bluuebunny/crossref_metadata_2025 huggingface dataset:
Demonstration of the Burkitt's lymphoma Epstein-Barr virus phenotype in dividing latently infected memory cellsin vivo
Authors: Hochberg Donna , Middeldorp Jaap M. , Catalina Michelle , Sullivan John L. , Luzuriaga Katherine , Thorley-Lawson David A.
Published: January 2004

Epstein-Barr virus (EBV) is a herpesvirus that establishes a lifelong, persistent infection. It was first discovered in the tumor Burkitt's lymphoma (BL). Despite intensive study, the role of EBV in BL remains enigmatic. One striking feature of the tumor is the unique pattern of viral latent protein expression, which is restricted to EBV-encoded nuclear antigen (EBNA) 1. EBNA1 is required to maintain the viral genome but is not recognized by cytotoxic T cells. Consequently, it was proposed that this expression pattern was used by latently infected B cellsin vivo. This would be the site of long-term, persistent infection by the virus and, by implication, the progenitor of BL. We now know that EBV persists in memory B cells in the peripheral blood and that BL is a tumor of memory cells. However, a normal B cell expressing EBNA1 alone has been elusive. Here we show that most infected cells in the blood express no detectable latent mRNA or proteins. The exception is that when infected cells divide they express EBNA1 only. This is the first detection of the BL viral phenotype in a normal, infected B cellin vivo. It suggests that BL may be a tumor of a latently infected memory B cell that is stuck proliferating because it is a tumor and, therefore, constitutively expressing only EBNA1.
Identification of many microRNAs that copurify with polyribosomes in mammalian neurons
Authors: Kim John , Krichevsky Anna , Grad Yonatan , Hayes Gabriel D. , Kosik Kenneth S. , Church George M. , Ruvkun Gary
Published: January 2004

Localized translation in mammalian dendrites may play a role in synaptic plasticity and contribute to the molecular basis for learning and memory. The regulatory mechanisms that control localized translation in neurons are not well understood. We propose a role for microRNAs (miRNAs), a class of noncoding RNAs, as mediators of neuronal translational regulation. We have identified 86 miRNAs expressed in mammalian neurons, of which 40 have not previously been reported. A subset of these miRNAs exhibits temporally regulated expression in cortical cultures. Moreover, all of the miRNAs that were tested cofractionate with polyribosomes, the sites of active translation. These findings indicate that a large, diverse population of miRNAs may function to regulate translation in mammalian neurons.
Reversing established sepsis with antagonists of endogenous high-mobility group box 1
Authors: Yang Huan , Ochani Mahendar , Li Jianhua , Qiang Xiaoling , Tanovic Mahira , Harris Helena E. , Susarla Srinivas M. , Ulloa Luis , Wang Hong , DiRaimo Robert , Czura Christopher J. , Wang Haichao , Roth Jesse , Warren H. Shaw , Fink Mitchell P. , Fenton Matthew J. , Andersson Ulf , Tracey Kevin J.
Published: January 2004

Despite significant advances in intensive care therapy and antibiotics, severe sepsis accounts for 9% of all deaths in the United States annually. The pathological sequelae of sepsis are characterized by a systemic inflammatory response, but experimental therapeutics that target specific early inflammatory mediators [tumor necrosis factor (TNF) and IL-1β] have not proven efficacious in the clinic. We recently identified high mobility group box 1 (HMGB1) as a late mediator of endotoxin-induced lethality that exhibits significantly delayed kinetics relative to TNF and IL-1β. Here, we report that serum HMGB1 levels are increased significantly in a standardized model of murine sepsis, beginning 18 h after surgical induction of peritonitis. Specific inhibition of HMGB1 activity [with either anti-HMGB1 antibody (600 μg per mouse) or the DNA-binding A box (600 μg per mouse)] beginning as late as 24 h after surgical induction of peritonitis significantly increased survival (nonimmune IgG-treated controls = 28% vs. anti-HMGB1 antibody group = 72%,
P
< 0.03; GST control protein = 28% vs. A box = 68%,
P
< 0.03). Animals treated with either HMGB1 antagonist were protected against the development of organ injury, as evidenced by improved levels of serum creatinine and blood urea nitrogen. These observations demonstrate that specific inhibition of endogenous HMGB1 therapeutically reverses lethality of established sepsis indicating that HMGB1 inhibitors can be administered in a clinically relevant time frame.
Structural, dynamic, and energetic aspects of long-range electron transfer in photosynthetic reaction centers
Authors: Kriegl Jan M. , Nienhaus G. Ulrich
Published: January 2004

Intramolecular electron transfer within proteins plays an essential role in biological energy transduction. Electron donor and acceptor cofactors are bound in the protein matrix at specific locations, and protein–cofactor interactions as well as protein conformational changes can markedly influence the electron transfer rates. To assess these effects, we have investigated charge recombination from the primary quinone acceptor to the special pair bacteriochlorophyll dimer in wild-type reaction centers of
Rhodobacter sphaeroides
and four mutants with widely modified free energy gaps. After light-induced charge separation, the recombination kinetics were measured in the light- and dark-adapted forms of the protein from 10 to 300 K. The data were analyzed by using the spin-boson model, which allowed us to self-consistently determine the electronic coupling energy, the distribution of energy gaps, the spectral density of phonons, and the reorganization energy. The analysis revealed slow changes of the energy gap after charge separation. Interesting correlations of the control parameters governing electron transfer were found and related to structural and dynamic properties of the protein.
Intracellular calmodulin availability accessed with two-photon cross-correlation
Authors: Kim Sally A. , Heinze Katrin G. , Waxham M. Neal , Schwille Petra
Published: January 2004

The availability and interactions of signaling proteins are tightly regulated in time and space to produce specific and localized effects. For calmodulin (CaM), a key transducer of intracellular Ca
2+
signaling, binding to its variety of targets initiates signaling cascades and regulates its subcellular localization, thereby making it unavailable for subsequent binding interactions. Among CaM's numerous targets, Ca
2+
/CaM-dependent protein kinase II is one of the most striking due to its unique ability to increase its affinity for CaM by autophosphorylation and to translocate when bound to Ca
2+
/CaM. Two-photon fluorescence correlation spectroscopy and cross-correlation spectroscopy were utilized to compare mobility and molecular interactions between CaM and Ca
2+
/CaM-dependent protein kinase II in solution and in living cells. These techniques revealed that CaM availability in cells could be altered by a change in intracellular conditions. Two-photon fluorescence cross-correlation spectroscopy exemplifies a generally applicable approach for studying protein–protein interactions in living cells that allows access to the behavior of signaling molecules within their native environment to probe for heterogeneities in signaling pathways in different cellular compartments.
Mono- and multisite phosphorylation enhances Bcl2's antiapoptotic function and inhibition of cell cycle entry functions
Authors: Deng Xingming , Gao Fengqin , Flagg Tammy , May W. Stratford
Published: January 2004

Bcl2 functions to suppress apoptosis and retard cell cycle entry. Single-site phosphorylation at serine 70 (S70) is required for Bcl2's antiapoptotic function, and multisite phosphorylation at threonine 69 (T69), S70, and S87 has been reported to inactivate Bcl2. To address this apparent conflict and identify the regulatory role for Bcl2 phosphorylation in cell death and cell cycle control, a series of serine/threonine (S/T) → glutamate/alanine (E/A) mutants including T69E/A, S70E/A, S87E/A, T69E/S70A/S87A (EAA), T69A/S70E/S87A (AEA), T69A/S70A/S87E (AAE), T69E/S70E/S87E (EEE), and T69A/S70A/S87A (AAA) was created to mimic or abrogate, respectively, either single-site or multisite phosphorylation. The survival and cell cycle status of cells expressing the phosphomimetic or nonphosphorylatable Bcl2 mutants were compared. Surprisingly, all of the E but not the A Bcl2 mutants potently enhance cell survival after stress and retard G
1
/S cell cycle transition. The EEE Bcl2 mutant is the most potent, indicating a possible cumulative advantage for multisite phosphorylation of Bcl2 in survival and retardation of G
1
/S transition functions. Because the E-containing Bcl2 mutants, but not the A-containing mutants, can more potently block cytochrome
c
release from mitochondria during apoptotic stress, even at times when steady-state expression levels are similar for all mutants, we conclude that phosphorylation at one or multiple sites within the flexible loop domain of Bcl2 not only stimulates antiapoptotic activity but also can regulate cell cycle entry.
Local complexity of amino acid interactions in a protein core
Authors: Jain Rajul K. , Ranganathan Rama
Published: January 2004

Atomic resolution structures of proteins indicate that the core is typically well packed, suggesting a densely connected network of interactions between amino acid residues. The combinatorial complexity of energetic interactions in such a network could be enormous, a problem that limits our ability to relate structure and function. Here, we report a case study of the complexity of amino acid interactions in a localized region within the core of the GFP, a particularly stable and tightly packed molecule. Mutations at three sites within the chromophore-binding pocket display an overlapping pattern of conformational change and are thermodynamically coupled, seemingly consistent with the dense network model. However, crystallographic and energetic analyses of coupling between mutations paint a different picture; pairs of mutations couple through independent “hotspots” in the region of structural overlap. The data indicate that, even in highly stable proteins, the core contains sufficient plasticity in packing to uncouple high-order energetic interactions of residues, a property that is likely general in proteins.
Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules
Authors: Laird Charles D. , Pleasant Nicole D. , Clark Aaron D. , Sneeden Jessica L. , Hassan K. M. Anwarul , Manley Nathan C. , Vary Jay C. , Morgan Todd , Hansen R. Scott , Stöger Reinhard
Published: January 2004

Epigenetic inheritance, the transmission of gene expression states from parent to daughter cells, often involves methylation of DNA. In eukaryotes, cytosine methylation is a frequent component of epigenetic mechanisms. Failure to transmit faithfully a methylated or an unmethylated state of cytosine can lead to altered phenotypes in plants and animals. A central unresolved question in epigenetics concerns the mechanisms by which a locus maintains, or changes, its state of cytosine methylation. We developed “hairpin-bisulfite PCR” to analyze these mechanisms. This method reveals the extent of methylation symmetry between the complementary strands of individual DNA molecules. Using hairpin-bisulfite PCR, we determined the fidelity of methylation transmission in the CpG island of the
FMR1
gene in human lymphocytes. For the hypermethylated CpG island of this gene, characteristic of inactive-X alleles, we estimate a maintenance methylation efficiency of ≈0.96 per site per cell division. For
de novo
methylation efficiency (E
d
), remarkably different estimates were obtained for the hypermethylated CpG island (E
d
= 0.17), compared with the hypomethylated island on the active-X chromosome (E
d
< 0.01). These results clarify the mechanisms by which the alternative hypomethylated and hypermethylated states of CpG islands are stably maintained through many cell divisions. We also analyzed a region of human L1 transposable elements. These L1 data provide accurate methylation patterns for the complementary strand of each repeat sequence analyzed. Hairpin-bisulfite PCR will be a powerful tool in studying other processes for which genetic or epigenetic information differs on the two complementary strands of DNA.
Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1
Authors: Kuhnert Frank , Davis Corrine R. , Wang Hsiao-Ting , Chu Pauline , Lee Mark , Yuan Jenny , Nusse Roel , Kuo Calvin J.
Published: January 2004

Whereas the adult gastrointestinal epithelium undergoes tremendous self-renewal through active proliferation in crypt stem cell compartments, the responsible growth factors regulating this continuous proliferation have not been defined. The exploration of physiologic functions of Wnt proteins in adult organisms has been hampered by functional redundancy and the necessity for conditional inactivation strategies. Dickkopf-1 (Dkk1) is a potent secreted Wnt antagonist that interacts with Wnt coreceptors of the LRP family. To address the contribution of Wnt signaling to gastrointestinal epithelial proliferation, adenoviral expression of Dkk1 was used to achieve stringent, conditional, and reversible Wnt inhibition in adult animals. Adenovirus Dkk1 (Ad Dkk1) treatment of adult mice repressed expression of the Wnt target genes CD44 and EphB2 within 2 days in both small intestine and colon, indicating an extremely broad role for Wnt signaling in the maintenance of adult gastrointestinal gene expression. In parallel, Ad Dkk1 markedly inhibited proliferation in small intestine and colon, accompanied by progressive architectural degeneration with the loss of crypts, villi, and glandular structure by 7 days. Whereas decreased Dkk1 expression at later time points (>10 days) was followed by crypt and villus regeneration, which was consistent with a reversible process, substantial mortality ensued from colitis and systemic infection. These results indicate the efficacy of systemic expression of secreted Wnt antagonists as a general strategy for conditional inactivation of Wnt signaling in adult organisms and illustrate a striking reliance on a single growth factor pathway for the maintenance of the architecture of the adult small intestine and colon.
T cell chemotaxis to lysophosphatidylcholine through the G2A receptor
Authors: Radu Caius G. , Yang Li V. , Riedinger Mireille , Au Matthew , Witte Owen N.
Published: January 2004

G2A is an immunoregulatory G protein-coupled receptor predominantly expressed in lymphocytes and macrophages. Ectopic overexpression studies have implicated G2A as a receptor for the bioactive lysophospholipid, lysophosphatidylcholine (LPC). However, the functional consequences of LPC–G2A interaction at physiological levels of receptor expression, and in a cellular context relevant to its immunological role, remain largely unknown. Here, we show impaired chemotaxis to LPC of a T lymphoid cell line in which G2A expression was chronically down-regulated by RNA interference technology. Rescuing this phenotype by reconstitution of the physiological level of receptor expression further supports a functional connection between LPC–G2A interaction and cellular motility. Overexpression of G2A in the T lymphoid cell line significantly enhanced chemotaxis to LPC. It also modified migration toward the LPC-related molecule, lysophosphatidic acid, indicating the possibility of crosstalk between G2A and endogenous lysophosphatidic acid receptors. The role of G2A in LPC-mediated cell migration may be relevant to the autoimmune syndrome associated with genetic inactivation of this G protein-coupled receptor in mice. The experimental system described here can be useful for understanding the structural requirements for LPC recognition by G2A and the signaling pathways regulated by this ligand-receptor pair.
Sequence determinants of amyloid fibril formation
Authors: López de la Paz Manuela , Serrano Luis
Published: January 2004

The establishment of rules that link sequence and amyloid feature is critical for our understanding of misfolding diseases. To this end, we have performed a saturation mutagenesis analysis on the
de novo
-designed amyloid peptide STVIIE (
1
). The positional scanning mutagenesis has revealed that there is a position dependence on mutation of amyloid fibril formation and that both very tolerant and restrictive positions to mutation can be found within an amyloid sequence. In this system, mutations that accelerate β-sheet polymerization do not always lead to an increase of amyloid products. On the contrary, abundant fibrils are typically found for mutants that polymerize slowly. From these experiments, we have extracted a sequence pattern to identify amyloidogenic stretches in proteins. The pattern has been validated experimentally.
In silico
sequence scanning of amyloid proteins also supports the pattern. Analysis of protein databases has shown that highly amyloidogenic sequences matching the pattern are less frequent in proteins than innocuous amino acid combinations and that, if present, they are surrounded by amino acids that disrupt their aggregating capability (amyloid breakers). This study provides the potential for a proteome-wide scanning to detect fibril-forming regions in proteins, from which molecules can be designed to prevent and/or disrupt this process.
Energetics of ion conduction through the gramicidin channel
Authors: Allen Toby W. , Andersen Olaf S. , Roux Benoît
Published: January 2004

The free energy governing K
+
conduction through gramicidin A channels is characterized by using over 0.1 μs of all-atom molecular dynamics simulations with explicit solvent and membrane. The results provide encouraging agreement with experiments and insights into the permeation mechanism. The free energy surface of K
+
, as a function of both axial and radial coordinates, is calculated. Correcting for simulation artifacts due to periodicity and the lack of hydrocarbon polarizability, the calculated single-channel conductance for K
+
ions is 0.8 pS, closer to experiment than any previous calculation. In addition, the estimated single ion dissociation constants are within the range of experimental determinations. The relatively small free energy barrier to ion translocation arises from a balance of large opposing contributions from protein, single-file water, bulk electrolyte, and membrane. Mean force decomposition reveals a remarkable ability of the single-file water molecules to stabilize K
+
by –40 kcal/mol, roughly half the bulk solvation free energy. The importance of the single-file water confirms the conjecture of Mackay
et al.
[Mackay, D. H. J., Berens, P. H., Wilson, K. R. & Hagler, A. T. (1984)
Biophys. J.
46, 229–248]. Ion association with the channel involves gradual dehydration from approximately six to seven water molecules in the first shell, to just two inside the narrow pore. Ion permeation is influenced by the orientation of the single-file water column, which can present a barrier to conduction and give rise to long-range coupling of ions on either side of the pore. Small changes in the potential function, including contributions from electronic polarization, are likely to be sufficient to obtain quantitative agreement with experiments.
Functional brain abnormalities in young adults at genetic risk for late-onset Alzheimer's dementia
Authors: Reiman Eric M. , Chen Kewei , Alexander Gene E. , Caselli Richard J. , Bandy Daniel , Osborne David , Saunders Ann M. , Hardy John
Published: January 2004

Fluorodeoxyglucose positron emission tomography (PET) studies have found that patients with Alzheimer's dementia (AD) have abnormally low rates of cerebral glucose metabolism in posterior cingulate, parietal, temporal, and prefrontal cortex. We previously found that cognitively normal, late-middle-aged carriers of the apolipoprotein E ε4 allele, a common susceptibility gene for late-onset Alzheimer's dementia, have abnormally low rates of glucose metabolism in the same brain regions as patients with probable AD. We now consider whether ε4 carriers have these regional brain abnormalities as relatively young adults. Apolipoprotein E genotypes were established in normal volunteers 20–39 years of age. Clinical ratings, neuropsychological tests, magnetic resonance imaging, and PET were performed in 12 ε4 heterozygotes, all with the ε3/ε4 genotype, and 15 noncarriers of the ε4 allele, 12 of whom were individually matched for sex, age, and educational level. An automated algorithm was used to generate an aggregate surface-projection map that compared regional PET measurements in the two groups. The young adult ε4 carriers and noncarriers did not differ significantly in their sex, age, educational level, clinical ratings, or neuropsychological test scores. Like previously studied patients with probable AD and late-middle-aged ε4 carriers, the young ε4 carriers had abnormally low rates of glucose metabolism bilaterally in the posterior cingulate, parietal, temporal, and prefrontal cortex. Carriers of a common Alzheimer's susceptibility gene have functional brain abnormalities in young adulthood, several decades before the possible onset of dementia.
Shifting foci of hematopoiesis during reconstitution from single stem cells
Authors: Cao Yu-An , Wagers Amy J. , Beilhack Andreas , Dusich Joan , Bachmann Michael H. , Negrin Robert S. , Weissman Irving L. , Contag Christopher H.
Published: January 2004

To reveal the early events and dynamics of hematopoietic reconstitution in living animals in real-time, we used bioluminescence imaging to monitor engraftment from single luciferase-labeled hematopoietic stem cells (HSC) in irradiated recipients. Transplanted HSC generated discrete foci in the spleen and bone marrow (BM), at a frequency that correlated with BM compartment size. Initially detected foci could expand locally, seed other sites in BM or spleen, and/or recede with different kinetics. These studies reveal dynamic and variable patterns of engraftment from highly purified HSC and indicate that the final overall contribution of individual HSC to hematopoietic chimerism does not depend on the specific site of initial engraftment and expansion.
Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells
Authors: Cambi Alessandra , de Lange Frank , van Maarseveen Noortje M. , Nijhuis Monique , Joosten Ben , van Dijk Erik M.H.P. , de Bakker Bärbel I. , Fransen Jack A.M. , Bovee-Geurts Petra H.M. , van Leeuwen Frank N. , Van Hulst Niek F. , Figdor Carl G.
Published: January 2004

The C-type lectin dendritic cell (DC)–specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.
IGF-I instructs multipotent adult neural progenitor cells to become oligodendrocytes
Authors: Hsieh Jenny , Aimone James B. , Kaspar Brian K. , Kuwabara Tomoko , Nakashima Kinichi , Gage Fred H.
Published: January 2004

Adult multipotent neural progenitor cells can differentiate into neurons, astrocytes, and oligodendrocytes in the mammalian central nervous system, but the molecular mechanisms that control their differentiation are not yet well understood. Insulin-like growth factor I (IGF-I) can promote the differentiation of cells already committed to an oligodendroglial lineage during development. However, it is unclear whether IGF-I affects multipotent neural progenitor cells. Here, we show that IGF-I stimulates the differentiation of multipotent adult rat hippocampus-derived neural progenitor cells into oligodendrocytes. Modeling analysis indicates that the actions of IGF-I are instructive. Oligodendrocyte differentiation by IGF-I appears to be mediated through an inhibition of bone morphogenetic protein signaling. Furthermore, overexpression of IGF-I in the hippocampus leads to an increase in oligodendrocyte markers. These data demonstrate the existence of a single molecule, IGF-I, that can influence the fate choice of multipotent adult neural progenitor cells to an oligodendroglial lineage.
Oriented clonal cell growth in the developing mouse myocardium underlies cardiac morphogenesis
Authors: Meilhac Sigolène M. , Esner Milan , Kerszberg Michel , Moss Julie E. , Buckingham Margaret E.
Published: January 2004

During heart morphogenesis, cardiac chambers arise by differential expansion of regions of the primitive cardiac tube. This process is under the control of specific transcription factors such as Tbx5 and dHAND. To gain insight into the cellular mechanisms that underlie cardiogenesis, we have used a retrospective clonal approach based on the spontaneous recombination of an nlaacZ reporter gene targeted to the murine α-cardiac actin locus. We show that clonal growth of myocardial cells is oriented. At embryonic day (E) 10.5, the shape of clones is characteristic of a given cardiac region and reflects its morphology. This is already detectable in the primitive cardiac tube at E8.5, and is maintained after septation at E14.5 with additional modulations. The clonal analysis reveals new subdivisions of the myocardium, including an interventricular boundary region. Our results show that the myocardium, from the time of its formation, is a polarized and regionalized tissue and point to the role of oriented clonal cell growth in cardiac chamber morphogenesis.
Apaf-1 and caspase-9 do not act as tumor suppressors in myc-induced lymphomagenesis or mouse embryo fibroblast transformation
Authors: Scott Clare L. , Schuler Martin , Marsden Vanessa S. , Egle Alex , Pellegrini Marc , Nesic Dobrila , Gerondakis Steve , Nutt Stephen L. , Green Douglas R. , Strasser Andreas
Published: January 2004

Based on experiments with cultured fibroblasts, the apoptosis regulators caspase-9 and Apaf-1 are hypothesized to function as tumor suppressors. To investigate their in vivo role in lymphomagenesis, an IgH enhancer-driven c-myc transgene was crossed onto Apaf-1−/− and caspase-9−/− mice. Due to perinatal lethality, Eμ-myc transgenic Apaf-1−/− or caspase-9−/− fetal liver cells were used to reconstitute lethally irradiated recipient mice. Surprisingly, no differences were seen in rate, incidence, or severity of lymphoma with loss of Apaf-1 or caspase-9, and Apaf-1 was not a critical determinant of anticancer drug sensitivity of c-myc–induced lymphomas. Moreover, loss of Apaf-1 did not promote oncogene-induced transformation of mouse embryo fibroblasts. Thus, Apaf-1 and caspase-9 do not suppress c-myc–induced lymphomagenesis and embryo fibroblast transformation.
Nuclear stress granules
Authors: Sandqvist Anton , Sistonen Lea
Published: January 2004

Nuclear stress granules are subnuclear compartments that form in response to heat shock and other stress stimuli. Although many components of nuclear stress granules have been identified, including HSF1 and pre-mRNA processing factors, their function remains a mystery. A paper in this issue describes the stress-induced transcriptional activation of one of the nuclear stress granule target sites, a heterochromatic region that has been considered silent (Jolly et al., 2004). These intriguing findings will certainly give the research of these structures a new twist.
Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum
Authors: Klemba Michael , Beatty Wandy , Gluzman Ilya , Goldberg Daniel E.
Published: January 2004

fA amily of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum food vacuole. To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II–GFP chimera. By taking advantage of green fluorescent protein fluorescence in live parasites, the ultrastructural resolution of immunoelectron microscopy, and inhibitors of trafficking and PM maturation, we have investigated the biosynthetic path leading to mature PM II in the food vacuole. Our data support a model whereby proPM II is transported through the secretory system to cytostomal vacuoles and then is carried along with its substrate hemoglobin to the food vacuole where it is proteolytically processed to mature PM II.
Sensitivity of Volume-regulated Anion Current to Cholesterol Structural Analogues
Authors: Romanenko Victor G. , Rothblat George H. , Levitan Irena
Published: December 2003

Depletion of membrane cholesterol and substitution of endogenous cholesterol with its structural analogues was used to analyze the mechanism by which cholesterol regulates volume-regulated anion current (VRAC) in endothelial cells. Depletion of membrane cholesterol enhanced the development of VRAC activated in a swelling-independent way by dialyzing the cells either with GTPγS or with low ionic strength solution. Using MβCD–sterol complexes, 50–80% of endogenous cholesterol was substituted with a specific analogue, as verified by gas-liquid chromatography. The effects of cholesterol depletion were reversed by the substitution of endogenous cholesterol with its chiral analogue, epicholesterol, or with a plant sterol, β-sitosterol, two analogues that mimic the effect of cholesterol on the physical properties of the membrane bilayer. Alternatively, when cholesterol was substituted with coprostanol that has only minimal effect on the membrane physical properties it resulted in VRAC enhancement, similar to cholesterol depletion. In summary, our data show that these channels do not discriminate between the two chiral analogues of cholesterol, as well as between the two cholesterols and β-sitosterol, but discriminate between cholesterol and coprostanol. These observations suggest that endothelial VRAC is regulated by the physical properties of the membrane.
Ca2+/Calmodulin Modulates TRPV1 Activation by Capsaicin
Authors: Rosenbaum Tamara , Gordon-Shaag Ariela , Munari Mika , Gordon Sharona E.
Published: December 2003

TRPV1 ion channels mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent extract from plants in the Capsicum family (hot chili peppers) (Szallasi, A., and P.M. Blumberg. 1999. Pharmacol. Rev. 51:159–212; Caterina, M.J., and D. Julius. 2001. Annu. Rev. Neurosci. 24:487–517). The convergence of these stimuli on TRPV1 channels expressed in peripheral sensory nerves underlies the common perceptual experience of pain due to hot temperatures, tissue damage and exposure to capsaicin. TRPV1 channels are nonselective cation channels (Caterina, M.J., M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, and D. Julius. 1997. Nature. 389:816–824). When activated, they produce depolarization through the influx of Na+, but their high Ca2+ permeability is also important for mediating the response to pain. In particular, Ca2+ influx is thought to be required for the desensitization to painful sensations over time (Cholewinski, A., G.M. Burgess, and S. Bevan. 1993. Neuroscience. 55:1015–1023; Koplas, P.A., R.L. Rosenberg, and G.S. Oxford. 1997. J. Neurosci. 17:3525–3537). Here we show that in inside-out excised patches from TRPV1 expressed in Xenopus oocytes and HEK 293 cells, Ca2+/calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by Mg2+, reflected a decrease in open probability, and was slowly reversible. Furthermore, increasing the calmodulin concentration in our patches by coexpression of wild-type calmodulin with TRPV1 produced inhibition by Ca2+ alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to Ca2+. Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a Ca2+-independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel NH2-terminal domain demonstrated that a stretch of ∼30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a Ca2+-dependent manner. The physiological response to pain involves an influx of Ca2+ through TRPV1. Our results indicate that this Ca2+ influx may feed back on the channels, inhibiting their gating. This type of feedback inhibition could play a role in the desensitization produced by capsaicin.
In Intact Mammalian Photoreceptors, Ca2+-dependent Modulation of cGMP-gated Ion Channels Is Detectable in Cones but Not in Rods
Authors: Rebrik Tatiana I. , Korenbrot Juan I.
Published: December 2003

In the mammalian retina, cone photoreceptors efficiently adapt to changing background light intensity and, therefore, are able to signal small differences in luminance between objects and backgrounds, even when the absolute intensity of the background changes over five to six orders of magnitude. Mammalian rod photoreceptors, in contrast, adapt very little and only at intensities that nearly saturate the amplitude of their photoresponse. In search of a molecular explanation for this observation we assessed Ca2+-dependent modulation of ligand sensitivity in cyclic GMP–gated (CNG) ion channels of intact mammalian rods and cones. Solitary photoreceptors were isolated by gentle proteolysis of ground squirrel retina. Rods and cones were distinguished by whether or not their outer segments bind PNA lectin. We measured membrane currents under voltage-clamp in photoreceptors loaded with Diazo-2, a caged Ca2+ chelator, and fixed concentrations of 8Br-cGMP. At 600 nM free cytoplasmic Ca2+ the midpoint of the cone CNG channels sensitivity to 8BrcGMP, 8BrcGMPK1/2, is ∼2.3 μM. The ligand sensitivity is less in rod than in cone channels. Instantly decreasing cytoplasmic Ca2+ to <30 nM activates a large inward membrane current in cones, but not in rods. Current activation arises from a Ca2+ -dependent modulation of cone CNG channels, presumably because of an increase in their affinity to the cyclic nucleotide. The time course of current activation is temperature dependent; it is well described by a single exponential process of ∼480 ms time constant at 20–21°C and 138 ms at 32°C. The absence of detectable Ca2+-dependent CNG current modulation in intact rods, in view of the known channel modulation by calmodulin in-vitro, affirms the modulation in intact rods may only occur at low Ca2+ concentrations, those expected at intensities that nearly saturate the rod photoresponse. The correspondence between Ca2+ dependence of CNG modulation and the ability to light adapt suggest these events are correlated in photoreceptors.
hRIP, a cellular cofactor for Rev function, promotes release of HIV RNAs from the perinuclear region
Authors: Sánchez-Velar Nuria , Udofia Enyeneama B. , Yu Zhong , Zapp Maria L.
Published: January 2004

Human immunodeficiency virus Rev facilitates the cytoplasmic accumulation of viral RNAs that contain a Rev binding site. A human Rev-interacting protein (hRIP) was originally identified based on its ability to interact with the Rev nuclear export signal (NES) in yeast two-hybrid assays. To date, however, the function of hRIP and a role for hRIP in Rev-directed RNA export have remained elusive. Here we ablate hRIP activity with a dominant-negative mutant or RNA interference and analyze Rev function by RNA in situ hybridization. We find, unexpectedly, that in the absence of functional hRIP, Rev-directed RNAs mislocalize and aberrantly accumulate at the nuclear periphery, where hRIP is localized. In contrast, in the absence of Rev or the Rev cofactor CRM1, Rev-directed RNAs remain nuclear. We further show that the RNA mislocalization pattern resulting from loss of hRIP activity is highly specific to Rev function: the intracellular distribution of cellular poly(A)+ mRNA, nuclear proteins, and, most important, NES-containing proteins, are unaffected. Thus, hRIP is an essential cellular Rev cofactor, which acts at a previously unanticipated step in HIV-1 RNA export: movement of RNAs from the nuclear periphery to the cytoplasm.
In vivo requirements for rDNA chromosome condensation reveal two cell-cycle-regulated pathways for mitotic chromosome folding
Authors: Lavoie Brigitte D. , Hogan Eileen , Koshland Doug
Published: January 2004

Chromosome condensation plays an essential role in the maintenance of genetic integrity. Using genetic, cell biological, and biochemical approaches, we distinguish two cell-cycle-regulated pathways for chromosome condensation in budding yeast. From G2 to metaphase, we show that the condensation of the ∼1-Mb rDNA array is a multistep process, and describe condensin-dependent clustering, alignment, and resolution steps in chromosome folding. We functionally define a further postmetaphase chromosome assembly maturation step that is required for the maintenance of chromosome structural integrity during segregation. This late step in condensation requires the conserved mitotic kinase Ipl1/aurora in addition to condensin, but is independent of cohesin. Consistent with this, the late condensation pathway is initiated during the metaphase-to-anaphase transition, supports de novo condensation in cohesin mutants, and correlates with the Ipl1/aurora-dependent phosphorylation of condensin. These data provide insight into the molecular mechanisms of higher-order chromosome folding and suggest that two distinct condensation pathways, one involving cohesins and the other Ipl1/aurora, are required to modulate chromosome structure during mitosis.
Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation
Authors: Timmerman Luika A. , Grego-Bessa Joaquín , Raya Angel , Bertrán Esther , Pérez-Pomares José María , Díez Juan , Aranda Sergi , Palomo Sergio , McCormick Frank , Izpisúa-Belmonte Juan Carlos , de la Pompa José Luis
Published: January 2004

Epithelial-to-mesenchymal transition (EMT) is fundamental to both embryogenesis and tumor metastasis. The Notch intercellular signaling pathway regulates cell fate determination throughout metazoan evolution, and overexpression of activating alleles is oncogenic in mammals. Here we demonstrate that Notch activity promotes EMT during both cardiac development and oncogenic transformation via transcriptional induction of the Snail repressor, a potent and evolutionarily conserved mediator of EMT in many tissues and tumor types. In the embryonic heart, Notch functions via lateral induction to promote a selective transforming growth factor-β (TGFβ)-mediated EMT that leads to cellularization of developing cardiac valvular primordia. Embryos that lack Notch signaling elements exhibit severely attenuated cardiac snail expression, abnormal maintenance of intercellular endocardial adhesion complexes, and abortive endocardial EMT in vivo and in vitro. Accordingly, transient ectopic expression of activated Notch1 (N1IC) in zebrafish embryos leads to hypercellular cardiac valves, whereas Notch inhibition prevents valve development. Overexpression of N1IC in immortalized endothelial cells in vitro induces EMT accompanied by oncogenic transformation, with corresponding induction of snail and repression of VE-cadherin expression. Notch is expressed in embryonic regions where EMT occurs, suggesting an intimate and fundamental role for Notch, which may be reactivated during tumor metastasis.
What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame?
Authors: Pöyry Tuija A.A. , Kaminski Ann , Jackson Richard J.
Published: January 2004

If the 5′-proximal AUG triplet in a mammalian mRNA is followed by a short open reading frame (sORF), a significant fraction of ribosomes resume scanning after termination of sORF translation, and reinitiate at a downstream AUG. To examine the underlying mechanism, we examined reinitiation in vitro using a series of mRNAs that differed only in the 5′-untranslated region (UTR). Efficient reinitiation was found to occur only if the eIF4F complex, or at a minimum the central one-third fragment of eIF4G, participated in the primary initiation event at the sORF initiation codon. It did not occur, however, when sORF translation was driven by the classical swine fever virus or cricket paralysis virus internal ribosome entry sites (IRESs), which do not use eIF4A, 4B, 4E, or 4G. A critical test was provided by an mRNA with an unstructured 5′-UTR, which is translated by scanning but does not absolutely need eIF4G and eIF4A: There was efficient reinitiation in a standard reticulocyte lysate, when initiation would be largely driven by eIF4F, but no reinitiation in an eIF4G-depleted lysate. These results suggest that resumption of scanning may depend on the interaction between eIF4F (or the eIF4G central domain) and the ribosome being maintained while the ribosome translates the sORF.
‘Sexy docs’ and ‘busty blondes’: press coverage of professional misconduct cases brought before the General Medical Council
Authors: Bradby Hannah , Gabe Jonathan , Bury Michael
Published: January 2004

This paper presents an analysis of press coverage of the General Medical Council Professional Conduct Committee (PCC) during 1990 and 1991. All tabloid press coverage during this period was compared with PCC minutes to establish what kinds of cases were reported. The analysis reveals that the press gave undue attention to hearings involving sexual misconduct by General Practitioners and that the majority of patients reported as suffering from such misconduct were young women. The paper goes on to document the ideological assumptions underpinning this coverage and considers the consequences for the doctor‐patient relationship.
Detecting disease and designing treatment. Duplex and the diagnosis of diseased leg vessels
Authors: Mol Annemarie , Elsman Bernard
Published: January 2004

In hospital Z, a Dutch university hospital, a new protocol for the diagnosis of vascular patients has been introduced. It requires vascular surgeons to rely sometimes on a non‐invasive diagnostic technique, called duplex, in place of a previously used invasive one, called angiography. This article is a multivocal account of the protocol's introduction. It focuses on the intricate ways in which two aspects of diagnosis, the detection of disease and the design of treatment, relate.
Meaning Theory and Autistic Speakers
Authors: Glüer Kathrin , Pagin Peter
Published: January 2004

Some theories of linguistic meaning, such as those of Paul Grice and David Lewis, make appeal to higher–order thoughts: thoughts about thoughts. Because of this, such theories run the risk of being empirically refuted by the existence of speakers who lack, completely or to a high degree, the capacity of thinking about thoughts. Research on autism during the past 15 years provides strong evidence for the existence of such speakers. Some persons with autism have linguistic abilities that qualify them as speakers, but manifest a severely impaired capacity to understand what it is to have beliefs
Connectionist Semantics and the Collateral Information Challenge
Authors: Garzóan Francisco Calvo
Published: January 2004

Jerry Fodor and Ernest Lepore have launched a powerful attack against Paul Churchland's connectionist theory of semantics—aka State Space Semantics. In one part of their overall attack, they exploit the potentially orthogonal histories of different individuals to introduce what they labeled ‘the collateral information problem’. Aarre Laakso and Gary Cottrell have recently put forward a mathematical technique for measuring conceptual similarity across neural networks. Churchland uses Laakso and Cottrell's tecnique to defend State Space Semantics. In this paper I shall highlight a potential problem for Laakso and Cottrell's technique, and for Churchland's subsequent defence of connectionist semantics that has been ignored in the connectionist literature. I shall argue that a connectionist sympathiser of Churchland could not make use of Laakso and Cottrell's neurosimulations to address Fodor and Lepore's collateral information challenge.
Inhibition of photosynthesis by heat stress: the activation state of Rubisco as a limiting factor in photosynthesis
Authors: Salvucci Michael E. , Crafts‐Brandner Steven J.
Published: January 2004

Although the catalytic activity of Rubisco increases with temperature, the low affinity of the enzyme for CO2 and its dual nature as an oxygenase limit the possible increase in net photosynthesis with temperature. For cotton, comparisons of measured rates of net photosynthesis with predicted rates that take into account limitations imposed by the kinetic properties of Rubisco indicate that direct inhibition of photosynthesis occurs at temperatures higher than about 30°C. Inhibition of photosynthesis by moderate heat stress (i.e. 30–42°C) is generally attributed to reduced rates of RuBP regeneration caused by disruption of electron transport activity, and specifically inactivation of the oxygen evolving enzymes of photosystem II. However, measurements of chlorophyll fluorescence and metabolite levels at air‐levels of CO2 indicate that electron transport activity is not limiting at temperatures that inhibit CO2 fixation. Instead, recent evidence shows that inhibition of net photosynthesis correlates with a decrease in the activation state of Rubisco in both C3 and C4 plants and that this decrease in the amount of active Rubisco can fully account for the temperature response of net photosynthesis. Biochemically, the decrease in Rubisco activation can be attributed to: (1) more rapid de‐activation of Rubisco caused by a faster rate of dead‐end product formation; and (2) slower re‐activation of Rubisco by activase. The net result is that as temperature increases activase becomes less effective in keeping Rubisco catalytically competent. In this opinionated review, we discuss how these processes limit photosynthetic performance under moderate heat stress.
Active site mutagenesis and phospholipid hydroperoxide reductase activity of poplar type II peroxiredoxin
Authors: Rouhier Nicolas , Gelhaye Eric , Corbier Catherine , Jacquot Jean Pierre
Published: January 2004

The nature of the active site and the substrate specificity of poplar type II peroxiredoxin, an enzyme which preferentially uses glutaredoxin as an electron donor, were investigated in this study. The type II peroxiredoxin is able to use phospholipid hydroperoxide nearly as efficiently as hydrogen peroxide. Two of the hyper‐conserved amino acid residues in peroxiredoxins have been altered, by site‐directed mutagenesis, generating the mutants T48V and R129Q. The two mutant proteins are inactive with hydrogen peroxide or tertiary butyl hydroperoxide as substrates. On the other hand, the mutant enzymes catalyse the degradation of cumene hydroperoxide with low efficiency. This suggests that the thiol‐dependent regeneration process of the catalytic cysteine is not affected by the mutations and that all substrates are not accommodated identically in the active site.