bluuebunny/crossref_metadata_2025 huggingface dataset:
Human cytomegalovirus latent gene expression in granulocyte-macrophage progenitors in culture and in seropositive individuals.
Authors: Kondo K , Xu J , Mocarski E S
Published: July 2002

Following infection with cytomegalovirus, human granulocyte-macrophage progenitors carry the viral genome but fail to support productive replication. Viral transcripts arise from a region encompassing the major regulatory gene locus; however, their structure differs significantly from productive phase transcripts. One class, sense transcripts, is encoded in the same direction as productive phase transcripts but uses two novel start sites in the ie1/ie2 promoter/enhancer region. These transcripts have the potential to encode a novel 94 aa protein. The other class, antisense transcript, is unspliced and complimentary to ie1 exons 2-4, and has the potential to encode novel 154 and 152 aa proteins. Consistent with a role in latency, these transcripts are present in bone marrow aspirates from naturally infected, healthy seropositive donors but are not present in seronegative controls. Sense latent transcripts are present in a majority of seropositive individuals. Consistent with the expression of latent transcripts, antibody to the 94 aa and 152 aa proteins is detectable in the serum of seropositive individuals. Thus, latent infection by cytomegalovirus is accompanied by the presence of latency-associated transcripts and expression of immunogenic proteins. Overall, these results suggest that bone marrow-derived myeloid progenitors are an important natural site of viral latency.
Applications of pox virus vectors to vaccination: an update.
Authors: Paoletti E
Published: July 2002

Recombinant pox viruses have been generated for vaccination against heterologous pathogens. Amongst these, the following are notable examples. (i) The engineering of the Copenhagen strain of vaccinia virus to express the rabies virus glycoprotein. When applied in baits, this recombinant has been shown to vaccinate the red fox in Europe and raccoons in the United States, stemming the spread of rabies virus infection in the wild. (ii) A fowlpox-based recombinant expressing the Newcastle disease virus fusion and hemagglutinin glycoproteins has been shown to protect commercial broiler chickens for their lifetime when the vaccine was administered at 1 day of age, even in the presence of maternal immunity against either the Newcastle disease virus or the pox vector. (iii) Recombinants of canarypox virus, which is restricted for replication to avian species, have provided protection against rabies virus challenge in cats and dogs, against canine distemper virus, feline leukemia virus, and equine influenza virus disease. In humans, canarypox virus-based recombinants expressing antigens from rabies virus, Japanese encephalitis virus, and HIV have been shown to be safe and immunogenic. (iv) A highly attenuated vaccinia derivative, NYVAC, has been engineered to express antigens from both animal and human pathogens. Safety and immunogenicity of NYVAC-based recombinants expressing the rabies virus glycoprotein, a polyprotein from Japanese encephalitis virus, and seven antigens from Plasmodium falciparum have been demonstrated to be safe and immunogenic in early human vaccine studies.
The cell surface metalloprotease/disintegrin Kuzbanian is required for axonal extension in
Drosophila
Authors: Fambrough Douglas , Pan Duojia , Rubin Gerald M. , Goodman Corey S.
Published: July 2002

It has long been suspected that proteolytic activity associated
with advancing growth cones may be required for axon extension. We have
isolated mutations in the
kuzbanian
(
kuz
) gene,
which is expressed in the nervous system and encodes a putative zinc
metalloprotease with a disintegrin domain.
Drosophila
embryos with loss-of-function mutations in
kuz
have dramatic defects in the development of central
nervous system axon pathways, with many axons stalling and failing to
extend through the nerve cord. This phenotype is rescued by panneural
expression of
kuz
mRNA in the embryo. These results show
that the Kuz metalloprotease is required for axon extension,
suggesting a requirement for proteolytic activity at the growth cone
surface.
Effects of productivity, consumers, competitors, and El Niño events on food chain patterns in a rocky intertidal community
Authors: Wootton J. T. , Power M. E. , Paine R. T. , Pfister C. A.
Published: July 2002

We experimentally manipulated nutrient input to a rocky intertidal
community, using nutrient-diffusing flowerpots, to determine
(
i
) whether nutrients limited intertidal productivity,
(
ii
) how a large-scale oceanographic disturbance (an El
Niño event) affected patterns of nutrient limitation,
(
iii
) the relative impacts of molluscan grazers and
nutrient limitation, and (
iv
) if responses to
experimental nutrient addition among trophic levels were more
consistent with prey-dependent or ratio-dependent food chain models.
Nutrients measurably increased the abundance of micrograzers (amphipods
and chironomid larvae), but not algal biomass, during the summer of an
El Niño year. Nutrients had no effects in two non-El Niño
years and during the autumn of an El Niño year. Adding nutrients
did not affect food chain stability as assessed by temporal variation
in algal biomass and micrograzer abundance. Large molluscan grazers
caused large reductions in micrograzers and smaller reductions in
algae, indicating consistent consumer effects. The results demonstrate
that in this intertidal community, nutrient limitation can occur under
conditions of nutrient stress, that top-down grazing effects are
typically stronger than bottom-up nutrient effects, and that
prey-dependent models are more appropriate than ratio-dependent models.
Cytoplasmically sequestered wild-type p53 protein in neuroblastoma is relocated to the nucleus by a C-terminal peptide
Authors: Ostermeyer Anne G. , Runko Erik , Winkfield Blonka , Ahn Brand , Moll Ute M.
Published: July 2002

Cytoplasmic sequestration of wild-type p53 protein occurs in a
subset of primary human tumors including breast cancer, colon cancer,
and neuroblastoma (NB). The sequestered p53 localizes to punctate
cytoplasmic structures that represent large protein aggregates. One
functional consequence of this blocked nuclear access is impairment of
the p53-mediated G
1
checkpoint after DNA damage. Here we
show that cytoplasmic p53 from NB cells is incompetent for specific DNA
binding, probably due to its sequestration. Importantly, the C-terminal
domain of sequestered p53 is masked, as indicated by the failure of a
C-terminally directed antibody to detect p53 in these structures. To
determine (
i
) which domain of p53 is involved in the
aggregation and (
ii
) whether this phenotype is
potentially reversible, we generated stable NB sublines that coexpress
the soluble C-terminal mouse p53 peptide DD1 (amino acids 302–390). A
dramatic phenotypic reversion occurred in five of five lines. The
presence of DD1 blocked the sequestration of wild-type p53 and
relocated it to the nucleus, where it accumulated. The nuclear
translocation is due to shuttling of wild-type p53 by
heteroligomerization to DD1, as shown by coimmunoprecipitation. As
expected, the nuclear heterocomplexes were functionally inactive, since
DD1 is a dominant negative inhibitor of wild-type p53. In summary, we
show that nuclear access of p53 can be restored in NB cells.
A regenerative link in the ionic fluxes through the
weaver
potassium channel underlies the pathophysiology of the mutation
Authors: Silverman Scott K. , Kofuji Paulo , Dougherty Dennis A. , Davidson Norman , Lester Henry A.
Published: July 2002

The homozygous
weaver
mouse displays neuronal
degeneration in several brain regions. Previous experiments in
heterologous expression systems showed that the G protein-gated inward
rectifier K
+
channel (GIRK2) bearing the
weaver
pore-region GYG-to-SYG mutation (i) is not
activated by G
βγ
subunits, but instead shows
constitutive activation, and (ii) is no longer a
K
+
-selective channel but conducts Na
+
as well.
The present experiments on
weaver
GIRK2
(
wv
GIRK2) expressed in
Xenopus
oocytes
show that the level of constitutive activation depends on intracellular
Na
+
concentration. In particular, manipulations that
decrease intracellular Na
+
produce a component of
Na
+
-permeable current activated via a G protein pathway.
Therefore, constitutive activation may not arise because the
weaver
mutation directly alters the gating transitions
of the channel protein. Instead, there may be a regenerative cycle of
Na
+
influx through the
wv
GIRK2 channel,
leading to additional Na
+
activation. We also show that the
wv
GIRK2 channel is permeable to Ca
2+
,
providing an additional mechanism for the degeneration that
characterizes the
weaver
phenotype. We further
demonstrate that the GIRK4 channel bearing the analogous
weaver
mutation has properties similar to those of the
wv
GIRK2 channel, providing a glimpse of the selective
pressures that have maintained the GYG sequence in nearly all known
K
+
channels.
A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells.
Authors: Andersen P S , Stryhn A , Hansen B E , Fugger L , Engberg J , Buus S
Published: July 2002

Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.
Nonlinear control of heart rate variability in human infants.
Authors: Sugihara G , Allan W , Sobel D , Allan K D
Published: July 2002

Nonlinear analyses of infant heart rhythms reveal a marked rise in the complexity of the electrocardiogram with maturation. We find that normal mature infants (gestation greater than or equal to 35 weeks) have complex and distinctly nonlinear heart rhythms (consistent with recent reports for healthy adults) but that such nonlinearity is lacking in preterm infants (gestation > or = to 27 weeks) where parasympathetic-sympathetic interaction and function are presumed to be less well developed. Our study further shows that infants with clinical brain death and those treated with atropine exhibit a similar lack of nonlinear feedback control. These three lines of evidence support the hypothesis championed by Goldberger et al. [Goldberger, A.L., Rigney, D.R. & West, B.J. (1990) Sci. Am. 262, 43-49] that autonomic nervous system control underlies the nonlinearity and possible chaos of normal heart rhythms. This report demonstrates the acquisition of nonlinear heart rate dynamics and possible chaos in developing human infants and its loss in brain death and with the administration of atropine. It parallels earlier work documenting changes in the variability of heart rhythms in each of these cases and suggests that nonlinearity may provide additional power in characterizing physiological states.
The genomic and physical organization of Ty1-copia-like sequences as a component of large genomes in Pinus elliottii var. elliottii and other gymnosperms.
Authors: Kamm A , Doudrick R L , Heslop-Harrison J S , Schmidt T
Published: July 2002

A DNA sequence, TPE1, representing the internal domain of a Ty1-copia retroelement, was isolated from genomic DNA of Pinus elliottii Engelm. var. elliottii (slash pine). Genomic Southern analysis showed that this sequence, carrying partial reverse transcriptase and integrase gene sequences, is highly amplified within the genome of slash pine and part of a dispersed element >4.8 kbp. Fluorescent in situ hybridization to metaphase chromosomes shows that the element is relatively uniformly dispersed over all 12 chromosome pairs and is highly abundant in the genome. It is largely excluded from centromeric regions and intercalary chromosomal sites representing the 18S-5.8S-25S rRNA genes. Southern hybridization with specific DNA probes for the reverse transcriptase gene shows that TPE1 represents a large subgroup of heterogeneous Ty1-copia retrotransposons in Pinus species. Because no TPE1 transcription could be detected, it is most likely an inactive element--at least in needle tissue. Further evidence for inactivity was found in recombinant reverse transcriptase and integrase sequences. The distribution of TPE1 within different gymnosperms that contain Ty1-copia group retrotransposons, as shown by a PCR assay, was investigated by Southern hybridization. The TPE1 family is highly amplified and conserved in all Pinus species analyzed, showing a similar genomic organization in the three- and five-needle pine species investigated. It is also present in spruce, bald cypress (swamp cypress), and in gingko but in fewer copies and a different genomic organization.
Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene.
Authors: Deconinck N , Ragot T , Maréchal G , Perricaudet M , Gillis J M
Published: July 2002

Fast skeletal muscles of mdx (X chromosome-linked muscular dystrophy) mice were injected after birth with a recombinant adenovirus containing a minidys- trophin gene, a 6.3-kbp cDNA coding for the N- and C-terminal ends of dystrophin. Adult muscles were challenged by forced lengthening during tetanic contractions. Stretch-induced mechanical and histological damages were much reduced in injected muscles, in direct proportion of the Miniber of fibers expressing minidystrophin. Damaged fibers were preferentially found among minidystrophin-negative regions. Minidystrostrophin confers an important functional and structural protection of limb muscles against high mechanical stress, even after a partial somatic gene transfer.
Nitric oxide synthase content of hypothalamic explants: increase by norepinephrine and inactivated by NO and cGMP.
Authors: Canteros G , Rettori V , Genaro A , Suburo A , Gimeno M , McCann S M
Published: July 2002

Release of luteinizing hormone (LH)-releasing hormone (LHRH), the hypothalamic peptide that controls release of LH from the adenohypophysis, is controlled by NO. There is a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers in the lateral median eminence, intermingled with terminals of the LHRH neurons. To study relations between NOS and LHRH in this brain region, we measured NOS activity in incubated medial basal hypothalamus (MBH). NOS converts [14C]arginine to equimolar quantities of [14C]citrulline plus NO, which rapidly decomposes. The [14C]citrulline serves as an index of the NO produced. NOS basal activity was suppressed by incubation of the tissue with an inhibitor of NOS, nitroarginine methyl ester (NAME) (10(-5) M). Furthermore, incubation of MBH explants for 30 min with norepinephrine (NE) increased NOS activity and the increase was prevented by prazosine (10(-5) M), an alpha 1-adrenergic receptor blocker; however, direct addition of NE to the tissue homogenate or to a preparation of MBH synaptosomes did not alter enzyme activity, which suggested that NE increased the content of NOS during incubation with the tissue. After purification of NOS, the increase in enzyme content induced by NE was still measurable. This indicates that within 30 min NE increased the synthesis of NOS in vitro. Incubation of MBH or the MBH homogenate with various concentrations of sodium nitroprusside (NP), a releaser of NO, reduced NOS activity at high concentrations (> or = 0.9 mM), which were associated with either a reduction of stimulation or a plateau of LHRH release. Finally, incubation of either MBH or the homogenate with cGMP, a major mediatior of NO action, at concentrations that increased LHRH release also reduced NOS activity. These results indicate that NO at high concentrations can inactivate NOS and that cGMP can also inhibit the enzyme directly. Therefore, the increased NOS activity induced by activation of alpha 1 receptors by NE is inhibited by NO itself and a principal product of its activity, cGMP, providing negative feedback on NOS. In central nervous system (CNS) infections with high concentrations of inducible NOS produced by glial elements, the high concentrations of NO and cGMP produced may suppress LHRH release, resulting in decreased gonadotropin and gonadal steroid release.
An
Arabidopsis
photolyase mutant is hypersensitive to ultraviolet-B radiation
Authors: Landry Laurie G. , Stapleton Ann E. , Lim Jackie , Hoffman Peter , Hays John B. , Walbot Virginia , Last Robert L.
Published: July 2002

Photolyases are DNA repair enzymes that use energy from blue light to repair pyrimidine dimers. We report the isolation of an
Arabidopsis thaliana
mutant (
uvr2-1
) that is defective in photorepair of cyclobutylpyrimidine dimers (CPDs). Whereas
uvr2-1
is indistinguishable from wild type in the absence of UV light, low UV-B levels inhibit growth and cause leaf necrosis.
uvr2-1
is more sensitive to UV-B than wild type when placed under white light after UV-B treatment. In contrast, recovery in darkness or in light lacking photoreactivating blue light results in equal injury in
uvr2-1
and wild type. The
uvr2-1
mutant is unable to remove CPDs
in vivo
, and plant extracts lack detectable photolyase activity. This recessive mutation segregates as a single gene located near the top of chromosome 1, and is a structural gene mutation in the type II CPD photolyase
PHR1
. This mutant provides evidence that CPD photolyase is required for plant survival in the presence of UV-B light.
Peptidyl prolyl cis-trans isomerase activity of cyclophilin A in functional homo-oligomeric receptor expression
Authors: Helekar Santosh A. , Patrick Jim
Published: July 2002

The functional expression of homo-oligomeric α7 neuronal nicotinic and type 3 serotonin receptors is dependent on the activity of a cyclophilin. In this paper we demonstrate that the mechanism of cyclophilin action during functional homo-oligomeric receptor expression in
Xenopus
oocytes is distinct from the calcineurin-dependent immunosuppressive mechanism by showing that a nonimmunosuppressive analog of cyclosporin A (CsA), SDZ 211–811, reduces functional receptor expression to the same extent as CsA. The cytoplasmic subtype of cyclophilin, cyclophilin A (CyPA), appears to be required for functional receptor expression. This is because overexpression of CyPA and a CyPA mutant that is deficient in CsA binding activity reverses CsA-induced reduction in functional receptor expression. The mechanism of action of CyPA is likely to involve its prolyl isomerase activity because a mutant CyPA with a single amino acid substitution (arginine 55 to alanine) that is predicted to produce a 1000-fold attenuation in isomerase activity fails to reverse the cyclosporin A effect. Our data also suggest that CyPA does not form a stable complex with receptor subunits.
Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus
Coccidioides immitis
Authors: Koufopanou Vassiliki , Burt Austin , Taylor John W.
Published: July 2002

Simple cladogenetic theory suggests that gene genealogies can be used to detect mixis in a population and delineate reproductively isolated groups within sexual taxa. We have taken this approach in a study of
Coccidioides immitis
, an ascomycete fungus responsible for a recent epidemic of coccidioidomycosis (Valley fever) in California. To test whether this fungus represents a single sexual species throughout its entire geographic range, we have compared genealogies from fragments of five nuclear genes. The five genealogies show multiple incompatibilities indicative of sex, but also share a branch that partitions the isolates into two reproductively isolated taxa, one centered in California and the other outside California. We conclude that coccidioidomycosis can be caused by two distinct noninterbreeding taxa. This result should aid the future study of the disease and illustrates the utility of the genealogical approach in population genetics.
Na,K-ATPase subunit isoforms in human reticulocytes: Evidence from reverse transcription–PCR for the presence of α1, α3, β2, β3, and γ
Authors: Stengelin Martin K. , Hoffman Joseph F.
Published: July 2002

The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium efflux mode between the human red blood cell Na,K-ATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoform-specific primers were used. The α1 and α3 isoforms of the α subunit, and the β2 and β3 isoforms of the β subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found β2 but not β1 in reticulocyte single-stranded cDNA, and β1 but not β2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that α1, α2, and α3 as well as all three β isoforms were present. The extent to which the kinetic properties of uncoupled sodium efflux might depend on different isoform combinations is not yet known.
Casein kinase II is a selective target of HIV-1 transcriptional inhibitors
Authors: Critchfield J. William , Coligan John E. , Folks Thomas M. , Butera Salvatore T.
Published: July 2002

The identification of cellular factors that are required to complete various steps of the HIV-1 life cycle may lead to the development of new therapeutics. One key step, transcription from the integrated provirus, is inhibited by members of two distinct classes of compounds, the flavonoids and the benzothiophenes, via an unknown mechanism, possibly involving a cellular factor. A marked specificity toward inhibiting HIV-1 transcription is evidenced by the ability of drug-treated cells to retain their proliferative and differentiation capabilities. In addition, the compounds do not impede the activation and function of the transcriptional factor NF-κB. Here we report on the identification of several cellular proteins that mediate the HIV-1 transcriptional inhibitory property of the flavonoid chrysin. Chemical and immunologic analyses identified these cellular proteins as the individual subunits of casein kinase II (CKII). Though structurally unrelated to chrysin, an HIV-1 inhibitory benzothiophene also bound selectively to CKII. Both chrysin and the benzothiophenes inhibited human recombinant CKII enzymatic activity and showed competitive kinetics with respect to ATP, analogous to the classic CKII inhibitor 5,6-dichloro-1-β-
d
-ribofuranosylbenzimidazole (DRB). Moreover, DRB potently inhibited HIV-1 expression in chronically infected cells. CKII may regulate HIV-1 transcription by phosphorylating cellular proteins involved in HIV-1 transactivation that contain multiple CKII phosphorylation consensus sequences.
The product of a thyroid hormone-responsive gene interacts with thyroid hormone receptors
Authors: Thompson Catherine C. , Bottcher Margaret C.
Published: July 2002

Thyroid hormone is a critical mediator of central nervous system (CNS) development, acting through nuclear receptors to modulate the expression of specific genes. Transcription of the rat
hairless
(
hr
) gene is highly up-regulated by thyroid hormone in the developing CNS; we show here that
hr
is directly induced by thyroid hormone. By identifying proteins that interact with the
hr
gene product (Hr), we find that Hr interacts directly and specifically with thyroid hormone receptor (TR)—the same protein that regulates its expression. Unlike previously described receptor-interacting factors, Hr associates with TR and not with retinoic acid receptors (RAR, RXR). Hr can act as a transcriptional repressor, suggesting that its interaction with TR is part of a novel autoregulatory mechanism.
Kinase domain of the muscle-specific receptor tyrosine kinase (MuSK) is sufficient for phosphorylation but not clustering of acetylcholine receptors: Required role for the MuSK ectodomain?
Authors: Glass David J. , Apel Elizabeth D. , Shah Sonal , Bowen David C. , DeChiara Thomas M. , Stitt Trevor N. , Sanes Joshua R. , Yancopoulos George D.
Published: July 2002

Formation of the neuromuscular junction (NMJ) depends upon a nerve-derived protein, agrin, acting by means of a muscle-specific receptor tyrosine kinase, MuSK, as well as a required accessory receptor protein known as MASC. We report that MuSK does not merely play a structural role by demonstrating that MuSK kinase activity is required for inducing acetylcholine receptor (AChR) clustering. We also show that MuSK is necessary, and that MuSK kinase domain activation is sufficient, to mediate a key early event in NMJ formation—phosphorylation of the AChR. However, MuSK kinase domain activation and the resulting AChR phosphorylation are not sufficient for AChR clustering; thus we show that the MuSK ectodomain is also required. These results indicate that AChR phosphorylation is not the sole trigger of the clustering process. Moreover, our results suggest that, unlike the ectodomain of all other receptor tyrosine kinases, the MuSK ectodomain plays a required role in addition to simply mediating ligand binding and receptor dimerization, perhaps by helping to recruit NMJ components to a MuSK-based scaffold.
Loss of protein kinase C inhibition in the β-T594M variant of the amiloride-sensitive Na
+
channel
Authors: Cui Yong , Su Yan Ru , Rutkowski Mark , Reif Max , Menon Anil G. , Pun R. Y. K.
Published: July 2002

We previously reported the presence of a novel variant (β-T594M) of the amiloride-sensitive Na
+
channel (ASSC) in which the threonine residue at position 594 in the β-subunit has been replaced by a methionine residue. Electrophysiological studies of the ASSC on Epstein–Barr virus (EBV)-transformed lymphocytes carrying this variant showed that the 8-(4-chlorophenylthio) adenosine 3′:5′-cyclic monophosphate (8cpt-cAMP)-induced responses were enhanced when compared to wild-type EBV-transformed lymphocytes. Furthermore, in wild-type EBV-transformed cells, the 8cpt-cAMP-induced response was totally blocked by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This inhibitory effect of PMA was blocked by a protein kinase C inhibitor, chelerythrine. We now have identified individuals who are homozygous for this variant, and showed that PMA had no effect on the 8cpt-cAMP-induced responses in the EBV-transformed lymphocytes from such individuals. Cells heterozygous for this variant showed mixed responses to PMA, with the majority of cells partially inhibited by PMA. Our results demonstrate that an alteration in a single amino acid residue in the β-subunit of the ASSC can lead to a total loss of inhibition to PMA, and establish the β-subunit as having an important role in conferring a regulatory effect on the ASSC of lymphocytes.
Membrane specific mapping and colocalization of malarial and host skeletal proteins in the
Plasmodium falciparum
infected erythrocyte by dual-color near-field scanning optical microscopy
Authors: Enderle Th. , Ha T. , Ogletree D. F. , Chemla D. S. , Magowan C. , Weiss S.
Published: July 2002

Accurate localization of proteins within the substructure of cells and cellular organelles enables better understanding of structure–function relationships, including elucidation of protein–protein interactions. We describe the use of a near-field scanning optical microscope (NSOM) to simultaneously map and detect colocalized proteins within a cell, with superresolution. The system we elected to study was that of human red blood cells invaded by the human malaria parasite
Plasmodium falciparum
. During intraerythrocytic growth, the parasite expresses proteins that are transported to the erythrocyte cell membrane. Association of parasite proteins with host skeletal proteins leads to modification of the erythrocyte membrane. We report on colocalization studies of parasite proteins with an erythrocyte skeletal protein. Host and parasite proteins were selectively labeled in indirect immunofluorescence antibody assays. Simultaneous dual-color excitation and detection with NSOM provided fluorescence maps together with topography of the cell membrane with subwavelength (100 nm) resolution. Colocalization studies with laser scanning confocal microscopy provided lower resolution (310 nm) fluorescence maps of cross sections through the cell. Because the two excitation colors shared the exact same near-field aperture, the two fluorescence images were acquired in perfect, pixel-by-pixel registry, free from chromatic aberrations, which contaminate laser scanning confocal microscopy measurements. Colocalization studies of the protein pairs of mature parasite-infected erythrocyte surface antigen (MESA)(parasite)/protein4.1(host) and
P. falciparum
histidine rich protein (PfHRP1)(parasite)/protein4.1(host) showed good real-space correlation for the MESA/protein4.1 pair, but relatively poor correlation for the PfHRP1/protein4.1 pair. These data imply that NSOM provides high resolution information on
in situ
interactions between proteins in biological membranes. This method of detecting colocalization of proteins in cellular structures may have general applicability in many areas of current biological research.
MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclX
L
and initiates cell death
Authors: Han David K. M. , Chaudhary Preet M. , Wright Michael E. , Friedman Cynthia , Trask Barbara J. , Riedel Rodney T. , Baskin Dale G. , Schwartz Stephen M. , Hood Leroy
Published: July 2002

Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems. We have isolated a gene, termed
MRIT
, that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclX
L
and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.
Interferon β transduction of peripheral blood lymphocytes from HIV-infected donors increases Th1-type cytokine production and improves the proliferative response to recall antigens
Authors: Vieillard Vincent , Cremer Isabelle , Lauret Evelyne , Rozenbaum Willy , Debré Patrice , Autran Brigitte , De Maeyer Edward
Published: July 2002

We are developing a gene therapy method of HIV infection based on the constitutive low production of interferon (IFN) β. Peripheral blood lymphocytes (PBL) from HIV-infected patients at different clinical stages of infection were efficiently transduced with the HMB-HbHuIFNβ retroviral vector. The constitutive low production of IFN-β in cultured PBL from HIV-infected patients resulted in a decreased viral production and an enhanced survival of CD4+cells, and this protective effect was observed only in the PBL derived from donors having a CD4+cell count above 200 per mm3. In IFN-β-transduced PBL from healthy and from HIV-infected donors, the production of the Th1-type cytokines IFN-γ and interleukin (IL)-12 was enhanced. In IFN-β-transduced PBL from HIV-infected donors, the production of IL-4, IL-6, IL-10, and tumor necrosis factor α was maintained at normal levels, contrary to the increased levels produced by the untransduced PBL. The proliferative response to recall antigens was partially restored in IFN-β-transduced PBL from donors with an impaired antigen response. Thus, in addition to inhibiting HIV replication, IFN-β transduction of PBL from HIV-infected donors improves several parameters of immune function.
Human deoxycytidine kinase is located in the cell nucleus
Authors: Johansson Magnus , Brismar Sophia , Karlsson Anna
Published: July 2002

Human deoxyribonucleoside kinases are required for the pharmacological activity of several clinically important anticancer and antiviral nucleoside analogs. Human deoxycytidine kinase and thymidine kinase 1 are described as cytosolic enzymes in the literature, whereas human deoxyguanosine kinase and thymidine kinase 2 are believed to be located in the mitochondria. We expressed the four human deoxyribonucleoside kinases as fusion proteins with the green fluorescent protein to study their intracellular locations
in vivo
. Our data showed that the human deoxycytidine kinase is located in the cell nucleus and the human deoxyguanosine kinase is located in the mitochondria. The fusion proteins between green fluorescent protein and thymidine kinases 1 and 2 were both predominantly located in the cytosol. Site-directed mutagenesis of a putative nuclear targeting signal, identified in the primary structure of deoxycytidine kinase, completely abolished nuclear import of the protein. Reconstitution of a deoxycytidine kinase-deficient cell line with the wild-type nuclear or the mutant cytosolic enzymes both restored sensitivity toward anticancer nucleoside analogs. This paper reports that a deoxyribonucleoside kinase is located in the cell nucleus and we discuss the implications for deoxyribonucleotide synthesis and phosphorylation of nucleoside analogs.
Cytokine-mediated enhancement of epidermal growth factor receptor expression provides an immunological approach to the therapy of pancreatic cancer
Authors: Schmiegel Wolff , Schmielau Jan , Henne-Bruns Doris , Juhl Hartmut , Roeder Christian , Buggisch Peter , Onur Aydin , Kremer Bernd , Kalthoff Holger , Jensen Elwood V.
Published: July 2002

The increased expression of epidermal growth factor receptor induced by tumor necrosis factor α renders pancreatic cancer cells more susceptible to antibody-dependent cellular cytotoxicity by a mAb specific for this receptor. Laboratory studies with athymic mice bearing xenografts of human pancreatic cancer cells demonstrated a cytokine-induced ability of the mAb to cause significant tumor regression. In a phase I/II clinical trial, 26 patients with unresectable pancreatic cancer were enrolled into three cohorts receiving variable amounts of the antibody together with a constant amount of tumor necrosis factor α. With increasing doses of antibody, the growth of the primary tumor was significantly inhibited. This was reflected by a longer median survival, with one complete remission lasting for 3 years obtained with the highest dose of antibody employed. Thus, a combination of the cytokine, tumor necrosis factor α, with a mAb to the epidermal growth factor receptor offers a potentially useful approach for the treatment of pancreatic cancer.
Loss of
p
21 increases sensitivity to ionizing radiation and delays the onset of lymphoma in
atm
-deficient mice
Authors: Wang Y. Alan , Elson Ari , Leder Philip
Published: July 2002

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by growth retardation, cerebellar ataxia, oculocutaneous telangiectasias, and a high incidence of lymphomas and leukemias. In addition, AT patients are sensitive to ionizing radiation.
Atm
-deficient mice recapitulate most of the AT phenotype. p21
cip1/waf1
(p21 hereafter), an inhibitor of cyclin-dependent kinases, has been implicated in cellular senescence and response to γ-radiation-induced DNA damage. To study the role of p21 in ATM-mediated signal transduction pathways, we examined the combined effect of the genetic loss of
atm
and
p21
on growth control, radiation sensitivity, and tumorigenesis. As might have been expected, our data provide evidence that p21 modifies the
in vitro
senescent response seen in AT fibroblasts. Further, it is a downstream effector of ATM-mediated growth control. In addition, however, we find that loss of
p21
in the context of an
atm
-deficient mouse leads to a delay in thymic lymphomagenesis and an increase in acute radiation sensitivity
in vivo
(the latter principally because of effects on the gut epithelium). Modification of these two crucial aspects of the ATM phenotype can be related to an apparent increase in spontaneous apoptosis seen in tumor cells and in the irradiated intestinal epithelium of mice doubly null for
atm
and
p21
. Thus, loss of p21 seems to contribute to tumor suppression by a mechanism that operates via a sensitized apoptotic response. These results have implications for cancer therapy in general and AT patients in particular.
Combined effects of insulin treatment and adipose tissue-specific agouti expression on the development of obesity
Authors: Mynatt R. L. , Miltenberger R. J. , Klebig M. L. , Zemel M. B. , Wilkinson J. E. , Wilkison W. O. , Woychik R. P.
Published: July 2002

The
agouti
gene product is a secreted protein that acts in a paracrine manner to regulate coat color in mammals. Several dominant mutations at the
agouti
locus in mice cause the ectopic, ubiquitous expression of
agouti
, resulting in a condition similar to adult-onset obesity and non-insulin-dependent diabetes mellitus. The human agouti protein is 85% homologous to mouse agouti; however, unlike the mouse
agouti
gene, human
agouti
is normally expressed in adipose tissue. To address whether expression of
agouti
in human adipose tissue is physiologically relevant, transgenic mice were generated that express
agouti
in adipose tissue. Similar to most humans, these mice do not become obese or diabetic. However, we found that daily insulin injections significantly increased weight gain in the transgenic lines expressing
agouti
in adipose tissue, but not in nontransgenic mice. These results suggest that insulin triggers the onset of obesity and that
agouti
expression in adipose tissue potentiates this effect. Accordingly, the investigation of agouti’s role in obesity and non-insulin-dependent diabetes mellitus in mice holds significant promise for understanding the pathophysiology of human obesity.
BNaC1 and BNaC2 constitute a new family of human neuronal sodium channels related to degenerins and epithelial sodium channels
Authors: García-Añoveros Jaime , Derfler Bruce , Neville-Golden Janine , Hyman Bradley T. , Corey David P.
Published: July 2002

The recently defined DEG/ENaC superfamily of sodium channels includes subunits of the amiloride-sensitive epithelial sodium channel (ENaC) of vertebrate colon, lung, kidney, and tongue, a molluscan FMRFamide-gated channel (FaNaC), and the nematode degenerins, which are suspected mechanosensory channels. We have identified two new members of this superfamily (BNaC1 and BNaC2) in a human brain cDNA library. Phylogenetic analysis indicates they are equally divergent from all other members of the DEG/ENaC superfamily and form a new branch or family. Human BNaC1 maps to 17q11.2-12 and hBNaC2 maps to 12q12. Northern blot and mouse brain
in situ
hybridizations indicate that both genes are coexpressed in most if not all brain neurons, although their patterns of expression vary slightly, and are expressed early in embryogenesis and throughout life. By analogy to the ENaCs and the degenerins, which form heteromultimeric channels, BNaC1 and BNaC2 may be subunits of the same channel.
Stabilization of ion selectivity filter by pore loop ion pairs in an inwardly rectifying potassium channel
Authors: Yang Jian , Yu Mei , Jan Yuh Nung , Jan Lily Yeh
Published: July 2002

Ion selectivity is critical for the biological functions of voltage-dependent cation channels and is achieved by specific ion binding to a pore region called the selectivity filter. In voltage-gated K+, Na+and Ca2+channels, the selectivity filter is formed by a short polypeptide loop (called the H5 or P region) between the fifth and sixth transmembrane segments, donated by each of the four subunits or internal homologous domains forming the channel. While mutagenesis studies on voltage-gated K+channels have begun to shed light on the structural organization of this pore region, little is known about the physical and chemical interactions that maintain the structural stability of the selectivity filter. Here we show that in an inwardly rectifying K+(IRK) channel, IRK1, short range interactions of an ion pair in the H5 pore loop are crucial for pore structure and ion permeation. The two residues, a glutamate and an arginine, appear to form exposed salt bridges in the tetrameric channel. Alteration or disruption of such ion pair interactions dramatically alters ion selectivity and permeation. Since this ion pair is conserved in all IRK channels, it may constitute a general mechanism for maintaining the stability of the pore structure in this channel superfamily.
Plastoquinol at the quinol oxidation site of reduced cytochrome
bf
mediates signal transduction between light and protein phosphorylation: Thylakoid protein kinase deactivation by a single-turnover flash
Authors: Vener Alexander V. , van Kan Paul J. M. , Rich Peter R. , Ohad Itzhak , Andersson Bertil
Published: July 2002

Redox-controlled phosphorylation of thylakoid membrane proteins represents a unique system for the regulation of light energy utilization in photosynthesis. The molecular mechanisms for this process remain unknown, but current views suggest that the plastoquinone pool directly controls the activation of the kinase. On the basis of enzyme activation by a pH shift in the darkness combined with flash photolysis, EPR, and optical spectroscopy we propose that activation occurs when plastoquinol occupies the quinol-oxidation (Qo) site of the cytochrome
bf
complex, having its high-potential path components in a reduced state. A linear correlation between kinase activation and accessibility of the Qo site to plastoquinol was established by quantification of the shift in the
g

y

EPR signal of the Rieske Fe–S center resulting from displacement of the Qo-site plastoquinol by a quinone analog. Activity persists as long as one plastoquinol per cytochrome
bf
is still available. Withdrawal of one electron from this plastoquinol after a single-turnover flash exciting photosystem I leads to deactivation of the kinase parallel with a decrease in the
g

z

EPR signal of the reduced Rieske Fe–S center. Cytochrome
f
, plastocyanin, and P
700
are rereduced after the flash, indicating that the plastoquinol at the Qo site is limiting in maintaining the kinase activity. These results give direct evidence for a functional cytochrome
bf
–kinase interaction, analogous to a signal transduction system where the cytochrome
bf
is the receptor and the ligand is the plastoquinol at the Qo site.
syk kinase activation by a src kinase-initiated activation loop phosphorylation chain reaction
Authors: El-Hillal O. , Kurosaki T. , Yamamura H. , Kinet J.-P. , Scharenberg A. M.
Published: July 2002

Activation of the syk tyrosine kinase occurs almost immediately following engagement of many types of antigen receptors, including Fc receptors, but the mechanism through which syk is activated is currently unclear. Here we demonstrate that Fc receptor-induced syk activation occurs as the result of phosphorylation of the syk activation loop by both src family kinases and other molecules of activated syk, suggesting that syk activation occurs as the result of a src kinase-initiated activation loop phosphorylation chain reaction. This type of activation mechanism predicts that syk activation would exhibit exponential kinetics, providing a potential explanation for its rapid and robust activation by even weak antigen receptor stimuli. We propose that a similar mechanism may be responsible for generating rapid activation of other cytoplasmic tyrosine kinases, such as those of the Bruton tyrosine kinase/tec family, as well.
The β subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin
Authors: Hanner Markus , Schmalhofer William A. , Munujos Petraki , Knaus Hans-Günther , Kaczorowski Gregory J. , Garcia Maria L.
Published: July 2002

Transient expression of either α or α+β subunits of the high-conductance Ca
2+
-activated K
+
(maxi-K) channel has been achieved in COS-1 cells. Expression has been studied using charybdotoxin (ChTX), a peptidyl inhibitor that binds in the pore on the α subunit. Although some properties of monoiodotyrosine-ChTX (
125
I-ChTX) binding to membranes derived from each type of transfected cells appear to be identical, other parameters of the binding reaction are markedly different. Under low ionic strength conditions, the affinity constant for
125
I-ChTX measured under equilibrium binding conditions is increased
ca.
50-fold in the presence of the β subunit. The rate constant for
125
I-ChTX association is enhanced
ca.
5-fold, whereas the dissociation rate constant is decreased more than 7-fold when the β subunit is present. These data indicate that functional coassembly of maxi-K channel subunits can be obtained in a transient expression system, and that the β subunit has profound effects on
125
I-ChTX binding. We postulate that certain negatively charged residues in the large extracellular loop of β attract the positively charged
125
I-ChTX to its binding site on α through electrostatic interactions, and account for effects observed on ligand association kinetics. Moreover, another residue(s) in the loop of β must contribute to stabilization of the toxin-bound state, either by a direct interaction with toxin, or through an allosteric effect on the α subunit. Certain regions in the extracellular loop of the β subunit may be in close proximity to the pore of the channel, and could play an important role in maxi-K channel function.
Interleukin 10 is induced by recombinant HIV-1 Nef protein involving the calcium/calmodulin-dependent phosphodiesterase signal transduction pathway
Authors: Brigino Emerita , Haraguchi Soichi , Koutsonikolis Angelos , Cianciolo George J. , Owens Una , Good Robert A. , Day Noorbibi K.
Published: July 2002

HIV-1 Nef protein shares a significant homology with the immunosuppressive and highly conserved retroviral transmembrane protein p15E. In the present study, extracellular Nef protein is shown to induce interleukin (IL)-10 mRNA expression in human peripheral blood mononuclear cells as well as in cells of H9 T and U937 promonocytic human cell lines. Release of IL-10 protein into supernatants of peripheral blood mononuclear cells stimulated with Nef is dose-dependent. Expression of cytokines IL-2, IL-4, IL-5, IL-12 p40, IL-13, and interferon γ is not affected by Nef stimulation. IL-10 protein production induced by Nef is inhibited by the calcium/calmodulin phosphodiesterase inhibitor W-7 but not by the protein kinase A inhibitor H-89 nor the protein kinase C inhibitors staurosporine and calphostin C. The calcium chelating agent EGTA also inhibits the IL-10 production induced by Nef, and this inhibition is reversed by the addition of calcium along with Nef. These findings indicate that extracellular Nef may contribute to the immunopathogenesis of HIV infection by inducing IL-10.