bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular localization of a group II chaperonin indicates a membrane-related function
Authors: Trent Jonathan D. , Kagawa Hiromi K. , Paavola Chad D. , McMillan R. Andrew , Howard Jeanie , Jahnke Linda , Lavin Colleen , Embaye Tsegereda , Henze Christopher E.
Published: December 2003

Chaperonins are protein complexes that are believed to function as part of a protein folding system in the cytoplasm of the cell. We observed, however, that the group II chaperonins known as rosettasomes in the hyperthermophilic archaeon
Sulfolobus shibatae
, are not cytoplasmic but membrane associated. This association was observed in cultures grown at 60°C and 76°C or heat-shocked at 85°C by using immunofluorescence microscopy and in thick sections of rapidly frozen cells grown at 76°C by using immunogold electron microscopy. We observed that increased abundance of rosettasomes after heat shock correlated with decreased membrane permeability at lethal temperature (92°C). This change in permeability was not seen in cells heat-shocked in the presence of the amino acid analogue azetidine 2-carboxylic acid, indicating functional protein synthesis influences permeability. Azetidine experiments also indicated that observed heat-induced changes in lipid composition in
S. shibatae
could not account for changes in membrane permeability. Rosettasomes purified from cultures grown at 60°C and 76°C or heat-shocked at 85°C bind to liposomes made from either the bipolar tetraether lipids of
Sulfolobus
or a variety of artificial lipid mixtures. The presence of rosettasomes did not significantly change the transition temperature of liposomes, as indicated by differential scanning calorimetry, or the proton permeability of liposomes, as indicated by pyranine fluorescence. We propose that these group II chaperonins function as a structural element in the natural membrane based on their intracellular location, the correlation between their functional abundance and membrane permeability, and their potential distribution on the membrane surface.
Genome evolution reveals biochemical networks and functional modules
Authors: von Mering Christian , Zdobnov Evgeny M. , Tsoka Sophia , Ciccarelli Francesca D. , Pereira-Leal Jose B. , Ouzounis Christos A. , Bork Peer
Published: December 2003

The analysis of completely sequenced genomes uncovers an astonishing variability between species in terms of gene content and order. During genome history, the genes are frequently rear-ranged, duplicated, lost, or transferred horizontally between genomes. These events appear to be stochastic, yet they are under selective constraints resulting from the functional interactions between genes. These genomic constraints form the basis for a variety of techniques that employ systematic genome comparisons to predict functional associations among genes. The most powerful techniques to date are based on conserved gene neighborhood, gene fusion events, and common phylogenetic distributions of gene families. Here we show that these techniques, if integrated quantitatively and applied to a sufficiently large number of genomes, have reached a resolution which allows the characterization of function at a higher level than that of the individual gene: global modularity becomes detectable in a functional protein network. In
Escherichia coli
, the predicted modules can be bench-marked by comparison to known metabolic pathways. We found as many as 74% of the known metabolic enzymes clustering together in modules, with an average pathway specificity of at least 84%. The modules extend beyond metabolism, and have led to hundreds of reliable functional predictions both at the protein and pathway level. The results indicate that modularity in protein networks is intrinsically encoded in present-day genomes.
Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells
Authors: Li Yi , Welm Bryan , Podsypanina Katrina , Huang Shixia , Chamorro Mario , Zhang Xiaomei , Rowlands Tracey , Egeblad Mikala , Cowin Pam , Werb Zena , Tan Lee K. , Rosen Jeffrey M. , Varmus Harold E.
Published: December 2003

Breast cancer is a genetically and clinically heterogeneous disease, and the contributions of different target cells and different oncogenic mutations to this heterogeneity are not well understood. Here we report that mammary tumors induced by components of the Wnt signaling pathway contain heterogeneous cell types and express early developmental markers, in contrast to tumors induced by other signaling elements. Expression of the
Wnt-1
protooncogene in mammary glands of transgenic mice expands a population of epithelial cells expressing progenitor cell markers, keratin 6 and Sca-1; subsequent tumors express these markers and contain luminal epithelial and myoepithelial tumor cells that share a secondary mutation, loss of
Pten
, implying that they arose from a common progenitor. Mammary tumors arising in transgenic mice expressing β-
catenin
and
c-Myc
, downstream components of the canonical Wnt signaling pathway, also contain a significant proportion of myoepithelial cells and cells expressing keratin 6. Progenitor cell markers and myoepithelial cells, however, are lacking in mammary tumors from transgenic mice expressing
Neu
, H-
Ras
, or
polyoma middle T antigen
. These results suggest that mammary stem cells and/or progenitors to mammary luminal epithelial and myoepithelial cells may be the targets for oncogenesis by Wnt-1 signaling elements. Thus, the developmental heterogeneity of different breast cancers is in part a consequence of differential effects of oncogenes on distinct cell types in the breast.
Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis
Authors: Uhlirova Mirka , Foy Brian D. , Beaty Barry J. , Olson Ken E. , Riddiford Lynn M. , Jindra Marek
Published: December 2003

The transcription factor Broad-Complex (BR-C) is required for differentiation of adult structures as well as for the programmed death of obsolete larval organs during metamorphosis of the fruit fly
Drosophila melanogaster
. Whether BR-C has a similar role in other holometabolous insects could not be proven without a loss-of-function genetic test, performed in a non-drosophilid species. Here we use a recombinant Sindbis virus as a tool to silence BR-C expression in the silkmoth
Bombyx mori
. The virus expressing a
BR-C
antisense RNA fragment reduced endogenous
BR-C
mRNA levels in infected tissues (adult wing and leg primordia) via RNA interference (RNAi). The RNAi knock-down of
BR-C
resulted in the failure of animals to complete the larval–pupal transition or in later morphogenetic defects, including differentiation of adult compound eyes, legs, and wings from their larval progenitors.
BR-C
RNAi also perturbed the programmed cell death of larval silk glands. These developmental defects correspond to loss-of-function phenotypes of
BR-C Drosophila
mutants in both the morphogenetic and degenerative aspects, suggesting that the critical role of
BR-C
in metamorphosis is evolutionarily conserved. We also demonstrate that the Sindbis virus is a useful vehicle for silencing of developmental genes in new insect models.
Transforming growth factor β1 induction of vascular endothelial growth factor receptor 1: Mechanism of pericyte-induced vascular survival
in vivo
Authors: Shih Shu-Ching , Ju Meihua , Liu Nan , Mo Jan-Rung , Ney Joshua J. , Smith Lois E. H.
Published: December 2003

Degeneration of vessels precedes and precipitates the devastating ischemia of many diseases, including retinopathy of prematurity and diabetic retinopathy. Ischemia then leads to proliferative retinopathy and blindness. Understanding the mechanisms of blood vessel degeneration is critical to prevention of these diseases. Vessel loss is associated with oxygen-induced suppression of vascular endothelial growth factor (VEGF) and with pericyte (vascular smooth muscle cell) dropout. The molecular mechanism of pericyte protection of the vasculature is unknown. We show that transforming growth factor β1 (TGF-β1)-expressing pericytes are specifically found on vessels resistant to oxygen-induced loss. TGF-β1 potently induces VEGF receptor 1 (VEGFR-1) expression in endothelial cells and thereby prevents oxygen-induced vessel loss
in vivo
. Vessel survival is further stimulated with a VEGFR-1-specific ligand, placental growth factor 1. TGF-β1 induction of VEGFR-1 in endothelial cells explains pericyte protection of vessels and the selective vulnerability of neonatal vessels to oxygen. These results implicate induction and activation of VEGFR-1 as critical targets to prevent vessel loss.
Targeting Schwann cells by nonlytic arenaviral infection selectively inhibits myelination
Authors: Rambukkana Anura , Kunz Stefan , Min Jenny , Campbell Kevin P. , Oldstone Michael B. A.
Published: December 2003

Members of the arenavirus family, famous for their hemorrhagic syndromes, cause distinct neurological disorders; however, cellular and molecular targets as well as pathogenesis of peripheral nervous system disorders associated with these viruses are unknown. Using noncytolytic lymphocytic choriomeningitis virus, the prototype arenavirus, and pseudotyped Lassa fever virus, we showed that the Schwann cells, but not the neurons, were preferentially targeted and harbored the virus. This permissiveness was caused by the viral glycoprotein usage of its receptor α-dystroglycan, which was highly abundant on Schwann cell membranes. Persistent lymphocytic choriomeningitis virus infection rendered immature Schwann cells defective or incapable of forming compact myelin sheathes when they differentiated to myelinating phenotype in anin vitrodifferentiation model of Schwann cells. Persistent infection did not cause Schwann cell apoptosis or cytopathic effect. Defects in myelination coincided with the down-regulation of dystroglycan expression and disruption of the laminin-2 organization and basal lamina assembly on Schwann cell-axon units. The data provide evidence for a selective perturbation of laminin-2-laminin-2 receptor communication pathway in the peripheral nervous system by a nonlytic virus and the resulting myelin defects, which may partly contribute to neurological abnormalities associated with arenaviral infection.
Increased phosphorylation of the neuronal L-type Ca
2+
channel Ca
v
1.2 during aging
Authors: Davare Monika A. , Hell Johannes W.
Published: December 2003

An increase in Ca
2+
influx through L-type Ca
2+
channels is thought to contribute to neuronal dysfunctions that underlie senile symptoms and Alzheimer's disease. The molecular basis of the age-dependent up-regulation in neuronal L-type Ca
2+
channel activity is largely unknown. We show that phosphorylation of the L-type channel Ca
v
1.2 by cAMP-dependent protein kinase is increased >2-fold in the hippocampus of aged rats. The hippocampus is critical for learning and is one of the first brain regions to be affected in Alzheimer's disease. Phosphorylation of Ca
v
1.2 by cAMP-dependent protein kinase strongly enhances its activity. Therefore, increased Ca
v
1.2 phosphorylation may account for a substantial portion of the age-related rise in neuronal Ca
2+
influx and its neuropathological consequences.
Molecular determinants of Ca
2+
/calmodulin-dependent regulation of Ca
v
2.1 channels
Authors: Lee Amy , Zhou Hong , Scheuer Todd , Catterall William A.
Published: December 2003

Ca
2+
-dependent facilitation and inactivation (CDF and CDI) of Ca
v
2.1 channels modulate presynaptic P/Q-type Ca
2+
currents and contribute to activity-dependent synaptic plasticity. This dual feedback regulation by Ca
2+
involves calmodulin (CaM) binding to the α
1
subunit (α
1
2.1). The molecular determinants for Ca
2+
-dependent modulation of Ca
v
2.1 channels reside in CaM and in two CaM-binding sites in the C-terminal domain of α
1
2.1, the CaM-binding domain (CBD) and the IQ-like domain. In transfected tsA-201 cells, CDF and CDI were both reduced by deletion of CBD. In contrast, alanine substitution of the first two residues of the IQ-like domain (IM-AA) completely prevented CDF but had little effect on CDI, and glutamate substitutions (IM-EE) greatly accelerated voltage-dependent inactivation but did not prevent CDI. Mutational analyses of the Ca
2+
binding sites of CaM showed that both the N- and C-terminal lobes of CaM were required for full development of facilitation, but only the N-terminal lobe was essential for CDI. In biochemical assays, CaM
12
and CaM
34
were unable to bind CBD, whereas CaM
34
but not CaM
12
retained Ca
2+
-dependent binding to the IQ-like domain. These findings support a model in which Ca
2+
binding to the C-terminal EF-hands of preassociated CaM initiates CDF via interaction with the IQ-like domain. Further Ca
2+
binding to the N-terminal EF-hands promotes secondary CaM interactions with CBD, which enhance facilitation and cause a conformational change that initiates CDI. This multifaceted mechanism allows positive regulation of Ca
v
2.1 in response to local Ca
2+
increases (CDF) and negative regulation during more global Ca
2+
increases (CDI).
Contribution of the H63D mutation in HFE to murine hereditary hemochromatosis
Authors: Tomatsu Shunji , Orii Koji O. , Fleming Robert E. , Holden Christopher C. , Waheed Abdul , Britton Robert S. , Gutierrez Monica A. , Velez-Castrillon Susana , Bacon Bruce R. , Sly William S.
Published: December 2003

Hereditary hemochromatosis (HH) is an autosomal recessive disease characterized by iron accumulation in several organs, followed by organ damage and failure. The C282Y mutation in the
HFE
gene explains 80-90% of all diagnosed cases of HH in populations of northwestern European ancestry. Targeted disruption of the mouse
Hfe
gene (or introduction of the murine mutation analogous to the C282Y human mutation) produces a murine model of HH. Another mutation in the
HFE
gene, H63D, is more prevalent than C282Y. However, the physiological consequences of the H63D mutation (as well as C282Y/H63D compound heterozygosity) on iron homeostasis are less well established. To evaluate the phenotypic consequences of the C282Y/H63D and H63D/H63D genotypes, we produced H67D (corresponding to H63D in humans) and C294Y (corresponding to C282Y in humans) knock-in mice. H67D homozygous mice, C294Y homozygous mice, and H67D/C294Y compound heterozygous mice each demonstrated hepatic iron loading. Even on a standard diet, by 10 weeks of age, hepatic iron levels in mice of these three genotypes were significantly higher than those of wild-type littermates. The relative severity of hepatic iron loading was C294Y/C294Y > C294Y/H67D > H67D/H67D. We conclude that the H67D allele, when homozygous or combined with a more consequential mutation like C294Y, leads to hepatic iron loading. These observations indicate that the H67D mutation leads to partial loss of Hfe function and can contribute to murine HH.
Ebola virus-like particles protect from lethal Ebola virus infection
Authors: Warfield Kelly L. , Bosio Catharine M. , Welcher Brent C. , Deal Emily M. , Mohamadzadeh Mansour , Schmaljohn Alan , Aman M. Javad , Bavari Sina
Published: December 2003

The filovirus Ebola causes hemorrhagic fever with 70–80% human mortality. High case-fatality rates, as well as known aerosol infectivity, make Ebola virus a potential global health threat and possible biological warfare agent. Development of an effective vaccine for use in natural outbreaks, response to biological attack, and protection of laboratory workers is a higher national priority than ever before. Coexpression of the Ebola virus glycoprotein (GP) and matrix protein (VP40) in mammalian cells results in spontaneous production and release of virus-like particles (VLPs) that resemble the distinctively filamentous infectious virions. VLPs have been tested and found efficacious as vaccines for several viruses, including papillomavirus, HIV, parvovirus, and rotavirus. Herein, we report that Ebola VLPs (eVLPs) were immunogenic
in vitro
as eVLPs matured and activated mouse bone marrow-derived dendritic cells, assessed by increases in cell-surface markers CD40, CD80, CD86, and MHC class I and II and secretion of IL-6, IL-10, macrophage inflammatory protein (MIP)-1α, and tumor necrosis factor α by the dendritic cells. Further, vaccinating mice with eVLPs activated CD4
+
and CD8
+
T cells, as well as CD19
+
B cells. After vaccination with eVLPs, mice developed high titers of Ebola virus-specific antibodies, including neutralizing antibodies. Importantly, mice vaccinated with eVLPs were 100% protected from an otherwise lethal Ebola virus inoculation. Together, our data suggest that eVLPs represent a promising vaccine candidate for protection against Ebola virus infections and a much needed tool to examine the genesis and nature of immune responses to Ebola virus.
Migration and differentiation of neural precursor cells can be directed by microglia
Authors: Aarum Johan , Sandberg Kristian , Haeberlein Samantha L. Budd , Persson Mats A. A.
Published: December 2003

Recent reports have supported the existence of neural stem cells in the adult mammalian CNS. Important features of such cells are self-renewal and multipotency, i.e., they can give rise to neurons, astrocytes, and oligodendrocytes and thus in principle replace lost cells in the CNS. Observations in several animal models of CNS diseases have shown that by unknown mechanisms endogenous as well as exogenous precursor cells preferentially migrate to damaged areas. Microglia are immunoreactive cells of nonneural lineage resident in the CNS. After injury to the CNS, microglia are rapidly activated and found concentrated at the sites of injury. In the present article we show, in two different assays, that soluble factors released from mouse microglial cells direct the migration of neural CNS precursor cells. We also provide evidence that microglia have the capacity to influence the differentiation of both adult and embryonic neural precursor cells toward a neuronal phenotype. Given that an invariant feature of pathological processes in CNS is the activation of microglia, these results indicate an important and unique role for microglia in directing the replacement of damaged or lost cells in the CNS.
Generating a synthetic genome by whole genome assembly: φX174 bacteriophage from synthetic oligonucleotides
Authors: Smith Hamilton O. , Hutchison Clyde A. , Pfannkoch Cynthia , Venter J. Craig
Published: December 2003

We have improved upon the methodology and dramatically shortened the time required for accurate assembly of 5- to 6-kb segments of DNA from synthetic oligonucleotides. As a test of this methodology, we have established conditions for the rapid (14-day) assembly of the complete infectious genome of bacteriophage φX174 (5,386 bp) from a single pool of chemically synthesized oligonucleotides. The procedure involves three key steps: (
i
) gel purification of pooled oligonucleotides to reduce contamination with molecules of incorrect chain length, (
ii
) ligation of the oligonucleotides under stringent annealing conditions (55°C) to select against annealing of molecules with incorrect sequences, and (
iii
) assembly of ligation products into full-length genomes by polymerase cycling assembly, a nonexponential reaction in which each terminal oligonucleotide can be extended only once to produce a full-length molecule. We observed a discrete band of full-length assemblies upon gel analysis of the polymerase cycling assembly product, without any PCR amplification. PCR amplification was then used to obtain larger amounts of pure full-length genomes for circularization and infectivity measurements. The synthetic DNA had a lower infectivity than natural DNA, indicating approximately one lethal error per 500 bp. However, fully infectious φX174 virions were recovered after electroporation into
Escherichia coli
. Sequence analysis of several infectious isolates verified the accuracy of these synthetic genomes. One such isolate had exactly the intended sequence. We propose to assemble larger genomes by joining separately assembled 5- to 6-kb segments; ≈60 such segments would be required for a minimal cellular genome.
Positional cloning and characterization of
Mei1
, a vertebrate-specific gene required for normal meiotic chromosome synapsis in mice
Authors: Libby Brian J. , Reinholdt Laura G. , Schimenti John C.
Published: December 2003

The mouse meiotic mutant
Mei1
was isolated in a screen for infertile mice descended from chemically mutagenized embryonic stem cells. Homozygotes of both sexes are sterile due to meiotic arrest caused by defects in chromosome synapsis. Notably, RAD51 protein does not load onto
Mei1
mutant meiotic chromosomes, suggesting that there is a defect in either recombinational repair or the production of double-strand breaks (DSBs) that require such repair. Here, we show that treatment of mutant males with cisplatin restores RAD51 loading, suggesting that mutant spermatocytes have intact recombinational repair mechanisms. Levels of histone H2AX phosphorylation (γH2AX) at leptonema are significantly reduced compared with wild-type controls but comparable to that seen in animals deficient for SPO11, the molecule required for catalyzing DSB formation during meiosis. These observations provide evidence that genetically programmed DSB induction is defective in
Mei1
leptotene spermatocytes. We also report the positional cloning of
Mei1
, which encodes a product without significant homology to any known protein. Expressed almost exclusively in gonads,
Mei1
has no apparent homologs in yeast, worms, or flies. However,
Mei1
orthologs are present in the genomes of mammals, chickens, and zebrafish. Thus,
Mei1
is required for vertebrate meiosis. To our knowledge,
Mei1
is the first meiosis-specific mutation identified by forward genetic approaches in mammals.
Borrelia burgdorferi
transcriptome in the central nervous system of non-human primates
Authors: Narasimhan Sukanya , Camaino Melissa J. , Liang Fang Ting , Santiago Felix , Laskowski Michelle , Philipp Mario T. , Pachner Andrew R. , Radolf Justin D. , Fikrig Erol
Published: December 2003

Neurological symptoms are common manifestations of Lyme disease; however, the paucibacillary nature of the spirochete in this environment has precluded a molecular analysis of the spirochete in the CNS. We have now adapted differential expression analysis by using a custom-amplified library (DECAL) in conjunction with
Borrelia burgdorferi
whole-genome and subgenome arrays to examine
in vivo
gene expression by
B. burgdorferi
in a non-human primate (NHP) model of neuroborreliosis. The expression profile of
B. burgdorferi
was examined in the CNS and heart of steroid-treated and immunocompetent NHPs. Eighty-six chromosomal genes and 80 plasmid-encoded genes were expressed at similar levels in the CNS and heart tissue of both immunocompetent and steroid-treated NHPs. The expression of 66 chromosomal genes and 32 plasmid-encoded genes was increased in the CNS of both immunocompetent and steroid-treated NHPs. It is likely that the expression of these genes is governed by physiological factors specific to the CNS milieu. However, 83 chromosomal and 114 plasmid-encoded genes showed contrasting expression profiles in steroid-treated and immunocompetent NHPs. The effect of dexamethasone on the immune status of the host as well as on the host metabolic pathways could contribute to these differences in the
B. burgdorferi
transcriptome. Results obtained herein underscore the complex interplay of host factors on
B. burgdorferi
gene expression
in vivo
. The results provide a global snapshot of the spirochetal transcriptome in the CNS and should spur the design of experiments aimed at understanding the molecular basis of neuroborreliosis.
An oxocarbenium-ion intermediate of a ribozyme reaction indicated by kinetic isotope effects
Authors: Unrau Peter J. , Bartel David P.
Published: December 2003

Many of the enzymes that catalyze reactions at nucleotide glycosidic linkages proceed through either a reactive oxocarbenium-ion intermediate or a transition state with considerable oxocarbenium character. To investigate how an RNA active site deals with the catalytic challenge of nucleotide synthesis, we probed the transition state of a ribozyme able to promote the formation of a pyrimidine nucleotide. Primary and secondary kinetic isotope effects indicate that this ribozyme stabilizes a highly dissociative reaction with considerable sp
2
hybridization and negligible bond order between the departing pyrophosphate leaving group and the anomeric carbon. The small primary
13
C isotope effect of 1.002 ± 0.003 indicates that the reaction is likely to be less concerted than that observed for protein nucleotide synthesis enzymes, which typically have primary
13
C isotope effects of 1.02–1.03. The dissociative nature of the ribozyme reaction most resembles the reaction of some hydrolytic enzymes, such as uracil DNA glycosylase, which uses the negative charges found in the phosphodiester backbone of its DNA substrate to transiently stabilize an oxocarbenium ion during hydrolysis. The detectable hydrolysis observed in the ribozyme reaction indicates that shielding of this reactive intermediate from water is a significant challenge for RNA, which protein enzymes that synthesize nucleotides have managed to overcome during evolution, apparently by the utilization of more concerted chemistry.
Hypervirulent mutant ofMycobacterium tuberculosisresulting from disruption of themce1operon
Authors: Shimono Nobuyuki , Morici Lisa , Casali Nicola , Cantrell Sally , Sidders Ben , Ehrt Sabine , Riley Lee W.
Published: December 2003

An estimated one-third of the world's population is latently infected withMycobacterium tuberculosis, the etiologic agent of tuberculosis. Here, we demonstrate that, unlike wild-typeM. tuberculosis, a strain ofM. tuberculosisdisrupted in themce1operon was unable to enter a stable persistent state of infection in mouse lungs. Instead, the mutant continued to replicate and killed the mice more rapidly than did the wild-type strain. Histological examination of mouse lungs infected with the mutant strain revealed diffusely organized granulomas with aberrant inflammatory cell migration. Murine macrophages infectedex vivowith the mutant strain were reduced in their ability to produce tumor necrosis factor α, IL-6, monocyte chemoattractant protein 1, and nitric oxide (NO), but not IL-4. Themce1mutant strain complemented with themce1genes stimulated tumor necrosis factor α and NO production by murine macrophages at levels stimulated by the wild-type strain. These observations indicate that themce1operon mutant is unable to stimulate T helper 1-type immunity in mice. The hypervirulence of the mutant strain may have resulted from its inability to stimulate a proinflammatory response that would otherwise induce organized granuloma formation and control the infection without killing the organism. Themce1operon ofM. tuberculosismay be involved in modulating the host inflammatory response in such a way that the bacterium can enter a persistent state without being eliminated or causing disease in the host.
Heterogeneity of rhythmic suprachiasmatic nucleus neurons: Implications for circadian waveform and photoperiodic encoding
Authors: Schaap Jeroen , Albus Henk , vanderLeest Henk Tjebbe , Eilers Paul H. C. , Détári László , Meijer Johanna H.
Published: December 2003

Circadian rhythms in neuronal ensemble, subpopulations, and single unit activity were recorded in the suprachiasmatic nuclei (SCN) of rat hypothalamic slices. Decomposition of the ensemble pattern revealed that neuronal subpopulations and single units within the SCN show surprisingly short periods of enhanced electrical activity of ≈5 h and show maximal activity at different phases of the circadian cycle. The summed activity accounts for the neuronal ensemble pattern of the SCN, indicating that circadian waveform of electrical activity is a composed tissue property. The recorded single unit activity pattern was used to simulate the responsiveness of SCN neurons to different photoperiods. We inferred predictions on changes in peak width, amplitude, and peak time in the multiunit activity pattern and confirmed these predictions with hypothalamic slices from animals that had been kept in a short or long photoperiod. We propose that the animals' ability to code for day length derives from plasticity in the neuronal network of oscillating SCN neurons.
Protection of
Cftr
knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing
Cftr
in airway epithelia
Authors: Koehler David R. , Sajjan Umadevi , Chow Yu-Hua , Martin Bernard , Kent Geraldine , Tanswell A. Keith , McKerlie Colin , Forstner Janet F. , Hu Jim
Published: December 2003

We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses
cystic fibrosis transmembrane conductance regulator
(
Cftr
) using control elements from
cytokeratin 18
. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice.
Cftr
RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of
Burkholderia cepacia
complex (Bcc).
Cftr
knockout mice (but not
Cftr
+/+
littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts.
Cftr
knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in
Cftr
+/+
littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens.
l
-Lysine acts like a partial serotonin receptor 4 antagonist and inhibits serotonin-mediated intestinal pathologies and anxiety in rats
Authors: Smriga Miro , Torii Kunio
Published: December 2003

The purpose of this investigation was to determine whether a nutritionally essential amino acid,
l
-lysine, acts like a serotonin receptor 4 (5-HT
4
) antagonist, and if
l
-lysine is beneficial in animal models of serotonin (5-HT)-induced anxiety, diarrhea, ileum contractions, and tachycardia and in stress-induced fecal excretion. The radioligand-binding assay was used to test the binding of
l
-lysine to various 5-HT receptors. The effects of
l
-lysine on 5-HT-induced contractions of isolated guinea pig ileum were studied
in vitro
. The effects of oral administration of
l
-lysine on diarrhea, stress-induced fecal excretion, and 5-HT-induced corticosterone release, tachycardia, and anxiety (an elevated plus maze paradigm) were studied in rats
in vivo
.
l
-Lysine (0.8 mmol/dl) inhibited (9.17%) binding of 5-HT to the 5-HT
4
receptor, without any effect on 5-HT
1A,2A,2B,2C,3
binding.
l
-Lysine (0.07 and 0.7 mmol/dl) blocked 5-HT-induced contractions of an isolated guinea pig ileum
in vitro
(
P
< 0.05 and
P
< 0.01). Orally applied
l
-lysine (1 g/kg of body weight) inhibited (
P
< 0.12) diarrhea triggered by coadministration of restraint stress and 5-hydroxytryptophane (10 mg/kg of body weight), and significantly blocked anxiety induced by the 5-HT
4
receptor agonist (3.0 mmol/liter) in rats
in vivo
. No effects of
l
-lysine or the 5-HT
4
receptor agonist on plasma corticosterone and heart rate were recorded.
l
-Lysine may be a partial 5-HT
4
receptor antagonist and suppresses 5-HT
4
receptor-mediated intestinal pathologies and anxiety in rats. An increase in nutritional load of
l
-lysine might be a useful tool in treating stress-induced anxiety and 5-HT-related diarrhea-type intestinal dysfunctions.
Molecular basis of calcium regulation in connexin-32 hemichannels
Authors: Gómez-Hernández Juan M. , de Miguel Marta , Larrosa Belen , González Daniel , Barrio Luis C.
Published: December 2003

In addition to forming gap-junction channels, a subset of connexins (Cxs) also form functional hemichannels. Most hemichannels are activated by depolarization, and opening depends critically on the external Ca
2+
concentration. Here we describe the mechanisms of action and the structural determinants underlying the Ca
2+
regulation of Cx32 hemichannels. At millimolar calcium concentrations, hemichannel voltage gating to the full open state of ≈90 pS is inhibited, and ion conduction at negative voltages of the partially open hemichannels (≈18 pS) is blocked. Thus, divalent cation blockage should be considered as a physiological mechanism to protect the cell from the potentially adverse effects of leaky hemichannels. A ring of 12 Asp residues within the external vestibule of the pore is responsible for the binding of Ca
2+
that accounts for both pore occlusion and blockage of gating. The residue Asp-169 of one subunit and the Asp-178 of an adjacent subunit must be arranged precisely to allow interactions with Ca
2+
to occur. Interestingly, a naturally occurring mutation (D178Y) that causes an inherited peripheral neuropathy induces a complete Ca
2+
deregulation of Cx32 hemichannel activity, suggesting that this dysfunction may be involved in the pathogenesis of the neuropathy.
Symmetry considerations and development of pinwheels in visual maps
Authors: Lee Ha Youn , Yahyanejad Mehdi , Kardar Mehran
Published: December 2003

Neurons in the visual cortex respond best to rod-like stimuli of given orientation. While the preferred orientation varies continuously across most of the cortex, there are prominent pinwheel centers around which all orientations are present. Oriented segments abound in natural images and tend to be collinear; neurons are also more likely to be connected if their preferred orientations are aligned to their topographic separation. These are indications of a reduced symmetry requiring joint rotations of both orientation preference and the underlying topography. We verify that this requirement extends to cortical maps of monkey and cat by direct statistical analysis. Furthermore, analytical arguments and numerical studies indicate that pinwheels are generically stable in evolving field models that couple orientation and topography.
Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes
Authors: Kroken Scott , Glass N. Louise , Taylor John W. , Yoder O. C. , Turgeon B. Gillian
Published: December 2003

Fungal type I polyketides (PKs) are synthesized by PK synthases (PKSs) and include well known secondary metabolites such as the anticholesterol drug lovastatin and the potent natural carcinogen aflatoxin. Other type I PKs are known to be virulence factors for some plant pathogens and pigments such as melanin. In this study, a phylogenomic approach was used to investigate the origin and diversity of fungal genes encoding putative PKSs that are predicted to synthesize type I PKs. The resulting genealogy, constructed by using the highly conserved PKS ketosynthase (KS) domain, indicated that: (
i
) Species within subphylum Pezizomycotina (phylum Ascomycota) but not early diverging ascomycetes, like
Saccharomyces cerevisiae
(Saccharomycotina) or
Schizosaccharomyces pombe
(Taphrinomycotina), had large numbers (7–25) of
PKS
genes. (
ii
) Bacteria and fungi had separate groups of
PKS
genes; the few exceptions are the likely result of horizontal gene transfer from bacteria to various sublineages of fungi. (
iii
) The bulk of genes encoding fungal PKSs fell into eight groups. Four groups were predicted to synthesize variously reduced PKs, and four groups were predicted to make unreduced PKs. (
iv
) Species within different classes of Pezizomycotina shared the same groups of
PKS
genes. (
v
) Different fungal genomes shared few putative orthologous
PKS
genes, even between closely related genomes in the same class or genus. (
vi
) The discontinuous distributions of orthologous
PKS
s among fungal species can be explained by gene duplication, divergence, and gene loss; horizontal gene transfer among fungi does not need to be invoked.
A general strategy for target-promoted alkylation in biological systems
Authors: Zhou Qibing , Rokita Steven E.
Published: December 2003

Selective alkylation of a chosen sequence of DNA typically relies on ligand-directed delivery of a compound that expresses an intrinsic reactivity. A significant and biologically relevant enhancement in specificity is theoretically possible if such an intrinsic reactivity could be replaced by a latent activity induced solely by the target of interest, but examples of this are rare and not easily emulated. A simple strategy for target-promoted alkylation is now illustrated by an intramolecular adduct formed by an oligonucleotide–quinone methide conjugate. This adduct persists in the absence of a complementary sequence of DNA for at least 8 days, yet remarkably is able to alkylate target DNA upon duplex hybridization. Neither formation of the intramolecular self-adduct nor transfer of the quinone methide to its target is significantly quenched by 450-fold excess 2-mercaptoethanol. Similarly, noncomplementary DNA is neither subject to alkylation by the self-adduct nor able to effect its consumption. Reversible trapping of the nascent quinone methide through an intramolecular reaction thus appears efficient enough to inhibit competing intermolecular reaction. Only complementary base pairing induces a conformational change necessary to promote intermolecular transfer of the quinone methide. Generalization of this approach based on reversible intramolecular trapping of a reactive intermediate by a ligand with multiple recognition subdomains has the potential for wide-ranging applications in targeting nucleic acids and proteins.
Fungal endophytes limit pathogen damage in a tropical tree
Authors: Arnold A. Elizabeth , Mejía Luis Carlos , Kyllo Damond , Rojas Enith I. , Maynard Zuleyka , Robbins Nancy , Herre Edward Allen
Published: December 2003

Every plant species examined to date harbors endophytic fungi within its asymptomatic aerial tissues, such that endophytes represent a ubiquitous, yet cryptic, component of terrestrial plant communities. Fungal endophytes associated with leaves of woody angiosperms are especially diverse; yet, fundamental aspects of their interactions with hosts are unknown. In contrast to the relatively species-poor endophytes that are vertically transmitted and act as defensive mutualists of some temperate grasses, the diverse, horizontally transmitted endophytes of woody angiosperms are thought to contribute little to host defense. Here, we document high diversity, spatial structure, and host affinity among foliar endophytes associated with a tropical tree (
Theobroma cacao
, Malvaceae) across lowland Panama. We then show that inoculation of endophyte-free leaves with endophytes isolated frequently from naturally infected, asymptomatic hosts significantly decreases both leaf necrosis and leaf mortality when
T. cacao
seedlings are challenged with a major pathogen (
Phytophthora
sp.). In contrast to reports of fungal inoculation inducing systemic defense, we found that protection was primarily localized to endophyte-infected tissues. Further, endophyte-mediated protection was greater in mature leaves, which bear less intrinsic defense against fungal pathogens than do young leaves.
In vitro
studies suggest that host affinity is mediated by leaf chemistry, and that protection may be mediated by direct interactions of endophytes with foliar pathogens. Together, these data demonstrate the capacity of diverse, horizontally transmitted endophytes of woody angiosperms to play an important but previously unappreciated role in host defense.
Plastid protein synthesis is required for plant development in tobacco
Authors: Ahlert Daniela , Ruf Stephanie , Bock Ralph
Published: December 2003

Chloroplasts fulfill important functions in cellular metabolism. The majority of plastid genome-encoded genes is involved in either photosynthesis or chloroplast gene expression. Whether or not plastid genes also can determine extraplastidic functions has remained controversial. We demonstrate here an essential role of plastid protein synthesis in tobacco leaf development. By using chloroplast transformation, we have developed an experimental system that produces recombination-based knockouts of chloroplast translation in a cell-line-specific manner. The resulting plants are chimeric and, in the presence of translational inhibitors, exhibit severe developmental abnormalities. In the absence of active plastid protein synthesis, leaf blade development is abolished because of an apparent arrest of cell division. This effect appears to be cell-autonomous in that adjacent sectors of cells with translating plastids are phenotypically normal but cannot complement for the absence of plastid translation in mutant sectors. Developmental abnormalities also are seen in flower morphology, indicating that the defects are not caused by inhibited expression of plastid photosynthesis genes. Taken together, our data point to an unexpected essential role of plastid genes and gene expression in plant development and cell division.
Host defense reinforces host–parasite cospeciation
Authors: Clayton Dale H. , Bush Sarah E. , Goates Brad M. , Johnson Kevin P.
Published: December 2003

Cospeciation occurs when interacting groups, such as hosts and parasites, speciate in tandem, generating congruent phylogenies. Cospeciation can be a neutral process in which parasites speciate merely because they are isolated on diverging host islands. Adaptive evolution may also play a role, but this has seldom been tested. We explored the adaptive basis of cospeciation by using a model system consisting of feather lice (Columbicola) and their pigeon and dove hosts (Columbiformes). We reconstructed phylogenies for both groups by using nuclear and mitochondrial DNA sequences. Both phylogenies were well resolved and well supported. Comparing these phylogenies revealed significant cospeciation and correlated evolution of host and parasite body size. The match in body size suggested that adaptive constraints limit the range of hosts lice can use. We tested this hypothesis by transferring lice among hosts of different sizes to simulate host switches. The results of these experiments showed that lice cannot establish viable populations on novel hosts that differ in size from the native host. To determine why size matters, we measured three components of louse fitness: attachment, feeding, and escape from host defense (preening). Lice could remain attached to, and feed on, hosts varying in size by an order of magnitude. However, they could not escape from preening on novel hosts that differed in size from the native host. Overall, our results suggest that host defense reinforces cospeciation in birds and feather lice by preventing lice from switching between hosts of different sizes.
Chemocyanin, a small basic protein from the lily stigma, induces pollen tube chemotropism
Authors: Kim Sunran , Mollet Jean-Claude , Dong Juan , Zhang Kangling , Park Sang-Youl , Lord Elizabeth M.
Published: December 2003

In plant reproduction, pollination is an essential process that delivers the sperm through specialized extracellular matrices (ECM) of the pistil to the ovule. Although specific mechanisms of guidance for pollen tubes through the pistil are not known, the female tissues play a critical role in this event. Many studies have documented the existence of diffusible chemotropic factors in the lily stigma that can induce pollen tube chemotropism
in vitro
, but no molecules have been isolated to date. In this study, we identified a chemotropic compound from the stigma by use of biochemical methods. We purified a lily stigma protein that is active in an
in vitro
chemotropism assay by using cation exchange, gel filtration, and HPLC. Tryptic digestion of the protein yielded peptides that identified the protein as a plantacyanin (basic blue protein), and this was confirmed by cloning the cDNA from the lily stigma. Plantacyanins are small cell wall proteins of unknown function. The measured molecular mass by electrospray ionization ion source MS is 9,898 Da, and the molecular mass of the mature protein (calculated from the cDNA) is 9,900.2 Da. Activity of the lily plantacyanin (named chemocyanin) is enhanced in the presence of stigma/stylar cysteine-rich adhesin, previously identified as a pollen tube adhesin in the lily style.
Direct observation of tiers in the energy landscape of a chromoprotein: A single-molecule study
Authors: Hofmann Clemens , Aartsma Thijs J. , Michel Hartmut , Köhler Jürgen
Published: December 2003

Single-molecule spectroscopic techniques were applied to individual pigments embedded in a chromoprotein. A sensitive tool to monitor structural fluctuations of the protein backbone in the local environment of the chromophore is provided by recording the changes of the spectral positions of the pigment absorptions as a function of time. The data provide information about the organization of the energy landscape of the protein in tiers that can be characterized by an average barrier height. Additionally, a correlation between the average barrier height within a distinct tier and the time scale of the structural fluctuations is observed.
In vivo
oligomerization and raft localization of Ebola virus protein VP40 during vesicular budding
Authors: Panchal Rekha G. , Ruthel Gordon , Kenny Tara A. , Kallstrom George H. , Lane Douglas , Badie Shirin S. , Li Limin , Bavari Sina , Aman M. Javad
Published: December 2003

The matrix protein VP40 plays a critical role in Ebola virus assembly and budding, a process that utilizes specialized membrane domains known as lipid rafts. Previous studies with purified protein suggest a role for oligomerization of VP40 in this process. Here, we demonstrate VP40 oligomers in lipid rafts of mammalian cells, virus-like particles, and in the authentic Ebola virus. By mutagenesis, we identify several critical C-terminal sequences that regulate oligomerization at the plasma membrane, association with detergent-resistant membranes, and vesicular release of VP40, directly linking these phenomena. Furthermore, we demonstrate the active recruitment of TSG101 into lipid rafts by VP40. We also report the successful application of the biarsenic fluorophore, FlAsH, combined with a tetracysteine tag for imaging of Ebola VP40 in live cells.
Visualizing lipid structure and raft domains in living cells with two-photon microscopy
Authors: Gaus Katharina , Gratton Enrico , Kable Eleanor P. W. , Jones Allan S. , Gelissen Ingrid , Kritharides Leonard , Jessup Wendy
Published: December 2003

The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell–cell contacts and cover 10–15% of the cell surface at 37°C. By deconvoluting the images, we demonstrate the existence of phase separation
in vivo
. We compare the properties of microscopically visible domains (<1 μm
2
), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis.