bluuebunny/crossref_metadata_2025 huggingface dataset:
Myoblasts transplanted into rat infarcted myocardium are functionally isolated from their host
Authors: Léobon Bertrand , Garcin Isabelle , Menasché Philippe , Vilquin Jean-Thomas , Audinat Etienne , Charpak Serge
Published: June 2003

Survival and differentiation of myogenic cells grafted into infarcted myocardium have raised the hope that cell transplantation becomes a new therapy for cardiovascular diseases. The approach was further supported by transplantation of skeletal myoblasts, which was shown to improve cardiac performance in several animal species. Despite the success of myoblast transplantation and its recent trial in human, the mechanism responsible for the functional improvement remains unclear. Here, we used intracellular recordings coupled to video and fluorescence microscopy to establish whether myoblasts, genetically labeled with enhanced GFP and transplanted into rat infarcted myocardium, retain excitable and contractile properties, and participate actively to cardiac function. Our results indicate that grafted myoblasts differentiate into peculiar hyperexcitable myotubes with a contractile activity fully independent of neighboring cardiomyocytes. We conclude that mechanisms other than electromechanical coupling between grafted and host cells are involved in the improvement of cardiac function.
Oncogenic potential of TASK3 (Kcnk9) depends on K+channel function
Authors: Pei Lin , Wiser Ofer , Slavin Anthony , Mu David , Powers Scott , Jan Lily Yeh , Hoey Timothy
Published: June 2003

TASK3 gene (Kcnk9) is amplified and overexpressed in several types of human carcinomas. In this report, we demonstrate that a point mutation (G95E) within the consensus K+filter of TASK3 not only abolished TASK3 potassium channel activity but also abrogated its oncogenic functions, including proliferation in low serum, resistance to apoptosis, and promotion of tumor growth. Furthermore, we provide evidence that TASK3G95Eis a dominant-negative mutation, because coexpression of the wild-type and the mutant TASK3 resulted in inhibition of K+current of wild-type TASK3 and its tumorigenicity in nude mice. These results establish a direct link between the potassium channel activity of TASK3 and its oncogenic functions and imply that blockers for this potassium channel may have therapeutic potential for the treatment of cancers.
A newly discovered cholesteryl galactoside from
Borrelia burgdorferi
Authors: Ben-Menachem Gil , Kubler-Kielb Joanna , Coxon Bruce , Yergey Alfred , Schneerson Rachel
Published: June 2003

Two major glycolipids, which comprise ≈36% of the total lipid mass from
Borrelia burgdorferi
, the etiological agent of Lyme disease, were investigated. We determined the fatty acid type, sugar identity, anomeric configuration, and substituent type and position. The structures were identified as cholesteryl 6-
O
-acyl-β-
d
-galactopyranoside (
B. burgdorferi
glycolipid 1, BbGL-I), and 1,2-di-
O
-acyl-3-
O
-α-
d
-galactopyranosyl-
sn
-glycerol (BbGL-II). The major fatty acids were palmitate and oleate. The structures were corroborated by gas–liquid chromatography MS, matrix-assisted laser desorption/ionization time-of-flight spectroscopy, fast atom bombardment MS, detailed NMR spectrometry, and metabolic labeling. This is a previously undescribed demonstration of a cholesteryl galactoside in bacteria. Lipopolysaccharide was not detected in
B. burgdorferi
. The two glycolipids have several properties suggesting they may function as lipopolysaccharide: both are main components of the bacterial membrane, surface exposed, and have a three-domain structure. BbGL-I elicited specific antibodies in mice and rabbits, and BbGL-II elicited antibodies that reacted with both glycolipids.
Evidence that mouse brain neuropathy target esterase is a lysophospholipase
Authors: Quistad Gary B. , Barlow Carrolee , Winrow Christopher J. , Sparks Susan E. , Casida John E.
Published: June 2003

Neuropathy target esterase (NTE) is inhibited by several organophosphorus (OP) pesticides, chemical warfare agents, lubricants, and plasticizers, leading to OP-induced delayed neuropathy in people (>30,000 cases of human paralysis) and hens (the best animal model for this demyelinating disease). The active site region of NTE as a recombinant protein preferentially hydrolyzes lysolecithin, suggesting that this enzyme may be a type of lysophospholipase (LysoPLA) with lysolecithin as its physiological substrate. This hypothesis is tested here in mouse brain by replacing the phenyl valerate substrate of the standard NTE assay with lysolecithin for an “NTE-LysoPLA” assay with four important findings. First, NTE-LysoPLA activity, as the NTE activity, is 41–45% lower inNte-haploinsufficient transgenic mice than in their wild-type littermates. Second, the potency of six delayed neurotoxicants or toxicants asin vitroinhibitors varies from IC500.02 to 13,000 nM and is essentially the same for NTE-LysoPLA and NTE (r2= 0.98). Third, the same six delayed toxicants administered i.p. to mice at multiple doses inhibit brain NTE-LysoPLA and NTE to the same extent (r2= 0.90). Finally, theirin vivoinhibition of brain NTE-LysoPLA generally correlates with delayed toxicity. Therefore, OP-induced delayed toxicity in mice, and possibly the hyperactivity associated with NTE deficiency, may be due to NTE-LysoPLA inhibition, leading to localized accumulation of lysolecithin, a known demyelinating agent and receptor-mediated signal transducer. This mouse model has some features in common with OP-induced delayed neuropathy in hens and people but differs in the neuropathological signs and apparently the requirement for NTE aging.
Direct molecular haplotyping of long-range genomic DNA with M1-PCR
Authors: Ding Chunming , Cantor Charles R.
Published: June 2003

Haplotypes, combinations of several phase-determined polymorphic markers, are extremely valuable for studies of disease association and chromosome evolution. Here we describe a technique called M1-PCR (M for ``multiplex'' and 1 for ``single-copy DNA molecules'') that enables direct molecular haplotyping of several polymorphic markers separated by as many as 24 kb. A genomic DNA sample first is diluted to approximately single-copy. The haplotype is directly determined by simultaneously genotyping several polymorphic markers in the same reaction with a multiplex PCR and base extension reaction. This approach does not rely on pedigree data and does not require previous amplification of the entire genomic region containing the selected markers.
Carbohydrate-dependent signaling from the phosphatidylglucoside-based microdomain induces granulocytic differentiation of HL60 cells
Authors: Nagatsuka Yasuko , Hara-Yokoyama Miki , Kasama Takeshi , Takekoshi Masataka , Maeda Fumiko , Ihara Seiji , Fujiwara Shigeyoshi , Ohshima Eriko , Ishii Kumiko , Kobayashi Toshihide , Shimizu Kazufumi , Hirabayashi Yoshio
Published: June 2003

Glycosphingolipids form glycosphingolipid signaling microdomains. Here, we report an unrecognized type of phosphatidylglucoside (PhGlc)-based lipid microdomain in HL60 cells. Treatment of cells with rGL-7, which preferentially reacts with PhGlc, induced differentiation of HL60 cells. This was manifested by the appearance of nitroblue tetrazolium-positive cells together with CD38 expression and c-Myc down-regulation. We determined the molecular mechanisms underlying early stages of signal transduction. rGL-7 treatment induced rapid tyrosine phosphorylation of Src family protein kinases Lyn and Hck. Reduction of endogenous cholesterol after application of methyl-β-cyclodextrin suppressed rGL-7-stimulated tyrosine phosphorylation. Phosphorylated proteins and PhGlc colocalized in the Triton X-100 insoluble, light buoyant density fraction after sucrose gradient ultracentrifugation of HL60 cell lysates. This suggests PhGlc-based microdomain is involved in GL-7 signaling. Ligation of known components of microdomains, such as sphingomyelin and ganglioside GM1, with corresponding antibodies failed to induce differentiation and tyrosine phosphorylation. These results show that PhGlc constitutes a previously undescribed lipid signaling domain, and the glucose residue of PhGlc is critical for organization of the carbohydrate-dependent signaling domain involved in cellular differentiation of HL60 cells.
The interplay of glycosylation and disulfide formation influences fibrillization in a prion protein fragment
Authors: Bosques Carlos J. , Imperiali Barbara
Published: June 2003

It is now accepted that the structural transition from cellular prion protein (PrP
C
) to proteinase K-resistant prion protein scrapie (PrP
Sc
) is the major event leading to transmissible spongiform encephalopathies. Although the mechanism of this transition remains elusive, glycosylation has been proposed to impede the PrP
C
to PrP
Sc
conversion. To address the role of glycosylation, we have prepared glycosylated and unglycosylated peptides derived from the 175–195 fragment of the human prion protein. Comparison of the structure, aggregation kinetics, fibril formation capabilities, and redox susceptibility of Cys-179 has shown that the N-linked glycan (at Asn-181) significantly reduces the rate of fibrillization by promoting intermolecular disulfide formation via Cys-179. Further-more, the aggressive fibrillization of a C179S mutant of this fragment highlights the significant role of disulfide stability in retarding the rate of fibril formation. The implications of these studies are discussed in the context of fibril formation in the intact prion protein.
Genes encoding putative natural killer cell C-type lectin receptors in teleostean fishes
Authors: Sato Akie , Mayer Werner E. , Overath Peter , Klein Jan
Published: June 2003

Mammalian natural killer (NK) cells are cytotoxic lymphocytes that express receptors specific for MHC class I molecules. The NK cell receptors belong to two structurally unrelated families, the killer cell Ig-like receptors and the killer cell C-type lectin receptors. We describe a cDNA clone derived from the bony (cichlid) fish
Paralabidochromis chilotes
and show that it encodes a protein related to the CD94/NK cell group 2 (NKG2) subfamily of the killer cell C-type lectin receptors. The gene encoding this receptor in a related species,
Oreochromis niloticus
, has a similar structure to the human
CD94
/
NKG2
genes and is a member of a multigene cluster that resembles the mammalian NK cell gene complex. Thus, the CD94/NKG2 subfamily of NK cell receptors must have arisen before the divergence of fish and tetrapods and may have retained its function (possibly monitoring the expression of MHC class I molecules) for >400 million years.
Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes
Authors: Beard Brian C. , Wilson Samuel H. , Smerdon Michael J.
Published: June 2003

The majority of DNA in eukaryotic cells exists in the highly condensed structural hierarchy of chromatin, which presents a challenge to DNA repair enzymes in that recognition, incision, and restoration of the original sequence at most sites must take place within these structural constraints. To test base excision repair (BER) activities on chromatin substrates, an
in vitro
system was developed that uses human uracil DNA glycosylase (UDG), apyrimidinic/apurinic endonuclease (APE), and DNA polymerase β (pol β) on homogeneously damaged, rotationally positioned DNA in nucleosomes. We find that UDG and APE carry out their combined catalytic activities with reduced efficiency on nucleosome substrates (≈10% of that on naked DNA). Furthermore, these enzymes distinguish between two different rotational settings of the lesion on the histone surface, showing a 2- to 3-fold difference in activity between uracil facing ``toward'' and ``away from'' the histones. However, UDG and APE will digest such substrates to completion in a concentration-dependent manner. Conversely, the synthesis activity of pol β is inhibited completely by nucleosome substrates and is independent of enzyme concentration. These results suggest that the first two steps of BER, UDG and APE, may occur ``unassisted'' in chromatin, whereas downstream factors in this pathway (i.e., pol β) may require nucleosome remodeling for efficient DNA BER in at least some regions of chromatin in eukaryotic cells.
Human and mouse genomic sequences reveal extensive breakpoint reuse in mammalian evolution
Authors: Pevzner Pavel , Tesler Glenn
Published: June 2003

The human and mouse genomic sequences provide evidence for a larger number of rearrangements than previously thought and reveal extensive reuse of breakpoints from the same short fragile regions. Breakpoint clustering in regions implicated in cancer and infertility have been reported in previous studies; we report here on breakpoint clustering in chromosome evolution. This clustering reveals limitations of the widely accepted random breakage theory that has remained unchallenged since the mid-1980s. The genome rearrangement analysis of the human and mouse genomes implies the existence of a large number of very short “hidden” synteny blocks that were invisible in the comparative mapping data and ignored in the random breakage model. These blocks are defined by closely located breakpoints and are often hard to detect. Our results suggest a model of chromosome evolution that postulates that mammalian genomes are mosaics of fragile regions with high propensity for rearrangements and solid regions with low propensity for rearrangements.
Expression of prestin-homologous solute carrier (SLC26) in auditory organs of nonmammalian vertebrates and insects
Authors: Weber Thomas , Göpfert Martin C. , Winter Harald , Zimmermann Ulrike , Kohler Hanni , Meier Alexandra , Hendrich Oliver , Rohbock Karin , Robert Daniel , Knipper Marlies
Published: June 2003

Prestin, the fifth member of the anion transporter family SLC26, is the outer hair cell molecular motor thought to be responsible for active mechanical amplification in the mammalian cochlea. Active amplification is present in a variety of other auditory systems, yet the prevailing view is that prestin is a motor molecule unique to mammalian ears. Here we identify prestin-related SLC26 proteins that are expressed in the auditory organs of nonmammalian vertebrates and insects. Sequence comparisons revealed the presence of SLC26 proteins in fish (
Danio
, GenBank accession no. AY278118, and
Anguilla
, GenBank accession no. BAC16761), mosquitoes (
Anopheles
, GenBank accession nos. EAA07232 and EAA07052), and flies (
Drosophila
, GenBank accession no. AAF49285). The fly and zebrafish homologues were cloned and, by using
in situ
hybridization, shown to be expressed in the auditory organs. In mosquitoes, in turn, the expression of prestin homologues was demonstrated for the auditory organ by using highly specific riboprobes against rat prestin. We conclude that prestin-related SLC26 proteins are widespread, possibly ancestral, constituents of auditory organs and are likely to serve salient roles in mammals and across taxa.
The coiled-coil of the human Rad50 DNA repair protein contains specific segments of increased flexibility
Authors: van Noort John , van der Heijden Thijn , de Jager Martijn , Wyman Claire , Kanaar Roland , Dekker Cees
Published: June 2003

Protein structural features are usually determined by defining regularities in a large population of homogeneous molecules. However, irregular features such as structural variation and flexibility are likely to be missed, despite their vital role for their biological function. In this paper, we report the observation of striking irregularities in the flexibility of the coiled-coil region of the human Rad50 DNA repair protein. Existing methods to quantitatively analyze flexibility are applicable to homogeneous polymers only. Because protein coiled-coils cannot be assumed to be homogeneous, we develop a method to quantify the
local
flexibility from high-resolution atomic force microscopy images. Indeed, in Rad50 coiled-coils, two positions of increased flexibility are observed. We discuss how this dynamic structural feature is integral to Rad50 function.
Crystal structure of the SOS cell division inhibitor SulA and in complex with FtsZ
Authors: Cordell Suzanne C. , Robinson Elva J. H. , Löwe Jan
Published: June 2003

SulA halts cell division in
Escherichia coli
by binding to the major component of the division machinery FtsZ. We have solved the crystal structure of SulA alone and in complex with FtsZ from
Pseudomonas aeruginosa
. SulA is expressed when the SOS response is induced. This is a mechanism to inhibit cell division and repair DNA in the event of DNA damage. FtsZ is a tubulin-like protein that forms polymers, with the active-site GTPase split across two monomers. One monomer provides the GTP-binding site and the other, through its T7 loop nucleotide hydrolysis. Our structures show that SulA is a dimer, and that SulA inhibits cell division neither by binding the nucleotide-binding site nor by inducing conformational changes in FtsZ. Instead, SulA binds the T7 loop surface of FtsZ, opposite the nucleotide-binding site, blocking polymer formation. These findings explain why GTP hydrolysis and polymer turnover are required for SulA inhibition.
An unusual type IB topoisomerase from African trypanosomes
Authors: Bodley Annette L. , Chakraborty Asit K. , Xie Suji , Burri Christian , Shapiro Theresa A.
Published: June 2003

African trypanosomes are ancient eukaryotes that cause lethal disease in humans and cattle. Available drugs are inadequate and the need for new therapeutic targets is great.
Trypanosoma brucei
and related pathogens differ strikingly from higher eukaryotes in many aspects of nucleic acid structure and metabolism. We find yet another example of this in their unusual DNA topoisomerase IB. Type IB topoisomerases relieve the supercoils that accumulate during DNA and RNA synthesis, and are of considerable importance as the target for antitumor camptothecins. Dozens of type IB topoisomerases sequenced from eukaryotes, bacteria, and pox viruses are all encoded by a single gene that predictably contains a highly conserved DNA binding domain and C-terminal catalytic domain, linked by a nonconserved hydrophilic region. We find that topoisomerase IB in
T. brucei
is encoded by two genes: one for the DNA-binding domain and a second for the C-terminal catalytic domain. In keeping with this, highly purified fractions of native
T. brucei
topoisomerase IB catalytic activity contain two proteins, of 90 and 36 kDa. The native enzyme is conventional in its Mg
2
+
-independence, ability to relax positive and negative supercoils, and inhibition by camptothecin. Camptothecin promotes the formation of a covalent complex between
32
P-labeled substrate DNA and the small subunit. This unusual structural organization may provide a missing link in the evolution of type IB enzymes, which are thought to have arisen over time from the fusion of two independent domains. It also provides another basis for the design of selectively toxic drug candidates.
Validation of the hexose transporter of
Plasmodium falciparum
as a novel drug target
Authors: Joët Thierry , Eckstein-Ludwig Ursula , Morin Christophe , Krishna Sanjeev
Published: June 2003

Chemotherapy of malaria parasites is limited by established drug resistance and lack of novel targets. Intraerythrocytic stages of
Plasmodium falciparum
are wholly dependent on host glucose for energy. Glucose uptake is mediated by a parasite-encoded facilitative hexose transporter (PfHT). We report that
O
-3 hexose derivatives inhibit uptake of glucose and fructose by PfHT when expressed in
Xenopus
oocytes. Selectivity of these derivatives for PfHT is confirmed by lack of inhibition of hexose transport by the major mammalian glucose and fructose transporters (Gluts) 1 and 5. A long chain
O
-3 hexose derivative is the most effective inhibitor of PfHT and also kills
P. falciparum
when it is cultured in medium containing either glucose or fructose as a carbon source. To extend our observations to the second most important human malarial pathogen, we have cloned and expressed the
Plasmodium vivax
orthologue of PfHT, and demonstrate inhibition of glucose uptake by the long chain
O
-3 hexose derivative. Furthermore, multiplication of
Plasmodium berghei
in a mouse model is significantly reduced by the
O
-3 derivative. Our robust expression system conclusively validates PfHT as a novel drug target and is an important step in the development of novel antimalarials directed against membrane transport proteins.
Caspase 8 small interfering RNA prevents acute liver failure in mice
Authors: Zender Lars , Hütker Sebastian , Liedtke Christian , Tillmann Hans Ludger , Zender Steffen , Mundt Bettina , Waltemathe Morlen , Gösling Thomas , Flemming Peer , Malek Nisar Peter , Trautwein Christian , Manns Michael Peter , Kühnel Florian , Kubicka Stefan
Published: June 2003

A major concern in therapy of acute liver failure is protection of hepatocytes to prevent apoptosis and maintain liver function. Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells. To evaluate the therapeutic efficacy of siRNA
in vivo
we used different mouse models of acute liver failure. We directed 21-nt siRNAs against caspase 8, which is a key enzyme in death receptor-mediated apoptosis. Systemic application of caspase 8 siRNA results in inhibition of caspase 8 gene expression in the liver, thereby preventing Fas (CD95)-mediated apoptosis. Protection of hepatocytes by caspase 8 siRNA significantly attenuated acute liver damage induced by agonistic Fas (CD95) antibody (Jo2) or by adenovirus expressing Fas ligand (AdFasL). However, in a clinical situation the siRNAs most likely would be applied after the onset of acute liver failure. Therefore we injected caspase 8 siRNA at a time point during AdFasL- and adenovirus wild type (Adwt)-mediated liver failure with already elevated liver transaminases. Improvement of survival due to RNA interference was significant even when caspase 8 siRNA was applied during ongoing acute liver failure. In addition, it is of particular interest that caspase 8 siRNA treatment was successful not only in acute liver failure mediated by specific Fas agonistic agents (Jo2 and AdFasL) but also in acute liver failure mediated by Adwt, which is an animal model reflecting multiple molecular mechanisms involved in human acute viral hepatitis. Consequently, our data raise hope for future successful application of siRNA in patients with acute liver failure.
Understanding folding and design: Replica-exchange simulations of ``Trp-cage'' miniproteins
Authors: Pitera Jed W. , Swope William
Published: June 2003

Replica-exchange molecular dynamics simulations in implicit solvent have been carried out to study the folding thermodynamics of a designed 20-residue peptide, or ``miniprotein.'' The simulations in this study used the
amber
(parm94) force field along with the generalized Born/solvent-accessible surface area implicit solvent model, and they spanned a range of temperatures from 273 to 630 K. Starting from a completely extended initial conformation, simulations of one peptide sequence sample conformations that are <1.0 Å C
α
rms positional deviation from structures in the corresponding NMR ensemble. These folded states are thermodynamically stable with a simulated melting temperature of ≈400 K, and they satisfy the majority of experimentally observed NMR restraints. Simulations of a related mutant peptide show a degenerate ensemble of states at low temperature, in agreement with experimental results.
Developing an energy landscape for the novel function of a (β/α)8barrel: Ammonia conduction through HisF
Authors: Amaro Rommie , Tajkhorshid Emad , Luthey-Schulten Zaida
Published: June 2003

HisH–hisF is a multidomain globular protein complex; hisH is a class I glutamine amidotransferase that hydrolyzes glutamine to form ammonia, and hisF is a (β/α)8barrel cyclase that completes the ring formation of imidizole glycerol phosphate synthase. Together, hisH and hisF form a glutamine amidotransferase that carries out the fifth step of the histidine biosynthetic pathway. Recently, it has been suggested that the (β/α)8barrel participates in a novel function: to channel ammonia from the active site of hisH to the active site of hisF. The present study presents a series of molecular dynamic simulations that investigate the channeling function of hisF. This article reconstructs potentials of mean force for the conduction of ammonia through the channel, and the entrance of ammonia through the strictly conserved channel gate, in both a closed and a hypothetical open conformation. The resulting energy landscape within the channel supports the idea that ammonia does indeed pass through the barrel, interacting with conserved hydrophilic residues along the way. The proposed open conformation, which involves an alternate rotamer state of one of the gate residues, presents only an ≈2.5-kcal energy barrier to ammonia entry. Another alternate open-gate conformation, which may play a role in non-nitrogen-fixing organisms, is deduced through bioinformatics.
Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2
Authors: Abbadie Catherine , Lindia Jill A. , Cumiskey Anne Marie , Peterson Larry B. , Mudgett John S. , Bayne Ellen K. , DeMartino Julie A. , MacIntyre D. Euan , Forrest Michael J.
Published: June 2003

Mice lacking the chemokine receptor chemotactic cytokine receptor 2 (CCR2) have a marked attenuation of monocyte recruitment in response to various inflammatory stimuli and a reduction of inflammatory lesions in models of demyelinating disease. In the present study, we compared nociceptive responses in inflammatory and neuropathic models of pain in CCR2 knockout and wild-type mice. In acute pain tests, responses were equivalent in CCR2 knockout and wild-type mice. In models of inflammatory pain, CCR2 knockout mice showed a 70% reduction in phase 2 of the intraplantar formalin-evoked pain response but only a modest (20–30%) and nonsignificant reduction of mechanical allodynia after intraplantar Freund's adjuvant (CFA). In a model of neuropathic pain, the development of mechanical allodynia was totally abrogated in CCR2 knockout mice. CFA administration induced marked up-regulation of CCR2 mRNA in the skin and a moderate increase in the sciatic nerve and dorsal root ganglia (DRG). In response to nerve ligation, persistent and marked up-regulation of CCR2 mRNA was evident in the nerve and DRG. Disruption of Schwann cells in response to nerve lesion resulted in infiltration of CCR2-positive monocytes/macrophages not only to the neuroma but also to the DRG. Chronic pain also resulted in the appearance of activated CCR2-positive microglia in the spinal cord. Collectively, these data suggest that the recruitment and activation of macrophages and microglia peripherally and in neural tissue may contribute to both inflammatory and neuropathic pain states. Accordingly, blockade of the CCR2 receptor may provide a novel therapeutic modality for the treatment of chronic pain.
Maspin expression inhibits osteolysis, tumor growth, and angiogenesis in a model of prostate cancer bone metastasis
Authors: Cher Michael L. , Biliran Hector R. , Bhagat Sunita , Meng Yonghong , Che Mingxin , Lockett Jaron , Abrams Judith , Fridman Rafael , Zachareas Michael , Sheng Shijie
Published: June 2003

Emerging evidence indicates that tumor-associated proteolytic remodeling of bone matrix may underlie the capacity of tumor cells to colonize and survive in the bone microenvironment. Of particular importance, urokinase-type plasminogen activator (uPA) has been shown to correlate with human prostate cancer (PC) metastasis. The importance of this protease may be related to its ability to initiate a proteolytic cascade, leading to the activation of multiple proteases and growth factors. Previously, we showed that maspin, a serine protease inhibitor, specifically inhibits PC-associated uPA and PC cell invasion and motility
in vitro
. In this article, we showed that maspin-expressing transfectant cells derived from PC cell line DU145 were inhibited in
in vitro
extracellular matrix and collagen degradation assays. To test the effect of tumor-associated maspin on PC-induced bone matrix remodeling and tumor growth, we injected the maspin-transfected DU145 cells into human fetal bone fragments, which were previously implanted in immunodeficient mice. These studies showed that maspin expression decreased tumor growth, reduced osteolysis, and decreased angiogenesis. Furthermore, the maspin-expressing tumors contained significant fibrosis and collagen staining, and exhibited a more glandular organization. These data represent evidence that maspin inhibits PC-induced bone matrix remodeling and induces PC glandular redifferentiation. These results support our current working hypothesis that maspin exerts its tumor suppressive role, at least in part, by blocking the pericellular uPA system and suggest that maspin may offer an opportunity to improve therapeutic intervention of bone metastasis.
A simple energy-conserving system: Proton reduction coupled to proton translocation
Authors: Sapra Rajat , Bagramyan Karine , Adams Michael W. W.
Published: June 2003

Oxidative phosphorylation involves the coupling of ATP synthesis to the proton-motive force that is generated typically by a series of membrane-bound electron transfer complexes, which ultimately reduce an exogenous terminal electron acceptor. This is not the case with
Pyrococcus furiosus
, an archaeon that grows optimally near 100°C. It has an anaerobic respiratory system that consists of a single enzyme, a membrane-bound hydrogenase. Moreover, it does not require an added electron acceptor as the enzyme reduces protons, the simplest of acceptors, to hydrogen gas by using electrons from the cytoplasmic redox protein ferredoxin. It is demonstrated that the production of hydrogen gas by membrane vesicles of
P. furiosus
is directly coupled to the synthesis of ATP by means of a proton-motive force that has both electrochemical and pH components. Such a respiratory system enables rationalization in this organism of an unusual glycolytic pathway that was previously thought not to conserve energy. It is now clear that the use of ferredoxin in place of the expected NAD as the electron acceptor for glyceraldehyde 3-phosphate oxidation enables energy to be conserved by hydrogen production. In addition, this simple respiratory mechanism readily explains why the growth yields of
P. furiosus
are much higher than could be accounted for if ATP synthesis occurred only by substrate-level phosphorylation. The ability of microorganisms such as
P. furiosus
to couple hydrogen production to energy conservation has important ramifications not only in the evolution of respiratory systems but also in the origin of life itself.
From latent disseminated cells to overt metastasis: Genetic analysis of systemic breast cancer progression
Authors: Schmidt-Kittler Oleg , Ragg Thomas , Daskalakis Angela , Granzow Martin , Ahr Andre , Blankenstein Thomas J. F. , Kaufmann Manfred , Diebold Joachim , Arnholdt Hans , Müller Peter , Bischoff Joachim , Harich Detlev , Schlimok Günter , Riethmüller Gert , Eils Roland , Klein Christoph A.
Published: June 2003

According to the present view, metastasis marks the end in a sequence of genomic changes underlying the progression of an epithelial cell to a lethal cancer. Here, we aimed to find out at what stage of tumor development transformed cells leave the primary tumor and whether a defined genotype corresponds to metastatic disease. To this end, we isolated single disseminated cancer cells from bone marrow of breast cancer patients and performed single-cell comparative genomic hybridization. We analyzed disseminated tumor cells from patients after curative resection of the primary tumor (stage M0), as presumptive progenitors of manifest metastasis, and from patients with manifest metastasis (stage M1). Their genomic data were compared with those from microdissected areas of matched primary tumors. Disseminated cells from M0-stage patients displayed significantly fewer chromosomal aberrations than primary tumors or cells from M1-stage patients (
P
< 0.008 and
P
< 0.0001, respectively), and their aberrations appeared to be randomly generated. In contrast, primary tumors and M1 cells harbored different and characteristic chromosomal imbalances. Moreover, applying machine-learning methods for the classification of the genotypes, we could correctly identify the presence or absence of metastatic disease in a patient on the basis of a single-cell genome. We suggest that in breast cancer, tumor cells may disseminate in a far less progressed genomic state than previously thought, and that they acquire genomic aberrations typical of metastatic cells thereafter. Thus, our data challenge the widely held view that the precursors of metastasis are derived from the most advanced clone within the primary tumor.
Small nucleolar RNA interference induced by antisense or double-stranded RNA in trypanosomatids
Authors: Liang Xue-hai , Liu Qing , Michaeli Shulamit
Published: June 2003

In trypanosomes the C/D- and H/ACA-like small nucleolar RNAs (snoRNAs) are clustered and repeated in the genome. The snoRNAs studied to date are transcribed as polycistronic transcripts by RNA polymerase II and then processed, resulting in mature snoRNAs. In this study we demonstrated that snoRNA genes can be silenced in three trypanosomatid species:
Leptomonas collosoma, Leishmania major
, and
Trypanosoma brucei
. Silencing was achieved in
L. collosoma
and
L. major
by the expressing of an antisense transcript complementary to the snoRNA gene and was accompanied by the accumulation of small interfering RNA. Silencing eliminated the mature snoRNA but not its precursor and abolished the specific 2′-O-methylation guided by the snoRNA. In
T. brucei
, silencing was achieved by using the inducible synthesis of double-stranded RNA from T7 opposing promoters. Silencing varied between the different snoRNA genes, which may reflect the accessibility of small interfering RNA to the target RNAs. This study suggests that RNA interference can degrade snoRNAs. This study has further implications in elucidating the function of nucleolar RNAs and specific modifications guided by these RNAs in trypanosomatids and perhaps in other eukaryotes as well.
The complete genome sequence of the carcinogenic bacterium
Helicobacter hepaticus
Authors: Suerbaum Sebastian , Josenhans Christine , Sterzenbach Torsten , Drescher Bernd , Brandt Petra , Bell Monica , Dröge Marcus , Fartmann Berthold , Fischer Hans-Peter , Ge Zhongming , Hörster Andrea , Holland Rudi , Klein Kerstin , König Jochen , Macko Ludwig , Mendz George L. , Nyakatura Gerald , Schauer David B. , Shen Zeli , Weber Jacqueline , Frosch Matthias , Fox James G.
Published: June 2003

Helicobacter hepaticus
causes chronic hepatitis and liver cancer
in mice. It is the prototype enterohepatic
Helicobacter
species and a
close relative of
Helicobacter pylori
, also a recognized carcinogen.
Here we report the complete genome sequence of
H. hepaticus
ATCC51449.
H. hepaticus
has a circular chromosome of 1,799,146 base
pairs, predicted to encode 1,875 proteins. A total of 938, 953, and 821
proteins have orthologs in
H. pylori, Campylobacter jejuni
, and both
pathogens, respectively.
H. hepaticus
lacks orthologs of most known
H. pylori
virulence factors, including adhesins, the VacA cytotoxin,
and almost all
cag
pathogenicity island proteins, but has orthologs
of the
C. jejuni
adhesin PEB1 and the cytolethal distending toxin
(CDT). The genome contains a 71-kb genomic island (HHGI1) and several genomic
islets whose G+C content differs from the rest of the genome. HHGI1 encodes
three basic components of a type IV secretion system and other virulence
protein homologs, suggesting a role of HHGI1 in pathogenicity. The genomic
variability of
H. hepaticus
was assessed by comparing the genomes of
12
H. hepaticus
strains with the sequenced genome by microarray
hybridization. Although five strains, including all those known to have caused
liver disease, were indistinguishable from ATCC51449, other strains lacked
between 85 and 229 genes, including large parts of HHGI1, demonstrating
extensive variation of genome content within the species.
Deletion of histidine triad nucleotide-binding protein 1/PKC-interacting protein in mice enhances cell growth and carcinogenesis
Authors: Su Tao , Suzui Masumi , Wang Lei , Lin Chyuan-Sheng , Xing Wang-Qiu , Weinstein I. Bernard
Published: June 2003

PKC-interacting protein (PKCI), also designated histidine triad nucleotide-binding protein 1, belongs to the histidine triad (HIT) family of proteins. Its structure is highly conserved from bacteria to humans and shares homology with the tumor-suppressor gene fragile histidine triad (FHIT). Although it was originally thought to inhibit PKC, its actual physiologic function is not known. Therefore, we used the technique of homologous recombination to generate homozygous deleted PKCI
-/-
mice. These mice display normal fetal and adult development. However, when mouse embryo fibroblasts were established from 13.5-day embryos and serially passaged the PKCI
-/-
cells displayed an increase in growth rate and underwent spontaneous immortalization, whereas the PKCI
+/+
cells senesced and ceased growing. Furthermore, the PKCI
-/-
mouse embryo fibroblasts displayed increased resistance to cytotoxicity by ionizing radiation. In view of these findings we examined possible effects of PKCI on susceptibility to carcinogenicity. Both PKCI
+/+
and PKCI
-/-
mice were treated with the chemical carcinogen
N
-nitrosomethylbenzylamine (NMBA) by intragastric administration and killed 12 weeks later. As expected with this protocol, NMBA induced squamous tumors (both papillomas and carcinomas) of the forestomach. The incidence, multiplicity per mouse, volume, and degree of malignancy of these tumors were significantly greater in the PKCI
-/-
than in the PKCI
+/+
mice. Furthermore, four adenomas and one adenocarcinoma of the glandular stomach were found in the NMBA-treated PKCI
-/-
mice but no tumors of the glandular stomach were found in the NMBA-treated PKCI
+/+
mice or in any of the untreated mice. Taken together, these findings suggest that, like FHIT, PKCI may normally play a tumor-suppressor role. The possible role of PKCI as a tumor suppressor in humans remains to be determined.
Spreading of Sir3 protein in cells with severe histone H3 hypoacetylation
Authors: Kristjuhan Arnold , Wittschieben Birgitte Ø. , Walker Jane , Roberts Douglas , Cairns Bradley R. , Svejstrup Jesper Q.
Published: June 2003

Heterochromatin formation in yeast involves deacetylation of histones, but the precise relationship between acetylation and the association of proteins such as Sir3, Sir4, and the histone deacetylase Sir2 with chromatin is still unclear. Here we show that Sir3 protein spreads to subtelomeric DNA in cells lacking the transcription-related histone acetyltransferases
GCN5
and
ELP3
. Spreading correlates with hypoacetylation of lysines in the histone H3 tail and results in deacetylation of lysine 16 in histone H4. De-repression of genes situated very close to the ends of the chromosomes in
gcn5 elp3
suggests that Sir3 spreads into subtelomeric DNA from the tip of the telomere. Interestingly, growth defects caused by
gcn5 elp3
mutation can be suppressed by
SIR
deletion, suggesting that Sir proteins become detrimental for growth when chromatin is severely hypoacetylated.
Fast gating in the
Shaker
K
+
channel and the energy landscape of activation
Authors: Sigg Daniel , Bezanilla Francisco , Stefani Enrico
Published: June 2003

An early component of the gating current in
Shaker
K
+
channels with a time constant of ≈12 μsec has been recorded with a high-speed patch–clamp setup. This fast component was found to be part of the gating current associated with the opening and closing of the channel. With regard to an energy-landscape interpretation of protein kinetics, the voltage and temperature dependence of the fast component may be explained by a combination of drift diffusion and barrier jumping in the initial stages of channel activation. The data were modeled by a gating particle undergoing Brownian motion in a one-dimensional diffusion landscape that featured diminishing electrical resistance and entropy in the direction of channel activation. The final open state of the channel was reasoned to be narrow and deep to account for successful subtraction of linear-charge displacements at positive potentials. The overall picture of gating that emerges from these studies is that the channel experiences incremental organization from a relaxed state in the early steps of activation to a rigidly structured open state.
Social interaction shapes babbling: Testing parallels between birdsong and speech
Authors: Goldstein Michael H. , King Andrew P. , West Meredith J.
Published: June 2003

Birdsong is considered a model of human speech development at behavioral and neural levels. Few direct tests of the proposed analogs exist, however. Here we test a mechanism of phonological development in human infants that is based on social shaping, a selective learning process first documented in songbirds. By manipulating mothers' reactions to their 8-month-old infants' vocalizations, we demonstrate that phonological features of babbling are sensitive to nonimitative social stimulation. Contingent, but not noncontingent, maternal behavior facilitates more complex and mature vocal behavior. Changes in vocalizations persist after the manipulation. The data show that human infants use social feedback, facilitating immediate transitions in vocal behavior. Social interaction creates rapid shifts to developmentally more advanced sounds. These transitions mirror the normal development of speech, supporting the predictions of the avian social shaping model. These data provide strong support for a parallel in function between vocal precursors of songbirds and infants. Because imitation is usually considered the mechanism for vocal learning in both taxa, the findings introduce social shaping as a general process underlying the development of speech and song.
Pronounced reduction in adenoma recurrence associated with aspirin use and a polymorphism in the ornithine decarboxylase gene
Authors: Martínez María Elena , O'Brien Thomas G. , Fultz Kimberly E. , Babbar Naveen , Yerushalmi Hagit , Qu Ning , Guo Yongjun , Boorman David , Einspahr Janine , Alberts David S. , Gerner Eugene W.
Published: June 2003

Most sporadic colon adenomas acquire mutations in the adenomatous polyposis coli gene (
APC
) and show defects in
APC
-dependent signaling.
APC
influences the expression of several genes, including the c-
myc
oncogene and its antagonist
Mad1
. Ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is a transcriptional target of c-
myc
and a modifier of
APC
-dependent tumorigenesis. A single-nucleotide polymorphism exists in intron 1 of the human
ODC
gene, which lies between two
myc
-binding domains. This region is known to affect
ODC
transcription, but no data exist on the relationship of this polymorphism to risk of colorectal neoplasia in humans. We show that individuals homozygous for the minor
ODC
A-allele who reported using aspirin are ≈0.10 times as likely to have an adenoma recurrence as non-aspirin users homozygous for the major G-allele.
Mad1
selectively suppressed the activity of the
ODC
promoter containing the A-allele, but not the G-allele, in a human colon cancer-derived cell line (HT29). Aspirin (≥10 μM) did not affect
ODC
allele-specific promoter activity but did activate polyamine catabolism and lower polyamine content in HT29 cells. We propose that the
ODC
polymorphism and aspirin act independently to reduce the risk of adenoma recurrence by suppressing synthesis and activating catabolism, respectively, of colonic mucosal polyamines. These findings confirm the hypothesis that the
ODC
polymorphism is a genetic marker for colon cancer risk, and support the use of ODC inhibitors and aspirin, or other nonsteroidal antiinflammatory drugs (NSAIDs), in combination as a strategy for colon cancer prevention.
Origin and evolutionary process of the CNS elucidated by comparative genomics analysis of planarian ESTs
Authors: Mineta Katsuhiko , Nakazawa Masumi , Cebrià Francesc , Ikeo Kazuho , Agata Kiyokazu , Gojobori Takashi
Published: June 2003

Among the bilateral animals, a centralized nervous system is found in both the deuterostome and protostome. To address the question of whether the CNS was derived from a common ancestor of deuterostomes and protostomes, it is essential to know kinds of genes existed in the CNS of the putative common ancestor and to trace the evolutionary divergence of genes expressed in the CNS. To answer these questions, we took a comparative approach using different species, particularly focusing on one of the lower bilateral animals, the planarian (Platyhelminthes, Tricladida), which is known to possess a CNS. We determined the nucleotide sequence of ESTs from the head portion of planarians, obtaining 3,101 nonredundant EST clones. As a result of homology searches, we found that 116 clones had significant similarity to known genes related to the nervous system. Here, we compared these 116 planarian EST clones with all ORFs of the complete genome sequences of the human, fruit fly, and nematode, and showed that >95% of these 116 nervous system-related genes, including genes involved in brain or neural morphogenesis, were commonly shared among these organisms, thus providing evidence at the molecular level for the existence of a common ancestral CNS. Interestingly, we found that ≈30% of planarian nervous system-related genes had homologous sequences in
Arabidopsis
and yeast, which do not possess a nervous system. This implies that the origin of nervous system-related genes greatly predated the emergence of the nervous system, and that these genes might have been recruited toward the nervous system.
Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts
Authors: Stump Craig S. , Short Kevin R. , Bigelow Maureen L. , Schimke Jill M. , Nair K. Sreekumaran
Published: June 2003

Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32–42%) in healthy humans (
P
< 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome
c
oxidase (COX) subunit IV] genes (164–180%) encoding mitochondrial proteins (
P
< 0.05). In addition, muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (
P
< 0.05). Further studies demonstrated no effect of low to high insulin levels on muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16–26% (
P
< 0.02) when four different substrate combinations were used. In conclusion, insulin stimulates mitochondrial oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels.