bluuebunny/crossref_metadata_2025 huggingface dataset:
The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface.
Authors: Chak K F , Safo M K , Ku W Y , Hsieh S Y , Yuan H S
Published: July 2002

The immunity protein of colicin E7 (ImmE7) can bind specifically to the DNase-type colicin E7 and inhibit its bactericidal activity. Here we report the 1.8-angstrom crystal structure of the ImmE7 protein. This is the first x-ray structure determined in the superfamily of colicin immunity proteins. The ImmE7 protein consists of four antiparallel alpha-helices, folded in a topology similar to the architecture of a four-helix bundle structure. A region rich in acidic residues is identified. This negatively charged area has the greatest variability within the family of DNase-type immunity proteins; thus, it seems likely that this area is involved in specific binding to colicin. Based on structural, genetic, and kinetic data, we suggest that all the DNase-type immunity proteins, as well as colicins, share a "homologous-structural framework" and that specific interaction between a colicin and its cognate immunity protein relies upon how well these two proteins' charged residues match on the interaction surface, thus leading to specific immunity of the colicin.
Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma.
Authors: Rencic A , Gordon J , Otte J , Curtis M , Kovatich A , Zoltick P , Khalili K , Andrews D
Published: July 2002

We describe molecular and clinical findings in an immunocompetent patient with an oligoastrocytoma and the concomitant presence of the human papovavirus, JC virus (JCV), which is the etiologic agent of the subacute, debilitating demyelinating disease, progressive multifocal leukoencephalopathy. Histologic review revealed a glial neoplasm consisting primarily of a moderately cellular oligodendroglioma with distinct areas of a fibrillary astrocytoma. Immunohistochemical analysis revealed nuclear staining of tumor cells with antibodies against the viral oncoprotein [tumor antigen (T antigen)], the proliferation marker (Ki67), and the cellular proliferation regulator (p53). Using primers specific to the JCV control region, PCR yielded amplified DNA that was identical to the control region of the Mad-4 strain of the virus. PCR analysis demonstrated the presence of the genome for the viral oncoprotein, T antigen, and results from primer extension studies revealed synthesis of the viral early RNA for T antigen in the tumor tissues. The presence of viral T antigen in the tumor tissue was further demonstrated by immunoblot assay. To our knowledge, this is the first report of the presence of JCV DNA, RNA, and T antigen in tissue in which viral T antigen is localized to tumor cell nuclei and suggests the possible association of JCV with some glial neoplasms.
High affinity type I interleukin 1 receptor antagonists discovered by screening recombinant peptide libraries.
Authors: Yanofsky S D , Baldwin D N , Butler J H , Holden F R , Jacobs J W , Balasubramanian P , Chinn J P , Cwirla S E , Peters-Bhatt E , Whitehorn E A , Tate E H , Akeson A , Bowlin T L , Dower W J , Barrett R W
Published: July 2002

Two families of peptides that specifically bind the extracellular domain of the human type I interleukin I (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-lalpha to the type I IL-1 receptor with IC50 values of 45-140 microM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 approximately 2 nM). These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor. This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.
Minimal definition of the imprinting center and fixation of chromosome 15q11-q13 epigenotype by imprinting mutations.
Authors: Saitoh S , Buiting K , Rogan P K , Buxton J L , Driscoll D J , Arnemann J , König R , Malcolm S , Horsthemke B , Nicholls R D
Published: July 2002

Patients with disorders involving imprinted genes such as Angelman syndrome (AS) and Prader-Willi syndrome (PWS) can have a mutation in the imprinting mechanism. Previously, we identified an imprinting center (IC) within chromosome 15q11-ql3 and proposed that IC mutations block resetting of the imprint, fixing on that chromosome the parental imprint (epigenotype) on which the mutation arose. We now describe four new microdeletions of the IC, the smallest (6 kb) of which currently defines the minimal region sufficient to confer an AS imprinting mutation. The AS deletions all overlap this minimal region, centromeric to the PWS microdeletions, which include the first exon of the SNRPN gene. None of five genes or transcripts in the 1.0 Mb vicinity of the IC (ZNF127, SNRPN, PAR-5, IPW, and PAR-1), each normally expressed only from the paternal allele, was expressed in cells from PWS imprinting mutation patients. In contrast, AS imprinting mutation patients show biparental expression of SNRPN and IPW but must lack expression of the putative AS gene 250-1000 kb distal of the IC. These data strongly support a model in which the paternal chromosome of these PWS patients carries an ancestral maternal epigenotype, and the maternal chromosome of these AS patients carries an ancestral paternal epigenotype. The IC therefore functions to reset the maternal and paternal imprints throughout a 2-Mb imprinted domain within human chromosome 15q11-q13 during gametogenesis.
APC mutations in colorectal tumors with mismatch repair deficiency.
Authors: Huang J , Papadopoulos N , McKinley A J , Farrington S M , Curtis L J , Wyllie A H , Zheng S , Willson J K , Markowitz S D , Morin P , Kinzler K W , Vogelstein B , Dunlop M G
Published: July 2002

We have investigated the influence of genetic instability [replication error (RER) phenotype] on APC (adenomatous polyposis coli), a gene thought to initiate colorectal tumorigenesis. The prevalence of APC mutations was similar in RER and non-RER tumors, indicating that both tumor types share this step in neoplastic transformation. However, in a total of 101 sequenced mutations, we noted a substantial excess of APC frameshift mutations in the RER cases (70% in RER tumors versus 47% in non-RER tumors, P < 0.04). These frameshifts were characteristic of mutations arising in cells deficient in DNA mismatch repair, with a predilection for mononucleotide repeats in the RER tumors (P < 0.0002), particularly (A)n tracts (P < 0.00007). These findings suggest that the genetic instability that is reflected by the RER phenotype precedes, and is responsible for, APC mutation in RER large bowel tumors and have important implications for understanding the very earliest stages of neoplasia in patients with tumors deficient in mismatch repair.
Agonist-selective endocytosis of mu opioid receptor by neurons in vivo.
Authors: Sternini C , Spann M , Anton B , Keith D E , Bunnett N W , von Zastrow M , Evans C , Brecha N C
Published: July 2002

Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the mu opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the delta or kappa receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesicle-like structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.
Ablation of suprachiasmatic nucleus alters timing of hibernation in ground squirrels.
Authors: Ruby N F , Dark J , Heller H C , Zucker I
Published: July 2002

Hibernation patterns were monitored continuously for 2.5 years in female squirrels that were neurologically intact or in which the hypothalamic suprachiasmatic nucleus (SCN) was completely ablated (SCNx). The number of hibernation bouts in SCNx squirrels increased by 159%, total hibernation time increased by 58%, and periodic arousals from hibernation were 47% longer in SCNx than in control squirrels; the duration of individual torpor bouts was 2 days shorter and far more variable in SCNx than in control animals. Some SCNx squirrels cycled through bouts of torpor continuously for nearly 2 years. The SCN appears to be part of the mechanism that controls the duration of the hibernation season and the temporal structure of individual torpor bouts.
Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia.
Authors: Lee P J , Alam J , Wiegand G W , Choi A M
Published: July 2002

Heme oxygenase (HO) catalyzes the rate-limiting step in the degradation of heme to biliverdin, which is reduced by biliverdin reductase to bilirubin. Heme oxygenase-1 (HO-1) is inducible not only by its heme substrate, but also by a variety of agents causing oxidative stress. Although much is known about the regulation of HO-1 expression, the functional significance of HO-1 induction after oxidant insult is still poorly understood. We hypothesize and provide evidence that HO-1 induction serves to protect cells against oxidant stress. Human pulmonary epithelial cells (A549 cells) stably transfected with the rat HO-1 cDNA exhibit marked increases of HO-1 mRNA levels which were correlated with increased HO enzyme activity. Cells that overexpress HO-1 (A549-A4) exhibited a marked decrease in cell growth compared with wild-type A549 (A549-WT) cells or A549 cells transfected with control DNA (A549-neo). This slowing of cell growth was associated with an increased number of cells in G0/G1 phase during the exponential growth phase and decreased entry into the S phase, as determined by flow cytometric analysis of propidium iodide-stained cells and pulse experiments with bromodeoxyuridine. Furthermore, the A549-A4 cells accumulated at the G2/M phase and failed to progress through the cell cycle when stimulated with serum, whereas the A549-neo control cells exhibited normal cell cycle progression. Interestingly, the A549-A4 cells also exhibited marked resistance to hyperoxic oxidant insult. Tin protoporphyrin, a selective inhibitor of HO, reversed the growth arrest and ablated the increased survival against hyperoxia observed in the A549-A4 cells overexpressing HO-1. Taken together, our data suggest that overexpression of HO-1 results in cell growth arrest, which may facilitate cellular protection against non-heme-mediated oxidant insult such as hyperoxia.
Moderate increase in histone acetylation activates the mouse mammary tumor virus promoter and remodels its nucleosome structure.
Authors: Bartsch J , Truss M , Bode J , Beato M
Published: July 2002

The mouse mammary tumor virus (MMTV) promoter is regulated by steroid hormones through a hormone-responsive region that is organized in a positioned nucleosome. Hormone induction leads to a structural change of this nucleosome which makes its DNA more sensitive to cleavage by DNase I and enables simultaneous binding of all relevant transcription factors. In cells carrying either episomal or chromosomally integrated MMTV promoters, moderate acetylation of core histones, generated by treatment with low concentrations of the histone deacetylase inhibitors sodium butyrate or trichostatin A, enhances transcription from the MMTV promoter in the absence of hormone and potentiates transactivation by either glucocorticoids or progestins. At higher concentrations, histone deacetylase inhibitors reduce basal and hormone induced MMTV transcription. Inducing inhibitor concentrations lead to the same type of nucleosomal DNase I hypersensitivity as hormone treatment, suggesting that moderate acetylation of core histone activates the MMTV promoter by mechanisms involving chromatin remodeling similar to that generated by the inducing hormones.
Rescue of abasic hammerhead ribozymes by exogenous addition of specific bases.
Authors: Peracchi A , Beigelman L , Usman N , Herschlag D
Published: July 2002

We have synthesized 13 hammerhead ribozyme variants, each containing an abasic residue at a specific position of the catalytic core. The activity of each of the variants is significantly reduced. In four cases, however, activity can be rescued by exogenous addition of the missing base. For one variant, the rescue is 300-fold; for another, the rescue is to the wild-type level. This latter abasic variant (G10.1X) has been characterized in detail. Activation is specific for guanine, the base initially removed. In addition, the specificity for guanine versus adenine is substantially altered by replacing C with U in the opposite strand of the ribozyme. These results show that a binding site for a small, noncharged ligand can be created in a preexisting ribozyme structure. This has implications for structure-function analysis of RNA, and leads to speculations about evolution in an "RNA world" and about the potential therapeutic use of ribozymes.
Multiple DNA and protein sequence alignment based on segment-to-segment comparison.
Authors: Morgenstern B , Dress A , Werner T
Published: July 2002

In this paper, a new way to think about, and to construct, pairwise as well as multiple alignments of DNA and protein sequences is proposed. Rather than forcing alignments to either align single residues or to introduce gaps by defining an alignment as a path running right from the source up to the sink in the associated dot-matrix diagram, we propose to consider alignments as consistent equivalence relations defined on the set of all positions occurring in all sequences under consideration. We also propose constructing alignments from whole segments exhibiting highly significant overall similarity rather than by aligning individual residues. Consequently, we present an alignment algorithm that (i) is based on segment-to-segment comparison instead of the commonly used residue-to-residue comparison and which (ii) avoids the well-known difficulties concerning the choice of appropriate gap penalties: gaps are not treated explicity, but remain as those parts of the sequences that do not belong to any of the aligned segments. Finally, we discuss the application of our algorithm to two test examples and compare it with commonly used alignment methods. As a first example, we aligned a set of 11 DNA sequences coding for functional helix-loop-helix proteins. Though the sequences show only low overall similarity, our program correctly aligned all of the 11 functional sites, which was a unique result among the methods tested. As a by-product, the reading frames of the sequences were identified. Next, we aligned a set of ribonuclease H proteins and compared our results with alignments produced by other programs as reported by McClure et al. [McClure, M. A., Vasi, T. K. & Fitch, W. M. (1994) Mol. Biol. Evol. 11, 571-592]. Our program was one of the best scoring programs. However, in contrast to other methods, our protein alignments are independent of user-defined parameters.
Hyperalgesic agents increase a tetrodotoxin-resistant Na+ current in nociceptors.
Authors: Gold M S , Reichling D B , Shuster M J , Levine J D
Published: July 2002

Sensitization of primary afferent neurons underlies much of the pain and tenderness associated with tissue injury and inflammation. The increase in excitability is caused by chemical agents released at the site of injury. Because recent studies suggest that an increase in voltage-gated Na+ currents may underlie increases in neuronal excitability associated with injury, we have tested the hypothesis that a tetrodotoxin-resistant voltage-gated Na+ current (TTX-R INa), selectively expressed in a subpopulation of sensory neurons with properties of nociceptors, is a target for hyperalgesic agents. Our results indicate that three agents that produce tenderness or hyperalgesia in vivo, prostaglandin E2, adenosine, and serotonin, modulate TTX-R INa. These agents increase the magnitude of the current, shift its conductance-voltage relationship in a hyperpolarized direction, and increase its rate of activation and inactivation. In contrast, thromboxane B2, a cyclooxygenase product that does not produce hyperalgesia, did not affect TTX-R INa. These results suggest that modulation of TTX-R INa is a mechanism for sensitization of mammalian nociceptors.
An induction gene trap screen in embryonic stem cells: Identification of genes that respond to retinoic acid in vitro.
Authors: Forrester L M , Nagy A , Sam M , Watt A , Stevenson L , Bernstein A , Joyner A L , Wurst W
Published: July 2002

We have developed a novel induction gene trap approach that preselects in vitro for integrations into genes that lie downstream of receptor/ligand-mediated signaling pathways. Using this approach, we have identified 20 gene trap integrations in embryonic stem cells, 9 of which were induced and 11 of which were repressed after exposure to exogenous retinoic acid (RA). All but one of these integrations showed unique spatially restricted or tissue-specific patterns of expression between 8.5 and 11.5 days of embryogenesis. Interestingly, expression was observed in tissues that are affected by alterations in RA levels during embryogenesis. Sequence analysis of fusion transcripts from six integrations revealed five novel gene sequences and the previously identified protooncogene c-fyn. To date, germ-line transmission and breeding has uncovered one homozygous embryonic lethal and three homozygous viable insertions. These studies demonstrate the potential of this induction gene trap approach for identifying and mutating genes downstream of signal transduction pathways.
A new high molecular weight immunoglobulin class from the carcharhine shark: implications for the properties of the primordial immunoglobulin.
Authors: Berstein R M , Schluter S F , Shen S , Marchalonis J J
Published: July 2002

All immunoglobulins and T-cell receptors throughout phylogeny share regions of highly conserved amino acid sequence. To identify possible primitive immunoglobulins and immunoglobulin-like molecules, we utilized 3' RACE (rapid amplification of cDNA ends) and a highly conserved constant region consensus amino acid sequence to isolate a new immunoglobulin class from the sandbar shark Carcharhinus plumbeus. The immunoglobulin, termed IgW, in its secreted form consists of 782 amino acids and is expressed in both the thymus and the spleen. The molecule overall most closely resembles mu chains of the skate and human and a new putative antigen binding molecule isolated from the nurse shark (NAR). The full-length IgW chain has a variable region resembling human and shark heavy-chain (VH) sequences and a novel joining segment containing the WGXGT motif characteristic of H chains. However, unlike any other H-chain-type molecule, it contains six constant (C) domains. The first C domain contains the cysteine residue characteristic of C mu1 that would allow dimerization with a light (L) chain. The fourth and sixth domains also contain comparable cysteines that would enable dimerization with other H chains or homodimerization. Comparison of the sequences of IgW V and C domains shows homology greater than that found in comparisons among VH and C mu or VL, or CL thereby suggesting that IgW may retain features of the primordial immunoglobulin in evolution.
Evidence that free polyunsaturated fatty acids modify Na+ channels by directly binding to the channel proteins.
Authors: Kang J X , Leaf A
Published: July 2002

The effects of free polyunsaturated fatty acids (PUFA) on the binding of ligands to receptors on voltage-sensitive Na+ channels of neonatal rat cardiac myocytes were assessed. The radioligand was [benzoyl-2,5-(3)H] batrachotoxinin A 20alpha-benzoate ([(3)H]BTXB), a toxin that binds to the Na+ channel. The PUFA that have been shown to be antiarrhythmic, including eicosapentaenoic acid (EPA; C20:5n-3), docosahexaenoic acid (DHA; C22:6n-3), eicosatetraynoic acid (ETYA), linolenic acid (C18:3n-3), and linoleic acid (C18:2n-6), inhibited [(3)H]BTXB binding in a dose-dependent fashion with IC50 values of 28-35 microM, whereas those fatty acids that have no antiarrhythmic effects including saturated fatty acid (stearic acid, C18:0), monounsaturated fatty acid (oleic acid; C18:1n-9), and EPA methyl ester did not have a significant effect on [(3)H]BTXB binding. Enrichment of the myocyte membrane with cholesterol neither affected [(3)H]BTXB binding when compared with control cells nor altered the inhibitory effects of PUFA on [(3)H]BTXB binding. Scatchard analysis of [(3)H]BTXB binding showed that EPA reduced the maximal binding without altering the Kd for [(3)H]BTXB binding, indicating allosteric inhibition. The inhibition by EPA of [(3)H]BTXB binding was reversible (within 30 min) when delipidated bovine serum albumin was added. The binding of the PUFA to this site on the Na+ channel is reversible and structure-specific and occurs at concentrations close to those required for apparent antiarrhythmic effects and a blocking effect on the Na+ current, suggesting that binding of the PUFA at this site relates to their antiarrhythmic action.
Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains.
Authors: Hesterkamp T , Hauser S , Lütcke H , Bukau B
Published: July 2002

Correct folding of newly synthesized proteins is proposed to be assisted by molecular chaperones and folding catalysts. To identify cellular factors involved in the initial stages of this process we searched for proteins associated with nascent polypeptide chains. In an Escherichia coli transcription/translation system synthesizing beta-galactosidase we identified a 58-kDa protein which associated with translating ribosomes but dissociated from these ribosomes upon release of nascent beta-galactosidase. N-terminal sequencing identified it as trigger factor, previously implicated in protein secretion. Direct evidence for association of trigger factor with nascent polypeptide chains was obtained by crosslinking. In a wheat germ translation system complemented with E. coli lysates, epsilon-4-(3-trifluoromethyldiazirino)benzoic acid-lysine residues were incorporated into nascent secretory preprolactin and a nonsecretory preprolactin mutant. Trigger factor crosslinked to both types of nascent chains, provided they were ribosome bound. Trigger factor contains key residues of the substrate-binding pocket of FK506-binding protein-type peptidyl-prolyl-cis/trans-isomerases and has prolyl isomerase activity in vitro. We propose that trigger factor is a folding catalyst acting cotranslationally.
Karyopherin β2 mediates nuclear import of a mRNA binding protein
Authors: Bonifaci Neris , Moroianu Junona , Radu Aurelian , Blobel Günter
Published: July 2002

We have cloned and sequenced cDNA for human karyopherin β2, also known as transportin. In a solution binding assay, recombinant β2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1’s previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin β1, karyopherin β2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both β1 and β2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, β2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of β2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of β2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin β2 inhibited karyopherin α/β1-mediated import of a classical NLS containing substrate and,
vice versa
, β1 inhibited β2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of β1 and β2 to repeat nucleoporins.
The origin and evolution of animal appendages
Authors: Panganiban Grace , Irvine Steven M. , Lowe Chris , Roehl Henry , Corley Laura S. , Sherbon Beverley , Grenier Jennifer K. , Fallon John F. , Kimble Judith , Walker Muriel , Wray Gregory A. , Swalla Billie J. , Martindale Mark Q. , Carroll Sean B.
Published: July 2002

Animals have evolved diverse appendages adapted for locomotion, feeding and other functions. The genetics underlying appendage formation are best understood in insects and vertebrates. The expression of the
Distal-less
(
Dll
) homeoprotein during arthropod limb outgrowth and of
Dll
orthologs (
Dlx
) in fish fin and tetrapod limb buds led us to examine whether expression of this regulatory gene may be a general feature of appendage formation in protostomes and deuterostomes. We find that
Dll
is expressed along the proximodistal axis of developing polychaete annelid parapodia, onychophoran lobopodia, ascidian ampullae, and even echinoderm tube feet.
Dll/Dlx
expression in such diverse appendages in these six coelomate phyla could be convergent, but this would have required the independent co-option of
Dll/Dlx
several times in evolution. It appears more likely that ectodermal
Dll/Dlx
expression along proximodistal axes originated once in a common ancestor and has been used subsequently to pattern body wall outgrowths in a variety of organisms. We suggest that this pre-Cambrian ancestor of most protostomes and the deuterostomes possessed elements of the genetic machinery for and may have even borne appendages.
The α1domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: Is HLA-G the public ligand for natural killer cell inhibitory receptors?
Authors: Rouas-Freiss Nathalie , Marchal Rachel E. , Kirszenbaum Marek , Dausset Jean , Carosella Edgardo D.
Published: July 2002

We have investigated the protective role of the membrane-bound HLA-G1 and HLA-G2 isoforms against natural killer (NK) cell cytotoxicity. For this purpose, HLA-G1 and HLA-G2 cDNAs were transfected into the HLA class I-negative human K562 cell line, a known reference target for NK lysis. The HLA-G1 protein, encoded by a full-length mRNA, presents a structure similar to that of classical HLA class I antigens. The HLA-G2 protein, deduced from an alternatively spliced transcript, consists of the α1domain linked to the α3domain. In this study we demonstrate that (i) HLA-G2 is present at the cell surface as a truncated class I molecule associated with β2-microglobulin; (ii) NK cytolysis, observed in peripheral blood mononuclear cells and in polyclonal CD3−CD16+CD56+NK cells obtained from 20 donors, is inhibited by both HLA-G1 and HLA-G2; this HLA-G-mediated inhibition is reversed by blocking HLA-G with a specific mAb; this led us to the conjecture that HLA-G is the public ligand for NK inhibitory receptors (NKIR) present in all individuals; (iii) the α1domain common to HLA-G1 and HLA-G2 could mediate this protection from NK lysis; and (iv) when transfected into the K562 cell line, both HLA-G1 and HLA-G2 abolish lysis by the T cell leukemia NK-like YT2C2 clone due to interaction between the HLA-G isoform on the target cell surface and a membrane receptor on YT2C2. Because NKIR1 and NKIR2, known to interact with HLA-G, were undetectable on YT2C2, we conclude that a yet-unknown specific receptor for HLA-G1 and HLA-G2 is present on these cells.
Elevated levels of cysteine protease activity in saliva and salivary glands of the nonobese diabetic (NOD) mouse model for Sjögren syndrome
Authors: Robinson Christopher P. , Yamachika Shigeo , Alford Connie E. , Cooper Christopher , Pichardo Edgar L. , Shah Niel , Peck Ammon B. , Humphreys-Beher Michael G.
Published: July 2002

Nonobese diabetic (NOD) mice develop an anti-exocrine gland pathology similar to human Sjögren syndrome. Recently, we demonstrated that NOD-
scid
mice develop severe loss of submandibular acinar cells with concomitant appearance of abnormal isoforms of salivary proteins suggesting
de novo
enzymatic cleavage. Because these changes may indicate activation of apoptotic proteases, we examined saliva and salivary tissue for cysteine protease activity. Cysteine protease activities were elevated in saliva and gland lysates from 20-week-old NOD and NOD-
scid
mice as compared with age- and sex-matched BALB/c or 8-week-old NOD mice. This activity appeared in the submandibular glands, but not in the parotid glands. Western blot analyses using antibodies directed against specific apoptotic proteases (interleukin 1β converting enzyme, Nedd-2, and Apopain/CPP 32) confirmed these findings. Submandibular glands from NOD-
scid
mice exhibited the greatest increase in proteolytic activity, indicating that infiltrating leukocytes are not responsible for these changes. Western blot analyses also failed to reveal changes in the levels of cystatins (saliva proteins that inhibit protease activity). Thus, increased cysteine protease activity appears to be directly related to submandibular acinar cell loss in NOD-
scid
mice involving the apoptotic pathway. Additional protease activity in saliva and gland lysates of older NOD and NOD-
scid
mice, apparently mutually distinct from cysteine proteases, generated an enzymatically cleaved parotid secretory protein. We suggest, therefore, that proteolytic enzyme activity contributes to loss of exocrine gland tolerance by generating abnormally processed protein constituents.
Agonist-induced closure of constitutively open γ-aminobutyric acid channels with mutated M2 domains
Authors: Pan Zhuo-Hua , Zhang Dongxian , Zhang Xishan , Lipton Stuart A.
Published: July 2002

Ligand-gated ion channels display a fundamental property—channels remain virtually closed at rest and open upon agonist binding. Here we show that substituting alanines for either of two amino acid residues (T314 or L317) in the M2 region of the γ-aminobutyric acid (GABA) ρ1 subunit results in spontaneous channel opening in the absence of ligand. Surprisingly, for two single point mutants (T314A or L317A), application of very low concentrations of agonist partially suppressed this spontaneous current, while higher concentrations re-activated the receptors. When both of these sites were mutated (T314A/L317A), GABA nearly completely suppressed the constitutive current and did not re-activate the current even at very high concentrations. This study provides important new insights into the structure–function relationship of ligand-gated ion channels, where modification of the structure of the channel pore region not only alters the allosteric transition of the receptor protein but also reverses the polarity of agonist regulation of channel gating. Our results suggest that the sites where these two residues are located are structurally critical for channel gating.
Group I allergens of grass pollen as cell wall-loosening agents
Authors: Cosgrove Daniel J. , Bedinger Patricia , Durachko Daniel M.
Published: July 2002

Group I allergens are the major allergens of grass pollen, but their biological function is unknown. These proteins are shown here to be structurally related to expansins, which are able to induce extension (creep) of plant cell walls. Extracts of maize pollen possess potent expansin-like activity, as measured in wall extension and wall stress-relaxation assays. This activity is selective for grass cell walls and is, at least partly, due to the action of maize group I allergens. We propose that group I allergens facilitate invasion of the pollen tube into the maternal tissues by loosening the cell walls of the grass stigma and style. Additionally, the presence of related mRNAs in vegetative tissues of rice,
Arabidopsis
, and soybean implies that allergen homologs may function to loosen walls in growing vegetative tissues as well.
Origin of Genes
Authors: Gilbert Walter , de Souza Sandro J. , Long Manyuan
Published: July 2002

We discuss two tests of the hypothesis that the first genes were
assembled from exons. The hypothesis of exon shuffling in the progenote
predicts that intron phases will be correlated so that exons will be an
integer number of codons and predicts that the exons will be correlated
with compact regions of polypeptide chain. These predictions have been
tested on ancient conserved proteins (proteins without introns in
prokaryotes but with introns in eukaryotes) and hold with high
statistical significance. We conclude that introns are correlated with
compact features of proteins 15-, 22-, or 30-amino acid residues long,
as was predicted by “The Exon Theory of Genes.”
Xeroderma pigmentosum and trichothiodystrophy are associated with different mutations in the
XPD
(
ERCC
2) repair/transcription gene
Authors: Taylor Elaine M. , Broughton Bernard C. , Botta Elena , Stefanini Miria , Sarasin Alain , Jaspers Nicolaas G. J. , Fawcett Heather , Harcourt Susan A. , Arlett Colin F. , Lehmann Alan R.
Published: July 2002

The xeroderma pigmentosum group D (XPD) protein has a dual function, both in nucleotide excision repair of DNA damage and in basal transcription. Mutations in the
XPD
gene can result in three distinct clinical phenotypes, XP, trichothiodystrophy (TTD), and XP with Cockayne syndrome. To determine if the clinical phenotypes of XP and TTD can be attributed to the sites of the mutations, we have identified the mutations in a large group of TTD and XP-D patients. Most sites of mutations differed between XP and TTD, but there are three sites at which the same mutation is found in XP and TTD patients. Since the corresponding patients were all compound heterozygotes with different mutations in the two alleles, the alleles were tested separately in a yeast complementation assay. The mutations which are found in both XP and TTD patients behaved as null alleles, suggesting that the disease phenotype was determined by the other allele. If we eliminate the null mutations, the remaining mutagenic pattern is consistent with the site of the mutation determining the phenotype.
Stimulation of neurite outgrowth using an electrically conducting polymer
Authors: Schmidt Christine E. , Shastri Venkatram R. , Vacanti Joseph P. , Langer Robert
Published: July 2002

Damage to peripheral nerves often cannot be repaired by the juxtaposition of the severed nerve ends. Surgeons have typically used autologous nerve grafts, which have several drawbacks including the need for multiple surgical procedures and loss of function at the donor site. As an alternative, the use of nerve guidance channels to bridge the gap between severed nerve ends is being explored. In this paper, the electrically conductive polymer—oxidized polypyrrole (PP)—has been evaluated for use as a substrate to enhance nerve cell interactions in culture as a first step toward potentially using such polymers to stimulate
in vivo
nerve regeneration. Image analysis demonstrates that PC-12 cells and primary chicken sciatic nerve explants attached and extended neurites equally well on both PP films and tissue culture polystyrene in the absence of electrical stimulation. In contrast, PC-12 cells interacted poorly with indium tin oxide (ITO), poly(
l
-lactic acid) (PLA), and poly(lactic acid-co-glycolic acid) surfaces. However, PC-12 cells cultured on PP films and subjected to an electrical stimulus through the film showed a significant increase in neurite lengths compared with ones that were not subjected to electrical stimulation through the film and tissue culture polystyrene controls. The median neurite length for PC-12 cells grown on PP and subjected to an electrical stimulus was 18.14 μm (
n
= 5643) compared with 9.5 μm (
n
= 4440) for controls. Furthermore, animal implantation studies reveal that PP invokes little adverse tissue response compared with poly(lactic acid-co-glycolic acid).
Cytokine suppression of protease activation in wild-type p53-dependent and p53-independent apoptosis
Authors: Lotem Joseph , Sachs Leo
Published: July 2002

M1 myeloid leukemic cells overexpressing wild-type p53 undergo apoptosis. This apoptosis can be suppressed by some cytokines, protease inhibitors, and antioxidants. We now show that induction of apoptosis by overexpressing wild-type p53 is associated with activation of interleukin-1β-converting enzyme (ICE)-like proteases, resulting in cleavage of poly(ADP- ribose) polymerase and the proenzyme of the ICE-like protease Nedd-2. Activation of these proteases and apoptosis were suppressed by the cytokine interleukin 6 or by a combination of the cytokine interferon γ and the antioxidant butylated hydroxyanisole, and activation of poly(ADP-ribose) polymerase and apoptosis were suppressed by some protease inhibitors. In a clone of M1 cells that did not express p53, vincristine or doxorubicin induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by interleukin 6. In another myeloid leukemia (7-M12) doxorubicin also induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by granulocyte–macrophage colony-stimulating factor. The results indicate that (
i
) overexpression of wild-type p53 by itself or treatment with cytotoxic compounds in wild-type p53-expressing or p53-nonexpressing myeloid leukemic cells is associated with activation of ICE-like proteases; (
ii
) cytokines exert apoptosis-suppressing functions upstream of protease activation; (
iii
) the cytotoxic compounds induce additional pathways in apoptosis; and (
iv
) cytokines can also suppress these other components of the apoptotic machinery.
Potent, protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein vaccination
Authors: Letvin Norman L. , Montefiori David C. , Yasutomi Yasuhiro , Perry Helen C. , Davies Mary-Ellen , Lekutis Christine , Alroy Marianne , Freed Daniel C. , Lord Carol I. , Handt Laurence K. , Liu Margaret A. , Shiver John W.
Published: July 2002

It is generally thought that an effective vaccine to prevent HIV-1 infection should elicit both strong neutralizing antibody and cytotoxic T lymphocyte responses. We recently demonstrated that potent, boostable, long-lived HIV-1 envelope (Env)-specific cytotoxic T lymphocyte responses can be elicited in rhesus monkeys using plasmid-encoded HIV-1envDNA as the immunogen. In the present study, we show that the addition of HIV-1 Env protein to this regimen as a boosting immunogen generates a high titer neutralizing antibody response in this nonhuman primate species. Moreover, we demonstrate in a pilot study that immunization with HIV-1envDNA (multiple doses) followed by a final immunization with HIV-1envDNA plus HIV-1 Env protein (envgene from HXBc2 clone of HIV IIIB; Env protein from parental HIV IIIB) completely protects monkeys from infection after i.v. challenge with a chimeric virus expressing HIV-1env(HXBc2) on a simian immmunodeficiency virusmacbackbone (SHIV-HXBc2). The potent immunity and protection seen in these pilot experiments suggest that a DNA prime/DNA plus protein boost regimen warrants active investigation as a vaccine strategy to prevent HIV-1 infection.
A vaccine for HIV type 1: The antibody perspective
Authors: Burton Dennis R.
Published: July 2002

Antibodies that bind well to the envelope spikes of immunodeficiency viruses such as HIV type 1 (HIV-1) and simian immunodeficiency virus (SIV) can offer protection or benefit if present at appropriate concentrations before viral exposure. The challenge in antibody-based HIV-1 vaccine design is to elicit such antibodies to the viruses involved in transmission in humans (primary viruses). At least two major obstacles exist. The first is that very little of the envelope spike surface of primary viruses appears accessible for antibody binding (low antigenicity), probably because of oligomerization of the constituent proteins and a high degree of glycosylation of one of the proteins. The second is that the mature oligomer constituting the spikes appears to stimulate only weak antibody responses (low immunogenicity). Viral variation is another possible obstacle that appears to present fewer problems than anticipated. Vaccine design should focus on presentation of an intact mature oligomer, increasing the immunogenicity of the oligomer and learning from the antibodies available that potently neutralize primary viruses.
Antibodies to ribosomal P proteins ofTrypanosoma cruziin Chagas disease possess functional autoreactivity with heart tissue and differ from anti-P autoantibodies in lupus
Authors: Kaplan Dan , Ferrari Ines , Bergami Pablo Lopez , Mahler Evelina , Levitus Gabriela , Chiale Pablo , Hoebeke Johan , Van Regenmortel Marc H. V. , Levin Mariano J.
Published: July 2002

Anti-P antibodies present in sera from patients with chronic Chagas heart disease (cChHD) recognize peptide R13, EEEDDDMGFGLFD, which encompasses the C-terminal region of theTrypanosoma cruziribosomal P1 and P2 proteins. This peptide shares homology with the C-terminal region (peptide H13 EESDDDMGFGLFD) of the human ribosomal P proteins, which is in turn the target of anti-P autoantibodies in systemic lupus erythematosus (SLE), and with the acidic epitope, AESDE, of the second extracellular loop of the β1-adrenergic receptor. Anti-P antibodies from chagasic patients showed a marked preference for recombinant parasite ribosomal P proteins and peptides, whereas anti-P autoantibodies from SLE reacted with human and parasite ribosomal P proteins and peptides to the same extent. A semi-quantitative estimation of the binding of cChHD anti-P antibodies to R13 and H13 using biosensor technology indicated that the average affinity constant was about 5 times higher for R13 than for H13. Competitive enzyme immunoassays demonstrated that cChHD anti-P antibodies bind to the acidic portions of peptide H13, as well as to peptide H26R, encompassing the second extracellular loop of the β1adrenoreceptor. Anti-P antibodies isolated from cChHD patients exert a positive chronotropic effectin vitroon cardiomyocytes from neonatal rats, which resembles closely that of anti-β1receptor antibodies isolated from the same patient. In contrast, SLE anti-P autoantibodies have no functional effect. Our results suggest that the adrenergic-stimulating activity of anti-P antibodies may be implicated in the induction of functional myocardial impairments observed in cChHD.
Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis
Authors: Yamada Tomoya , De Souza Angus T. , Finkelstein Sydney , Jirtle Randy L.
Published: July 2002

This report shows that loss of heterozygosity at the mannose 6-phosphate/insulin-like growth factor II receptor (
M6P/IGF2R
) locus occurred in 5/8 (63%) dysplastic liver lesions and 11/18 (61%) hepatocellular carcinomas (HCCs) associated with the high risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in 6/11 (55%) HCCs, including deletions in a polydeoxyguanosine region known to be a target of microsatellite instability.
M6P/IGF2R
allele loss was also found in cirrhotic tissue of clonal origin adjacent to these dysplastic lesions and HCCs, demonstrating that
M6P/IGF2R
inactivation occurs early in liver carcinogenesis. In conclusion, HCCs frequently develop from clonal expansions of phenotypically normal,
M6P/IGF2R-
mutated hepatocytes, providing further support for the idea that
M6P/IGF2R
functions as a liver tumor-suppressor gene.
Genetic definition of a protein-splicing domain: Functional mini-inteins support structure predictions and a model for intein evolution
Authors: Derbyshire Victoria , Wood David W. , Wu Wei , Dansereau John T. , Dalgaard Jacob Z. , Belfort Marlene
Published: July 2002

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the
Mycobacterium tuberculosis recA
intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu
recA
intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.