bluuebunny/crossref_metadata_2025 huggingface dataset:
Skin wounds and severed nerves heal normally in mice lacking tenascin-C.
Authors: Forsberg E , Hirsch E , Fröhlich L , Meyer M , Ekblom P , Aszodi A , Werner S , Fässler R
Published: July 2002

A large number of functions have been demonstrated for tenascin-C by antibody perturbation assays and in vitro cell culture experiments. However, these results contrast sharply with the lack of any apparent phenotype in mice with a genetic deletion of tenascin-C. A possible explanation for the lack of phenotype would be expression of some altered but functional tenascin-C in the mutant. We report the generation of an independent tenascin-C null mouse and conclude that the original tenascin-C knockout, which is genetically very similar to ours, is also a true null. As found previously, the absence of tenascin-C has no influence on development, adulthood, life span, and fecundity. We have studied in detail two models of wound healing. After axotomy, the regeneration of the sciatic nerve is not altered without tenascin-C. During healing of cutaneous wounds, deposition of collagen I, fibulin-2, and nidogen is identical in mutant and wild-type mice. In contrast. fibronectin appears diminished in wounds of tenascin-C-deficient mice. However, the lack of tenascin-C together with the reduced amount of fibronectin has no influence on the quality of the healing process.
Mouse mast cell gp49B1 contains two immunoreceptor tyrosine-based inhibition motifs and suppresses mast cell activation when coligated with the high-affinity Fc receptor for IgE.
Authors: Katz H R , Vivier E , Castells M C , McCormick M J , Chambers J M , Austen K F
Published: July 2002

Mouse mast cells express gp49B1, a cell-surface member of the Ig superfamily encoded by the gp49B gene. We now report that by ALIGN comparison of the amino acid sequence of gp49B1 with numerous receptors of the Ig superfamily, a newly recognized family has been established that includes gp49B1, the human myeloid cell Fc receptor for IgA, the bovine myeloid cell Fc receptor for IgG2, and the human killer cell inhibitory receptors expressed on natural killer cells and T lymphocyte subsets. Furthermore, the cytoplasmic domain of gp49B1 contains two immunoreceptor tyrosine-based inhibition motifs that are also present in killer cell inhibitory receptors; these motifs downregulate natural killer cell and T-cell activation signals that lead to cytotoxic activity. As assessed by flow cytometry with transfectants that express either gp49B1 or gp49A, which are 89% identical in the amino acid sequences of their extracellular domains, mAb B23.1 was shown to recognize only gp49B1. Coligation of mAb B23.1 bound to gp49B1 and IgE fixed to the high-affinity Fc receptor for IgE on the surface of mouse bone marrow-derived mast cells inhibited exocytosis in a dose-related manner, as defined by the release of the secretory granule constituent beta-hexosaminidase, as well as the generation of the membrane-derived lipid mediator, leukotriene C4. Thus, gp49B1 is an immunoreceptor tyrosine-based inhibition motif-containing integral cell-surface protein that downregulates the high-affinity Fc receptor for IgE-mediated release of proinflammatory mediators from mast cells. Our findings establish a novel counterregulatory transmembrane pathway by which mast cell activation can be inhibited.
Activation of mouse sperm T-type Ca
2+
channels by adhesion to the egg zona pellucida
Authors: Arnoult Christophe , Cardullo Richard A. , Lemos Jose R. , Florman Harvey M.
Published: July 2002

The sperm acrosome reaction is a Ca
2+
-dependent
exocytotic event that is triggered by adhesion to the mammalian egg’s
zona pellucida. Previous studies using ion-selective fluorescent probes
suggested a role of voltage-sensitive Ca
2+
channels in
acrosome reactions. Here, whole-cell patch clamp techniques are used to
demonstrate the expression of functional T-type Ca
2+
channels during mouse spermatogenesis. The germ cell T current is
inhibited by antagonists of T-type channels (pimozide and amiloride) as
well as by antagonists whose major site of action is the somatic cell
L-type Ca
2+
channel (1,4-dihydropyridines, arylalkylamines,
benzothiazapines), as has also been reported for certain somatic cell T
currents. In sperm, inhibition of T channels during gamete interaction
inhibits zona pellucida-dependent Ca
2+
elevations, as
demonstrated by ion-selective fluorescent probes, and also inhibits
acrosome reactions. These studies directly link sperm T-type
Ca
2+
channels to fertilization. In addition, the kinetics
of channel inhibition by 1,4-dihydropyridines suggests a mechanism for
the reported contraceptive effects of those compounds in human males.
The subcellular localization of E2F-4 is cell-cycle dependent
Authors: Lindeman Geoffrey J. , Gaubatz Stefan , Livingston David M. , Ginsberg Doron
Published: July 2002

The E2F family of transcription factors plays a crucial role in cell cycle progression. E2F activity is tightly regulated by a number of mechanisms, which include the timely synthesis and degradation of E2F, interaction with retinoblastoma protein family members (“pocket proteins”), association with DP heterodimeric partner proteins, and phosphorylation of the E2F/DP complex. Here we report that another mechanism, subcellular localization, is important for the regulation of E2F activity. Unlike E2F-1, -2, or -3, which are constitutively nuclear, ectopic E2F-4 and -5 were predominantly cytoplasmic. Cotransfection of expression vectors encoding p107, p130, or DP-2, but not DP-1, resulted in the nuclear localization of E2F-4 and -5. Moreover, the transcriptional activity of E2F-4 was markedly enhanced when it was invariably nuclear. Conversely, it was reduced when the protein was excluded from the nucleus, implying that E2F-4 transcription function depends upon its cytological location. In keeping with this, the nuclear/cytoplasmic ratios of endogenous E2F-4 changed as cells exited G
0
, with high ratios in G
0
and early G
1
and a progressive increase in cytoplasmic E2F-4 as cells approached S phase. Thus, the subcellular location of E2F-4 is regulated in a cell cycle-dependent manner, providing another potential mechanism for its functional regulation.
Three-dimensional solution structure of the complex of α-bungarotoxin with a library-derived peptide
Authors: Scherf Tali , Balass Moshe , Fuchs Sara , Katchalski-Katzir Ephraim , Anglister Jacob
Published: July 2002

The solution structure of the complex between α-bungarotoxin (α-BTX) and a 13-residue library-derived peptide (MRYYESSLKSYPD) has been solved using two-dimensional proton–NMR spectroscopy. The bound peptide adopts an almost-globular conformation resulting from three turns that surround a hydrophobic core formed by Tyr-11 of the peptide. The peptide fills an α-BTX pocket made of residues located at fingers I and II, as well as at the C-terminal region. Of the peptide residues, the largest contact area is formed by Tyr-3 and Tyr-4. These findings are in accord with the previous data in which it had been shown that substitution of these aromatic residues by aliphatic amino acids leads to loss of binding of the modified peptide with α-BTX. Glu-5 and Leu-8, which also remarkably contribute to the contact area with the toxin, are present in all the library-derived peptides that bind strongly to α-BTX. The structure of the complex may explain the fact that the library-derived peptide binds α-BTX with a 15-fold higher affinity than that shown by the acetylcholine receptor peptide (α185–196). Although both peptides bind to similar sites on α-BTX, the latter adopts an extended conformation when bound to the toxin [Basus, V., Song, G. & Hawrot, E. (1993)
Biochemistry
32, 12290–12298], whereas the library peptide is nearly globular and occupies a larger surface area of α-BTX binding site.
Intercellular adhesion molecule 1 knockout abrogates radiation induced pulmonary inflammation
Authors: Hallahan Dennis E. , Virudachalam Subbulakshmi
Published: July 2002

Increased expression of intercellular adhesion molecule 1 (ICAM-1; CD54) is induced by exposure to ionizing radiation. The lung was used as a model to study the role of ICAM-1 in the pathogenesis of the radiation-induced inflammation-like response. ICAM-1 expression increased in the pulmonary microvascular endothelium and not in the endothelium of larger pulmonary vessels following treatment of mice with thoracic irradiation. To quantify radiation-induced ICAM-1 expression, we utilized fluorescence-activated cell sorting analysis of anti-ICAM-1 antibody labeling of pulmonary microvascular endothelial cells from human cadaver donors (HMVEC-L cells). Fluorochrome conjugates and UV microscopy were used to quantify the fluorescence intensity of ICAM in the irradiated lung. These studies showed a dose- and time-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium. Peak expression occurred at 24 h, while threshold dose was as low as 2 Gy. To determine whether ICAM-1 is required for inflammatory cell infiltration into the irradiated lung, the anti-ICAM-1 blocking antibody was administered by tail vein injection to mice following thoracic irradiation. Inflammatory cells were quantified by immunofluorescence for leukocyte common antigen (CD45). Mice treated with the anti-ICAM-1 blocking antibody showed attenuation of inflammatory cell infiltration into the lung in response to ionizing radiation exposure. To verify the requirement of ICAM-1 in the inflammation-like radiation response, we utilized the ICAM-1 knockout mouse. ICAM-1 was not expressed in the lungs of ICAM-1-deficient mice following treatment with thoracic irradiation. ICAM-1 knockout mice had no increase in the inflammatory cell infiltration into the lung in response to thoracic irradiation. These studies demonstrate a radiation dose-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium, and show that ICAM-1 is required for inflammatory cell infiltration into the irradiated lung.
Interaction of human apurinic endonuclease and DNA polymerase β in the base excision repair pathway
Authors: Bennett Richard A. O. , Wilson David M. , Wong Donny , Demple Bruce
Published: July 2002

Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase β (Polβ) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein–protein contact between Polβ and human AP endonuclease (Ape protein).
In vitro
, binding of Ape protein to uncleaved AP sites loads Polβ into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Polβ exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5′-terminal deoxyribose 5-phosphate by Polβ. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.
High-affinity binding of the
Drosophila
Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif
Authors: Li Shun-Cheng , Songyang Zhou , Vincent Sébastien J. F. , Zwahlen Catherine , Wiley Sandra , Cantley Lewis , Kay Lewis E. , Forman-Kay Julie , Pawson Tony
Published: July 2002

The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid). We describe here a novel peptide sequence recognized by the PTB domain from
Drosophila
Numb (dNumb), a protein involved in cell fate determination and asymmetric cell division during the development of the
Drosophila
nervous system. Using a Tyr-oriented peptide library to screen for ligands, the dNumb PTB domain was found to bind selectively to peptides containing a YIGPYφ motif (φ represents a hydrophobic residue). A synthetic peptide containing this sequence bound specifically to the isolated dNumb PTB domain in solution with a dissociation constant (
K
d
) of 5.78 ± 0.74 μM. Interestingly, the affinity of this peptide for the dNumb PTB domain was increased (
K
d
= 1.41 ± 0.10 μM) when the second tyrosine in the sequence was phosphorylated. Amino acid substitution studies of the phosphopeptide demonstrated that a core motif of sequence GP(p)Y is required for high-affinity binding to the dNumb PTB domain. Nuclear magnetic resonance experiments performed on isotopically labeled protein complexed with either Tyr- or pTyr-containing peptides suggest that the same set of amino acids in the dNumb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the peptide. The
in vitro
selectivity of the dNumb PTB domain is therefore markedly different from those of the Shc and IRS-1 PTB domains, in that it interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. Our results suggest that the PTB domain is a versatile protein module, capable of exhibiting varied binding specificities.
Mouse mammary tumor virus/v-Ha-
ras
transgene-induced mammary tumors exhibit strain-specific allelic loss on mouse chromosome 4
Authors: Radany Eric H. , Hong Karen , Kesharvarzi Sima , Lander Eric S. , Bishop J. Michael
Published: July 2002

Hybrid mice carrying oncogenic transgenes afford powerful systems for investigating loss of heterozygosity (LOH) in tumors. Here, we apply this approach to a neoplasm of key importance in human medicine: mammary carcinoma. We performed a whole genome search for LOH using the mouse mammary tumor virus/v-Ha-
ras
mammary carcinoma model in female (FVB/N ×
Mus musculus castaneus
)F
1
mice. Mammary tumors developed as expected, as well as a few tumors of a second type (uterine leiomyosarcoma) not previously associated with this transgene. Genotyping of 94 anatomically independent tumors revealed high-frequency LOH (≈38%) for markers on chromosome 4. A marked allelic bias was observed, with
M. musculus castaneus
alleles almost exclusively being lost. No evidence of genomic imprinting effects was noted. These data point to the presence of a tumor suppressor gene(s) on mouse chromosome 4 involved in mammary carcinogenesis induced by mutant H-
ras
expression, and for which a significant functional difference may exist between the
M. musculus castaneus
and FVB/N alleles. Provisional subchromosomal localization of this gene, designated
Loh-3
, can be made to a distal segment having syntenic correspondence to human chromosome 1p; LOH in this latter region is observed in several human malignancies, including breast cancers. Evidence was also obtained for a possible second locus associated with LOH with less marked allele bias on proximal chromosome 4.
Essential role of POU–domain factor Brn-3c in auditory and vestibular hair cell development
Authors: Xiang Mengqing , Gan Lin , Li Daqing , Chen Zhi-Yong , Zhou Lijuan , O’Malley Bert W. , Klein William , Nathans Jeremy
Published: July 2002

The
Brn-3
subfamily of POU–domain transcription factor genes consists of three highly homologous members—Brn-3a, Brn-3b, and Brn-3c—that are expressed in sensory neurons and in a small number of brainstem nuclei. This paper describes the role of
Brn-3c
in auditory and vestibular system development. In the inner ear, the Brn-3c protein is found only in auditory and vestibular hair cells, and the Brn-3a and Brn-3b proteins are found only in subsets of spiral and vestibular ganglion neurons. Mice carrying a targeted deletion of the
Brn-3c
gene are deaf and have impaired balance. These defects reflect a complete loss of auditory and vestibular hair cells during the late embryonic and early postnatal period and a secondary loss of spiral and vestibular ganglion neurons. Together with earlier work demonstrating a loss of trigeminal ganglion neurons and retinal ganglion cells in mice carrying targeted disruptions in the
Brn-3a
and
Brn-3b
genes, respectively, the
Brn-3c
phenotype reported here demonstrates that each of the
Brn-3
genes plays distinctive roles in the somatosensory, visual, and auditory/vestibular systems.
The phosphorylation state of the FIGQY tyrosine of neurofascin determines ankyrin-binding activity and patterns of cell segregation
Authors: Tuvia Shmuel , Garver Timothy D. , Bennett Vann
Published: July 2002

Cell–cell recognition and patterning of cell contacts have a critical role in mediating reversible assembly of a variety of transcellular complexes in the nervous system. This study provides evidence for regulation of cell interactions through modulation of ankyrin binding to neurofascin, a member of the L1CAM family of nervous system cell adhesion molecules. The phosphorylation state of the conserved FIGQY tyrosine in the cytoplasmic domain of neurofascin regulates ankyrin binding and governs neurofascin-dependent cell aggregation as well as cell sorting when neurofascin is expressed in neuroblastoma cells. These findings suggest a general mechanism for the patterning of cell contact based on external signals that regulate tyrosine phosphorylation of L1CAM members and modulate their binding to ankyrin.
Impaired motor coordination and persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking Gαq
Authors: Offermanns Stefan , Hashimoto Kouichi , Watanabe Masahiko , Sun William , Kurihara Hideo , Thompson Richard F. , Inoue Yoshiro , Kano Masanobu , Simon Melvin I.
Published: July 2002

Mice lacking the α-subunit of the heterotrimeric guanine nucleotide binding protein G
q
(Gα
q
) are viable but suffer from ataxia with typical signs of motor discoordination. The anatomy of the cerebellum is not overtly disturbed, and excitatory synaptic transmission from parallel fibers to cerebellar Purkinje cells (PCs) and from climbing fibers (CFs) to PCs is functional. However, about 40% of adult Gα
q
mutant PCs remain multiply innervated by CFs because of a defect in regression of supernumerary CFs in the third postnatal week. Evidence is provided suggesting that Gα
q
is part of a signaling pathway that is involved in the elimination of multiple CF innervation during this period.
Phloem sap proteins from
Cucurbita maxima
and
Ricinus communis
have the capacity to traffic cell to cell through plasmodesmata
Authors: Balachandran Suchandra , Xiang Yu , Schobert Christian , Thompson Gary A. , Lucas William J.
Published: July 2002

In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of fluorescently labeled dextrans along with size-fractionated
Cucurbita maxima
phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual fluorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20–100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from
Ricinus communis
, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.
Classification of mononuclear zinc metal sites in protein structures
Authors: Karlin Samuel , Zhu Zhan-Yang
Published: July 2002

Our study of the extended metal environment, particularly of the second shell, focuses in this paper on zinc sites. Key findings include: (
i
) The second shell of mononuclear zinc centers is generally more polar than hydrophobic and prominently features charged residues engaged in an abundance of hydrogen bonding with histidine ligands. Histidine–acidic or histidine–tyrosine clusters commonly overlap the environment of zinc ions. (
ii
) Histidine tautomeric metal bonding patterns in ligating zinc ions are mixed. For example, carboxypeptidase A, thermolysin, and sonic hedgehog possess the same ligand group (two histidines, one unibidentate acidic ligand, and a bound water), but their histidine tautomeric geometries markedly differ such that the carboxypeptidase A makes only N
δ1
contacts, thermolysin makes only N
ɛ2
contacts, and sonic hedgehog uses one of each. Thus the presence of a similar ligand cohort does not necessarily imply the same topology or function at the active site. (
iii
) Two close histidine ligands HX

m

H,
m
≤ 5, rarely both coordinate a single metal ion in the N
δ1
tautomeric conformation, presumably to avoid steric conflicts. Mononuclear zinc sites can be classified into six types depending on the ligand composition and geometry. Implications of the results are discussed in terms of divergent and convergent evolution.
A mutation in the putative RNA polymerase gene inhibits nonhomologous, but not homologous, genetic recombination in an RNA virus
Authors: Figlerowicz M. , Nagy P. D. , Bujarski J. J.
Published: July 2002

Brome mosaic bromovirus (BMV), a positive-stranded RNA virus, supports both homologous and nonhomologous RNA recombinations. Two BMV (temperature-sensitive) mutants with alterations in the 2a protein, the putative RNA polymerase component of the viral replicase, were tested for their ability to support both types of recombination. Here we report that one of these mutants with the Leu-486 substituted by Phe did not support nonhomologous recombination. Effect on homologous recombination was mainly on the location and precision of crossover events. The other 2a mutant with Asn-458 substituted by Asp did not negatively affect either type of recombination. Apparently, BMV RNA polymerase participates differently in the two types of recombination events.
A cloned
Erwinia chrysanthemi
Hrp (type III protein secretion) system functions in
Escherichia coli
to deliver
Pseudomonas syringae
Avr signals to plant cells and to secrete Avr proteins in culture
Authors: Ham Jong Hyun , Bauer David W. , Fouts Derrick E. , Collmer Alan
Published: July 2002

The Hrp (type III protein secretion) system is essential for the plant parasitic ability of
Pseudomonas syringae
and most Gram-negative bacterial plant pathogens. AvrB and AvrPto are two
P. syringae
proteins that have biological activity when produced via heterologous gene expression inside plant cells or when produced by Hrp
+
bacteria. Avr-like proteins, presumably injected by the Hrp system on bacterial contact with plant cells, appear to underlie pathogenic interactions, but none has been observed outside of the bacterial cytoplasm, and identifying novel genes encoding them is tedious and uncertain without a phenotype in culture. Here we describe a cloned Hrp secretion system that functions heterologously in
Escherichia coli
to secrete AvrB and AvrPto in culture and to promote AvrB and AvrPto biological activity in inoculated plants. The
hrp
gene cluster, carried on cosmid pCPP2156, was cloned from
Erwinia chrysanthemi
, a pathogen that differs from
P. syringae
in being host promiscuous.
E. coli
DH5α carrying pCPP2156, but not related Hrp-deficient cosmids, elicited a hypersensitive response in
Nicotiana clevelandii
only when also expressing
avrB
in trans. The use of pAVRB-FLAG2 and pAVRPTO-FLAG, which produce Avr proteins with a C-terminal FLAG-epitope fusion, enabled immunoblot detection of the secretion of these proteins to
E. coli
(pCPP2156) culture media. Secretion was Hrp dependent, occurred without leakage of a cytoplasmic marker, and did not occur with
E. coli
(pHIR11), which encodes a functional
P. syringae
Hrp system.
E. coli
(pCPP2156) will promote investigation of Avr protein secretion and systematic prospecting for the effector proteins underlying bacterial plant pathogenicity.
The
APC
variants I1307K and E1317Q are associated with colorectal tumors, but not always with a family history
Authors: Frayling Ian M. , Beck Nicholas E. , Ilyas Mohammad , Dove-Edwin Isis , Goodman Peter , Pack Kevin , Bell Jennifer A. , Williams Christopher B. , Hodgson Shirley V. , Thomas Huw J. W. , Talbot Ian C. , Bodmer Walter F. , Tomlinson Ian P. M.
Published: July 2002

Classical familial adenomatous polyposis (FAP) is a high-penetrance autosomal dominant disease that predisposes to hundreds or thousands of colorectal adenomas and carcinoma and that results from truncating mutations in the
APC
gene. A variant of FAP is attenuated adenomatous polyposis coli, which results from germ-line mutations in the 5′ and 3′ regions of the
APC
gene. Attenuated adenomatous polyposis coli patients have “multiple” colorectal adenomas (typically fewer than 100) without the florid phenotype of classical FAP. Another group of patients with multiple adenomas has no mutations in the
APC
gene, and their phenotype probably results from variation at a locus, or loci, elsewhere in the genome. Recently, however, a missense variant of
APC
(I1307K) was described that confers an increased risk of colorectal tumors, including multiple adenomas, in Ashkenazim. We have studied a set of 164 patients with multiple colorectal adenomas and/or carcinoma and analyzed codons 1263–1377 (exon 15G) of the
APC
gene for germ-line variants. Three patients with the I1307K allele were detected, each of Ashkenazi descent. Four patients had a germ-line E1317Q missense variant of
APC
that was not present in controls; one of these individuals had an unusually large number of metaplastic polyps of the colorectum. There is increasing evidence that there exist germ-line variants of the
APC
gene that predispose to the development of multiple colorectal adenomas and carcinoma, but without the florid phenotype of classical FAP, and possibly with importance for colorectal cancer risk in the general population.
An ERp60-like protein from the filarial parasiteDirofilaria immitishas both transglutaminase and protein disulfide isomerase activity
Authors: Chandrashekar Ramaswamy , Tsuji Naotoshi , Morales Tony , Ozols Victor , Mehta Kapil
Published: July 2002

Transglutaminases (TGases; EC2.3.2.13) are a family of enzymes that catalyze calcium-dependent covalent cross-linking of cellular proteins by establishing ɛ-(γ-glutamyl)lysine isopeptide bonds. These covalent isopeptide bonds are of great physiological significance because they are highly resistant to proteolysis, denaturants, and reducing agents. Prior studies have demonstrated the presence of isopeptide bonds in the sheath and cuticle of filarial parasites, suggesting an important role for TGase-catalyzed reactions during the growth and development of filarial nematodes. Herein we report the identification and cloning of a cDNA encoding a TGase from the dog heartwormDirofilaria immitis(DiTG). The DiTG expressed inEscherichia coli(recombinant DiTG) was able to catalyze calcium-dependent cross-linking reactions. The derived amino acid sequence of the DiTG cDNA (pDiTG) predicts a protein of 57.1 kDa and includes an N-terminal hydrophobic signal peptide. The pDiTG has no sequence similarity with any of the known TGases, but it has significant homology to protein disulfide isomerase (PDI) and, particularly, to the PDI-related endoplasmic reticulum protein ERp60, a PDI isoform found in the lumen of endoplasmic reticulum. As predicted from the amino acid sequence homology, recombinant DiTG catalyzed the isomerization of intramolecular disulfide/sulfhydryl bonds in denatured RNasein vitroas effectively as did mammalian PDI. Conversely, purified PDI from bovine liver could catalyze protein cross-linking reactions in a Ca2+-dependent manner. This report describes the dual catalytic activity of TGase and PDI in post- and/or cotranslational modification of newly synthesized proteins. These TGase-catalyzed posttranslational modifications may play a pivotal role in the synthesis of new cuticle during the growth and maturation of filarial parasites.
Peptide design by artificial neural networks and computer-based evolutionary search
Authors: Schneider Gisbert , Schrödl Wieland , Wallukat Gerd , Müller Johannes , Nissen Eberhard , Rönspeck Wolfgang , Wrede Paul , Kunze Rudolf
Published: July 2002

A technique for systematic peptide variation by a combination of rational and evolutionary approaches is presented. The design scheme consists of five consecutive steps: (
i
) identification of a “seed peptide” with a desired activity, (
ii
) generation of variants selected from a physicochemical space around the seed peptide, (
iii
) synthesis and testing of this biased library, (
iv
) modeling of a quantitative sequence-activity relationship by an artificial neural network, and (
v
)
de novo
design by a computer-based evolutionary search in sequence space using the trained neural network as the fitness function. This strategy was successfully applied to the identification of novel peptides that fully prevent the positive chronotropic effect of anti-β
1
-adrenoreceptor autoantibodies from the serum of patients with dilated cardiomyopathy. The seed peptide, comprising 10 residues, was derived by epitope mapping from an extracellular loop of human β
1
-adrenoreceptor. A set of 90 peptides was synthesized and tested to provide training data for neural network development.
De novo
design revealed peptides with desired activities that do not match the seed peptide sequence. These results demonstrate that computer-based evolutionary searches can generate novel peptides with substantial biological activity.
Transgenic
Drosophila
expressing human amyloid precursor protein show γ-secretase activity and a blistered-wing phenotype
Authors: Fossgreen Anke , Brückner Bodo , Czech Christian , Masters Colin L. , Beyreuther Konrad , Paro Renato
Published: July 2002

The importance of the amyloid precursor protein (APP) in the pathogenesis of Alzheimer’s disease (AD) became apparent through the identification of distinct mutations in the APP gene, causing early onset familial AD with the accumulation of a 4-kDa peptide fragment (βA4) in amyloid plaques and vascular deposits. However, the physiological role of APP is still unclear. In this work,
Drosophila melanogaster
is used as a model system to analyze the function of APP by expressing wild-type and various mutant forms of human APP in fly tissue culture cells as well as in transgenic fly lines. After expression of full-length APP forms, secretion of APP but not of βA4 was observed in both systems. By using SPA4CT, a short APP form in which the signal peptide was fused directly to the βA4 region, transmembrane domain, and cytoplasmic tail, we observed βA4 release in flies and fly-tissue culture cells. Consequently, we showed a γ-secretase activity in flies. Interestingly, transgenic flies expressing full-length forms of APP have a blistered-wing phenotype. As the wing is composed of interacting dorsal and ventral epithelial cell layers, this phenotype suggests that human APP expression interferes with cell adhesion/signaling pathways in
Drosophila
, independently of βA4 generation.
Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome
Authors: Nichols Kim E. , Harkin D. Paul , Levitz Seth , Krainer Michael , Kolquist Kathryn Ann , Genovese Cameo , Bernard Amy , Ferguson Martin , Zuo Lin , Snyder Eric , Buckler Alan J. , Wise Carol , Ashley Jennifer , Lovett Michael , Valentine Marcus B. , Look A. Thomas , Gerald William , Housman David E. , Haber Daniel A.
Published: July 2002

X-linked lymphoproliferative syndrome (XLP) is an inherited immunodeficiency characterized by increased susceptibility to Epstein–Barr virus (EBV). In affected males, primary EBV infection leads to the uncontrolled proliferation of virus-containing B cells and reactive cytotoxic T cells, often culminating in the development of high-grade lymphoma. TheXLPgene has been mapped to chromosome band Xq25 through linkage analysis and the discovery of patients harboring large constitutional genomic deletions. We describe here the presence of small deletions and intragenic mutations that specifically disrupt a gene namedDSHPin 6 of 10 unrelated patients with XLP. This gene encodes a predicted protein of 128 amino acids composing a single SH2 domain with extensive homology to the SH2 domain of SHIP, an inositol polyphosphate 5-phosphatase that functions as a negative regulator of lymphocyte activation.DSHPis expressed in transformed T cell lines and is induced followingin vitroactivation of peripheral blood T lymphocytes. Expression ofDSHPis restrictedin vivoto lymphoid tissues, and RNAin situhybridization demonstratesDSHPexpression in activated T and B cell regions of reactive lymph nodes and in both T and B cell neoplasms. These observations confirm the identity ofDSHPas the gene responsible for XLP, and suggest a role in the regulation of lymphocyte activation and proliferation. Induction ofDSHPmay sustain the immune response by interfering withSHIP-mediated inhibition of lymphocyte activation, while its inactivation in XLP patients results in a selective immunodeficiency to EBV.
Perceptual learning reflects external noise filtering and internal noise reduction through channel reweighting
Authors: Dosher Barbara Anne , Lu Zhong-Lin
Published: July 2002

To investigate the nature of plasticity in the adult visual system,
perceptual learning was measured in a peripheral orientation
discrimination task with systematically varying amounts of external
(environmental) noise. The signal contrasts required to achieve
threshold were reduced by a factor or two or more after training at all
levels of external noise. The strong quantitative regularities revealed
by this novel paradigm ruled out changes in multiplicative internal
noise, changes in transducer nonlinearites, and simple attentional
tradeoffs. Instead, the regularities specify the mechanisms of
perceptual learning at the behavioral level as a combination of
external noise exclusion and stimulus enhancement via additive internal
noise reduction. The findings also constrain the neural architecture of
perceptual learning. Plasticity in the weights between basic visual
channels and decision is sufficient to account for perceptual learning
without requiring the retuning of visual mechanisms.
Perturbation of the T cell repertoire in rheumatoid arthritis
Authors: Wagner Ulf G. , Koetz Kerstin , Weyand Cornelia M. , Goronzy Jörg J.
Published: July 2002

Aberrations in the T cell repertoire with the emergence of oligoclonal populations have been described in patients with rheumatoid arthritis (RA). However, the extent of the repertoire perturbations as well as the underlying mechanisms are not known. We now have examined the diversity of the peripheral CD4 T cell repertoire by determining the frequencies of arbitrarily selected T cell receptor (TCR) β-chain sequences. Healthy individuals displayed a highly diverse repertoire, with a median frequency of individual TCR β-chain sequences of 1 in 2.4 × 107CD4 T cells. In RA patients, the median TCR β-chain frequency was increased 10-fold, indicating marked contraction of the repertoire (P< 0.001). The loss in TCR diversity was not limited to CD4 memory T cells but also involved the compartment of naive T cells, suggesting that it reflected an abnormality in T cell repertoire formation and not a consequence of antigen recognition in the synovium. Also, control patients with chronic inflammatory disease such as hepatitis C expressed a diverse repertoire indistinguishable from that of normals. Telomere length studies indicated an increased replicative history of peripheral CD4 T cells in RA patients, suggesting an enhanced turnover within the CD4 compartment. Compared with age-matched controls, terminal restriction fragment sizes were 1.7 kilobases shorter (P< 0.001). These data demonstrate an altered CD4 T cell homeostasis in RA that may contribute to the autoimmune response as well as to the immunodeficiency in these patients.
Isotope exchange studies on the
Escherichia coli
selenophosphate synthetase mechanism
Authors: Walker Heidi , Ferretti James A. , Stadtman Thressa C.
Published: July 2002

Selenophosphate synthetase, the
Escherichia coli selD
gene product, is a 37-kDa protein that catalyzes the synthesis of selenophosphate from ATP and selenide. In the absence of selenide, ATP is converted quantitatively to AMP and two orthophosphates in a very slow partial reaction. A monophosphorylated enzyme derivative containing the γ-phosphoryl group of ATP has been implicated as an intermediate from the results of positional isotope exchange studies. Conservation of the phosphate bond energy in the final selenophosphate product is indicated by its ability to phosphorylate alcohols and amines to form O-phosphoryl- and N-phosphoryl-derivatives. To further probe the mechanism of action of selenophosphate synthetase, isotope exchange studies with [8-
14
C]ADP or [8-
14
C]AMP and unlabeled ATP were carried out, and
31
P NMR analysis of reaction mixtures enriched in H
2
18
O was performed. A slow enzyme-catalyzed exchange of ADP with ATP observed in the absence of selenide implies the existence of a phosphorylated enzyme and further supports an intermediary role of ADP in the reaction. Under these conditions ADP is slowly converted to AMP. Incorporation of
18
O from H
2
18
O exclusively into orthophosphate in the overall selenide-dependent reaction indicates that the β-phosphoryl group of the enzyme-bound ADP is attacked by water with liberation of orthophosphate and formation of AMP. Based on these results and the failure of the enzyme to catalyze an exchange of labeled AMP with ATP, the existence of a pyrophosphorylated enzyme intermediate that was postulated earlier can be excluded.
Anticancer action of cubé insecticide: Correlation for rotenoid constituents between inhibition of NADH:ubiquinone oxidoreductase and induced ornithine decarboxylase activities
Authors: Fang Nianbai , Casida John E.
Published: July 2002

Rotenone and rotenoid-containing botanicals, important insecticides and fish poisons, are reported to have anticancer activity in rats and mice. The toxic action of rotenone is attributed to inhibition of NADH:ubiquinone oxidoreductase activity and the purported cancer chemopreventive effect of deguelin analogs has been associated with inhibition of phorbol ester-induced ornithine decarboxylase (ODC) activity. This study defines a possible relationship between these two types of activity important in evaluating the toxicology of rotenoid pesticides and the suitability of the anticancer model. Fractionation of cubé resin (the commercial rotenoid pesticide) establishes that the activity in both assays is due primarily to rotenone (IC
50
= 0.8–4 nM), secondarily to deguelin, and in small part to rotenolone and tephrosin. In addition, the potency of 29 rotenoids from cubé insecticide for inhibiting NADH:ubiquinone oxidoreductase
in vitro
assayed with bovine heart electron transport particles satisfactorily predicts their potency
in vivo
in the induced ODC assay using noncytotoxic rotenoid concentrations with cultured MCF-7 human breast cancer cells (
r
= 0.86). Clearly the molecular features of rotenoids essential for inhibiting NADH:ubiquinone oxidoreductase are similar to those for blocking ODC induction. This apparent correlation extends to 11 flavonoids and stilbenoids from cubé resin (
r
= 0.98) and genistein and resveratrol except for lower potency and less selectivity than the rotenoids relative to cytotoxicity. These findings on cubé insecticide constituents and our earlier study comparing rotenone and pyridaben miticide indicate that inhibition of NADH:ubiquinone oxidoreductase activity lowers the level of induced ODC activity leading to the antiproliferative effect and anticancer action.
Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—similarities to astrocyte grafts
Authors: Azizi S. Ausim , Stokes David , Augelli Brian J. , DiGirolamo Carla , Prockop Darwin J.
Published: July 2002

Neurotransplantation has been used to explore the development of the central nervous system and for repair of diseased tissue in conditions such as Parkinson’s disease. Here, we examine the effects of direct injection into rat brain of human marrow stromal cells (MSCs), a subset of cells from bone marrow that include stem-like precursors for nonhematopoietic tissues. Human MSCs isolated by their adherence to plastic were infused into the corpus striatum. Five to 72 days later, brain sections were examined for the presence of the donor cells. About 20% of the infused cells had engrafted. There was no evidence of an inflammatory response or rejection. The cells had migrated from the injection site along known pathways for migration of neural stem cells to successive layers of the brain. After infusion into the brain, the human MSCs lost their immunoreactivity to antibodies for collagen I. Initially, the human cells continued to stain with antibodies to fibronectin but the region of staining with fibronectin was significantly decreased at 30 and 72 days. The results suggest that MSCs may be useful vehicles for autotransplantation in both cell and gene therapy for a variety of diseases of the central nervous system.
Down-regulation of
Leishmania donovani
trypanothione reductase by heterologous expression of a trans-dominant mutant homologue: Effect on parasite intracellular survival
Authors: Tovar Jorge , Cunningham Mark L. , Smith Aden C. , Croft Simon L. , Fairlamb Alan H.
Published: July 2002

A trans-dominant mutational strategy was used to down-regulate trypanothione reductase (TR) activity levels in
Leishmania donovani,
the causative agent of visceral leishmaniasis in humans. TR, regarded as an ideal drug target against trypanosomatid infections, is a homodimeric flavoprotein oxidoreductase unique to these organisms that plays a central role in the enzymatic regeneration of the thiol pool. Extrachromosomal, heterologous expression of a trans-dominant mutant version of the
Trypanosoma cruzi
enzyme in
L. donovani
resulted in the formation of inactive cross-species heterodimers and in a dramatic decrease of endogenous TR activity levels. Recombinant cells depleted of up to 85% of TR activity were significantly impaired in their ability to regenerate dihydrotrypanothione from trypanothione disulfide following oxidation with diamide. Nonetheless trans-dominant mutant recombinants were still capable of maintaining a reduced intracellular environment during cell growth in culture and were able to metabolize hydrogen peroxide at wild-type rates
in vitro
. Importantly, however, cells expressing the trans-dominant mutant enzyme displayed a decreased ability to survive inside activated macrophages in a murine model of
Leishmania
infection. The apparent inability of
Leishmania
to modulate the expression of active TR homodimers in response to the expression of trans-dominant mutant protein suggests that specific inhibitors of this enzyme should be useful anti-leishmanial agents.
A novel mechanism for
P
element homing in
Drosophila
Authors: Taillebourg Emmanuel , Dura Jean-Maurice
Published: July 2002

P
element insertion is essentially random at the scale of the genome. However,
P
elements containing regulatory sequences from
Drosophila engrailed
and
polyhomeotic
genes and from the
Bithorax
and
Antennapedia
complexes show some insertional specificity by frequently inserting near the parent gene (homing) and/or near genes containing
Polycomb
group response elements (preferential insertion). This phenomenon is thought to be mediated by
Polycomb
group proteins. In this report, we describe a case of homing of
P
elements containing regulatory sequences of the
linotte
gene. This homing occurs with high frequency (up to 20% of the lines) and high precision (inserted into a region of <1 kilobase).We present evidence showing that it is not mediated by
Polycomb
group proteins but by a new, as yet unknown, mechanism. We also suggest that
P
element homing could be a more frequent phenomenon than generally assumed and that it could become a powerful tool of
Drosophila
reverse genetics, for which there is no other described gene targeting technique.
Dynamics of
Helicobacter pylori
colonization in relation to the host response
Authors: Blaser Martin J. , Kirschner Denise
Published: July 2002

The dynamics of
Helicobacter pylori
colonization from its acquisition through the development of steady-state are examined through a mathematical model that includes the host response. The model encompasses both host and microbiological variation. The individual capacity of the host response is shown to be a key model parameter, leading to either transient or persistent colonization, whereas the growth rate of that response has little effect. Analyses of competing strains indicate that each must occupy a specific niche, otherwise exclusion occurs. The model implies that there exists a lower bound on the host response to the indigenous microflora that is consistent with current biological views of
H. pylori
. Parallel models may be useful in understanding other persistent host–microbial interactions.
Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting
Authors: Sun Xiujun , Funk Colin D. , Deng Chengjun , Sahu Arvind , Lambris John D. , Song Wen-Chao
Published: July 2002

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that inhibits both the classical and the alternative pathways of complement activation. DAF has been studied extensively in humans under two clinical settings: when absent from the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) patients, who suffer from complement-mediated hemolytic anemia, and in transgenic pigs expressing human DAF, which have been developed to help overcome complement-mediated hyperacute rejection in xenotransplantation. Nevertheless, the exact role of DAF in regulating complement activationin vivoon the cell surface and the species specificity of this molecule remain to be fully characterized. To address these issues, we have used gene targeting to produce mice lacking GPI-anchored DAF. We found that erythrocytes from mice deficient in GPI-anchored DAF showed no increase in spontaneous complement activationin vivobut exhibited impaired regulation of zymosan-initiated bystander and antibody-triggered classical pathway complement activationin vitro, resulting in enhanced complement deposition. Despite a high level of C3 fixation, no homologous hemolysis occurred. It is noteworthy that GPI-linked DAF knockout erythrocytes, when tested with human and guinea pig sera, were more susceptible to heterologous complement lysis than were normal erythrocytes. These results suggest that DAF is capable of regulating homologous as well as heterologous complement activation via the alternative or the classical pathway. They also indicate that DAF deficiency alone is not sufficient to cause homologous hemolysis. In contrast, when the assembly of the membrane-attack complex is not properly regulated, as in the case of heterologous complement activation or in PNH patients, impaired erythrocyte DAF activity and enhanced C3 deposition could lead to increased hemolytic reaction.