bluuebunny/crossref_metadata_2025 huggingface dataset:
A role for intracellular pH in membrane IgM-mediated cell death of human B lymphomas
Authors: Marches Radu , Vitetta Ellen S. , Uhr Jonathan W.
Published: July 2002

We show that anti-IgM-induced cell death in a human B lymphoma cell line, B104, is associated with early intracellular acidification and cell shrinkage. In contrast, another human B cell lymphoma line, Daudi, less susceptible to B cell antigen receptor-mediated cell death, responded to anti-IgM with an early increase in intracellular pH (pHi). The anti-IgM-induced changes of pHiwere associated with different levels of activation of the Na+/H+exchanger isoform 1 (NHE1) as judged by its phosphorylation status. Prevention of anti-IgM-induced cell death in B104 cells by the calcineurin phosphatase inhibitor, cyclosporin A, abrogated both intracellular acidification and cell shrinkage and was associated with an increase in the phosphorylation level of NHE1 within the first 60 min of stimulation. This indicates a key role for calcineurin in regulating pHiand cell viability. The potential role of pHiin cell viability was confirmed in Daudi cells treated with an Na+/H+exchanger inhibitor 5-(N,N-hexamethylene)amiloride. These observations indicate that the outcome of the anti-IgM treatment depends on NHE1-controlled pHi. We suggest that inactivation of the NHE1 in anti-IgM-stimulated cells results in intracellular acidification and subsequently triggers or amplifies cell death.
Efficient use of a small genome to generate antigenic diversity in tick-borne ehrlichial pathogens
Authors: Brayton Kelly A. , Knowles Donald P. , McGuire Travis C. , Palmer Guy H.
Published: July 2002

Ehrlichiae are responsible for important tick-transmitted diseases,
including anaplasmosis, the most prevalent tick-borne infection of
livestock worldwide, and the emerging human diseases monocytic and
granulocytic ehrlichiosis. Antigenic variation of major surface
proteins is a key feature of these pathogens that allows persistence in
the mammalian host, a requisite for subsequent tick transmission. In
Anaplasma marginale
pseudogenes for two antigenically
variable gene families,
msp
2 and
msp
3,
appear in concert. These pseudogenes can be recombined into the
functional expression site to generate new antigenic variants.
Coordinated control of the recombination of these genes would allow
these two gene families to act synergistically to evade the host immune
response.
The immunosuppressive macrolide RAD inhibits growth of human Epstein–Barr virus-transformed B lymphocytes
in vitro
and
in vivo
: A potential approach to prevention and treatment of posttransplant lymphoproliferative disorders
Authors: Majewski Miroslaw , Korecka Magdalena , Kossev Plamen , Li Shiyong , Goldman June , Moore Jonni , Silberstein Leslie E. , Nowell Peter C. , Schuler Walter , Shaw Leslie M. , Wasik Mariusz A.
Published: July 2002

Whereas the standard immunosuppressive agents foster development of posttransplant lymphoproliferative disorders (PTLDs), the impact of RAD, a macrolide with potent immunosuppressive properties, and other immunosuppressive macrolides on these disorders remains undetermined. We found that RAD had a profound inhibitory effect on
in vitro
growth of six different PTLD-like Epstein–Barr virus+ lymphoblastoid B cell lines. Similar to normal T cells, RAD blocked cell-cycle progression in PTLD-like B cells in the early (G
0
/G
1
) phase. Furthermore, RAD increased the apoptotic rate in such cells. The drug also had a profound inhibitory effect on the growth of PTLD-like Epstein–Barr virus+ B cells xenotransplanted s.c. into SCID mice. The degree of the RAD effect varied among the three B cell lines tested and was proportional to its effects on the cell lines
in vitro
. In this
in vivo
xenotransplant model, RAD markedly delayed growth or induced regression of the established tumors. In one line, it was able to eradicate the tumor in four of eight mice. When RAD treatment was initiated before tumor cell injection, a marked inhibition of tumor growth was seen in all three lines. In two of them, the drug prevented tumor establishment in approximately 50% of mice (5/11 and 5/8). In summary, RAD is a potent inhibitor of PTLD-like cells
in vitro
and
in vivo
. These findings indicate that, in contrast to the standard immunosuppressive agents, macrolides such as RAD may be effective in prevention and treatment of PTLDs.
Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2
Authors: Deniz Ashok A. , Laurence Ted A. , Beligere Gangamani S. , Dahan Maxime , Martin Andrew B. , Chemla Daniel S. , Dawson Philip E. , Schultz Peter G. , Weiss Shimon
Published: July 2002

We report single-molecule folding studies of a small, single-domain protein, chymotrypsin inhibitor 2 (CI2). CI2 is an excellent model system for protein folding studies and has been extensively studied, both experimentally (at the ensemble level) and theoretically. Conformationally assisted ligation methodology was used to synthesize the proteins and site-specifically label them with donor and acceptor dyes. Folded and denatured subpopulations were observed by fluorescence resonance energy transfer (FRET) measurements on freely diffusing single protein molecules. Properties of these subpopulations were directly monitored as a function of guanidinium chloride concentration. It is shown that new information about different aspects of the protein folding reaction can be extracted from such subpopulation properties. Shifts in the mean transfer efficiencies are discussed, FRET efficiency distributions are translated into potentials, and denaturation curves are directly plotted from the areas of the FRET peaks. Changes in stability caused by mutation also are measured by comparing pseudo wild-type CI2 with a destabilized mutant (K17G). Current limitations and future possibilities and prospects for single-pair FRET protein folding investigations are discussed.
Surface expression and function of p75/AIRM-1 or CD33 in acute myeloid leukemias: Engagement of CD33 induces apoptosis of leukemic cells
Authors: Vitale Chiara , Romagnani Chiara , Puccetti Antonio , Olive Daniel , Costello Regis , Chiossone Laura , Pitto Anna , Bacigalupo Andrea , Moretta Lorenzo , Mingari Maria Cristina
Published: July 2002

p75/AIRM-1 is a recently identified inhibitory receptor expressed by natural killer and myeloid cells displaying high homology with CD33. Crosslinking of p75/AIRM-1 or CD33 has been shown to sharply inhibit thein vitroproliferation of both normal myeloid cells and chronic myeloid leukemias. In this study, we analyzed acute myeloid leukemic cells for the expression of p75/AIRM-1. p75/AIRM-1 marked the M5 (11/12) and M4 (2/2) but not the M1, M2, and M3 subtypes according to the French–American–British classification. Cell samples from 12 acute myeloid leukemias were cultured in the presence of granulocyte/macrophage colony-stimulating factor. Addition to these cultures of anti-CD33 antibody resulted in ≈70% inhibition of cell proliferation as assessed by [3H]thymidine uptake or by the recovery of viable cells. Anti-p75/AIRM-1 antibody exerted a strong inhibitory effect only in two cases characterized by a highin vitroproliferation rate. After crosslinking of CD33 (but not of p75/AIRM-1), leukemic cells bound Annexin V and displayed changes in their light-scattering properties and nucleosomal DNA fragmentation, thus providing evidence for the occurrence of apoptotic cell death. Remarkably, when anti-CD33 antibody was used in combination with concentrations of etoposide insufficient to induce apoptosis when used alone, a synergistic effect could be detected in the induction of leukemic cell death. These studies provide the rationale for new therapeutic approaches in myeloid leukemias by using both chemotherapy and apoptosis-inducing mAbs.
Population maintenance among tropical reef fishes: Inferences from small-island endemics
Authors: Robertson D. Ross
Published: July 2002

To what extent do local populations of tropical reef fishes persist
through the recruitment of pelagic larvae to their natal reef? Endemics
from small, isolated islands can help answer that question by
indicating whether special biological attributes are needed for
long-term survival under enforced localization in high-risk situations.
Taxonomically and biologically, the endemics from seven such islands
are broadly representative of their regional faunas. As natal-site
recruitment occurs among reef fishes in much less isolated situations,
these characteristics of island endemics indicate that a wide range of
reef fishes could have persistent self-sustaining local populations.
Because small islands regularly support substantial reef fish faunas,
regional systems of small reserves could preserve much of the diversity
of these fishes.
Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions
Authors: Thornton Joseph W.
Published: July 2002

The evolution of novelty in tightly integrated biological systems, such as hormones and their receptors, seems to challenge the theory of natural selection: it has not been clear how a new function for any one part (such as a ligand) can be selected for unless the other members of the system (e.g., a receptor) are already present. Here I show—based on identification and phylogenetic analysis of steroid receptors in basal vertebrates and reconstruction of the sequences and functional attributes of ancestral proteins—that the first steroid receptor was an estrogen receptor, followed by a progesterone receptor. Genome mapping and phylogenetic analyses indicate that the full complement of mammalian steroid receptors evolved from these ancient receptors by two large-scale genome expansions, one before the advent of jawed vertebrates and one after. Specific regulation of physiological processes by androgens and corticoids are relatively recent innovations that emerged after these duplications. These findings support a model of ligand exploitation in which the terminal ligand in a biosynthetic pathway is the first for which a receptor evolves; selection for this hormone also selects for the synthesis of intermediates despite the absence of receptors, and duplicated receptors then evolve affinity for these substances. In this way, novel hormone-receptor pairs are created, and an integrated system of increasing complexity elaborated. This model suggests that ligands for some “orphan” receptors may be found among intermediates in the synthesis of ligands for phylogenetically related receptors.
Top-down free-energy minimization on protein potential energy landscapes
Authors: Church Bruce W. , Shalloway David
Published: July 2002

The hierarchical properties of potential energy landscapes have
been used to gain insight into thermodynamic and kinetic properties of
protein ensembles. It also may be possible to use them to direct
computational searches for thermodynamically stable macroscopic states,
i.e., computational protein folding. To this end, we have developed a
top-down search procedure in which conformation space is recursively
dissected according to the intrinsic hierarchical structure of a
landscape's effective-energy barriers. This procedure generates an
inverted tree similar to the disconnectivity graphs generated by local
minima-clustering methods, but it fundamentally differs in the manner
in which the portion of the tree that is to be computationally explored
is selected. A key ingredient is a branch-selection algorithm that
takes advantage of statistically predictive properties of the landscape
to guide searches down the tree branches that are most likely to lead
to the physically relevant macroscopic states. Using the computational
folding of a β-hairpin-forming peptide as an example, we show that
such predictive properties indeed exist and can be used for structure
prediction by free-energy global minimization.
A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ
Authors: Hashimoto Yuichi , Niikura Takako , Tajima Hirohisa , Yasukawa Takashi , Sudo Haruka , Ito Yuko , Kita Yoshiko , Kawasumi Masaoki , Kouyama Keisuke , Doyu Manabu , Sobue Gen , Koide Takashi , Tsuji Shoji , Lang Jochen , Kurokawa Kiyoshi , Nishimoto Ikuo
Published: July 2002

Through functional expression screening, we identified a
gene, designated Humanin (HN) cDNA, which encodes a short polypeptide
and abolishes death of neuronal cells caused by multiple different
types of familial Alzheimer's disease genes and by Aβ amyloid,
without effect on death by Q79 or superoxide dismutase-1 mutants.
Transfected HN cDNA was transcribed to the corresponding polypeptide
and then was secreted into the cultured medium. The rescue action
clearly depended on the primary structure of HN. This polypeptide would
serve as a molecular clue for the development of new therapeutics for
Alzheimer's disease targeting neuroprotection.
Insulin-like growth factor 2 and potential regulators of hemangioma growth and involution identified by large-scale expression analysis
Authors: Ritter Matthew R. , Dorrell Michael I. , Edmonds Joseph , Friedlander Sheila Fallon , Friedlander Martin
Published: July 2002

Hemangiomas are benign tumors of the vascular endothelium and are the most common tumors of infancy. These tumors are characterized by an initial phase of rapid proliferation, which is followed, in most cases, by spontaneous involution. Although most lesions resolve without complication, there are some cases in which hemangiomas can be life threatening when occurring near a vital structure. Treatment for these aggressive tumors represents an unmet clinical need. In addition, this characteristic progression of hemangiomas through distinct phases provides a unique opportunity for studying endothelial cell biology and angiogenesis. Using DNA microarrays representing approximately 10,000 human genes, we identified insulin-like growth factor 2 (IGF-2) as a potentially important regulator of hemangioma growth. IGF-2 was highly expressed during the proliferative phase and substantially decreased during involution. This finding was confirmed at the message level by quantitative reverse transcription–PCR and at the protein level by immunohistochemistry. IGF-2 protein was localized primarily to tumor vessels or vascular channels. Using a human hemangioma explant model, we show that IGF-2 promotes sprouting from intact hemangioma tissue. In addition, several angiogenesis-related factors, including integrins αvβ3 and α5β1, are present in proliferating hemangiomas. During the involuting phase, an increase in several IFN-induced genes was observed. These studies identify potential regulators of hemangioma growth and involution and provide a foundation on which to build further mechanistic investigations into angiogenesis, using hemangiomas as a model.
Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled
Authors: McGlynn Peter , Lloyd Robert G. , Marians Kenneth J.
Published: July 2002

Replication forks formed at bacterial origins often encounter template roadblocks in the form of DNA adducts and frozen protein–DNA complexes, leading to replication-fork stalling and inactivation. Subsequent correction of the corrupting template lesion and origin-independent assembly of a new replisome therefore are required for survival of the bacterium. A number of models for replication-fork restart under these conditions posit that nascent strand regression at the stalled fork generates a Holliday junction that is a substrate for subsequent processing by recombination and repair enzymes. We show here that early replication intermediates containing replication forks stalled
in vitro
by the accumulation of excess positive supercoils could be cleaved by the Holliday junction resolvases RusA and RuvC. Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecG, confirming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent strand regression at stalled replication forks. Furthermore, RecG promoted Holliday junction formation when replication intermediates in which the replisome had been inactivated were negatively supercoiled, suggesting that under intracellular conditions, the action of RecG, or helicases with similar activities, is necessary for the catalysis of nascent strand regression.
Effects of forced diving on the spleen and hepatic sinus in northern elephant seal pups
Authors: Thornton Sheila J. , Spielman Daniel M. , Pelc Norbert J. , Block Wally F. , Crocker Daniel E. , Costa Daniel P. , LeBoeuf Burney J. , Hochachka Peter W.
Published: July 2002

In phocid seals, an increase in hematocrit (Hct) accompanies diving and periods of apnea. The variability of phocid Hct suggests that the total red cell mass is not always in circulation, leading researchers to speculate on the means of blood volume partitioning. The histology and disproportionate size of the phocid spleen implicates it as the likely site for RBC storage. We used magnetic resonance imaging on Northern elephant seals to demonstrate a rapid contraction of the spleen and a simultaneous filling of the hepatic sinus during forced dives (
P
< 0.0001,
R
2
= 0.97). The resulting images are clear evidence demonstrating a functional relationship between the spleen and hepatic sinus. The transfer of blood from the spleen to the sinus provides an explanation for the disparity between the timing of diving-induced splenic contraction (≈1–3 min) and the occurrence of peak Hct (15–25 min). Facial immersion was accompanied by an immediate and profound splenic contraction, with no further significant decrease in splenic volume after min 2 (Tukey–Kramer HSD,
P
= 0.05). At the conclusion of the dive, the spleen had contracted to 16% of its predive volume (mean resting splenic volume = 3,141 ml ± 68.01 ml; 3.54% of body mass). In the postdive period, the spleen required 18–22 min to achieve resting volume, indicating that this species may not have sufficient time to refill the spleen when routinely diving at sea, which is virtually continuous with interdive surface intervals between 1 and 3 min.
TARP: A nuclear protein expressed in prostate and breast cancer cells derived from an alternate reading frame of the T cell receptor γ chain locus
Authors: Wolfgang Curt D. , Essand Magnus , Vincent James J. , Lee Byungkook , Pastan Ira
Published: July 2002

Previously, we identified the expression of a prostate-specific form of T cell receptor γ chain (
TCRγ
) mRNA in the human prostate and demonstrated that it originates from epithelial cells and not from infiltrating T lymphocytes. Here, we show that this prostate-specific transcript is also expressed in three breast cancer cell lines and breast cancer tissues. Analysis of the cDNA sequence predicts that this transcript can encode two protein products of 7 and 13 kDa, and
in vitro
translation experiments showed that both proteins were made. The longer ORF encodes a 13-kDa truncated version of TCRγ, whereas the shorter alternative reading frame encodes a 7-kDa protein with five leucine residues in heptad repeats followed by a basic region. Studies with specific antibodies against each protein product revealed that both prostate and breast cancer cells contain only the 7-kDa protein, which is located in the nucleus. We have named this protein TCRγ alternate reading frame protein (TARP). These results demonstrate that an alternative protein product is encoded by the
TCRγ
locus in cells other than T lymphocytes.
A soluble class II cytokine receptor, IL-22RA2, is a naturally occurring IL-22 antagonist
Authors: Xu Wenfeng , Presnell Scott R. , Parrish-Novak Julia , Kindsvogel Wayne , Jaspers Steve , Chen Zhi , Dillon Stacey R. , Gao Zeren , Gilbert Teresa , Madden Karen , Schlutsmeyer Stacy , Yao Lena , Whitmore Theodore E. , Chandrasekher Yasmin , Grant Francis J. , Maurer Mark , Jelinek Laura , Storey Harold , Brender Ty , Hammond Angie , Topouzis Stavros , Clegg Christopher H. , Foster Donald C.
Published: July 2002

IL-22 is an IL-10 homologue that binds to and signals through the class II cytokine receptor heterodimer IL-22RA1/CRF2–4. IL-22 is produced by T cells and induces the production of acute-phase reactantsin vitroandin vivo, suggesting its involvement in inflammation. Here we report the identification of a class II cytokine receptor designated IL-22RA2 (IL-22 receptor-α 2) that appears to be a naturally expressed soluble receptor. IL-22RA2 shares amino acid sequence homology with IL-22RA1 (also known as IL-22R, zcytor11, and CRF2–9) and is physically adjacent to IL-20Rα and IFN-γR1 on chromosome 6q23.3–24.2. We demonstrate that IL-22RA2 binds specifically to IL-22 and neutralizes IL-22-induced proliferation of BaF3 cells expressing IL-22 receptor subunits. IL-22RA2 mRNA is highly expressed in placenta and spleen by Northern blotting. PCR analysis using RNA from various tissues and cell lines showed that IL-22RA2 was expressed in a range of tissues, including those in the digestive, female reproductive, and immune systems.In situhybridization revealed the dominant cell types expressing IL-22RA2 were mononuclear cells and epithelium. Because IL-22 induces the expression of acute phase reactants, IL-22RA2 may play an important role as an IL-22 antagonist in the regulation of inflammatory responses.
The interplay of biology and technology
Authors: Fields Stanley
Published: July 2002

Technologies for biological research arise in multiple ways—through serendipity, through inspired insights, and through incremental advances—and they are tightly coupled to progress in engineering. Underlying the complex dynamics of technology and biology are the different motivations of those who work in the two realms. Consideration of how methodologies emerge has implications for the planning of interdisciplinary centers and the training of the next generation of scientists.
β-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins
Authors: Chen Wei , Hu Liaoyuan A. , Semenov Mikhail V. , Yanagawa Shinichi , Kikuchi Akira , Lefkowitz Robert J. , Miller William E.
Published: July 2002

One aspect of the function of the β-arrestins is to serve as
scaffold or adapter molecules coupling G-protein coupled receptors
(GPCRs) to signal transduction pathways distinct from traditional
second messenger pathways. Here we report the identification of
Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as β-arrestin1
(βarr1) interacting proteins. Dvl proteins participate as key
intermediates in signal transmission from the seven membrane-spanning
Frizzled receptors leading to inhibition of glycogen synthase
kinase-3β (GSK-3β), stabilization of β-catenin, and activation of
the lymphoid enhancer factor (LEF) transcription factor. We find that
phosphorylation of Dvl strongly enhances its interaction with βarr1,
suggesting that regulation of Dvl phosphorylation and subsequent
interaction with βarr1 may play a key role in the activation of the
LEF transcription pathway. Because coexpression of the Dvl kinases,
CK1ɛ and PAR-1, with Dvl synergistically activates LEF reporter gene
activity, we reasoned that coexpression of βarr1 with Dvl might also
affect LEF-dependent gene activation. Interestingly, whereas βarr1 or
Dvl alone leads to low-level stimulation of LEF (2- to 5-fold),
coexpression of βarr1 with either Dvl1 or Dvl2 leads to a synergistic
activation of LEF (up to 16-fold). Additional experiments with LiCl as
an inhibitor of GSK-3β kinase activity indicate that the step
affected by βarr1 is upstream of GSK-3β and most likely at the
level of Dvl. These results identify βarr1 as a regulator of
Dvl-dependent LEF transcription and suggest that βarr1 might serve as
an adapter molecule that can couple Frizzled receptors and perhaps
other GPCRs to these important transcription pathways.
Origin of mitochondria in relation to evolutionary history of eukaryotic alanyl-tRNA synthetase
Authors: Chihade Joseph W. , Brown James R. , Schimmel Paul R. , Ribas de Pouplana Lluís
Published: July 2002

The origin of the eukaryotic cell remains an unsolved question.
Numerous experimental and phylogenetic observations support the
symbiotic origin of the modern eukaryotic cell, with its nucleus and
(typically) mitochondria. Incorporation of mitochondria has been
proposed to precede development of the nucleus, but it is still unclear
whether mitochondria were initially part of basal eukaryotes. Data on
alanyl-tRNA synthetase from an early eukaryote and other sources are
presented and analyzed here. These data are consistent with the notion
that mitochondrial genesis did not significantly precede nucleus
formation. Moreover, the data raise the possibility that diplomonads
are primary amitochondriates that radiated from the eukaryotic lineage
before mitochondria became fully integrated as a cellular organelle.
A molecular profile of the mouse gastric parietal cell with and without exposure to
Helicobacter pylori
Authors: Mills Jason C. , Syder Andrew J. , Hong Chieu V. , Guruge Janaki L. , Raaii Farhang , Gordon Jeffrey I.
Published: July 2002

The parietal cell (PC) plays an important role in normal gastric physiology and in common diseases of the stomach. Although the genes involved in acid secretion are well known, there is limited molecular information about other aspects of PC function. We have generated a comprehensive database of genes expressed preferentially in PCs relative to other gastric mucosal cell lineages. PCs were purified from FVB/N mouse stomachs by lectin panning. cRNA generated from PC-enriched (PC
+
) and PC-depleted (PC
−
) populations were used to query oligonucleotide-based microarrays. False-positive signals were filtered by using a new algorithm for noise reduction and selected results independently audited by real-time quantitative reverse transcription (RT)-PCR. The annotated database of 240 genes reveals previously unappreciated aspects of cellular function, including factors that may mediate PC regulation of gastric stem cell proliferation. PC
+
and PC
−
expression profiles were also prepared from germ-free mice 2 and 8 weeks after colonization with a clinical isolate of
Helicobacter pylori
(Hp)—the pathogen that produces acid-peptic disease (gastritis, ulcers) in humans. Whereas PC
+
gene expression was remarkably constant, the PC
−
fractions demonstrated a robust, evolving host response, with increased expression of genes involved in cell motility/migration, extracellular matrix interactions, and IFN responses. The consistency of PC
+
gene expression allowed identification of a cohort of 92 genes enriched in PCs under
all
conditions studied. These genes provide a molecular profile that can be used to define this epithelial lineage under a variety of physiologic, pharmacologic, and pathologic stimuli.
Antisense imaging of gene expression in the brain
in vivo
Authors: Shi Ningya , Boado Ruben J. , Pardridge William M.
Published: July 2002

Antisense radiopharmaceuticals could be used to image gene
expression in the brain
in vivo
, should these polar
molecules be made transportable through the blood–brain barrier. The
present studies describe an antisense imaging agent comprised of an
iodinated peptide nucleic acid (PNA) conjugated to a monoclonal
antibody to the rat transferrin receptor by using avidin–biotin
technology. The PNA was a 16-mer antisense to the sequence around the
methionine initiation codon of the luciferase mRNA. C6 rat glioma cells
were permanently transfected with a luciferase expression plasmid, and
C6 experimental brain tumors were developed in adult rats. The
expression of the luciferase transgene in the tumors
in
vivo
was confirmed by measurement of luciferase enzyme activity
in the tumor extract. The [
125
I]PNA conjugate was
injected intravenously in anesthetized animals with brain tumors and
killed 2 h later for frozen sectioning of brain and film
autoradiography. No image of the luciferase gene expression was
obtained after the administration of either the unconjugated
antiluciferase PNA or a PNA conjugate that was antisense to the mRNA of
a viral transcript. In contrast, tumors were imaged in all rats
administered the [
125
I]PNA that was antisense to the
luciferase sequence and was conjugated to the targeting antibody. In
conclusion, these studies demonstrate gene expression in the brain
in vivo
can be imaged with antisense
radiopharmaceuticals that are conjugated to a brain drug-targeting
system.
Genomewide studies of histone deacetylase function in yeast
Authors: Bernstein Bradley E. , Tong Jeffrey K. , Schreiber Stuart L.
Published: July 2002

The trichostatin A (TSA)-sensitive histone deacetylase (HDAC) Rpd3p
exists in a complex with Sin3p and Sap30p in yeast that is recruited to
target promoters by transcription factors including Ume6p. Sir2p is a
TSA-resistant HDAC that mediates yeast silencing. The transcription
profile of
rpd3
is similar to the profiles of
sin3
,
sap30
,
ume6
, and
TSA-treated wild-type yeast. A Ume6p-binding site was identified in the
promoters of genes up-regulated in the
sin3
strain. Two
genes appear to participate in feedback loops that modulate HDAC
activity:
ZRT1
encodes a zinc transporter and is
repressed by
RPD3
(Rpd3p is zinc-dependent);
BNA1
encodes a nicotinamide adenine dinucleotide
(NAD)-biosynthesis enzyme and is repressed by
SIR2
(Sir2p is NAD-dependent). Although HDACs are transcriptional
repressors, deletion of
RPD3
down-regulates certain
genes. Many of these are down-regulated rapidly by TSA, indicating that
Rpd3p may also activate transcription. Deletion of
RPD3
previously has been shown to repress (“silence”) reporter genes
inserted near telomeres. The profiles demonstrate that 40% of
endogenous genes located within 20 kb of telomeres are
down-regulated by
RPD3
deletion. Rpd3p appears to
activate telomeric genes sensitive to histone depletion indirectly by
repressing transcription of histone genes. Rpd3p also appears to
activate telomeric genes repressed by the silent information regulator
(SIR) proteins directly, possibly by deacetylating lysine 12 of histone
H4. Finally, bioinformatic analyses indicate that the yeast HDACs
RPD3
,
SIR2
, and
HDA1
play
distinct roles in regulating genes involved in cell cycle progression,
amino acid biosynthesis, and carbohydrate transport and utilization,
respectively.
Phototransduction in transgenic mice after targeted deletion of the rod transducin α-subunit
Authors: Calvert P. D. , Krasnoperova N. V. , Lyubarsky A. L. , Isayama T. , Nicoló M. , Kosaras B. , Wong G. , Gannon K. S. , Margolskee R. F. , Sidman R. L. , Pugh E. N. , Makino C. L. , Lem J.
Published: July 2002

Retinal photoreceptors use the heterotrimeric G protein
transducin to couple rhodopsin to a biochemical cascade that underlies
the electrical photoresponse. Several isoforms of each transducin
subunit are present in the retina. Although rods and cones seem to
contain distinct transducin subunits, it is not known whether
phototransduction in a given cell type depends strictly on a single
form of each subunit. To approach this question, we have deleted the
gene for the rod transducin α-subunit in mice. In hemizygous knockout
mice, there was a small reduction in retinal transducin α-subunit
content but retinal morphology and the physiology of single rods were
largely normal. In homozygous knockout mice, a mild retinal
degeneration occurred with age. Rod-driven components were absent from
the electroretinogram, whereas cone-driven components were retained.
Every photoreceptor examined by single-cell recording failed to respond
to flashes, with one exception. The solitary responsive cell was
insensitive, as expected for a cone, but had a rod-like spectral
sensitivity and flash response kinetics that were slow, even for rods.
These results indicate that most if not all rods use a single
transducin type in phototransduction.
Active barnase variants with completely random hydrophobic cores.
Authors: Axe D D , Foster N W , Fersht A R
Published: July 2002

The central structural feature of natural proteins is a tightly packed and highly ordered hydrophobic core. If some measure of exquisite, native-like core packing is necessary for enzymatic function, this would constitute a significant obstacle to the development of novel enzymes, either by design or by natural or experimental evolution. To test the minimum requirements for a core to provide sufficient structural integrity for enzymatic activity, we have produced mutants of the ribonuclease barnase in which 12 of the 13 core residues have together been randomly replaced by hydrophobic alternatives. Using a sensitive biological screen, we find that a strikingly high proportion of these mutants (23%) retain enzymatic activity in vivo. Further substitution at the 13th core position shows that a similar proportion of completely random hydrophobic cores supports enzyme function. Of the active mutants produced, several have no wild-type core residues. These results imply that hydrophobicity is nearly a sufficient criterion for the construction of a functional core and, in conjunction with previous studies, that refinement of a crudely functional core entails more stringent sequence constraints than does the initial attainment of crude core function. Since attainment of crude function is the critical initial step in evolutionary innovation, the relatively scant requirements contributed by the hydrophobic core would greatly reduce the initial hurdle on the evolutionary pathway to novel enzymes. Similarly, experimental development of novel functional proteins might be simplified by limiting core design to mere specification of hydrophobicity and using iterative mutation-selection to optimize core structure.
Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases.
Authors: Meyer K , Cusumano J C , Somerville C , Chapple C C
Published: July 2002

The fah1 mutant of Arabidopsis is defective in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. Earlier results indicated that the FAH1 locus encodes ferulate-5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase (P450) of the general phenylpropanoid pathway. We have cloned the gene encoding this P450 by T-DNA tagging and have confirmed the identity of the cloned gene by complementation of the mutant phenotype. F5H shows 34% amino acid sequence identity with the avocado ripening-induced P450 CYP71A1 and 32% identity with the flavonoid-3',5'-hydroxylases of Petunia hybrida. In contrast, it shares much less homology with cinnamate-4-hydroxylase, a P450 that catalyzes the hydroxylation of cinnamic acid three steps earlier in the general phenylpropanoid pathway. Since the highest degree of identity between F5H and previously sequenced P450s is only 34%, F5H identifies a new P450 subfamily that has been designated CYP84.
Selection for genes encoding secreted proteins and receptors.
Authors: Klein R D , Gu Q , Goddard A , Rosenthal A
Published: July 2002

Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.
A common origin for woody Sonchus and five related genera in the Macaronesian islands: molecular evidence for extensive radiation.
Authors: Kim S C , Crawford D J , Francisco-Ortega J , Santos-Guerra A
Published: July 2002

Woody Sonchus and five related genera (Babcockia, Taeckholmia, Sventenia, Lactucosonchus, and Prenanthes) of the Macaronesian islands have been regarded as an outstanding example of adaptive radiation in angiosperms. Internal transcribed spacer region of the nuclear rDNA (ITS) sequences were used to demonstrate that, despite the extensive morphological and ecological diversity of the plants, the entire alliance in insular Macaronesia has a common origin. The sequence data place Lactucosonchus as sister group to the remainder of the alliance and also indicate that four related genera are in turn sister groups to subg. Dendrosonchus and Taeckholmia. This implies that the woody members of Sonchus were derived from an ancestor similar to allied genera now present on the Canary Islands. It is also evident that the alliance probably occurred in the Canary Islands during the late Miocene or early Pliocene. A rapid radiation of major lineages in the alliance is consistent with an unresolved polytomy near the base and low ITS sequence divergence. Increase of woodiness is concordant with other insular endemics and refutes the relictural nature of woody Sonchus in the Macaronesian islands.
Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.
Authors: Hosaka N , Nose M , Kyogoku M , Nagata N , Miyashima S , Good R A , Ikehara S
Published: July 2002

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.
Mutations affecting conserved cysteine residues within the extracellular domain of Neu promote receptor dimerization and activation.
Authors: Siegel P M , Muller W J
Published: July 2002

Overexpression of the Neu/ErbB-2 receptor tyrosine kinase has been implicated in the genesis of human breast cancer. Indeed, expression of either activated or wild-type neu in the mammary epithelium of transgenic mice results in the induction of mammary tumors. Previously, we have shown that many of the mammary tumors arising in transgenic mice expressing wild-type neu occur through somatic activating mutations within the neu transgene itself. Here we demonstrate that these mutations promote dimerization of the Neu receptor through the formation of disulfide bonds, resulting in its constitutive activation. To explore the role of conserved cysteine residues within the region deleted in these altered Neu proteins, we examined the transforming potential of a series of Neu receptors in which the individual cysteine residues were mutated. These analyses indicated that mutation of certain cysteine residues resulted in the oncogenic activation of Neu. The increased transforming activity displayed by the altered receptors correlated with constitutive dimerization that occurred in a disulfide bond-dependent manner. We further demonstrate that addition of 2-mercaptoethanol to the culture medium interfered with the specific transforming activity of the mutant Neu receptors. These observations suggest that oncogenic activation of Neu results from constitutive disulfide bond-dependent dimerization.
Transloading of tumor cells with foreign major histocompatibility complex class I peptide ligand: a novel general strategy for the generation of potent cancer vaccines.
Authors: Schmidt W , Steinlein P , Buschle M , Schweighoffer T , Herbst E , Mechtler K , Kirlappos H , Birnstiel M L
Published: July 2002

The major hurdle to be cleared in active immunotherapy of cancer is the poor immunogenicity of cancer cells. In previous attempts to overcome this problem, whole tumor cells have been used as vaccines, either admixed with adjuvant(s) or genetically engineered to express nonself proteins or immunomodulatory factors before application. We have developed a novel approach to generate an immunogeneic, highly effective vaccine: major histocompatibility complex (MHC) class I-positive cancer cells are administered together with MHC class I-matched peptide ligands of foreign, nonself origin, generated by a procedure we term transloading. Murine tumor lines of the H2-Kd or the H2-Db haplotype, melanoma M-3 and B16-F10, respectively, as well as colon carcinoma CT-26 (H2-Kd), were transloaded with MHC-matched influenza virus-derived peptides and applied as irradiated vaccines. Mice bearing a deposit of live M-3 melanoma cells were efficiently cured by this treatment. In the CT-26 colon carcinoma and the B16-F10 melanoma, high efficacies were obtained against tumor challenge, suggesting the universal applicability of this new type of vaccine. With foreign peptide ligands adapted to the requirements of a desired MHC class I haplotype, this concept may be used for the treatment of human cancers.
Human prostate tumor growth in athymic mice: inhibition by androgens and stimulation by finasteride.
Authors: Umekita Y , Hiipakka R A , Kokontis J M , Liao S
Published: July 2002

When the human prostate cancer cell line, LNCaP 104-S, the growth of which is stimulated by physiological levels of androgen, is cultured in androgen-depleted medium for > 100 passages, the cells, now called LNCaP 104-R2, are proliferatively repressed by low concentrations of androgens. LNCaP 104-R2 cells formed tumors in castrated male athymic nude mice. Testosterone propionate (TP) treatment prevented LNCaP 104-R2 tumor growth and caused regression of established tumors in these mice. Such a tumor-suppressive effect was not observed with tumors derived from LNCaP 104-S cells or androgen receptor-negative human prostate cancer PC-3 cells. 5 alpha-Dihydrotestosterone, but not 5 beta-dihydrotestosterone, 17 beta-estradiol, or medroxyprogesterone acetate, also inhibited LNCaP 104-R2 tumor growth. Removal of TP or implantation of finasteride, a 5 alpha-reductase inhibitor, in nude mice bearing TP implants resulted in the regrowth of LNCaP 104-R2 tumors. Within 1 week after TP implantation, LNCaP 104-R2 tumors exhibited massive necrosis with severe hemorrhage. Three weeks later, these tumors showed fibrosis with infiltration of chronic inflammatory cells and scattered carcinoma cells exhibiting degeneration. TP treatment of mice with LNCaP 104-R2 tumors reduced tumor androgen receptor and c-myc mRNA levels but increased prostate-specific antigen in serum- and prostate-specific antigen mRNA in tumors. Although androgen ablation has been the standard treatment for metastatic prostate cancer for > 50 years, our study shows that androgen supplementation therapy may be beneficial for treatment of certain types of human prostate cancer and that the use of 5 alpha-reductase inhibitors, such as finasteride or anti-androgens, in the general treatment of metastatic prostate cancer may require careful assessment.
The challenge of contracting for technological information
Authors: Zeckhauser Richard
Published: July 2002

Contracting to provide technological information (TI) is a
significant challenge. TI is an unusual commodity in five ways.
(
i
) TI is difficult to count and value; conventional
indicators, such as patents and citations, hardly indicate value. TI is
often sold at different prices to different parties.
(
ii
) To value TI, it may be necessary to “give away
the secret.” This danger, despite nondisclosure agreements, inhibits
efforts to market TI. (
iii
) To prove its value, TI is
often bundled into complete products, such as a computer chip or
pharmaceutical product. Efficient exchange, by contrast, would involve
merely the raw information. (
iv
) Sellers’ superior
knowledge about TI’s value make buyers wary of overpaying.
(
v
) Inefficient contracts are often designed to secure
rents from TI. For example, licensing agreements charge more than
marginal cost. These contracting difficulties affect the way TI is
produced, encouraging self-reliance. This should be an advantage to
large firms. However, small research and development firms spend more
per employee than large firms, and nonprofit universities are major
producers. Networks of organizational relationships, particularly
between universities and industry, are critical in transmitting TI.
Implicit barter—money for guidance—is common. Property rights for TI
are hard to establish. Patents, quite suitable for better mousetraps,
are inadequate for an era when we design better mice. Much TI is not
patented, and what is patented sets fuzzy demarcations. New
organizational forms are a promising approach to contracting
difficulties for TI. Webs of relationships, formal and informal,
involving universities, start-up firms, corporate giants, and venture
capitalists play a major role in facilitating the production and spread
of TI.
“Agrolistic” transformation of plant cells: Integration of T-strands generated
in planta
Authors: Hansen Geneviève , Chilton Mary-Dell
Published: July 2002

We describe a novel plant transformation technique, termed
“agrolistic,” that combines the advantages of the
Agrobacterium
transformation system with the high
efficiency of biolistic DNA delivery. Agrolistic transformation allows
integration of the gene of interest without undesired vector sequence.
The virulence genes
vir
D1 and
vir
D2 from
Agrobacterium tumefaciens
that are required in bacteria
for excision of T-strands from the tumor-inducing plasmid were placed
under the control of the CaMV35S promoter and codelivered with a target
plasmid containing border sequences flanking the gene of interest.
Transient expression assays in tobacco and in maize cells indicated
that
vir
gene products caused strand-specific nicking
in planta
at the right border sequence, similar to
VirD1/VirD2-catalyzed T-strand excision observed in
Agrobacterium
. Agrolistically transformed tobacco calli
were obtained after codelivery of
vir
D1 and
vir
D2 genes together with a selectable marker flanked by
border sequences. Some inserts exhibited right junctions with plant DNA
that corresponded precisely to the sequence expected for T-DNA (portion
of the tumor-inducing plasmid that is transferred to plant cells)
insertion events. We designate these as “agrolistic” inserts, as
distinguished from “biolistic” inserts. Both types of inserts
were found in some transformed lines. The frequency of agrolistic
inserts was 20% that of biolistic inserts.