bluuebunny/crossref_metadata_2025 huggingface dataset:
The origin and efficient derivation of embryonic stem cells in the mouse
Authors: Brook F. A. , Gardner R. L.
Published: July 2002

By explanting tissues isolated microsurgically from implanting strain 129 mouse blastocysts individually on STO feeder cells we have established that embryonic stem (ES) cells originate from the epiblast (primitive ectoderm). Isolated early epiblasts yielded ES cell lines at a substantially higher frequency than intact blastocysts regardless of whether they were explanted whole or as strictly single-cell suspensions. When explanted from delayed-implanting 129 blastocysts, epiblasts gave lines consistently in 100% of cases. If primary embryonic fibroblasts rather than STO cells were used as feeders, germline-competent ES cell lines were obtained readily from epiblasts of delayed-implanting blastocysts of several hitherto refractory strains, particularly when recombinant leukemia inhibitory factor was included in the medium during the initial period of culture. Because lines were obtained from the nonpermissive CBA/Ca strain at a rate of up to 56%, this approach to the derivation of germline-competent ES cell lines may not only prove generic for the mouse but also worth pursuing in other species of mammal.
Blocking activator protein-1 activity, but not activating retinoic acid response element, is required for the antitumor promotion effect of retinoic acid
Authors: Huang Chuanshu , Ma Wei-Ya , Dawson Marcia I. , Rincon Mercedes , Flavell Richard A. , Dong Zigang
Published: July 2002

Retinoic acid is one of the most promising drugs for chemotherapy and chemoprevention of cancer. Either blocking activator protein-1 (AP-1) activity or activating retinoic acid response element (RARE) have been proposed to be responsible for its antitumor activity. However, evidence for this hypothesis is lacking
in vivo
studies. To address this issue, we used an AP-1-luciferase transgenic mouse as a carcinogenesis model and new synthetic retinoids that are either selective inhibitors of AP-1 activation or selective activators of the RARE. The results showed that the SR11302, an AP-1 inhibition-specific retinoid, and other AP-1 inhibitors such as trans-retinoic acid and fluocinolone acetonide, markedly inhibit both 12-
O
-tetradecanoylphorbol-13-acetate-induced papilloma formation and AP-1 activation in 7,12-dimethyl benz(a)anthracene-initiated mouse skin (
P
< 0.05). In contrast, repeated applications of SR11235, a retinoid with RARE transactivating activity, but devoid of AP-1 inhibiting effect, did not cause significant inhibition of papilloma formation and AP-1 activation (
P
> 0.05). These results provide the first
in vivo
evidence that the antitumor effect of retinoids is mediated by blocking AP-1 activity, but not by activation of RARE.
Neuregulins stimulate the functional expression of Ca
2+
-activated K
+
channels in developing chicken parasympathetic neurons
Authors: Subramony Priya , Dryer Stuart E.
Published: July 2002

The developmental expression of macroscopic Ca
2+
-activated K
+
currents (
I
K[Ca]
) in chicken ciliary ganglion (CG) neurons is dependent in part on trophic factors released from preganglionic nerve terminals. Neuregulins are expressed in the preganglionic neurons that innervate the chicken CG and are therefore plausible candidates for this activity. Application of 1 nM β1-neuregulin peptide for 12 hr evokes a large (7- to 10-fold) increase in
I
K[Ca]
in embryonic day 9 CG neurons, even in the presence of a translational inhibitor. A similar posttranslational effect is produced by high concentrations (10 nM) of epidermal growth factor and type α transforming growth factor but not by 10 nM α2-neuregulin peptide or by neurotrophins at 40 ng⋅ml
−1
. β1-neuregulin treatment for 12 hr also confers Ca
2+
sensitivity onto large-conductance (285 pS) K
+
channels observed in inside–out patches. β-Neuregulins have no effect on voltage-activated Ca
2+
currents of CG neurons. These data support the hypothesis that β-neuregulins mediate the trophic effects of preganglionic nerve terminals on the electrophysiological differentiation of developing CG neurons.
RAB22 and RAB163/mouse BRCA2: Proteins that specifically interact with the RAD51 protein
Authors: Mizuta Ryushin , LaSalle Janine M. , Cheng Hwei-Ling , Shinohara Akira , Ogawa Hideyuki , Copeland Neal , Jenkins Nancy A. , Lalande Marc , Alt Frederick W.
Published: July 2002

The human RAD51 protein is a homologue of the bacteria RecA and yeast RAD51 proteins that are involved in homologous recombination and DNA repair. RAD51 interacts with proteins involved in recombination and also with tumor suppressor proteins p53 and breast cancer susceptibility gene 1 (BRCA1). We have used the yeast two-hybrid system to clone murine cDNA sequences that encode two RAD51-associated molecules, RAB22 and RAB163. RAB163 encodes the C-terminal portion of mouse BRCA2, the homologue of the second breast cancer susceptibility gene protein in humans, demonstrating an
in vitro
association between RAD51 and BRCA2. RAB22 is a novel gene product that also interacts with RAD51
in vitro
. To detect RAD51 interactions
in vivo
, we developed a transient nuclear focus assay that was used to demonstrate a complete colocalization of RAB22 with RAD51 in large nuclear foci.
B cell receptor-associated protein α4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A
Authors: Murata Kohei , Wu Jun , Brautigan David L.
Published: July 2002

Recently, TAP42 was isolated as a high copy suppressor ofsit4−, a yeast phosphatase related to protein phosphatase 2A (PP2A). TAP42 is related to the murine α4 protein, which was discovered independently by its association with Ig-α in the B cell receptor complex. Herein we show that a glutathioneS-transferase (GST)–α4 fusion protein bound the catalytic subunit (C) of human PP2A from monomeric or multimeric preparations of PP2A in a “pull-down” assay. In an overlay assay, the GST–α4 protein bound to the phosphorylated and unphosphorylated forms of C that were separated in two-dimensional gels and immobilized on filters. The results show direct and exclusive binding of α4 to C. This is unusual because all known regulatory B subunits, or tumor virus antigens, bind stably only to the AC dimer of PP2A. The α4–C form of PP2A had an increased activity ratio compared with the AC form of PP2A when myelin basic protein phosphorylated by mitogen-activated protein kinase and phosphorylaseawere used as substrates. Recombinant α4 cleaved from GST was phosphorylated by p56lcktyrosine kinase and protein kinase C. A FLAG-tagged α4 expressed in COS7 cells was recovered as a protein containing phosphoserine and coimmunoprecipitated with the C but not the A subunit of PP2A. Treatment of cells with rapamycin prevented the association of PP2A with FLAG-α4. The results reveal a novel heterodimer α4–C form of PP2A that may be involved in rapamycin-sensitive signaling pathways in mammalian cells.
Heme oxygenase 1 is required for mammalian iron reutilization
Authors: Poss Kenneth D. , Tonegawa Susumu
Published: July 2002

The majority of iron for essential mammalian biological activities such as erythropoiesis is thought to be reutilized from cellular hemoproteins. Here, we generated mice lacking functional heme oxygenase 1 (Hmox1; EC
1.14.99.3
), which catabolizes heme to biliverdin, carbon monoxide, and free iron, to assess its participation in iron homeostasis. Hmox1-deficient adult mice developed an anemia associated with abnormally low serum iron levels, yet accumulated hepatic and renal iron that contributed to macromolecular oxidative damage, tissue injury, and chronic inflammation. Our results indicate that Hmox1 has an important recycling role by facilitating the release of iron from hepatic and renal cells, and describe a mouse model of human iron metabolic disorders.
p53-dependent regulation of
MDR1
gene expression causes selective resistance to chemotherapeutic agents
Authors: Thottassery Jaideep V. , Zambetti Gerard P. , Arimori Kazuhiko , Schuetz Erin G. , Schuetz John D.
Published: July 2002

Loss of functional p53 paradoxically results in either increased or decreased resistance to chemotherapeutic drugs. The inconsistent relationship between p53 status and drug sensitivity may reflect p53’s selective regulation of genes important to cytotoxic response of chemotherapeutic agents. We reasoned that the discrepant effects of p53 on chemotherapeutic cytotoxicity is due to p53-dependent regulation of the multidrug resistance gene (
MDR1
) expression in tumors that normally express
MDR1
. To test the hypothesis that wild-type p53 regulates the endogenous
mdr1
gene we stably introduced a trans-dominant negative (TDN) p53 into rodent H35 hepatoma cells that express P-glycoprotein (Pgp) and have wild-type p53. Levels of Pgp and
mdr1a
mRNA were markedly elevated in cells expressing TDN p53 and were linked to impaired p53 function (both transactivation and transrepression) in these cells. Enhanced
mdr1a
gene expression in the TDN p53 cells was not secondary to
mdr1
gene amplification and Pgp was functional as demonstrated by the decreased uptake of vinblastine. Cytotoxicity assays revealed that the TDN p53 cell lines were selectively insensitive to Pgp substrates. Sensitivity was restored by the Pgp inhibitor reserpine, demonstrating that only drug retention was the basis for loss of drug sensitivity. Similar findings were evident in human LS180 colon carcinoma cells engineered to overexpress TDN p53. Therefore, the p53 inactivation seen in cancers likely leads to selective resistance to chemotherapeutic agents because of up-regulation of
MDR1
expression.
Src-induced activation of inducible T cell kinase (ITK) requires phosphatidylinositol 3-kinase activity and the Pleckstrin homology domain of inducible T cell kinase
Authors: August Avery , Sadra Ali , Dupont Bo , Hanafusa Hidesaburo
Published: July 2002

The Tec family of tyrosine kinases are involved in signals emanating from cytokine receptors, antigen receptors, and other lymphoid cell surface receptors. One family member, ITK (inducible T cell kinase), is involved in T cell activation and can be activated by the T cell receptor and the CD28 cell surface receptor. This stimulation of tyrosine phosphorylation and activation of ITK can be mimicked by the Src family kinase Lck. We have explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that coexpression of ITK and Src results in increased membrane association, tyrosine phosphorylation and activation of ITK, which could be blocked by inhibitors of the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase) as well as overexpression of the p85 subunit of PI 3-kinase. Removal of the Pleckstrin homology domain (PH) of ITK resulted in a kinase that could no longer be induced to localize to the membrane or be activated by Src. The PH of ITK was also able to bind inositol phosphates phosphorylated at the D3 position. Membrane targeting of ITK without the PH recovered its ability to be activated by Src. These results suggest that ITK can be activated by a combination of Src and PI 3-kinase.
Molecular immune responses of the mosquito
Anopheles gambiae
to bacteria and malaria parasites
Authors: Dimopoulos George , Richman Adam , Müller Hans-Michael , Kafatos Fotis C.
Published: July 2002

Immune responses of the malaria vector mosquito
Anopheles gambiae
were monitored systematically by the induced expression of five RNA markers after infection challenge. One newly isolated marker encodes a homologue of the moth Gram-negative bacteria-binding protein (GNBP), and another corresponds to a serine protease-like molecule. Additional previously described markers that respond to immune challenge encode the antimicrobial peptide defensin, a putative galactose lectin, and a putative serine protease. Specificity of the immune responses was indicated by differing temporal patterns of induction of specific markers in bacteria-challenged larvae and adults, and by variations in the effectiveness of different microorganisms and their components for marker induction in an immune-responsive cell line. The markers exhibit spatially distinct patterns of expression in the adult female mosquito. Two of them are highly expressed in different regions of the midgut, one in the anterior and the other in the posterior midgut. Marker induction indicates a significant role of the midgut in insect innate immunity. Immune responses to the penetration of the midgut epithelium by a malaria parasite occur both within the midgut itself and elsewhere in the body, suggesting an immune-related signaling process.
Atomic structure of a thermostable subdomain of HIV-1 gp41
Authors: Tan Kemin , Liu Jin-huan , Wang Jia-huai , Shen Steven , Lu Min
Published: July 2002

Infection by HIV-1 involves the fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. The HIV-1 envelope glycoprotein that mediates fusion consists of the surface subunit gp120 and the transmembrane subunit gp41. gp120 directs virion attachment to the cell–surface receptors, and gp41 then promotes viral–cell membrane fusion. A soluble, α-helical, trimeric complex within gp41 composed of N-terminal and C-terminal extraviral segments has been proposed to represent the core of the fusion-active conformation of the HIV-1 envelope. A thermostable subdomain denoted N34(L6)C28 can be formed by the N-34 and C-28 peptides connected by a flexible linker in place of the disulfide-bonded loop region. Three-dimensional structure of N34(L6)C28 reveals that three molecules fold into a six-stranded helical bundle. Three N-terminal helices within the bundle form a central, parallel, trimeric coiled coil, whereas three C-terminal helices pack in the reverse direction into three hydrophobic grooves on the surface of the N-terminal trimer. This thermostable subdomain displays the salient features of the core structure of the isolated gp41 subunit and thus provides a possible target for therapeutics designed selectively to block HIV-1 entry.
Ultrastructural localization of zinc transporter-3 (ZnT-3) to synaptic vesicle membranes within mossy fiber boutons in the hippocampus of mouse and monkey
Authors: Wenzel H. Jürgen , Cole Toby B. , Born Donald E. , Schwartzkroin Philip A. , Palmiter Richard D.
Published: July 2002

Zinc transporter-3 (ZnT-3), a member of a growing family of mammalian zinc transporters, is expressed in regions of the brain that are rich in histochemically reactive zinc (as revealed by the Timm’s stain), including entorhinal cortex, amygdala, and hippocampus. ZnT-3 protein is most abundant in the zinc-enriched mossy fibers that project from the dentate granule cells to hilar and CA3 pyramidal neurons. We show here by electron microscopy that ZnT-3 decorates the membranes of all clear, small, round synaptic vesicles (SVs) in the mossy fiber boutons of both mouse and monkey. Furthermore, up to 60–80% of these SVs contain Timm’s-stainable zinc. The coincidence of ZnT-3 on the membranes of SVs that accumulate zinc, and its homology with known zinc transporters, suggest that ZnT-3 is responsible for the transport of zinc into SVs, and hence for the ability of these neurons to release zinc upon excitation.
Relative mutation rates at di-, tri-, and tetranucleotide microsatellite loci
Authors: Chakraborty Ranajit , Kimmel Marek , Stivers David N. , Davison Leslea J. , Deka Ranjan
Published: July 2002

Using the generalized stepwise mutation model, we propose a method of estimating the relative mutation rates of microsatellite loci, grouped by the repeat motif. Applying ANOVA to the distributions of the allele sizes at microsatellite loci from a set of populations, grouped by repeat motif types, we estimated the effect of population size differences and mutation rate differences among loci. This provides an estimate of motif-type-specific mutation rates up to a multiplicative constant. Applications to four different sets of di-, tri-, and tetranucleotide loci from a number of human populations reveal that, on average, the non-disease-causing microsatellite loci have mutation rates inversely related to their motif sizes. The dinucleotides appear to have mutation rates 1.5–2 times higher than the tetranucleotides, and the non-disease-causing trinucleotides have mutation rates intermediate between the di- and tetranucleotides. In contrast, the disease-causing trinucleotides have mutation rates 3.9–6.9 times larger than the tetranucleotides. Comparison of these estimates with the direct observations of mutation rates at microsatellites indicates that the earlier suggestion of higher mutation rates of tetranucleotides in comparison with the dinucleotides may stem from a nonrandom sampling of tetranucleotide loci in direct mutation assays.
Characterization of two scleroderma autoimmune antigens that copurify with human ribonuclease P
Authors: Eder Paul S. , Kekuda Ramesh , Stolc Viktor , Altman Sidney
Published: July 2002

Human RNase P has been purified more than 2000-fold from HeLa cells. In addition to the RNA component, H1 RNA, polypeptides of molecular masses 14, 20, 25, 30, 38, and 40 kDa copurify with the enzyme activity. Sera from two different patients with the autoimmune disease scleroderma were used to immunodeplete human RNase P activity. These same sera cross-reacted on immunoblots with two of the copurifying polypeptides, p30 and p38, whereas an autoimmune serum that does not immunodeplete RNase P activity did not react with these proteins. Peptide fragments derived from purified p30 and p38 facilitated the molecular cloning and sequencing of cDNAs coding for these two polypeptides, which are now designated as Rpp30 and Rpp38, respectively.RPP38cDNA encodes a polypeptide that may be identical to a previously identified antigen of ≈40 kDa, which is immunoprecipitated by Th and To autoimmune antisera, and that has been implicated as a protein subunit of human RNase P by virtue of its ability to bind to H1 RNAin vitro. The second autoimmune antigen, Rpp30, as such, has not been described previously.
The strength of indirect selection on female mating preferences
Authors: Kirkpatrick M. , Barton N. H.
Published: July 2002

An important but controversial class of hypotheses concerning the evolution of female preferences for extreme male mating displays involves “indirect selection.” Even in the absence of direct fitness effects, preference for males with high overall fitness can spread via a genetic correlation that develops between preference alleles and high fitness genotypes. Here we develop a quantitative expression for the force of indirect selection that (i) applies to any female mating behavior, (ii) is relatively insensitive to the underlying genetics, and (iii) is based on measurable quantities. In conjunction with the limited data now available, it suggests that the evolutionary force generated by indirect selection on preferences is weak in absolute terms. This finding raises the possibility that direct selection on preference genes may often be more important than indirect selection, but more data on the quantities identified by our model and on direct selection are needed to decide the question.
In vivoaccumulation of the same anti-melanoma T cell clone in two different metastatic sites
Authors: Hishii Makoto , Andrews David , Boyle Lenora A. , Wong Johnson T. , Pandolfi Franco , van den Elsen Peter J. , Kurnick James T.
Published: July 2002

In a patient with progressing metastatic melanoma, we showed that the same autologous tumor-cytolytic CD8+tumor infiltrating lymphocyte (TIL) clone accumulated in two separate metastatic sites. This clone, which represented three of eight independently derived clones from a tumor deposit on the skin of the abdomen, also represented two of eight clones derived from a skin lesion on the shoulder. This clone could be identified by its use of a unique TCRBV2-nD1n-J1S6 sequence, and could also be detected by single-stranded conformational polymorphism (SSCP) as the dominant TCRBV2-expressing clone among CD8+TILs propagated from both shoulder and abdominal lesions. Using SSCP analysis, we also demonstrated that this clone was dominant in the fresh tumor tissue and in all TILs in which CD8+were strongly represented, including several separate but parallel cultures. The SSCP pattern for this clone was not apparent among CD4+TILs or CD8+peripheral blood mononuclear cells. The SSCP analysis of the tumor tissue prior toin vitroculture is an indication that the selection for this anti-tumor cytotoxic T cell clone was a reflection of itsin vivoaccumulation. Thus, we provide evidence that melanomas are immunogenic and able to select for cytotoxic anti-tumor-specific TIL clones that are expandedin vivoand can circulate to accumulate in different tumor sites. However, because these clones were isolated from progressing tumor metastases, the accumulation of these specific cytotoxic T cells was not sufficient to contain tumor growth.
UV-induced mutagenesis of human
p53
in a vector replicated in
Saccharomyces cerevisiae
Authors: Moshinsky Deborah J. , Wogan Gerald N.
Published: July 2002

Mutation of the
p53
tumor suppressor gene is the most common genetic alteration identified to date in human cancers. Similarities of
p53
mutations found in human cancers with those induced in experimental systems have been interpreted as evidence supporting a causative role for environmental carcinogens in certain tumor types. We have developed and validated a method for generation of mutation spectra and measurement of mutation frequency directly on human
p53
cDNA in a vector following treatment with mutagens and replication in yeast. Mutants that had lost the DNA binding/transcription activation function of p53 were detected by yeast colony color, isolated, and sequenced. UV light was used to characterize and validate the system, and a dose-dependent increase in mutation frequency was seen following exposure of the plasmid to increasing doses of UV, resulting in an 18-fold increase over the spontaneous frequency (3.2 × 10
−4
) at the highest level tested (300 J/m
2
). Sequence analysis of
p53
in the mutants revealed that the types of mutations induced were similar to those obtained in previous studies of UV mutagenesis in other model systems, and the types and positions of mutations were also similar to those found in human skin tumors. This experimental system will be useful in further evaluation of the importance of environmental agents as risk factors for cancer.
Specific chromosomal imbalances in human papillomavirus-transfected cells during progression toward immortality
Authors: Solinas-Toldo Sabina , Dürst Matthias , Lichter Peter
Published: July 2002

High risk human papillomaviruses (HPVs) known to be closely associated with cervical cancer, such as HPV16 and HPV18, have the potential to immortalize human epithelial cells in culture. Four lines of HPV-transfected keratinocytes were analyzed by comparative genomic hybridization at different time points after transfection. A number of chromosomal imbalances was found to be highly characteristic for the cultures progressing toward immortality. Whereas several of these were new and previously not found as recurrent aberrations in cervical tumors, some were identical to chromosomal changes observed during cervical carcinogenesis. The data put new emphasis on the studied cell system as a relevant model for HPV-induced pathogenesis.
Structural basis for heterogeneous kinetics: Reengineering the hairpin ribozyme
Authors: Esteban José A. , Walter Nils G. , Kotzorek Gerd , Heckman Joyce E. , Burke John M.
Published: July 2002

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inactive form of the complex was trapped by the addition of T4 RNA ligase to a cleavage reaction, resulting in covalent linkage of the 5′ end of the substrate to the 3′ end of the ribozyme and in selective and quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two-way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.
Abnormal transport along the lysosomal pathway in Mucolipidosis, type IV disease
Authors: Chen Chii-Shiarng , Bach Gideon , Pagano Richard E.
Published: July 2002

Mucolipidosis, type IV (ML-IV) is an autosomal recessive storage disease that is characterized by lysosomal accumulation of sphingolipids, phospholipids, and acid mucopolysaccharides. Unlike most other storage diseases, the lysosomal hydrolases participating in the catabolism of the stored molecules appear to be normal. In the present study, we examined the hypothesis that the ML-IV phenotype might arise from abnormal transport along the lysosomal pathway. By using various markers for endocytosis, we found that plasma membrane internalization and recycling were nearly identical in ML-IV and normal fibroblasts. A fluorescent analog of lactosylceramide (LacCer) was used to study plasma membrane lipid internalization and subsequent transport. Lipid internalization at 19°C was similar in both cell types; however, 40–60 min after raising the temperature to 37°C, the fluorescent lipid accumulated in the lysosomes of ML-IV cells but was mainly concentrated at the Golgi complex of normal fibroblasts. Biochemical studies demonstrated that at these time points, hydrolysis of the lipid analog was minimal (∼7%) in both cell types. A fluorescence ratio imaging assay was developed to monitor accumulation of fluorescent LacCer in the lysosomes and showed that the apparent concentration of the lipid increased more rapidly and to a greater extent in ML-IV cells than in normal fibroblasts. By 60 min, LacCer apparently decreased in the lysosomes of normal fibroblasts but not in ML-IV cells, suggesting that lipid efflux from the lysosomes was also impaired. These results demonstrate that there is a defect in ML-IV fibroblasts that affects membrane sorting and/or late steps of endocytosis.
Precisely full length, circularizable, complementary RNA: An infectious form of potato spindle tuber viroid
Authors: Feldstein P. A. , Hu Y. , Owens R. A.
Published: July 2002

The replication of many viral and subviral pathogens as well as the amplification of certain cellular genes proceeds via a rolling circle mechanism. For potato spindle tuber (PSTVd) and related viroids, the possible role of a circular (−)strand RNA as a template for synthesis of (+)strand progeny is unclear. Infected plants appear to contain only multimeric linear (−)strand RNAs, and attempts to initiate infection with multimeric (−)PSTVd RNAs generally have failed. To examine critically the infectivity of monomeric (−)strand viroid RNAs, we have developed a ribozyme-based expression system for the production of precisely full length (−)strand RNAs whose termini are capable of undergoing facile circularization
in vitro
. Mechanical inoculation of tomato seedlings with electrophoretically purified (−)PSTVd RNA led to a small fraction of plants becoming infected whereas parallel assays with an analogous tomato planta macho viroid (−)RNA resulted in a much larger fraction of infected plants. Ribozyme-mediated production of (−)PSTVd RNA in transgenic plants led to the appearance of monomeric circular (−)PSTVd RNA and large amounts of (+)PSTVd progeny. No monomeric circular (−)PSTVd RNA could be detected in naturally infected plants by using either ribonuclease protection or electrophoresis under partially denaturing conditions. Although not a component of the normal replicative pathway, precisely full length (−)PSTVd RNA appears to contain all of the structural and regulatory elements necessary for initiation of viroid replication.
Ozonolysis for selectively depolymerizing polysaccharides containing β-d-aldosidic linkages
Authors: Wang Ying , Hollingsworth Rawle I. , Kasper Dennis L.
Published: July 2002

The depolymerization of polysaccharides, particularly those containing acid-sensitive components, into intact constituent repeating units can be very difficult. We describe a method using ozonolysis for depolymerizing polysaccharides containing β-d-aldosidic linkages into short-chain polysaccharides and oligosaccharides. This method is carried out on polysaccharides that have been fully acetylated whereby β-d-aldosidic linkages are selectively oxidized by ozone to form esters, from which the polysaccharides are subsequently cleaved with a nucleophile. Ozone oxidation of aldosidic linkages proceeds under strong stereoelectronic control, and reaction rates depend on the conformations of glycosidic linkages. Thus, β-d-aldosidic linkages with different conformations can have very different reaction rates even in the absence of substantial chemical differences. These rate differences allowed for very high selectivity in cleaving β-d-linkages of polysaccharides. Several polysaccharides from group BStreptococcusand other bacterial species were selectively depolymerized with this method. The repeating units of the group BStreptococcuspolysaccharides all contain an acid-sensitive sialic acid residue in a terminal position on a side chain and several β-d-residues including galactose, glucose, andN-acetylglucosamine; however, with each polysaccharide, one type of linkage was more reactive than others. Selective cleavage of the most sensitive linkage occurs randomly throughout the polymer chain, yielding fragments of controllable and narrowly distributed sizes and the same repeating-unit structure. The average size of the molecules decreases exponentially, and desired sizes can be obtained by stopping the reaction at appropriate time points. With this method the labile sialic acid residue was not affected.
Ligands for peroxisome proliferator-activated receptorγ and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells
in vitro
and in BNX mice
Authors: Elstner Elena , Müller Carsten , Koshizuka Kozo , Williamson Elizabeth A. , Park Dorothy , Asou Hiroya , Shintaku Peter , Said Jonathan W. , Heber David , Koeffler H. Phillip
Published: July 2002

Induction of differentiation and apoptosis in cancer cells through ligands of nuclear hormone receptors (NHRs) is a novel and promising approach to cancer therapy. All-
trans
-retinoic acid (ATRA), an RA receptor-specific NHR ligand, is now used for selective cancers. The NHR, peroxisome proliferator-activated receptor γ (PPARγ) is expressed in breast cancer cells. Activation of PPARγ through a synthetic ligand, troglitazone (TGZ), and other PPARγ-activators cause inhibition of proliferation and lipid accumulation in cultured breast cancer cells. TGZ (10
−5
M, 4 days) reversibly inhibits clonal growth of MCF7 breast cancer cells and the combination of TGZ (10
−5
M) and ATRA (10
−6
M, 4 days) synergistically and irreversibly inhibits growth and induces apoptosis of MCF7 cells, associated with a dramatic decrease of their bcl-2 protein levels. Similar effects are noted with
in vitro
cultured breast cancer tissues from patients, but not with normal breast epithelial cells. The observed apoptosis mediated by TGZ and ATRA may be related to the striking down-regulation of bcl-2, because forced over-expression of bcl-2 in MCF7 cells cultured with TGZ and ATRA blocks their cell death. TGZ significantly inhibits MCF7 tumor growth in triple immunodeficient mice. Combined administration of TGZ and ATRA causes prominent apoptosis and fibrosis of these tumors without toxic effects on the mice. Taken together, this combination may provide a novel, nontoxic and selective therapy for human breast cancers.
DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone
Authors: Balasubramanian Bhavani , Pogozelski Wendy K. , Tullius Thomas D.
Published: July 2002

Despite extensive study, there is little experimental information available as to which of the deoxyribose hydrogen atoms of duplex DNA reacts most with the hydroxyl radical. To investigate this question, we prepared a set of double-stranded DNA molecules in which deuterium had been incorporated specifically at each position in the deoxyribose of one of the four nucleotides. We then measured deuterium kinetic isotope effects on the rate of cleavage of DNA by the hydroxyl radical. These experiments demonstrate that the hydroxyl radical reacts with the various hydrogen atoms of the deoxyribose in the order 5′ H > 4′ H > 3′ H ≈ 2′ H ≈ 1′ H. This order of reactivity parallels the exposure to solvent of the deoxyribose hydrogens. Our work therefore reveals the structural basis of the reaction of the hydroxyl radical with DNA. These results also provide information on the mechanism of DNA damage caused by ionizing radiation as well as atomic-level detail for the interpretation of hydroxyl radical footprints of DNA-protein complexes and chemical probe experiments on the structure of RNA and DNA in solution.
In vivo
activities of GroEL minichaperones
Authors: Chatellier Jean , Hill Fergal , Lund Peter A. , Fersht Alan R.
Published: July 2002

Fragments encompassing the apical domain of GroEL, called minichaperones, facilitate the refolding of several proteins
in vitro
without requiring GroES, ATP, or the cage-like structure of multimeric GroEL. We have identified the smallest minichaperone that is active
in vitro
in chaperoning the refolding of rhodanese and cyclophilin A: GroEL(193–335). This finding raises the question of whether the minichaperones are active under more stringent conditions
in vivo
. The smallest minichaperones complement two temperature-sensitive
Escherichia coli groEL
alleles,
EL44
and
EL673,
at 43°C. Although they cannot replace GroEL in cells in which the chromosomal
groEL
gene has been deleted by P1 transduction, GroEL(193–335) enhances the colony-forming ability of such cells when limiting amounts of GroEL are expressed from a tightly regulated plasmid. Surprisingly, we found that overexpression of GroEL prevents plaque formation by bacteriophage λ and inhibits replication of the λ origin-dependent plasmid, Lorist6. The minichaperones also inhibit Lorist6 replication, but less markedly. The complex quaternary structure of GroEL, its central cavity, and the structural allosteric changes that take place on the binding of nucleotides and GroES are not essential for all of its functions
in vivo
.
Replicational and transcriptional selection on codon usage in
Borrelia burgdorferi
Authors: McInerney James O.
Published: July 2002

With more than 10 fully sequenced, publicly available prokaryotic genomes, it is now becoming possible to gain useful insights into genome evolution. Before the genome era, many evolutionary processes were evaluated from limited data sets and evolutionary models were constructed on the basis of small amounts of evidence. In this paper, I show that genes on the
Borrelia burgdorferi
genome have two separate, distinct, and significantly different codon usages, depending on whether the gene is transcribed on the leading or lagging strand of replication. Asymmetrical replication is the major source of codon usage variation. Replicational selection is responsible for the higher number of genes on the leading strands, and transcriptional selection appears to be responsible for the enrichment of highly expressed genes on these strands. Replicational–transcriptional selection, therefore, has an influence on the codon usage of a gene. This is a new paradigm of codon selection in prokaryotes.
A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl–tRNA synthetase in
Arabidopsis thaliana
Authors: Menand B. , Maréchal-Drouard L. , Sakamoto W. , Dietrich A. , Wintz H.
Published: July 2002

One-fifth of the tRNAs used in plant mitochondrial translation is coded for by chloroplast-derived tRNA genes. To understand how aminoacyl–tRNA synthetases have adapted to the presence of these tRNAs in mitochondria, we have cloned an
Arabidopsis thaliana
cDNA coding for a methionyl–tRNA synthetase. This enzyme was chosen because chloroplast-like elongator tRNA
Met
genes have been described in several plant species, including
A. thaliana
. We demonstrate here that the isolated cDNA codes for both the chloroplastic and the mitochondrial methionyl–tRNA synthetase (MetRS). The protein is transported into isolated chloroplasts and mitochondria and is processed to its mature form in both organelles. Transient expression assays using the green fluorescent protein demonstrated that the N-terminal region of the MetRS is sufficient to address the protein to both chloroplasts and mitochondria. Moreover, characterization of MetRS activities from mitochondria and chloroplasts of pea showed that only one MetRS activity exists in each organelle and that both are indistinguishable by their behavior on ion exchange and hydrophobic chromatographies. The high degree of sequence similarity between
A. thaliana
and
Synechocystis
MetRS strongly suggests that the
A. thaliana
MetRS gene described here is of chloroplast origin.
Rapid hybrid speciation in wild sunflowers
Authors: Ungerer Mark C. , Baird Stuart J. E. , Pan Jean , Rieseberg Loren H.
Published: July 2002

Hybrid or “recombinational” speciation refers to the origin of a new homoploid species via hybridization between chromosomally or genetically divergent parental species. Theory predicts that this mode of speciation is punctuated, but there has been little empirical evidence to support this claim. Here, we test the hypothesis of rapid hybrid speciation by estimating the sizes of parental species chromosomal blocks inHelianthus anomalus, a wild sunflower species derived via hybridization betweenH. annuusandH. petiolaris. Analysis of the frequency spectrum of parental species chromosomal blocks with respect to predictions based on R. A. Fisher’s [Fisher, R. A. (1953)Heredity8, 187–197] junctions approach, suggests thatH. anomalusarose rapidly, probably in fewer than 60 generations. This result is corroborated by independent lines of evidence demonstrating (i) a significant concordance between the genomes ofH. anomalusand early generationH. annuus×H. petiolarissynthetic hybrids, and (ii) a rapid recovery of pollen fertility in these synthetic hybrid lineages. These results are not only consistent with theory but also provide a new and general method for estimating the tempo of hybrid speciation and dating the origin of hybrid zones.
Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in
Saccharomyces cerevisiae
Authors: Miret Juan José , Pessoa-Brandão Luis , Lahue Robert S.
Published: July 2002

A quantitative and selective genetic assay was developed to monitor expansions of trinucleotide repeats (TNRs) in yeast. A promoter containing 25 repeats allows expression of a
URA3
reporter gene and yields sensitivity to the drug 5-fluoroorotic acid. Expansion of the TNR to 30 or more repeats turns off
URA3
and provides drug resistance. When integrated at either of two chromosomal loci, expansion rates were 1 × 10
−5
to 4 × 10
−5
per generation if CTG repeats were replicated on the lagging daughter strand. PCR analysis indicated that 5–28 additional repeats were present in 95% of the expanded alleles. No significant changes in CTG expansion rates occurred in strains deficient in the mismatch repair gene
MSH2
or the recombination gene
RAD52
. The frequent nature of CTG expansions suggests that the threshold number for this repeat is below 25 in this system. In contrast, expansions of the complementary repeat CAG occurred at 500- to 1,000-fold lower rates, similar to a randomized (C,A,G) control sequence. When the reporter plasmid was inverted within the chromosome, switching the leading and lagging strands of replication, frequent expansions were observed only when CTG repeats resided on the lagging daughter strand. Among the rare CAG expansions, the largest gain in tract size was 38 repeats. The control repeats CTA and TAG showed no detectable rate of expansions. The orientation-dependence and sequence-specificity data support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments.
The role of alternative mRNA splicing in generating heterogeneity within the
Anopheles gambiae
class I glutathione
S-
transferase family
Authors: Ranson Hilary , Collins Frank , Hemingway Janet
Published: July 2002

The class I glutathione
S
-transferases (GSTs) of
Anopheles gambiae
are encoded by a complex gene family. We describe the genomic organization of three members of this family, which are sequentially arranged on the chromosome in divergent orientations. One of these genes,
aggst1-2
, is intronless and has been described. In contrast, the two
A. gambiae
GST genes (
aggst1α
and
aggst1β
) reported within are interrupted by introns. The gene
aggst1α
contains five coding exons that are alternatively spliced to produce four mature GST transcripts, each of which contains a common 5′ exon encoding the N termini of the GST protein spliced to one of four distinct 3′ exons encoding the carboxyl termini. All four of the alternative transcripts of
aggst1α
are expressed in
A. gambiae
larvae, pupae, and adults. We report on the involvement of alternative RNA splicing in generating multiple functional GST transcripts. A cDNA from the
aggst1β
gene was detected in adult mosquitoes, demonstrating that this GST gene is actively transcribed. The percentage similarity of the six cDNAs transcribed from the three GST genes range from 49.5% to 83.1% at the nucleotide level.
Vascular endothelial growth factor C induces angiogenesis
in vivo
Authors: Cao Yihai , Linden Philip , Farnebo Jacob , Cao Renhai , Eriksson Anna , Kumar Vijay , Qi Jian-Hua , Claesson-Welsh Lena , Alitalo Kari
Published: July 2002

Vascular endothelial growth factor C (VEGF-C) recently has been described to be a relatively specific growth factor for the lymphatic vascular system. Here we report that ectopic application of recombinant VEGF-C also has potent angiogenic effects
in vivo
. VEGF-C is sufficiently potent to stimulate neovascularization from limbal vessels in the mouse cornea. Similar to VEGF, the angiogenic response of corneas induced by VEGF-C is intensive, with a high density of new capillaries. However, the outgrowth of microvessels stimulated by VEGF-C was significantly longer than that induced by VEGF. In the developing embryo, VEGF-C was able to induce branch sprouts from the established blood vessels. VEGF-C also induced an elongated, spindle-like cell shape change and actin reorganization in both VEGF receptor (VEGFR)-2 and VEGFR-3-overexpressing endothelial cells, but not in VEGFR-1-expressing cells. Further, both VEGFR-2 and VEGFR-3 could mediate proliferative and chemotactic responses in endothelial cells on VEGF-C stimulation. Thus, VEGF-C may regulate physiological angiogenesis and participate in the development and progression of angiogenic diseases in addition to lymphangiogenesis.
Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy
Authors: Nijhuis Monique , Boucher Charles A. B. , Schipper Pauline , Leitner Thomas , Schuurman Rob , Albert Jan
Published: July 2002

It has long been assumed that HIV-1 evolution is best described by deterministic evolutionary models because of the large population size. Recently, however, it was suggested that the effective population size (
N
e
) may be rather small, thereby allowing chance to influence evolution, a situation best described by a stochastic evolutionary model. To gain experimental evidence supporting one of the evolutionary models, we investigated whether the development of resistance to the protease inhibitor ritonavir affected the evolution of the
env
gene. Sequential serum samples from five patients treated with ritonavir were used for analysis of the protease gene and the V3 domain of the
env
gene. Multiple reverse transcription–PCR products were cloned, sequenced, and used to construct phylogenetic trees and to calculate the genetic variation and
N
e
. Genotypic resistance to ritonavir developed in all five patients, but each patient displayed a unique combination of mutations, indicating a stochastic element in the development of ritonavir resistance. Furthermore, development of resistance induced clear bottleneck effects in the
env
gene. The mean intrasample genetic variation, which ranged from 1.2% to 5.7% before treatment, decreased significantly (
P
< 0.025) during treatment. In agreement with these findings,
N
e
was estimated to be very small (500–15,000) compared with the total HIV-1 RNA copy number. This study combines three independent observations, strong population bottlenecking, small
N
e
, and selection of different combinations of protease-resistance mutations, all of which indicate that HIV-1 evolution is best described by a stochastic evolutionary model.
Structural assignments to the
Mycoplasma genitalium
proteins show extensive gene duplications and domain rearrangements
Authors: Teichmann Sarah A. , Park Jong , Chothia Cyrus
Published: July 2002

The parasitic bacterium
Mycoplasma genitalium
has a small, reduced genome with close to a basic set of genes. As a first step toward determining the families of protein domains that form the products of these genes, we have used the multiple sequence programs
psi-blast
and
geanfammer
to match the sequences of the 467 gene products of
M. genitalium
to the sequences of the domains that form proteins of known structure [Protein Data Bank (PDB) sequences]. PDB sequences (274) match all of 106
M. genitalium
sequences and some parts of another 85; thus, 41% of its total sequences are matched in all or part. The evolutionary relationships of the PDB domains that match
M. genitalium
are described in the structural classification of proteins (SCOP) database. Using this information, we show that the domains in the matched
M. genitalium
sequences come from 114 superfamilies and that 58% of them have arisen by gene duplication. This level of duplication is more than twice that found by using pairwise sequence comparisons. The PDB domain matches also describe the domain structure of the matched sequences: just over a quarter contain one domain and the rest have combinations of two or more domains.
Patches of synchronized activity in the cerebellar cortex evoked by mossy-fiber stimulation: Questioning the role of parallel fibers
Authors: Cohen Dana , Yarom Yosef
Published: July 2002

The discrepancy between the structural longitudinal organization of the parallel-fiber system in the cerebellar cortex and the functional mosaic-like organization of the cortex has provoked controversial theories about the flow of information in the cerebellum. We address this issue by characterizing the spatiotemporal organization of neuronal activity in the cerebellar cortex by using optical imaging of voltage-sensitive dyes in isolated guinea-pig cerebellum. Parallel-fiber stimulation evoked a narrow beam of activity, which propagated along the parallel fibers. Stimulation of the mossy fibers elicited a circular, nonpropagating patch of synchronized activity. These results strongly support the hypothesis that a beam of parallel fibers, activated by a focal group of granule cells, fails to activate the Purkinje cells along most of its length. It is thus the ascending axon of the granule cell, and not its parallel branches, that activates and defines the basic functional modules of the cerebellar cortex.