bluuebunny/crossref_metadata_2025 huggingface dataset:
Bamboo, fire and flood: regeneration of Bambusa arnhemica (Bambuseae: Poaceae) after mass-flowering and die-off at contrasting sites in monsoonal northern Australia
Authors: Franklin Donald C. , Bowman David J. M. S.
Published: October 2003

Bambusa arnhemica F.Muell., a long-lived, gregarious-flowering and semelparous bamboo endemic to north-western Australia, occurs in remarkably disparate but somewhat fire-sheltered flood-prone riparian forest and rocky hillside vine-thickets, but not in adjacent fire-prone savannas. We investigated the response of B. arnhemica seedlings to fire and flood at two contrasting sites over 2.5 years following a mass-flowering and die-off event. Seedlings grew vigorously notwithstanding either prolonged inundation or total loss of above-ground parts to fire within their first year. However, there was no evidence that such disturbance promoted regeneration, and several veins of evidence suggest that B. arnhemica is fire-retardant and refugial rather than fire-promoting. We suggest that creation of canopy gaps by parental death is a more parsimonious and generalisable hypothesis for the evolution of gregarious semelparity in bamboos than the recently advanced bamboo fire-cycle hypothesis. However, both hypotheses are potentially group selectionist, and resolution of dispersal distances and/or the spatial genetics of relatedness are required to resolve the problem.
Essential oil composition of the seasonal heterophyllous leaves of Eryngium vesiculosum from Australia
Authors: Palá-Paúl Jesús , Brophy Joseph J. , Goldsack Robert J. , Copeland Lachlan M. , Pérez-Alonso M. José , Velasco-Negueruela Arturo
Published: October 2003

Eryngium vesiculosum Labill. is seasonally heterophyllous. The essential oils of its aerial parts gathered during summer and winter from New South Wales (Australia) have been examined by analytical gas chromatography and gas chromatography/mass spectrometry. We have found quantitative but not qualitative differences between the two samples. The principal compounds of the winter leaves were identified as β-caryophyllene (20.3%), germacrene D (19.2%) and α-humulene (8.8%), while the summer leaves (SL) contained bicyclogermacrene (22.2%), β-caryophyllene (15.6%), germacrene D (15.8%) and α-humulene (8.1%) as major constituents. The amount of bicyclogermacrene changes by a factor of ten, ranging from 2.4% in winter leaves to being the principal compound (22.2%) in summer leaves. The oil of the flowers and fruits contained almost the same composition as the summer leaves. This is the first comparative study on the chemistry of both kinds of leaves of E. vesiculosum.
The history of the family Onagraceae in Australia and its relevance to biogeography
Authors: Martin Helene A.
Published: October 2003

The family Onagraceae is a relatively minor part of the Australian flora but it has a long history in Australia: a probable Ludwigia dates from the Eocene; Fuchsia, not native to Australia today, is present from early Oligocene times; and Epilobium is found only in the Pleistocene. Onagraceae first appears in the Late Cretaceous in northern South America and southern North America, where it is thought to have originated, and Ludwigia dates from the Palaeocene. It is thought that Ludwigia migrated into Australia via a northern route. Fuchsia in Australia predates its first appearance in New Zealand, suggesting that New Zealand Fuchsia may have been derived from the Australian Fuchsia.
Sexual dimorphism in reproductive allocation and water use efficiency in Maireana pyramidata (Chenopodiaceae), a dioecious, semi-arid shrub
Authors: Leigh Andrea , Nicotra Adrienne B.
Published: October 2003

Sexual dimorphism in dioecious plant species is widely attributed to the differential impacts of reproduction on male v. female plants. We investigated sexual dimorphism in reproductive, morphological and physiological traits of Maireana pyramidata (Benth.) Paul G.Wilson (Chenopodiaceae), a dioecious, semi-arid shrub endemic to Australia. We estimated reproductive allocation for each sex by calculating the relative biomass allocated to flowers and fruits per gram of leaf tissue, based on one branch per sample plant. Morphological measurements included leaf mass, stem mass, specific leaf area, plant height and plant leaf area index. We also measured leaf nitrogen and chlorophyll, gas exchange and Δ13C. Reproductive allocation was nine times greater in females than in males. No significant difference between the sexes in photosynthetic rate or transpiration could be detected but instantaneous water use efficiency (photosynthesis/transpiration) was significantly lower in females than in males during the fruiting period. Δ13C did not differ between the sexes. The results indicate that greater reproductive allocation in females has an immediate impact on their capacity for conservative water use but does not lead to long-term differences in water use efficiency.
Nodulation and performance of exotic and native legumes in Australian soils
Authors: Pérez-Fernández María A. , Lamont Byron B.
Published: October 2003

Six Spanish legumes, Cytisus balansae, C. multiflorus, C. scoparius, C. striatus, Genista hystrix and Retama sphaerocarpa, were able to form effective nodules when grown in six south-western Australian soils. Soils and nodules were collected from beneath natural stands of six native Australian legumes, Jacksonia floribunda, Gompholobium tomentosum, Bossiaea aquifolium, Daviesia horrida, Gastrolobium spinosum and Templetonia retusa. Four combinations of soils and bacterial treatments were used as the soil treatments: sterile soil (S), sterile inoculated soils (SI), non-treated soil (N) and non-treated inoculated soils (NI). Seedlings of the Australian species were inoculated with rhizobia cultured from nodules of the same species, while seedlings of the Spanish species were inoculated with cultures from each of the Australian species. All Australian rhizobia infected all the Spanish species, suggesting a high degree of 'promiscuity' among the bacteria and plant species. The results from comparing six Spanish and six Australian species according to their biomass and total nitrogen in the presence (NI) or absence (S) of rhizobia showed that all species benefitted from nodulation (1.02–12.94 times), with R.�sphaerocarpa and C. striatus benefiting more than the native species. Inoculation (SI and NI) was just as effective as, or more effective than the non-treated soil (i.e. non-sterile) in inducing nodules. Nodules formed on the Spanish legumes were just as efficient at fixing N2 as were those formed on the Australian legumes. Inoculation was less effective than non-treated soil at increasing biomass but just as effective as the soil at increasing nitrogen content. Promiscuity in the legume–bacteria symbiosis should increase the ability of legumes to spread into new habitats throughout the world.
A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets
Authors: Schultz Nikolaus , Hamra F. Kent , Garbers David L.
Published: October 2003

Understanding mammalian spermatozoan development and the events surrounding fertilization has grown slowly, in part because of uncertainty about the number and identity of the cellular components involved. Determination of those transcripts expressed specifically by germ cells should provide an inclusive list of probable critical proteins. Here, total mouse testis transcript profiles were trimmed of transcripts found in cultures enriched in Sertoli or interstitial cells to yield a germ cell-enriched transcript profile. Monitoring of changes of this profile in the developing testis identified 1,652 genes whose transcript abundance increased markedly coincident with the onset of meiosis. Remarkably, 351 of these genes (≈20%) appear to be expressed only in the male germline. Germ cell-specific transcripts are much less common earlier in testis development. Further analysis of the UniGene EST database coupled with quantitative PCR indicates that ≈4% of the mouse genome is dedicated to expression in postmeiotic male germ cells. Most or many of the protein products of these transcripts are probably retained in mature spermatozoa. Targeted disruption of 19 of these genes has indicated that a majority have roles critical for normal fertility. Thus, we find an astonishing number of genes expressed specifically by male germ cells late in development. This extensive group provides a plethora of potential targets for germ cell-directed contraception and a staggering number of candidate proteins that could be critical for fertilization.
DcpS can act in the 5′–3′ mRNA decay pathway in addition to the 3′–5′ pathway
Authors: van Dijk Erwin , Le Hir Hervé , Séraphin Bertrand
Published: October 2003

Eukaryotic mRNA degradation proceeds through two main pathways, both involving mRNA cap breakdown. In the 3′–5′ mRNA decay pathway, mRNA body degradation generates free m7GpppN that is hydrolyzed by DcpS generating m7GMP. In the 5′–3′ pathway, the recently identified human Dcp2 decapping enzyme cleaves the cap of deadenylated mRNAs to produce m7GDP and 5′-phosphorylated mRNA. We investigated mRNA decay in human cell extracts by using a new assay for decapping. We observed that 5′-phosphorylated intermediates resulting from decapping appear after incubation of a substrate RNA in human cell extracts, indicating the presence of an active 5′–3′ mRNA decay pathway. Surprisingly, however, the cognate m7GDP product was not detected, whereas abundant amounts of m7GMP were generated. Additional experiments revealed that m7GDP is, unexpectedly, efficiently converted to m7GMP in extracts from various organisms. The factor necessary and sufficient for this reaction was identified as DcpS in both yeast and human. m7GMP is thus a general, pathway-independent, by-product of eukaryotic mRNA decay. m7GDP breakdown should prevent misincorporation of methylated nucleotides in nucleic acids and could generate a unique indicator allowing the cell to monitor mRNA decay.
Dewetting-induced collapse of hydrophobic particles
Authors: Huang X. , Margulis C. J. , Berne B. J.
Published: October 2003

A molecular dynamics study of the depletion of water (drying) around a single and between two hydrophobic nanoscale oblate plates in explicit water as a function of the distance of separation between them, their size, and the strength of the attraction between the plates and the water molecules is presented. A simple macroscopic thermodynamic model based on Young's law successfully predicts drying between the stacked plates and accounts for the free-energy barriers to this drying. However, because drying around a single plate is not macroscopic, a molecular theory is required to describe it. The data are consistent with the rate-determining step in the hydrophobic collapse of the two plates being a large-scale drying fluctuation, characterized by a free-energy barrier that grows with particle size.
Genome-scale functional profiling of the mammalian AP-1 signaling pathway
Authors: Chanda Sumit K. , White Suhaila , Orth Anthony P. , Reisdorph Richard , Miraglia Loren , Thomas Russell S. , DeJesus Paul , Mason Daniel E. , Huang Qihong , Vega Raquel , Yu De-Hua , Nelson Christian G. , Smith Brendan M. , Terry Robert , Linford Alicia S. , Yu Yang , Chirn Gung-wei , Song Chuanzheng , Labow Mark A. , Cohen Dalia , King Frederick J. , Peters Eric C. , Schultz Peter G. , Vogt Peter K. , Hogenesch John B. , Caldwell Jeremy S.
Published: October 2003

Large-scale functional genomics approaches are fundamental to the characterization of mammalian transcriptomes annotated by genome sequencing projects. Although current high-throughput strategies systematically survey either transcriptional or biochemical networks, analogous genome-scale investigations that analyze gene function in mammalian cells have yet to be fully realized. Through transient overexpression analysis, we describe the parallel interrogation of ≈20,000 sequence annotated genes in cancer-related signaling pathways. For experimental validation of these genome data, we apply an integrative strategy to characterize previously unreported effectors of activator protein-1 (AP-1) mediated growth and mitogenic response pathways. These studies identify the ADP-ribosylation factor GTPase-activating protein Centaurin α1 and a Tudor domain-containing hypothetical protein as putative AP-1 regulatory oncogenes. These results provide insight into the composition of the AP-1 signaling machinery and validate this approach as a tractable platform for genome-wide functional analysis.
Activation of NF-κB in cells productively infected with HSV-1 depends on activated protein kinase R and plays no apparent role in blocking apoptosis
Authors: Taddeo Brunella , Luo Ting Rong , Zhang Weiran , Roizman Bernard
Published: October 2003

Microarray data reported elsewhere indicated that herpes simplex virus 1 induces the up-regulation of nuclear factor κB (NF-κB)-regulated genes, including that of its inhibitor, IκBα, consistent with the reports that wild-type virus induces the activation of NF-κB. In this report we show that activation of NF-κB in infected cells is linked to the activation of protein kinase R (PKR). Specifically: (
i
) PKR is activated in infected cells although the effects of the activated enzyme on protein synthesis are negated by the viral gene γ
1
34.5, which encodes a protein phosphatase 1α accessory factor that enables the dephosphorylation of the α subunit of eukaryotic translation initiation factor 2. NF-κB is activated in wild-type murine embryonic fibroblasts but not in related PKR-null cells. (
ii
) In cells infected with a replication-competent Δγ
1
34.5 mutant (R5104), but carrying a U
S
11 gene expressed early in infection, eukaryotic translation initiation factor 2α is not phosphorylated, and in
in vitro
assays, PKR bound to the U
S
11 protein is not phosphorylated on subsequent addition of double-stranded RNA. Here we report that this mutant does not activate PKR, has no effect on the accumulation of IκBα, and does not cause the translocation of NF-κB in infected cells. (
iii
) One hypothesis advanced for the activation of NF-κB is that it blocks apoptosis induced by viral gene products. The replication-competent R5104 mutant does not induce the programmed cell's death. We conclude that in herpes simplex virus 1-infected cells, activation of NF-κB depends on activation of PKR and that NF-κB is not required to block apoptosis in productively infected cells.
Nuclear localization of pyrrole–imidazole polyamide–fluorescein conjugates in cell culture
Authors: Best Timothy P. , Edelson Benjamin S. , Nickols Nicholas G. , Dervan Peter B.
Published: October 2003

A series of hairpin pyrrole–imidazole polyamide–fluorescein conjugates were synthesized and assayed for cellular localization. Thirteen cell lines, representing 11 human cancers, one human transformed kidney cell line, and one murine leukemia cell line, were treated with 5 μM polyamide–fluorescein conjugates for 10–14 h, then imaged by confocal laser scanning microscopy. A conjugate containing a β-alanine residue at the C terminus of the polyamide moiety showed no nuclear localization, whereas an analogous compound lacking the β-alanine residue was strongly localized in the nuclei of all cell lines tested. The localization profiles of several other conjugates suggest that pyrrole–imidazole sequence and content, dye choice and position, linker composition, and molecular weight are determinants of nuclear localization. The attachment of fluorescein to the C terminus of a hairpin polyamide results in an ≈10-fold reduction in DNA-binding affinity, with no loss of binding specificity with reference to mismatch binding sites.
Biosilica formation in diatoms: Characterization of native silaffin-2 and its role in silica morphogenesis
Authors: Poulsen Nicole , Sumper Manfred , Kröger Nils
Published: October 2003

The biological formation of inorganic materials with complex form (biominerals) is a widespread phenomenon in nature, yet the molecular mechanisms underlying biomineral morphogenesis are not well understood. Among the most fascinating examples of biomineral structures are the intricately patterned, silicified cell walls of diatoms, which contain tightly associated organic macromolecules. From diatom biosilica a highly polyanionic phosphoprotein, termed native silaffin-2 (natSil-2), was isolated that carries unconventional amino acid modifications. natSil-2 lacked intrinsic silica formation activity but was able to regulate the activities of the previously characterized silica-forming biomolecules natSil-1A and long-chain polyamines. Combining natSil-2 and natSil-1A (or long-chain polyamines) generated an organic matrix that mediated precipitation of porous silica within minutes after the addition of silicic acid. Remarkably, the precipitate displayed pore sizes in the range 100–1000 nm, which is characteristic for diatom biosilica nanopatterns.
Unique conformation of cancer autoantigen B23 in hepatoma: A mechanism for specificity in the autoimmune response
Authors: Ulanet Danielle B. , Torbenson Michael , Dang Chi V. , Casciola-Rosen Livia , Rosen Antony
Published: October 2003

The association of a specific autoantibody response with distinct disease phenotypes is observed in both autoimmune diseases and cancer. Although the underlying mechanisms remain unclear, it is likely that unique properties of disease-specific autoantigens expressed in the relevant target cells play a role. It has recently been observed that the majority of autoantigens targeted across the spectrum of systemic autoimmune diseases (but not nonautoantigens) are selectively cleaved by the cytotoxic lymphocyte granule protease granzyme B (GB), generating unique fragments not observed during other forms of cell death. Although susceptibility of a molecule to cleavage by GB strongly predicts autoantigen status, the significance of this association is unclear. We used hepatocellular carcinoma and the hepatocellular carcinoma autoantigen, nucleophosmin/B23, as a model system to define the unique features of disease-specific autoantigens in the relevant disease microenvironment. These studies revealed a striking, selective susceptibility of B23 to cleavage by GB in extracts of neoplastic liver. The increased sensitivity of tumor B23 to proteolysis by GB was accompanied by slightly increased mobility on SDS/PAGE, altered subcellular localization, enrichment of an SDS-stable oligomeric form of B23, and recognition by a conformation-specific antibody detecting a B23 epitope ending at the GB cleavage site.In vitrostudies demonstrated that this unique B23 conformation and resultant increased susceptibility to cleavage by GB arise when B23 translation is initiated at methionine-7. We propose that unique features of autoantigens in the disease-relevant microenvironment may regulate susceptibility to cleavage by GB and their selection by the specific autoimmune response.
Predicting aberrant CpG island methylation
Authors: Feltus F. A. , Lee E. K. , Costello J. F. , Plass C. , Vertino P. M.
Published: October 2003

Epigenetic silencing associated with aberrant methylation of promoter region CpG islands is one mechanism leading to loss of tumor suppressor function in human cancer. Profiling of CpG island methylation indicates that some genes are more frequently methylated than others, and that each tumor type is associated with a unique set of methylated genes. However, little is known about why certain genes succumb to this aberrant event. To address this question, we used Restriction Landmark Genome Scanning to analyze the susceptibility of 1,749 unselected CpG islands to
de novo
methylation driven by overexpression of DNA cytosine-5-methyltransferase 1 (DNMT1). We found that although the overall incidence of CpG island methylation was increased in cells overexpressing DNMT1, not all loci were equally affected. The majority of CpG islands (69.9%) were resistant to
de novo
methylation, regardless of DNMT1 overexpression. In contrast, we identified a subset of methylation-prone CpG islands (3.8%) that were consistently hypermethylated in multiple DNMT1 overexpressing clones. Methylation-prone and methylation-resistant CpG islands were not significantly different with respect to size, C+G content, CpG frequency, chromosomal location, or promoter association. We used DNA pattern recognition and supervised learning techniques to derive a classification function based on the frequency of seven novel sequence patterns that was capable of discriminating methylation-prone from methylation-resistant CpG islands with 82% accuracy. The data indicate that CpG islands differ in their intrinsic susceptibility to
de novo
methylation, and suggest that the propensity for a CpG island to become aberrantly methylated can be predicted based on its sequence context.
Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid
Authors: Tangvoranuntakul Pam , Gagneux Pascal , Diaz Sandra , Bardor Muriel , Varki Nissi , Varki Ajit , Muchmore Elaine
Published: October 2003

Humans are genetically unable to produce the sialic acidN-glycolylneuraminic acid (Neu5Gc), because of a mutation that occurred after our last common ancestor with great apes. Although Neu5Gc is presumed absent from normal humans, small amounts have been claimed to exist in human tumors and fetal meconium. We have generated an antibody with high specificity and avidity for Neu5Gc. Fetal tissues, normal adult tissues, and breast carcinomas from humans showed reactivity to this antibody, primarily within secretory epithelia and blood vessels. The presence of small amounts of Neu5Gc was confirmed by MS. Absent any known alternate pathway for its synthesis, we reasoned that these small amounts of Neu5Gc might originate from exogenous sources. Indeed, human cells fed with Neu5Gc incorporated it into endogenous glycoproteins. When normal human volunteers ingested Neu5Gc, a portion was absorbed and eliminated in urine, and small quantities were incorporated into newly synthesized glycoproteins. Neu5Gc has never been reported in plants or microbes to our knowledge. We found that Neu5Gc is rare in poultry and fish, common in milk products, and enriched in red meats. Furthermore, normal humans have variable amounts of circulating IgA, IgM, and IgG antibodies against Neu5Gc, with the highest levels comparable to those of the previously known anti-α-galactose xenoreactive antibodies. This finding represents an instance wherein humans absorb and metabolically incorporate a nonhuman dietary component enriched in foods of mammalian origin, even while generating xenoreactive, and potentially autoreactive, antibodies against the same molecule. Potential implications for human diseases are briefly discussed.
GABA uptake into astrocytes is not associated with significant metabolic cost: Implications for brain imaging of inhibitory transmission
Authors: Chatton Jean-Yves , Pellerin Luc , Magistretti Pierre J.
Published: October 2003

Synaptically released glutamate has been identified as a signal coupling excitatory neuronal activity to increased glucose utilization. The proposed mechanism of this coupling involves glutamate uptake into astrocytes resulting in increased intracellular Na
+
(Na
i
+
) and activation of the Na
+
/K
+
-ATPase. Increased metabolic demand linked to disruption of Na
i
+
homeostasis activates glucose uptake and glycolysis in astrocytes. Here, we have examined whether a similar neurometabolic coupling could operate for the inhibitory neurotransmitter γ-aminobutyric acid (GABA), also taken up by Na
+
-dependent transporters into astrocytes. Thus, we have compared the Na
i
+
response to GABA and glutamate in mouse astrocytes by microspectrofluorimetry. The Na
i
+
response to GABA consisted of a rapid rise of 4–6 mM followed by a plateau that did not, however, significantly activate the pump. Indeed, the GABA transporter-evoked Na
+
influxes are transient in nature, almost totally shutting off within ≈30 sec of GABA application. The metabolic consequences of the GABA-induced Na
i
+
response were evaluated by monitoring cellular ATP changes indirectly in single cells and measuring 2-deoxyglucose uptake in astrocyte populations. Both approaches showed that, whereas glutamate induced a robust metabolic response in astrocytes (decreased ATP levels and glucose uptake stimulation), GABA did not cause any measurable metabolic response, consistent with the Na
i
+
measurements. Results indicate that GABA does not couple inhibitory neuronal activity with glucose utilization, as does glutamate for excitatory neurotransmission, and suggest that GABA-mediated synaptic transmission does not contribute directly to brain imaging signals based on deoxyglucose.
Membrane association of myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain and a coiled-coil dimerization module
Authors: Berger Philipp , Schaffitzel Christiane , Berger Imre , Ban Nenad , Suter Ueli
Published: October 2003

Mutations in the myotubularin (MTM)-related protein 2 (
MTMR2
) gene are responsible for the severe autosomal recessive neuropathy Charcot–Marie–Tooth disease type 4B1. MTMR2 belongs to the MTM family of dual-specific phosphatases that use phosphatidylinositol (PI) 3,5-bisphosphate [PI(3,5)P
2
] and PI 3-phosphate [PI(3)P] as their substrate. Because these substrates are localized in the membrane bilayer, membrane targeting of Mtmr2 is an important regulatory mechanism. In hypoosmotically stressed COS cells with increased levels of PI(3,5)P
2
, Mtmr2 is bound to the membrane of vacuoles formed under these conditions. Using several mutant forms of Mtmr2, we identified two domains that are necessary for membrane association: (
i
) A pleckstrin homology-GRAM domain; and (
ii
) a coiled-coil module. Protein-lipid overlay assays show that the pleckstrin homology-GRAM domain binds to PI(3,5)P
2
and PI(5)P, a substrate and a product of the Mtmr2 enzyme, respectively. We also demonstrate that Mtmr2 forms a dimer and that the C-terminal coiled-coil is responsible for homodimerization, in addition to membrane association. Our data indicate that phosphoinositide–protein interactions, as well as protein–protein interactions, are necessary for the correct regulation of MTMR2.
Enzyme screening with synthetic multifunctional pores: Focus on biopolymers
Authors: Sordé Nathalie , Das Gopal , Matile Stefan
Published: October 2003

This report demonstrates that a single set of identical synthetic multifunctional pores can detect the activity of many different enzymes. Enzymes catalyzing either synthesis or degradation of DNA (exonuclease III or polymerase I), RNA (RNase A), polysaccharides (heparinase I, hyaluronidase, and galactosyltransferase), and proteins (papain, ficin, elastase, subtilisin, and pronase) are selected to exemplify this key characteristic of synthetic multifunctional pore sensors. Because anionic, cationic, and neutral substrates can gain access to the interior of complementarily functionalized pores, such pores can be the basis for very user-friendly screening of a broad range of enzymes.
Structure and function of the feed-forward loop network motif
Authors: Mangan S. , Alon U.
Published: October 2003

Engineered systems are often built of recurring circuit modules that carry out key functions. Transcription networks that regulate the responses of living cells were recently found to obey similar principles: they contain several biochemical wiring patterns, termed network motifs, which recur throughout the network. One of these motifs is the feed-forward loop (FFL). The FFL, a three-gene pattern, is composed of two input transcription factors, one of which regulates the other, both jointly regulating a target gene. The FFL has eight possible structural types, because each of the three interactions in the FFL can be activating or repressing. Here, we theoretically analyze the functions of these eight structural types. We find that four of the FFL types, termed incoherent FFLs, act as sign-sensitive accelerators: they speed up the response time of the target gene expression following stimulus steps in one direction (e.g., off to on) but not in the other direction (on to off). The other four types, coherent FFLs, act as sign-sensitive delays. We find that some FFL types appear in transcription network databases much more frequently than others. In some cases, the rare FFL types have reduced functionality (responding to only one of their two input stimuli), which may partially explain why they are selected against. Additional features, such as pulse generation and cooperativity, are discussed. This study defines the function of one of the most significant recurring circuit elements in transcription networks.
Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins
Authors: Martin-Serrano Juan , Yaravoy Anton , Perez-Caballero David , Bieniasz Paul D.
Published: October 2003

The release of enveloped viruses from infected cells often requires a virally encoded activity, termed a late-budding domain (L domain), encoded by essential PTAP, PPXY, or YPDL sequence motifs. PTAP-type L domains recruit one of three endosomal sorting complexes required for transport (ESCRT-I). However, subsequent events in viral budding are poorly defined, and neither YPDL nor PPXY-type L domains require ESCRT-I. Here, we show that ESCRT-I and other class E vacuolar protein sorting (VPS) factors are linked by a complex series of protein–protein interactions. In particular, interactions between ESCRT-I and ESCRT-III are bridged by AIP-1/ALIX, a mammalian orthologue of the yeast class E VPS factor, Bro1. Expression of certain ESCRT-III components as fusion proteins induces a late budding defect that afflicts all three L-domain types, suggesting that ESCRT-III integrity is required in a general manner. Notably, the prototype YPDL-type L domain encoded by equine infectious anemia virus (EIAV) acts by recruiting AIP-1/ALIX and expression of a truncated form of AIP-1/ALIX or small interfering RNA-induced AIP-1/ALIX depletion specifically inhibits EIAV YPDL-type L-domain function. Overall, these findings indicate that L domains subvert a subset of class E VPS factors to mediate viral budding, some of which are required for each of the L-domain types, whereas others apparently act as adaptors to physically link specific L-domain types to the class E VPS machinery.
Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase η
Authors: Washington M. Todd , Prakash Louise , Prakash Satya
Published: October 2003

DNA polymerase η (Polη) has the unique ability to replicate through UV-light-induced cyclobutane pyrimidine dimers. Here we use pre-steady-state kinetic analyses to examine the mechanism of nucleotide incorporation opposite a
cis
-
syn
thymine-thymine (TT) dimer and an identical nondamaged sequence by yeast Polη. Polη displayed “burst” kinetics for nucleotide incorporation opposite both the damaged and nondamaged templates. Although a slight decrease occurred in the affinity (
K
d
) of nucleotide binding opposite the TT dimer relative to the nondamaged template, the rate (
k
pol
) of nucleotide incorporation was the same whether the template was damaged or nondamaged. These results strongly support a mechanism in which the nucleotide is directly inserted opposite the TT dimer by using its intrinsic base-pairing ability without any hindrance from the distorted geometry of the lesion.
Distribution of NF-κB-binding sites across human chromosome 22
Authors: Martone Rebecca , Euskirchen Ghia , Bertone Paul , Hartman Stephen , Royce Thomas E. , Luscombe Nicholas M. , Rinn John L. , Nelson F. Kenneth , Miller Perry , Gerstein Mark , Weissman Sherman , Snyder Michael
Published: October 2003

We have mapped the chromosomal binding site distribution of a transcription factor in human cells. The NF-κB family of transcription factors plays an essential role in regulating the induction of genes involved in several physiological processes, including apoptosis, immunity, and inflammation. The binding sites of the NF-κB family member p65 were determined by using chromatin immunoprecipitation and a genomic microarray of human chromosome 22 DNA. Sites of binding were observed along the entire chromosome in both coding and noncoding regions, with an enrichment at the 5′ end of genes. Strikingly, a significant proportion of binding was seen in intronic regions, demonstrating that transcription factor binding is not restricted to promoter regions. NF-κB binding was also found at genes whose expression was regulated by tumor necrosis factor α, a known inducer of NF-κB-dependent gene expression, as well as adjacent to genes whose expression is not affected by tumor necrosis factor α. Many of these latter genes are either known to be activated by NF-κB under other conditions or are consistent with NF-κB's role in the immune and apoptotic responses. Our results suggest that binding is not restricted to promoter regions and that NF-κB binding occurs at a significant number of genes whose expression is not altered, thereby suggesting that binding alone is not sufficient for gene activation.
MAL regulates clathrin-mediated endocytosis at the apical surface of Madin–Darby canine kidney cells
Authors: Martín-Belmonte Fernando , Martínez-Menárguez José A. , Aranda Juan F. , Ballesta José , de Marco María C. , Alonso Miguel A.
Published: October 2003

MAL is an integral protein component of the machinery for apical transport in epithelial Madin–Darby canine kidney (MDCK) cells. To maintain its distribution, MAL cycles continuously between the plasma membrane and the Golgi complex. The clathrin-mediated route for apical internalization is known to differ from that at the basolateral surface. Herein, we report that MAL depends on the clathrin pathway for apical internalization. Apically internalized polymeric Ig receptor (pIgR), which uses clathrin for endocytosis, colocalized with internalized MAL in the same apical vesicles. Time-lapse confocal microscopic analysis revealed cotransport of pIgR and MAL in the same endocytic structures. Immunoelectron microscopic analysis evidenced colabeling of MAL with apically labeled pIgR in pits and clathrin-coated vesicles. Apical internalization of pIgR was abrogated in cells with reduced levels of MAL, whereas this did not occur either with its basolateral entry or the apical internalization of glycosylphosphatidylinositol-anchored proteins, which does not involve clathrin. Therefore, MAL is critical for efficient clathrin-mediated endocytosis at the apical surface in MDCK cells.
Actin cytoskeleton remodeling during early Drosophila furrow formation requires recycling endosomal components Nuclear-fallout and Rab11
Authors: Riggs Blake , Rothwell Wendy , Mische Sarah , Hickson Gilles R.X. , Matheson Johanne , Hays Thomas S. , Gould Gwyn W. , Sullivan William
Published: October 2003

Cytokinesis requires a dramatic remodeling of the cortical cytoskeleton as well as membrane addition. The Drosophila pericentrosomal protein, Nuclear-fallout (Nuf), provides a link between these two processes. In nuf-derived embryos, actin remodeling and membrane recruitment during the initial stages of metaphase and cellular furrow formation are disrupted. Nuf is a homologue of arfophilin-2, an ADP ribosylation factor effector that binds Rab11 and influences recycling endosome (RE) organization. Here, we show that Nuf is an important component of the RE, and that these phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a key RE component. GST pull-downs and the presence of a conserved Rab11-binding domain in Nuf demonstrate that Nuf and Rab11 physically associate. In addition, Nuf and Rab11 are mutually required for their localization to the RE. Embryos with reduced levels of Rab11 produce membrane recruitment and actin remodeling defects strikingly similar to nuf-derived embryos. These analyses support a common role for Nuf and Rab11 at the RE in membrane trafficking and actin remodeling during the initial stages of furrow formation.
Ligand crowding at a nascent signal sequence
Authors: Eisner Gottfried , Koch Hans-Georg , Beck Konstanze , Brunner Joseph , Müller Matthias
Published: October 2003

We have systematically analyzed the molecular environment of the signal sequence of a growing secretory protein from Escherichia coli using a stage- and site-specific cross-linking approach. Immediately after emerging from the ribosome, the signal sequence of pOmpA is accessible to Ffh, the protein component of the bacterial signal recognition particle, and to SecA, but it remains attached to the surface of the ribosome via protein L23. These contacts are lost upon further growth of the nascent chain, which brings the signal sequence into sole proximity to the chaperone Trigger factor (TF). In its absence, nascent pOmpA shows extended contacts with L23, and even long chains interact in these conditions proficiently with Ffh. Our results suggest that upon emergence from the ribosome, the signal sequence of an E. coli secretory protein gradually becomes sequestered by TF. Although TF thereby might control the accessibility of pOmpA's signal sequence to Ffh and SecA, it does not influence interaction of pOmpA with SecB.
Common Adverse Events Associated with the Use of Ribavirin for Severe Acute Respiratory Syndrome in Canada
Authors: Knowles Sandra R. , Phillips Elizabeth J. , Dresser Linda , Matukas Larissa
Published: October 2003

Although information on efficacy and adverse drug reactions is lacking, ribavirin has been used empirically for the treatment of severe acute respiratory syndrome (SARS). We report common adverse events in 110 patients with suspected or probable SARS who were treated with ribavirin. Sixty-one percent of the patients had evidence of hemolytic anemia, and hypocalcemia and hypomagnesmia were reported in 58% and 46% of patients, respectively.
A strongly diagonal power of algebraic order bounded disjointness preserving operators
Authors: Boulabiar Karim , Buskes Gerard , Sirotkin Gleb
Published: October 2003

An order bounded disjointness preserving operator

T
T

on an Archimedean vector lattice is algebraic if and only if the restriction of

T

n
!

T^{n!}

to the vector sublattice generated by the range of

T

m

T^{m}

is strongly diagonal, where

n
n

is the degree of the minimal polynomial of

T
T

and

m
m

is its ‘valuation’.
Tyrosine Phosphatase Epsilon Is a Positive Regulator of Osteoclast Function in Vitro and In Vivo
Authors: Chiusaroli Riccardo , Knobler Hilla , Luxenburg Chen , Sanjay Archana , Granot-Attas Shira , Tiran Zohar , Miyazaki Tsuyoshi , Harmelin Alon , Baron Roland , Elson Ari
Published: October 2003

Protein tyrosine phosphorylation is a major regulator of bone metabolism. Tyrosine phosphatases participate in regulating phosphorylation, but roles of specific phosphatases in bone metabolism are largely unknown. We demonstrate that young (<12 weeks) female mice lacking tyrosine phosphatase epsilon (PTPϵ) exhibit increased trabecular bone mass due to cell-specific defects in osteoclast function. These defects are manifested in vivo as reduced association of osteoclasts with bone and as reduced serum concentration of C-terminal collagen telopeptides, specific products of osteoclast-mediated bone degradation. Osteoclast-like cells are generated readily from PTPϵ-deficient bone-marrow precursors. However, cultures of these cells contain few mature, polarized cells and perform poorly in bone resorption assays in vitro. Podosomes, structures by which osteoclasts adhere to matrix, are disorganized and tend to form large clusters in these cells, suggesting that lack of PTPϵ adversely affects podosomal arrangement in the final stages of osteoclast polarization. The gender and age specificities of the bone phenotype suggest that it is modulated by hormonal status, despite normal serum levels of estrogen and progesterone in affected mice. Stimulation of bone resorption by RANKL and, surprisingly, Src activity and Pyk2 phosphorylation are normal in PTPϵ-deficient osteoclasts, indicating that loss of PTPϵ does not cause widespread disruption of these signaling pathways. These results establish PTPϵ as a phosphatase required for optimal structure, subcellular organization, and function of osteoclasts in vivo and in vitro.
Roles of Phosphoinositides and of Spo14p (phospholipase D)-generated Phosphatidic Acid during Yeast Sporulation
Authors: Rudge Simon A. , Sciorra Vicki A. , Iwamoto Michelle , Zhou Chun , Strahl Thomas , Morris Andrew J. , Thorner Jeremy , Engebrecht JoAnne
Published: October 2003

During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P2is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P2is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P2formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P2synthesis.
Vacuole Size Control: Regulation of PtdIns(3,5)P2Levels by the Vacuole-associated Vac14-Fig4 Complex, a PtdIns(3,5)P2-specific Phosphatase
Authors: Rudge Simon A. , Anderson Deborah M. , Emr Scott D.
Published: October 2003

In the budding yeast Saccharomyces cerevisiae, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is synthesized by a single phosphatidylinositol 3-phosphate 5-kinase, Fab1. Cells deficient in PtdIns(3,5)P2synthesis exhibit a grossly enlarged vacuole morphology, whereas increased levels of PtdIns(3,5)P2provokes the formation of multiple small vacuoles, suggesting a specific role for PtdIns(3,5)P2in vacuole size control. Genetic studies have indicated that Fab1 kinase is positively regulated by Vac7 and Vac14; deletion of either gene results in ablation of PtdIns(3,5)P2synthesis and the formation of a grossly enlarged vacuole. More recently, a suppressor of vac7Δ mutants was identified and shown to encode a putative phosphoinositide phosphatase, Fig4. We demonstrate that Fig4 is a magnesium-activated PtdIns(3,5)P2-selective phosphoinositide phosphatase in vitro. Analysis of a Fig4-GFP fusion protein revealed that the Fig4 phosphatase is localized to the limiting membrane of the vacuole. Surprisingly, in the absence of Vac14, Fig4-GFP no longer localizes to the vacuole. However, Fig4-GFP remains localized to the grossly enlarged vacuoles of vac7 deletion mutants. Consistent with these observations, we found that Fig4 physically associates with Vac14 in a common membrane–associated complex. Our studies indicate that Vac14 both positively regulates Fab1 kinase activity and directs the localization/activation of the Fig4 PtdIns(3,5)P2phosphatase.
Endocytosis of Epithelial Apical Junctional Proteins by a Clathrin-mediated Pathway into a Unique Storage Compartment
Authors: Ivanov Andrei I. , Nusrat Asma , Parkos Charles A.
Published: October 2003

The adherens junction (AJ) and tight junction (TJ) are key regulators of epithelial polarity and barrier function. Loss of epithelial phenotype is accompanied by endocytosis of AJs and TJs via unknown mechanisms. Using a model of calcium depletion, we defined the pathway of internalization of AJ and TJ proteins (E-cadherin, p120 and β-catenins, occludin, JAM-1, claudins 1 and 4, and ZO-1) in T84 epithelial cells. Proteinase protection assay and immunocytochemistry revealed orchestrated internalization of AJs and TJs into a subapical cytoplasmic compartment. Disruption of caveolae/lipid rafts did not prevent endocytosis, nor did caveolin-1 colocalize with internalized junctional proteins. Furthermore, AJ and TJ proteins did not colocalize with the macropinocytosis marker dextran. Inhibitors of clathrin-mediated endocytosis blocked internalization of AJs and TJs, and junctional proteins colocalized with clathrin and α-adaptin. AJ and TJ proteins were observed to enter early endosomes followed by movement to organelles that stained with syntaxin-4 but not with markers of late and recycling endosomes, lysosomes, or Golgi. These results indicate that endocytosis of junctional proteins is a clathrin-mediated process leading into a unique storage compartment. Such mechanisms may mediate the disruption of intercellular contacts during normal tissue remodeling and in pathology.
Dynamic and Regulated Association of Caveolin with Lipid Bodies: Modulation of Lipid Body Motility and Function by a Dominant Negative Mutant
Authors: Pol Albert , Martin Sally , Fernandez Manuel A. , Ferguson Charles , Carozzi Amanda , Luetterforst Robert , Enrich Carlos , Parton Robert G.
Published: October 2003

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cavDGV) to study LB formation and to examine its effect on LB function. We now show that the cavDGVmutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.