bluuebunny/crossref_metadata_2025 huggingface dataset:
The nitric oxide/cyclic GMP pathway in organ transplantation: critical role in successful lung preservation.
Authors: Pinsky D J , Naka Y , Chowdhury N C , Liao H , Oz M C , Michler R E , Kubaszewski E , Malinski T , Stern D M
Published: May 2006

Reestablishment of vascular homeostasis following ex vivo preservation is a critical determinant of successful organ transplantation. Because the nitric oxide (NO) pathway modulates pulmonary vascular tone and leukocyte/endothelial interactions, we hypothesized that reactive oxygen intermediates would lead to decreased NO (and hence cGMP) levels following pulmonary reperfusion, leading to increased pulmonary vascular resistance and leukostasis. Using an orthotopic rat model of lung transplantation, a porphyrinic microsensor was used to make direct in vivo measurements of pulmonary NO. NO levels measured at the surface of the transplanted lung plummeted immediately upon reperfusion, with levels moderately increased by topical application of superoxide dismutase. Because cGMP levels declined in preserved lungs after reperfusion, this led us to buttress the NO pathway by adding a membrane-permeant cGMP analog to the preservation solution. Compared with grafts stored in its absence, grafts stored with supplemental 8-Br-cGMP and evaluated 30 min after reperfusion demonstrated lower pulmonary vascular resistances with increased graft blood flow, improved arterial oxygenation, decreased neutrophil infiltration, and improved recipient survival. These beneficial effects were dose dependent, mimicked by the type V phosphodiesterase inhibitor 2-o-propoxyphenyl-8-azapurin-6-one, and inhibited by a cGMP-dependent protein kinase antagonist, the R isomer of 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate. Augmenting the NO pathway at the level of cGMP improves graft function and recipient survival following lung transplantation.
Detection of conserved segments in proteins: iterative scanning of sequence databases with alignment blocks.
Authors: Tatusov R L , Altschul S F , Koonin E V
Published: May 2006

We describe an approach to analyzing protein sequence databases that, starting from a single uncharacterized sequence or group of related sequences, generates blocks of conserved segments. The procedure involves iterative database scans with an evolving position-dependent weight matrix constructed from a coevolving set of aligned conserved segments. For each iteration, the expected distribution of matrix scores under a random model is used to set a cutoff score for the inclusion of a segment in the next iteration. This cutoff may be calculated to allow the chance inclusion of either a fixed number or a fixed proportion of false positive segments. With sufficiently high cutoff scores, the procedure converged for all alignment blocks studied, with varying numbers of iterations required. Different methods for calculating weight matrices from alignment blocks were compared. The most effective of those tested was a logarithm-of-odds, Bayesian-based approach that used prior residue probabilities calculated from a mixture of Dirichlet distributions. The procedure described was used to detect novel conserved motifs of potential biological importance.
Cloning of ELL, a gene that fuses to MLL in a t(11;19)(q23;p13.1) in acute myeloid leukemia.
Authors: Thirman M J , Levitan D A , Kobayashi H , Simon M C , Rowley J D
Published: May 2006

To characterize the functions of MLL fusion transcripts, we cloned the gene that fuses to MLL in the translocation t(11;19)(q23;p13.1). This translocation is distinct from another type of 11;19 translocation with a 19p13.3 breakpoint that results in the fusion of MLL to the ENL gene. By PCR screening of a cDNA library prepared from a patient's leukemia cells with this translocation, we obtained a fusion transcript containing exon 7 of MLL and sequence of an unknown gene. The sequence of this gene was amplified and used as a probe to screen a fetal brain cDNA library. On Northern blot analysis, this cDNA detected a 4.4-kb transcript that was abundant in peripheral blood leukocytes, skeletal muscle, placenta, and testis and expressed at lower levels in spleen, thymus, heart, brain, lung, kidney, liver, and ovary. In addition, a 2.8-kb transcript was present in peripheral blood, testis, and placenta. On "zoo blots," this gene was shown to be evolutionarily conserved in 10 mammalian species as well as in chicken, frog, and fish. We have named this gene ELL (for eleven-nineteen lysine-rich leukemia gene). A highly basic, lysine-rich motif of the predicted ELL protein is homologous to similar regions of several proteins, including the DNA-binding domain of poly(ADP-ribose) polymerase. The characterization of the normal functions of ELL as well as its altered function when fused to MLL will be critical to further our understanding of the mechanisms of leukemogenesis.
Tobacco smoke tumor promoters, catechol and hydroquinone, induce oxidative regulation of protein kinase C and influence invasion and metastasis of lung carcinoma cells.
Authors: Gopalakrishna R , Chen Z H , Gundimeda U
Published: May 2006

Cigarette smoke polyphenolic agents (catechol and hydroquinone) that generate oxidants have been shown to be tumor promoters. Furthermore, oxidants can influence protein kinase C (PKC)-mediated signal transduction. Since terpenoid tumor promoters, phorbol esters, increase invasion and metastasis by activating PKC, we have determined whether polyphenolic agents present in the cigarette smoke condensate (CSC) could also influence these events. Hydroquinone (50 microM), catechol (500 microM), or CSC (50 micrograms/ml) induced an initial cytosol-to-membrane translocation of PKC in LL/2 lung carcinoma cells, followed by a later down-regulation of the enzyme. LL/2 cells treated with these CSC-related agents for a limited time (45 min) and exhibiting high membrane-associated PKC activity, when injected into mice through the tail vein, produced an increase in metastatic nodules in the lungs after 20 days. However, cells treated with CSC-related agents for a prolonged period did not exhibit an increase in metastasis. Agents that decrease the rate of production of reactive oxygen species, such as catalase either alone or in combination with superoxide dismutase, and a cell-permeable iron-chelator, o-phenanthroline, inhibited CSC-mediated membrane association of PKC and metastasis. Prior treatment of CSC with tyrosinase to modify polyphenols resulted in a partial loss of CSC stimulation of metastasis. Furthermore, a cell-permeable Ca2+ chelator and diverse PKC inhibitors, such as calphostin C, hypericin, chelerythrine, and bisindolylmaleimide, inhibited CSC-enhanced metastasis. CSC increased in vitro tumor cell adhesion to endothelial monolayers and to reconstituted basement membrane (Matrigel) and also enhanced the invasion through Matrigel coated on the polycarbonate filters in Transwells. All these CSC effects were found to be temporary and were blocked by the above mentioned antioxidant systems and PKC inhibitors. Thus, these results suggest that the oxidants generated by autooxidation of polyphenolic agents present in tobacco smoke increase tumor cell invasion and metastasis, at least in part by activation of Ca2+/PKC signal transduction. Conceivably, cigarette smoke constituents not only promote tumorigenesis but also may increase the spread of cancer in the body.
Transcriptional activation of a ras-like gene (kir) by oncogenic tyrosine kinases.
Authors: Cohen L , Mohr R , Chen Y Y , Huang M , Kato R , Dorin D , Tamanoi F , Goga A , Afar D , Rosenberg N
Published: May 2006

We report the characterization of a member of the ras gene family that is overexpressed in cells transformed by abl tyrosine kinase oncogenes. The gene, named kir (for kinase-inducible ras-like), is induced at the transcriptional level. kir mRNA has a rapid turnover and encodes a protein of 33 kDa with guanine nucleotide-binding activity but undetectable intrinsic GTPase activity. kir was cloned by differential screening of genes present in fully malignant versus growth factor-independent cell lines expressing wild-type or mutant forms of BCR/ABL. BCR/ABL and v-Abl induce transcription of the kir gene via specific signaling pathway(s), but kir overexpression alone is not sufficient to mediate transformation.
A predominant role of integrin alpha 4 in the spontaneous development of autoimmune diabetes in nonobese diabetic mice.
Authors: Yang X D , Michie S A , Tisch R , Karin N , Steinman L , McDevitt H O
Published: May 2006

To elucidate the role of cell adhesion molecules in the pathogenesis of insulin-dependent diabetes mellitus and to determine the predominant lymphocytic homing pathway(s) involved in the selective lymphocytic infiltration of pancreatic islets (insulitis), nonobese diabetic mice were treated with monoclonal antibodies specific for the L-selectin and integrin alpha 4 lymphocyte adhesion molecules. Treatment of neonatal mice with either anti-L-selectin or anti-integrin alpha 4 monoclonal antibodies for the first 4 weeks of life led to a significant and long-term protection against spontaneous occurrence of insulitis and diabetes. The same treatment failed to inhibit lymphocytic infiltration of the salivary glands (sialadenitis). This tissue-specific inhibition of inflammation may be attributed to differences between the pancreas and salivary gland in their expression of endothelial ligands for L-selectin (peripheral vascular addressin) and for integrin alpha 4 (mucosal addressin cell adhesion molecule 1 and vascular cell adhesion molecule 1). Mucosal addressin cell adhesion molecule 1 is highly expressed by vessels within the inflamed islets but was not detected in the salivary glands. In contrast, peripheral vascular addressin- and vascular cell adhesion molecule 1-expressing vessels can be found in almost every area of inflammation within the salivary glands but are seen in only 40-50% of inflamed islets. Anti-L-selectin and anti-integrin alpha 4 treatment had no demonstrable effect on anti-beta-cell autoimmunity or on the immune responses to foreign antigens. Therapeutic treatment with anti-L-selectin after the onset of insulitis from 10 to 14 weeks of age delayed the onset but failed to prevent spontaneous insulin-dependent diabetes mellitus, whereas anti-integrin alpha 4 treatment resulted in a significant and long-lasting suppression of the disease. These data strongly suggest that integrin alpha 4 plays a prominent role in the spontaneous development of insulitis and diabetes in nonobese diabetic mice.
Characteristics of peptide and major histocompatibility complex class I/beta 2-microglobulin binding to the transporters associated with antigen processing (TAP1 and TAP2).
Authors: Androlewicz M J , Ortmann B , van Endert P M , Spies T , Cresswell P
Published: May 2006

The transporter proteins associated with antigen processing (TAP proteins) transport antigenic peptides across the endoplasmic reticulum membrane where they can assemble with newly synthesized major histocompatibility complex (MHC) class I/beta 2-microglobulin (beta 2m) dimers. We have shown previously that TAP possesses a peptide-recognition site with broad specificity and that MHC class I/beta 2m dimers physically associate with TAP. Here, we further characterize the nature of the peptide-binding site on TAP, and the site of interaction of TAP with MHC class I/beta 2m dimers. TAP photoaffinity labeling experiments revealed that both TAP1 and TAP2 are photolabeled by two distinct photopeptide analogues, suggesting that elements of both TAP1 and TAP2 compose the peptide-recognition site. TAP photolabeling analysis on transfectant cell lines that express TAP1 and TAP2 both individually and together revealed that efficient formation of the peptide-binding site occurs only when TAP1 and TAP2 are coexpressed, which correlates with the finding that peptide translocation via TAP occurs only in the presence of both TAP1 and TAP2. These data strongly support the notion that TAP functions as a heterodimer. MHC class I/beta 2m dimers were shown to associate with individual TAP1 chains but were not detectable with individual TAP2 chains. This result suggests that the site of interaction for MHC class I/beta 2m dimers with TAP is on TAP1.
Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma.
Authors: Hagen T M , Huang S , Curnutte J , Fowler P , Martinez V , Wehr C M , Ames B N , Chisari F V
Published: May 2006

A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in the liver was used to determine the extent of oxidative DNA damage that occurs during chronic HBV infection. This mouse strain develops a chronic necroinflammatory liver disease that mimics the inflammation, cellular hyperplasia, and increased risk for cancer that is evident in human chronic active hepatitis. When perfused in situ with nitroblue tetrazolium, an indicator for superoxide formation, the liver of transgenic mice displayed intense formazan deposition in Kupffer cells, indicating oxygen radical production, and S-phase hepatocytes were commonly seen adjacent to the stained Kupffer cells. Similar changes were not observed in nontransgenic control livers. To determine whether these events were associated with oxidative DNA damage, genomic DNA from the livers of transgenic mice and nontransgenic controls was isolated and examined for 8-oxo-2'-deoxyguanosine, an oxidatively modified adduct of deoxyguanosine. Results showed a significant, sustained accumulation in steady-state 8-oxo-2'-deoxyguanosine that started early in life exclusively in the transgenic mice and increased progressively with advancing disease. The most pronounced increase occurred in livers exhibiting microscopic nodular hyperplasia, adenomas, and hepatocellular carcinoma. Thus, HBV transgenic mice with chronic active hepatitis display greatly increased hepatic oxidative DNA damage. Moreover, the DNA damage occurs in the presence of heightened hepatocellular proliferation, increasing the probability of fixation of the attendant genetic and chromosomal abnormalities and the development of hepatocellular carcinoma.
Plethystic algebras and vector symmetric functions.
Authors: Rota G C , Stein J A
Published: May 2006

An isomorphism is established between the plethystic Hopf algebra Pleth(Super[L]) and the algebra of vector symmetric functions. The Hall inner product of symmetric function theory is extended to the Hopf algebra Pleth(Super[L]).
Mitochondrial DNA "clock" for the Amerinds and its implications for timing their entry into North America.
Authors: Torroni A , Neel J V , Barrantes R , Schurr T G , Wallace D C
Published: May 2006

Students of the time of entry of the ancestors of the Amerinds into the New World are divided into two camps, one favoring an "early" entry [more than approximately 30,000 years before the present (YBP)], the other favoring a "late" entry (less than approximately 13,000 YBP). An "intermediate" date is unlikely for geological reasons. The correlation of the appropriate data on mtDNA variation in Amerinds with linguistic, archaeological, and genetic data offers the possibility of establishing a time frame for mtDNA evolution in Amerinds. In this paper, we estimate that the separation of the Chibcha-speaking tribes of Central America from other linguistic groups/nascent tribes began approximately 8000-10,000 YBP. Characterization of the mtDNA of 110 Chibcha speakers with 14 restriction enzymes leads on the basis of their time depth to an estimated mtDNA nucleotide substitution rate for Amerinds of 0.022-0.029% per 10,000 years. As a first application of this rate, we consider the mtDNA variation observed in 18 Amerind tribes widely dispersed throughout the Americas and studied by ourselves with the same techniques, and we estimate that if the Amerinds entered the New World as a single group, that entry occurred approximately 22,000-29,000 YBP. This estimate carries a large but indeterminate error. The mtDNA data are thus at present equivocal with respect to the most likely times of entry of the Amerind into the New World mentioned above but favor the "early" entry hypothesis.
A simpler sort of antibody.
Authors: Masat L , Wabl M , Johnson J P
Published: May 2006

The monoclonal antibody NEMO is directed against a molecule expressed by human cells of the melanocytic lineage. Although obtained by conventional immunization and fusion procedures, NEMO consists solely of kappa light chain. SDS/PAGE analysis indicates that the kappa chains are present as both monomers and dimers. When these two forms were separated by gel filtration, only the monomeric form bound antigen. As kappa light chains from the myeloma MOPC-41 and the hybridoma MORK do not bind to the melanocytic cells, we conclude that the binding specificity of NEMO resides in the variable region.
Two-site binding of C5a by its receptor: analternative binding paradigm for G protein-coupled receptors.
Authors: Siciliano S J , Rollins T E , DeMartino J , Konteatis Z , Malkowitz L , Van Riper G , Bondy S , Rosen H , Springer M S
Published: May 2006

The guanine nucleotide-binding protein-coupled
receptor superfamily binds a vast array of biological messengers including
lipids, odorants, catecholamines, peptides, and proteins. While some small
molecules bind to these receptors at a single interhelical site, we find that
the binding domain on the receptor for the inflammatory protein C5a is more
complex and consists of two distinct subsites. This more elaborate motif appears
to be an evolutionary adaptation of the simpler paradigm to which a second
interaction site has been added in the receptor N terminus. Surprisingly,
occupation of only one of the subsites is required for receptor activation. The
two-site motif is not unique to the C5a receptor but appears to be widely used
by the superfamily to accommodate macromolecular ligands.
Enzymatic characteristics of the c-Raf-1 proteinkinase.
Authors: Force T , Bonventre J V , Heidecker G , Rapp U , Avruch J , Kyriakis J M
Published: May 2006

The c-Raf-1 protein kinase plays a central role in
the mitogenic response of cells to growth factors, cytokines, and many
oncogenes. Despite the critical importance of this enzyme, very little is known
of its biochemical properties or mechanisms of regulation. In these experiments,
we used the only candidate physiologic substrate identified as yet for c-Raf-1,
mitogen-activated protein kinase kinase (MAPKK), to examine enzymatic
characteristics and candidate modulators of c-Raf-1, c-Raf-1 was purified from
Sf9 cells infected with recombinant baculovirus encoding a histidine-tagged
c-Raf-1. The Km values of c-Raf-1 for ATP and MAPKK were 11.6 microM and 0.8
microM, respectively, and the stoichiometry of phosphorylation of MAPKK by
c-Raf-1 was 1.67 mol of phosphate per mol of MAPKK. In contrast to prior
reports, Mg2+ was the preferred cation at Mg2+ and Mn2+ concentrations > 5
mM. c-Raf-1 substrate specificity was extremely restricted, consistent with the
identification of only one candidate physiologic substrate to date and
highlighting the necessity of using MAPKK rather than artificial substrates in
c-Raf-1 activity assays. Of multiple potential substrates tested, the only one
phosphorylated to > 20% of the level of MAPKK phosphorylation was myelin
basic protein (22%). Heat-denatured MAPKK was phosphorylated at only 2% the
level of native MAPKK, indicating that the restricted substrate specificity may
be due to tertiary-structural requirements. We also examined whether c-Raf-1
activity is modulated by lipid binding to the cysteine finger region in its
regulatory domain. Of multiple mitogen-stimulated or cell-membrane lipids
tested, only phosphatidylserine and diacylglycerol in the presence of Ca2+ (2.5
mM) increased c-Raf-1 kinase activity significantly (1.5-fold). The increase is
probably not of physiologic significance because it was about two orders of
magnitude less than the stimulation of protein kinase C by these lipids. On
gel-filtration chromatography, the peak of c-Raf-1 kinase activity and
immunoreactivity eluted at a predicted molecular mass of > 150 kDa,
suggesting that active c-Raf-1 (but not inactive c-Raf-1) exists as a multimeric
complex. This complex may not include p21ras, however, because immunoreactive
p21ras was not identified in the active fractions.
In vivo and in vitro analysis of electricalactivity-dependent expression of muscle acetylcholine receptor genes usingadenovirus.
Authors: Bessereau J L , Stratford-Perricaudet L D , Piette J , Le Poupon C , Changeux J P
Published: May 2006

Acetylcholine receptor (AChR) genes are repressed
in extrajunctional domains of adult muscle fiber by neurally evoked electrical
activity. Denervation elicits upregulation of AChR gene transcription in
extrasynaptic areas. We have used an adenovirus (Ad)-based strategy to analyze
in vitro and in vivo the electrical activity-dependent transcription of the
chicken AChR alpha 1 subunit gene. The luciferase gene placed under the control
of wild-type and mutated fragments of the alpha 1 subunit promoter was inserted
in a defective Ad vector designed for the study of transcriptional regulation.
Animals were infected by intramuscular injection and in vivo luciferase levels
were normalized by coinfection with an Ad vector containing the chloramphenicol
acetyltransferase gene driven by an electrical activity-insensitive promoter.
Our results demonstrate that although both proximal MyoD binding sites of the
alpha 1 promoter are required for muscle-specific expression of the alpha 1
gene, only one is necessary, albeit insufficient, to enhance alpha 1 promoter
activity after denervation. Parallel results were obtained with cultured muscle
cells in vitro following tetrodotoxin blocking of spontaneous electrical
activity. These results substantiate a direct contribution of MyoD factors in
electrical activity-dependent regulation of AChR expression and further indicate
that Ad-based vectors constitute a powerful tool in the field of transcriptional
regulation.
Activation of the human mitochondrial transcriptionfactor A gene by nuclear respiratory factors: a potential regulatory linkbetween nuclear and mitochondrial gene expression in organellebiogenesis.
Authors: Virbasius J V , Scarpulla R C
Published: May 2006

Mitochondrial transcription factor A (mtTFA), the
product of a nuclear gene, stimulates transcription from the two divergent
mitochondrial promoters and is likely the principal activator of mitochondrial
gene expression in vertebrates. Here we establish that the proximal promoter of
the human mtTFA gene is highly dependent upon recognition sites for the nuclear
respiratory factors, NRF-1 and NRF-2, for activity. These factors have been
previously implicated in the activation of numerous nuclear genes that
contribute to mitochondrial respiratory function. The affinity-purified factors
from HeLa cells specifically bind to the mtTFA NRF-1 and NRF-2 sites through
guanine nucleotide contacts that are characteristic for each site. Mutations in
these contacts eliminate NRF-1 and NRF-2 binding and also dramatically reduce
promoter activity in transfected cells. Although both factors contribute, NRF-1
binding appears to be the major determinant of promoter function. This
dependence on NRF-1 activation is confirmed by in vitro transcription using
highly purified recombinant proteins that display the same binding specificities
as the HeLa cell factors. The activation of the mtTFA promoter by both NRF-1 and
NRF-2 therefore provides a link between the expression of nuclear and
mitochondrial genes and suggests a mechanism for their coordinate regulation
during organelle biogenesis.
Evidence supporting a tethered tracking model forhelicase activity of Escherichia coli Rho factor.
Authors: Steinmetz E J , Platt T
Published: May 2006

Transcription termination factor Rho of
Escherichia coli has an ATP-dependent RNA.DNA helicase activity that presumably
facilitates RNA transcript release from the elongation complex. This helicase
activity is unidirectional (5' to 3') and is stoichiometric, with one
RNA molecule released per Rho hexamer in vitro. A simple RNA tracking model
postulates that after Rho's initial binding, it translocates preferentially
toward the 3' end of the RNA. Nitrocellulose filter binding studies
combined with RNase H cleavage are inconsistent with this simple tracking model.
Instead, they support a model in which Rho forms tight primary binding
interactions with the recognition region of the RNA and remains bound there
while transient secondary RNA binding interactions coupled to ATP hydrolysis
serve to scan along the RNA to contact the RNA.DNA helix. This "tethered
tracking" model is consistent with other properties of Rho factor,
including the presence of two classes of RNA binding sites on the Rho hexamer
and the 1:1 stoichiometry in the Rho helicase assay.
n-Sec1: a neural-specific syntaxin-bindingprotein.
Authors: Pevsner J , Hsu S C , Scheller R H
Published: May 2006

We have identified n-Sec1, a rat brain homolog of
the yeast Sec1p protein that participates in the constitutive secretory pathway
between the Golgi apparatus and the plasma membrane. The rat brain cDNA is
predicted to encode a 68-kDa protein with 65% amino acid identity to Drosophila
rop, 59% identity to Caenorhabditis elegans unc-18, and 27% identity to
Saccharomyces cerevisiae Sec1p. By RNA blot analysis, n-Sec1 mRNA expression is
neural-specific. An anti-peptide antiserum directed against the n-Sec1 carboxyl
terminus detects a 68-kDa protein in rat brain cytosol and membranes, but not in
peripheral tissues. In the presence of syntaxin 1a, a plasma membrane protein
implicated in synaptic vesicle docking, n-Sec1 becomes membrane-associated.
n-Sec1 binds to syntaxin 1a, 2, and 3 fusion proteins coupled to agarose beads,
but not to syntaxin 4 fusion protein or beads coupled to a variety of other
proteins. These findings indicate that n-Sec1 is a neural-specific,
syntaxin-binding protein that may participate in the regulation of synaptic
vesicle docking and fusion.
Heparinase inhibitsneovascularization.
Authors: Sasisekharan R , Moses M A , Nugent M A , Cooney C L , Langer R
Published: May 2006

Neovascularization is associated with the
regulation of tissue development, wound healing, and tumor metastasis. A number
of studies have focused on the role of heparin-like molecules in
neovascularization; however, little is known about the role of heparin-degrading
enzymes in neovascularization. We report here that the heparin-degrading
enzymes, heparinases I and III, but not heparinase II, inhibited both
neovascularization in vivo and proliferation of capillary endothelial cells
mediated by basic fibroblast growth factor in vitro. We suggest that the role of
heparinases in inhibition of neovascularization is through depletion of heparan
sulfate receptors that are critical for growth factor-mediated endothelial cell
proliferation and hence neovascularization. The differences in the effects of
the three heparinases on neovascularization could be due to different substrate
specificities for the enzymes, influencing the availability of specific heparin
fragments that modulate heparin-binding cytokines involved in
angiogenesis.
A subset of HLA-B27 molecules contains peptides muchlonger than nonamers.
Authors: Urban R G , Chicz R M , Lane W S , Strominger J L , Rehm A , Kenter M J , UytdeHaag F G , Ploegh H , Uchanska-Ziegler B , Ziegler A
Published: May 2006

An unusual monoclonal antibody (MARB4) directed
against HLA-B27 that reacts with only approximately 5-20% of the cell surface
HLA-B27 was used for large-scale purification of these molecules. Subsequent
mass spectrometry of HLA-B27-bound peptides showed that the minor MARB4-reactive
population contained peptides primarily from 900 to 4000 Da in size
(approximately 8-33 amino acid residues), whereas the major HLA-B27 population
contained peptides in the mass range of 900-1400 Da (approximately 8-12 amino
acid residues). Thus, a subset of HLA-B27 molecules binds to peptides much
longer than nonamers. Typical HLA-B27-binding peptides contain arginine in
position 2. Further analysis by Edman sequencing of the pooled bound peptides
revealed that the major population contained substantial amounts of arginine at
positions 1 and 9 (40-50%) and exclusively arginine at position 2, as expected.
The minor population of peptides also contained detectable amounts of arginine
at these positions, but at the level of only approximately 10%; no marked
enrichment at any position was observed. These long HLA-B27-bound peptides could
represent either intermediates in the formation of nonamers or adventitiously
bound peptides. Lastly, in the TAP2 mutant cell line BM36.1 transfected with
HLA-B*2705, MARB4-reactive HLA-B27 molecules were absent from the cell surface,
indicating that the peptide transporter was required for delivery of the long
peptides. Thus, during the folding of class I heavy chains, peptides of diverse
lengths are available and participating.
The role of charge and multiple faces of the CD8alpha/alpha homodimer in binding to major histocompatibility complex class Imolecules: support for a bivalent model.
Authors: Giblin P A , Leahy D J , Mennone J , Kavathas P B
Published: May 2006

The CD8 dimer interacts with the alpha 3 domain of
major histocompatibility complex class I molecules through two immunoglobulin
variable-like domains. In this study a crystal structure-informed mutational
analysis has been performed to identify amino acids in the CD8 alpha/alpha
homodimer that are likely to be involved in binding to class I. Several key
residues are situated on the top face of the dimer within loops analogous to the
complementarity-determining regions (CDRs) of immunoglobulin. In addition, other
important amino acids are located in the A and B beta-strands on the sides of
the dimer. The potential involvement of amino acids on both the top and the side
faces of the molecule is consistent with a bivalent model for the interaction
between a single CD8 alpha/alpha homodimer and two class I molecules and may
have important implications for signal transduction in class I-expressing cells.
This study also demonstrates a role for the positive surface potential of CD8 in
class I binding and complements previous work demonstrating the importance of a
negatively charged loop on the alpha 3 domain of class I for CD8
alpha/alpha-class I interaction. We propose a model whereby residues located on
the CDR-like loops of the CD8 homodimer interact with the alpha 3 domain of MHC
class I while amino acids on the side of the molecule containing the A and B
beta-strands contact the alpha 2 domain of class I.
Molecular dynamics simulation of protein denaturation:solvation of the hydrophobic cores and secondary structure ofbarnase.
Authors: Caflisch A , Karplus M
Published: May 2006

The transition in barnase from the native state to
a compact globule has been studied with high-temperature molecular dynamics
simulations. A partial destruction of the alpha-helices and the outer strands of
the beta-sheet is observed with water molecules replacing the hydrogen bonds of
the secondary structural elements. Simultaneously, the main alpha-helix moves
away from the beta-sheet and exposes the principal hydrophobic core, many of
whose nonpolar side chains, beginning with the ones near the surface, become
solvated by hydrogen-bonded water molecules. This step involves a significant
increase in the solvent-exposed surface area; the resulting loss of stability
due to the hydrophobic effect may be the major source of the activation barrier
in the unfolding reaction. The detailed mechanism described here for the first
stage of the denaturation of barnase, including the essential role of water
molecules, is likely to be representative of protein denaturation, in
general.
Complementation of an Arabidopsis thaliana mutant thatlacks complex asparagine-linked glycans with the human cDNA encodingN-acetylglucosaminyltransferase I.
Authors: Gomez L , Chrispeels M J
Published: May 2006

N-Acetylglucosaminyltransferase I (EC 2.4.1.101)
initiates the conversion of high-mannose asparagine-linked glycans to complex
asparagine-linked glycans in plant as well as in animal cells. This Golgi enzyme
is missing in the cgl mutant of Arabidopsis thaliana, and the mutant cells are
unable to synthesize complex glycans. Transformation of cells from the mutant
plants with the cDNA encoding human N-acetylglucosaminyltransferase I restores
the wild-type phenotype of the plant cells. Fractionation of the subcellular
organelles on isopycnic sucrose gradients shows that the human enzyme in the
complemented cells bands at the same density, 1.14 g/cm3, typical of Golgi
cisternae, as the enzyme in the wild-type plant cells. These results demonstrate
that complementation results from the presence of the human enzyme in the plant
Golgi apparatus, where it is functionally integrated into the biosynthetic
machinery of the plant cell. In addition, given the evolutionary distance
between plants and mammals and the great diversity of glycoproteins that are
modified in each, there is probably no specific recognition between this Golgi
enzyme and the polypeptide domains of the proteins it modifies.
Alternative splicing of class Ib majorhistocompatibility complex transcripts in vivo leads to the expression ofsoluble Qa-2 molecules in murine blood.
Authors: Tabaczewski P , Shirwan H , Lewis K , Stroynowski I
Published: May 2006

Class Ib Qa-2 molecules are expressed in tissue
culture cells as approximately 40-kDa membrane-bound,
glycophosphatidylinositol-linked antigens and as approximately 39-kDa soluble
polypeptides. Recently, alternative splicing events which delete exon 5 from a
portion of Qa-2 transcripts were demonstrated to give rise to truncated secreted
Qa-2 molecules in transfected cell lines. To determine whether this mechanism
operates in vivo and to find out whether Qa-2 can be detected in soluble form in
circulation, murine blood samples were analyzed. Critical to these experiments
was preparation of an anti-peptide antiserum against an epitope encoded by a
junction of exon 4 and exon 6. We find that supernatants of splenocytes cultured
in vitro as well as serum or plasma contain two forms of soluble Qa-2 molecules.
One form corresponds to a secreted molecule translated from transcripts from
which exon 5 has been deleted; the other is derived from membrane-bound antigens
or their precursors. The levels of both soluble forms of Qa-2 are inducible upon
stimulation of the immune system, suggesting an immunoregulatory role for these
molecules or for the mechanism leading to the reduction of cell-associated Qa-2
antigens in vivo.
Intestinal absorption and excretion of octapeptidescomposed of D amino acids.
Authors: Pappenheimer J R , Dahl C E , Karnovsky M L , Maggio J E
Published: May 2006

Octapeptides synthesized from D amino acids were
absorbed from the intestine and excreted in urine of normal rats drinking 5%
glucose/1% creatinine containing the 125I-labeled peptides at 0.1-25 mg/dl. The
rats ingested fluid at the rate of about 20 ml/hr and produced urine at 15 ml/hr
for several hours during the nocturnal feeding period. Sixty-one +/- 4% of the
ingested creatinine and 50 +/- 3% of a lipid-insoluble D octapeptide (EASASYSA,
784 Da) were excreted intact in the urine. The steady-state molar rate of
absorption-excretion of creatinine equaled or exceeded the maximum rate of
carrier-mediated intestinal transport of glucose, suggesting that both the
creatinine and the D octapeptide were transported paracellularly by solvent drag
through absorptive cell junctions that were dilated by the glucose. More than
70% of the ingested glucose was also absorbed paracellularly. The results
demonstrate that intact oligopeptides can be absorbed efficiently from the
intestine when they are not hydrolyzed by membrane-bound peptidases of the brush
border. The results also provide support for recent theories proposing that
coupling of membrane digestion with paracellular solvent drag accounts for a
major fraction of normal intestinal absorption of nutrients.
Self-incompatibility: how plants avoid illegitimate offspring.
Authors: Matton D P , Nass N , Clarke A E , Newbigin E
Published: May 2006

In some families of flowering plants, a single self-incompatibility (S) locus prevents the fertilization of flowers by pollen from the same plant. Self-incompatibility of this type involves the interaction of molecules produced by the S locus in pollen with those present in the female tissues (pistil). Until recently, the pistil products of the S locus were known in only two families, the Brassicaceae (which includes the cabbages and mustards) and Solanaceae (potatoes and tomatoes). A paper in this issue of the Proceedings describes the molecules associated with self-incompatibility in a third family, the Papaveraceae (poppies). We review current research on self-incompatibility in these three families and discuss the implications of the latest findings in poppy on the likely evolution of self-incompatibility in flowering plants. We also compare research into self-incompatibility with recent progress in understanding the mechanisms by which plants overcome infection by certain pathogens.
Sequence-specific transcriptional activation is essential for growth suppression by p53.
Authors: Pietenpol J A , Tokino T , Thiagalingam S , el-Deiry W S , Kinzler K W , Vogelstein B
Published: May 2006

Although several biochemical features of p53 have been described, their relationship to tumor suppression remains uncertain. We have compared the ability of p53-derived proteins to act as sequence-specific transcriptional (SST) activators with their ability to suppress tumor cell growth, using an improved growth-suppression assay. Both naturally occurring and in vitro derived mutations that abrogated the SST activity of p53 lost the ability to suppress tumor cell growth. Additionally, the N- and C-terminal ends of p53 were shown to be functionally replaceable with foreign transactivation and dimerization domains, respectively, with concordant preservation of both SST and tumor-suppressive properties. Only the central region of p53, conferring specific DNA binding, was required to suppress growth by such hybrid proteins. The SST activity of p53 thus appeared to be essential for the protein to function as a tumor suppressor.
Abrogation of oncogene-associated apoptosis allows transformation of p53-deficient cells.
Authors: Lowe S W , Jacks T , Housman D E , Ruley H E
Published: May 2006

p53-deficient mouse embryonic fibroblasts were used to establish a direct mechanism of tumor suppression by p53 involving the destruction of oncogene-expressing cells by apoptosis. The absence of p53 enhanced cell growth, appeared sufficient for immortalization, and allowed a single oncogene [adenovirus early region 1A (E1A)] to transform cells to a tumorigenic state. p53 suppressed transformation of E1A-expressing cells by apoptosis. Apoptosis was associated with p53 stabilization and was triggered by environmental signals that normally suppress cell growth. Absence of even a single p53 allele significantly enhanced cell growth and survival. Although abrogation of apoptosis allowed transformation by E1A alone, escape from apoptosis susceptibility was not a prerequisite for tumor growth. Consequently, p53 mutation could enhance the survival of malignant cells expressing oncogenes activated early in tumor progression.
Noninvasive measurement of microvascular and interstitial oxygen profiles in a human tumor in SCID mice.
Authors: Torres Filho I P , Leunig M , Yuan F , Intaglietta M , Jain R K
Published: May 2006

Simultaneous measurements of intravascular and interstitial oxygen partial pressure (PO2) in any tissue have not previously been reported, despite the importance of oxygen in health and in disease. This is due to the limitations of current techniques, both invasive and noninvasive. We have optically measured microscopic profiles of PO2 with high spatial resolution in subcutaneous tissue and transplanted tumors in mice by combining an oxygen-dependent phosphorescence quenching method and a transparent tissue preparation. The strengths of our approach include the ability to follow PO2 in the same location for several weeks and to relate these measurements to local blood flow and vascular architecture. Our results show that (i) PO2 values in blood vessels in well-vascularized regions of a human colon adenocarcinoma xenograft are comparable to those in surrounding arterioles and venules, (ii) carbogen (95% O2/5% CO2) breathing increases microvascular PO2 in tumors, and (iii) in unanesthetized and anesthetized mice PO2 drops to hypoxic values at < 200 microns from isolated vessels but drops by < 5 mmHg (1 mmHg = 133 Pa) in highly vascularized tumor regions. Our method should permit noninvasive evaluations of oxygen-modifying agents and offer further mechanistic information about tumor pathophysiology in tissue preparations where the surface of the tissue can be observed.
Molecular analysis of cell surface beta-1,4-galactosyltransferase function during cell migration.
Authors: Appeddu P A , Shur B D
Published: May 2006

Despite the identification and characterization of cell surface receptors for the extracellular matrix, it is unknown how their relative expression and cytoskeletal association regulate cell migration. Previous studies have identified beta-1,4-galactosyltransferase (GalTase; EC 2.4.1.38) on the surface of migrating cells, where it mediates cell migration on basal lamina matrices by associating with the cytoskeleton and binding to N-linked oligosaccharides in the E8 domain of laminin. In this study, the function of GalTase during cell migration was examined directly by analyzing the migration rate of stably transfected cell lines in which the relative level of surface GalTase and its ability to associate with the cytoskeleton were altered. We show here that the cytoskeleton contains a limiting, saturable, number of binding sites for surface GalTase. Furthermore, the rate of cell migration was inversely related to the ability of surface GalTase to associate with the cytoskeleton. Elevating surface GalTase in excess of the number of cytoskeleton-binding sites reduced the rate of cell migration, whereas decreasing the amount of surface GalTase available to bind the cytoskeleton increased migration rates. These results show that the rate of cell migration on basal lamina is directly dependent upon the expression of surface GalTase and the ability of this protein to associate with a limiting number of cytoskeleton-binding sites.
Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice.
Authors: Callow M J , Stoltzfus L J , Lawn R M , Rubin E M
Published: May 2006

The atherogenic macromolecule lipoprotein(a) [Lp(a)] has resisted in vivo analyses partly because it is found in a limited number of experimental animals. Although transgenic mice expressing human apolipoprotein (a) [apo(a)] have previously been described, they failed to assemble Lp(a) particles because of the inability of human apo(a) to associate with mouse apolipoprotein B (apoB). We isolated a 90-kilobase P1 phagemid containing the human apoB gene and with this DNA generated 13 lines of transgenic mice of which 11 expressed human apoB. The human apoB transcript was expressed and edited in the liver of the transgenic mice. Plasma concentrations of human apoB, as well as low density lipoprotein (LDL), were related to transgene copy number; the transgenic line with the most copies of human apoB had a > 4-fold increase in LDL cholesterol compared with nontransgenics and a lipoprotein profile similar to that of humans. When human apoB and apo(a) transgenic mice were bred together, plasma apo(a) in mice expressing both human proteins was tightly associated with lipoproteins in the LDL density region. These studies demonstrate the successful expression of human apoB and the efficient assembly of Lp(a) in mice.
On Liapunov's convexity theorem.
Authors: Dvoretzky A
Published: May 2006

A necessary and sufficient condition is given for the range of a finite Rn-valued measure to be convex. A related result is given for Banach-valued set functions.
Redirection of tryptophan leads to production of low indole glucosinolate canola.
Authors: Chavadej S , Brisson N , McNeil J N , De Luca V
Published: May 2006

Cruciferous plants are known to produce over a hundred different mustard oil glycosides, which are derived from methionine, phenylalanine, or tryptophan. In oil-producing crops like Brassica napus (canola), the presence of indole glucosinolates in seed protein meals has decreased meal palatability and has limited their value as animal feed. We have transformed canola plants with a gene that encodes tryptophan decarboxylase (TDC) in an attempt to redirect tryptophan into tryptamine rather than into indole glucosinolates. Transgenic plants that expressed this decarboxylase activity accumulated tryptamine while correspondingly lower levels of tryptophan-derived indole glucosinolates were produced in all plant parts compared with nontransformed controls. Of particular significance, the indole glucosinolate content of mature seeds from transgenic plants was only 3% of that found in nontransformed seeds. These results demonstrate how the creation of artificial metabolic sinks could divert metabolite flow and be used to remove these undesirable indole glucosinolates, thereby increasing the value of the oilseed meals, which are produced after extraction of oil from the seed.
Developmentally excised sequences in micronuclear DNA of Paramecium.
Authors: Steele C J , Barkocy-Gallagher G A , Preer L B , Preer J R
Published: May 2006

DNA processing occurs in ciliates at autogamy and conjugation when new macronuclei are formed from micronuclei and old macronuclei degrade. Processing of micronuclear DNA consists of removal of certain internal sequences, chromosomal fragmentation, addition of new telomeres, and amplification. Aside from a recent brief report, internal eliminated sequences have not been described in Paramecium. In this paper we characterize nine internal eliminated sequences found within and near the gene that codes for surface protein A in Paramecium tetraurelia. Of these nine, seven are located within the translated portion of the gene, and all include short, inverted terminal repeats. The characteristic sequence, TA, appears at the boundaries of all of the internal eliminated sequences.