bluuebunny/crossref_metadata_2025 huggingface dataset:
Seasonal reproductive patterns and effectiveness as teasers (ram effect) of Corriedale and Milchschaf rams
Authors: Ungerfeld R.
Published: July 2012

The first objective of this study was to compare the seasonal reproductive pattern of Corriedale and Milchschaf rams. Because seasonal patterns were similar, a second objective was to compare the effectiveness of rams from both breeds to induce oestrus in seasonally anoestrous Corriedale ewes using the ram effect. In the first experiment, the seasonal changes of bodyweight, scrotal circumference, testosterone concentrations, sexual behaviour towards oestrous ewes (ano-genital sniffing, lateral approaches, flehmen, mount attempts, mounts, and copulation), and semen characteristics (total spermatozoa in the ejaculate, mass motility, and percentage of alive and normal spermatozoa) were recorded during 13 months in five Corriedale and seven Milchschaf rams. All recorded variables changed through the year, with maximum reproductive patterns occurring in summer and autumn. Milchschaf rams were heavier than Corriedale rams, but no difference was observed in their reproductive patterns. Experiment 2 was performed during the non-breeding season with 169 Corriedale and Polwarth mulitparous ewes. All ewes were isolated from the rams so that they could not see, hear or smell them (minimum distance: 1000 m) from Day –40. They were primed over 8 days with an intravaginal sponge impregnated with 50 mg of medroxiprogesterone. Four Corriedale rams were introduced to 83 ewes, and four Milchschaf rams to the remaining 86 ewes at sponge withdrawal, and oestrus was recorded for 5 days. On Day 5, no ewe of the group bred with Milchschaf rams was marked. By contrast, 46 out of 83 (55.4%) of the ewes bred with Corriedale rams were marked (P < 0.0001). It was concluded that reproductive seasonal pattern of Corriedale and Milchschaf rams, including scrotal circumference, testosterone concentrations, sexual behaviour and semen characteristics, was similar. However, Corriedale rams were more effective than Milchschaf rams in inducing oestrus in anoestrus ewes (the ram effect).
Reconstitution of the central and peripheral nervous system during salamander tail regeneration
Authors: Mchedlishvili Levan , Mazurov Vladimir , Grassme Kathrin S. , Goehler Kerstin , Robl Bernhard , Tazaki Akira , Roensch Kathleen , Duemmler Annett , Tanaka Elly M.
Published: July 2012

We show that after tail amputation in
Ambystoma mexicanum
(Axolotl) the correct number and spacing of dorsal root ganglia are regenerated. By transplantation of spinal cord tissue and nonclonal neurospheres, we show that the central spinal cord represents a source of peripheral nervous system cells. Interestingly, melanophores migrate from preexisting precursors in the skin. Finally, we demonstrate that implantation of a clonally derived spinal cord neurosphere can result in reconstitution of all examined cell types in the regenerating central spinal cord, suggesting derivation of a cell with spinal cord stem cell properties.
Unconventional initiator tRNAs sustain
Escherichia coli
Authors: Samhita Laasya , Shetty Sunil , Varshney Umesh
Published: July 2012

Of all tRNAs, initiator tRNA is unique in its ability to start protein synthesis by directly binding the ribosomal P-site. This ability is believed to derive from the almost universal presence of three consecutive G-C base (3G-C) pairs in the anticodon stem of initiator tRNA. Consistent with the hypothesis, a plasmid-borne initiator tRNA with one, two, or all 3G-C pairs mutated displays negligible initiation activity when tested in a WT
Escherichia coli
cell. Given this, the occurrence of unconventional initiator tRNAs lacking the 3G-C pairs, as in some species of
Mycoplasma
and
Rhizobium
, is puzzling. We resolve the puzzle by showing that the poor activity of unconventional initiator tRNAs in
E. coli
is because of competition from a large pool of the endogenous WT initiator tRNA (possessing the 3G-C pairs). We show that
E. coli
can be sustained on an initiator tRNA lacking the first and third G-C pairs; thereby reducing the 3G-C rule to a mere middle G-C requirement. Two general inferences following from our findings, that the activity of a mutant gene product may depend on its abundance in the cell relative to that of the WT, and that promiscuous initiation with elongator tRNAs has the potential to enhance phenotypic diversity without affecting genomic integrity, have been discussed.
Correlation of enterohemorrhagic
Escherichia coli
O157 prevalence in feces, hides, and carcasses of beef cattle during processing
Authors: Elder Robert O. , Keen James E. , Siragusa Gregory R. , Barkocy-Gallagher Genevieve A. , Koohmaraie Mohammad , Laegreid William W.
Published: July 2012

A survey was performed to estimate the frequency of enterohemorrhagic
Escherichia coli
O157:H7 or O157:nonmotile (EHEC O157) in feces and on hides within groups of fed cattle from single sources (lots) presented for slaughter at meat processing plants in the Midwestern United States, as well as frequency of carcass contamination during processing from cattle within the same lots. Of 29 lots sampled, 72% had at least one EHEC O157-positive fecal sample and 38% had positive hide samples. Overall, EHEC O157 prevalence in feces and on hides was 28% (91 of 327) and 11% (38 of 355), respectively. Carcass samples were taken at three points during processing: preevisceration, postevisceration before antimicrobial intervention, and postprocessing after carcasses entered the cooler. Of 30 lots sampled, 87% had at least one EHEC O157-positive preevisceration sample, 57% of lots were positive postevisceration, and 17% had positive postprocessing samples. Prevalence of EHEC O157 in the three postprocessing samples was 43% (148 of 341), 18% (59 of 332) and 2% (6 of 330), respectively. Reduction in carcass prevalence from preevisceration to postprocessing suggests that sanitary procedures were effective within the processing plants. Fecal and hide prevalence were significantly correlated with carcass contamination (
P
= 0.001), indicating a role for control of EHEC O157 in live cattle.
Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the
cag
pathogenicity island of
Helicobacter pylori
share similar hexameric ring structures
Authors: Krause Sabine , Bárcena Montserrat , Pansegrau Werner , Lurz Rudi , Carazo José María , Lanka Erich
Published: July 2012

RP4 TrbB, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4, is a member of the PulE protein superfamily involved in multicomponent machineries transporting macromolecules across the bacterial envelope. PulE-like proteins share several well conserved motifs, most notable a nucleoside triphosphate binding motif (P-loop).
Helicobacter pylori
HP0525 also belongs to the PulE superfamily and is encoded by the pathogenicity island
cag
, involved in the inflammatory response of infected gastric epithelial cells in mammals. The native molecular masses of TrbB and HP0525 as determined by gel filtration and glycerol gradient centrifugation suggested a homohexameric structure in the presence of ATP and Mg
2+
. In the absence of nucleotides and bivalent cations, TrbB behaved as a tetramer whereas the hexameric state of HP0525 remained unaffected. Electron microscopy and image processing demonstrated that TrbB and HP0525 form ring-shaped complexes (diameter: 12 nm) with a central region (diameter: 3 nm) of low electron density when incubated in the presence of ATP and Mg
2+
. However, the TrbB average image appeared to be more elliptical with strong twofold rotational symmetry whereas HP0525 complexes are regular hexagons. Six well defined triangle-shaped areas of high electron density were distinguishable in both cases. Covalent crosslinking of TrbB suggests that the hexameric ring is composed from a trimer of dimers, because only dimeric, tetrameric, and hexameric species were detectable. The toroidal structure of TrbB and HP0525 suggests that both proteins catalyze a repetitive process, most probably translocating a cognate substrate across the inner membrane.
Design and properties of human
d
-amino acid oxidase with covalently attached flavin
Authors: Raibekas Andrei A. , Fukui Kiyoshi , Massey Vincent
Published: July 2012

An “artificial flavinylation” approach was developed to replace a native noncovalent flavin prosthetic group with a covalently attached flavin analogue in recombinant human
d
-amino acid oxidase. The protein residue Gly-281 was replaced with Cys by site-directed mutagenesis, followed by reaction between mutated apoenzyme and the thiol-reactive flavin analogue, 8-methylsulfonyl FAD. The stoichiometric process of flavin attachment was accompanied by gain in enzymatic activity, reaching up to 26% activity of the recombinant native enzyme. The steady-state kinetic data together with the results of limited proteolysis and benzoate-binding studies suggest that, although mutation perturbs protein structural and catalytic properties, the flavinylation alone does not have any negative impact. We conclude that, despite the implemented restraints on its mobility, the covalently attached flavin is properly positioned within the protein active site and acts efficiently during
d
-amino acid oxidase catalytic turnover.
Recognition of a conserved class of RNA tetraloops by
Saccharomyces cerevisiae
RNase III
Authors: Chanfreau Guillaume , Buckle Malcolm , Jacquier Alain
Published: July 2012

Ribonucleases III are double-stranded RNA (dsRNA) endonucleases required for the processing of a large number of prokaryotic and eukaryotic transcripts. Although the specificity of bacterial RNase III cleavage relies on antideterminants in the dsRNA, the molecular basis of eukaryotic RNase III specificity is unknown. All substrates of yeast RNase III (Rnt1p) are capped by terminal tetraloops showing the consensus AGNN and located within 13–16 bp to Rnt1p cleavage sites. We show that these tetraloops are essential for Rnt1p cleavage and that the distance to the tetraloop is the primary determinant of cleavage site selection. The presence of AGNN tetraloops also enhances Rnt1p binding, as shown by surface plasmon resonance monitoring and modification interference studies. These results define a paradigm of RNA loops and show that yeast RNase III behaves as a helical RNA ruler that recognizes these tetraloops and cleaves the dsRNA at a fixed distance to this RNA structure. These results also indicate that proteins belonging to the same class of RNA endonucleases require different structural elements for RNA cleavage.
Millisecond time scale conformational flexibility in a hyperthermophile protein at ambient temperature
Authors: Hernández Griselda , Jenney Francis E. , Adams Michael W. W. , LeMaster David M.
Published: July 2012

Rubredoxin from the hyperthermophile
Pyrococcus furiosus
is the most thermostable protein characterized to date with an estimated global unfolding rate of 10
−6
s
−1
at 100°C. In marked contrast to these slow global dynamics, hydrogen exchange experiments here demonstrate that conformational opening for solvent access occurs in the ≈millisecond time frame or faster at 28°C for all amide positions. Under these conditions all backbone amides with exchange protection factors between 10
4
and 10
6
, for which EX
2
exchange kinetics were directly verified, have exchange activation energy values within 2–3 kcal/mol of that observed for unstructured peptides. The conformational flexibility of this protein is thus sufficient for water and base catalyst access to the exchanging amide with quite limited structural disruption. The common hypothesis that enhanced conformational rigidity in the folded native state underlies the increased thermal stability of hyperthermophile proteins is not supported by these data.
Structures of recombinant human and mouse NAD(P)H:quinone oxidoreductases: Species comparison and structural changes with substrate binding and release
Authors: Faig Margarita , Bianchet Mario A. , Talalay Paul , Chen Shiuan , Winski Shannon , Ross David , Amzel L. Mario
Published: July 2012

NAD(P)H/quinone acceptor oxidoreductase (QR1, NQO1, formerly DT-diaphorase; EC
1.6.99.2
) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. In this paper we report the apoenzyme structures of human (at 1.7-Å resolution) and mouse (2.8 Å) QR1 and the complex of the human enzyme with the substrate duroquinone (2.5 Å) (2,3,5,6-tetramethyl-
p
-benzoquinone). In addition to providing a description and rationale of the structural and catalytic differences among several species, these structures reveal the changes that accompany substrate or cofactor (NAD) binding and release. Tyrosine-128 and the loop spanning residues 232–236 close the binding site, partially occupying the space left vacant by the departing molecule (substrate or cofactor). These changes highlight the exquisite control of access to the catalytic site that is required by the ping-pong mechanism in which, after reducing the flavin, NAD(P)
+
leaves the catalytic site and allows substrate to bind at the vacated position. In the human QR1-duroquinone structure one ring carbon is significantly closer to the flavin N5, suggesting a direct hydride transfer to this atom.
αβ protomers of Na
+
,K
+
-ATPase from microsomes of duck salt gland are mostly monomeric: Formation of higher oligomers does not modify molecular activity
Authors: Martin Dwight W. , Marecek James , Scarlata Suzanne , Sachs John R.
Published: July 2012

The distance that separates αβ protomers of the Na
+
,K
+
-ATPase in microsomes and in purified membranes prepared from duck nasal salt glands was estimated by measuring fluorescence resonance energy transfer between anthroylouabain bound to a population of αβ protomers and either
N-
[7-nitrobenz-2-oxa-1,3-diazol-4-yl]-6-aminohexyl ouabain or 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain bound to the rest. Energy transfer between probes bound in the microsomal preparation was less than in the purified membranes. The efficiency of energy transfer between anthroylouabain and
N-
[7-nitrobenz-2-oxa-1,3-diazol-4-yl]-6-aminohexyl ouabain was 29.2% in the microsomes compared with 62.6% in the purified preparation. Similar results were obtained with 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain as acceptor. We calculate that either the protomer bound probes were on the average 13 Å farther apart in the microsomes than in the purified membranes, or that 53% of the protomers are monomeric in the microsome preparation. Microsomes prepared in the presence of phalloidin (a toxin that binds to F actin and stabilizes the actin-based cytoskeleton) showed less quench than those prepared in its absence. The data support the hypothesis that protomers are kept apart by their association with the cytoskeleton. The turnover rate while hydrolyzing ATP is the same in the microsomal and purified preparations; higher oligomer formation has no significant effect on the enzyme reaction mechanism.
Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast
Authors: Bagnat Michel , Keränen Sirkka , Shevchenko Anna , Shevchenko Andrej , Simons Kai
Published: July 2012

Lipid rafts, formed by lateral association of sphingolipids and
cholesterol, have been implicated in membrane traffic and cell
signaling in mammalian cells. Sphingolipids also have been shown to
play a role in protein sorting in yeast. Therefore, we wanted to
investigate whether lipid rafts exist in yeast and whether these
membrane microdomains have an analogous function to their mammalian
counterparts. We first developed a protocol for isolating
detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast
cells. Sequencing of the major protein components of the isolated DIGs
by mass spectrometry allowed us to identify, among others, Gas1p,
Pma1p, and Nce2p. Using lipid biosynthetic mutants we could demonstrate
that conditions that impair the synthesis of sphingolipids and
ergosterol also disrupt raft association of Gas1p and Pma1p but not the
secretion of acid phosphatase. That endoplasmic reticulum (ER)-to-Golgi
transport of Gas1p is blocked in the sphingolipid mutant
lcb1–100
raised the question of whether proteins
associate with lipid rafts in the ER or later as shown in mammalian
cells. Using the
sec18–1
mutant we found that DIGs are
present already in the ER. Taken together, our results suggest that
lipid rafts are involved in the biosynthetic delivery of proteins to
the yeast plasma membrane.
Drosophila
female sexual behavior induced by sterile males showing copulation complementation
Authors: Xue Lei , Noll Markus
Published: July 2012

Females of most animal species are usually inseminated by more than one male, which allows sperm from different males to compete for fertilization. To prevent invasion of sperm from other males,
Drosophila
males elicit a rejection behavior in their mates after copulation. Using
paired
mutant males that, for the lack of accessory glands, are sterile, we show that this rejection behavior is induced exclusively by the secreted accessory gland products transferred to the female during copulation. Moreover, the activities of sperm and accessory gland products are complementary and interdependent: both sperm fertility and rejection behavior depend on accessory gland products whose prolonged activities, in turn, require the presence of sperm. Fertility of sperm from
paired
males can be restored by accessory gland products of spermless males in “copulation complementation” experiments. Our observations may have important implications for the role of sexual behavior in evolution and for the treatment of male sexual dysfunction in humans.
Dictyosteliumamoebae lacking an F-box protein form spores rather than stalk in chimeras with wild type
Authors: Ennis Herbert L. , Dao Dee N. , Pukatzki Stefan U. , Kessin Richard H.
Published: July 2012

Using a selection forDictyosteliummutants that preferentially form spores, we have recovered a mutant calledCheaterA.In chimeras with isogenic wild-type cells, theCheaterAmutant preferentially forms viable spores rather than inviable stalk cells. The mutant causes wild-type cells that have begun to express spore-specific genes to accumulate in the prestalk compartment of the developing organism. In the wild-type cells, thechtAtranscript is absent in growing cells and appears early in development. No transcript was detected in the mutant by Northern blot. ThechtAgene codes for a protein with an F-box and WD40 domains. This class of protein usually forms part of anSkp1,cullin,F-box (SCF) complex that targets specific protein substrates for ubiquitination and degradation.
Evolution and assembly of an extremely scrambled gene
Authors: Landweber Laura F. , Kuo Tai-Chih , Curtis Edward A.
Published: July 2012

The process of gene unscrambling in hypotrichous ciliates
represents one of nature's ingenious solutions to the problem of gene
assembly. With some essential genes scrambled in as many as 51 pieces,
these ciliates rely on sequence and structural cues to rebuild their
fragmented genes and genomes. Here we report the complex pattern of
scrambling in the DNA polymerase α gene of
Stylonychia
lemnae
. The germline (micronuclear) copy of this gene is broken
into 48 pieces with 47 dispersed over two loci, with no asymmetry in
the placement of coding segments on either strand. Direct repeats
present at the boundaries between coding and noncoding sequences
provide pointers to help guide assembly of the functional
(macronuclear) gene. We investigate the evolution of this complex gene
in three hypotrichous species.
Experimental evolution of aging, growth, and reproduction in fruitflies
Authors: Stearns S. C. , Ackermann M. , Doebeli M. , Kaiser M.
Published: July 2012

We report in this paper an evolutionary experiment on
Drosophila
that tested life-history theory and the evolutionary theory of aging. As theory predicts, higher extrinsic mortality rates did lead to the evolution of higher intrinsic mortality rates, to shorter lifespans, and to decreased age and size at eclosion; peak fecundity also shifted earlier in life. These results confirm the key role of extrinsic mortality rates in the evolution of growth, maturation, reproduction, and aging, and they do so with a selection regime that maintained selection on fertility throughout life while holding population densities constant.
Whole-genome expression analysis of
snf/swi
mutants of
Saccharomyces
cerevisiae
Authors: Sudarsanam Priya , Iyer Vishwanath R. , Brown Patrick O. , Winston Fred
Published: July 2012

The
Saccharomyces cerevisiae
Snf/Swi complex has been previously demonstrated to control transcription and chromatin structure of particular genes
in vivo
and to remodel nucleosomes
in vitro
. We have performed whole-genome expression analysis, using DNA microarrays, to study mutants deleted for a gene encoding one conserved (Snf2) or one unconserved (Swi1) Snf/Swi component. This analysis was performed on cells grown in both rich and minimal media. The microarray results, combined with Northern blot, computational, and genetic analyses, show that
snf2
Δ and
swi1
Δ mutations cause similar effects on mRNA levels, that Snf/Swi controls some genes differently in rich and minimal media, and that Snf/Swi control is exerted at the level of individual genes rather than over larger chromosomal domains. In addition, this work shows that Snf/Swi controls mRNA levels of
MAT
α-specific genes, likely via controlling transcription of the regulators
MAT
α
1
and
MCM1
.
Finally, we provide evidence that Snf/Swi acts both as an activator and as a repressor of transcription, and that neither mode of control is an indirect effect of the other.
Modification of P-selectin glycoprotein ligand-1 with a natural killer cell-restricted sulfated lactosamine creates an alternate ligand for L-selectin
Authors: André Pascale , Spertini Olivier , Guia Sophie , Rihet Pascal , Dignat-George Françoise , Brailly Hervé , Sampol José , Anderson Paul J. , Vivier Eric
Published: July 2012

Natural killer (NK) cells are components of the innate immune system that can recognize and kill virally infected cells, tumor cells, and allogeneic cells without prior sensitization. NK cells also elaborate cytokines (e.g., interferon-γ and tumor necrosis factor-α) and chemokines (e.g., macrophage inflammatory protein-1α) that promote the acquisition of antigen-specific immunity. NK cell differentiation is accompanied by the cell surface expression of a mucin-like glycoprotein bearing an NK cell-restricted keratan sulfate-related lactosamine carbohydrate, the PEN5 epitope. Here, we report that PEN5 is a post-translational modification of P-selectin glycoprotein ligand-1 (PSGL-1). The PEN5 epitope creates on PSGL-1 a unique binding site for L-selectin, which is independent of PSGL-1 tyrosine sulfation. On the surface of NK cells, the expression of PEN5 is coordinated with the disappearance of L-selectin and the up-regulation of Killer cell Ig-like Receptors (KIR). These results indicate that NK cell differentiation is accompanied by the acquisition of a unique carbohydrate, PEN5, that can serve as part of a combination code to deliver KIR+NK cells to specific tissues.
Potent induction of long-term CD8+T cell memory by short-term IL-4 exposure during T cell receptor stimulation
Authors: Huang Li-Rung , Chen Fen-Ling , Chen Yi-Ting , Lin Ya-Min , Kung John T.
Published: July 2012

An important goal of vaccination is to achieve long-term survival of functional memory T cells. Using a MHC-compatible adoptive transfer system, we show here that a short, 3-day IL-4 but not IL-2 or IL-12 exposure duringin vitroT cell receptor stimulation of naive CD8+T cells induced long-lastingin vivomemory. Such long-term memory CD8+T cells expressed antigen-specific cytotoxicity and the potential for IFN-γ and IL-4 production. Our results support the concept that functional T cell longevity can be regulated by cytokines during initial antigen encounter and provide a rational foundation for vaccine development. They also may have implications in formulating optimal therapeutic regimens ofex vivoexpanded autologous cancer- and HIV-specific CD8+T cells. In addition, the availability of large numbers of memory CD8+T cells generated through our high-efficiency system should facilitate progress in the molecular dissection of CD8+T cell memory development.
Transplantation ofex vivoexpanded endothelial progenitor cells for therapeutic neovascularization
Authors: Kalka Christoph , Masuda Haruchika , Takahashi Tomono , Kalka-Moll Wiltrud M. , Silver Marcy , Kearney Marianne , Li Tong , Isner Jeffrey M. , Asahara Takayuki
Published: July 2012

Animal studies and preliminary results in humans suggest that lower extremity and myocardial ischemia can be attenuated by treatment with angiogenic cytokines. The resident population of endothelial cells that is competent to respond to an available level of angiogenic growth factors, however, may potentially limit the extent to which cytokine supplementation enhances tissue neovascularization. Accordingly, we transplanted human endothelial progenitor cells (hEPCs) to athymic nude mice with hindlimb ischemia. Blood flow recovery and capillary density in the ischemic hindlimb were markedly improved, and the rate of limb loss was significantly reduced.Ex vivoexpanded hEPCs may thus have utility as a “supply-side” strategy for therapeutic neovascularization.
Recombinant adeno-associated virus type 2, 4, and 5 vectors: Transduction of variant cell types and regions in the mammalian central nervous system
Authors: Davidson Beverly L. , Stein Colleen S. , Heth Jason A. , Martins Inês , Kotin Robert M. , Derksen Todd A. , Zabner Joseph , Ghodsi Abdi , Chiorini John A.
Published: July 2012

Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in the central nervous system (CNS), but it is not known how other rAAV serotypes perform as CNS gene transfer vectors. Serotypes 4 and 5 are distinct from rAAV2 and from each other in their capsid regions, suggesting that they may direct binding and entry into different cell types. In this study, we examined the tropisms and transduction efficiencies of β-galactosidase-encoding vectors made from rAAV4 and rAAV5 compared with similarly designed rAAV2-based vectors. Injection of rAAV5 β-galactosidase (βgal) or rAAV4βgal into the lateral ventricle resulted in stable transduction of ependymal cells, with approximately 10-fold more positive cells than in mice injected with rAAV2βgal. Major differences between the three vectors were revealed upon striatal injections. Intrastriatal injection of rAAV4βgal resulted again in striking ependyma-specific expression of transgene, with a notable absence of transduced cells in the parenchyma. rAAV2βgal and rAAV5βgal intrastriatal injections led to β-gal-positive parenchymal cells, but, unlike rAAV2βgal, rAAV5βgal transduced both neurons and astrocytes. The number of transgene-positive cells in rAAV5βgal-injected brains was 130 and 5,000 times higher than in rAAV2βgal-injected brains at 3 and 15 wk, respectively. Moreover, transgene-positive cells were widely dispersed throughout the injected hemisphere in rAAV5βgal-transduced animals. Together, our data provide
in vivo
support for earlier
in vitro
work, suggesting that rAAV4 and rAAV5 gain cell entry by means of receptors distinct from rAAV2. These differences could be exploited to improve gene therapy for CNS disorders.
Induction of basal cell carcinomas and trichoepitheliomas in mice overexpressing GLI-1
Authors: Nilsson Mats , Undèn Anne Birgitte , Krause Darren , Malmqwist Ulrica , Raza Karima , Zaphiropoulos Peter G. , Toftgård Rune
Published: July 2012

Basal cell carcinoma is the most prevalent cancer in the western world, showing a rapid increase in incidence. Activation of the Sonic hedgehog/Patched (PTCH) signaling pathway because of PTCH1 inactivation is a key event in sporadic and familial basal cell carcinoma development in humans and is associated with transcriptional activation of specific target genes, including
PTCH1
itself. These changes are analogous to the situation in
Drosophila
where hedgehog activates the zinc-finger transcription factor Cubitus interruptus, leading to increased transcription of target genes. In the present study, we show that mice ectopically expressing the human Cubitus interruptus homolog GLI-1 in the skin develop tumors closely resembling human BCCs as well as other hair follicle-derived neoplasias, such as trichoepitheliomas, cylindromas, and trichoblastomas. Furthermore, examination of the tumors revealed wild-type
p53
and Ha
ras
genes. These findings firmly establish that increased GLI-1 expression is central and probably sufficient for tumor development and suggest that GLI-1-induced tumor development does not depend on additional
p53
or Ha
ras
mutations.
Amplification of the
neu/erbB-2
oncogene in a mouse model of mammary tumorigenesis
Authors: Andrechek Eran R. , Hardy William R. , Siegel Peter M. , Rudnicki Michael A. , Cardiff Robert D. , Muller William J.
Published: July 2012

The
neu
(
c-erbB-2
,
Her-2
) protooncogene is amplified and overexpressed in 20–30% of human breast cancers. Although transgenic mouse models have illustrated the role of Neu in the induction of mammary tumors, Neu expression in these models is driven by a strong viral promoter of questionable relevance to the human disease. To ascertain whether expression of activated Neu under the control of the endogenous promoter in the mammary gland could induce mammary tumors we have generated mice that conditionally express activated Neu under the transcriptional control of the intact endogenous Neu promoter. Expression of oncogenic
neu
in the mammary gland resulted in accelerated lobulo-alveolar development and formation of focal mammary tumors after a long latency period. However, expression of activated Neu under the normal transcriptional control of the endogenous promoter was not sufficient for the initiation of mammary carcinogenesis. Strikingly, all mammary tumors bear amplified copies (2–22 copies) of the activated
neu
allele relative to the wild-type allele and express highly elevated levels of
neu
transcript and protein. Thus, like human
erbB-2
-positive breast tumors, mammary tumorigenesis in this mouse model requires the amplification and commensurate elevated expression of the
neu
gene.
Synthesis and antitumor activity of an inhibitor of fatty acid synthase
Authors: Kuhajda Francis P. , Pizer Ellen S. , Li Ji Nong , Mani Neelakandha S. , Frehywot Gojeb L. , Townsend Craig A.
Published: July 2012

Compared to normal human tissues, many common human cancers, including carcinoma of the colon, prostate, ovary, breast, and endometrium, express high levels of fatty acid synthase (FAS, EC
2.3.1.85
), the primary enzyme responsible for the synthesis of fatty acids. This differential expression of FAS between normal tissues and cancer has led to the notion that FAS is a target for anticancer drug development. Recent studies with C75, an inhibitor of fatty acid synthesis, have shown significant antitumor activity with concomitant inhibition of fatty acid synthesis in tumor tissue and normal liver. Importantly, histopathological analysis of normal tissues after C75 treatment showed no adverse effects on proliferating cellular compartments, such as bone marrow, gastrointestinal tract, skin, or lymphoid tissues. In this study, we describe the
de novo
synthesis of C75 based on the known mechanism of action of cerulenin and the theoretical reaction intermediates of the β-ketoacyl synthase moiety of FAS. In addition, we demonstrate that C75 is a synthetic, chemically stable inhibitor of FAS. C75 inhibits purified mammalian FAS with characteristics of a slow-binding inhibitor and also inhibits fatty acid synthesis in human cancer cells. Treatment of human breast cancer cells with [5-
3
H]C75 demonstrates that C75 reacts preferentially with FAS in whole cells. Therefore, we have shown that the primary mechanism of the antitumor activity of C75 is likely mediated through its interaction with, and inhibition of, FAS. This development will enable the
in vivo
study of FAS inhibition in human cancer and other metabolic diseases.
Tumorigenesis in the multiple intestinal neoplasia mouse: Redundancy of negative regulators and specificity of modifiers
Authors: Halberg Richard B. , Katzung Darren S. , Hoff Peter D. , Moser Amy R. , Cole Carolyn E. , Lubet Ronald A. , Donehower Lawrence A. , Jacoby Russell F. , Dove William F.
Published: July 2012

The interaction between mutations in the tumor-suppressor genes
Apc
and
p53
was studied in congenic mouse strains to minimize the influence of polymorphic modifiers. The multiplicity and invasiveness of intestinal adenomas of
Apc
Min/+
(Min) mice was enhanced by deficiency for
p53
. In addition, the occurrence of desmoid fibromas was strongly enhanced by
p53
deficiency. The genetic modifier
Mom1
and the pharmacological agents piroxicam and difluoromethylornithine each reduced intestinal adenoma multiplicity in the absence of
p53
function.
Mom1
showed no influence on the development of desmoid fibromas, whereas the combination of piroxicam and difluoromethylornithine exerted a moderate effect. The ensemble of tumor suppressors and modifiers of a neoplastic process can be usefully analyzed in respect to tissue specificity and synergy.
Functional polymorphisms of the human multidrug-resistance gene: Multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity
in
vivo
Authors: Hoffmeyer S. , Burk O. , von Richter O. , Arnold H. P. , Brockmöller J. , Johne A. , Cascorbi I. , Gerloff T. , Roots I. , Eichelbaum M. , Brinkmann U.
Published: July 2012

To evaluate whether alterations in the multidrug-resistance (MDR)-1 gene correlate with intestinal MDR-1 expression and uptake of orally administered P-glycoprotein (PGP) substrates, we analyzed the MDR-1 sequence in 21 volunteers whose PGP expression and function in the duodenum had been determined by Western blots and quantitative immunohistology (
n
= 21) or by plasma concentrations after orally administered digoxin (
n
= 8 + 14). We observed a significant correlation of a polymorphism in exon 26 (C3435T) of MDR-1 with expression levels and function of MDR-1. Individuals homozygous for this polymorphism had significantly lower duodenal MDR-1 expression and the highest digoxin plasma levels. Homozygosity for this variant was observed in 24% of our sample population (
n
= 188). This polymorphism is expected to affect the absorption and tissue concentrations of numerous other substrates of MDR-1.
The RheA repressor is the thermosensor of the HSP18 heat shock response in
Streptomyces albus
Authors: Servant Pascale , Grandvalet Cosette , Mazodier Philippe
Published: July 2012

Microorganisms have mechanisms to sense their environment and rapidly adapt to survive changes in conditions. In
Streptomyces albus
, various transcriptional repressors mediate the induction of heat shock genes. The RheA repressor regulates the synthesis of HSP18, a small heat shock protein, which plays a role in thermotolerance. The RheA protein was purified to determine how it responds rapidly to temperature. Gel retardation assays and footprinting experiments identified the specific target of RheA as an inverted repeat (TGTCATC 5N GATGACA) located in P
hsp18
, P
rheA
which is the common promoter region of the divergon. Gel retardation assays detected RheA-complexes formed with the
hsp18-rheA
promoters. The complexes did not form at higher temperature.
In vitro
transcription experiments showed that RheA is an autoregulatory protein and that its activity is inhibited by high temperature. The temperature-induced derepression by RheA is reversible. Dichroism circular spectroscopy revealed a reversible change of RheA conformation in relation with the temperature that could represent a transition between an active and an inactive form. Our experiments demonstrate that RheA acts as a cellular thermometer in
hsp18
regulation.
Functional neuroanatomy of executive processes involved in dual-task performance
Authors: Adcock R. Alison , Constable R. Todd , Gore John C. , Goldman-Rakic Patricia S.
Published: July 2012

The subjective experience of allocating one's attentional resources among competing tasks is nearly universal, and most current models of cognition include a mechanism that performs this allocation; examples include the central executive system and the supervisory attentional system. Yet, the exact form that an executive system might take and even its necessity for cognition are controversial. Dual-task paradigms have commonly been used to investigate executive function. The few neuroimaging studies of these paradigms have yielded contradictory findings. Using functional MRI, we imaged brain function during two dual-task paradigms, each with a common auditory component task (NOUN task) but varying with respect to a visual component task (SPACE or FACE tasks). In each of the two dual-task paradigms, the results showed that the activated areas varied with the component tasks, that all of the areas activated during dual task performance were also activated during the component tasks, and that surplus activation within activated areas during DUAL conditions was parsimoniously accounted for by the addition of the second task. These findings suggest that executive processes may be mediated by interactions between anatomically and functionally distinct systems engaged in performance of component tasks, as opposed to an area or areas dedicated to a generic executive system.
The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus
Authors: Bi Ruifen , Broutman Greg , Foy Mike R. , Thompson Richard F. , Baudry Michel
Published: July 2012

Estrogen replacement therapy in women is associated with improvement of cognitive deficits and reduced incidence of Alzheimer's disease. The present study indicates that estrogen is neuroprotective against
N
-methyl-
d
-aspartate (NMDA)- and kainate-mediated neurotoxicity, an effect mediated by tyrosine kinase/mitogen-activated protein kinase (MAPK) pathways. Estrogen also stimulates tyrosine phosphorylation of NMDA receptors via an src tyrosine kinase/MAPK pathway. Finally, estrogen-mediated enhancement of long-term potentiation in hippocampal slices is mediated by activation of an src tyrosine kinase pathway. Thus, estrogen, by activating an src tyrosine kinase and the extracellular signal-related protein kinase/MAPK signaling pathway, both enhances NMDA receptor function and long-term potentiation and retains neuroprotective properties against excitotoxicity. These findings warrant further evaluation of the usefulness of estrogenic compounds for the treatment of Alzheimer's disease and other neurodegenerative diseases.
Differential regulation of circadian pacemaker output by separate clock genes in
Drosophila
Authors: Park Jae H. , Helfrich-Förster Charlotte , Lee Gyunghee , Liu Li , Rosbash Michael , Hall Jeffrey C.
Published: July 2012

Regulation of the
Drosophila
pigment-dispersing factor (
pdf
) gene products was analyzed in wild-type and clock mutants. Mutations in the transcription factors CLOCK and CYCLE severely diminish
pdf
RNA and neuropeptide (PDF) levels in a single cluster of clock-gene-expressing brain cells, called small ventrolateral neurons (
s-
LN
v
s). This clock-gene regulation of specific cells does not operate through an E-box found within
pdf
regulatory sequences. PDF immunoreactivity exhibits daily cycling, but only within terminals of axons projecting from the
s
-LN
v
s. This posttranslational rhythm is eliminated by
period
or
timeless
null mutations, which do not affect PDF staining in cell bodies or
pdf
mRNA levels. Therefore, within these chronobiologically important neurons, separate elements of the central pacemaking machinery regulate
pdf
or its product in novel and different ways. Coupled with contemporary results showing a
pdf-
null mutant to be severely defective in its behavioral rhythmicity, the present results reveal PDF as an important circadian mediator whose expression and function are downstream of the clockworks.
Progesterone-metabolite prevents protein kinase C-dependent modulation of γ-aminobutyric acid type A receptors in oxytocin neurons
Authors: Brussaard Arjen B. , Wossink Juriaan , Lodder Johannes C. , Kits Karel S.
Published: July 2012

Gonadal steroid feedback to oxytocin neurons during pregnancy is in part mediated via the neurosteroid allopregnanolone (3α-OH-DHP), acting as allosteric modulator of postsynaptic γ-aminobutyric acid type A (GABA
A
) receptors. We describe here a form of nongenomic progesterone signaling by showing that 3α-OH-DHP not only potentiates GABA
A
receptor-channel activity but also prevents its modulation by protein kinase C (PKC). Application of oxytocin or stimulation of PKC suppressed the postsynaptic GABA responses of oxytocin neurons in the absence, but not in the presence of 3α-OH-DHP. This finding was true at the juvenile stage and during late pregnancy, when the GABA
A
receptor is sensitive to 3α-OH-DHP. In contrast, after parturition, when the GABA
A
receptors expressed by oxytocin neurons are less sensitive to 3α-OH-DHP, this neurosteroid no longer counteracts PKC. The change in GABA
A
-receptor responsiveness to 3α-OH-DHP helps to explain the onset of firing activity and thus the induction of oxytocin release at parturition.
Stanniocalcin: A molecular guard of neurons during cerebral ischemia
Authors: Zhang Ke-zhou , Lindsberg Perttu J. , Tatlisumak Turgut , Kaste Markku , Olsen Henrik S. , Andersson Leif C.
Published: July 2012

Stanniocalcin (STC) is a glycoprotein hormone originally found in bony fish, in which it regulates calcium/phosphate homeostasis and protects against hypercalcemia. The recently characterized human STC shows about 70% homology with fish STC. We previously reported a constitutive expression of STC in terminally differentiated neurons. Here, we show that exposure of human neural-crest-derived cell line Paju to hypercalcemic culture medium induced expression of STC. Treatment of Paju cells with recombinant human STC increased their uptake of inorganic phosphate. Paju cells expressing STC by cDNA transfection displayed increased resistance to ischemic challenge and to elevated intracellular free calcium induced by treatment with thapsigargin. An up-regulated and redistributed expression of STC was observed in neurons surrounding the core of acute infarcts in human and rat brains. Given that mobilization and influx of calcium is considered a main neurotoxic mechanism following ischemia, our results suggest that the altered expression of STC contributes to the protection of cerebral neurons against hypoxic/ischemic damage. Manipulation of the STC expression may therefore offer a therapeutic approach to limit the injury after ischemic brain insults.
A retinitis pigmentosa GTPase regulator (RPGR)- deficient mouse model for X-linked retinitis pigmentosa (RP3)
Authors: Hong Dong-Hyun , Pawlyk Basil S. , Shang Jingzi , Sandberg Michael A. , Berson Eliot L. , Li Tiansen
Published: July 2012

The X-linked RP3 locus codes for retinitis pigmentosa GTPase regulator (RPGR), a protein of unknown function with sequence homology to the guanine nucleotide exchange factor for Ran GTPase. We created an RPGR-deficient murine model by gene knockout. In the mutant mice, cone photoreceptors exhibit ectopic localization of cone opsins in the cell body and synapses and rod photoreceptors have a reduced level of rhodopsin. Subsequently, both cone and rod photoreceptors degenerate. RPGR was found normally localized to the connecting cilia of rod and cone photoreceptors. These data point to a role for RPGR in maintaining the polarized protein distribution across the connecting cilium by facilitating directional transport or restricting redistribution. The function of RPGR is essential for the long-term maintenance of photoreceptor viability.
Activation of the human estrogen receptor by the antiestrogens ICI 182,780 and tamoxifen in yeast genetic systems: Implications for their mechanism of action
Authors: Dudley Mark W. , Sheeler Cameron Q. , Wang Hong , Khan Sohaib
Published: July 2012

The antiestrogens tamoxifen and ICI 182,780 have been portrayed as competitive antagonists of the estrogen binding site of the α-form of the human estrogen receptor (ER). However, in functional studies, neither compound has consistently been able to block estradiol-induced transcription. In this report, three yeast genetic systems were used to investigate the effects of tamoxifen and ICI 182,780 on ER dimerization, transcriptional activation, and the interaction of the receptor with a coactivator, RIP140. Tamoxifen and ICI 182,780 were able to induce ER dimerization and ER-dependent transcription, albeit at up to 15,000-fold higher concentrations than that of estradiol. In the presence of RIP140, the transcription response maximum was increased up to 30-fold for estradiol and both antiestrogens. Whole yeast cell [
3
H]estradiol binding studies demonstrated that tamoxifen could displace the estradiol from the ER, whereas ICI 182,780 treatment resulted in a 4-fold increase in [
3
H]estradiol binding to the receptor. No antagonism of estradiol was observed with tamoxifen or ICI 182,780 in any of the yeast models employed. We have concluded that the antiestrogen activity of compounds like tamoxifen and ICI 182,780 is not caused by their ability to competitively antagonize estradiol binding to the hormone binding site, but possibly by their ability to induce ER-dependent transcription, which in mammalian systems would result in receptor down-regulation. Compounds such as tamoxifen act through the hormone binding site, whereas ICI 182,780 may cause receptor activation through an allosteric binding site.
Mating experience and juvenile hormone enhance sexual signaling and mating in male Caribbean fruit flies
Authors: Teal P. E. A. , Gomez-Simuta Y. , Proveaux A. T.
Published: July 2012

Young mated male Caribbean fruit flies [
Anastrepha suspensa
(Loew)] have greater sexual prowess than their virgin counterparts. After mating for the first time, 6- to 7-day-old males released twice as much sex pheromone and acquired another mate in less than half the time required by virgin males of the same age. Mass spectroscopic analysis of extracts of hemolymph from mated and virgin 7-day-old males resulted in identification of juvenile hormone III bisepoxide and juvenile hormone III in a ratio of 2.5:1. Extracts from mated males contained 3-fold more juvenile hormone than did extracts from virgins. Enhancement of sexual signaling, pheromone release, and mating was induced by topical application of juvenile hormone, methoprene, or fenoxycarb. Newly eclosed adult males treated with juvenoids engaged in sexual signaling, released pheromone, and mated at significantly earlier ages than control males. We conclude that juvenile hormone mediated a positive feedback system that imparted a competitive advantage, guaranteeing that males who mated at an early age would out-compete virgins of the same age for mating opportunities. Additionally, the results support the hypothesis that juvenile hormone is a pivotal hormone coordinating the development of sexual signaling and reproductive maturity in these flies.