bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth and viability of macrophages continuously stimulated to produce nitric oxide
Authors: Zhuang John C. , Wogan Gerald N.
Published: July 2002

Deregulated production of nitric oxide (NO) has been implicated in the development of certain human diseases, including cancer. We sought to assess the damaging potential of NO produced under long-term conditions through the development of a suitable model cell culture system. In this study, we report that when murine macrophage-like RAW264.7 cells were exposed continuously to bacterial lipopolysaccharide (LPS) or mouse recombinant interferon-γ (IFN-γ) over periods of 21–23 days, they continued to grow, but with doubling times 2 to 4 times, respectively, longer than the doubling time of unstimulated cells. Stimulated cells produced NO at rates of 30 to 70 nmol per million cells per day throughout the stimulation period. Within 24 hr after removal of stimulant, cells resumed exponential growth. Simultaneous exposure to LPS and IFN-γ resulted in decreased cell number, which persisted for 2 days after removal of the stimulants. Exponential growth was attained only after an additional 4 days. Addition ofN-methyl-l-arginine (NMA), an NO synthase inhibitor, to the medium inhibited NO production by 90% of all stimulated cells, partially reduced doubling time of cells stimulated with LPS or IFN-γ, and partially increased viability and growth rates in those exposed to both LPS and IFN-γ. However, when incubated with LPS and IFN-γ at low densities both in the presence and in the absence of NMA, cells grew at a rate slower than that of unstimulated cells, with no cell death, and they resumed exponential growth 24 hr after removal of stimulants. Results from cell density experiments suggest that macrophages are protected from intracellularly generated NO; much of the NO damaging activity occurred outside of the producer cells. Collectively, results presented in this study suggest that the type of cellular toxicity observed in macrophages is markedly influenced by rate of exposure to NO: at low rates of exposure, cells exhibit slower growth; at higher rates, cells begin to die; at even higher rates, cells undergo growth arrest or die. The ability of RAW264.7 cells to produce NO over many cell generations makes the cell line a useful system for the study of other aspects of cellular damage, including genotoxicity, resulting from exposure to NO under long-term conditions.
The interaction between cytoplasmic dynein and dynactin is required for fast axonal transport
Authors: Waterman-Storer Clare M. , Karki Sher B. , Kuznetsov Sergei A. , Tabb Joel S. , Weiss Dieter G. , Langford George M. , Holzbaur Erika L. F.
Published: July 2002

Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic dynein is necessary but not sufficient for retrograde transport directed from the synapse to the cell body. Dynactin is a heteromultimeric protein complex, enriched in neurons, that binds to both microtubules and cytoplasmic dynein. To determine whether dynactin is required for retrograde axonal transport, we examined the effects of anti-dynactin antibodies on organelle transport in extruded axoplasm. Treatment of axoplasm with antibodies to the p150
Glued
subunit of dynactin resulted in a significant decrease in the velocity of microtubule-based organelle transport, with many organelles bound along microtubules. We examined the molecular mechanism of the observed inhibition of motility, and we demonstrated that antibodies to p150
Glued
disrupted the binding of cytoplasmic dynein to dynactin and also inhibited the association of cytoplasmic dynein with organelles. In contrast, the anti-p150
Glued
antibodies had no effect on the binding of dynactin to microtubules nor on cytoplasmic dynein-driven microtubule gliding. These results indicate that the interaction between cytoplasmic dynein and the dynactin complex is required for the axonal transport of membrane-bound vesicles and support the hypothesis that dynactin may function as a link between the organelle, the microtubule, and cytoplasmic dynein during vesicle transport.
Determining divergence times with a protein clock: Update and reevaluation
Authors: Feng Da-Fei , Cho Glen , Doolittle Russell F.
Published: July 2002

A recent study of the divergence times of the major groups of organisms as gauged by amino acid sequence comparison has been expanded and the data have been reanalyzed with a distance measure that corrects for both constraints on amino acid interchange and variation in substitution rate at different sites. Beyond that, the availability of complete genome sequences for several eubacteria and an archaebacterium has had a great impact on the interpretation of certain aspects of the data. Thus, the majority of the archaebacterial sequences are not consistent with currently accepted views of the Tree of Life which cluster the archaebacteria with eukaryotes. Instead, they are either outliers or mixed in with eubacterial orthologs. The simplest resolution of the problem is to postulate that many of these sequences were carried into eukaryotes by early eubacterial endosymbionts about 2 billion years ago, only very shortly after or even coincident with the divergence of eukaryotes and archaebacteria. The strong resemblances of these same enzymes among the major eubacterial groups suggest that the cyanobacteria and Gram-positive and Gram-negative eubacteria also diverged at about this same time, whereas the much greater differences between archaebacterial and eubacterial sequences indicate these two groups may have diverged between 3 and 4 billion years ago.
Rapid decline of fitness in panmictic populations of
Drosophila melanogaster
maintained under relaxed natural selection
Authors: Shabalina Svetlana A. , Yampolsky Lev Yu. , Kondrashov Alexey S.
Published: July 2002

The parameters of the spontaneous deleterious mutation process remain poorly known, despite their importance. Here, we report the results of a mutation accumulation experiment performed on panmictic populations of
Drosophila melanogaster
without any genetic manipulations. Two experimental populations were kept for 30 generations under relaxed natural selection. Each generation, 100 pairs were formed randomly, and every fecund pair contributed a son and a daughter to the next generation. Comparison with two controls, one cryopreserved and the other kept as the experimental populations but with long generation time, showed that the number of surviving offspring per female declined by 0.2% and 2.0% per generation under benign and harsh, competitive conditions, respectively. Thus, the mutational pressure on fitness may be strong and depends critically on the conditions under which fitness is assayed.
Direct repeat sequences in the
Streptomyces
chitinase-63 promoter direct both glucose repression and chitin induction
Authors: Ni Xiangyang , Westpheling Janet
Published: July 2002

The
chi63
promoter directs glucose-sensitive, chitin-dependent transcription of a gene involved in the utilization of chitin as carbon source. Analysis of 5′ and 3′ deletions of the promoter region revealed that a 350-bp segment is sufficient for wild-type levels of expression and regulation. The analysis of single base changes throughout the promoter region, introduced by random and site-directed mutagenesis, identified several sequences to be important for activity and regulation. Single base changes at −10, −12, −32, −33, −35, and −37 upstream of the transcription start site resulted in loss of activity from the promoter, suggesting that bases in these positions are important for RNA polymerase interaction. The sequences centered around −10 (TATTCT) and −35 (TTGACC) in this promoter are, in fact, prototypical of eubacterial promoters. Overlapping the RNA polymerase binding site is a perfect 12-bp direct repeat sequence. Some base changes within this direct repeat resulted in constitutive expression, suggesting that this sequence is an operator for negative regulation. Other base changes resulted in loss of glucose repression while retaining the requirement for chitin induction, suggesting that this sequence is also involved in glucose repression. The fact that cis-acting mutations resulted in glucose resistance but not inducer independence rules out the possibility that glucose repression acts exclusively by inducer exclusion. The fact that mutations that affect glucose repression and chitin induction fall within the same direct repeat sequence module suggests that the direct repeat sequence facilitates both chitin induction and glucose repression.
Transcription factor Mts1/Mts2 (Atf1/Pcr1, Gad7/Pcr1) activates the
M
26 meiotic recombination hotspot in
Schizosaccharomyces pombe
Authors: Kon Ning , Krawchuk Michelle D. , Warren B. Greg , Smith Gerald R. , Wahls Wayne P.
Published: July 2002

Homologous recombination hotspots increase the frequency of recombination in nearby DNA. The
M26
hotspot in the
ade6
gene of
Schizosaccharomyces pombe
is a meiotic hotspot with a discrete, cis-acting nucleotide sequence (5′-ATGACGT-3′) defined by extensive mutagenesis. A heterodimeric
M26
DNA binding protein, composed of subunits Mts1 and Mts2, has been identified and purified 40,000-fold. Cloning, disruption, and genetic analyses of the
mts
genes demonstrate that the Mts1/Mts2 heterodimer is essential for hotspot activity. This provides direct evidence that a specific trans-acting factor, binding to a cis-acting site with a unique nucleotide sequence, is required to activate this meiotic hotspot. Intriguingly, the Mts1/Mts2 protein subunits are identical to the recently described transcription factors Atf1 (Gad7) and Pcr1, which are required for a variety of stress responses. However, we report differential dependence on the Mts proteins for hotspot activation and stress response, suggesting that these proteins are multifunctional and have distinct activities. Furthermore,
ade6
mRNA levels are equivalent in hotspot and nonhotspot meioses and do not change in
mts
mutants, indicating that hotspot activation is not a consequence of elevated transcription levels. These findings suggest an intimate but separable link between the regulation of transcription and meiotic recombination. Other studies have recently shown that the Mts1/Mts2 protein and
M26
sites are involved in meiotic recombination elsewhere in the
S. pombe
genome, suggesting that these factors help regulate the timing and distribution of homologous recombination.
RGS2/G0S8 is a selective inhibitor of Gqα function
Authors: Heximer Scott P. , Watson Ned , Linder Maurine E. , Blumer Kendall J. , Hepler John R.
Published: July 2002

RGS (
r
egulators of
G
protein
s
ignaling) proteins are GTPase activating proteins that inhibit signaling by heterotrimeric G proteins. All RGS proteins studied to date act on members of the Giα family, but not Gsα or G12α. RGS4 regulates Giα family members and Gqα. RGS2 (G0S8) is exceptional because the G proteins it regulates have not been identified. We report that RGS2 is a selective and potent inhibitor of Gqα function. RGS2 selectively binds Gqα, but not other Gα proteins (Gi, Go, Gs, G12/13) in brain membranes; RGS4 binds Gqα and Giα family members. RGS2 binds purified recombinant Gqα, but not Goα, whereas RGS4 binds either. RGS2 does not stimulate the GTPase activities of Gsα or Giα family members, even at a protein concentration 3000-fold higher than is sufficient to observe effects of RGS4 on Giα family members. In contrast, RGS2 and RGS4 completely inhibit Gq-directed activation of phospholipase C in cell membranes. When reconstituted with phospholipid vesicles, RGS2 is 10-fold more potent than RGS4 in blocking Gqα-directed activation of phospholipase Cβ1. These results identify a clear physiological role for RGS2, and describe the first example of an RGS protein that is a selective inhibitor of Gqα function.
A
Xenopus
oocyte β subunit: Evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit
Authors: Tareilus Erwin , Roux Michel , Qin Ning , Olcese Riccardo , Zhou Jianming , Stefani Enrico , Birnbaumer Lutz
Published: July 2002

Two closely related β subunit mRNAs (xo28 and xo32) were identified in
Xenopus
oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (
i
) injection of
Xenopus
β antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca
2+
channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian α
1
cRNAs (α
1C
and α
1E
); (
ii
) coinjection of a
Xenopus
β antisense oligonucleotide and excess rat β cRNA rescued expression of α
1
Ca
2+
channel currents; and (
iii
) coinjection of mammalian α
1
cRNA with cRNA encoding either of the two
Xenopus
β subunits facilitated both activation and inactivation of Ca
2+
channel currents by voltage, as happens with most mammalian β subunits. The
Xenopus
β subunit cDNAs (β3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 β subunit. We propose that “α
1
alone” channels are in fact tightly associated α
1
β3xo channels, and that effects of exogenous β subunits are due to formation of higher-order [α
1
β]β
n
complexes with an unknown contribution of β3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition α
1
β, are [α
1
β]β
n
complexes that associate with α
2
δ and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of α
1E
have a β subunit binding domain. This β binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel α
1
subunits.
Sodium salicylate induces apoptosis via p38 mitogen-activated protein kinase but inhibits tumor necrosis factor-induced c-Jun N-terminal kinase/stress-activated protein kinase activation
Authors: Schwenger Paul , Bellosta Paola , Vietor Ilja , Basilico Claudio , Skolnik Edward Y. , Vilček Jan
Published: July 2002

In a previous study, we demonstrated that sodium salicylate (NaSal) selectively inhibits tumor necrosis factor (TNF)-induced activation of the p42 and p44 mitogen-activated protein kinases (MAPKs) (known as extracellular signal-regulated kinases). Here we show that in normal human FS-4 fibroblasts NaSal inhibits TNF-induced activation of another member of the MAPK family, the c-Jun N-terminal kinase/stress-activated protein kinase. c-Jun N-terminal kinase activation induced by interleukin 1 or epidermal growth factor was less strongly inhibited by NaSal. Unexpectedly, treatment of FS-4 cells with NaSal alone produced a strong activation of p38 MAPK and cell death by apoptosis. NaSal-induced apoptosis was blocked by the selective p38 MAPK inhibitor SB-203580, indicating that p38 MAPK serves as a mediator of NaSal-induced apoptosis in human fibroblasts. Activation of p38 MAPK and the resulting induction of apoptosis may be important in the demonstrated antineoplastic actions of nonsteroidal anti-inflammatory drugs.
CREB-binding protein/p300 are transcriptional coactivators of p65
Authors: Gerritsen Mary E. , Williams Amy J. , Neish Andrew S. , Moore Sarah , Shi Yang , Collins Tucker
Published: July 2002

CBP (CREB-binding protein) and p300 are versatile coactivators that link transcriptional activators to the basal transcriptional apparatus. In the present study, we identify CBP and p300 as coactivators of the nuclear factor-κB (NF-κB) component p65 (RelA). Consistent with their role as coactivators, both CBP and p300 potentiated p65-activated transcription of E-selectin and VCAM-1–CAT reporter constructs. The N- and C-terminal domains of both CBP/p300 functionally interact with a region of p65 containing the transcriptional activation domain as demonstrated by mammalian two-hybrid assays. Direct physical interactions of CBP/p300 with p65 were demonstrated by glutathioneS-transferase fusion protein binding, and coimmunoprecipitation/Western blot studies. The adenovirus E1A 12S protein, which complexes with CBP and p300, inhibited p65-dependent gene expression. Reporter gene expression could be rescued from E1A inhibition by overexpression of CBP or p300. CBP and p300 act as coactivators of p65-driven gene activation and may play an important role in the cytokine-induced expression of various immune and inflammatory genes.
A peptide composed of tandem analogs of two myasthenogenic T cell epitopes interferes with specific autoimmune responses
Authors: Katz-Levy Yael , Paas-Rozner Miri , Kirshner Susan , Dayan Molly , Zisman Einat , Fridkin Mati , Wirguin Itzhak , Sela Michael , Mozes Edna
Published: July 2002

Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor α-subunit, p195-212 and p259-271, were previously shown to stimulate peripheral blood lymphocytes of patients with MG and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Single amino acid substituted analogs of p195-212 (analog Ala-207) and p259-271 (analog Lys-262) were synthesized. We showed that analogs Ala-207 and Lys-262 inhibited,in vitroandin vivo, the proliferative responses of T cell lines specific to the relevant peptide and lymph node cells of mice immunized to p195-212 and p259-271, respectively. To inhibit T cell responses to both peptides (p195-212 and p259-271), we synthesized dual analogs composed of the tandemly arranged two single (Ala-207 and Lys-262) analogs (dual analog) either sequentially (Ala-207–Lys-262) or reciprocally (Lys-262–Ala-207). In the present study, we report that both dual analogs could bind to major histocompatibility complex class II molecules on antigen-presenting cells of SJL and BALB/c mice. Analog Lys-262–Ala-207, which bound more efficiently to major histocompatibility complex class II molecules, was found to inhibit the proliferative responses of both p195-212- and p259-271-specific T cell lines. Furthermore, the analog inhibited thein vivopriming of lymph node cells of both SJL and BALB/c mice when administered i.v., i.p., orper os. The dual analog Lys-262–Ala-207 could also immunomodulate myasthenogenic manifestations in mice with experimental autoimmune MG induced by inoculation of a pathogenic T cell line. Thus, a single peptide that is composed of analogs to two epitope specificities can be used to regulate T cell responses and disease associated with each epitope.
Microcolinearity in
sh2
-homologous regions of the maize, rice, and sorghum genomes
Authors: Chen M. , SanMiguel P. , de Oliveira A. C. , Woo S.-S. , Zhang H. , Wing R. A. , Bennetzen J. L.
Published: July 2002

Large regions of genomic colinearity have been demonstrated among
grass species by recombinational mapping, but the degree of chromosomal
conservation at the sub-centimorgan level has not been extensively
investigated. We cloned the rice and sorghum genes homologous to the
sh2
locus of maize on bacterial artificial chromosomes
(BACs), and observed that a homologue of the maize
a1
gene was also present on each of these BACs. In sorghum, we found a
direct duplication of
a1
homologues separated by about
10 kb. In maize,
sh2
and
a1
are
approximately 140 kb apart and transcribed in the same direction, with
sh2
upstream of
a1
. In rice and sorghum,
this arrangement is fully conserved. However, the
sh2
and
a1
homologues are separated by about 19 kb in both
rice and sorghum. We found low-copy-number and repetitive DNAs between
the
sh2
and
a1
homologues of sorghum and
rice. The
sh2
and
a1
homologues
cross-hybridized, but the repetitive DNA and most low-copy-number
sequences between these genes did not. These results indicate that
maize, sorghum, and rice have conserved gene order and composition in
the
sh2
-
a1
region, but have acquired
extensive qualitative and quantitative differences in the sequences
between these genes.
Rhythms ofDrosophila periodgene expression in culture
Authors: Emery Ivette F. , Noveral Jocelyne M. , Jamison Creston F. , Siwicki Kathleen King
Published: July 2002

TheDrosophilaclock genesperiod(per) andtimeless(tim) have been studied behaviorally and biochemically, but to date there has been no viable culture system for studying the cell biology of theDrosophilaclock. We have cultured pupal ring glands attached to the central nervous system and observed rhythms ofperiodgene expression in the prothoracic gland for 4–7 days. A daily rhythm of Per protein can be entrained by light in culture, even when neural activity is blocked by tetrodotoxin. In cultures maintained for a week in constant darkness, aper-luciferasereporter gene revealed circadian rhythms of bioluminescence. As the first circadian culture system fromDrosophila, the prothoracic gland provides unique advantages for investigating the interactions between clock genes and cellular physiology.
The cyanobacterium
Synechococcus
resists UV-B by exchanging photosystem II reaction-center D1 proteins
Authors: Campbell Douglas , Eriksson Mats-Jerry , Öquist Gunnar , Gustafsson Petter , Clarke Adrian K.
Published: July 2002

Current ambient UV-B levels can significantly depress productivity in aquatic habitats, largely because UV-B inhibits several steps of photosynthesis, including the photooxidation of water catalyzed by photosystem II. We show that upon UV-B exposure the cyanobacterium
Synechococcus
sp. PCC 7942 rapidly changes the expression of a family of three
psbA
genes encoding photosystem II D1 proteins. In wild-type cells the
psbAI
gene is expressed constitutively, but strong accumulations of
psbAII
and
psbAIII
transcripts are induced within 15 min of moderate UV-B exposure (0.4 W/m
2
). This transcriptional response causes an exchange of two distinct photosystem II D1 proteins. D1:1 is encoded by
psbAI
, but on UV-B exposure, it is largely replaced by the alternate D1:2 form, encoded by both
psbAII
and
psbAIII
. The total content of D1 and other photosystem II reaction center protein, D2, remained unchanged throughout the UV exposure, as did the content and composition of the phycobilisome. Wild-type cells suffered only slight transient inhibition of photosystem II function under UV-B exposure. In marked contrast, under the same UV-B treatment, a mutant strain expressing only
psbAI
suffered severe (40%) and sustained inhibition of photosystem II function. Another mutant strain with constitutive expression of
psbAII
and
psbAIII
was almost completely resistant to the UV-B treatment, showing no inhibition of photosystem II function and only a slight drop in electron transport. In
Synechococcus
the rapid exchange of alternate D1 forms, therefore, accounts for much of the cellular resistance to UV-B inhibition of photosystem II activity and photosynthetic electron transport. This molecular plasticity may be an important element in community-level responses to UV-B, where susceptibility to UV-B inhibition of photosynthesis changes diurnally.
Directed evolution of an aspartate aminotransferase with new substrate specificities
Authors: Yano Takato , Oue Shinya , Kagamiyama Hiroyuki
Published: July 2002

The substrate specificity of aspartate aminotransferase was successfully modified by directed molecular evolution using a combination of DNA shuffling and selection in an auxotrophic
Escherichia coli
strain. After five rounds of selection, one of the evolved mutants showed a 10
5
-fold increase in the catalytic efficiency (
k
cat
/
K
m
) for β-branched amino and 2-oxo acids and a 30-fold decrease in that for the native substrates compared with the wild-type enzyme. The mutant had 13 amino acid substitutions, 6 of which contributed 80–90% to the total effect. Five of these six substitutions were conserved among the five mutants that showed the highest activity for β-branched substrates. Interestingly, only one of the six functionally important residues is located within a distance of direct interaction with the substrate, supporting the idea that rational design of the substrate specificity of an enzyme is very difficult. The present results show that directed molecular evolution is a powerful technique for enzyme redesign if an adequate selection system is applied.
Assessing sequence comparison methods with reliable structurally identified distant evolutionary relationships
Authors: Brenner Steven E. , Chothia Cyrus , Hubbard Tim J. P.
Published: July 2002

Pairwise sequence comparison methods have been assessed using proteins whose relationships are known reliably from their structures and functions, as described in the
scop
database [Murzin, A. G., Brenner, S. E., Hubbard, T. & Chothia C. (1995)
J. Mol. Biol.
247, 536–540]. The evaluation tested the programs
blast
[Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990).
J. Mol. Biol.
215, 403–410],
wu-blast
2 [Altschul, S. F. & Gish, W. (1996)
Methods Enzymol.
266, 460–480],
fasta
[Pearson, W. R. & Lipman, D. J. (1988)
Proc. Natl. Acad. Sci. USA
85, 2444–2448], and
ssearch
[Smith, T. F. & Waterman, M. S. (1981)
J. Mol. Biol.
147, 195–197] and their scoring schemes. The error rate of all algorithms is greatly reduced by using statistical scores to evaluate matches rather than percentage identity or raw scores. The E-value statistical scores of
ssearch
and
fasta
are reliable: the number of false positives found in our tests agrees well with the scores reported. However, the P-values reported by
blast
and
wu-blast
2 exaggerate significance by orders of magnitude.
ssearch
,
fasta
ktup = 1, and
wu-blast
2 perform best, and they are capable of detecting almost all relationships between proteins whose sequence identities are >30%. For more distantly related proteins, they do much less well; only one-half of the relationships between proteins with 20–30% identity are found. Because many homologs have low sequence similarity, most distant relationships cannot be detected by any pairwise comparison method; however, those which are identified may be used with confidence.
Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens
Authors: Sheets Michael D. , Amersdorfer Peter , Finnern Ricarda , Sargent Peter , Lindqvist Ericka , Schier Robert , Hemingsen Grete , Wong Cindy , Gerhart John C. , Marks James D.
Published: July 2002

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 10
9
members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody–antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the
Chlamydia trachomatis
elementary bodies stained
Chlamydia
-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.
Eotaxin is required for the baseline level of tissue eosinophils
Authors: Matthews Angela N. , Friend Daniel S. , Zimmermann Nives , Sarafi Mindy N. , Luster Andrew D. , Pearlman Eric , Wert Susan E. , Rothenberg Marc E.
Published: July 2002

Eotaxin is an eosinophil-selective chemokine that is constitutively expressed in a variety of organs such as the intestine. Previous studies have demonstrated that the recruitment of eosinophils during inflammation is partially dependent on eotaxin, but the function of constitutive eotaxin during homeostasis has not been examined. To elucidate the biological role of this molecule, we now examine tissue levels of eosinophils in healthy states in wild-type and eotaxin-deficient mice. The lamina propria of the jejunum of wild-type mice is demonstrated to express eotaxin mRNA, but not mRNA for the related monocyte chemoattractant proteins. Wild-type mice contained readily detectable eosinophils in the lamina propria of the jejunum. In contrast, mice genetically deficient in eotaxin had a large selective reduction in the number of eosinophils residing in the jejunum. The reduction of tissue eosinophils was not limited to the jejunum, because a loss of thymic eosinophils was also observed in eotaxin-deficient mice. These studies demonstrate that eotaxin is a fundamental regulator of the physiological trafficking of eosinophils during healthy states. Because a variety of chemokines are constitutively expressed, their involvement in the baseline trafficking of leukocytes into nonhematopoietic tissue should now be considered.
Jasmonate-induced responses are costly but benefit plants under attack in native populations
Authors: Baldwin Ian T.
Published: July 2002

Herbivore attack is widely known to reduce food quality and to increase chemical defenses and other traits responsible for herbivore resistance. Inducible defenses are commonly thought to allow plants to forgo the costs of defense when not needed; however, neither their defensive function (increasing a plant’s fitness) nor their cost-savings function have been demonstrated in nature. The root-produced toxin nicotine increases after herbivore attack in the native, postfire annual
Nicotiana attenuata
and is internally activated by the wound hormone, jasmonic acid. I treated the roots of plants with the methyl ester of this hormone (MeJA) to elicit a response in one member of each of 745 matched pairs of plants growing in native populations with different probabilities of attack from herbivores, and measured the lifetime production of viable seed. In populations with intermediate rates of attack, induced plants were attacked less often by herbivores and survived to produce more seed than did their uninduced counterparts. Previous induction did not significantly increase the fitness of plants suffering high rates of attack. However, if plants had not been attacked, induced plants produced less seed than did their uninduced counterparts. Jasmonate-induced responses function as defenses but are costly, and inducibility allows this species to forgo these costs when the defenses are unnecessary.
A cloning method for caspase substrates that uses the yeast two-hybrid system: Cloning of the antiapoptotic gene gelsolin
Authors: Kamada Shinji , Kusano Hajime , Fujita Hisakazu , Ohtsu Makoto , Koya Richard Chikara , Kuzumaki Noboru , Tsujimoto Yoshihide
Published: July 2002

Caspase-mediated proteolysis is a critical and central element of the apoptotic process; therefore, it is important to identify the downstream molecular targets of caspases. We established a method for cloning the genes of caspase substrates by two major modifications of the yeast two-hybrid system: (
i
) both large and small subunits of active caspases were expressed in yeast under
ADH1
promoters and the small subunit was fused to the LexA DNA-binding domain; and (
ii
) a point mutation was introduced that substituted serine for the active site cysteine and thereby prevented proteolytic cleavage of the substrates, possibly stabilizing the enzyme–substrate complexes in yeast. After screening a mouse embryo cDNA expression library by using the bait plasmid for caspase-3, we obtained 13 clones that encoded proteins binding to caspase-3, and showed that 10 clones including gelsolin, an actin-regulatory protein implicated in apoptosis, were cleaved by recombinant caspase-3
in vitro
. Using the same bait, we also isolated human gelsolin cDNA from a human thymus cDNA expression library. We showed that human gelsolin was cleaved during Fas-mediated apoptosis
in vivo
and that the caspase-3 cleavage site of human gelsolin was at D
352
of DQTD
352
G, findings consistent with previous observations on murine gelsolin. In addition, we ascribed the antiapoptotic activity of gelsolin (which we previously reported) to prevention of a step leading to cytochrome
c
release from the mitochondria into the cytosol. Our results indicate that this cloning method is useful for identification of the substrates of caspases and possibly also of other enzymes.
The superior colliculus–pretectum mediates the direct effects of light on sleep
Authors: Miller Ann M. , Obermeyer William H. , Behan Mary , Benca Ruth M.
Published: July 2002

Light and dark have immediate effects on sleep and wakefulness in mammals, but the neural mechanisms underlying these effects are poorly understood. Lesions of the visual cortex or the superior colliculus–pretectal area were performed in albino rats to determine retinorecipient areas that mediate the effects of light on behavior, including rapid eye movement sleep triggering by lights-off and redistribution of non-rapid eye movement sleep in short light–dark cycles. Acute responses to changes in light conditions were virtually eliminated by superior colliculus-pretectal area lesions but not by visual cortex lesions. Circadian entrainment was evident in both groups with lesions and in normal controls. Thus, acute light-dark effects on sleep and wakefulness appear to be mediated independently from cortical vision or circadian rhythms.
CTLA-4 blockade synergizes with tumor-derived granulocyte– macrophage colony-stimulating factor for treatment of an experimental mammary carcinoma
Authors: Hurwitz Arthur A. , Yu Tina F.-Y. , Leach Dana R. , Allison James P.
Published: July 2002

Generation of a T cell-mediated antitumor response depends on T cell receptor engagement by major histocompatibility complex/antigen as well as CD28 ligation by B7. CTLA-4 is a second B7 receptor expressed by T cells upon activation that, unlike CD28, appears to deliver an inhibitory signal to T cells. Recently, we and others demonstrated that administration of an anti-CTLA-4 antibody was sufficient to promote regression of several murine tumors. However, certain tumors, such as the SM1 mammary carcinoma, remain refractory to this type of immunotherapy. In the present study, we report that the combination of both CTLA-4 blockade and a vaccine consisting of granulocyte–macrophage colony-stimulating factor-expressing SM1 cells resulted in regression of parental SM1 tumors, despite the ineffectiveness of either treatment alone. This synergistic therapy resulted in long-lasting immunity to SM1 and depended on both CD4+and CD8+T cells. Interestingly, synergy was not observed between CTLA-4 and a B7-expressing SM1 vaccine. Given that granulocyte–macrophage colony-stimulating factor promotes differentiation and activation of dendritic cells as well as enhances cross-priming of T cells to tumor-derived antigens and that SM1 is major histocompatibility complex class II-negative, our findings suggest that CTLA-4 blockade acts at the level of a host-derived antigen-presenting cell. In addition, these results also support the idea that the most effective and synergistic vaccine strategy targets treatments that enhance T cell priming at the level of host-derived antigen-presenting cells.
A viral gene that activates lytic cycle expression of Kaposi’s sarcoma-associated herpesvirus
Authors: Sun Ren , Lin Su-Fang , Gradoville Lyndle , Yuan Yan , Zhu Fanxiu , Miller George
Published: July 2002

Herpesviruses exist in two states, latency and a lytic productive cycle. Here we identify an immediate-early gene encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome. The gene is a homologue of Rta, a transcriptional activator encoded by Epstein–Barr virus (EBV). KSHV/Rta activated KSHV early lytic genes, including virus-encoded interleukin 6 and polyadenylated nuclear RNA, and a late gene, small viral capsid antigen. In cells dually infected with Epstein–Barr virus and KSHV, each Rta activated only autologous lytic cycle genes. Expression of viral cytokines under control of the KSHV/Rta gene is likely to contribute to the pathogenesis of KSHV-associated diseases.
Clustering of low-energy conformations near the native structures of small proteins
Authors: Shortle David , Simons Kim T. , Baker David
Published: July 2002

Recent experimental studies of the denatured state and theoretical analyses of the folding landscape suggest that there are a large multiplicity of low-energy, partially folded conformations near the native state. In this report, we describe a strategy for predicting protein structure based on the working hypothesis that there are a greater number of low-energy conformations surrounding the correct fold than there are surrounding low-energy incorrect folds. To test this idea, 12 ensembles of 500 to 1,000 low-energy structures for 10 small proteins were analyzed by calculating the rms deviation of the Cα coordinates between each conformation and every other conformation in the ensemble. In all 12 cases, the conformation with the greatest number of conformations within 4-Å rms deviation was closer to the native structure than were the majority of conformations in the ensemble, and in most cases it was among the closest 1 to 5%. These results suggest that, to fold efficiently and retain robustness to changes in amino acid sequence, proteins may have evolved a native structure situated within a broad basin of low-energy conformations, a feature which could facilitate the prediction of protein structure at low resolution.
Linkage disequilibrium mapping in isolated populations: The example of Finland revisited
Authors: de la Chapelle Albert , Wright Fred A.
Published: July 2002

Linkage disequilibrium analysis can provide high resolution in the mapping of disease genes because it incorporates information on recombinations that have occurred during the entire period from the mutational event to the present. A circumstance particularly favorable for high-resolution mapping is when a single founding mutation segregates in an isolated population. We review here the population structure of Finland in which a small founder population some 100 generations ago has expanded into 5.1 million people today. Among the 30-odd autosomal recessive disorders that are more prevalent in Finland than elsewhere, several appear to have segregated for this entire period in the “panmictic” southern Finnish population. Linkage disequilibrium analysis has allowed precise mapping and determination of genetic distances at the 0.1-cM level in several of these disorders. Estimates of genetic distance have proven accurate, but previous calculations of the confidence intervals were too small because sampling variation was ignored. In the north and east of Finland the population can be viewed as having been “founded” only after 1500. Disease mutations that have undergone such a founding bottleneck only 20 or so generations ago exhibit linkage disequilibrium and haplotype sharing over long genetic distances (5–15 cM). These features have been successfully exploited in the mapping and cloning of many genes. We review the statistical issues of fine mapping by linkage disequilibrium and suggest that improved methodologies may be necessary to map diseases of complex etiology that may have arisen from multiple founding mutations.
The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation
Authors: Chung Ung-il , Lanske Beate , Lee Kaechoong , Li En , Kronenberg Henry
Published: July 2002

During vertebrate limb development, growth plate chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (−/−) cells, and analyzed cell–cell interactions in the growth plate
in vivo
. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic chondrocytes. Further, ectopic hypertrophic chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.
Pharmacological analysis of cyclooxygenase-1 in inflammation
Authors: Smith Christopher J. , Zhang Yan , Koboldt Carol M. , Muhammad Jerry , Zweifel Ben S. , Shaffer Alex , Talley John J. , Masferrer Jaime L. , Seibert Karen , Isakson Peter C.
Published: July 2002

The enzymes cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin (PG) H2, the precursor of PGs and thromboxane. These lipid mediators play important roles in inflammation and pain and in normal physiological functions. While there are abundant data indicating that the inducible isoform, COX-2, is important in inflammation and pain, the constitutively expressed isoform, COX-1, has also been suggested to play a role in inflammatory processes. To address the latter question pharmacologically, we used a highly selective COX-1 inhibitor, SC-560 (COX-1 IC50= 0.009 μM; COX-2 IC50= 6.3 μM). SC-560 inhibited COX-1-derived platelet thromboxane B2, gastric PGE2, and dermal PGE2production, indicating that it was orally active, but did not inhibit COX-2-derived PGs in the lipopolysaccharide-induced rat air pouch. Therapeutic or prophylactic administration of SC-560 in the rat carrageenan footpad model did not affect acute inflammation or hyperalgesia at doses that markedly inhibitedin vivoCOX-1 activity. By contrast, celecoxib, a selective COX-2 inhibitor, was anti-inflammatory and analgesic in this model. Paradoxically, both SC-560 and celecoxib reduced paw PGs to equivalent levels. Increased levels of PGs were found in the cerebrospinal fluid after carrageenan injection and were markedly reduced by celecoxib, but were not affected by SC-560. These results suggest that, in addition to the role of peripherally produced PGs, there is a critical, centrally mediated neurological component to inflammatory pain that is mediated at least in part by COX-2.
Activation of human monocytes induces differential resistance to apoptosis with rapid down regulation of caspase-8/FLICE
Authors: Perera Liyanage P. , Waldmann Thomas A.
Published: July 2002

Cells of the monocyte/macrophage lineage play a central role in both innate and acquired immunity of the host. However, the acquisition of functional competence and the ability to respond to a variety of activating or modulating signals require maturation and differentiation of circulating monocytes and entail alterations in both biochemical and phenotypic profiles of the cells. The process of activation also confers survival signals essential for the functional integrity of monocytes enabling the cells to remain viable in microenvironments of immune or inflammatory lesions that are rich in cytotoxic inflammatory mediators and reactive free-radical species. However, the molecular mechanisms of activation-induced survival signals in monocytes remain obscure. To define the mechanistic basis of activation-induced resistance to apoptosis in human monocytes at the molecular level, we evaluated the modulation of expression profiles of genes associated with the cellular apoptotic pathways upon activation and demonstrate the following: (i) activation results in selective resistance to apoptosis particularly to that induced by signaling via death receptors and DNA damage; (ii) concurrent with activation, the most apical protease in the death receptor pathway, caspase-8/FLICE is rapidly down-regulated at the mRNA level representing a novel regulatory mechanism; and (iii) activation of monocytes also leads to dramatic induction of theBfl-1gene, an anti apoptotic member of the Bcl-2 family. Our findings thus provide a potential mechanistic basis for the activation-induced resistance to apoptosis in human monocytes.
Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus
Authors: Lelièvre Sophie A. , Weaver Valerie M. , Nickerson Jeffrey A. , Larabell Carolyn A. , Bhaumik Ankan , Petersen Ole W. , Bissell Mina J.
Published: July 2002

What determines the nuclear organization within a cell and whether this organization itself can impose cellular function within a tissue remains unknown. To explore the relationship between nuclear organization and tissue architecture and function, we used a model of human mammary epithelial cell acinar morphogenesis. When cultured within a reconstituted basement membrane (rBM), HMT-3522 cells form polarized and growth-arrested tissue-like acini with a central lumen and deposit an endogenous BM. We show that rBM-induced morphogenesis is accompanied by relocalization of the nuclear matrix proteins NuMA, splicing factor SRm160, and cell cycle regulator Rb. These proteins had distinct distribution patterns specific for proliferation, growth arrest, and acini formation, whereas the distribution of the nuclear lamina protein, lamin B, remained unchanged. NuMA relocalized to foci, which coalesced into larger assemblies as morphogenesis progressed. Perturbation of histone acetylation in the acini by trichostatin A treatment altered chromatin structure, disrupted NuMA foci, and induced cell proliferation. Moreover, treatment of transiently permeabilized acini with a NuMA antibody led to the disruption of NuMA foci, alteration of histone acetylation, activation of metalloproteases, and breakdown of the endogenous BM. These results experimentally demonstrate a dynamic interaction between the extracellular matrix, nuclear organization, and tissue phenotype. They further show that rather than passively reflecting changes in gene expression, nuclear organization itself can modulate the cellular and tissue phenotype.
PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G
1
cell cycle arrest in human glioblastoma cells
Authors: Li Da-Ming , Sun Hong
Published: July 2002

PTEN/MMAC1/TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G
1
phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27
KIP1
and a concomitant decrease in the activities of the G
1
cyclin-dependent kinases. PTEN expression also led to the inhibition of Akt/protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27
KIP1
and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G
1
cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27
KIP1
.