bluuebunny/crossref_metadata_2025 huggingface dataset:
Simian virus 40 large tumor antigen is unable to transform mouse embryonic fibroblasts lacking type 1 insulin-like growth factor receptor.
Authors: Sell C , Rubini M , Rubin R , Liu J P , Efstratiadis A , Baserga R
Published: May 2006

Fibroblast cell lines were established from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 receptor for insulin-like growth factor I (IGF-I) and from their wild-type littermates. The cells from the wild-type embryos (W cells) grow in serum-free medium supplemented with platelet-derived growth factor, epidermal growth factor, and IGF-I, whereas the cells from Igf1r(-/-) embryos (R- cells) do not, although they grow at a reduced rate in 10% fetal calf serum. The simian virus 40 (SV40) large T antigen, expressed from a transfected plasmid, can transform W cells, which form foci in monolayer cultures and colonies in soft agar (anchorage-independent growth). In contrast, the SV40 large tumor antigen, although normally expressed from the transfected template, is unable to transform R- cells, which remain contact-inhibited and fail to grow in soft agar. The transformed phenotype is restored if the R- cells carrying the SV40 large tumor antigen are stably transfected with a plasmid expressing the human IGF-I receptor. These results demonstrate that signaling via the IGF-I receptor is an indispensable component of the SV40 transformation pathway. This conclusion is further supported from the results of antisense RNA experiments with tumor cell lines showing that interference with the function of the IGF-I receptor has a profound effect on anchorage-independent growth, even under conditions that only modestly affect growth in monolayers.
High-affinity RNA ligands to basic fibroblast growth factor inhibit receptor binding.
Authors: Jellinek D , Lynott C K , Rifkin D B , Janjić N
Published: May 2006

We have isolated RNA ligands with low-nanomolar affinity and high specificity to basic fibroblast growth factor from a pool of 10(14) molecules containing 30 randomized positions by the systematic evolution of ligands by exponential enrichment (SELEX) procedure. High-affinity ligands could be classified into two families based on sequence and secondary structure similarities. Representative RNA ligands from the two families compete with one another as well as with heparin for binding to the protein. Furthermore, we show that these ligands inhibit the first step in the signaling pathway of basic fibroblast growth factor: binding of the growth factor to its cell-surface receptors. These findings emphasize the general usefulness of SELEX as a tool for discovering potent, specific oligonucleotide antagonists of target proteins.
Effective immunization against cutaneous leishmaniasis with recombinant bacille Calmette-Guérin expressing the Leishmania surface proteinase gp63.
Authors: Connell N D , Medina-Acosta E , McMaster W R , Bloom B R , Russell D G
Published: May 2006

Leishmania parasites cause a spectrum of diseases that afflict the populations of 86 countries in the world. The parasites can survive within the lysosomal compartments of the host's macrophages, unless those macrophages are appropriately activated. Despite the fact that protective immunity can be induced by vaccination with crude parasite preparations, little progress has been made toward a defined vaccine for humans. In this study the gene encoding the Leishmania surface proteinase gp63 was cloned and expressed as a cytoplasmic protein in a bacille Calmette-Guérin (BCG) vaccine strain. BALB/c and CBA/J mice were inoculated with a single dose of recombinant BCG and challenged with infective Leishmania major or Leishmania mexicana promastigotes. Significant protection was observed in both mouse strains against L. mexicana and in CBA/J against L. major, whereas only a delay in L. major growth was seen in BALB/c mice. Recombinant BCG also engendered a strong protective response against challenge with amastigotes of L. mexicana, demonstrating that the induced immune response recognized the intracellular form of the parasite. The results support the view that recombinant BCG expressing gp63 may prove a useful vaccine for inducing protective cell-mediated immune responses to Leishmania species causing American cutaneous leishmaniasis.
The min K channel underlies the cardiac potassium current IKs and mediates species-specific responses to protein kinase C.
Authors: Varnum M D , Busch A E , Bond C T , Maylie J , Adelman J P
Published: May 2006

A clone encoding the guinea pig (gp) min K potassium channel was isolated and expressed in Xenopus oocytes. The currents, gpIsK, exhibit many of the electrophysiological and pharmacological properties characteristic of gpIKs, the slow component of the delayed rectifier potassium conductance in guinea pig cardiac myocytes. Depolarizing commands evoke outward potassium currents that activate slowly, with time constants on the order of seconds. The currents are blocked by the class III antiarrhythmic compound clofilium but not by the sotalol derivative E4031 or low concentrations of lanthanum. Like IKs in guinea pig myocytes, gpIsK is modulated by stimulation of protein kinase A and protein kinase C (PKC). In contrast to rat and mouse IsK, which are decreased upon stimulation of PKC, myocyte IK and gpIsK in oocytes are increased after PKC stimulation. Substitution of an asparagine residue at position 102 by serine (N102S), the residue found in the analogous position of the mouse and rat min K proteins, results in decreased gpIsK in response to PKC stimulation. These results support the hypothesis that the min K protein underlies the slow component of the delayed rectifier potassium current in ventricular myocytes and account for the species-specific responses to stimulation of PKC.
Animals and fungi are each other's closest relatives: congruent evidence from multiple proteins.
Authors: Baldauf S L , Palmer J D
Published: May 2006

Phylogenetic relationships among plants, animals, and fungi were examined by using sequences from 25 proteins. Four insertions/deletions were found that are shared by two of the three taxonomic groups in question, and all four are uniquely shared by animals and fungi relative to plants, protists, and bacteria. These include a 12-amino acid insertion in translation elongation factor 1 alpha and three small gaps in enolase. Maximum-parsimony trees were constructed from published data for four of the most broadly sequenced of the 25 proteins, actin, alpha-tubulin, beta-tubulin, and elongation factor 1 alpha, with the latter supplemented by three new outgroup sequences. All four proteins place animals and fungi together as a monophyletic group to the exclusion of plants and a broad diversity of protists. In all cases, bootstrap analyses show no support for either an animal-plant or fungal-plant clade. This congruence among multiple lines of evidence strongly suggests, in contrast to traditional and current classification, that animals and fungi are sister groups while plants constitute an independent evolutionary lineage.
Enhancer activity of light-responsive regulatory elements in the untranslated leader regions of cyanobacterial psbA genes.
Authors: Li R , Golden S S
Published: May 2006

Three psbA genes encoding the D1 protein of the photosystem II reaction center are differentially expressed under different light intensities in the cyanobacterium Synechococcus sp. strain PCC 7942. Two of the three psbA genes, psbAII and psbAIII, are induced rapidly when light intensity is increased from 125 x 10(-6) mol.m-2.s-1 to 750 x 10(-6) mol.m-2.s-1. A recombinational cloning vector that carries a transcriptional lacZ reporter gene was used to characterize the controlling elements responsible for light induction. At least three distinct cis elements are present in the regulatory regions of pbsAII and psbAIII: basal promoters, comparable to Escherichia coli sigma 70 promoters in position and sequence, confer constitutive expression of the genes under both low and high light intensities; negative elements upstream of the promoters down-regulate the expression of the corresponding gene; and sequences downstream of the promoters that correspond to the untranslated leader regions of the mRNAs (+1 to +41 in psbAII and +1 to +39 in psbAIII) are responsible for increased expression under high light. When these light-responsive elements were combined with an E. coli promoter (conII) in different positions and orientations, the expression of the lacZ gene was induced 4- to 11-fold. The induction of gene expression under high light by these enhancers was position independent but orientation dependent. When the elements were combined with the conII promoter in the correct orientation, they also conferred a small but reproducible level of light-responsive expression on this E. coli promoter.
Corticosterone in the range of stress-induced levels possesses reinforcing properties: implications for sensation-seeking behaviors.
Authors: Piazza P V , Deroche V , Deminière J M , Maccari S , Le Moal M , Simon H
Published: May 2006

In both humans and animals certain individuals seek stimuli or situations that are considered stressful and consequently avoided by others. A common feature of such situations is an activation of the hypothalamo-pituitary-adrenal axis leading to secretion of glucocorticoids. Since glucocorticoids have euphoric effects in some individuals and have been shown to potentiate the reinforcing properties of drugs of abuse in animals, we hypothesized that corticosterone secretion during stress-like situations may have reinforcing effects and that a higher sensitivity to the reinforcing effects of glucocorticoids might be a biological basis of sensation seeking. In this report we show that (i) corticosterone has reinforcing properties, as evidenced by the development of intravenous self-administration, (ii) self-administration of corticosterone is observed at plasma levels that are comparable to those induced by stress, and (iii) there are individual differences in corticosterone self-administration, which are related to individual reactivity to novelty and sensitivity to drugs of abuse, behavioral features akin to certain traits of high-sensation seekers. These findings provide insight into the physiological role of glucocorticoids and the biology of sensation seeking and may have clinical implications.
Crystal structure of murine cyclophilin C complexed with immunosuppressive drug cyclosporin A.
Authors: Ke H , Zhao Y , Luo F , Weissman I , Friedman J
Published: May 2006

Cyclophilin is a cellular receptor for the immunosuppressive drug cyclosporin A (CsA). Cyclophilin C (CyPC) is highly expressed in murine kidney, making it a potential mediator of the nephrotoxic effects of CsA. The structure of murine CyPC complexed with CsA has been solved and refined to an R factor of 0.197 at a 1.64-A resolution. Superposition of the CyPC-CsA structure with the unligated cyclophilin A (CyPA) revealed significant migration of three loops: Gln-179 to Thr-189, Asp-47 to Lys-49, and Met-170 to Ile-176. The proximity of the loop Gln-179 to Thr-189 to the CsA binding site may account for the unique binding of a 77-kDa glycoprotein, CyPC binding protein (CyCAP), to CyPC. The binding of CsA to CyPC is similar to that of CsA to human T-cell cyclophilin A (CyPA). However, the conformation of CsA when bound to CyPC is significantly different from that when bound to CyPA. These differences may reflect conformational variation of CsA when bound to different proteins. Alternatively, the previous CyPA-CsA structure at low resolution may not provide sufficient details for a comparison with the CyPC-CsA structure.
Antibody-catalyzed reversal of chemoselectivity.
Authors: Sinha S C , Keinan E , Reymond J L
Published: May 2006

A monoclonal antibody, 14D9, which has been elicited against a cationic hapten, N-alkyl-N-methyl-3-glutarylamidomethyl piperidinium, in which alkyl = [4-(2-hydroxyethylamido)carbonyl]phenylmethyl, is capable of inverting the intrinsic order of reactivity in a series of structurally related enol ethers and ketals towards hydrolysis. The order of reactivity of compounds 2 (1-methoxy-2-alkylcyclopent-1-ene), 3 (1-methoxy-5-alkylcylopent-1-ene), and 4 (1,1-dimethoxy-2-alkylcyclopentane) has changed from 0.09:0.17:1 in the uncatalyzed reaction to 1100:25:1 under antibody catalysis. Also, the order of reactivity of the three chemically similar ketals, 6a (1-alkyl-2,2-dimethoxypropane), 6b (1-alkyl-1-methyl-2,2-dimethoxypropane), and 4, has changed from 0.23:0.38:1 in the uncatalyzed hydrolysis to 100:9:1 within the antibody active site. As all compounds bind the antibody with very similar affinities, these effects cannot be simply attributed to selective binding by the antibody. In fact, ketal 4, which shows no measurable catalysis, acts as a competitive inhibitor of 14D9-catalyzed hydrolysis of 6a. Both the solution and the antibody-catalyzed hydrolysis of the ketal substrates are shown to be specific acid catalyzed, involving the unimolecular cleavage of the protonated substrate or antibody-substrate complex in the rate-determining step. Reactivity effects from the acid catalyst itself on ketal hydrolysis (reagent-controlled reactivity) are ruled out under this mechanistic scheme.
Signal efficacy and the evolution of male dimorphism in the jumping spider, Maevia inclemens.
Authors: Clark D L , Uetz G W
Published: May 2006

An extreme form of male dimorphism occurs in the jumping spider, Maevia inclemens, where two male morphs differ dramatically in both morphology and behavior. We examined the visual signals transmitted to females by the two different morphs during courtship display. Even though the display of each male is different, as a function of behavior and courtship distance, the males present females with images that are almost identical in height and visual target area. By substituting live males with computer-animated sequences of male courtship behavior, the male morphs were superimposed upon each other, and females were determined to have two distinct male-recognition templates. We hypothesize that the dimorphic males of this species evolved through sensory exploitation and selection for alternative male strategies that stimulate different fields of the female visual range.
Arachidonic acid status correlates with first year growth in preterm infants.
Authors: Carlson S E , Werkman S H , Peeples J M , Cooke R J , Tolley E A
Published: May 2006

Diets deficient in the omega-6 fatty acid linoleic acid reduce arachidonic acid (Ach) concentrations and retard growth of developing animals and humans. Nevertheless, plasma phosphatidylcholine Ach concentrations declined from 84 +/- 23 mg/liter at birth to a nadir of 38 +/- 11 mg/liter at 4 mo of age in preterm infants fed commercial formulas with linoleic acid, and weight normalized to that of term infants fell progressively beginning at 2 mo of age. The nadir of plasma phosphatidylcholine Ach (31 +/- 7 mg/liter) and growth were further reduced by formula containing marine oil compared with the commercial formulas. Ach status (defined as the mean plasma phosphatidylcholine Ach concentration at 2, 4, and 6.5 mo) correlated with one or more measures of normalized growth through 12 mo. Ach status and maternal height accounted for as much as 59% of the weight variance and 68% of the length variance in infants fed standard formulas. Better Ach status was not from higher energy intakes. A conditional Ach deficiency in preterm infants may contribute to growth over the first year of life. On the strength of the relationship between Ach status and growth, we hypothesize that dietary Ach could improve first year growth of preterm infants.
Do all programmed cell deaths occur via apoptosis?
Authors: Schwartz L M , Smith S W , Jones M E , Osborne B A
Published: May 2006

During development, large numbers of cells die by a nonpathological process referred to as programmed cell death. In many tissues, dying cells display similar changes in morphology and chromosomal DNA organization, which has been termed apoptosis. Apoptosis is such a widely documented phenomenon that many authors have assumed all programmed cell deaths occur by this process. Two well-characterized model systems for programmed cell death are (i) the death of T cells during negative selection in the mouse thymus and (ii) the loss of intersegmental muscles of the moth Manduca sexta at the end of metamorphosis. In this report we compare the patterns of cell death displayed by T cells and the intersegmental muscles and find that they differ in terms of cell-surface morphology, nuclear ultrastructure, DNA fragmentation, and polyubiquitin gene expression. Unlike the T cells, which are known to die via apoptosis, we find that the intersegmental muscles display few of the features that characterize apoptosis. These data suggest that more than one cell death mechanism is used during development.
Ia-antigen-T-cell interactions for a thymus-independent antigen composed of D amino acids.
Authors: Zisman E , Dayan M , Sela M , Mozes E
Published: May 2006

Synthetic polypeptide antigens of L amino acids, although bearing repeating sequences, are thymus-dependent (L-TD), whereas the same polymers composed of D amino acids are thymus-independent (D-TI), probably due to a slower rate of metabolism. Yet we found that lymph-node cells of BALB/c mice immunized with D-TI proliferate in response to it in vitro. To follow T-cell activation by D-TI, we established T-cell hybridomas to D-TI and to its analog composed of L isomers, L-TD, for comparison. The T-cell hybridomas express membrane alpha/beta T-cell receptors and secrete interleukin 2 upon stimulation with the respective antigen. In addition, D-TI-specific hybridomas are stimulated, to a lesser extent, by the L-TD antigen, whereas only some L-TD-specific hybridomas recognize D-TI. Moreover, biotinylated analogs of D-TI and L-TD bind to splenic antigen-presenting cells (APCs) from BALB/c mice. Binding is inhibited by an excess of nonbiotinylated L-TD, and by an excess of a peptide comprising residues 259-271 of the human acetylcholine receptor alpha subunit, which binds to I-Ad and I-Ed molecules without prior processing. Analysis of APC lysates following incubation of the APCs with biotinylated D-TI and L-TD reveals that the biotinylated antigen moiety is associated with Ia molecules. D-TI and L-TD bind to Ia molecules on intact APCs with similar KD values, 5 x 10(-8) M and 3 x 10(-8) M, respectively. However, D-TI has faster kinetics of binding than L-TD, probably due to different processing requirements. Hence, we have demonstrated a major histocompatibility complex class II-mediated T-cell response to a thymus-independent antigen.
Kinetics and mechanism in RNA cleavage.
Authors: Breslow R
Published: May 2006

Experimental studies on the cleavage of various RNA molecules--poly(U), 3',5''-UpU, 2',5''-UpU, and 3',5''-ApA--catalyzed by imidazole buffers show that there is a sequential bifunctional mechanism. One catalyst species converts the substrate to an intermediate phosphorane, and the second catalyst converts the phosphorane to cleavage products. Detailed steady-state kinetics are presented to determine all mechanisms that are consistent with the findings. Choice among these possibilities can be made considering other experimental evidence about the catalysis of an isomerization reaction that accompanies cleavage. It is concluded that all acceptable mechanisms involve action of the second catalyst on a phosphorane monoanion; it can be formed directly from the substrate in the first step or by proton equilibrations involving either the substrate or the phosphorane intermediate. The relationship of these conclusions to the likely mechanism of action of the enzyme ribonuclease is briefly discussed.
Displacement-clamp measurement of the forces exerted by gating springs in the hair bundle.
Authors: Jaramillo F , Hudspeth A J
Published: May 2006

Mechanical stimuli applied to the hair bundle of a hair cell are communicated to the transduction channels by gating springs, elastic elements that are stretched when the bundle is displaced toward its tall edge. To quantify the magnitude and time dependence of the forces exerted by gating springs, we have developed a displacement-clamp system that constrains a bundle's motion while measuring the forces that the bundle produces during adaptation to mechanical stimuli, in response to channel blockage, and upon destruction of the gating springs. Our results suggest that each gating spring exerts a tension of approximately 8 pN in the resting bundle and can sustain at least 4-13 pN of additional tension. The experiments provide further evidence that the gating springs account for at least one-third of the hair bundle's dynamic stiffness and that a force of approximately 100 fN is sufficient to open a single transduction channel.
Formation of large deletions by illegitimate recombination in the HPRT gene of primary human fibroblasts.
Authors: Morris T , Thacker J
Published: May 2006

HPRT gene mutants were isolated from untreated and x-irradiated cultures of primary human fibroblasts, and mutants carrying large deletions were identified. The breakpoints of the deletions were mapped by methods based on the polymerase chain reaction, and the deletion junctions of four different mutants were sequenced. Alu repeats were associated with one end of three of these junctions, but in each case repeat sequences were not found at the other end. Sequence features found at the deletion breakpoints included in particular short direct and inverted repeats, which may mispair to promote illegitimate recombination. One mutant had additional bases inserted at the deletion junction; these bases formed a direct repeat with a sequence immediately adjacent to the junction, suggesting a mechanism of templated repair of broken DNA in deletion formation.
mRNA processing independent of RNase III and RNase E in the expression of the F1845 fimbrial adhesin of Escherichia coli.
Authors: Bilge S S , Apostol J M , Aldape M A , Moseley S L
Published: May 2006

F1845, the fimbrial adhesin of a diarrhea-associated Escherichia coli, confers upon the bacteria the ability to adhere to cultured epithelial cells in a diffuse pattern. The fimbrial subunit gene, daaE, is encoded on a polycistronic mRNA which is processed endoribonucleolytically to produce a stable message encoding only daaE. The processing event occurs in bacterial strains with mutations in RNase III or RNase E, the only endoribonucleases which have been implicated in the processing of E. coli mRNA. Sequences encoding a stem-loop structure downstream of daaE play an essential role in determining the stability of the daaE mRNA. Rapid degradation of the sequences upstream of the cleavage site occurs upon processing, suggesting that processing of the F1845 polycistronic mRNA results in differential expression of genes involved in the biogenesis of fimbriae.
Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy.
Authors: Swain A L , Jaskólski M , Housset D , Rao J K , Wlodawer A
Published: May 2006

The crystal structure of Escherichia coli asparaginase II (EC 3.5.1.1), a drug (Elspar) used for the treatment of acute lymphoblastic leukemia, has been determined at 2.3 A resolution by using data from a single heavy atom derivative in combination with molecular replacement. The atomic model was refined to an R factor of 0.143. This enzyme, active as a homotetramer with 222 symmetry, belongs to the class of alpha/beta proteins. Each subunit has two domains with unique topological features. On the basis of present structural evidence consistent with previous biochemical studies, we propose locations for the active sites between the N- and C-terminal domains belonging to different subunits and postulate a catalytic role for Thr-89.
Using three-color chromosome painting to test chromosome aberration models.
Authors: Lucas J N , Sachs R K
Published: May 2006

Ionizing radiation induces DNA double-strand breaks (DSB), which interact pairwise to produce chromosome aberrations. There have long been two main competing theories of such pairwise DSB-DSB interactions. The "classical" theory asserts that an unrepaired DSB makes two ends that separate within the cell nucleus, with each end subsequently able to join any similar (nontelomeric) end. The "exchange" theory asserts that at a DSB the chromatin does not separate completely; rather the DSB ends remain associated until repair, or an illegitimate recombination involving another DSB, occurs. The DSB-DSB interaction mechanism was tested by using three-color fluorescence in situ hybridization to paint chromosomes and observe "three-color triplets": three broken and misrejoined chromosomes having cyclically permuted colors. We observed 18 "three-color triplets" in 2000 cells after 2.25 Gy of gamma-irradiation. On the exchange model in its standard form such three-color triplets cannot occur, so this model is inconsistent with the observations. On the classical model, formalized as a discrete time Markov chain embedded at the transitions of a continuous time Markov chain, the frequency of occurrence of three-color triplets can be computed by Monte Carlo simulations. The number of three-color triplets predicted mathematically by the classical model was found to be slightly larger than the observed number. Thus our data, together with our computer simulations, exclude the standard form of the exchange model but are compatible with the classical model. The results are also compatible with other, more complicated models.
Plasticity in chromosome number and testing of essential genes in Leishmania by targeting.
Authors: Cruz A K , Titus R , Beverley S M
Published: May 2006

We attempted to generate homozygous dhfr-ts (dihydrofolate reductase-thymidylate synthase) knockouts in virulent Leishmania major, an asexual diploid protozoan parasite. Transfection of a neo (neomycin phosphotransferase) targeting fragment yielded heterozygous replacement lines with high efficiency. However, second transfections with a hyg (hygromycin B phosphotransferase) targeting fragment in the presence of metabolites shown to rescue homozygous knockouts in attenuated Leishmania did not yield the expected dhfr-ts- thymidine auxotrophs obtained previously with attenuated lines. Molecular karyotype, Southern blot, and flow cytometric DNA content analysis of clonal transfectants revealed three classes: (i) genomic tetraploids, containing two wild-type dhfr-ts chromosomes and one neo and one hyg replacement chromosome; (ii) aneuploid trisomic lines with one wild-type dhfr-ts and one neo and one hyg replacement chromosome; (iii) diploids bearing homologous integration of the targeting fragment without replacement. Aneuploid and tetraploid lines predominated. This confirms the common impression that natural populations of Leishmania are often aneuploid. The remarkable ability of these parasites to undergo and tolerate changes in chromosome number suggests a general method for testing whether genes are essential for growth in vitro, as the ability of Leishmania to simultaneously undergo homologous gene replacement while retaining wild-type genes by increasing chromosome number provides a diagnostic and positive experimental result. Our results show that virulent Leishmania require at least one copy of dhfr-ts and argue that DHFR-TS plays an unanticipated role in addition to its role in the de novo synthesis of thymidine. These results also have implications for genetic tests of the organization of Leishmania populations.
Stimulation of human thyroid growth via the inhibitory guanine nucleotide binding (G) protein Gi: constitutive expression of the G-protein alpha subunit Gi alpha-1 in autonomous adenoma.
Authors: Selzer E , Wilfing A , Schiferer A , Hermann M , Grubeck-Loebenstein B , Freissmuth M
Published: May 2006

The alpha subunits of the stimulatory and inhibitory G proteins, Gs alpha and Gi alpha, activate transmembrane-signaling systems involved in the control of cell proliferation. We have investigated the pattern of expression of Gi alpha subtypes and Gi alpha-mediated proliferative responses in the human thyroid. Human thyroid membranes contain two subtypes of Gi alpha, Gi alpha-1 and Gi alpha-2, as assessed by using specific antibodies. The expression of Gi alpha-1 is under tight control by thyrotropin in vivo and in primary cultures of thyroid epithelial cells. In contrast, Gi alpha-1 is expressed in the absence of thyrotropin in thyroid autonomous adenoma, an endocrine-active tumor, and its levels are not regulated by thyrotropin in thyroid epithelial cells prepared from these tumors. If thyroid epithelial cells are treated with pertussis toxin to block signal transduction via Gi, the mitogenic response to serum factors is reduced. These observations demonstrate that Gi subtypes transmit growth stimuli in the human thyroid. The constitutive expression of Gi alpha-1 in autonomous adenoma may allow for the unregulated stimulation of thyroid cell proliferation by a yet unidentified signaling pathway and, thus, be causally related to autonomous growth of thyroid cells.
Maturation of thylakoid lumen proteins proceeds post-translationally through an intermediate in vivo.
Authors: Howe G , Merchant S
Published: May 2006

Many thylakoid lumen proteins are synthesized outside the chloroplast as larger molecular weight precursors and then processed to their mature size during transport to the lumenal space. We have examined the post-translational processing of thylakoid lumen proteins in vivo by pulse-radiolabeling experiments with Chlamydomonas reinhardtii. Antibodies against the lumenal protein cytochrome c6 specifically immunoprecipitated three polypeptides from extracts of briefly pulse-radiolabeled cells. The molecular weights and kinetics of synthesis and turnover indicate that these three polypeptides are (i) the full-length cytochrome c6 precursor, (ii) a partially processed precursor (intermediate), and (iii) the completely processed mature protein. Identification of analogous forms of two other lumenal proteins, plastocyanin and the oxygen evolving enhancer 1 protein, indicates that the maturation of thylakoid lumen proteins occurs post-translationally in vivo and that the partially processed intermediate is a general feature of the pathway. The intermediate form of cytochrome c6 accumulated to a greater extent in cells incubated at 10 degrees C, relative to cells incubated at 22 degrees C, concomitantly with a decrease in the accumulation of the mature protein. The intermediate accumulating at 10 degrees C is quantitatively converted to the mature protein upon incubation at higher temperature, thus demonstrating a precursor-product relationship between the intermediate and mature forms of cytochrome c6. Our results prove the model [Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K. & Weisbeek, P. (1986) Cell 46, 365-375] that precursors of lumenal proteins are post-translationally converted to their mature forms in two steps through a distinct intermediate.
GD2 ganglioside on human T-lymphotropic virus type I-infected T cells: possible activation of beta-1,4-N-acetylgalactosaminyltransferase gene by p40tax.
Authors: Furukawa K , Akagi T , Nagata Y , Yamada Y , Shimotohno K , Cheung N K , Shiku H , Furukawa K
Published: May 2006

Ganglioside expression on adult T-cell leukemia (ATL) and human T-cell lymphotropic virus type I (HTLV-I)-infected cells was determined by using a panel of monoclonal antibodies. ATL lines and HTLV-I-infected cells specifically expressed GD2. Leukemia cells from ATL patients generally expressed low levels of GD2 but the percentage of GD2+ cells increased up to 40-70% after in vitro culture in the presence of interleukin 2 for about a week. No other type of leukemia cells and normal peripheral T cells expressed GD2 during in vitro culture under the same conditions. The appearance of GD2 in the cultured ATL cells corresponded with the expression of p40tax, a product of the HTLV-I gene. Peripheral lymphocytes infected with a p40tax-expressing retroviral vector expressed high levels of GD2 in comparison with control lymphocytes containing the neomycin-resistance gene alone. The apparently increased levels of beta-1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) mRNA in these cells were demonstrated by reverse transcription-polymerase chain reaction analysis. Concordance between mRNA expression for the HTLV-I tax1/rex1 genes and the beta-1,4-N-acetylgalactosaminyltransferase gene was also observed in uncultured ATL cells. These results suggest that high GD2 expression was due to neosynthesis from precursor GD3 by increased expression of this enzyme induced by p40tax in vitro and in vivo.
In vitro culture of primary plasmacytomas requires stromal cell feeder layers.
Authors: Degrassi A , Hilbert D M , Rudikoff S , Anderson A O , Potter M , Coon H G
Published: May 2006

Attempts to grow primary murine plasmacytomas in vitro have, to date, been largely unsuccessful. In this study, we demonstrate that long-term in vitro growth of primary plasmacytomas is accomplished by using feeder layers composed of stromal cells from the initial site of plasmacytomagenesis. The early neoplastic lines established in this manner are dependent on physical contact with the stromal layer, which is mediated in part by CD44, for growth and survival. The stromal cells provide at least two stimuli for the plasma cells, one being interleukin 6 and the second, of unknown nature, resulting from direct physical interaction that cannot be replaced by soluble factors. These plasma cell lines have been passaged for as long as 20 months yet still maintain characteristics associated with primary plasmacytomas as they will grow in vivo only in pristane-primed animals, indicating a continued dependence on the pristane-induced microenvironment characteristic of early-stage tumors. The ability to grow primary plasmacytomas in culture and maintain their "primary" properties provides a model system for detailed analysis of early events in plasma cell tumor progression involving neoplastic cells completely dependent on physical contact with a stromal feeder layer for survival and expansion.
In vivo evidence for endogenous DNA alkylation damage as a source of spontaneous mutation in eukaryotic cells.
Authors: Xiao W , Samson L
Published: May 2006

Three genes that participate in the repair of DNA alkylation damage were recently cloned from Saccharomyces cerevisiae: the MGT1 O6-methylguanine DNA methyltransferase gene, the MAG 3-methyladenine DNA glycosylase gene, and the APN1 apurinic/apyrimidinic (AP) endonuclease gene. Altering the expression levels of these three genes produced significant changes in the S. cerevisiae spontaneous mutation rate. Spontaneous mutation increased in the absence of the MGT1 DNA methyltransferase, presumably because unrepaired, spontaneously produced, O6-alkylguanine lesions mispair during replication. Moreover, changing the ratios of the MAG 3-methyladenine DNA glycosylase and the APN1 AP endonuclease had profound effects on spontaneous mutation rates. In the absence of APN1, the overexpression of MAG increased spontaneous mutation, and the underexpression of MAG decreased spontaneous mutation. We infer that the MAG glycosylase acts upon spontaneously produced 3-alkyladenine and 7-alkylguanine DNA lesions to produce mutagenic abasic sites, and that if the repair of these abasic sites is not initiated by the APN1 AP endonuclease they cause mutations during replication. Our results indicate that eukaryotic cells harbor endogenous metabolites that alkylate nuclear DNA at both oxygens and nitrogens.
An HLA-A11-specific motif in nonamer peptides derived from viral and cellular proteins.
Authors: Zhang Q J , Gavioli R , Klein G , Masucci M G
Published: May 2006

T lymphocytes recognize their antigenic targets as peptides associated with major histocompatibility complex molecules. The HLA-A11 allele, a preferred restriction element for Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocyte responses, presents an immunodominant epitope derived from the EBV nuclear antigen 4. Subpicomolar concentrations of a synthetic nonamer peptide, IVTDFSVIK, corresponding to amino acids 416-424 of the EBV nuclear antigen 4 sequence, can sensitize phytohemagglutinin-stimulated blasts to lysis by EBV-specific HLA-A11-restricted cytotoxic T-lymphocytes. We show that micromolar concentrations of this peptide induce assembly and surface expression of HLA-A11 in an A11-transfected subline of the peptide transporter mutant cell line T2. Using the IVTDFSVIK peptide and a series of synthetic nonamer peptides, differing from the original sequence by single amino acid substitutions, we have defined a motif for HLA-A11-binding peptides. This predicts the presence of a hydrophobic amino acid in position 2, amino acids with small side chains in positions 3 and 6, and a lysine in position 9. Using this motif, we have identified a peptide in the carboxyl-terminal end of wild-type p53, ELNEALELK, which is able to induce HLA-A11 assembly as efficiently as the IVTDFSVIK viral peptide.
Comparative reproductive success of communally breeding burying beetles as assessed by PCR with randomly amplified polymorphic DNA.
Authors: Scott M P , Williams S M
Published: May 2006

To understand the evolution of alternative reproductive strategies such as communal breeding, it is important to recognize the options open to individuals and to evaluate their consequences. The relative reproductive success of individuals taking each option is one of the most important of these consequences. Burying beetles, Nicrophorus, are an excellent model system for the investigation of reproductive cooperation because they can breed in pairs or communally and provide extensive parental care. In this study, we examine the relationship of the duration of care and the reproductive success of each potentially communally breeding adult. Ten experimental broods reared on mouse carcasses were buried by two males and two females. Using PCR with single short primers that randomly amplify polymorphic DNA, we determined the maternity and paternity of 98.2% and 99.5% of the offspring (n = 217), respectively. In 70% of the broods, both females produced larvae, and in 70%, both males inseminated one or both females. The male and female providing longer care, usually the larger of each sex, were the mother and father of most larvae (50-100%).
Retroviral infection coupled with tissue transplantation limits gene transfer in the chicken embryo.
Authors: Fekete D M , Cepko C L
Published: May 2006

Gene transfer into early embryos is a powerful methodology for unraveling the molecular bases of developmental processes. One can attempt to minimize widespread effects of an exogenous gene by using tissue- or region-specific promoters in the few instances where they are available. We have developed a method that bypasses the requirement for specific targeting sequences to achieve regionally restricted gene transfer. Intraspecific chimeras have been created by transplantation of restricted portions of a chicken embryo from a donor strain to a host strain. The donor cells are infectable with a recombinant retroviral vector that carries the exogenous gene, whereas the host cells are not. We have demonstrated the feasibility of this approach using a histochemically distinct reporter gene, human placental alkaline phosphatase. The expression of retrovirally transduced alkaline phosphatase was limited to a transplanted hemiprosencephalon (forebrain and eye) in embryonic chickens. This technique can be applied to many other organ systems during avian embryogenesis to test the function(s) of molecules that are normally controlled through spatial and/or temporal regulation, such as many of the growth factor receptors or homeobox-containing proteins.
Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3'-->5' exonuclease activity.
Authors: Frey M W , Nossal N G , Capson T L , Benkovic S J
Published: May 2006

Bacteriophage T4 DNA polymerase has a proofreading 3'-->5' exonuclease that plays an important role in maintaining the accuracy of DNA replication. We have constructed a T4 DNA polymerase deficient in this exonuclease by converting Asp-219 to Ala. The exonuclease activity of the mutant T4 DNA polymerase has been reduced by a factor of at least 10(7), but it retains a polymerase activity whose kinetic parameters, kcat, Kd DNA, and Kd dATP, are very close to those of the wild-type enzyme. Bacteriophage T4 with the mutant polymerase gene has a markedly increased mutation frequency. Asp-219 in T4 DNA polymerase is within a sequence similar to those surrounding Asp residues previously shown to be essential for the exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I (Asp-424), bacteriophage phi 29 DNA polymerase (Asp-66), and Saccharomyces cerevisiae DNA polymerase delta (Asp-405). Thus, these studies support the proposal that there are similar sequences in the active sites for the proofreading exonucleases of these and related DNA polymerases.
Technique for in situ measurement of calcium in intracellular inositol 1,4,5-trisphosphate-sensitive stores using the fluorescent indicator mag-fura-2.
Authors: Hofer A M , Machen T E
Published: May 2006

Stimulation of cells with calcium-mobilizing agonists frequently results in inositol 1,4,5-trisphosphate (InsP3)-mediated discharge of Ca from an internal store. We report here a technique for directly monitoring Ca within this and other stores in gastric epithelial cells. This technique takes advantage of the propensity of the acetoxymethyl ester derivative of the fluorescent dye mag-fura-2 (which is sensitive to Ca concentrations above 5 microM) to accumulate in subcellular compartments where it can report changes in the free Ca concentration. Intact dye-loaded cells responded to cholinergic stimulation with a decrease in the 350 nm/385 nm excitation ratio, as measured in individual cells with a digital imaging microscope, consistent with reduced Ca concentration in one or more cellular compartments. When cells were permeabilized with digitonin and incubated in an "intracellular buffer," the cytoplasmic dye was released, leaving the mag-fura-2 in the internal store. InsP3 caused the ratio from the trapped indicator to decrease (i.e., Ca was released) in a dose-dependent manner, and this effect was blocked by the InsP3 receptor antagonist heparin. Ca sequestration into the internal store was ATP-dependent, and reuptake into the InsP3-sensitive pool was blocked by thapsigargin, a specific inhibitor of the Ca-ATPase of the internal store. We used this technique to investigate the role of Cl on the release and reloading of the InsP3-sensitive internal store and found that Ca uptake was reduced in Cl-free solutions, suggesting an important function for Cl in the refilling of this pool.
Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration.
Authors: Saito K , Elce J S , Hamos J E , Nixon R A
Published: May 2006

Calcium-activated neutral proteinases (CANPs or calpains) are believed to be key enzymes in intracellular signaling cascades and potential mediators of calcium-induced neuronal degeneration. To investigate their involvement in Alzheimer disease, we identified three isoforms of muCANP (calpain I) in human postmortem brain corresponding to an 80-kDa precursor and two autolytically activated isoforms (78 and 76 kDa). As an index of changes in the in vivo activity of muCANP in Alzheimer disease, the ratio of the 76-kDa activated isoform of muCANP to its 80-kDa precursor was measured by immunoassay in selected brain regions from 22 individuals with Alzheimer disease and 18 normal controls. This muCANP activation ratio was elevated 3-fold in the prefrontal cortex from patients with Alzheimer disease but not from patients with Huntington disease. The activation ratio was also significantly elevated, but to a lesser degree, in brain regions where Alzheimer pathology is milder and has not led to overt neuronal degeneration. These findings indicate that muCANP activation is not simply a consequence of cellular degeneration but may be associated with dysfunction in many neurons before gross structural changes occur. The known influences of CANPs on cytoskeleton and membrane dynamics imply that persistent CANP activation may contribute to neurofibrillary pathology and abnormal amyloid precursor protein processing prior to causing synapse loss or cell death in the most vulnerable neuronal populations. Pharmacological modulation of the CANP system may merit consideration as a potential therapeutic strategy in Alzheimer disease.
Cholesterol deprivation affects the fluorescence properties of a ceramide analog at the Golgi apparatus of living cells.
Authors: Martin O C , Comly M E , Blanchette-Mackie E J , Pentchev P G , Pagano R E
Published: May 2006

Previous studies have established that a fluorescent analog of ceramide, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)] -6-aminohexanoyl-D-erythro-sphingosine (C6-NBD-Cer), is a vital stain for the Golgi apparatus and a useful tool for studying the sorting and transport of sphingolipids along the secretory pathway in animal cells. Here, we examine the effects of various culture conditions on labeling of the Golgi apparatus of human skin fibroblasts by C6-NBD-Cer and demonstrate that cholesterol deprivation affects the fluorescence properties of the probe at this organelle. Labeling of the Golgi apparatus by C6-NBD-Cer was dramatically reduced in cells grown in medium containing lipoprotein-deficient serum compared to cells grown in medium containing normal serum. Quantitative fluorescence microscopy showed that this apparent reduction in labeling resulted from accelerated photo-bleaching of the fluorescent analog. C6-NBD-Cer labeling of the Golgi apparatus was restored in cholesterol-deprived cells by stimulating endogenous cholesterol biosynthesis with mevalonic acid or by adding exogenous nonlipoprotein cholesterol or low density lipoprotein to the culture medium. In addition, when cells grown in medium containing normal serum were perforated and treated with cholesterol oxidase, an apparent reduction in labeling resulted, further implicating an intracellular pool of cholesterol in the potentiation of C6-NBD-Cer fluorescence. These results demonstrate that cytological studies using C6-NBD-Cer are affected by cholesterol deprivation and suggest that this fluorescent lipid may be used to monitor cholesterol at the Golgi apparatus of living cells.
Rate variation of protein and mitochondrial DNA evolution as revealed by sea urchins separated by the isthmus of Panama.
Authors: Bermingham E , Lessios H A
Published: May 2006

Acceptance of the rough constancy of rates of molecular evolution, averaged over tens of millions of years, is widely used to date the splitting between taxa. However, for the study of speciation a hypothesis of rough constancy over tens of millions of years is of little use. In order to date the splitting of congeneric species within defined ranges of uncertainty, we need to know the variation of evolutionary rates over shorter periods of time. Such estimates of uncertainty are particularly useful if they apply to techniques of molecular comparisons that lend themselves to the assessment of intraspecific variation. We have measured protein divergence by electrophoresis and mitochondrial DNA differentiation by restriction fragment length polymorphism analysis in three pairs of sea urchin species believed to have resulted from the simultaneous fragmentation of ranges of marine species by the emergence of the Isthmus of Panama, about 3 million years ago. Transisthmian isozyme divergence in these pairs varies by an order of magnitude; mitochondrial DNA divergence, on the other hand, is equivalent in all pairs, suggesting that this molecule, assayed by endonucleases, can provide fairly accurate estimates of times since separation in the 3-million-year range.