bluuebunny/crossref_metadata_2025 huggingface dataset:
Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.
Authors: Jones S A , Wolf M , Qin S , Mackay C R , Baggiolini M
Published: July 2002

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.
Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins.
Authors: Anderson M T , Tjioe I M , Lorincz M C , Parks D R , Herzenberg L A , Nolan G P , Herzenberg L A
Published: July 2002

Green fluorescent protein (GFP) is widely used as a reporter gene in both prokaryotes and eukaryotes. However, the fluorescence levels of wild-type GFP (wtGFP) are not bright enough for fluorescence-activated cell sorting or flow cytometry. Several GFP variants were generated that are brighter or have altered excitation spectra when expressed in prokaryotic cells. We engineered two GFP genes with different combinations of these mutations, GFP(S65T,V163A) termed GFP-Bex1, and GFP(S202F,T203I,V163A) termed GFP-Vex1. Both show enhanced brightness and improved signal-to-noise ratios when expressed in mammalian cells and appropriately excited, compared with wtGFP. Each mutant retains only one of the two excitation peaks of the wild-type protein. GFP-Bex1 excites at 488 nm (blue) and GFP-Vex1 excites at 406 nm (violet), both of which are available laser lines. Excitation at these wavelengths allows for the independent analyses of these mutants by fluorescence-activated cell sorting, permitting simultaneous, quantitative detection of expression from two different genes within single mammalian cells.
Primordial emergence of the recombination activating gene 1 (RAG1): sequence of the complete shark gene indicates homology to microbial integrases.
Authors: Bernstein R M , Schluter S F , Bernstein H , Marchalonis J J
Published: July 2002

The rearrangement of antibody and T-cell receptor gene segments is indispensable to the vertebrate immune response. All extant jawed vertebrates can rearrange these gene segments. This ability is conferred by the recombination activating genes I and II (RAG I and RAG II). To elucidate their origin and function, the cDNA encoding RAG I from a member of the most ancient class of extant gnathostomes, the Carcharhine sharks, was characterized. Homology domains identified within shark RAG I prompted sequence comparison analyses that suggested similarity of the RAG I and II genes, respectively, to the integrase family genes and integration host factor genes of the bacterial site-specific recombination system. Thus, the apparent explosive evolution (or "big bang") of the ancestral immune system may have been initiated by a transfer of microbial site-specific recombinases.
Conformational dependence of electron transfer across de novo designed metalloproteins.
Authors: Mutz M W , McLendon G L , Wishart J F , Gaillard E R , Corin A F
Published: July 2002

Flash photolysis and pulse radiolysis measurements demonstrate a conformational dependence of electron transfer rates across a 16-mer helical bundle (three-helix metalloprotein) modified with a capping CoIII(bipyridine)3 electron acceptor at the N terminus and a 1-ethyl-1'-ethyl-4,4'- bipyridinium donor at the C terminus. For the CoIII(peptide)3-1-ethyl-1'-ethyl-4,4'-bipyridinium maquettes, the observed transfer is a first order, intramolecular process, independent of peptide concentration or laser pulse energy. In the presence of 6 M urea, the random coil bundle (approximately 0% helicity) has an observed electron transfer rate constant of kobs = 900 +/- 100 s-1. In the presence of 25% trifluoroethanol (TFE), the helicity of the peptide is 80% and the kobs increases to 2000 +/- 200 s-1. Moreover, the increase in the rate constant in TFE is consistent with the observed decrease in donor-acceptor distance in this solvent. Such bifunctional systems provide a class of molecules for testing the effects of conformation on electron transfer in proteins and peptides.
Irreversibility of cellular aging and neoplastic transformation: a clonal analysis.
Authors: Chow M , Rubin H
Published: July 2002

Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in a persistent impairment of proliferation when the cells are subcultured at low density and a greatly increased probability of neoplastic transformation in assays for transformation. These properties, along with the large accumulation of age pigment bodies in the confluent cells, are cardinal cellular characteristics of aging in organisms and validate the system as a model of cellular aging. Two cultures labeled alpha and beta were obtained after prolonged confluence; both were dominated by cells that were both slowed in growth at low population density and enhanced in growth capacity at high density, a marker of neoplastic transformation. An experiment was designed to study the reversibility of these age-related properties by serial subculture at low density of the two uncloned cultures and their progeny clones derived from assuredly single cells. Both uncloned cultures had many transformed cells and a reduced growth rate on subculture. Serial subculture resulted in a gradual increase in growth rates of both populations, but a reversal of transformation only in the alpha population. The clones originating from both populations varied in the degree of growth impairment and neoplastic transformation. None of the alpha clones increased in growth rate on low density passage nor did the transformed clones among them revert to normal growth behavior. The fastest growing beta clone was originally slower than the control clone, but caught up to it after four weekly subcultures. The other beta clones retained their reduced growth rates. Four of the five beta clones, including the fastest grower, were transformed, and none reverted on subculture. We conclude that the apparent reversal of impaired growth and transformation in the uncloned parental alpha population resulted from the selective growth at low density of fast growing nontransformed clones. The reversal of impaired growth in the uncloned parental beta population was also the result of selective growth of fast growing clones, but in this case they were highly transformed so no apparent reversal of transformation occurred. The clonal results indicate that neither the impaired growth nor the neoplastic transformation found in aging cells is reversible. We discuss the possible contribution of epigenetic and genetic processes to these irreversible changes.
Trypanothione overproduction and resistance to antimonials and arsenicals in Leishmania.
Authors: Mukhopadhyay R , Dey S , Xu N , Gage D , Lightbody J , Ouellette M , Rosen B P
Published: July 2002

Leishmania resistant to arsenicals and antimonials extrude arsenite. Previous results of arsenite uptake into plasma membrane-enriched vesicles suggested that the transported species is a thiol adduct of arsenite. In this paper, we demonstrate that promastigotes of arsenite-resistant Leishmania tarentolae have increased levels of intracellular thiols. High-pressure liquid chromatography of the total thiols showed that a single peak of material was elevated almost 40-fold. The major species in this peak was identified by matrix-assisted laser desorption/ionization mass spectrometry as N1,N8-bis-(glutathionyl)spermidine (trypanothione). The trypanothione adduct of arsenite was effectively transported by the As-thiol pump. No difference in pump activity was observed in wild type and mutants. A model for drug resistance is proposed in which Sb(V)/As(V)-containing compounds, including the antileishmanial drug Pentostam, are reduced intracellularly to Sb(III)/As(III), conjugated to trypanothione, and extruded by the As-thiol pump. The rate-limiting step in resistance is proposed to be formation of the metalloid-thiol pump substrates, so that increased synthesis of trypanothione produces resistance. Increased synthesis of the substrate rather than an increase in the number of pump molecules is a novel mechanism for drug resistance.
Localization of the mouse gene releasing sex-limited expression of Slp.
Authors: Jiang P P , Frederick K , Hansen T H , Miller R D
Published: July 2002

To probe genetic variation in the regulation of sexual dimorphism, we have characterized the mouse protein Slp, coded by the gene sex-limited protein (Slp). Slp expression in many strains is limited to males and is androgen-dependent. However, female expression is also observed in rare strains, due to nonlinked gene(s) termed regulator of sex-limitation (rsl). In this report we demonstrate that female expression of Slp results from homozygous recessive allele(s) at a single autosomal locus that maps to a 2.2-centimorgan interval on chromosome 13. This conclusion was supported by extensive genetic analyses including the use of polymorphic microsatellites to type numerous backcross progeny and a recombinant inbred series and to identify the congenic interval in three independently derived congenic strains. Four attractive candidate genes were identified by the localization of rsl. Interestingly, rsl was found not only to enable expression in females but to also increase expression in males. The findings suggest that the expression of Slp and perhaps other sexually dimorphic proteins is regulated by two pathways, one that is dependent upon rsl but not androgens and another that is rsl-independent but requires androgens.
Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene.
Authors: Gnarra J R , Zhou S , Merrill M J , Wagner J R , Krumm A , Papavassiliou E , Oldfield E H , Klausner R D , Linehan W M
Published: July 2002

The VHL tumor suppressor gene is inactivated in patients with von Hippel-Lindau disease and in most sporadic clear cell renal carcinomas. Although VHL protein function remains unclear, VHL does interact with the elongin BC subunits in vivo and regulates RNA polymerase II elongation activity in vitro by inhibiting formation of the elongin ABC complex. Expression of wild-type VHL in renal carcinoma cells with inactivated endogenous VHL resulted in unaltered in vitro cell growth and decreased vascular endothelial growth factor (VEGF) mRNA expression and responsiveness to serum deprivation. VEGF is highly expressed in many tumors, including VHL-associated and sporadic renal carcinomas, and it stimulates neoangiogenesis in growing solid tumors. Despite 5-fold differences in VEGF mRNA levels, VHL overexpression did not affect VEGF transcription initiation or elongation as would have been suggested by VHL-elongin association. These results suggest that VHL regulates VEGF expression at a post-transcriptional level and that VHL inactivation in target cells causes a loss of VEGF suppression, leading to formation of a vascular stroma.
Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade.
Authors: Enslen H , Tokumitsu H , Stork P J , Davis R J , Soderling T R
Published: July 2002

Membrane depolarization of NG108 cells gives rapid (< 5 min) activation of Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV), as well as activation of c-Jun N-terminal kinase (JNK). To investigate whether the Ca2+-dependent activation of mitogen-activated protein kinases (ERK, JNK, and p38) might be mediated by the CaM kinase cascade, we have transfected PC12 cells, which lack CaM-KIV, with constitutively active mutants of CaM kinase kinase and/or CaM-KIV (CaM-KKc and CaM-KIVc, respectively). In the absence of depolarization, CaM-KKc transfection had no effect on Elk-dependent transcription of a luciferase reporter gene, whereas CaM-KIVc alone or in combination with CaM-KKc gave 7- to 10-fold and 60- to 80-fold stimulations, respectively, which were blocked by mitogen-activated protein (MAP) kinase phosphatase cotransfection. When epitope-tagged constructs of MAP kinases were co-transfected with CaM-KKc plus CaM-KIVc, the immunoprecipitated MAP kinases were activated 2-fold (ERK-2) and 7- to 10-fold (JNK-1 and p38). The JNK and p38 pathways were further investigated using specific c-Jun or ATF2-dependent transcriptional assays. We found that c-Jun/ATF2-dependent transcriptions were enhanced 7- to 10-fold by CaM-KIVc and 20- to 30-fold by CaM-KKc plus CaM-KIVc. In the case of the Jun-dependent transcription, this effect was not due to direct phosphorylation of c-Jun by activated CaM-KIV, since transcription was blocked by a dominant-negative JNK and by two MAP kinase phosphatases. Mutation of the phosphorylation site (Thr196) in CaM-KIV, which mediates its activation by CaM-KIV kinase, prevented activation of Elk-1, c-Jun, and ATF2 by the CaM kinase cascade. These results establish a new Ca2+-dependent mechanism for regulating MAP kinase pathways and resultant transcription.
Early events in poliovirus infection: virus-receptor interactions.
Authors: Racaniello V R
Published: July 2002

The interaction of poliovirus with its cell receptor initiates conformational changes that lead to uncoating of the viral RNA. Three types of genetic analyses have been used to study the poliovirus-receptor interaction: (i) mutagenesis of the poliovirus receptor (PVR), (ii) selection of viral mutants resistant to neutralization with soluble PVR, and (iii) selection of viral variants adapted to use mutant PVRs. The results of these studies show that a small portion of the first immunoglobulin-like domain of PVR contacts viral residues within a deep depression on the surface of the capsid that encircles the fivefold axis of symmetry. Viral capsid residues that influence the interaction with PVR are also found in locations such as the capsid interior that cannot directly contact PVR. These mutations might influence the ability of the capsid to undergo receptor-mediated conformational transitions that are necessary for high-affinity interactions with PVR.
Use of virion DNA as a cloning vector for the construction of mutant and recombinant herpesviruses.
Authors: Duboise S M , Guo J , Desrosiers R C , Jung J U
Published: July 2002

We have developed improved procedures for the isolation of deletion mutant, point mutant, and recombinant herpesvirus saimiri. These procedures take advantage of the absence of NotI and AscI restriction enzyme sites within the viral genome and use reporter genes for the identification of recombinant viruses. Genes for secreted engineered alkaline phosphatase and green fluorescent protein were placed under simian virus 40 early promoter control and flanked by NotI and AscI restriction sites. When permissive cells were cotransfected with herpesvirus saimiri virion DNA and one of the engineered reporter genes cloned within herpesvirus saimiri sequences, recombinant viruses were readily identified and purified on the basis of expression of the reporter gene. Digestion of recombinant virion DNA with NotI or AscI was used to delete the reporter gene from the recombinant herpesvirus saimiri. Replacement of the reporter gene can be achieved by NotI or AscI digestion of virion DNA and ligation with a terminally matched fragment or, alternatively, by homologous recombination in cotransfected cells. Any gene can, in theory, be cloned directly into the virion DNA when flanked by the appropriate NotI or AscI sites. These procedures should be widely applicable in their general form to most or all herpesviruses that replicate permissively in cultured cells.
Reactive oxygen species are downstream mediators of p53-dependent apoptosis.
Authors: Johnson T M , Yu Z X , Ferrans V J , Lowenstein R A , Finkel T
Published: July 2002

Reactive oxygen species (ROS) have been implicated as potential modulators of apoptosis. Conversely, experiments under hypoxic conditions have suggested that apoptosis could occur in the absence of ROS. We sought to determine whether a central modulator of apoptosis, p53, regulates the levels of intracellular ROS and whether a rise in ROS levels is required for the induction of p53-dependent apoptosis. We transiently overexpressed wild-type p53, using adenoviral gene transfer, and identified cell types that were sensitive or resistant to p53-mediated apoptosis. Cells sensitive to p53-mediated apoptosis produced ROS concomitantly with p53 overexpression, whereas cells resistant to p53 failed to produce ROS. In sensitive cells, both ROS production and apoptosis were inhibited by antioxidant treatment. These results suggest that p53 acts to regulate the intracellular redox state and induces apoptosis by a pathway that is dependent on ROS production.
An economic analysis of unilateral refusals to license intellectual  property
Authors: Gilbert Richard J. , Shapiro Carl
Published: July 2002

The intellectual property laws in the United States provide the
owners of intellectual property with discretion to license the right to
use that property or to make or sell products that embody the
intellectual property. However, the antitrust laws constrain the use of
property, including intellectual property, by a firm with market power
and may place limitations on the licensing of intellectual property.
This paper focuses on one aspect of antitrust law, the so-called
“essential facilities doctrine,” which may impose a duty upon
firms controlling an “essential facility” to make that facility
available to their rivals. In the intellectual property context, an
obligation to make property available is equivalent to a requirement
for compulsory licensing. Compulsory licensing may embrace the
requirement that the owner of software permit access to the underlying
code so that others can develop compatible application programs.
Compulsory licensing may undermine incentives for research and
development by reducing the value of an innovation to the inventor.
This paper shows that compulsory licensing also may reduce economic
efficiency in the short run by facilitating the entry of inefficient
producers and by promoting licensing arrangements that result in higher
prices.
Novel dimeric interface and electrostatic recognition in bacterial Cu,Zn superoxide dismutase
Authors: Bourne Yves , Redford Susan M. , Steinman Howard M. , Lepock James R. , Tainer John A. , Getzoff Elizabeth D.
Published: July 2002

Eukaryotic Cu,Zn superoxide dismutases (CuZnSODs) are antioxidant
enzymes remarkable for their unusually stable β-barrel fold and dimer
assembly, diffusion-limited catalysis, and electrostatic guidance of
their free radical substrate. Point mutations of CuZnSOD cause the
fatal human neurodegenerative disease amyotrophic lateral sclerosis. We
determined and analyzed the first crystallographic structure (to our
knowledge) for CuZnSOD from a prokaryote,
Photobacterium
leiognathi
, a luminescent symbiont of Leiognathid fish. This
structure, exemplifying prokaryotic CuZnSODs, shares the active-site
ligand geometry and the topology of the Greek key β-barrel common to
the eukaryotic CuZnSODs. However, the β-barrel elements recruited to
form the dimer interface, the strategy used to forge the channel for
electrostatic recognition of superoxide radical, and the connectivity
of the intrasubunit disulfide bond in
P. leiognathi
CuZnSOD are discrete and strikingly dissimilar from those highly
conserved in eukaryotic CuZnSODs. This new CuZnSOD structure broadens
our understanding of structural features necessary and sufficient for
CuZnSOD activity, highlights a hitherto unrecognized adaptability of
the Greek key β-barrel building block in evolution, and reveals that
prokaryotic and eukaryotic enzymes diverged from one primordial CuZnSOD
and then converged to distinct dimeric enzymes with electrostatic
substrate guidance.
Mössbauer and electron paramagnetic resonance studies of chloroperoxidase following mechanism-based inactivation with allylbenzene
Authors: Debrunner Peter G. , Dexter Annette F. , Schulz Charles E. , Xia Yao-Min , Hager Lowell P.
Published: July 2002

We have used Mössbauer and electron paramagnetic
resonance (EPR) spectroscopy to study a heme-
N
-alkylated
derivative of chloroperoxidase (CPO) prepared by mechanism-based
inactivation with allylbenzene and hydrogen peroxide. The freshly
prepared inactivated enzyme (“green CPO”) displayed a nearly pure
low-spin ferric EPR signal with
g
= 1.94, 2.15,
2.31. The Mössbauer spectrum of the same species recorded at 4.2
K showed magnetic hyperfine splittings, which could be simulated in
terms of a spin Hamiltonian with a complete set of hyperfine parameters
in the slow spin fluctuation limit. The EPR spectrum of green CPO was
simulated using a three-term crystal field model including g-strain.
The best-fit parameters implied a very strong octahedral field in which
the three
2
T
2
levels of the (3d)
5
configuration in green CPO were lowest in energy, followed by a
quartet. In native CPO, the
6
A
1
states follow
the
2
T
2
ground state doublet. The
alkene-mediated inactivation of CPO is spontaneously reversible.
Warming of a sample of green CPO to 22°C for increasing times before
freezing revealed slow conversion of the novel EPR species to two
further spin
S
= ½ ferric species. One of
these species displayed
g
= 1.82, 2.25, 2.60
indistinguishable from native CPO. By subtracting spectral components
due to native and green CPO, a third species with
g
= 1.86, 2.24, 2.50 could be generated. The EPR spectrum of this
“quasi-native CPO,” which appears at intermediate times during
the reactivation, was simulated using best-fit parameters similar to
those used for native CPO.
Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells
Authors: Cai Jinsong , Uhlmann Frank , Gibbs Emma , Flores-Rozas Hernan , Lee Chee-Gun , Phillips Barbara , Finkelstein Jeff , Yao Nina , O’Donnell Michael , Hurwitz Jerard
Published: July 2002

Human replication factor C (RFC, also called activator 1) is a
five-subunit protein complex (p140, p40, p38, p37, and p36) required
for proliferating cell nuclear antigen (PCNA)-dependent processive DNA
synthesis catalyzed by DNA polymerase δ or ɛ. Here we report the
reconstitution of the RFC complex from its five subunits simultaneously
overexpressed in baculovirus-infected insect cells. The purified
baculovirus-produced RFC appears to contain equimolar levels of each
subunit and was shown to be functionally identical to its native
counterpart in (
i
) supporting DNA polymerase
δ-catalyzed PCNA-dependent DNA chain elongation; (
ii
)
catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and
human single-stranded DNA binding protein; (
iii
) binding
preferentially to DNA primer ends; and (
iv
)
catalytically loading PCNA onto singly nicked circular DNA and
catalytically removing PCNA from these DNA molecules.
Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living
Escherichia coli
cells by using green fluorescent protein
Authors: Ma Xiaolan , Ehrhardt David W. , Margolin William
Published: July 2002

In the current model for bacterial cell division, FtsZ protein
forms a ring that marks the division plane, creating a cytoskeletal
framework for the subsequent action of other proteins such as FtsA.
This putative protein complex ultimately generates the division septum.
Herein we report that FtsZ and FtsA proteins tagged with green
fluorescent protein (GFP) colocalize to division-site ring-like
structures in living bacterial cells in a visible space between the
segregated nucleoids. Cells with higher levels of FtsZ–GFP or with
FtsA–GFP plus excess wild-type FtsZ were inhibited for cell division
and often exhibited bright fluorescent spiral tubules that spanned the
length of the filamentous cells. This suggests that FtsZ may switch
from a septation-competent localized ring to an unlocalized spiral
under some conditions and that FtsA can bind to FtsZ in both
conformations. FtsZ–GFP also formed nonproductive but localized
aggregates at a higher concentration that could represent FtsZ
nucleation sites. The general domain structure of FtsZ–GFP resembles
that of tubulin, since the C terminus of FtsZ is not required for
polymerization but may regulate polymerization state. The N-terminal
portion of
Rhizobium
FtsZ polymerized in
Escherichia coli
and appeared to copolymerize with
E. coli
FtsZ, suggesting a degree of interspecies
functional conservation. Analysis of several deletions of FtsA–GFP
suggests that multiple segments of FtsA are important for its
localization to the FtsZ ring.
Pleiotropic defects in ataxia-telangiectasia protein-deficient mice
Authors: Elson Ari , Wang Yaoqi , Daugherty Cathie J. , Morton Cynthia C. , Zhou Fen , Campos-Torres Juanita , Leder Philip
Published: July 2002

We have generated a mouse model for ataxia-telangiectasia by using
gene targeting to generate mice that do not express the Atm protein.
Atm-deficient mice are retarded in growth, do not produce mature sperm,
and exhibit severe defects in T cell maturation while going on to
develop thymomas. Atm-deficient fibroblasts grow poorly in culture and
display a high level of double-stranded chromosome breaks.
Atm-deficient thymocytes undergo spontaneous apoptosis
in vitro
significantly more than controls. Atm-deficient
mice then exhibit many of the same symptoms found in
ataxia-telangiectasia patients and in cells derived from them.
Furthermore, we demonstrate that the Atm protein exists as two discrete
molecular species, and that loss of one or of both of these can lead to
the development of the disease.
Absence of the β subunit (
cchb1
) of the skeletal muscle dihydropyridine receptor alters expression of the α
1
subunit and eliminates excitation-contraction coupling
Authors: Gregg Ronald G. , Messing Albee , Strube Caroline , Beurg Maryline , Moss Richard , Behan Mary , Sukhareva Manana , Haynes Susan , Powell Jeanne A. , Coronado Roberto , Powers Patricia A.
Published: July 2002

The multisubunit (α
1S
, α
2
/δ,
β
1
, and γ) skeletal muscle dihydropyridine receptor
transduces transverse tubule membrane depolarization into release of
Ca
2+
from the sarcoplasmic reticulum, and also acts as an
L-type Ca
2+
channel. The α
1S
subunit contains
the voltage sensor and channel pore, the kinetics of which are modified
by the other subunits. To determine the role of the β
1
subunit in channel activity and excitation-contraction coupling we have
used gene targeting to inactivate the β
1
gene.
β
1
-null mice die at birth from asphyxia. Electrical
stimulation of β
1
-null muscle fails to induce twitches,
however, contractures are induced by caffeine. In isolated
β
1
-null myotubes, action potentials are normal, but fail
to elicit a Ca
2+
transient. L-type Ca
2+
current
is decreased 10- to 20-fold in the β
1
-null cells compared
with littermate controls. Immunohistochemistry of cultured myotubes
shows that not only is the β
1
subunit absent, but the
amount of α
1S
in the membrane also is undetectable. In
contrast, the β
1
subunit is localized appropriately in
dysgenic,
mdg/mdg
, (α
1S
-null) cells.
Therefore, the β
1
subunit may not only play an important
role in the transport/insertion of the α
1S
subunit into
the membrane, but may be vital for the targeting of the muscle
dihydropyridine receptor complex to the transverse
tubule/sarcoplasmic reticulum junction.
Efficient gene transfer by a human immunodeficiency virus type 1 (HIV-1)-derived vector utilizing a stable HIV packaging cell line
Authors: Corbeau Pierre , Kraus Günter , Wong-Staal Flossie
Published: July 2002

By transfecting fibroblast cells with an HIV-1-MN molecular clone
with a deletion of the major packaging sequence, we have developed a
stable HIV-1 packaging cell line, Ψ422. Ψ422 cells form syncytia
with CD4-positive cells, correctly express HIV-1 structural proteins,
and produce a large amount of mature particles with normal reverse
transcriptase activity. Yet these particles, in which RNA was not
detected by reverse transcriptase–PCR, are not infectious. When stably
transfected with an HIV-1-based retroviral vector, the Ψ422 cell line
produces virions capable of transducing CD4-positive cells with high
efficiency (up to 10
5
cells per ml). The availability of
this stable noninfectious HIV-1 packaging cell line capable of
generating high-titer HIV-1 vectors represents a new step toward the
use of an HIV-1 delivery system in gene therapy.
Protein engineering of
Bacillus thuringiensis
δ-endotoxin: Mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae
Authors: Rajamohan Francis , Alzate Oscar , Cotrill Jeffrey A. , Curtiss April , Dean Donald H.
Published: July 2002

Substitutions or deletions of domain II loop residues of
Bacillus thuringiensis
δ-endotoxin CryIAb were
constructed using site-directed mutagenesis techniques to investigate
their functional roles in receptor binding and toxicity toward gypsy
moth (
Lymantria dispar
). Substitution of loop 2 residue
N372 with Ala or Gly (N372A, N372G) increased the toxicity against
gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth
midgut brush border membrane vesicles (BBMV) ≈4-fold. Deletion of
N372 (D3), however, substantially reduced toxicity (>21 times) as well
as binding affinity, suggesting that residue N372 is involved in
receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G
and L283S), has a 36-fold increase in toxicity to gypsy moth neonates
compared with wild-type toxin. The enhanced activity of DF-1 was
correlated with higher binding affinity (18-fold) and binding site
concentrations. Dissociation binding assays suggested that the off-rate
of the BBMV-bound mutant toxins was similar to that of the wild type.
However, DF-1 toxin bound 4 times more than the wild-type and N372A
toxins, and it was directly correlated with binding affinity and
potency. Protein blots of gypsy moth BBMV probed with labeled N372A,
DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating
that the increased activity of the mutants was not caused by binding to
additional receptor(s). The improved binding affinity of N372A and DF-1
suggest that a shorter side chain at these loops may fit the toxin more
efficiently to the binding pockets. These results offer an excellent
model system for engineering δ-endotoxins with higher potency and
wider spectra of target pests by improving receptor binding
interactions.
Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin
Authors: Nagasawa Takashi , Nakajima Toshihiro , Tachibana Kazunobu , Iizasa Hisashi , Bleul Conrad C. , Yoshie Osamu , Matsushima Kouji , Yoshida Nobuaki , Springer Timothy A. , Kishimoto Tadamitsu
Published: July 2002

Pre-B-cell growth-stimulating factor/stromal cell-derived factor
1 (PBSF/SDF-1) is a member of the CXC group of chemokines that is
initially identified as a bone marrow stromal cell-derived factor and
as a pre-B-cell stimulatory factor. Although most chemokines are
thought to be inducible inflammatory mediators, PBSF/SDF-1 is
essential for perinatal viability, B lymphopoiesis, bone marrow
myelopoiesis, and cardiac ventricular septal formation, and it has
chemotactic activities on resting lymphocytes and monocytes. In this
paper, we have isolated a cDNA that encodes a seven
transmembrane-spanning-domain receptor, designated pre-B-cell-derived
chemokine receptor (PB-CKR) from a murine pre-B-cell clone, DW34. The
deduced amino acid sequence has 90% identity with that of a
HUMSTSR/fusin, a human immunodeficiency virus 1 (HIV-1) entry
coreceptor. However, the second extracellular region has lower identity
(67%) compared with HUMSTSR/fusin. PB-CKR is expressed during embryo
genesis and in many organs and T cells of adult mice. Murine
PBSF/SDF-1 induced an increase in intracellular free Ca
2+
in DW34 cells and PB-CKR-transfected Chinese hamster ovary (CHO) cells,
suggesting that PB-CKR is a functional receptor for murine
PBSF/SDF-1. Murine PBSF/SDF-1 also induced Ca
2+
influx
in fusin-transfected CHO cells. On the other hand, considering previous
results that HIV-1 does not enter murine T cells that expressed human
CD4, PB-CKR may not support HIV-1 infection. Thus, PB-CKR will be an
important tool for functional mapping of HIV-1 entry coreceptor fusin
and for understanding the function of PBSF/SDF-1 further.
Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin
Authors: Green Carla B. , Besharse Joseph C.
Published: July 2002

Photoreceptors of the
Xenopus laevis
retina are the
site of a circadian clock. As part of a differential display screen for
rhythmic gene products in this system, we have identified a
photoreceptor-specific mRNA expressed in peak abundance at night. cDNA
cloning revealed an open reading frame encoding a putative 388 amino
acid protein that we have named “nocturnin” (for night-factor).
This protein has strong sequence similarity to the C-terminal domain of
the yeast transcription factor, CCR4, as well as a leucine zipper-like
dimerization motif. Nocturnin mRNA levels exhibit a high amplitude
circadian rhythm and nuclear run-on analysis indicates that it is
controlled by the retinal circadian clock at the level of
transcription. Our observations suggest that nocturnin may function
through protein–protein interaction either as a component of the
circadian clock or as a downstream effector of clock function.
Androgen-repressed phenotype in human prostate cancer
Authors: Zhau Hai Yen E. , Chang Shi-Ming , Chen Bao-Qi , Wang Yunling , Zhang Hongquan , Kao Chinghai , Sang Qingxiang Amy , Pathak Sen J. , Chung Leland W. K.
Published: July 2002

An androgen-repressed human prostate cancer cell line, ARCaP, was
established and characterized. This cell line was derived from the
ascites fluid of a patient with advanced metastatic disease. In
contrast to the behavior of androgen-dependent LNCaP and its
androgen-independent C4-2 subline, androgen and estrogen suppress the
growth of ARCaP cells in a dose-dependent manner
in vivo
and
in vitro
. ARCaP is tumorigenic and highly
metastatic. It metastasizes to the lymph node, lung, pancreas, liver,
kidney, and bone, and forms ascites fluid in athymic hosts. ARCaP cells
express low levels of androgen receptor mRNA and prostate-specific
antigen mRNA and protein. Immunohistochemical staining shows that ARCaP
cells stain intensely for epidermal growth factor receptor,
c-erb
B2/
neu
, and
c-erb
B3. Staining is negative for chromogranin A and positive for bombesin,
serotonin, neuron-specific enolase, and the
c-met
protooncogene (a hepatic growth factor/scatter factor receptor).
ARCaP cells also secrete high levels of gelatinase A and B and some
stromelysin, which suggests that this cell line may contain markers
representing invasive adenocarcinoma with selective neuronendocrine
phenotypes. Along with its repression of growth, androgen is also found
to repress the expression of prostate-specific antigen in ARCaP cells
as detected by a prostate-specific antigen promoter–β-galactosidase
reporter assay. Our results suggest that the androgen-repressed state
may be central to prostate cancer progression and that advanced
prostate cancer can progress from an androgen-independent to an
androgen-repressed state.
Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA–liposome complexes
Authors: Nabel Gary J. , Gordon David , Bishop D. Keith , Nickoloff Brian J. , Yang Zhi-yong , Aruga Atsushi , Cameron Mark J. , Nabel Elizabeth G. , Chang Alfred E.
Published: July 2002

Analysis of the antitumor immune response after gene transfer of a foreign major histocompatibility complex class I protein, HLA-B7, was performed. Ten HLA-B7-negative patients with stage IV melanoma were treated in an effort to stimulate local tumor immunity. Plasmid DNA was detected within treated tumor nodules, and RNA encoding recombinant HLA-B7 or HLA-B7 protein was demonstrated in 9 of 10 patients. T cell migration into treated lesions was observed and tumor-infiltrating lymphocyte reactivity was enhanced in six of seven and two of two patients analyzed, respectively. In contrast, the frequency of cytotoxic T lymphocyte against autologous tumor in circulating peripheral blood lymphocytes was not altered significantly, suggesting that peripheral blood lymphocyte reactivity is not indicative of local tumor responsiveness. Local inhibition of tumor growth was detected after gene transfer in two patients, one of whom showed a partial remission. This patient subsequently received treatment with tumor-infiltrating lymphocytes derived from gene-modified tumor, with a complete regression of residual disease. Thus, gene transfer with DNA–liposome complexes encoding an allogeneic major histocompatibility complex protein stimulated local antitumor immune responses that facilitated the generation of effector cells for immunotherapy of cancer.
Reconstitution of repair-gap UV mutagenesis with purified proteins from Escherichia coli: a role for DNA polymerases III and II.
Authors: Tomer G , Cohen-Fix O , O'Donnell M , Goodman M , Livneh Z
Published: July 2002

Using a cell-free system for UV mutagenesis, we have previously demonstrated the existence of a mutagenic pathway associated with nucleotide-excision repair gaps. Here, we report that this pathway can be reconstituted by using six purified proteins: UvrA, UvrB, UvrC, DNA helicase II, DNA polymerase III core, and DNA ligase. This establishes the minimal requirements for repair-gap UV mutagenesis. DNA polymerase II could replace DNA polymerase III, although less effectively, whereas DNA polymerase I, the major repair polymerase, could not. DNA sequence analysis of mutations generated in the in vitro reaction revealed a spectrum typical of mutations targeted to UV lesions. These observations suggest that repair-gap UV mutagenesis is performed by DNA polymerase III, and to a lesser extent by DNA polymerase II, by filling-in of a rare class of excision gaps that contain UV lesions.
A large-capacity memory system that recognizes the calls and songs of individual birds.
Authors: Chew S J , Vicario D S , Nottebohm F
Published: July 2002

Auditory responses in the caudomedial neostriatum (NCM) of the zebra finch (Taeniopygia guttata) forebrain habituate to repeated presentations of a novel conspecific song. This habituation is long lasting and specific to individual stimuli. We here test the acoustic and ethological basis of this stimulus-specific habituation by recording extracellular multiunit activity in the NCM of awake male and female zebra finches presented with a variety of conspecific and heterospecific vocalizations, white noise, and tones. Initial responses to conspecific song and calls and to human speech were higher than responses to the other stimuli. Immediate habituation rates were high for all novel stimuli except tones, which habituated at a lower rate. Habituation to conspecific calls and songs outlasted habituation to other stimuli. The extent of immediate habituation induced by a particular novel song was not diminished when other conspecific songs were presented in alternation. In addition, the persistence of habituation was not diminished by exposure to other songs before testing, nor was it influenced by gender or laterality. Our results suggest that the NCM is specialized for remembering the calls and songs of many individual conspecifics.
An ATP-dependent As(III)-glutathione transport system in membrane vesicles of Leishmania tarentolae.
Authors: Dey S , Ouellette M , Lightbody J , Papadopoulou B , Rosen B P
Published: July 2002

Membrane preparations enriched in plasma membrane vesicles prepared from promastigotes of Leishmania tarentolae were shown to accumulate thiolate derivatives of 73As(III). Free arsenite was transported at a low rate, but rapid accumulation was observed after reaction with reduced glutathione (GSH) conditions that favor the formation of As(GS)3. Accumulation required ATP but not electrochemical energy, indicating that As(GS)3 is transported by an ATP-coupled pump. Pentostam, a Sb(V)-containing drug that is one of the first-line therapeutic agents for treatment of leishmaniasis, inhibited uptake after reaction with GSH. Vesicles prepared from a strain in which both copies of the pgpA genes were disrupted accumulated As(GS)3 at wild-type levels, demonstrating that the PgpA protein is not the As(GS)3 pump. These results have important implications for the mechanism of drug resistance in the trypanosomatidae, suggesting that a plasma membrane As(GS)3 pump catalyzes active extrusion of metal thiolates, including the Pentostam-glutathione conjugate.
Human myosin VIIA responsible for the Usher 1B syndrome: a predicted membrane-associated motor protein expressed in developing sensory epithelia.
Authors: Weil D , Levy G , Sahly I , Levi-Acobas F , Blanchard S , El-Amraoui A , Crozet F , Philippe H , Abitbol M , Petit C
Published: July 2002

The gene encoding human myosin VIIA is responsible for Usher syndrome type III (USH1B), a disease which associates profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. The reconstituted cDNA sequence presented here predicts a 2215 amino acid protein with a typical unconventional myosin structure. This protein is expected to dimerize into a two-headed molecule. The C terminus of its tail shares homology with the membrane-binding domain of the band 4.1 protein superfamily. The gene consists of 48 coding exons. It encodes several alternatively spliced forms. In situ hybridization analysis in human embryos demonstrates that the myosin VIIA gene is expressed in the pigment epithelium and the photoreceptor cells of the retina, thus indicating that both cell types may be involved in the USH1B retinal degenerative process. In addition, the gene is expressed in the human embryonic cochlear and vestibular neuroepithelia. We suggest that deafness and vestibular dysfunction in USH1B patients result from a defect in the morphogenesis of the inner ear sensory cell stereocilia.
Axotomized neonatal motoneurons overexpressing the bcl2 proto-oncogene retain functional electrophysiological properties.
Authors: Alberi S , Raggenbass M , de Bilbao F , Dubois-Dauphin M
Published: July 2002

Bcl2 overexpression prevents axotomy-induced neuronal death of neonatal facial motoneurons, as defined by morphological criteria. However, the functional properties of these surviving lesioned transgenic neurons are unknown. Using transgenic mice overexpressing the protein Bcl2, we have investigated the bioelectrical properties of transgenic facial motoneurons from 7 to 20 days after neonatal unilateral axotomy using brain-stem slices and whole cell patch-clamp recording. Nonaxotomized facial motoneurons from wild-type and transgenic mice had similar properties; they had an input resistance of 38 +/- 6 M omega and fired repetitively after injection of positive current pulses. When cells were voltage-clamped at or near their resting membrane potential, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartic acid (NMDA), or vasopressin generated sustained inward currents. In transgenic axotomized mice, facial motoneurons could be found located ipsilaterally to the lesion; they had an input resistance of 150 +/- 30 M omega, indicating that they were smaller in size, fired repetitively, and were also responsive to AMPA, NMDA, and vasopressin. Morphological measurements achieved 1 week after the lesion have shown that application of brain-derived neurotrophic factor prevented the reduction in size of axotomized transgenic motoneurons. These data indicate that Bcl2 not only prevents morphological apoptotic death of axotomized neonatal transgenic motoneurons but also permits motoneurons to conserve functional electrophysiological properties.
4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.
Authors: von Manteuffel S R , Gingras A C , Ming X F , Sonenberg N , Thomas G
Published: July 2002

It has previously been argued that the repressor of protein synthesis initiation factor 4E, 4E-BP1, is a direct in vivo target of p42mapk. However, the immunosuppressant rapamycin blocks serum-induced 4E-BP1 phosphorylation and, in parallel, p70s6k activation, with no apparent effect on p42mapk activation. Consistent with this finding, the kinetics of serum-induced 4E-BP1 phosphorylation closely follow those of p70s6k activation rather than those of p42mapk. More striking, insulin, which does not induce p42mapk activation in human 293 cells or Swiss mouse 3T3 cells, induces 4E-BP1 phosphorylation and p70s6k activation in both cell types. Anisomycin, which, like insulin, does not activate p42mapk, promotes a small parallel increase in 4E-BP1 phosphorylation and p70s6k activation. The insulin effect on 4E-BP1 phosphorylation and p70s6k activation in both cell types is blocked by SQ20006, wortmannin, and rapamycin. These three inhibitors have no effect on p42mapk activation induced by phorbol 12-tetradecanoate 13-acetate, though wortmannin partially suppresses both the p70s6k response and the 4E-BP1 response. Finally, in porcine aortic endothelial cells stably transfected with either the wild-type platelet-derived growth factor receptor or a mutant receptor bearing the double point mutation 740F/751F, p42mapk activation in response to platelet-derived growth factor is unimpaired, but increased 4E-BP1 phosphorylation is ablated, as previously reported for p70s6k. The data presented here demonstrate that 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.