here we provide evidence that Ca2+ is implicated in the process of transduction of the neuralizing signal. We find that an increase in intracellular Ca2+ concentration [Ca2+]i occurs during neural induction provoked in vitro by the lectin Con A in Pleurodeles waltl embryo. We demonstrate that specific L-type Ca2+ channel agonists also trigger neural induction. Conversely, noninducing lectins do not raise [Ca2+]i. Ryanodine and caffeine trigger neural induction. An increase in [Ca2+]i was also observed after treatment with the phorbol 12-myristate 13-acetate, which has been reported to be inductive. The [Ca2+]i increase triggered by phorbol ester and Con A was abolished by staurosporine and by L-type Ca2+ channel antagonists. Our findings demonstrate that the [Ca2+]i increase occurs via L-type Ca2+ channels. We suggest an amplification of this increase by a Ca(2+)-induced Ca2+ release mechanism which involves intracellular ryanodine-sensitive stores. We propose that Ca(2+)-dependent processes controlled by protein kinase C are implicated in the regulation of gene expression in response to neural induction.
Tumor necrosis factor alpha-induced phosphorylation of I kappa B alpha is a signal for its degradation but not dissociation from NF-kappa B.
Authors: Miyamoto S , Maki M , Schmitt M J , Hatanaka M , Verma I M
Published: May 2006

Activation of the NF-kappa B/Rel family of transcription factors is regulated by a cytoplasmic inhibitor, I kappa B alpha. Activity of I kappa B alpha is in turn modulated by phosphorylation and proteolysis. It has been postulated that phosphorylation of I kappa B alpha leads to its dissociation from NF-kappa B, and free I kappa B alpha is targeted for rapid degradation. However, this phosphorylation-mediated dissociation event has not been demonstrated in vivo. We demonstrate that, contrary to this hypothesis, phosphorylation of I kappa B alpha induced by tumor necrosis factor alpha in HeLa cells does not induce dissociation. We propose a model in which (i) induced phosphorylation of I kappa B alpha does not result in its dissociation from NF-kappa B, (ii) phosphorylation of I kappa B alpha serves as a signal for degradation, and (iii) degradation of I kappa B alpha occurs while it is still complexed with NF-kappa B.
Signal transmission in the parallel fiber-Purkinje cell system visualized by high-resolution imaging.
Authors: Vranesic I , Iijima T , Ichikawa M , Matsumoto G , Knöpfel T
Published: May 2006

We investigated the synaptic transmission in the parallel fiber-Purkinje cell system at high spatio-temporal resolution by using voltage-sensitive dyes and an imaging system. In rat cerebellar slices, cut in the frontal plane or in a plane of the cerebellar surface, local electrical stimulation induced volleys of action potentials in the parallel fibers; subsequent postsynaptic responses from Purkinje cells were observed along the volleys' entire trajectories. Furthermore, the formation of an ordered spatial gradient in parallel fiber conduction velocity across the depth of the molecular layer during postnatal development was observed. In preparations of adult, but not of immature rats, the conduction velocity of parallel fibers in the deep molecular layer was faster than in its more superficial regions. Our observations demonstrate that parallel fibers can mediate Purkinje cell excitation effectively and over considerable distances in a well-organized spatio-temporal manner, thus supporting the classical view of the physiological role assigned to the parallel fibers.
Phylogenetic subtypes of human T-lymphotropic virus type I and their relations to the anthropological background.
Authors: Miura T , Fukunaga T , Igarashi T , Yamashita M , Ido E , Funahashi S , Ishida T , Washio K , Ueda S , Hashimoto K
Published: May 2006

Isolates of human T-lymphotropic virus type I (HTLV-I) were phylogenetically analyzed from native inhabitants in India and South America (Colombia and Chile) and from Ainu (regarded as pure Japanese descendants from the preagricultural "Jomon" period). Their genomes were partially sequenced together with isolates from Gabon in central Africa and from Ghana in West Africa. The phylogenetic tree was constructed from the sequence data obtained and those of previously reported HTLV-I isolates and simian T-lymphotropic virus type I (STLV-I) isolates. The heterogeneity of HTLV-I was recently recognized, and one major type, generally called the "cosmopolitan" type, contained Japanese, Caribbean, and West African isolates. The phylogenetic tree constructed in the present study has shown that this cosmopolitan type can be further grouped into three lineages (subtypes A, B, and C). Subtype A consists of some Caribbean, two South American, and some Japanese isolates, including that from the Ainu, in addition to an Indian isolate, and subtype B consists of other Japanese isolates in addition to another Indian isolate, suggesting that there might be at least two ancestral lineages of the Japanese HTLV-I. Subtype A implies a close connection of the Caribbean and South American natives with the Japanese and thereby a possible migration of the lineage to the American continent via Beringia in the Paleolithic era. Subtype C consists of the West African and other Caribbean isolates, indicating that not all but part of the Caribbean strains directly originated from West Africa probably during the period of slave trade. The tree also has shown that the HTLV-I isolate from Gabon in central Africa forms a cluster with STLV-I from a chimpanzee, suggesting a possible interspecies transmission between man and the chimpanzee in the past. No specific clustering was observed in the tree in relation to manifestations of the disease such as adult T-cell leukemia and HTLV-I-related neurological disorders. Thus, the topology of the phylogenetic tree reflects the movement of people carrying the virus in the past.
DNA class organization on maize Adh1 yeast artificial chromosomes.
Authors: Springer P S , Edwards K J , Bennetzen J L
Published: May 2006

The organization of higher plant genomes is poorly understood. These genomes are typified by their large size and extensive repetitive DNA component. To further our understanding of the composition and arrangement of genomic DNA sequences, we have performed a detailed analysis of a contiguous interval of 280 kb surrounding the Adh1 locus of maize. A series of overlapping lambda subclones was isolated, and individual fragments were characterized with respect to their genomic copy number. Cross-hybridization analyses were used to define a minimum of 37 repetitive DNA classes within the 280-kb interval. Hybridizations with highly repetitive DNAs cloned from other regions of the maize genome suggested that > 50% of all highly repetitive elements in maize are represented on this single yeast artificial chromosome. These repeated sequences were found in an organizational pattern not previously observed; individual repetitive elements are interspersed with one another in an apparently random fashion and are spatially separate from single copy number sequences. Extensive tandem arrays were not found. Sequences from one end of the 280-kb interval were used to isolate overlapping yeast artificial chromosome clones, representing the first step in a chromosome walk.
Regulation of metallothionein genes by heavy metalsappears to be mediated by a zinc-sensitive inhibitor that interacts with aconstitutively active transcription factor, MTF-1.
Authors: Palmiter R D
Published: May 2006

A construct, MRE-beta Geo, with five metal
response elements fused to a selectable reporter gene was transfected into BHK
cells and a stable clone that could be induced up to 100-fold by zinc, cadmium,
bismuth, silver, cobalt, copper, mercury, or nickle was isolated. Some, and
perhaps all, of these metals induce MRE-beta Geo by displacing zinc.
Transfection of these cells with a construct encoding the transcriptional
activator MTF-1 resulted in constitutive expression of MRE-beta Geo, whereas
expression of an antisense MTF-1 construct in these cells prevented induction by
all of the metals. A variant cell line with high constitutive expression in the
absence of added metals was isolated; normal regulation was restored by cell
fusion. These results suggest that regulation of metallothionein genes by metals
is mediated by MTF-1 interacting with metal response elements and that zinc
functions to release MTF-1 from an inhibitor.
Impact melting of frozen oceans on the early Earth: Implications for the origin of life
Authors: Bada J. L. , Bigham C. , Miller S. L.
Published: May 2006

Without sufficient greenhouse gases in the atmosphere, the early Earth would have become a permanently frozen planet because the young Sun was less luminous than it is today. Several resolutions to this faint young Sun-frozen Earth paradox have been proposed, with an atmosphere rich in CO
2
being the one generally favored. However, these models assume that there were no mechanisms for melting a once frozen ocean. Here we show that bolide impacts between about 3.6 and 4.0 billion years ago could have episodically melted an ice-covered early ocean. Thaw-freeze cycles associated with bolide impacts could have been important for the initiation of abiotic reactions that gave rise to the first living organisms.
Switch recombination in normal IgA1+ Blymphocytes.
Authors: Irsch J , Irlenbusch S , Radl J , Burrows P D , Cooper M D , Radbruch A H
Published: May 2006

Most B lymphocytes in normal individuals express
two classes of cell-surface immunoglobulins, IgM and IgD. The specificity of the
two antigen receptors is identical since they are produced by transcription and
differential splicing of the same variable region gene segment to the
heavy-chain constant region gene segments for both mu and delta heavy chains. B
lymphocytes expressing other immunoglobulin isotypes, IgG, IgA, or IgE, are rare
and not well characterized. Particularly controversial is the molecular
mechanism of their isotype switch. Here we use high-gradient magnetic cell
sorting and fluorescence-activated cell sorting to purify surface IgA1-bearing B
lymphocytes from human blood for cellular and molecular analysis. These cells
express no immunoglobulin class other than IgA1 and are a relatively uniform
population with regard to expression of other cell-surface molecules. They are
resting cells in terms of cell cycle and activation marker analysis. The
molecular basis for class switching in the IgA1+ cells is not differential
transcription or splicing. Rather, switch recombination involving deletion of
DNA has occurred on both immunoglobulin heavy-chain gene loci, including the
allelically excluded one, and appears to have been directed to IgA1 under normal
physiological conditions.
Mobile inverted-repeat elements of the Tourist familyare associated with the genes of many cereal grasses.
Authors: Bureau T E , Wessler S R
Published: May 2006

Tourist was originally described as a 128-bp
insertion mutation in the maize wx-B2 allele. Subsequent analysis revealed that
Tourist elements are in the introns or flanking sequences of 11 maize genes and
a single barley gene. In this study we report that Tourist elements are
frequently associated with the wild-type genes of two other grasses, rice and
sorghum. Six of 35 rice and 5 of 8 sorghum complete gene sequences reported to
date contain Tourist elements. Furthermore, 11 additional maize genes have been
found to contain Tourist elements, bringing the current total of elements
associated with maize genes to 23. Sequence comparison of Tourist elements has
led to the identification of four subfamilies, designated A-D. Evidence is
presented for the recent mobility of elements in three of these subfamilies and
in three of the four grass species. These data suggest that Tourist elements are
highly repetitive in the genomes of some and perhaps all members of the
grasses.
Polarized secretion of beta-amyloid precursor proteinand amyloid beta-peptide in MDCK cells.
Authors: Haass C , Koo E H , Teplow D B , Selkoe D J
Published: May 2006

The beta-amyloid precursor protein (beta APP) is a
widely expressed integral membrane protein that is proteolytically processed to
yield several secreted derivatives, including soluble APP (APPs), the 4-kDa
amyloid beta-peptide (A beta), and a related 3-kDa peptide (p3). To understand
beta APP trafficking and processing, we analyzed the sorting of beta APP in
Madin-Darby canine kidney (MDCK) cells, an epithelial cell known to possess
physiologically distinct apical and basolateral plasma membranes. Processing of
beta APP resulted in highly polarized secretion of APPs. More than 90% of APPs
was detected in the basolateral compartment, and less than 10% was found in the
apical compartment. This was associated with a preferential localization of beta
APP on the basolateral cell surface. Activation of protein kinase C, which is
known to enhance the secretion of APPs, did not change the polarity of APPs
release but significantly increased the amount secreted. A beta and p3 peptides
were also secreted predominantly basolaterally. In addition, MDCK cells secreted
a truncated form of A beta beginning at Arg-5. These data show that the
proteolytic processing products of beta APP undergo polarized secretion.
Moreover, the results suggest that the amyloidogenic A beta peptide is generated
following the polarized sorting of beta APP. The polarized basolateral secretion
of A beta in these epithelial cells provides a potential mechanism for the
accumulation of A beta in the abluminal basement membrane of brain microvessels
during Alzheimer disease.
Primary structure of a beta subunit ofalpha-dendrotoxin-sensitive K+ channels from bovine brain.
Authors: Scott V E , Rettig J , Parcej D N , Keen J N , Findlay J B , Pongs O , Dolly J O
Published: May 2006

Voltage-dependent cation channels are large
heterooligomeric proteins. Heterologous expression of cDNAs encoding the alpha
subunits alone of K+, Na+, or Ca2+ channels produces functional multimeric
proteins; however, coexpression of those for the latter two with their auxiliary
proteins causes dramatic changes in the resultant membrane currents.
Fast-activating, voltage-sensitive K+ channels from brain contain four alpha and
beta subunits, tightly associated in a 400-kDa complex; although molecular
details of the alpha-subunit proteins have been determined, little is known
about the beta-subunit constituent. Proteolytic fragments of a beta subunit from
bovine alpha-dendrotoxin-sensitive neuronal K+ channels yielded nine different
sequences. In the polymerase chain reaction, primers corresponding to two of
these peptides amplified a 329-base-pair fragment in a lambda gt10 cDNA library
from bovine brain; a full-length clone subsequently isolated encodes a protein
of 367 amino acids (M(r) approximately 40,983). It shows no significant homology
with any known protein. Unlike the channels' alpha subunits, the hydropathy
profile of this sequence failed to reveal transmembrane domains. Several
consensus phosphorylation motifs are apparent and, accordingly, the beta subunit
could be phosphorylated in the intact K+ channels. These results, including the
absence of a leader sequence and N-glycosylation, are consistent with the beta
subunit being firmly associated on the inside of the membrane with alpha
subunits, as speculated in a simplified model of these authentic K+ channels.
Importantly, this first primary structure of a K(+)-channel beta subunit
indicates that none of the cloned auxiliary proteins of voltage-dependent cation
channels, unlike their alpha subunits, belong to a super-family of
genes.
Human transcription factor IIIC box B bindingsubunit.
Authors: L'Etoile N D , Fahnestock M L , Shen Y , Aebersold R , Berk A J
Published: May 2006

Transcription factor IIIC (TFIIIC) is a
multisubunit basic TF for RNA polymerase III. It initiates transcription complex
assembly on tRNA and related genes by binding to the internal box B promoter
element and is also required for transcription of 5S rRNA and other stable
nuclear and cytoplasmic RNAs transcribed by polymerase III. In mammalian cells,
regulation of TFIIIC activity controls overall polymerase III transcription in
response to growth factors and viral infection. Here, we report the cloning and
sequencing of a full-length cDNA (and genomic DNA from the transcription
initiation region) encoding the box B binding subunit of human TFIIIC, the
243-kDa alpha subunit. Specific antisera raised against the encoded protein
super shifts a TFIIIC-box B DNA complex during an electrophoretic mobility shift
assay and immunodepletes TFIIIC transcriptional activity from a partially
purified TFIIIC fraction, proving that the cDNA encodes a component of TFIIIC.
The human protein shows surprisingly little similarity to the box B binding
subunit of yeast TFIIIC.