bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular basis for defective glycosylation and
Pseudomonas
pathogenesis in cystic fibrosis lung
Authors: Poschet J. F. , Boucher J. C. , Tatterson L. , Skidmore J. , Van Dyke R. W. , Deretic V.
Published: July 2002

The
CFTR
gene encodes a transmembrane conductance regulator, which is dysfunctional in patients with cystic fibrosis (CF). The mechanism by which defective CFTR (CF transmembrane conductance regulator) leads to undersialylation of plasma membrane glycoconjugates, which in turn promote lung pathology and colonization with
Pseudomonas aeruginosa
causing lethal bacterial infections in CF, is not known. Here we show by ratiometric imaging with lumenally exposed pH-sensitive green fluorescent protein that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network (TGN) in CF lung epithelial cells. The hyperacidification of TGN, glycosylation defect of plasma membrane glycoconjugates, and increased
P. aeruginosa
adherence were corrected by incubating CF respiratory epithelial cells with weak bases. Studies with pharmacological agents indicated a role for sodium conductance, modulated by CFTR regulatory function, in determining the pH of TGN. These studies demonstrate the molecular basis for defective glycosylation of lung epithelial cells and bacterial pathogenesis in CF, and suggest a cure by normalizing the pH of intracellular compartments.
Targeting
Plasmodium
ligands on mosquito salivary glands and midgut with a phage display peptide library
Authors: Ghosh Anil K. , Ribolla Paulo E. M. , Jacobs-Lorena Marcelo
Published: July 2002

Despite vast efforts and expenditures in the past few decades, malaria continues to kill millions of persons every year, and new approaches for disease control are urgently needed. To complete its life cycle in the mosquito,
Plasmodium
, the causative agent of malaria, has to traverse the epithelia of the midgut and salivary glands. Although strong circumstantial evidence indicates that parasite interactions with the two organs are specific, hardly any information is available about the interacting molecules. By use of a phage display library, we identified a 12-aa peptide—salivary gland and midgut peptide 1 (SM1)—that binds to the distal lobes of the salivary gland and to the luminal side of the midgut epithelium, but not to the midgut surface facing the hemolymph or to ovaries. The coincidence of the tissues with which parasites and the SM1 peptide interact suggested that the parasite and peptide recognize the same surface ligand. In support of this hypothesis, the SM1 peptide strongly inhibited
Plasmodium
invasion of salivary gland and midgut epithelia. These experiments suggest a new strategy for the genetic manipulation of mosquito vectorial capacity.
Activity of the TonEBP/OREBP transactivation domain varies directly with extracellular NaCl concentration
Authors: Ferraris Joan D. , Williams Chester K. , Persaud Prita , Zhang Zheng , Chen Ye , Burg Maurice B.
Published: July 2002

Hypertonicity-induced binding of the transcription factor TonEBP/OREBP to its cognate DNA element, ORE/TonE, is associated with increased transcription of several osmotically regulated genes. Previously, it was found that hypertonicity rapidly causes nuclear translocation and phosphorylation of TonEBP/OREBP and, more slowly, increases TonEBP/OREBP abundance. Also, the C terminus of TonEBP/OREBP was found to contain a transactivation domain (TAD). We have now tested for tonicity dependence of the TAD activity of the 983 C-terminal amino acids of TonEBP/OREBP. HepG2 cells were cotransfected with a reporter construct and one of several TAD expression vector constructs. The reporter construct contained GAL4 DNA binding elements, a minimal promoter, and the
Photinus
luciferase gene. TAD expression vectors generate chimeras comprised of the GAL4 DNA binding domain fused to (
i
) the 983 C-terminal amino acids of TonEBP/OREBP, (
ii
) 17 glutamine residues, (
iii
) the TAD of c-Jun, or (
iv
) no TAD. All TAD-containing chimeras were functional at normal extracellular osmolality (300 mosmol/kg), but the activity only of the chimera containing the 983 C-terminal amino acids of TonEBP/OREBP varied with extracellular NaCl concentration, decreasing by >80% at 200 mosmol/kg and increasing 8-fold at 500 mosmol/kg. The chimera containing the 983 C-terminal amino acids of TonEBP/OREBP was constitutively localized to the nucleus and showed tonicity-dependent posttranslational modification consistent with phosphorylation. The activity at 500 mosmol/kg was reduced by herbimycin, a tyrosine kinase inhibitor and by 5,6-dichloro-1-β-
d
-ribofuranosylbenzimidazole, a protein kinase CK2 inhibitor. Thus, the 983 C-terminal amino acids of TonEBP/OREBP contain a TAD that is regulated osmotically, apparently by tonicity-dependent phosphorylation.
Analysis of mitotic microtubule-associated proteins using mass spectrometry identifies astrin, a spindle-associated protein
Authors: Mack Gary J. , Compton Duane A.
Published: July 2002

We purified microtubules from a mammalian mitotic extract
and obtained an amino acid sequence from each microtubule-associated
protein by using mass spectrometry. Most of these proteins are known
spindle-associated components with essential functional roles in
spindle organization. We generated antibodies against a protein
identified in this collection and refer to it as astrin because of its
association with astral microtubule arrays assembled
in
vitro
. Astrin is ≈134 kDa, and except for a large predicted
coiled-coil domain in its C-terminal region it lacks any known
functional motifs. Astrin associates with spindle microtubules as early
as prophase where it concentrates at spindle poles. It localizes
throughout the spindle in metaphase and anaphase and associates with
midzone microtubules in anaphase and telophase. Astrin also localizes
to kinetochores but only on those chromosomes that have congressed.
Deletion analysis indicates that astrin's primary spindle-targeting
domain is at the C terminus, although a secondary domain in the N
terminus can target some of the protein to spindle poles. Thus, we have
generated a comprehensive list of major mitotic microtubule-associated
proteins, among which is astrin, a nonmotor spindle protein.
Neuregulins with an Ig-like domain are essential for mouse myocardial and neuronal development.
Authors: Kramer R , Bucay N , Kane D J , Martin L E , Tarpley J E , Theill L E
Published: July 2002

Neuregulins are ligands for the erbB family of receptor tyrosine kinases and mediate growth and differentiation of neural crest, muscle, breast cancer, and Schwann cells. Neuregulins contain an epidermal growth factor-like domain located C-terminally to either an Ig-like domain or a cysteine-rich domain specific to the sensory and motor neuron-derived isoform. Here it is shown that elimination of the Ig-like domain-containing neuregulins by homologous recombination results in embryonic lethality associated with a deficiency of ventricular myocardial trabeculation and impairment of cranial ganglion development. The erbB receptors are expressed in myocardial cells and presumably mediate the neuregulin signal originating from endocardial cells. The trigeminal ganglion is reduced in size and lacks projections toward the brain stem and mandible. We conclude that IgL-domain-containing neuregulins play a major role in cardiac and neuronal development.
Whole body positron emission tomography imaging of simian immunodeficiency virus-infected rhesus macaques.
Authors: Scharko A M , Perlman S B , Hinds PW2nd  , Hanson J M , Uno H , Pauza C D
Published: July 2002

Pathogenesis of simian immunodeficiency virus (SIV) infection in rhesus macaques begins with acute viremia and then progresses to a distributed infection in the solid lymphoid tissues, which is followed by a process of cellular destruction leading to terminal disease and death. Blood and tissue specimens show the progress of infection at the cellular level but do not reveal the pattern of infection and host responses occurring throughout the body. The purpose of this investigation was to determine whether positron emission tomography (PET) imaging with intravenous 2-18F-2-deoxyglucose (FDG) could identify activated lymphoid tissues in a living animal and whether this pattern would reflect the extent of SIV infection. PET images from SIV-infected animals were distinguishable from uninfected controls and revealed a pattern consistent with widespread lymphoid tissue activation. Significant FDG accumulation in colon along with mesenteric and ileocaecal lymph nodes was found in SIV infection, especially during terminal disease stages. Areas of elevated FDG uptake in the PET images were correlated with productive SIV infection using in situ hybridization as a test for virus replication. PET-FDG images of SIV-infected animals correlated sites of virus replication with high FDG accumulation. These data show that the method can be used to evaluate the distribution and activity of infected tissues in a living animal without biopsy. Fewer tissues had high FDG uptake in terminal animals than midstage animals, and both were clearly distinguishable from uninfected animal scans.
Interleaflet clear space is reduced in the membrane of COP I and COP II-coated buds/vesicles.
Authors: Orci L , Schekman R , Perrelet A
Published: July 2002

Intracellular transfers between membrane-bound compartments occur through vesicles that bud from a donor compartment to fuse subsequently with an acceptor membrane. We report that the membrane that delimits COP I or COP II-coated buds/vesicles from the endoplasmic reticulum and the Golgi complex has a thinner interleaflet clear space as compared with the surrounding, noncoated parental membrane. This change is compatible with a compositional change of the membrane bilayer during the budding process.
YY1 and c-Myc associate in vivo in a manner that depends on c-Myc levels.
Authors: Shrivastava A , Yu J , Artandi S , Calame K
Published: July 2002

The c-Myc oncoprotein has previously been shown to associate with transcription regulator YY1 and to inhibit its activity. We show herein that endogenous c-Myc and YY1 associate in vivo and that changes in c-Myc levels, which accompany mitogenic stimulation or differentiation of cultured cells, affect the ratio of free to c-Myc-associated YY1. We have also investigated the mechanism by which association with c-Myc inhibits YY1's ability to regulate transcription. c-Myc does not block binding of YY1 to DNA. However, protein association studies suggest that c-Myc interferes with the ability of YY1 to contact basal transcription proteins TATA-binding protein and TFIIB.
The evolution of the vertebrate Dlx gene family.
Authors: Stock D W , Ellies D L , Zhao Z , Ekker M , Ruddle F H , Weiss K M
Published: July 2002

The vertebrate Dlx gene family consists of homeobox-containing transcription factors distributed in pairs on the same chromosomes as the Hox genes. To investigate the evolutionary history of Dlx genes, we have cloned five new zebrafish family members and have provided additional sequence information for two mouse genes. Phylogenetic analyses of Dlx gene sequences considered in the context of their chromosomal arrangements suggest that an initial tandem duplication produced a linked pair of Dlx genes after the divergence of chordates and arthropods but prior to the divergence of tunicates and vertebrates. This pair of Dlx genes was then duplicated in the chromosomal events that led to the four clusters of Hox genes characteristic of bony fish and tetrapods. It is possible that a pair of Dlx genes linked to the Hoxc cluster has been lost from mammals. We were unable to distinguish between independent duplication and retention of the ancestral state of bony vertebrates to explain the presence of a greater number of Dlx genes in zebrafish than mammals. Determination of the linkage relationship of these additional zebrafish Dlx genes to Hox clusters should help resolve this issue.
Differential regulation of beta 1 and beta 2 integrin avidity by chemoattractants in eosinophils.
Authors: Weber C , Kitayama J , Springer T A
Published: July 2002

The CC chemokines regulated on activation normal T expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), and the anaphylatoxin C5a, induce activation, degranulation, chemotaxis, and transendothelial migration of eosinophils. Adhesion assays on purified ligands showed differential regulation of beta 1 and beta 2 integrin avidity in eosinophils. Adhesiveness of VLA-4 (alpha 4 beta 1, CD29/CD49d) for vascular cell adhesion molecule 1 or fibronectin was rapidly increased but subsequently reduced by RANTES, MCP-3, or C5a. The deactivation of VLA-4 lead to cell detachment, whereas phorbol 12-myristate 13-acetate induced sustained activation of VLA-4. In contrast, chemoattractants stimulated a prolonged increase in the adhesiveness of Mac-1 (alpha M beta 2, CD11b/CD18) for intercellular adhesion molecule 1. Inhibition by pertussis toxin confirmed signaling via G protein-coupled receptors. Chemoattractants induced transient, while phorbol 12-myristate 13-acetate induced sustained actin polymerization. Disruption of actin filaments by cytochalasins inhibited increases in avidity of VLA-4 but not of Mac-1. Chemoattractants did not upregulate a Mn2+-inducible beta 1 neoepitope defined by the mAb 9EG7, but induced prolonged expression of a Mac-1 activation epitope recognized by the mAb CBRM1/5. This mAb inhibited chemoattractant-stimulated adhesion of eosinophils to intercellular adhesion molecule 1. Thus, regulation of VLA-4 was dependent on the actin cytoskeleton, whereas conformational changes appeared to be crucial for activation of Mac-1. To our knowledge, this is the first demonstration that physiological agonists, such as chemoattractants, can differentially regulate the avidity of a beta 1 and a beta 2 integrin expressed on the same leukocyte.
Guanylyl cyclase C is a selective marker for metastatic colorectal tumors in human extraintestinal tissues
Authors: Carrithers S. L. , Barber M. T. , Biswas S. , Parkinson S. J. , Park P. K. , Goldstein S. D. , Waldman S. A.
Published: July 2002

Guanylyl cyclase C (GCC) has been detected only in intestinal
mucosa and colon carcinoma cells of placental mammals. However, this
receptor has been identified in several tissues in marsupials, and its
expression has been suggested in tissues other than intestine in
placental mammals. Selective expression of GCC by colorectal tumor
cells in extraintestinal tissues would permit this receptor to be
employed as a selective marker for metastatic disease. Thus, expression
of GCC was examined in human tissues and tumors, correlating receptor
function with detection by PCR. GCC was detected by ligand binding and
catalytic activation in normal intestine and primary and metastatic
colorectal tumors, but not in extraintestinal tissues or tumors.
Similarly, PCR yielded GCC-specific amplification products with
specimens from normal intestine and primary and metastatic colorectal
tumors, but not from extraintestinal tissues or tumors. Northern blot
analysis employing GCC-specific probes revealed an ≈4-kb transcript,
corresponding to recombinant GCC, in normal intestine and primary and
metastatic colorectal tumors, but not in extraintestinal tissues. Thus,
GCC is selectively expressed in intestine and colorectal tumors in
humans and appears to be a relatively specific marker for metastatic
cancer cells in normal tissues. Indeed, PCR of GCC detected tumor cells
in blood from some patients with Dukes B colorectal cancer and all
patients examined with Dukes C and D colorectal cancer, but not in that
from normal subjects or patients with Dukes A colon carcinoma or other
nonmalignant intestinal pathologies.
Unusual dynamics of extinction in a simple ecological model.
Authors: Sinha S , Parthasarathy S
Published: July 2002

Studies on natural populations and harvesting biological resources have led to the view, commonly held, that (i) populations exhibiting chaotic oscillations run a high risk of extinction; and (ii) a decrease in emigration/exploitation may reduce the risk of extinction. Here we describe a simple ecological model with emigration/depletion that shows behavior in contrast to this. This model displays unusual dynamics of extinction and survival, where populations growing beyond a critical rate can persist within a band of high depletion rates, whereas extinction occurs for lower depletion rates. Though prior to extinction at lower depletion rates the population exhibits chaotic dynamics with large amplitudes of variation and very low minima, at higher depletion rates the population persists at chaos but with reduced variation and increased minima. For still higher values, within the band of persistence, the dynamics show period reversal leading to stability. These results illustrate that chaos does not necessarily lead to population extinction. In addition, the persistence of populations at high depletion rates has important implications in the considerations of strategies for the management of biological resources.
A stationary-phase stress-response sigma factor from Mycobacterium tuberculosis.
Authors: DeMaio J , Zhang Y , Ko C , Young D B , Bishai W R
Published: July 2002

Alternative RNA polymerase sigma factors are a common means of coordinating gene regulation in bacteria. Using PCR amplification with degenerate primers, we identified and cloned a sigma factor gene, sigF, from Mycobacterium tuberculosis. The deduced protein encoded by sigF shows significant similarity to SigF sporulation sigma factors from Streptomyces coelicolor and Bacillus subtilis and to SigB, a stress-response sigma factor, from B. subtilis. Southern blot surveys with a sigF-specific probe identified cross-hybridizing bands in other slow-growing mycobacteria, Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mycobacterium avium, but not in the rapid-growers Mycobacterium smegmatis or Mycobacterium abscessus. RNase protection assays revealed that M. tuberculosis sigF mRNA is not present during exponential-phase growth in M. bovis BCG cultures but is strongly induced during stationary phase, nitrogen depletion, and cold shock. Weak expression of M. tuberculosis sigF was also detected during late-exponential phase, oxidative stress, anaerobiasis, and alcohol shock. The specific expression of M. tuberculosis sigF during stress or stationary phase suggests that it may play a role in the ability of tubercle bacilli to adapt to host defenses and persist during human infection.
Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an
in vivo
substrate for protein kinase C
Authors: Walter Jochen , Grünberg Jürgen , Capell Anja , Pesold Brigitte , Schindzielorz Alice , Citron Martin , Mendla Klaus , George-Hyslop Peter St , Multhaup Gerd , Selkoe Dennis J. , Haass Christian
Published: July 2002

The majority of familial Alzheimer disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). It was shown that the full-length PS-2 protein is phosphorylated constitutively within its N-terminal domain by casein kinases, whereas the PS-1 protein is not. Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of ≈20-kDa C-terminal fragments (CTF) and ≈30-kDa N-terminal fragments [Thinakaran, G.,
et al.
(1996)
Neuron
17, 181–190]. Here we describe the surprising finding that the CTF of PS-1 is phosphorylated by protein kinase C (PKC). Stimulation of PKC causes a 4- to 5-fold increase of the phosphorylation of the ≈20-kDa CTF of PS-1 resulting in reduced mobility in SDS gels. PKC-stimulated phosphorylation occurs predominantly on serine residues and can be induced either by direct stimulation of PKC with phorbol-12,13-dibutyrate or by activation of the m1 acetylcholine receptor-signaling pathway with the muscarinic agonist carbachol. However, phosphorylation of full-length PS-1 and PS-2 is not altered upon PKC stimulation. In addition, a mutant form of PS-1 lacking exon 10, which does not undergo endoproteolytic cleavage [Thinakaran, G.,
et al.
(1996)
Neuron
17, 181–190] is not phosphorylated by PKC, although it still contains all PKC phosphorylation sites conserved between different species. These results show that PKC phosphorylates the PS-1 CTF. Therefore, endoproteolytic cleavage of full-length PS-1 results in the generation of an
in vivo
substrate for PKC. The selective phosphorylation of the PS-1 CTF indicates that the physiological and/or pathological properties of the CTF are regulated by PKC activity.
Congestive heart failure in rats is associated with increased expression and targeting of aquaporin-2 water channel in collecting duct
Authors: Nielsen Søren , Terris James , Andersen Ditte , Ecelbarger Carolyn , Frøkiær Jørgen , Jonassen Thomas , Marples David , Knepper Mark A. , Petersen Jørgen S.
Published: July 2002

We tested whether severe congestive heart failure (CHF), a condition associated with excess free-water retention, is accompanied by altered regulation of the vasopressin-regulated water channel, aquaporin-2 (AQP2), in the renal collecting duct. CHF was induced by left coronary artery ligation. Compared with sham-operated animals, rats with CHF had severe heart failure with elevated left ventricular end-diastolic pressures (LVEDP): 26.9 ± 3.4 vs. 4.1 ± 0.3 mmHg, and reduced plasma sodium concentrations (142.2 ± 1.6 vs. 149.1 ± 1.1 mEq/liter). Quantitative immunoblotting of total kidney membrane fractions revealed a significant increase in AQP2 expression in animals with CHF (267 ± 53%,
n
= 12) relative to sham-operated controls (100 ± 13%,
n
= 14). In contrast, immunoblotting demonstrated a lack of an increase in expression of AQP1 and AQP3 water channel expression, indicating that the effect on AQP2 was selective. Furthermore, postinfarction animals without LVEDP elevation or plasma Na reduction showed no increase in AQP2 expression (121 ± 28% of sham levels,
n
= 6). Immunocytochemistry and immunoelectron microscopy demonstrated very abundant labeling of the apical plasma membrane and relatively little labeling of intracellular vesicles in collecting duct cells from rats with severe CHF, consistent with enhanced trafficking of AQP2 to the apical plasma membrane. The selective increase in AQP2 expression and enhanced plasma membrane targeting provide an explanation for the development of water retention and hyponatremia in severe CHF.
In vivorepopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus
Authors: Yoder Mervin C. , Hiatt Kelly , Mukherjee Pinku
Published: July 2002

The murine yolk sac, being the first site of embryonic blood cell production, has long been theorized to contain the migrating hematopoietic stem cells (HSC) that seed the liver and initiate hematopoiesis on day 10.0 postcoitus (pc). However, it remains controversial whether yolk sac cells isolated before day 11.0 pc possess any long-term repopulating HSC activity upon transplantation into adult recipient mice. We hypothesized that failure to demonstrate engraftment of day <11.0 yolk sac cells in adult hosts may result from an inability of yolk sac cells to home to the active adult hematopoietic sites (spleen and bone marrow). In the present studies, we transplanted yolk sac cells into conditioned newborn mice in whom the liver, as well as the spleen and bone marrow, concomitantly function as a site of blood cell formation. We report that yolk sac cells isolated from day 9.0 pc embryos provide long-term multilineage reconstitution for at least 11 months in primary conditioned newborn mice and for at least 6 months in secondary recipients. Donor yolk sac HSC progeny repopulated mature peripheral blood, thymus, spleen, and bone marrow lymphoid, myeloid, and erythroid compartments. Thus, day 9.0 pc yolk sac HSC can contribute to definitive multilineage hematopoiesis in transplanted recipients. Determination of HSC activity in the day 9.0 pc murine yolk sac suggests that yolk sac HSC are available to seed the liver on day 10.0 pc when definitive hematopoiesis is initiated.
Photorepair mutants of
Arabidopsis
Authors: Jiang C.-Z. , Yee J. , Mitchell D. L. , Britt A. B.
Published: July 2002

UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6–4) pyrimidinone dimer (6–4 product). Although
Escherichia coli
and
Saccharomyces cerevisiae
produce a CPD-specific photolyase that eliminates only this class of dimer,
Arabidopsis thaliana
,
Drosophila melanogaster
,
Crotalus atrox
, and
Xenopus laevis
have recently been shown to photoreactivate both CPDs and 6–4 products. We describe the isolation and characterization of two new classes of mutants of
Arabidopsis
, termed
uvr2
and
uvr3
, that are defective in the photoreactivation of CPDs and 6–4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6–4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system.
What initiates speciation in passion-vine butterflies?
Authors: McMillan W. Owen , Jiggins Chris D. , Mallet James
Published: July 2002

Studies of the continuum between geographic races and species provide the clearest insights into the causes of speciation. Here we report on mate choice and hybrid viability experiments in a pair of warningly colored butterflies,Heliconius eratoandHeliconius himera, that maintain their genetic integrity in the face of hybridization. Hybrid sterility and inviability have been unimportant in the early stages of speciation of these twoHeliconius. We find no evidence of reduced fecundity, egg hatch, or larval survival nor increases in developmental time in three generations of hybrid crosses. Instead, speciation in this pair appears to have been catalyzed by the association of strong mating preferences with divergence in warning coloration and ecology. In mate choice experiments, matings between the two species are a tenth as likely as matings within species. F1 hybrids of both sexes mate frequently with both pure forms. However, male F1 progeny from crosses betweenH. himeramothers andH. eratofathers have somewhat reduced mating success. The strong barrier to gene flow provided by divergence in mate preference is probably enhanced by frequency-dependent predation against hybrids similar to the type known to occur across interracial hybrid zones ofH. erato. In addition, the transition between this pair falls at the boundary between wet and dry forest, and rare hybrids may also be selected against because they are poorly adapted to either biotope. These results add to a growing body of evidence that challenge the importance of genomic incompatibilities in the earliest stages of speciation.
Polyvalent binding to carbohydrates immobilized on an insoluble resin
Authors: Liang Rui , Loebach Jennifer , Horan Nina , Ge Min , Thompson Chris , Yan Lin , Kahne Daniel
Published: July 2002

Numerous studies have established that polyvalency is a critical feature of cell surface carbohydrate recognition. Nevertheless, carbohydrate–protein interactions are typically evaluated by using assays that focus on the behavior of monovalent carbohydrate ligands in solution. It has generally been assumed that the relative affinities of monovalent carbohydrate ligands in solution correlate with their polyvalent avidities. In this paper we show that carbohydrate ligands synthesized directly on TentaGel beads interact with carbohydrate-binding proteins in a polyvalent manner. The carbohydrate-derivatized beads can, therefore, be used as model systems for cell surfaces to evaluate polyvalent carbohydrate–protein interactions. By using a combinatorial approach to synthesize solid-phase libraries of polyvalent carbohydrates, one can rapidly address key issues in the area of cell surface carbohydrate recognition. For example, studies reported herein demonstrate that there is an unanticipated degree of specificity in recognition processes involving polyvalent carbohydrates. However, the correlation between polyvalent avidities and solution affinities is poor. Apparently, the presentation of carbohydrates on the polymer surface has a profound influence on the interaction of the ligand with the protein receptor. These findings have implications for how carbohydrates function as recognition signals in nature, as well as for how polyvalent carbohydrate–protein interactions should be studied.
Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses
Authors: Shi Zengdun , Wakil Adil E. , Rockey Don C.
Published: July 2002

Hepatic fibrosis represents the generalized response of the liver to injury and is characterized by excessive deposition of extracellular matrix. The cellular basis of this process is complex and involves interplay of many factors, of which cytokines are prominent. We have identified divergent fibrosing responses to injury among mouse strains and taken advantage of these differences to examine and contrast T helper (Th)-derived cytokines during fibrogenesis. Liver injury was induced with carbon tetrachloride, fibrosis was quantitated, and Th1/Th2 cytokine mRNAs measured. Liver injury in BALB/c mice resulted in severe fibrosis, whereas C57BL/6 mice developed comparatively minimal fibrosis. Fibrogenesis was significantly modified in T and B cell-deficient BALB/c and C57BL/6 severe combined immunodeficient (SCID) mice compared with wild-type counterparts, suggesting a role of Th subsets. Fibrogenic BALB/c mice exhibited a Th2 response during the wounding response, whereas C57BL/6 mice displayed a Th1 response, suggesting that hepatic fibrosis is influenced by different T helper subsets. Moreover, mice lacking interferon γ, which default to the Th2 cytokine pathway, exhibited more pronounced fibrotic lesions than did wild-type animals. Finally, shifting of the Th2 response toward a Th1 response by treatment with neutralizing anti-interleukin 4 or with interferon γ itself ameliorated fibrosis in BALB/c mice. These data support a role for immune modulation of hepatic fibrosis and suggest that Th cytokine subsets can modulate the fibrotic response to injury.
A deletion in an indole synthase gene is responsible for the DIMBOA-deficient phenotype ofbxbx maize
Authors: Melanson Denise , Chilton Mary-Dell , Masters-Moore David , Chilton W. Scott
Published: July 2002

The biosynthesis of DIMBOA, a pesticidal secondary metabolite of maize, branches off the tryptophan pathway. We have previously demonstrated that indole is the last intermediate common to both the tryptophan and hydroxamic acid pathways. The earliest discovered mutant in the DIMBOA pathway,bxbx(benzoxazineless), is deficient in the production of DIMBOA and related compounds. This paper presents evidence that a gene identified by Kramer and Koziel [Kramer, V. C. & Koziel, M. G. (1995)Plant Mol. Biol.27, 1183–1188] as maize tryptophan synthase α (TSA) is the site of the genetic lesion in the DIMBOA-deficient mutant maize linebxbx. We demonstrate that theTSAgene has sustained a 924-bp deletion inbxbxcompared with its counterpart in wild-type maize. We report that theTSAgene maps to the same location as thebxbxmutation, on the short arm of chromosome 4. We present evidence that the very early and very high level of expression ofTSAcorresponds to the timing and level of DIMBOA biosynthesis but is strikingly different from the expression of the maize tryptophan synthase β (TSB) genes. We show that feeding indole tobxbxseedlings restores their ability to synthesize DIMBOA. We conclude that the maize enzyme initially named tryptophan synthase α in fact is a DIMBOA biosynthetic enzyme, and we propose that it be renamed indole synthase. This work confirms and enlarges upon the findings of Freyet al.[Frey, M. Chomet, P., Glawischniq, E., Stettner, C., Grün, S., Winklmair, A., Eisenreich, W., Bacher, A., Meeley, R. B., Briggs, S. P., Simcox, K. & Gierl, A. (1997)Science277, 696–699], which appeared while the present paper was in review.
β subunits influence the biophysical and pharmacological differences between P- and Q-type calcium currents expressed in a mammalian cell line
Authors: Moreno Herman , Rudy Bernardo , Llinás Rodolfo
Published: July 2002

Human epithelial kidney cells (HEK) were prepared to coexpress α1A, α2δ with different β calcium channel subunits and green fluorescence protein. To compare the calcium currents observed in these cells with the native neuronal currents, electrophysiological and pharmacological tools were used conjointly. Whole-cell current recordings of human epithelial kidney α1A-transfected cells showed small inactivating currents in 80 mM Ba
2+
that were relatively insensitive to calcium blockers. Coexpression of α1A, βIb, and α2δ produced a robust inactivating current detected in 10 mM Ba
2+
, reversibly blockable with low concentration of ω-agatoxin IVA (ω-Aga IVA) or synthetic funnel-web spider toxin (sFTX). Barium currents were also supported by α1A, β2a, α2δ subunits, which demonstrated the slowest inactivation and were relatively insensitive to ω-Aga IVA and sFTX. Coexpression of β3 with the same combination as above produced inactivating currents also insensitive to low concentration of ω-Aga IVA and sFTX. These data indicate that the combination α1A, βIb, α2δ best resembles P-type channels given the rate of inactivation and the high sensitivity to ω-Aga IVA and sFTX. More importantly, the specificity of the channel blocker is highly influenced by the β subunit associated with the α1A subunit.
Genetically determined chloride-sensitive hypertension and stroke
Authors: Tanaka Masae , Schmidlin Olga , Yi Sai-Li , Bollen Andrew W. , Morris R. Curtis
Published: July 2002

The stroke-prone spontaneously hypertensive rat (SHRSP) is a genetically determined model of “salt-sensitive” stroke and hypertension whose full phenotypic expression is said to require a diet high in Na
+
and low in K
+
. We tested the hypothesis that dietary Cl
−
determines the phenotypic expression of the SHRSP. In the SHRSP fed a normal NaCl diet, supplementing dietary K
+
with KCl exacerbated hypertension, whereas supplementing either KHCO
3
or potassium citrate (KB/C) attenuated hypertension, when blood pressure (BP) was measured radiotelemetrically, directly and continually. Supplemental KCl, but not KB/C, induced strokes, which occurred in all and only those rats in the highest quartiles of both BP and plasma renin activity (PRA). PRA was higher with KCl than with KB/C. These observations demonstrate that with respect to both severity of hypertension and frequency of stroke the phenotypic expression of the SHRSP is (
i
) either increased or decreased, depending on whether the anionic component of the potassium salt supplemented is, or is not, Cl
−
; (
ii
) increased by supplementing Cl
−
without supplementing Na
+
, and despite supplementing K
+
; and hence (
iii
) both selectively Cl
−
-sensitive and Cl
−
-determined. The observations suggest that in the SHRSP selectively supplemented with Cl
−
the likelihood of stroke depends on the extent to which both BP and PRA increase.
Scaling laws for fully developed turbulent flow in pipes: Discussion of experimental data
Authors: Barenblatt G. I. , Chorin A. J. , Prostokishin V. M.
Published: July 2002

We compare mean velocity profiles measured in turbulent pipe flows (and also in boundary layer flows) with the predictions of a recently proposed scaling law; in particular, we examine the results of the Princeton “superpipe” experiment and assess their range of validity.
Morphological plasticity of dendritic spines in central neurons is mediated by activation of cAMP response element binding protein
Authors: Murphy Diane D. , Segal Menahem
Published: July 2002

While evidence has accumulated in favor of cAMP-associated genomic involvement in long-term synaptic plasticity, the mechanisms downstream of the activated nucleus that underlie these changes in neuronal function remain mostly unknown. Dendritic spines, the locus of excitatory interaction among central neurons, are prime candidates for long-term synaptic modifications. We now present evidence that links phosphorylation of the cAMP response element binding protein (CREB) to formation of new spines; exposure to estradiol doubles the density of dendritic spines in cultured hippocampal neurons, and concomitantly causes a large increase in phosphorylated CREB and in CREB binding protein. Blockade of cAMP-regulated protein kinase A eliminates estradiol-evoked spine formation, as well as the CREB and CREB binding protein responses. A specific antisense oligonucleotide eliminates the phosphorylated CREB response to estradiol as well as the formation of new dendritic spines. These results indicate that CREB phosphorylation is a necessary step in the process leading to generation of new dendritic spines.
STAT3 complements defects in an interferon-resistant cell line: Evidence for an essential role for STAT3 in interferon signaling and biological activities
Authors: Yang Chuan-He , Murti Aruna , Pfeffer Lawrence M.
Published: July 2002

STAT proteins play critical roles in the signal transduction pathways for various cytokines. The type I interferons (IFNα/β) promote the DNA-binding activity of the transcription factors STAT1, STAT2, and STAT3. Although the requirement for STAT1 and STAT2 in IFNα/β signaling and action is well documented, the biological importance of STAT3 to IFN action has not yet been addressed. We found that STAT3 plays a critical role in signal transduction by IFNα/β. A human cell line that is resistant to the antiviral and antiproliferative activities of IFN but is still IFN-responsive by virtue of STAT1 and STAT2 activation was found to be defective in STAT3 activation and in induction of NF-κB DNA-binding activity. Expression of STAT3 in these resistant cells complemented these signaling defects and also markedly increased cellular sensitivity to the antiviral and antiproliferative effects of IFN. Because STAT3 is involved in the induction of NF-κB DNA-binding activity and in the induction of antiviral and antiproliferative activity, our results place STAT3 as an important upstream element in type I IFN signal transduction and in the induction of biological activities. Therefore, our results indicate that STAT1 and STAT2 are not the only STATs required for the expression of the key biological activities of IFNα/β.
Estimation of evolutionary distances under stationary and nonstationary models of nucleotide substitution
Authors: Gu Xun , Li Wen-Hsiung
Published: July 2002

Estimation of evolutionary distances has always been a major issue
in the study of molecular evolution because evolutionary distances are
required for estimating the rate of evolution in a gene, the divergence
dates between genes or organisms, and the relationships among genes or
organisms. Other closely related issues are the estimation of the
pattern of nucleotide substitution, the estimation of the degree of
rate variation among sites in a DNA sequence, and statistical testing
of the molecular clock hypothesis. Mathematical treatments of these
problems are considerably simplified by the assumption of a stationary
process in which the nucleotide compositions of the sequences under
study have remained approximately constant over time, and there now
exist fairly extensive studies of stationary models of nucleotide
substitution, although some problems remain to be solved. Nonstationary
models are much more complex, but significant progress has been
recently made by the development of the paralinear and LogDet
distances. This paper reviews recent studies on the above issues and
reports results on correcting the estimation bias of evolutionary
distances, the estimation of the pattern of nucleotide substitution,
and the estimation of rate variation among the sites in a sequence.
The vibrational energy flow transition in organic molecules: Theory meets experiment
Authors: Bigwood R. , Gruebele M. , Leitner D. M. , Wolynes P. G.
Published: July 2002

Most large dynamical systems are thought to have ergodic dynamics, whereas small systems may not have free interchange of energy between degrees of freedom. This assumption is made in many areas of chemistry and physics, ranging from nuclei to reacting molecules and on to quantum dots. We examine the transition to facile vibrational energy flow in a large set of organic molecules as molecular size is increased. Both analytical and computational results based on local random matrix models describe the transition to unrestricted vibrational energy flow in these molecules. In particular, the models connect the number of states participating in intramolecular energy flow to simple molecular properties such as the molecular size and the distribution of vibrational frequencies. The transition itself is governed by a local anharmonic coupling strength and a local state density. The theoretical results for the transition characteristics compare well with those implied by experimental measurements using IR fluorescence spectroscopy of dilution factors reported by Stewart and McDonald [Stewart, G. M. & McDonald, J. D. (1983)
J. Chem. Phys.
78, 3907–3915].
The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil
Authors: Weissenhorn Winfried , Calder Lesley J. , Wharton Stephen A. , Skehel John J. , Wiley Don C.
Published: July 2002

The ectodomain of the Ebola virus Gp2 glycoprotein was solubilized with a trimeric, isoleucine zipper derived from GCN4 (pIIGCN4) in place of the hydrophobic fusion peptide at the N terminus. This chimeric molecule forms a trimeric, highly α-helical, and very thermostable molecule, as determined by chemical crosslinking and circular dichroism. Electron microscopy indicates that Gp2 folds into a rod-like structure like influenza HA2 and HIV-1 gp41, providing further evidence that viral fusion proteins from diverse families such as Orthomyxoviridae (Influenza), Retroviridae (HIV-1), and Filoviridae (Ebola) share common structural features, and suggesting a common membrane fusion mechanism.
Absence of cytokine receptor-dependent specificity in red blood cell differentiation
in vivo
Authors: Goldsmith Mark A. , Mikami Aki , You Yun , Liu Kathleen D. , Thomas Liza , Pharr Pamela , Longmore Gregory D.
Published: July 2002

Erythropoietin (EPO) is required for red blood cell development, but whether EPO-specific signals directly instruct erythroid differentiation is unknown. We used a dominant system in which constitutively active variants of the EPO receptor were introduced into erythroid progenitors in mice. Chimeric receptors were constructed by replacing the cytoplasmic tail of constitutively active variants of the EPO receptor with tails of diverse cytokine receptors. Receptors linked to granulocyte or platelet production supported complete erythroid development
in vitro
and
in vivo
, as did the growth hormone receptor, a nonhematopoietic receptor. Therefore, EPOR-specific signals are not required for terminal differentiation of erythrocytes. Furthermore, we found that cellular context can influence cytokine receptor signaling.
Cloning and characterization of a cAMP-specific cyclic nucleotide phosphodiesterase
Authors: Soderling S. H. , Bayuga S. J. , Beavo J. A.
Published: July 2002

Cyclic nucleotide phosphodiesterases (PDEs) regulate intracellular levels of cAMP and cGMP by hydrolyzing them to their corresponding 5′ monophosphates. We report here the cloning and characterization of a novel cAMP-specific PDE from mouse testis. This unique phosphodiesterase contains a catalytic domain that overall shares <40% sequence identity to the catalytic domain of all other known PDEs. Based on this limited homology, this new PDE clearly represents a previously unknown PDE gene family designated as PDE8. The cDNA for PDE8 is 3,678 nucleotides in length and is predicted to encode an 823 amino acid enzyme. The cDNA includes a full ORF as it contains an in-frame stop codon before the start methionine. PDE8 is specific for the hydrolysis of cAMP and has aKmof 0.15 μM. Most common PDE inhibitors are ineffective antagonists of PDE8, including the nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine. Dipyridamole, however, an inhibitor that is generally considered to be relatively specific for the cGMP selective PDEs, does inhibit PDE8 with an IC50of 4.5 μM. Tissue distribution studies of 22 different mouse tissues indicates that PDE8 has highest expression in testis, followed by eye, liver, skeletal muscle, heart, 7-day embryo, kidney, ovary, and brain in decreasing order.In situhybridizations in testis, the tissue of highest expression, shows that PDE8 is expressed in the seminiferous epithelium in a stage-specific manner. Highest levels of expression are seen in stages 7–12, with little or no expression in stages 1–6.
RNA-binding site in T7 RNA polymerase
Authors: Sastry Srin , Ross Barbara M.
Published: July 2002

Recent models of RNA polymerase transcription complexes have invoked the idea that enzyme-nascent RNA contacts contribute to the stability of the complexes. Although much progress on this topic has been made with the multisubunit
Escherichia coli
RNA polymerase, there is a paucity of information regarding the structure of single-subunit phage RNA polymerase transcription complexes. Here, we photo-cross-linked the RNA in a T7 RNA polymerase transcription complex and mapped a major contact site between amino acid residues 144 and 168 and probably a minor contact between residues 1 and 93. These regions of the polymerase are proposed to interact with the emerging RNA during transcription because the 5′ end of the RNA was cross-linked. The contacts are both ionic and nonionic (hydrophobic). The specific inhibitor of T7 transcription, T7 lysozyme, does not compete with T7 RNA polymerase for RNA cross-linking, implying that the RNA does not bind the lysozyme. However, lysozyme may act indirectly via a conformational change in the polymerase. In the current model, the DNA template lies in the polymerase cleft and the fingers subdomain may contact or maintain a template bubble, and a region in the N terminus forms a partly solvent-accessible binding channel for the emerging RNA.
Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzyme
Authors: Ohno Kinji , Brengman Joan , Tsujino Akira , Engel Andrew G.
Published: July 2002

In skeletal muscle, acetylcholinesterase (AChE) exists in homomeric globular forms of type T catalytic subunits (ACHE
T
) and heteromeric asymmetric forms composed of 1, 2, or 3 tetrameric ACHE
T
attached to a collagenic tail (ColQ). Asymmetric AChE is concentrated at the endplate (EP), where its collagenic tail anchors it into the basal lamina. The
ACHE
T
gene has been cloned in humans;
COLQ
cDNA has been cloned in
Torpedo
and rodents but not in humans. In a disabling congenital myasthenic syndrome, EP AChE deficiency (EAD), the normal asymmetric species of AChE are absent from muscle. EAD could stem from a defect that prevents binding of ColQ to ACHE
T
or the insertion of ColQ into the basal lamina. In six EAD patients, we found no mutations in
ACHE
T
. We therefore cloned human
COLQ
cDNA, determined the genomic structure and chromosomal localization of
COLQ
, and then searched for mutations in this gene. We identified six recessive truncation mutations of
COLQ
in six patients. Coexpression of each
COLQ
mutant with wild-type
ACHE
T
in SV40-transformed monkey kidney fibroblast (COS) cells reveals that a mutation proximal to the ColQ attachment domain for ACHE
T
prevents association of ColQ with ACHE
T
; mutations distal to the attachment domain generate a mutant ≈10.5S species of AChE composed of one ACHE
T
tetramer and a truncated ColQ strand. The ≈10.5S species lack part of the collagen domain and the entire C-terminal domain of ColQ, or they lack only the C-terminal domain, which is required for formation of the triple collagen helix, and this likely prevents their insertion into the basal lamina.