bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic basis for queen–worker dimorphism in a social insect
Authors: Volny Veronica P. , Gordon Deborah M.
Published: July 2002

Eusocial insects are characterized by reproductive division of labor, cooperative brood care, and the presence of a sterile worker caste. It is generally accepted that caste determination, including the differentiation of females into sterile workers and reproductive queens, is determined by environmental factors. In contrast, we find that in the red harvester ant,Pogonomyrmex barbatus, an individual's genotype at a particular microsatellite locus predicts its caste. We propose that this microsatellite locus is in tight linkage disequilibrium with at least one locus that plays an important role in caste determination. We call this thecastelocus. We hypothesize that the system of caste determination we observe segregates the population into two distinct genetic lineages, each of which has distinct alleles at the microsatellite locus and also has distinct alleles, we propose, atcaste. Workers are the offspring of parents from different lineages, and are thus heterozygous atcaste, whereas queens are the offspring of parents from the same lineage, and are, therefore, homozygous atcaste. This mode of caste determination has important consequences for the evolution of multiple mating by females and for control of the sex ratio and reproductive allocation in social insect colonies.
Mechanical stress is communicated between different cell types to elicit matrix remodeling
Authors: Swartz M. A. , Tschumperlin D. J. , Kamm R. D. , Drazen J. M.
Published: July 2002

Tissue remodeling often reflects alterations in local mechanical
conditions and manifests as an integrated response among the different
cell types that share, and thus cooperatively manage, an extracellular
matrix. Here we examine how two different cell types, one that
undergoes the stress and the other that primarily remodels the matrix,
might communicate a mechanical stress by using airway cells as a
representative
in vitro
system. Normal stress is imposed
on bronchial epithelial cells in the presence of unstimulated lung
fibroblasts. We show that (
i
) mechanical stress can be
communicated from stressed to unstressed cells to elicit a remodeling
response, and (
ii
) the integrated response of two cell
types to mechanical stress mimics key features of airway remodeling
seen in asthma: namely, an increase in production of fibronectin,
collagen types III and V, and matrix metalloproteinase type 9
(MMP-9) (relative to tissue inhibitor of metalloproteinase-1,
TIMP-1). These observations provide a paradigm to use in understanding
the management of mechanical forces on the tissue level.
Cysteine-3635 is responsible for skeletal muscle ryanodine receptor modulation by NO
Authors: Sun Junhui , Xin Chunlin , Eu Jerry P. , Stamler Jonathan S. , Meissner Gerhard
Published: July 2002

We have shown previously that at physiologically relevant oxygen
tension (pO
2
≈ 10 mmHg), NO S-nitrosylates 1 of
≈50 free cysteines per ryanodine receptor 1 (RyR1) subunit and
transduces a calcium-sensitizing effect on the channel by means of
calmodulin (CaM). It has been suggested that cysteine-3635
is part of a CaM-binding domain, and its reactivity is attenuated by
CaM [Porter Moore, C., Zhang, J. Z., Hamilton, S. L. (1999)
J. Biol. Chem
. 274, 36831–36834]. Therefore, we
tested the hypothesis that the effect of NO was mediated by C3635. The
full-length RyR1 single-site C3635A mutant was generated and expressed
in HEK293 cells. The mutation resulted in the loss of CaM-dependent NO
modulation of channel activity and reduced S-nitrosylation by NO to
background levels but did not affect NO-independent channel modulation
by CaM or the redox sensitivity of the channel to O
2
and
glutathione. Our results reveal that different cysteines within the
channel have been adapted to serve in nitrosative and oxidative
responses, and that S-nitrosylation of the cysteine-containing
CaM-binding domain underlies the mechanism of CaM-dependent regulation
of RyR1 by NO.
Bacteriocin AS-48, a microbial cyclic polypeptide structurally and functionally related to mammalian NK-lysin
Authors: González Carlos , Langdon Grant M. , Bruix Marta , Gálvez Antonio , Valdivia Eva , Maqueda Mercedes , Rico Manuel
Published: July 2002

The solution structure of bacteriocin AS-48, a 70-residue cyclic
polypeptide from
Enterococcus faecalis
, consists of a
globular arrangement of five α-helices enclosing a compact
hydrophobic core. The head-to-tail union lies in the middle of helix 5,
a fact that is shown to have a pronounced effect on the stability of
the three-dimensional structure. Positive charges in the side chains of
residues in helix 4 and in the turn linking helix 4 to helix 5 form a
cluster that most probably determine its antibacterial activity by
promoting pore formation in cell membranes. A similar five-helix
structural motif has been found in the antimicrobial NK-lysin, an
effector polypeptide of T and natural killer (NK) cells. Bacteriocin
AS-48 lacks the three disulfide bridges characteristic of the saposin
fold present in NK-lysin, and has no sequence homology with it.
Nevertheless, the similar molecular architecture and high positive
charge strongly suggest a common mechanism of antibacterial action.
Observation of the noncovalent assembly and disassembly pathways of the chaperone complex MtGimC by mass spectrometry
Authors: Fändrich Marcus , Tito Mark A. , Leroux Michel R. , Rostom Adam A. , Hartl F. Ulrich , Dobson Christopher M. , Robinson Carol V.
Published: July 2002

We have analyzed a newly described archaeal GimC/prefoldin
homologue, termed MtGimC, by using nanoflow electrospray coupled with
time-of-flight MS. The molecular weight of the complex from
Methanobacterium thermoautotrophicum
corresponds to a
well-defined hexamer of two α subunits and four β subunits.
Dissociation of the complex within the gas phase reveals a quaternary
arrangement of two central subunits, both α, and four peripheral β
subunits. By constructing a thermally controlled nanoflow device, we
have monitored the thermal stability of the complex by MS. The results
of these experiments demonstrate that a significant proportion of the
MtGimC hexamer remains intact under low-salt conditions at elevated
temperatures. This finding is supported by data from CD spectroscopy,
which show that at physiological salt concentrations, the complex
remains stable at temperatures above 65°C. Mass spectrometric methods
were developed to monitor in real time the assembly of the MtGimC
hexamer from its component subunits. By using this methodology, the
mass spectra recorded throughout the time course of the experiment
showed the absence of any significantly populated intermediates,
demonstrating that the assembly process is highly cooperative. Taken
together, these data show that the complex is stable under the elevated
temperatures that are appropriate for its hyperthermophile host and
demonstrate that the assembly pathway leads exclusively to the hexamer,
which is likely to be a structural unit
in vivo
.
Pulse confinement in optical fibers with random dispersion
Authors: Chertkov M. , Gabitov I. , Moeser J.
Published: July 2002

Short-range correlated uniform noise in the dispersion coefficient,
inherent in many types of optical fibers, broadens and eventually
destroys all initially ultra-short pulses. However, under the
constraint that the integral of the random component of the dispersion
coefficient is set to zero (pinned), periodically or quasi-periodically
along the fiber, the dynamics of the pulse propagation changes
dramatically. For the case that randomness is present in addition to
constant positive dispersion, the pinning restriction significantly
reduces average pulse broadening. If the randomness is present in
addition to piece-wise constant periodic dispersion with positive
residual value, the pinning may even provide probability distributions
of pulse parameters that are numerically indistinguishable from the
statistically steady case. The pinning method can be used to both
manufacture better fibers and upgrade existing fiber links.
Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.
Authors: Fan J , Shuba Y M , Morad M
Published: July 2002

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.
Studies on the biosynthesis of taxol: the taxane carbon skeleton is not of mevalonoid origin.
Authors: Eisenreich W , Menhard B , Hylands P J , Zenk M H , Bacher A
Published: July 2002

A cell culture of Taxus chinensis was established to produce the diterpene 2alpha,5alpha,10beta,14beta-tetra-acetoxy4 ++ +(20),11-taxadiene (taxuyunnanine C) in 2.6% (dry weight) yield. The incorporation of [U-13C6]glucose, [1-13C]glucose, and [1,2-13C2]acetate into this diterpene was analyzed by NMR spectroscopy. Label from [1,2-13C2]acetate was diverted to the four acetyl groups of taxuyunnanine C, but not to the taxane ring system. Label from [1-13C]glucose and [U-13C6]glucose was efficiently incorporated into both the taxane ring system and the acetyl groups. The four isoprenoid moieties of the diterpene showed identical labeling patterns. The analysis of long-range 13C13C couplings in taxuyunnanine C obtained from an experiment with [U-13C6]glucose documents the involvement of an intramolecular rearrangement in the biosynthesis of the isoprenoid precursor. The labeling patterns are inconsistent with the mevalonate pathway. The taxoid data share important features with the alternative pathway of isoprenoid biosynthesis operating in certain eubacteria Rohmer, M., Knani, M., Simonin, P., Sutter, B. & Sahm, H. (1993) Biochem. J. 295, 517-524].
Clonal analysis of stably transduced human epidermal stem cells in culture.
Authors: Mathor M B , Ferrari G , Dellambra E , Cilli M , Mavilio F , Cancedda R , De Luca M
Published: July 2002

We have transduced normal human keratinocytes with retroviral constructs expressing a bacterial beta-galactosidase (beta-gal) gene or a human interleukin-6 (hIL-6) cDNA under control of a long terminal repeat. Efficiency of gene transfer averaged approximately 50% and 95% of clonogenic keratinocytes for beta-gal and hIL-6, respectively. Both genes were stably integrated and expressed for more than 150 generations. Clonal analysis showed that both holoclones and their transient amplifying progeny expressed the transgene permanently. Southern blot analysis on isolated clones showed that many keratinocyte stem cells integrated multiple proviral copies in their genome and that the synthesis of the exogenous gene product in vitro was proportional to the number of proviral integrations. When cohesive epidermal sheets prepared from stem cells transduced with hIL-6 were grafted on athymic animals, the serum levels of hIL-6 were strictly proportional to the rate of secretion in vitro and therefore to the number of proviral integrations. The possibility of specifying the level of transgene expression and its permanence in a homogeneous clone of stem cell origin opens new perspectives in the long-term treatment of genetic disorders.
Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.
Authors: Ford A M , Bennett C A , Healy L E , Towatari M , Greaves M F , Enver T
Published: July 2002

We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBP alpha exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBP beta is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP delta expression and functional recruitment of C-EBP delta and C-EBP beta to the nucleus. Our results implicate Pu1 and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes.
Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains.
Authors: Jones T R , Wiertz E J , Sun L , Fish K N , Nelson J A , Ploegh H L
Published: July 2002

The human cytomegalovirus (HCMV) early glycoprotein products of the US11 and US2 open reading frames cause increased turnover of major histocompatibility complex (MHC) class I heavy chains. Since US2 is homologous to another HCMV gene (US3), we hypothesized that the US3 gene product also may affect MHC class I expression. In cells constitutively expressing the HCMV US3 gene, MHC class I heavy chains formed a stable complex with beta 2-microglobulin. However, maturation of the N-linked glycan of MHC class I heavy chains was impaired in US3+ cells. The glycoprotein product of US3 (gpUS3) occurs mostly in a high-mannose form and coimmunoprecipitates with beta 2-microglobulin associated class I heavy chains. Mature class I molecules were detected at steady state on the surface of US3+ cells, as in control cells. Substantial perinuclear accumulation of heavy chains was observed in US3+ cells. The data suggest that gpUS3 impairs egress of MHC class I heavy chains from the endoplasmic reticulum.
Evaluating the federal role in financing health-related research
Authors: Garber Alan M. , Romer Paul M.
Published: July 2002

This paper considers the appropriate role for government in the support of scientific and technological progress in health care; the information the federal government needs to make well-informed decisions about its role; and the ways that federal policy toward research and development should respond to scientific advances, technology trends, and changes in the political and social environment. The principal justification for government support of research rests upon economic characteristics that lead private markets to provide inappropriate levels of research support or to supply inappropriate quantities of the products that result from research. The federal government has two basic tools for dealing with these problems: direct subsidies for research and strengthened property rights that can increase the revenues that companies receive for the products that result from research. In the coming years, the delivery system for health care will continue to undergo dramatic changes, new research opportunities will emerge at a rapid pace, and the pressure to limit discretionary federal spending will intensify. These forces make it increasingly important to improve the measurement of the costs and benefits of research and to recognize the tradeoffs among alternative policies for promoting innovation in health care.
Betidamino acids: versatile and constrained scaffolds for drug discovery.
Authors: Rivier J E , Jiang G , Koerber S C , Porter J , Simon L , Craig A G , Hoeger C A
Published: July 2002

Betidamino acids (a contraction of "beta" position and "amide") are N'-monoacylated (optionally, N'-monoacylated and N-mono- or N,N'-dialkylated) aminoglycine derivatives in which each N'acyl/alkyl group may mimic naturally occurring amino acid side chains or introduce novel functionalities. Betidamino acids are most conveniently generated on solid supports used for the synthesis of peptides by selective acylation of one of the two amino functions of orthogonally protected aminoglycine(s) to generate the side chain either prior to or after the elongation of the main chain. We have used unresolved Nalpha-tert-butyloxycarbonyl-N'alpha-fluorenylmethoxycarbonyl++ + aminoglycine, and Nalpha-(Nalpha-methyl)-tert-butyloxycarbonyl-N'alpha-fluo renylmethoxycarbonyl aminoglycine as the templates for the introduction of betidamino acids in Acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(A c)-Leu-Ilys-Pro-DAla-NH2, where 2Nal is 2-naphthylalanine, 4Cpa is 4-chlorophenylalanine, 3Pal is 3-pyridylalanine, Aph is 4-aminophenylalanine, and Ilys is Nepsilon-isopropyllysine], a potent gonadotropin-releasing hormone antagonist, in order to test biocompatibility of these derivatives. Diasteremneric peptides could be separated in most cases by reverse-phase HPLC. Biological results indicated small differences in relative potencies (<5-fold) between the D and L nonalkylated betidamino acid-containing Acyline derivatives. Importantly, most betide diastereomers were equipotent with Acyline. In an attempt to correlate structure and observed potency, Ramachandran-type plots were calculated for a series of betidamino acids and their methylated homologs. According to these calculations, betidamino acids have access to a more limited and distinct number of conformational states (including those associated with alpha-helices, beta-sheets, or turn structures), with deeper minima than those observed for natural amino acids.
The active oligomeric state of the minimalistic influenza virus M
2
ion channel is a tetramer
Authors: Sakaguchi Takemasa , Tu Qiang , Pinto Lawrence H. , Lamb Robert A.
Published: July 2002

The influenza A virus M
2
integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes and also modulates the pH of the trans-Golgi network in virus-infected cells. The M
2
protein is a homo-oligomer of 97 residues, and analysis by chemical cross-linking and SDS/PAGE indicates M
2
forms a tetramer. However, a higher order molecular form is sometimes observed and, thus, it is necessary to determine the active form of the molecule. This was done by studying the currents of oocytes that expressed mixtures of the wild-type M
2
protein (epitope tagged) and the mutant protein M
2
-V
27
S, which is resistant to the inhibitor amantadine. The composition of mixed oligomers of the two proteins expressed at the plasma membrane of individual oocytes was quantified after antibody capture of the cell surface expressed molecules and it was found that the subunits mixed freely. When the ratio of wild-type to mutant protein subunits was 0.85:0.15, the amantadine sensitivity was reduced to 50% and for a ratio of 0.71:0.29 to 20%. These results are consistent with the amantadine-resistant mutant being dominant and the oligomeric state being a tetramer.
CD27, a member of the tumor necrosis factor receptor family, induces apoptosis and binds to Siva, a proapoptotic protein
Authors: Prasad K. V. S. , Ao Zhaohui , Yoon Yoosik , Wu Mei X. , Rizk Majida , Jacquot Serge , Schlossman Stuart F.
Published: July 2002

Members of the tumor necrosis factor receptor (TNFR) superfamily are important for cell growth and survival. In addition to providing costimulatory signals for cell proliferation, ligation of both TNFR1 and Fas can result in programmed cell death or apoptosis. The underlying mechanism requires an intact 80-aa stretch present in the cytoplasmic tails of both TNFR1 and Fas, termed the death domain (DD). Here we show that CD27, a member of the TNFR family, expressed on discrete subpopulations of T and B cells and known to provide costimulatory signals for T and B cell proliferation and B cell Ig production, can also induce apoptosis. Co-crosslinking of surface Ig receptors along with ligation of CD27 augments CD27-mediated apoptosis. Unlike TNFR1 and Fas, the cytoplasmic tail of CD27 is relatively short and lacks the DD. Using the yeast two-hybrid system, we have cloned a novel protein (Siva) that binds to the CD27 cytoplasmic tail. It has a DD homology region, a box-B-like ring finger, and a zinc finger-like domain. Overexpression of Siva in various cell lines induces apoptosis, suggesting an important role for Siva in the CD27-transduced apoptotic pathway.
Proximo–distal specification in the wing disc of
Drosophila
by the nubbin gene
Authors: Cifuentes Francisco J. , García-Bellido Antonio
Published: July 2002

Mutations in the nubbin (
nub
) gene have a phenotype consisting of a severe wing size reduction and pattern alterations, such as transformations of distal elements into proximal ones.
nub
expression is restricted to the wing pouch cells in wing discs since early larval development. These effects are also observed in genetic mosaics where cell proliferation is reduced in all wing blade regions autonomously, and transformation into proximal elements is observed in distal clones. Clones located in the proximal region of the wing blade cause in addition nonautonomous reduction of the whole wing. Cell lineage experiments in a
nub
mutant background show that clones respect neither the anterior–posterior nor the dorsal–ventral boundary but that the selector genes have been correctly expressed since early larval development. The phenotypes of
nub el
and
nub dpp
genetic combinations are synergistic and the overexpression of
dpp
in clones in
nub
wings does not result in overproliferation of the surrounding wild-type cells. We discuss the role of
nub
in the wing’s proximo–distal axis and in the formation of compartment boundaries.
Phenotypic knockout of HIV type 1 chemokine coreceptor CCR-5 by intrakines as potential therapeutic approach for HIV-1 infection
Authors: Yang An-Gang , Bai Xuefan , Huang Xue F. , Yao Changping , Chen Si.-Yi
Published: July 2002

A genetic defect in a CC-chemokine receptor (CCR)-5, the principal coreceptor for the macrophage-tropic HIV type 1 (HIV-1), recently was found to naturally protect CCR-5-defective, but healthy, individuals from HIV-1 infection. In this study, we mimic the natural resistance of the CCR-5-defective individuals by designing a strategy to phenotypically knock out CCR-5. The inactivation of the CCR-5 coreceptor is accomplished by targeting a modified CC-chemokine to the endoplasmic reticulum to block the surface expression of newly synthesized CCR-5. The lymphocytes transduced to express the intracellular chemokine, termed “intrakine,” were found to be viable and resistant to macrophage-tropic HIV-1 infection. Thus, this gene-based intrakine strategy targeted at the conserved cellular receptor for the prevention of HIV-1 entry should have significant advantages over currently described approaches for HIV-1 therapy.
Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection
Authors: Browning Jessie , Horner James W. , Pettoello-Mantovani Massimo , Raker Christina , Yurasov Sergey , DePinho Ronald A. , Goldstein Harris
Published: July 2002

HIV entry into human cells is mediated by CD4 acting in concert with one of several members of the chemokine receptor superfamily. The resistance to HIV infection observed in individuals with defective CCR5 alleles indicated that this particular chemokine receptor plays a crucial role in the initiation ofin vivoHIV infection. Expression of human CD4 transgene does not render mice susceptible to HIV infection because of structural differences between human and mouse CCR5. To ascertain whether expression of human CD4 and CCR5 is sufficient to make murine T lymphocytes susceptible to HIV infection, thelckpromoter was used to direct the T cell-specific expression of human CD4 and CCR5 in transgenic mice. Peripheral blood mononuclear cells and splenocytes isolated from these mice expressed human CD4 and CCR5 and were infectible with selected M-tropic HIV isolates. Afterin vivoinoculation, HIV-infected cells were detected by DNA PCR in the spleen and lymph nodes of these transgenic mice, but HIV could not be cultured from these cells. This indicated that although transgenic expression of human CD4 and CCR5 permitted entry of HIV into the mouse cells, significant HIV infection was prevented by other blocks to HIV replication present in mouse cells. In addition to providingin vivoverification for the important role of CCR5 in T lymphocyte HIV infection, these transgenic mice represent a newin vivomodel for understanding HIV pathogenesis by delineating species-specific cellular factors required for productivein vivoHIV infection. These mice should also prove useful for the assessment of potential therapeutic and preventative modalities, particularly vaccines.
Effect of protein dynamics on biological electron transfer
Authors: Daizadeh Iraj , Medvedev Emile S. , Stuchebrukhov Alexei A.
Published: July 2002

Computer simulations of the effect of protein dynamics on the long
distance tunneling mediated by the protein matrix have been carried out
for a Ru-modified (His 126) azurin molecule. We find that the tunneling
matrix element is a sensitive function of the atomic configuration of
the part of the protein matrix in which tunneling currents (pathways)
are localized. Molecular dynamics simulations show that fluctuations of
the matrix element can occur on a time scale as short as 10 fs. These
short time fluctuations are an indication of a strong dynamic coupling
of a tunneling electron to vibrational motions of the protein nuclear
coordinates. The latter results in a modification of the conventional
Marcus picture of electron transfer in proteins. The new element in the
modified theory is that the tunneling electron is capable of emitting
or absorbing vibrational energy (phonons) from the medium. As a result,
some biological reactions may occur in an activationless fashion. An
analytical theoretical model is proposed to account for thermal
fluctuations of the medium in long distance electron transfer
reactions. The model shows that, at long distances, the phonon-modified
inelastic tunneling always dominates over the conventional elastic
tunneling.
Crystal structure of a recombinant α
E
C domain from human fibrinogen-420
Authors: Spraggon Glen , Applegate Dianne , Everse Stephen J. , Zhang Jian-Zhong , Veerapandian Leela , Redman Colvin , Doolittle Russell F. , Grieninger Gerd
Published: July 2002

The crystal structure of a recombinant α
E
C domain from human fibrinogen-420 has been determined at a resolution of 2.1 Å. The protein, which corresponds to the carboxyl domain of the α
E
chain, was expressed in and purified from
Pichia pastoris
cells. Felicitously, during crystallization an amino-terminal segment was removed, apparently by a contaminating protease, allowing the 201-residue remaining parent body to crystallize. An x-ray structure was determined by molecular replacement. The electron density was clearly defined, partly as a result of averaging made possible by there being eight molecules in the asymmetric unit related by noncrystallographic symmetry (P1 space group). Virtually all of an asparagine-linked sugar cluster is present. Comparison with structures of the β- and γ-chain carboxyl domains of human fibrinogen revealed that the binding cleft is essentially neutral and should not bind Gly-Pro-Arg or Gly-His-Arg peptides of the sort bound by those other domains. Nonetheless, the cleft is clearly evident, and the possibility of binding a carbohydrate ligand like sialic acid has been considered.
Oxygen and evolutionary patterns in the sea: Onshore/offshore trends and recent recruitment of deep-sea faunas
Authors: Jacobs David K. , Lindberg David R.
Published: July 2002

Over the last 15 years a striking pattern of diversification has been documented in the fossil record of benthic marine invertebrates. Higher taxa (orders) tend to originate onshore, diversify offshore, and retreat into deep-water environments. Previous studies attribute this macroevolutionary pattern to a variety of causes, foremost among them the role of nearshore disturbance in providing opportunities for the evolution of novel forms accorded ordinal rank. Our analysis of the post-Paleozoic record of ordinal first appearances indicates that the onshore preference of ordinal origination occurred only in the Mesozoic prior to the Turonian stage of the Cretaceous, a period characterized by relatively frequent anoxic/dysoxic bottom conditions in deeper marine environments. Later, in the Cretaceous and Cenozoic, ordinal origination of benthic organisms did not occur exclusively, or even preferentially, in onshore environments. This change in environmental pattern of ordinal origination roughly correlates with Late Cretaceous: (
i
) decline in anoxia/dysoxia in offshore benthic environments; (
ii
) extinction of faunas associated with dysoxic conditions; (
iii
) increase in bioturbation with the expansion of deep burrowing forms into offshore environments; and (
iv
) offshore expansion of bryozoan diversity. We also advance a separate argument that the Cenomanian/Turonian and latest Paleocene global events eliminated much of the deep-water benthos. This requires a more recent origin of modern vent and deep-sea faunas, from shallower water refugia, than the Paleozoic or early Mesozoic origin of these faunas suggested by other workers.
Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responsesin vitroandin vivo
Authors: Fields R. C. , Shimizu K. , Mulé J. J.
Published: July 2002

The highly efficient nature of dendritic cells (DC) as antigen-presenting cells raises the possibility of uncovering in tumor-bearing hosts very low levels of T cell reactivity to poorly immunogenic tumors that are virtually undetectable by other means. Here, we demonstrate thein vitroandin vivocapacities of murine bone marrow-derived, cytokine-driven DC to elicit potent and specific anti-tumor responses when pulsed with whole tumor lysates. Stimulation of naive spleen-derived T cells by tumor lysate-pulsed DC generated tumor-specific proliferative cytokine release and cytolytic reactivitiesin vitro. In addition, in two separate strains of mice with histologically distinct tumors, s.c. injections of DC pulsed with whole tumor lysates effectively primed these animals to reject subsequent lethal challenges with viable parental tumor cells and, important to note, also mediated significant reductions in the number of metastases established in the lungs. Tumor rejection depended on host-derived CD8+T cells and, to a lesser extent, CD4+T cells. Spleens from mice that had rejected their tumors contained specific precursor cytotoxic T lymphocytes. The use of whole tumor lysates as a source of tumor-associated antigen(s) for pulsing of DC circumvents several limitations encountered with other methods as well as provides certain distinct advantages, which are discussed. These data serve as rationale for our recent initiation of a phase I clinical trial of immunization with autologous tumor lysate-pulsed DC in adult and pediatric cancer patients.
Release of epigenetic gene silencing by trans-acting mutations in
Arabidopsis
Authors: Scheid Ortrun Mittelsten , Afsar Karin , Paszkowski Jerzy
Published: July 2002

Gene silencing in plants inactivates transgenes introduced into plants and/or endogenous homologous genes. This stable but potentially reversible loss of gene activity resembles epigenetic changes that occur in normal development. The stability of silencing implies the involvement of trans-acting components, although none of them have been identified so far. Here we report the finding of second-site mutations interfering with maintenance of the silent state. We mutagenized
Arabidopsis thaliana
plants carrying a silent transgene encoding hygromycin phosphotransferase (
hpt
) and therefore show a heritable hygromycin-sensitive phenotype. The M2 generation was screened for hygromycin resistance. Eight putative mutants
(som1
to
8)
were found that expressed the transgene and transmitted the expressed state to their progeny. All mutations were shown to reactivate a silent transgenic test locus
in trans
. The level of DNA methylation at the
hpt
locus and at centromeric repeats was found to be reduced in the
som
mutants. Complementation crosses indicated complex epigenetic interactions among the
som
mutant alleles and with the previously described
ddm1
allele, which elicits DNA hypomethylation [Vongs, A., Kakutani, T., Martienssen, R. A. & Richards, E. J. (1993)
Science
260, 1926–1928].
Som
mutants can be classified into three groups: (
i
) allelic or interacting with
ddm1
and with each other (
som 1, 4,
and
5
), (
ii
) nonallelic with
ddm1
and
som
mutants of group A (
som2
), and (
iii
) mutants with slow resilencing after outcrosses, which hinders their classification (
som 3, 6, 7,
and
8
).
Patterns of prehistoric human mobility in Polynesia indicated by mtDNA from the Pacific rat
Authors: Matisoo-Smith E. , Roberts R. M. , Irwin G. J. , Allen J. S. , Penny D. , Lambert D. M.
Published: July 2002

Human settlement of Polynesia was a major event in world prehistory. Despite the vastness of the distances covered, research suggests that prehistoric Polynesian populations maintained spheres of continuing interaction for at least some period of time in some regions. A low level of genetic variation in ancestral Polynesian populations, genetic admixture (both prehistoric and post-European contact), and severe population crashes resulting from introduction of European diseases make it difficult to trace prehistoric human mobility in the region by using only human genetic and morphological markers. We focus instead on an animal that accompanied the ancestral Polynesians on their voyages. DNA phylogenies derived from mitochondrial control-region sequences of Pacific rats (
Rattus exulans
) from east Polynesia are presented. A range of specific hypotheses regarding the degree of interaction within Polynesia are tested. These include the issues of multiple contacts between central east Polynesia and the geographically distinct archipelagos of New Zealand and Hawaii. Results are inconsistent with models of Pacific settlement involving substantial isolation after colonization and confirm the value of genetic studies on commensal species for elucidating the history of human settlement.
Experimentally determined chaotic phase synchronization in a neuronal system
Authors: Makarenko Vladimir , Llinás Rodolfo
Published: July 2002

Mathematical analysis of the subthreshold oscillatory properties of inferior olivary neurons
in vitro
indicates that the oscillation is nonlinear and supports low dimensional chaotic dynamics. This property leads to the generation of complex functional states that can be attained rapidly via phase coherence that conform to the category of “generalized synchronization.” Functionally, this translates into neuronal ensemble properties that can support maximum functional permissiveness and that rapidly can transform into robustly determined multicellular coherence.
Latent varicella–zoster virus is located predominantly in neurons in human trigeminal ganglia
Authors: Kennedy Peter G. E. , Grinfeld Esther , Gow John W.
Published: July 2002

Varicella–zoster virus (VZV) is a human herpesvirus that causes varicella (chicken pox) as a primary infection and, after a variable period of latency in trigeminal and dorsal root ganglia, reactivates to cause herpes zoster (shingles). Both of these conditions may be followed by a variety of neurological complications, especially in immunocompromised individuals such as those with human immunodeficiency virus (HIV) infection. There have been a number of conflicting reports regarding the cellular location of latent VZV within human ganglia. To address this controversy we examined fixed wax-embedded trigeminal ganglia from 30 individuals obtained at autopsy, including 11 with HIV infection, 2 neonates, and 17 immunocompetent individuals, for the presence of latent VZV. Polymerase chain reaction (PCR),
in situ
hybridization, and PCR
in situ
amplification techniques with oligonucleotide probes and primer sequences to VZV genes 18, 21, 29, and 63 were used. VZV DNA in ganglia was detected in 15 individuals by using PCR alone, and in 12 individuals (6 normal non-HIV and 6 positive HIV individuals, but not neonatal ganglia) by using PCR
in situ
amplification. When
in situ
hybridization alone was used, 5 HIV-positive individuals and only 1 non-HIV individual showed VZV nucleic acid signals in ganglia. In all of the VZV-positive ganglia examined, VZV nucleic acid was detected in neuronal nuclei. Only occasional nonneuronal cells contained VZV DNA. We conclude from these studies that the neuron is the predominant site of latent VZV in human trigeminal ganglia.
Interactions of G
h
/transglutaminase with phospholipase Cδ1 and with GTP
Authors: Murthy S. N. Prasanna , Lomasney Jon W. , Mak Eric C. , Lorand Laszlo
Published: July 2002

The inositol phosphate hydrolyzing activity of human phospholipase Cδ1 (PLCδ1) is markedly inhibited when the enzyme is coexpressed with the human heart G
h
/transglutaminase (TG) in human embryonic kidney cells. Because the cotransfection does not affect the amount of PLCδ1 in the cells, the depression of phospholipase activity probably is a result of a direct interaction between the two proteins. An ELISA procedure was employed to document the associations of purified TG preparations from a variety of tissues (human red cells, rabbit lens, guinea pig liver) with PLCδ1. Nucleotides (GTP > GDP > ATP > GMP = ADP, in order of decreasing efficiency) interfered with the formation of the PLCδ1:TG complex. A conformational change in the TG partner, occurring with nucleotide binding, is thought to be responsible for dissociating the two proteins. The structural rearrangement produces a remarkable shift in the anodic mobility of TG in electrophoresis: TG
slow
+ GTP ⇆ [TG:GTP]
fast
. Altogether, our findings indicate that GTP controls PLCδ1 activity by releasing this protein from an inhibitory association with G
h
/transglutaminase.
Mg-chelatase of tobacco: The role of the subunit CHL D in the chelation step of protoporphyrin IX
Authors: Gräfe Susanna , Saluz Hans-Peter , Grimm Bernhard , Hänel Frank
Published: July 2002

The Mg-chelation is found to be a prerequisite to direct protoporphyrin IX into the chlorophyll (Chl)-synthesizing branch of the tetrapyrrol pathway. The ATP-dependent insertion of magnesium into protoporphyrin IX is catalyzed by the enzyme Mg-chelatase, which consists of three protein subunits (CHL D, CHL I, and CHL H). We have chosen the Mg-chelatase from tobacco to obtain more information about the mode of molecular action of this complex enzyme by elucidating the interactions
in vitro
and
in vivo
between the central subunit CHL D and subunits CHL I and CHL H. We dissected CHL D in defined peptide fragments and assayed for the essential part of CHL D for protein–protein interaction and enzyme activity. Surprisingly, only a small part of CHL D, i.e., 110 aa, was required for interaction with the partner subunits and maintenance of the enzyme activity. In addition, it could be demonstrated that CHL D is capable of forming homodimers. Moreover, it interacted with both CHL I and CHL H. Our data led to the outline of a two-step model based on the cooperation of the subunits for the chelation process.
Yeast flavin-containing monooxygenase is induced by the unfolded protein response
Authors: Suh Jung-Keun , Robertus Jon D.
Published: July 2002

Flavin-containing monooxygenase from yeast (yFMO) carries out the O
2
- and NADPH-dependent oxidation of biological thiols, including oxidizing glutathione to glutathione disulfide. FMO provides a large fraction of the oxidizing necessary for proper folding of disulfide bond-containing proteins; deletion of the enzyme reduces proper folding of endogenous carboxypeptidase Y by about 40%. The enzyme is not essential to cell viability because other enzymes can generate a significant fraction of the oxidizing equivalents required by the cell. However, yFMO is vital to the yeast response to reductive stress.
FMO1
deletion mutants grow poorly under reductive stress, and carboxypeptidase Y activity is less than 10% of that in a stressed wild type. The
FMO1
gene appears to be under control of an unfolded protein response element and is inducible by factors, such as reductive stress, that elicit the unfolded protein response. Reductive stress can increase yFMO activity at least 6-fold. This increased activity allows the cell to process endogenous disulfide bond-containing proteins and also to allow correct folding of disulfide-bonded proteins expressed from multicopy plasmids. The unfolded protein response is mediated by the Hac1p transcription factor that mediates virtually all of the induction of yFMO triggered by exogenous reducing agents.
Solution structure of the RNA polymerase subunit RPB5 from
Methanobacterium thermoautotrophicum
Authors: Yee Adelinda , Booth Valerie , Dharamsi Akil , Engel Asaph , Edwards Aled M. , Arrowsmith Cheryl H.
Published: July 2002

RPB5 is an essential subunit of eukaryotic and archaeal RNA
polymerases. It is a proposed target for transcription activator
proteins in eukaryotes, but the mechanism of interaction is not known.
We have determined the solution structure of the RPB5 subunit from the
thermophilic archeon,
Methanobacterium
thermoautotrophicum
. MtRBP5 contains a four-stranded β-sheet
platform supporting two α-helices, one on each side of the β-sheet,
resulting in an overall mushroom shape that does not appear to have any
structural homologues in the structural database. The position and
conservation of charged surface residues suggests possible modes of
interaction with other proteins, as well as a rationale for the thermal
stability of this protein.
TEP1
, the yeast homolog of the human tumor suppressor gene PTEN/MMAC1/TEP1, is linked to the phosphatidylinositol pathway and plays a role in the developmental process of sporulation
Authors: Heymont Jennifer , Berenfeld Ludmilla , Collins Jennifer , Kaganovich Alexandra , Maynes Bradford , Moulin Aaron , Ratskovskaya Irina , Poon Pak P. , Johnston Gerald C. , Kamenetsky Margarita , DeSilva John , Sun Hong , Petsko Gregory A. , Engebrecht JoAnne
Published: July 2002

PTEN/MMAC1/TEP1 (PTEN, phosphatase deleted on chromosome
ten; MMAC1, mutated in multiple advanced cancers; TEP1, tensin-like
phosphatase) is a major human tumor suppressor gene whose suppressive
activity operates on the phosphatidylinositol pathway. A single
homologue of this gene,
TEP1 (YNL128w)
, exists in the
budding yeast
Saccharomyces cerevisiae
. Yeast strains
deleted for
TEP1
exhibit essentially no phenotype in
haploids; however, diploids exhibit resistance to the
phosphatidylinositol-3-phosphate kinase inhibitor wortmannin and to
lithium ions. Although rates of cancer increase with age, neither
tep1
haploids nor diploids have altered life spans.
TEP1
RNA is present throughout the cell cycle, and
levels are dramatically up-regulated during meiotic development.
Although homozygous
tep1
mutants initiate the meiotic
program and form spores with wild-type kinetics, analysis of the spores
produced in
tep1
mutants indicates a specific defect in
the trafficking or deposition of dityrosine, a major component of yeast
spore walls, to the surface. Introduction of a common PTEN mutation
found in human tumors into the analogous position in Tep1p produces a
nonfunctional protein based on
in vivo
activity. These
studies implicate Tep1p in a specific developmental trafficking or
deposition event and suggest that Tep1p, like its mammalian
counterpart, impinges on the phosphatidylinositol pathway.
Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases
Authors: Deng Xingming , Ruvolo Peter , Carr Boyd , May W. Stratford
Published: July 2002

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 μM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation
in vitro
. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both
in vitro
and
in vivo
. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.
Identification of hepatitis B virus indigenous to chimpanzees
Authors: Hu Xiaolei , Margolis Harold S. , Purcell Robert H. , Ebert James , Robertson Betty H.
Published: July 2002

Hepatitis B viruses (HBV) and related viruses, classified in the
Hepadnaviridae
family, are found in a wide variety of mammals and birds. Although the chimpanzee has been the primary experimental model of HBV infection, this species has not been considered a natural host for the virus. Retrospective analysis of 13 predominantly wild-caught chimpanzees with chronic HBV infection identified a unique chimpanzee HBV strain in 11 animals. Nucleotide and derived amino acid analysis of the complete HBV genome and the gene coding for the hepatitis B surface antigen (S gene) identified sequence patterns that could be used to reliably identify chimpanzee HBV. This analysis indicated that chimpanzee HBV is distinct from known human HBV genotypes and is closely related to HBVs previously isolated from a chimpanzee, gibbons, gorillas, and orangutans.