bluuebunny/crossref_metadata_2025 huggingface dataset:
Transition to the open state of the TolC periplasmic tunnel entrance
Authors: Andersen Christian , Koronakis Eva , Bokma Evert , Eswaran Jeyanthy , Humphreys Daniel , Hughes Colin , Koronakis Vassilis
Published: September 2002

The TolC channel-tunnel spans the bacterial outer membrane and periplasm, providing a large exit duct for protein export and multidrug efflux when recruited by substrate-engaged inner membrane complexes. The sole constriction in the single pore of the homotrimeric TolC is the periplasmic tunnel entrance, which in its resting configuration is closed by dense packing of the 12 tunnel-forming α-helices. Recruitment of TolC must trigger opening for substrate transit to occur, but the mechanism underlying transition from the closed to the open state is not known. The high resolution structure of TolC indicates that the tunnel helices are constrained at the entrance by a circular network of intra- and intermonomer hydrogen bonds and salt bridges. To assess how opening is achieved, we disrupted these connections and monitored changes in the aperture size by measuring the single channel conductance of TolC derivatives in black lipid bilayers. Elimination of individual connections caused incremental weakening of the circular network, accompanied by gradual relaxation from the closed state and increased flexibility of the entrance. Simultaneous abolition of the key links caused a substantial increase in conductance, generating an aperture that corresponds to the modeled open state, with the capacity to allow access and passage of diverse substrates. The results support a model in which transition to the open state of TolC is achieved by an iris-like realignment of the tunnel entrance helices.
Roles of mutation and recombination in the evolution of protein thermodynamics
Authors: Xia Yu , Levitt Michael
Published: September 2002

We present a comprehensive study of the evolutionary origin of the thermodynamic behavior of proteins. With the use of a simplified model, we exhaustively enumerate the space of all sequences and the space of all structures, simulate the evolutionary relationship between sequences and structures, and characterize the steady-state sequence distribution for all structures in terms of several thermodynamic variables. We assess the effects of two major forces of evolution: mutation and recombination. Three simplifications are made. First, a two-dimensional lattice model is used to represent protein sequences and structures. Second, proteins undergo neutral evolution so that the fitness landscape has a flat allowed region inside of which all sequences are equally fit. Third, we ignore otherwise important factors such as finite population size and evolutionary time. Two scenarios emerge from our study. The first occurs when evolution is dominated by mutation events. Even though the prototype sequence that is most mutationally robust is preferred by evolution, the preference is not strong enough to offset the huge size of sequence space. Most native sequences are located near the boundary of the fitness region and are marginally compatible with the native structure. The second scenario occurs when evolution is dominated by recombination events. Now evolutionary preference for prototype sequence is strong enough to overcome the size of sequence space so that most native sequences are located near the center of sequence-structure compatibility. We conclude that the relative frequency of mutation and recombination events is a major determinant of how optimal protein sequences are for their structures.
Inhibition of calcineurin-NFAT hypertrophy signaling by cGMP-dependent protein kinase type I in cardiac myocytes
Authors: Fiedler Beate , Lohmann Suzanne M. , Smolenski Albert , Linnemüller Stephan , Pieske Burkert , Schröder Frank , Molkentin Jeffery D. , Drexler Helmut , Wollert Kai C.
Published: September 2002

Recent investigation has focused on identifying signaling pathways that inhibit cardiac hypertrophy, a major risk factor for cardiovascular morbidity and mortality. In this context, nitric oxide (NO), signaling via cGMP and cGMP-dependent protein kinase type I (PKG I), has been recognized as a negative regulator of cardiac myocyte (CM) hypertrophy. However, the underlying mechanisms are poorly understood. Here, we show that PKG I inhibits CM hypertrophy by targeting the calcineurin-NFAT signaling pathway. Calcineurin, a Ca
2+
-dependent phosphatase, promotes hypertrophy in part by activating NFAT transcription factors which induce expression of hypertrophic genes, including brain natriuretic peptide (BNP). Activation of PKG I by NO/cGMP in CM suppressed NFAT transcriptional activity, BNP induction, and cell enlargement in response to α
1
-adrenoreceptor stimulation but not in response to adenoviral expression of a Ca
2+
-independent, constitutively active calcineurin mutant, thus demonstrating NO-cGMP-PKG I inhibition of calcineurin-NFAT signaling upstream of calcineurin. PKG I suppressed single L-type Ca
2+
-channel open probability, [Ca
2+
]
i
transient amplitude, and, most importantly, L-type Ca
2+
-channel current-induced NFAT activation, indicating that PKG I targets Ca
2+
-dependent steps upstream of calcineurin. Adenoviral expression of PKG I enhanced NO/cGMP inhibitory effects upstream of calcineurin, confirming that PKG I mediates NO/cGMP inhibition of calcineurin-NFAT signaling. In CM overexpressing PKG I, NO/cGMP also suppressed BNP induction and cell enlargement but not NFAT activation elicited by constitutively active calcineurin, which is consistent with additional, NFAT-independent inhibitory effect(s) of PKG I downstream of calcineurin. Inhibition of calcineurin-NFAT signaling by PKG I provides a framework for understanding how NO inhibits cardiac myocyte hypertrophy.
Repression of stress-responsive genes by FIERY2, a novel transcriptional regulator in
Arabidopsis
Authors: Xiong Liming , Lee Hojoung , Ishitani Manabu , Tanaka Yuko , Stevenson Becky , Koiwa Hisashi , Bressan Ray A. , Hasegawa Paul M. , Zhu Jian-Kang
Published: September 2002

Low temperature, drought, and high salinity induce the expression of many plant genes. To understand the mechanisms for the transcriptional activation of these genes, we conducted a reporter gene-aided genetic screen in
Arabidopsis
. Seven allelic mutations in the FIERY2 (
FRY2
) locus result in significant increases in the expression of stress-responsive genes with the DRE/CRT (drought-responsive/C-repeat) cis element but non-DRE/CRT type stress-responsive genes were less affected. The specific regulation of DRE/CRT class of genes by FRY2 appears to be caused by repression of stress induction of the upstream CBF/DREB transcription factor genes.
fry2
mutants show increased tolerance to salt stress and to abscisic acid during seed germination but are more sensitive to freezing damage at the seedling stage.
FRY2
/
CPL1
encodes a novel transcriptional repressor harboring two double-stranded RNA-binding domains and a region homologous to the catalytic domain of RNA polymerase II C-terminal domain phosphatases found in yeast and in animals that regulate gene transcription. These data indicate that FRY2 is an important negative regulator of stress gene transcription and suggest that structured RNA may regulate hormone and stress responses in plants as it does in animals.
Soft, conformable electrical contacts for organic semiconductors: High-resolution plastic circuits by lamination
Authors: Loo Yueh-Lin , Someya Takao , Baldwin Kirk W. , Bao Zhenan , Ho Peter , Dodabalapur Ananth , Katz Howard E. , Rogers John A.
Published: September 2002

Soft, conformable electrical contacts provide efficient, noninvasive probes for the transport properties of chemically and mechanically fragile, ultrathin organic semiconducting films. When combined with high-resolution printing and lamination techniques, these soft contacts also form the basis of a powerful technique for fabricating flexible plastic circuits. In this approach, a thin elastomeric film on a plastic substrate supports the electrodes and interconnections; laminating this substrate against another plastic substrate that supports the gate, dielectric and semiconductor levels establishes effective electrical contacts and completes the circuits. In addition to eliminating many of the problems associated with traditional layer-by-layer fabrication strategies, this lamination scheme possesses other attractive features: the transistors and circuit elements are naturally and efficiently encapsulated, and the active organic semiconductor layer is placed near the neutral mechanical plane. We demonstrate the features of soft, laminated contacts by fabricating large arrays of high-performance thin film transistors on plastic substrates by using a wide variety of organic semiconductors.
Caspase 3 activity is required for skeletal muscle differentiation
Authors: Fernando Pasan , Kelly John F. , Balazsi Kim , Slack Ruth S. , Megeney Lynn A.
Published: September 2002

The cellular alterations associated with skeletal muscle differentiation share a high degree of similarity with key phenotypic changes usually ascribed to apoptosis. For example, actin fiber disassembly/reorganization is a conserved feature of both apoptosis and differentiating myoblasts and the conserved muscle contractile protein, myosin light chain kinase, is required for the apoptotic feature of membrane blebbing. As such, these observations suggest that the induction of differentiation and apoptosis in the myogenic lineage may use overlapping cellular mechanisms. Here, we report that skeletal muscle differentiation depends on the activity of the key apoptotic protease, caspase 3. Peptide inhibition of caspase 3 activity or homologous deletion of caspase 3 leads to dramatic reduction in both myotube/myofiber formation and expression of muscle-specific proteins. Subsequently, we have identified Mammalian Sterile Twenty-like kinase as a crucial caspase 3 effector in this cellular process. Mammalian Sterile Twenty-like kinase is cleavage-activated by caspase 3, and restoration of this truncated kinase in caspase 3 null myoblasts restores the differentiation phenotype. Taken together, these results confirm a unique and unanticipated role for a caspase 3-mediated signal cascade in the promotion of myogenesis.
The chloroplast and mitochondrial genome sequences of the charophyte
Chaetosphaeridium globosum
: Insights into the timing of the events that restructured organelle DNAs within the green algal lineage that led to land plants
Authors: Turmel Monique , Otis Christian , Lemieux Claude
Published: September 2002

The land plants and their immediate green algal ancestors, the charophytes, form the Streptophyta. There is evidence that both the chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) underwent substantial changes in their architecture (intron insertions, gene losses, scrambling in gene order, and genome expansion in the case of mtDNA) during the evolution of streptophytes; however, because no charophyte organelle DNAs have been sequenced completely thus far, the suite of events that shaped streptophyte organelle genomes remains largely unknown. Here, we have determined the complete cpDNA (131,183 bp) and mtDNA (56,574 bp) sequences of the charophyte
Chaetosphaeridium globosum
(Coleochaetales). At the levels of gene content (124 genes), intron composition (18 introns), and gene order,
Chaetosphaeridium
cpDNA is remarkably similar to land-plant cpDNAs, implying that most of the features characteristic of land-plant lineages were gained during the evolution of charophytes. Although the gene content of
Chaetosphaeridium
mtDNA (67 genes) closely resembles that of the bryophyte
Marchantia polymorpha
(69 genes), this charophyte mtDNA differs substantially from its land-plant relatives at the levels of size, intron composition (11 introns), and gene order. Our finding that it shares only one intron with its land-plant counterparts supports the idea that the vast majority of mitochondrial introns in land plants appeared after the emergence of these organisms. Our results also suggest that the events accounting for the spacious intergenic spacers found in land-plant mtDNAs took place late during the evolution of charophytes or coincided with the transition from charophytes to land plants.
Global analysis of stress-regulated mRNA turnover by using cDNA arrays
Authors: Fan Jinshui , Yang Xiaoling , Wang Wengong , Wood William H. , Becker Kevin G. , Gorospe Myriam
Published: September 2002

cDNA array technology has proven to be a powerful way to monitor global changes in gene expression patterns. Here, we present an approach that extends the current utility of cDNA arrays to allow the evaluation of the relative roles of transcription and mRNA turnover in governing gene expression on a global basis, compared with current individual gene-by-gene analyses. This method, which involves comparison of large-scale hybridization patterns generated with steady-state mRNA versus newly transcribed (nuclear run-on) RNA, was used to demonstrate the importance of mRNA turnover in regulating gene expression following several conditions of stress.
Experimental evaluation of topological parameters determining protein-folding rates
Authors: Miller Erik J. , Fischer Kael F. , Marqusee Susan
Published: September 2002

Recent work suggests that structural topology plays a key role in determining protein-folding rates and pathways. The refolding rates of small proteins that fold without intermediates are found to correlate with simple structural parameters such as relative contact order, long-range order, or the fraction of short-range contacts. To test and evaluate the role of structural topology experimentally, a set of circular permutants of the ribosomal protein S6 from
Thermus thermophilus
was analyzed. Despite a wide range of relative contact order, the permuted proteins all fold with similar rates. These results suggest that alternative topological parameters may better describe the role of topology in protein-folding rates.
Clusterin promotes amyloid plaque formation and is critical for neuritic toxicity in a mouse model of Alzheimer's disease
Authors: DeMattos Ronald B. , O'dell Mark A. , Parsadanian Maia , Taylor Jennie W. , Harmony Judith A. K. , Bales Kelly R. , Paul Steven M. , Aronow Bruce J. , Holtzman David M.
Published: September 2002

Studies have shown that clusterin (also called apolipoprotein J) can influence the structure and toxicity of amyloid-β (Aβ)
in vitro
. To determine whether endogenous clusterin plays a role in influencing Aβ deposition, structure, and toxicity
in vivo
, we bred PDAPP mice, a transgenic mouse model of Alzheimer's disease, to clusterin
−/−
mice. By 12 months of age, PDAPP, clusterin
−/−
mice had similar levels of brain Aβ deposition as did PDAPP, clusterin
+/+
mice. Although Aβ deposition was similar, PDAPP, clusterin
−/−
mice had significantly fewer fibrillar Aβ (amyloid) deposits than PDAPP mice expressing clusterin. In the absence of clusterin, neuritic dystrophy associated with the deposited amyloid was markedly reduced, resulting in a dissociation between fibrillar amyloid formation and neuritic dystrophy. These findings demonstrate that clusterin markedly influences Aβ structure and neuritic toxicity
in vivo
and is likely to play an important role in Alzheimer's disease pathogenesis.
Increased expression of urokinase during atherosclerotic lesion development causes arterial constriction and lumen loss, and accelerates lesion growth
Authors: Falkenberg Mårten , Tom Clifford , DeYoung Mary Beth , Wen Shan , Linnemann Ruth , Dichek David A.
Published: September 2002

Overexpression of urokinase plasminogen activator (uPA) in endothelial cells can decrease intravascular thrombosis. However, expression of uPA is increased in atherosclerotic human arteries, which suggests that uPA might accelerate atherogenesis. To investigate whether elevated uPA expression accelerates atherogenesis, we cloned a rabbit uPA cDNA and expressed it in carotid arteries of cholesterol-fed rabbits. uPA gene transfer increased artery-wall uPA activity for at least 1 week, with a return to baseline by 2 weeks. One week after gene transfer, uPA-transduced arteries were constricted, with significantly smaller lumens and thicker walls, but no difference in intimal or medial mass. Two weeks after gene transfer, uPA- and control-transduced arteries were morphologically indistinguishable. By 4 weeks, however, uPA-transduced arteries had 70% larger intimas than control-transduced arteries (
P
< 0.01) and smaller lumens (
P
< 0.05). Intimal lesions appeared to be of similar composition in uPA- and control-transduced arteries, except that degradation of elastic laminae was evident in uPA-transduced arteries. These data suggest that elevated uPA expression in atherosclerotic arteries contributes to intimal growth and constrictive remodeling leading to lumen loss. Antagonists of uPA activity might, therefore, be useful in limiting intimal growth and preventing constrictive remodeling. Overexpression of uPA in endothelial cells to prevent intravascular thrombosis must be reconsidered, because this intervention could worsen underlying vascular disease.
Neuregulin-1 promotes formation of the murine cardiac conduction system
Authors: Rentschler Stacey , Zander Jennifer , Meyers Kathleen , France David , Levine Rebecca , Porter George , Rivkees Scott A. , Morley Gregory E. , Fishman Glenn I.
Published: September 2002

The cardiac conduction system is a network of cells responsible for the rhythmic and coordinated excitation of the heart. Components of the murine conduction system, including the peripheral Purkinje fibers, are morphologically indistinguishable from surrounding cardiomyocytes, and a paucity of molecular markers exists to identify these cells. The murine conduction system develops in close association with the endocardium. Using the recently identified CCS-
lacZ
line of reporter mice, in which
lacZ
expression delineates the embryonic and fully mature conduction system, we tested the ability of several endocardial-derived paracrine factors to convert contractile cardiomyocytes into conduction-system cells as measured by ectopic reporter gene expression in the heart. In this report we show that neuregulin-1, a growth and differentiation factor essential for ventricular trabeculation, is sufficient to induce ectopic expression of the
lacZ
conduction marker. This inductive effect of neuregulin-1 was restricted to a window of sensitivity between 8.5 and 10.5 days postcoitum. Using the whole mouse embryo culture system, neuregulin-1 was shown to regulate
lacZ
expression within the embryonic heart, whereas its expression in other tissues remained unaffected. We describe the electrical activation pattern of the 9.5-days postcoitum embryonic mouse heart and show that treatment with neuregulin-1 results in electrophysiological changes in the activation pattern consistent with a recruitment of cells to the conduction system. This study supports the hypothesis that endocardial-derived neuregulins may be the major endogenous ligands responsible for inducing murine embryonic cardiomyocytes to differentiate into cells of the conduction system.
Increasing plant susceptibility to
Agrobacterium
infection by overexpression of the
Arabidopsis
nuclear protein VIP1
Authors: Tzfira Tzvi , Vaidya Manjusha , Citovsky Vitaly
Published: September 2002

Agrobacterium
is a unique model system as well as a major biotechnological tool for genetic manipulation of plant cells. It is still unknown, however, whether host cellular factors exist that are limiting for infection, and whether their overexpression in plant cells can increase the efficiency of the infection. Here, we examined the effect of overexpression in tobacco plants of an
Arabidopsis
gene,
VIP1
, which encodes a recently discovered cellular protein required for
Agrobacterium
infection. Our results indicate that VIP1 is imported into the plant cell nucleus via the karyopherin αdependent pathway and that elevated intracellular levels of VIP1 render the host plants significantly more susceptible to transient and stable genetic transformation by
Agrobacterium
, probably because of the increased nuclear import of the transferred-DNA.
Three-dimensional stellarator codes
Authors: Garabedian P. R.
Published: September 2002

Three-dimensional computer codes have been used to develop quasisymmetric stellarators with modular coils that are promising candidates for a magnetic fusion reactor. The mathematics of plasma confinement raises serious questions about the numerical calculations. Convergence studies have been performed to assess the best configurations. Comparisons with recent data from large stellarator experiments serve to validate the theory.
Prominent neurodegeneration and increased plaque formation in complement-inhibited Alzheimer's mice
Authors: Wyss-Coray Tony , Yan Fengrong , Lin Amy Hsiu-Ti , Lambris John D. , Alexander Jessy J. , Quigg Richard J. , Masliah Eliezer
Published: September 2002

Abnormal accumulation of β-amyloid (Aβ) in Alzheimer's disease (AD) is associated with prominent brain inflammation. Whereas earlier studies concluded that this inflammation is detrimental, more recent animal data suggest that at least some inflammatory processes may be beneficial and promote Aβ clearance. Consistent with these observations, overproduction of transforming growth factor (TGF)-β1 resulted in a vigorous microglial activation that was accompanied by at least a 50% reduction in Aβ accumulation in human amyloid precursor protein (hAPP) transgenic mice. In a search for inflammatory mediators associated with this reduced pathology, we found that brain levels of C3, the central component of complement and a key inflammatory protein activated in AD, were markedly higher in hAPP/TGF-β1 mice than in hAPP mice. To assess the importance of complement in the pathogenesis of AD-like disease in mice, we inhibited C3 activation by expressing soluble complement receptor-related protein y (sCrry), a complement inhibitor, in the brains of hAPP mice. Aβ deposition was 2- to 3-fold higher in 1-year-old hAPP/sCrry mice than in age-matched hAPP mice and was accompanied by a prominent accumulation of degenerating neurons. These results indicate that complement activation products can protect against Aβ-induced neurotoxicity and may reduce the accumulation or promote the clearance of amyloid and degenerating neurons. These findings provide evidence for a role of complement and innate immune responses in AD-like disease in mice and support the concept that certain inflammatory defense mechanisms in the brain may be beneficial in neurodegenerative disease.
Severe block in processing of proinsulin to insulin accompanied by elevation of des-64,65 proinsulin intermediates in islets of mice lacking prohormone convertase 1/3
Authors: Zhu Xiaorong , Orci Lelio , Carroll Raymond , Norrbom Christina , Ravazzola Mariella , Steiner Donald F.
Published: September 2002

The neuroendocrine processing endoproteases PC2 and PC1/3 are expressed in the β cells of the islets of Langerhans and participate in the processing of proinsulin to insulin and C-peptide. We have previously shown that disruption of PC2 (SPC2) expression significantly impairs proinsulin processing. Here we report that disruption of the expression of PC1/3 (SPC3) produces a much more severe block in proinsulin conversion. In nulls, pancreatic and circulating proinsulin-like components comprise 87% and 91%, respectively, of total insulin-related immunoreactivity. Heterozygotes also show a more than 2-fold elevation in proinsulin levels to ≈12%. Immunocytochemical and ultrastructural studies of the β cells reveal the nearly complete absence of mature insulin immunoreactivity and its replacement by that of proinsulin in abundant immature-appearing secretory granules. In contrast, α cell morphology and glucagon processing are normal, and there is also no defect in somatostatin-14 generation. Pulse–chase labeling studies confirm the existence of a major block in proinsulin processing in PC1/3 nulls with prolongation of half-times of conversion by 7- and 10-fold for proinsulins I and II, respectively. Lack of PC1/3 also results in increased levels of des-64,65 proinsulin intermediates generated by PC2, in contrast to PC2 nulls, in which des- 31,32 proinsulin intermediates predominate. These results confirm that PC1/3 plays a major role in processing proinsulin, but that its coordinated action with PC2 is necessary for the most efficient and complete processing of this prohormone.
Strongly enhanced field-dependent single-molecule electroluminescence
Authors: Lee Tae-Hee , Gonzalez Jose I. , Dickson Robert M.
Published: September 2002

Individual, strongly electroluminescent Ag

n

molecules (
n
= 2∼8 atoms) have been electrically written within otherwise nonemissive silver oxide films. Exhibiting characteristic single-molecule behavior, these individual room-temperature molecules exhibit extreme electroluminescence enhancements (>10
4
vs. bulk and dc excitation on a per molecule basis) when excited with specific ac frequencies. Occurring through field extraction of electrons with subsequent reinjection and radiative recombination, single-molecule electroluminescence is enhanced by a general mechanism that avoids slow bulk material response. Thus, while we detail strong electroluminescence from single, highly fluorescent Ag

n

molecules, this mechanism also yields strong ac-excited electroluminescence from similarly prepared, but otherwise nonemissive, individual Cu nanoclusters.
The search for local native-like nucleation centers in the unfolded state of β-sheet proteins
Authors: Nikiforovich Gregory V. , Frieden Carl
Published: September 2002

An approach involving the systematic computational conformational analysis of all overlapping hexapeptide segments in the protein sequence has found fragments with the higher than average propensity to adopt the native-like three-dimensional structure and other regular nonrandom structures in the unfolded states of four β-sheet proteins, namely IFABP (intestinal fatty acid-binding protein), ILBP (ileal fatty acid-binding protein), CRABP I (cellular retinoic acid-binding protein), and CRBP II (cellular retinal binding protein). The native three-dimensional structures of these four proteins are very similar even though they possess as little as ≈30% sequence similarity. The computational results were validated by comparison with the experimental data of the heteronuclear sequential quantum correlation NMR spectroscopy obtained earlier for IFABP at high urea concentrations. On this basis, a molecular model of the unfolded state of IFABP has been developed. The model presumes a dynamic equilibrium between various nonrandom structures (including the native-like structure) and random coil in the local segments of the protein sequence. The model explains experimental observations obtained earlier for folding of several mutants of IFABP, as well as the observed differences in molecular mechanisms of folding for the four β-sheet proteins. Because the computational approach itself does not employ any experimentally derived information in advance, it is not necessarily limited to the β-sheet proteins.
Absolute requirement of spermidine for growth and cell cycle progression of fission yeast (
Schizosaccharomyces
pombe
)
Authors: Chattopadhyay Manas K. , Tabor Celia White , Tabor Herbert
Published: September 2002

Schizosaccharomyces pombe
cells that cannot synthesize spermidine or spermine because of a deletion–insertion in the gene coding for
S
-adenosylmethionine decarboxylase (Δ
spe2
) have an absolute requirement for spermidine for growth. Flow cytometry studies show that in the absence of spermidine an overall delay of the cell cycle progression occurs with some accumulation of cells in the G
1
phase; as little as 10
−6
M spermidine is sufficient to maintain normal cell cycle distribution and normal growth. Morphologically some of the spermidine-deprived cells become spherical at an early stage with little evidence of cell division. On further incubation in the spermidine-deprived medium, growth occurs in most of the cells, not by cell division but rather by cell elongation, with an abnormal distribution of the actin cytoskeleton, DNA (4′, 6-diamidino-2-phenylindole staining), and calcofluor-staining moieties. More prolonged incubation in the spermidine-deficient medium leads to profound morphological changes including nuclear degeneration.
An anti-transferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells
Authors: Ng Patrick P. , Dela Cruz Jay S. , Sorour David N. , Stinebaugh James M. , Shin Seung-Uon , Shin Daniel S. , Morrison Sherie L. , Penichet Manuel L.
Published: September 2002

We have developed an antibody fusion protein (anti-rat TfR IgG3-Av) with the ability to deliver different molecules into cancer cells. It consists of avidin genetically fused to the CH3 region of a human IgG3 specific for the rat transferrin receptor. It forms strong, noncovalent interactions with biotinylated molecules such as glucose oxidase and β-galactosidase, and delivers them into the rat myeloma cell line Y3-Ag1.2.3 through receptor-mediated endocytosis. Importantly, the β-galactosidase retains activity after internalization. Furthermore, we have unexpectedly discovered that anti-rat TfR IgG3-Av, but not a recombinant anti-rat TfR IgG3 or a nonspecific IgG3-Av, possesses proapoptotic activities against Y3-Ag1.2.3 and the rat T cell lymphoma cell line C58 (NT) D.1.G.OVAR.1. These activities were not observed in two rat cell lines of nonhematopoietic lineage (bladder carcinoma BC47 and gliosarcoma 9L). Anti-human TfR IgG3-Av also demonstrated proapoptotic activity against the human erythroleukemia cell line K562. Studies showed that anti-rat TfR IgG3-Av exists as a dimer, suggesting that cross-linking of the surface transferrin receptor may be responsible for the cytotoxic activity. These findings demonstrate that it is possible to transform an antibody specific for a growth factor receptor that does not exhibit inhibitory activity into a drug with significant intrinsic cytotoxic activity against selected cells by fusing it with avidin. The antitumor activity may be enhanced by delivering biotinylated therapeutics into cancer cells. Further development of this technology may lead to effective therapeutics forin vivoeradication of hematological malignancies, andex vivopurging of cancer cells in autologous transplantation.
On the calculation of absolute macromolecular binding free energies
Authors: Luo Hengbin , Sharp Kim
Published: September 2002

The standard framework for calculating the absolute binding free energy of a macromolecular association reaction A + B → AB with an association constantKABis to equate chemical potentials of the species on the left- and right-hand sides of this reaction and evaluate the chemical potentials from theory. This theory involves (usually hidden) assumptions about what constitutes the bound species, AB, and where the contribution of the solvent appears. We present here an alternative derivation that can be traced back to Bjerrum, in which the expectation value ofKABis obtained directly through the statistical mechanical method of evaluating its ensemble (Boltzmann-weighted) average. The generalized Bjerrum approach more clearly delineates: (i) the different contributions to binding; (ii) the origin of the much-discussed and somewhat controversial association entropy term; and (iii) where the solvent contribution appears. This approach also allows approximations required for practical evaluation of the binding constant in complex macromolecular systems, to be introduced in a well defined way. We provide an example, with application to test cases that illustrate a range of binding behavior.
Hydration at the surface of the protein Monellin: Dynamics with femtosecond resolution
Authors: Peon Jorge , Pal Samir Kumar , Zewail Ahmed H.
Published: September 2002

We have studied the femtosecond hydration dynamics of Monellin, a protein with a single tryptophan residue at its surface. Tryptophan was selectively used as a probe of the dynamics, and through monitoring of its fluorescence Stokes shift with time we obtained the hydration correlation function, which decays due to rotational and translational motions of water at the protein surface and in bulk. The decay exhibits a “bimodal” behavior with time constants of 1.3 and 16 ps, mirroring relaxation of the free/quasifree water molecules and surface-bound water layer (minimum binding energy of 1–2 kcal/mol). The observed slow decay of 16 ps for tryptophan in the native protein differs by more than an order of magnitude from that of bulk water because of the dynamical exchange in the layer. To examine the effect of unfolding, we also studied hydration dynamics when Monellin was denatured in a 6 M guanidine hydrochloride solution and obtained a totally different behavior: 3.5 and 56 ps. Comparing with the results of experiments on free tryptophan in the same concentration of the denaturing solution, we conclude that the fast component of 3.5 ps comes from bulk-type solvation in the 6 M guanidine hydrochloride. However, the absence of the 16-ps decay and appearance of the 56-ps component reflects a more “rigid solvation,” which is likely to involve the motions of the protein backbone in the random-coiled state. With the help of polymer theory, this time scale is reproduced in agreement with experimental observations.
A trophic cascade regulates salt marsh primary production
Authors: Silliman Brian Reed , Bertness Mark D.
Published: September 2002

Nutrient supply is widely thought to regulate primary production of many ecosystems including salt marshes. However, experimental manipulation of the dominant marsh grazer (the periwinkle,
Littoraria irrorata
) and its consumers (e.g., blue crabs,
Callinectes sapidus
, terrapins,
Malaclemys terrapin
) demonstrates plant biomass and production are largely controlled by grazers and their predators. Periwinkle grazing can convert one of the most productive grasslands in the world into a barren mudflat within 8 months. Marine predators regulate the abundance of this plant-grazing snail. Thus, top-down control of grazer density is a key regulatory determinant of marsh grass growth. The discovery of this simple trophic cascade implies that over-harvesting of snail predators (e.g., blue crabs) may be an important factor contributing to the massive die-off (tens of km
2
) of salt marshes across the southeastern United States. In addition, our results contribute to a growing body of evidence indicating widespread, predator regulation of marine macrophyte production via trophic cascades (kelps, seagrasses, intertidal algae).
The total influenza vaccine failure of 1947 revisited: Major intrasubtypic antigenic change can explain failure of vaccine in a post-World War II epidemic
Authors: Kilbourne Edwin D. , Smith Catherine , Brett Ian , Pokorny Barbara A. , Johansson Bert , Cox Nancy
Published: September 2002

Although vaccine-induced immunity to influenza A virus is continually challenged by progressively selected mutations in the virus's major antigens (antigenic drift), virus strains within a subtype (e.g., H1N1) are antigenically cross-reactive. Although cross-immunity diminishes as further mutations accumulate, necessitating frequent changes in vaccine strains, older vaccines are usually partially protective. The post-World War II epidemic of 1947 is notable for the total failure of a vaccine previously effective in the 1943–44 and 1944–45 seasons. We have combined extensive antigenic characterization of the hemagglutinin and neuraminidase antigens of the 1943 and 1947 viruses with analysis of their nucleotide and amino acid sequences and have found marked antigenic and amino acid differences in viruses of the two years. Furthermore, in a mouse model, vaccination with the 1943 vaccine had no effect on infection with the 1947 strain. These findings are important, because complete lack of cross-immunogenicity has been found previously only with antigenic shift, in which antigenically novel antigens have been captured by reassortment of human and animal strains, sometimes leading to pandemics. Although the 1947 epidemic lacked the usual hallmarks of pandemic disease, including an extensive increase in mortality, it warns of the possibility that extreme intrasubtypic antigenic variation (if coupled with an increase in disease severity) could produce pandemic disease without the introduction of animal virus antigens.
In vivo
quantification of localized neuronal activation and inhibition in the rat brain using a dedicated high temporal-resolution β
+
-sensitive microprobe
Authors: Pain Frédéric , Besret Laurent , Vaufrey Françoise , Grégoire Marie-Claude , Pinot Laurent , Gervais Philippe , Ploux Lydie , Bloch Gilles , Mastrippolito Roland , Lanièce Philippe , Hantraye Philippe
Published: September 2002

Understanding brain disorders, the neural processes implicated in cognitive functions and their alterations in neurodegenerative pathologies, or testing new therapies for these diseases would benefit greatly from combined use of an increasing number of rodent models and neuroimaging methods specifically adapted to the rodent brain. Besides magnetic resonance (MR) imaging and functional MR, positron-emission tomography (PET) remains a unique methodology to study
in vivo
brain processes. However, current high spatial-resolution tomographs suffer from several technical limitations such as high cost, low sensitivity, and the need of restraining the animal during image acquisition. We have developed a β
+
-sensitive high temporal-resolution system that overcomes these problems and allows the
in vivo
quantification of cerebral biochemical processes in rodents. This β-MICROPROBE is an
in situ
technique involving the insertion of a fine probe into brain tissue in a way very similar to that used for microdialysis and cell electrode recordings. In this respect, it provides information on molecular interactions and pathways, which is complementary to that produced by these technologies as well as other modalities such as MR or fluorescence imaging. This study describes two experiments that provide a proof of concept to substantiate the potential of this technique and demonstrate the feasibility of quantifying brain activation or metabolic depression in individual living rats with 2-[
18
F]fluoro-2-deoxy-
d
-glucose and standard compartmental modeling techniques. Furthermore, it was possible to identify correctly the origin of variations in glucose consumption at the hexokinase level, which demonstrate the strength of the method and its adequacy for
in vivo
quantitative metabolic studies in small animals.
Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of
Drosophila
melanogaster
Authors: Warren James T. , Petryk Anna , Marqués Guillermo , Jarcho Michael , Parvy Jean-Philippe , Dauphin-Villemant Chantal , O'Connor Michael B. , Gilbert Lawrence I.
Published: September 2002

Five different enzymatic activities, catalyzed by both microsomal and mitochondrial cytochrome P450 monooxygenases (CYPs), are strongly implicated in the biosynthesis of ecdysone (E) from cholesterol. However, none of these enzymes have been characterized completely. The present data show that the wild-type genes of two members of the Halloween family of embryonic lethals,
disembodied
(
dib
) and
shadow
(
sad
), code for mitochondrial cytochromes P450 that mediate the last two hydroxylation reactions in the ecdysteroidogenic pathway in
Drosophila
, namely the C
22
- and C
2
-hydroxylases. When
sad
(CYP315A1) is transfected into
Drosophila
S2 cells, the cells metabolize 2-deoxyecdysone (2dE) to E and the [
3
H]ketotriol (2,22-dideoxyecdysone) to 22-deoxyecdysone. In contrast,
dib
(CYP302A1) is responsible for the conversion of the [
3
H]ketotriol to [
3
H]2dE. When cells are transfected with both
dib
and
sad
, they metabolize the [
3
H]ketotriol to [
3
H]E in high yield. The expression of
sad
and
dib
is concentrated within the individual segments of the developing epidermis when there is a surge of ecdysteroid midway through embryogenesis. This result occurs before the ring gland has developed and suggests that the embryonic epidermis is a site of ecdysteroid biosynthesis. This pattern then diminishes, and, during late embryogenesis, expression of both genes is concentrated in the prothoracic gland cells of the developing ring gland. Expression of
dib
and
sad
continues to be localized in this endocrine compartment during larval development, being maximal in both the late second and third instar larvae, about the time of the premolt peaks in the ecdysteroid titer.
Identification of quantitative trait loci controlling acute virulence inToxoplasma gondii
Authors: Su Chunlei , Howe Daniel K. , Dubey J. P. , Ajioka James W. , Sibley L. David
Published: September 2002

Strains ofToxoplasma gondiican be grouped into three predominant clonal lineages with members of the type I group being uniformly lethal in mice. To elucidate the basis of this extreme virulence, a genetic cross was performed between a highly virulent type I strain (GT-1) and a less-virulent type III strain (CTG), and the phenotypes of resulting progeny were analyzed by genetic linkage mapping. Analysis of independent recombinant progeny identified several quantitative trait loci that contributed to acute virulence. A major quantitative trait locus located on chromosomeVIIaccounted for ≈50% of the virulence phenotype, whereas a minor locus on chromosomeIV, linked to theROP1gene, accounted for ≈10%. These loci are conserved in other type I strains, indicating that acute virulence is controlled by discrete genes common to the type I lineage.
Addition of a photocrosslinking amino acid to the genetic code of
Escherichia
coli
Authors: Chin Jason W. , Martin Andrew B. , King David S. , Wang Lei , Schultz Peter G.
Published: September 2002

Benzophenones are among the most useful photocrosslinking agents in biology. We have evolved an orthogonal aminoacyl-tRNA synthetase/tRNA pair that makes possible the
in vivo
incorporation of
p
-benzoyl-
l
-phenylalanine into proteins in
Escherichia coli
in response to the amber codon, TAG. This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione
S
-transferase. Irradiation resulted in efficient crosslinking (>50%) of the protein subunits. This methodology may prove useful for discovering and defining protein interactions
in vitro
and
in vivo
.
DNA-dependent divalent cation binding in the nucleosome core particle
Authors: Davey Curt A. , Richmond Timothy J.
Published: September 2002

Sequence-specific binding of divalent cations to nucleosomal DNA can potentially influence nucleosome position and mobility, as well as modulate interactions with nuclear factors. We define the bonding and specificity of divalent cation interaction with nucleosomal DNA by characterizing Mn
2+
binding in the x-ray structure of the nucleosome core particle at 1.9-Å resolution. Manganese ions are found ligated with high occupancy in the major groove to 12 of 22 GG and GC base pair steps. The specific location and mode of metal binding is the consequence of unambiguous conformational differences between dinucleotide sites, owing to their sequence context and orientation in the nucleosome core.
Genealogical evidence for epidemics of selfish genes
Authors: Ingvarsson Pär K. , Taylor Douglas R.
Published: September 2002

Some genetic elements spread infectiously in populations by increasing their rate of genetic transmission at the expense of other genes in the genome. These so-called selfish genetic elements comprise a substantial portion of eukaryotic genomes and have long been viewed as a potent evolutionary force. Despite this view, little is known about the evolutionary history of selfish genetic elements in natural populations, or their genetic effects on other portions of the genome. Here we use nuclear and chloroplast gene genealogies in two species of
Silene
to show the historical pattern of selection on a well known selfish genetic element, cytoplasmic male sterility. We provide evidence that evolution of cytoplasmic male sterility has been characterized by frequent turnovers of mutations in natural populations, thus supporting an epidemic model for the evolution of selfish genes, where new mutations repeatedly arise and rapidly sweep through populations.
Inducible expression of FGF-3 in mouse mammary gland
Authors: Ngan Elly S. W. , Ma Zhi-Qing , Chua Steven S. , DeMayo Francesco J. , Tsai Sophia Y.
Published: September 2002

Fibroblast growth factor-3 (FGF-3) is a crucial developmental regulator. Aberrant activation of this gene by mouse mammary tumor virus insertion results in pregnancy-responsive mammary tumorigenesis. To characterize better FGF-3 function in postnatal mammary gland development and cancer initiation/progression, we used a mifepristone (RU486)-inducible regulatory system to express conditionally FGF-3 in the mammary epithelium of transgenic mice. Ectopic overexpression of FGF-3 in pubescent mammary glands elicited severe perturbations in early mammary gland development leading to mammary hyperplasia. Ductal elongation was retarded, multiple cysts persisted in the virgin ducts, and ductal epithelium was expanded and multilayered. The altered ductal architecture and the persistence of hyperplastic multilayered epithelium reflect a defect in growth regulation, which resulted from an imbalance between mitogenic and apoptotic signals. By altering the duration of RU486 treatment, we showed that the persistence of mitogenic signal elicited by FGF-3 is crucial for the initiation, progression, and maintenance of the hyperplastic characteristic of the mammary epithelium. The manifestations elicited by FGF-3 could be reversed by RU486 withdrawal. In addition, synergism between the stimulus from estrogen and FGF-3 mitogenic pathways was evident and likely contributes to the pregnancy-dependent tumorigenesis of FGF-3. Taken together, the mifepristone-inducible regulatory system provides a powerful means for understanding the diverse roles of FGF-3 and its interactions with hormones in mammary gland tumorigenesis.
Identification of HLA-E-specific alloreactive T lymphocytes: A cell subset that undergoes preferential expansion in mixed lymphocyte culture and displays a broad cytolytic activity against allogeneic cells
Authors: Romagnani Chiara , Pietra Gabriella , Falco Michela , Millo Enrico , Mazzarino Paola , Biassoni Roberto , Moretta Alessandro , Moretta Lorenzo , Mingari Maria Cristina
Published: September 2002

Previous studies showed that a subset of CD8+cytolytic T lymphocytes expressing HLA class I-specific natural killer inhibitory receptors (iNKR) is characterized by the ability to recognize HLA-E and to mediate T cell receptor-dependent killing of different NK cell-susceptible tumor target cells. In this study, we show that this CD8+T cell subset can also undergo extensive proliferation in mixed lymphocyte cultures in response to allogeneic cells. Analysis of their cytolytic activity revealed a broad specificity against a panel of allogeneic phytohemagglutinin-induced blasts derived from HLA-unmatched donors. On the other hand, autologous and certain allogeneic phytohemagglutinin blasts were resistant to lysis. We used as target cells the transporter associated with antigen processing (TAP)-2−/−murine RMA-S cell line cotransfected with β2-microglobulin and HLA-E*01033. On loading these cells with different HLA-E-binding peptides derived either from HLA class I leader sequences or viral proteins, we could show that HLA-E-specific cytolytic T lymphocytes recognized many, but not all, peptides analyzed. These data suggest that these cells may recognize, on allogeneic cells, HLA-E with peptides that are not present in the host of origin. In addition to their ability to proliferate in response to allogeneic stimulation and to lyse, most allogeneic cells may have important implications in transplantation and in antitumor immune responses.