bluuebunny/crossref_metadata_2025 huggingface dataset:
Modifying the sequence of an immunoglobulin V-gene alters the resulting pattern of hypermutation
Authors: Goyenechea Beatriz , Milstein César
Published: July 2002

Affinity maturation of antibodies requires localized hypermutation and antigen selection. Hypermutation is particularly active in certain regions (notably the CDRs of light and heavy chains) due to the local accumulation of hot spots. We have now analyzed the role of individual nucleotides in the origin of hot spots and show that mutability is largely defined by the nucleotide sequence. We compared the mutability profile of wild-type and modified κ transgenes that contain silent mutations in the CDR1 segment. We found a new hot spot created at the third base of Ser-31 when its wild-type AGT codon was substituted by AGC. Two major hot spots associated with this AGC vanished when Ser-31 was encoded by the synonymous TCA. In addition to these, which were the most prominent changes, there were compensatory alterations in mutability of residues not directly related to the introduced silent mutations, so that the average hypermutation remained constant. Thus, mutations arising early in the immune response, even silent ones, could affect the mutability of critical residues and alter the pattern of affinity maturation. When analyzing hybridomas, we detected such alterations, but they seemed to better correlate with changes in average rather than local mutation rates. Overall, this paper shows how evolution could have optimized the mutability of individual residues to minimize deleterious mutations. Thus, the optimal strategy for affinity maturation may involve the incorporation of multiple point mutations before antigen selection of the relevant cells.
Crystal structure of the breakpoint cluster region-homology domain from phosphoinositide 3-kinase p85α subunit
Authors: Musacchio Andrea , Cantley Lewis C. , Harrison Stephen C.
Published: July 2002

Proteins such as the product of the breakpoint cluster region,
chimaerin, and the Src homology 3-binding protein 3BP1, are GTPase
activating proteins (GAPs) for members of the Rho subfamily of small
GTP-binding proteins (G proteins or GTPases). A 200-residue region,
named the breakpoint cluster region-homology (BH) domain, is
responsible for the GAP activity. We describe here the crystal
structure of the BH domain from the p85 subunit of phosphatidylinositol
3-kinase at 2.0 Å resolution. The domain is composed of seven
helices, having a previously unobserved arrangement. A core of four
helices contains most residues that are conserved in the BH family.
Their packing suggests the location of a G-protein binding site. This
structure of a GAP-like domain for small GTP-binding proteins provides
a framework for analyzing the function of this class of molecules.
Mutagenesis associated with nitric oxide production in transgenic SJL mice
Authors: Gal Aharon , Wogan Gerald N.
Published: July 2002

We recently reported development of an experimental model for the study of nitric oxide (NO·) toxicologyin vivo. SJL mice were injected with superantigen-bearing RcsX (pre-B-cell lymphoma) cells, which migrated to the spleen and lymph nodes, where their rapid growth induced activation of macrophages to produce large amounts of NO·over a period of several weeks. In the experiments described here, we used this model to investigate mutagenesis in splenocytes exposed to NO·during RcsX cell growth. Transgenic mice were produced by crossbreeding animals of the pUR288 transgenic C57BL/6 and SJL strains. RcsX cells were injected into F1mice and NO·production was confirmed by quantification of urinary nitrate, the ultimate metabolite of NO·. Mutant frequency in thelacZgene of the pUR288 plasmid was determined in DNA isolated from spleen (target) and kidney (nontarget) tissues. A significant elevation in mutant frequency was found in the spleen, but not in the kidney, of tumor-bearing mice. Furthermore, increases in mutant frequency in the spleen as well as NO·production were abrogated by administration ofN-methylarginine, a NO·inhibitor, to mice following injection of RcsX cells. These results indicate that NO·had mutagenic activity in RcsX tumor-bearing mice and thus support a possible role for its involvement in the carcinogenic process.
Krüppel-associated box-mediated repression of RNA polymerase II promoters is influenced by the arrangement of basal promoter elements.
Authors: Pengue G , Lania L
Published: July 2002

The evolutionarily conserved Krüppel-associated box (KRAB) is present in the N-terminal regions of more than one-third of all Krüppel-class zinc finger proteins. Recent experiments have demonstrated that the KRAB-A domain tethered to a promoter DNA by connecting to heterologous DNA-binding protein domain or targeted to a promoter-proximal RNA sequence acts as a transcriptional silencing of RNA polymerase II promoters. Here we show that expression of KRAB domain suppresses in vivo the activating function of various defined activating transcription factors, and we demonstrate that the KRAB domain specifically silences the activity of promoters whose initiation is dependent on the presence of a TATA box. Promoters whose accurate transcription initiation is directed by a pyrimidine-rich initiator element, however, are relatively unaffected. We also report in vitro transcription experiments indicating that the KRAB domain is able to repress both activated and basal promoter activity. Thus, the KRAB domain appears to repress the activity of certain promoters through direct communication with TATA box-dependent basal transcription machinery.
Identification of a minimal sequence of the mouse pro-alpha 1(I) collagen promoter that confers high-level osteoblast expression in transgenic mice and that binds a protein selectively present in osteoblasts.
Authors: Rossert J A , Chen S S , Eberspaecher H , Smith C N , de Crombrugghe B
Published: July 2002

Based on our previous transgenic mice results, which strongly suggested that separate cell-specific cis-acting elements of the mouse pro-alpha 1(I) collagen promoter control the activity of the gene in different type I collagen-producing cells, we attempted to delineate a short segment in this promoter that could direct high-level expression selectively in osteoblasts. By generating transgenic mice harboring various fragments of the promoter, we identified a 117-bp segment (-1656 to -1540) that is a minimal sequence able to confer high-level expression of a lacZ reporter gene selectively in osteoblasts when cloned upstream of the proximal 220-bp pro-alpha 1(I) promoter. This 220-bp promoter by itself was inactive in transgenic mice and unable to direct osteoblast-specific expression. The 117-bp enhancer segment contained two sequences that appeared to have different functions. The A sequence (-1656 to -1628) was required to obtain expression of the lacZ gene in osteoblasts, whereas the C sequence (-1575 to -1540) was essential to obtain consistent and high-level expression of the lacZ gene in osteoblasts. Gel shift assays showed that the A sequence bound a nuclear protein present only in osteoblastic cells. A mutation in the A segment that abolished the binding of this osteoblast-specific protein also abolished lacZ expression in osteoblasts of transgenic mice.
Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid.
Authors: Lopez J M , Bennett M K , Sanchez H B , Rosenfeld J M , Osborne T E
Published: July 2002

Transcription from the housekeeping promoter for the acetyl coenzyme A carboxylase (ACC) gene, which encodes the rate-controlling enzyme of fatty acid biosynthesis, is shown to be regulated by cellular sterol levels through novel binding sites for the sterol-sensitive sterol regulatory element binding protein (SREBP)-1 transcription factor. The position of the SREBP sites relative to those for the ubiquitous auxiliary transcription factor Sp1 is reminiscent of that previously described for the sterol-regulated low density lipoprotein receptor promoter. The experiments provide molecular evidence that the metabolism of fatty acids and cholesterol, two different classes of essential cellular lipids, are coordinately regulated by cellular lipid levels.
HLH106, a Drosophila transcription factor with similarity to the vertebrate sterol responsive element binding protein.
Authors: Theopold U , Ekengren S , Hultmark D
Published: July 2002

We cloned a Drosophila homolog to the sterol responsive element binding proteins (SREBPs). In vertebrates, the SREBPs are regulated by a mechanism that involves cleavage of the protein that normally residues in the cellular membranes and translocation of the released transcription factor into the nucleus. Regulation of the Drosophila factor HLH106 apparently follows the same mechanism, and we find the full-length gene product in the membrane fraction and a shorter cross-reacting form in the nuclear fraction. This nuclear form, which may correspond to proteolytically activated HLH106, is abundant in the blood cell line mbn-2. The general domain structure of HLH106 is very similar to that in SREBP. HLH106 is expressed throughout development, and it is present at high levels in Drosophila cell lines. In contrast to the rat homolog, HLH106 transcripts are not more abundant in adipose tissue than in other tissues.
Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.
Authors: Askanas V , McFerrin J , Baqué S , Alvarez R B , Sarkozi E , Engel W K
Published: July 2002

As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain.
An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae.
Authors: Philipp W J , Poulet S , Eiglmeier K , Pascopella L , Balasubramanian V , Heym B , Bergh S , Bloom B R , Jacobs W R , Cole S T
Published: July 2002

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom.
A natural transactivation mutation in the thyroid hormone β receptor: Impaired interaction with putative transcriptional mediators
Authors: Collingwood T. N. , Rajanayagam O. , Adams M. , Wagner R. , Cavaillès V. , Kalkhoven E. , Matthews C. , Nystrom E. , Stenlof K. , Lindstedt G. , Tisell L. , Fletterick R. J. , Parker M. G. , Chatterjee V. K. K.
Published: July 2002

The syndrome of resistance to thyroid hormone is characterized by elevated serum free thyroid hormones, failure to suppress pituitary thyrotropin secretion, and variable peripheral refractoriness to hormone action. Here we describe a novel leucine to valine mutation in codon 454 (L454V) of the thyroid hormone β receptor (TRβ) in this disorder, resulting in a mutant receptor with unusual functional properties. Although the mutant protein binds ligand comparably to wild-type receptor and forms homo- and heterodimers on direct repeat, everted repeat, or palindromic thyroid response elements, its ability to activate transcription via these elements is markedly impaired. The hydrophobic leucine residue lies within an amphipathic α-helix at the carboxyl terminus of TRβ and the position of the homologous residue in the crystal structure of TRα indicates that its side chain is solvent-exposed and might interact with other proteins. We find that two putative transcriptional mediators (RIP140 and SRC-1) exhibit hormone-dependent association with wild-type TR. In comparison, the interaction of this natural mutant (L454V) and artificial mutants (L454A, E457A) with RIP140 and SRC-1 is markedly reduced. Furthermore, coexpression of SRC-1 is able to restore the transcriptional activity of the L454V mutant receptor, indicating that the interaction of this residue with accessory proteins is critical for transcriptional activation. Finally, the occurrence of the L454V mutation in resistance to thyroid hormone, together with impaired negative regulation of the thyroid-stimulating hormone α promoter by this mutant, suggests that the amphipathic α-helix also mediates hormone-dependent transcriptional inhibition, perhaps via interaction with these or other accessory factors.
A single response mechanism is responsible for evolutionary adaptive variation in a bird’s laying date
Authors: Lambrechts Marcel M. , Blondel Jacques , Maistre Marie , Perret Philippe
Published: July 2002

Many proximate factors determine a bird’s laying date, including environmental and social stimuli as well as individual responses to internal and external factors. However, the relative importance of these factors has not been experimentally demonstrated. Here we show that (i) large differences in the onset of first clutches between different populations result from variation in different responses to photoperiod and not from variation in responses to any other proximate factors and (ii) the same response mechanism causes maladaptive laying dates in habitats modified by humans. We present, to our knowledge, the first experimental demonstration that a single response mechanism is responsible for evolutionary adaptive intraspecific variation in a vertebrate life history trait.
Radiation and stress-induced apoptosis: A role for Fas/Fas ligand interactions
Authors: Reap Elizabeth A. , Roof Kevin , Maynor Kenrick , Borrero Michelle , Booker Jessica , Cohen Philip L.
Published: July 2002

Thelprgene encodes a defective form of Fas, a cell surface protein that mediates apoptosis. This defect blocks apoptotic deletion of autoreactive T and B cells, leading to lymphoproliferation and lupus-like autoantibody production. The effects of thelprFas mutation on other kinds of physiologically relevant apoptosis are largely undocumented. To assess whether some of the apoptosis known to occur after ionizing radiation might be mediated by Fas/Fas ligand (FasL) interactions, we quantitatedin vitroapoptosis by flow cytometry measurement of DNA content in splenic T and B cells from irradiated 5- to 8-month-old B6/lprmice. Total apoptosis of bothlprand control cells was substantial after treatment; however there was a significant difference between B6 (73%) andlpr(25%) lymphocyte apoptosis. Thy1, CD4, CD8, and IgM cells fromlprshowed much lower levels of apoptosis than control cells after irradiation. Apoptosis induced by heat shock was also impaired inlpr. The finding that γ-irradiation increased Fas expression on B6 cells and that irradiation-induced apoptosis could be blocked with a Fas–Fc fusion protein further supported the possible involvement of Fas in this form of apoptosis. Fas/FasL interactions may thus play an important role in identifying and eliminating damaged cells after γ-irradiation and other forms of injury.
Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells
Authors: Oorschot A. A. A. M. Danen-Van , Fischer D. F. , Grimbergen J. M. , Klein B. , Zhuang S.-M. , Falkenburg J. H. F. , Backendorf C. , Quax P. H. A. , Van der Eb A. J. , Noteborn M. H. M.
Published: July 2002

The chicken anemia virus protein apoptin induces a p53-independent, Bcl-2-insensitive type of apoptosis in various human tumor cells. Here, we show that,
in vitro
, apoptin fails to induce programmed cell death in normal lymphoid, dermal, epidermal, endothelial, and smooth-muscle cells. However, when normal cells are transformed they become susceptible to apoptosis by apoptin. Long-term expression of apoptin in normal human fibroblasts revealed that apoptin has no toxic or transforming activity in these cells. In normal cells, apoptin was found predominantly in the cytoplasm, whereas in transformed and malignant cells it was located in the nucleus, suggesting that the localization of apoptin is related to its activity. These properties make apoptin a potential agent for the treatment of a large number of tumors, also those lacking p53 and/or overexpressing Bcl-2.
Physiological response to long-term peripheral and central leptin infusion in lean and obese mice
Authors: Halaas Jeffrey L. , Boozer Carol , Blair-West John , Fidahusein Naseem , Denton Derek A. , Friedman Jeffrey M.
Published: July 2002

Recent data have identified leptin as an afferent signal in a negative-feedback loop regulating the mass of the adipose tissue. High leptin levels are observed in obese humans and rodents, suggesting that, in some cases, obesity is the result of leptin insensitivity. This hypothesis was tested by comparing the response to peripherally and centrally administered leptin among lean and three obese strains of mice: diet-induced obese AKR/J, New Zealand Obese (NZO), and A
y
. Subcutaneous leptin infusion to lean mice resulted in a dose-dependent loss of body weight at physiologic plasma levels. Chronic infusions of leptin intracerebroventricularly (i.c.v.) at doses of 3 ng/hr or greater resulted in complete depletion of visible adipose tissue, which was maintained throughout 30 days of continuous i.c.v. infusion. Direct measurement of energy balance indicated that leptin treatment did not increase total energy expenditure but prevented the decrease that follows reduced food intake. Diet-induced obese mice lost weight in response to peripheral leptin but were less sensitive than lean mice. NZO mice were unresponsive to peripheral leptin but were responsive to i.c.v. leptin. A
y
mice did not respond to subcutaneous leptin and were 1/100 as sensitive to i.c.v. leptin. The decreased response to leptin in diet-induced obese, NZO, and A
y
mice suggests that obesity in these strains is the result of leptin resistance. In NZO mice, leptin resistance may be the result of decreased transport of leptin into the cerebrospinal fluid, whereas in A
y
mice, leptin resistance probably results from defects downstream of the leptin receptor in the hypothalamus.
Mitochondrial DNA rearrangements of
Podospora anserina
are under the control of the nuclear gene grisea
Authors: Borghouts Corina , Kimpel Erik , Osiewacz Heinz D.
Published: July 2002

Podospora anserina
is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5′ upstream region of the grisea ORF. One mutation is a G → A transition at the 5′ splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome
c
oxidase subunit I gene (
COI
) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of
P. anserina
cultures.
Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca
2+
channel activity by cAMP-dependent protein kinase
Authors: Burton Kimberly A. , Johnson Barry D. , Hausken Zachary E. , Westenbroek Ruth E. , Idzerda Rejean L. , Scheuer Todd , Scott John D. , Catterall William A. , McKnight G. Stanley
Published: July 2002

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca
2+
channel, and the K
Ca
channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIα subunit would reveal those tissues and signaling events that require anchored PKA. RIIα knockout mice appear normal and healthy. In adult skeletal muscle, RIα protein levels increased to partially compensate for the loss of RIIα. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca
2+
channel in RIIα knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA–AKAP interactions. The C subunit colocalized with the L-type Ca
2+
channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RIα subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RIIα subunit. The potentiation of the L-type Ca
2+
channel in RIIα knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIα is capable of physiologically relevant anchoring interactions.
Operative GABAergic inhibition in hippocampal CA1 pyramidal neurons in experimental epilepsy
Authors: Esclapez M. , Hirsch J. C. , Khazipov R. , Ben-Ari Y. , Bernard C.
Published: July 2002

Patch–clamp recordings of CA1 interneurons and pyramidal cells were performed in hippocampal slices from kainate- or pilocarpine-treated rat models of temporal lobe epilepsy. We report that γ-aminobutyric acid (GABA)ergic inhibition in pyramidal neurons is still functional in temporal lobe epilepsy because: (
i
) the frequency of spontaneous GABAergic currents is similar to that of control and (
ii
) focal electrical stimulation of interneurons evokes a hyperpolarization that prevents the generation of action potentials. In paired recordings of interneurons and pyramidal cells, synchronous interictal activities were recorded. Furthermore, large network-driven GABAergic inhibitory postsynaptic currents were present in pyramidal cells during interictal discharges. The duration of these interictal discharges was increased by the GABA type A antagonist bicuculline. We conclude that GABAergic inhibition is still present and functional in these experimental models and that the principal defect of inhibition does not lie in a complete disconnection of GABAergic interneurons from their glutamatergic inputs.
The Epstein–Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-κB
Authors: Izumi Kenneth M. , Kieff Elliott D.
Published: July 2002

The Epstein–Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxyl-terminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacted with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucine-mutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-κB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-κB activation also appears to be a critical component of long-term outgrowth.
Gene therapy
Authors: Kay Mark A. , Liu Dexi , Hoogerbrugge Peter M.
Published: July 2002

In recent years, there have been a number of technological
breakthroughs that have allowed for clinical trials in gene therapy to
be initiated. In combination with the genome initiative, the potential
for new therapeutics is limitless. Although an enormous amount of
information has been obtained in a relatively short period of time,
gene therapy is not yet ready for wide-scale practice. Some of the
successes and obstacles that remain are summarized in this report.
All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in
Saccharomyces cerevisiae
Authors: Dolinski Kara , Muir Scott , Cardenas Maria , Heitman Joseph
Published: July 2002

The cyclophilins and FK506 binding proteins (FKBPs) bind to cyclosporin A, FK506, and rapamycin and mediate their immunosuppressive and toxic effects, but the physiological functions of these proteins are largely unknown. Cyclophilins and FKBPs are ubiquitous and highly conserved enzymes that catalyze peptidyl-prolyl isomerization, a rate-limiting step during
in vitro
protein folding. We have addressed their functions by a genetic approach in the yeast
Saccharomyces cerevisiae
. Five cyclophilins and three FKBPs previously were identified in yeast. We identified four additional enzymes: Cpr6 and Cpr7, which are homologs of mammalian cyclophilin 40 that have also recently been independently isolated by others, Cpr8, a homolog of the secretory pathway cyclophilin Cpr4, and Fpr4, a homolog of the nucleolar FKBP, Fpr3. None of the eight cyclophilins or four FKBPs were essential. Surprisingly, yeast mutants lacking all 12 immunophilins were viable, and the phenotype of the dodecuplet mutant resulted from simple addition of the subtle phenotypes of each individual mutation. We conclude that cyclophilins and FKBPs do not play an essential general role in protein folding and find little evidence of functional overlap between the different enzymes. We propose that each cyclophilin and FKBP instead regulates a restricted number of unique partner proteins that remain to be identified.
Ascorbate recycling in human neutrophils: Induction by bacteria
Authors: Wang Yaohui , Russo Thomas A. , Kwon Oran , Chanock Stephen , Rumsey Steven C. , Levine Mark
Published: July 2002

Ascorbate (vitamin C) recycling occurs when extracellular ascorbate is oxidized, transported as dehydroascorbic acid, and reduced intracellularly to ascorbate. We investigated microorganism induction of ascorbate recycling in human neutrophils and in microorganisms themselves. Ascorbate recycling was determined by measuring intracellular ascorbate accumulation. Ascorbate recycling in neutrophils was induced by both Gram-positive and Gram-negative pathogenic bacteria, and the fungal pathogenCandida albicans. Induction of recycling resulted in as high as a 30-fold increase in intracellular ascorbate compared with neutrophils not exposed to microorganisms. Recycling occurred at physiologic concentrations of extracellular ascorbate within 20 min, occurred over a 100-fold range of effector/target ratios, and depended on oxidation of extracellular ascorbate to dehydroascorbic acid. Ascorbate recycling did not occur in bacteria nor inC. albicans. Ascorbate did not enter microorganisms, and dehydroascorbic acid entry was less than could be accounted for by diffusion. Because microorganism lysates reduced dehydroascorbic acid to ascorbate, ascorbate recycling was absent because of negligible entry of the substrate dehydroascorbic acid. Because ascorbate recycling occurs in human neutrophils but not in microorganisms, it may represent a eukaryotic defense mechanism against oxidants with possible clinical implications.
Pharmacological and immunohistochemical evidence for a functional nitric oxide synthase system in rat peritoneal eosinophils
Authors: Zanardo Renata C. O. , Costa Edmar , Ferreira Heloisa H. A. , Antunes Edson , Martins Antonio R. , Murad Ferid , De Nucci Gilberto
Published: July 2002

Eosinophil migrationin vivois markedly attenuated in rats treated chronically with the NO synthase (NOS) inhibitorNω-nitro-l-arginine methyl ester (l-NAME). In this study, we investigated the existence of a NOS system in eosinophils. Our results demonstrated that rat peritoneal eosinophils strongly express both type II (30.2 ± 11.6% of counted cells) and type III (24.7 ± 7.4% of counted cells) NOS, as detected by immunohistochemistry using affinity purified mouse mAbs. Eosinophil migrationin vitrowas evaluated by using 48-well microchemotaxis chambers and the chemotactic agents used wereN-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 × 10−8M) and leukotriene B4(LTB4, 10−8M).l-NAME (but notd-NAME) significantly inhibited the eosinophil migration induced by both fMLP (54% reduction for 1.0 mM;P< 0.05) and LTB4(61% reduction for 1.0 mM;P< 0.05). In addition, the type II NOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine and the type I/II NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole also markedly (P< 0.05) attenuated fMLP- (52% and 38% reduction for 1.0 mM, respectively) and LTB4- (52% and 51% reduction for 1.0 mM, respectively) induced migration. The inhibition of eosinophil migration byl-NAME was mimicked by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (0.01 and 0.1 mM) and reversed by either sodium nitroprusside (0.1 mM) or dibutyryl cyclic GMP (1 mM). We conclude that eosinophils do express NO synthase(s) and that nitric oxide plays an essential role in eosinophil locomotion by acting through a cyclic GMP transduction mechanism.
Leaf-specifically expressed genes for polypeptides destined for chloroplasts with domains of σ
70
factors of bacterial RNA polymerases in
Arabidopsis thaliana
Authors: Isono Kyoichi , Shimizu Masanori , Yoshimoto Kohki , Niwa Yasuo , Satoh Kimiyuki , Yokota Akiho , Kobayashi Hirokazu
Published: July 2002

Genes for σ-like factors of bacterial-type RNA polymerase have not been characterized from any multicellular eukaryotes, although they probably play a crucial role in the expression of plastid photosynthesis genes. We have cloned three distinct cDNAs, designated
SIG1
,
SIG2
, and
SIG3
, for polypeptides possessing amino acid sequences for domains conserved in σ
70
factors of bacterial RNA polymerases from the higher plant
Arabidopsis thaliana
. Each gene is present as one copy per haploid genome without any additional sequences hybridized in the genome. Transient expression assays using green fluorescent protein demonstrated that N-terminal regions of the
SIG2
and
SIG3
ORFs could function as transit peptides for import into chloroplasts. Transcripts for all three
SIG
genes were detected in leaves but not in roots, and were induced in leaves of dark-adapted plants in rapid response to light illumination. Together with results of our previous analysis of tissue-specific regulation of transcription of plastid photosynthesis genes, these results indicate that expressed levels of the genes may influence transcription by regulating RNA polymerase activity in a green tissue-specific manner.
Regulation of E2F through ubiquitin–proteasome-dependent degradation: Stabilization by the pRB tumor suppressor protein
Authors: Campanero Miguel R. , Flemington Erik K.
Published: July 2002

The E2F family of transcription factors plays a key role in regulating cell-cycle progression. Accordingly, E2F is itself tightly controlled by a series of transcriptional and posttranscriptional events. Here we provide evidence that E2F1 protein levels are regulated by the ubiquitin–proteasome-dependent degradation pathway. An analysis of E2F1 mutants identified a conserved carboxyl-terminal region, which is required for eliciting ubiquitination and protein turnover. Fusion of this E2F1 carboxyl-terminal sequence to a heterologous protein, GAL4, resulted in destabilization of GAL4. Previous studies identified an overlapping region of E2F1 that facilitates complex formation with retinoblastoma tumor suppressor protein, pRB, and we found that pRB blocks ubiquitination and stabilizes E2F1. These results suggest a new mechanism for controlling the cell-cycle regulatory activity of E2F1.
Regulated bidirectional motility of melanophore pigment granules along microtubules
in vitro
Authors: Rogers Stephen L. , Tint Irina S. , Fanapour Philip C. , Gelfand Vladimir I.
Published: July 2002

Although many types of membrane-bound organelles rely upon microtubule-based transport for their proper placement within the cytoplasm, the molecular mechanisms that regulate intracellular motility remain largely unknown. To address this problem, we have studied the microtubule-dependent dispersion and aggregation of pigment granules from an immortalized
Xenopus
melanophore cell line. We have reconstituted pigment granule motility along bovine brain microtubules
in vitro
using a microscope-based motility assay. Pigment granules, or melanosomes, move along single microtubules bidirectionally; however, analysis of the polarities of this movement shows that melanosomes that have been purified from dispersed cells exhibit mostly plus end-directed motility, while movement of organelles from aggregating cells is biased toward the minus end. Removal of all soluble proteins from the melanosome fractions by density gradient centrifugation does not diminish organelle motility, demonstrating that all the components required for transport have a stable association with the melanosome membranes. Western blotting shows the presence of the plus end-directed motor, kinesin-II, and the minus end-directed motor, cytoplasmic dynein in highly purified melanosomes. Therefore, purified melanosomes retain their ability to move along microtubules as well as their regulated state. Direct biochemical comparison of melanosomes from aggregated and dispersed cells may elucidate the molecular mechanisms that regulate organelle transport in melanophores.
Creation of libraries with long ORFs by polymerization of a microgene
Authors: Shiba Kiyotaka , Takahashi Yuki , Noda Tetsuo
Published: July 2002

We describe a novel method for constructing pools of DNA sequences that encode large proteins with molecular diversity. Sets of primer pairs that form 8 to 10 complementary base pairs in the 3′ region and have double mismatch pairs at their 3′-OH ends were designed so that primer dimers recreated short stretches of DNA (microgenes) devoid of termination codons. Cycles of denaturation and elongation reactions with a pair of primers, four dNTPs, and 3′–5′ exo+thermostable DNA polymerase gave head-to-tail polymers of the primer dimer unit (microgene) whose sizes exceeded 12 kb. No template was required in this reaction, but mismatched nucleotides at 3′-OH ends of the primers were critical for efficient polymerization. At end-joining junctions of a microgene, nucleotide insertions and deletions randomly occurred, resulting in combinatorial libraries of three reading frames from a single microgene. Further molecular diversity could be incorporated by using a mixture of primers. The resultant polymers have long ORFs whose products have a repetitious nature that could facilitate the formation of higher structures of translated products. Thus, microgene polymers may be used as a source of libraries forin vitroprotein evolution experiments. Ligation of a microgene is apparently related to the nonhomologous recombination of double-strand breaks in DNA that has been shown to be catalyzed by DNA polymerases. We named this polymerization reaction the “microgene polymerization reaction.”
Nuclear ribosomal DNA evidence for a western North American origin of Hawaiian and South American species of
Sanicula
(Apiaceae)
Authors: Vargas Pablo , Baldwin Bruce G. , Constance Lincoln
Published: July 2002

Results from phylogenetic analysis of nuclear rDNA internal transcribed spacer (ITS) sequences from a worldwide sample of
Sanicula
indicate that Hawaiian sanicles (
Sanicula
sect.
Sandwicenses
) constitute a monophyletic group that descended from a western North American ancestor in
Sanicula
sect.
Sanicoria
, a paraphyletic assemblage of mostly Californian species. A monophyletic group comprising representatives of all 15 species of
S
. sect.
Sanicoria
and the three sampled species of
S
. sect.
Sandwicenses
was resolved in all maximally parsimonious trees, rooted with sequences from species of
Astrantia
and
Eryngium
. All sequences sampled from eastern North American, European, and Asian species of
Sanicula
fell outside the ITS clade comprising
S
. sect.
Sanicoria
and
S
. sect.
Sandwicenses
. A lineage comprising the Hawaiian taxa and three species endemic to coastal or near-coastal habitats in western North America (
Sanicula arctopoides
,
Sanicula arguta
, and
Sanicula laciniata
) is diagnosed by nucleotide substitutions and a 24-bp deletion in ITS2. The hooked fruits in
Sanicula
lead us to conclude that the ancestor of Hawaiian sanicles arrived from North America by external bird dispersal; similar transport has been hypothesized for the North American tarweed ancestor of the Hawaiian silversword alliance (Asteraceae). Two additional long-distance dispersal events involving members of
S
. sect.
Sanicoria
can be concluded from the ITS phylogeny: dispersal of
Sanicula crassicaulis
and
Sanicula graveolens
from western North America to southern South America.
Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain
Authors: Nishi Nozomu , Matsushita Osamu , Yuube Kouichi , Miyanaka Hiroshi , Okabe Akinobu , Wada Fumio
Published: July 2002

The autocrine/paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives
in vivo
. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from
Clostridium histolyticum
collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF/CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB/c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect
in vivo
, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting
in vivo
.
Activation of ɛ protein kinase C correlates with a cardioprotective effect of regular ethanol consumption
Authors: Miyamae Masami , Rodriguez Manuel M. , Camacho S. Albert , Diamond Ivan , Mochly-Rosen Daria , Figueredo Vincent M.
Published: July 2002

In addition to decreasing the incidence of myocardial infarction, recent epidemiological data suggest that regular alcohol consumption improves survival after myocardial infarction. We recently found that chronic ethanol exposure induces long-term protection against cardiac ischemia–reperfusion injury, which improves myocardial recovery after infarction. Furthermore, this cardioprotection by ethanol is mediated through myocyte adenosine A
1
receptors. We now determine the role of protein kinase C (PKC) in ethanol’s protective effect against ischemia–reperfusion injury. Using perfused hearts of ethanol-fed guinea pigs, we find that improved contractile recovery and creatine kinase release after ischemia–reperfusion are abolished by PKC inhibition with chelerythrine. Western blot analysis and immunofluorescence localization demonstrate that regular ethanol consumption causes sustained translocation (activation) of ɛPKC, but not δ or αPKC. This same isozyme is directly implicated in ischemic preconditioning’s protection against ischemia–reperfusion injury. Our findings suggest (
i
) that regular ethanol consumption induces long-term cardioprotection through sustained translocation of ɛPKC and (
ii
) that PKC activity is necessary at the time of ischemia to mediate ethanol’s protective effect against ischemia–reperfusion injury. Studying this selective effect of ethanol on ɛPKC activation may lead to new therapies to protect against ischemia–reperfusion injury in the heart and other organ systems.
Biallelic inactivation of

hMLH
1

by epigenetic gene silencing, a novel mechanism causing human MSI cancers
Authors: Veigl Martina L. , Kasturi Lakshmi , Olechnowicz Joseph , Ma AiHong , Lutterbaugh James D. , Periyasamy Sumudra , Li Guo-Min , Drummond James , Modrich Paul L. , Sedwick W. David , Markowitz Sanford D.
Published: July 2002

Mutations of DNA mismatch repair genes, including the
hMLH1
gene, have been linked to human colon and other cancers in which defective DNA repair is evidenced by the associated instability of DNA microsatellite sequences (MSI). Germ-line
hMLH1
mutations are causally associated with inherited MSI colon cancer, and somatic mutations are causally associated with sporadic MSI colon cancer. Previously however, we demonstrated that in many sporadic MSI colon cancers
hMLH1
and all other DNA mismatch repair genes are wild type. To investigate this class of tumors further, we examined a group of MSI cancer cell lines, most of which were documented as established from antecedent MSI-positive malignant tumors. In five of six such cases we found that hMLH1 protein was absent, even though
hMLH1
-coding sequences were wild type. In each such case, absence of hMLH1 protein was associated with the methylation of the
hMLH1
gene promoter. Furthermore, in each case, treatment with the demethylating agent 5-azacytidine induced expression of the absent hMLH1 protein. Moreover, in single cell clones,
hMLH1
expression could be turned on, off, and on again by 5-azacytidine exposure, washout, and reexposure. This epigenetic inactivation of
hMLH1
additionally accounted for the silencing of both maternal and paternal tumor
hMLH1
alleles, both of which could be reactivated by 5-azacytidine. In summary, substantial numbers of human MSI cancers appear to arise by
hMLH1
silencing via an epigenetic mechanism that can inactivate both of the
hMLH1
alleles. Promoter methylation is intimately associated with this epigenetic silencing mechanism.
The Upf3 protein is a component of the surveillance complex that monitors both translation and mRNA turnover and affects viral propagation
Authors: Ruiz-Echevarría Maria J. , Yasenchak Jason M. , Han Xia , Dinman Jonathan D. , Peltz Stuart W.
Published: July 2002

The nonsense-mediated mRNA decay pathway functions to degrade aberrant mRNAs that contain premature translation termination codons. In
Saccharomyces cerevisiae
, the Upf1, Upf2, and Upf3 proteins have been identified as
trans
-acting factors involved in this pathway. Recent results have demonstrated that the Upf proteins may also be involved in maintaining the fidelity of several aspects of the translation process. Certain mutations in the
UPF1
gene have been shown to affect the efficiency of translation termination at nonsense codons and/or the process of programmed −1 ribosomal frameshifting used by viruses to control their gene expression. Alteration of programmed frameshift efficiencies can affect virus assembly leading to reduced viral titers or elimination of the virus. Here we present evidence that the Upf3 protein also functions to regulate programmed −1 frameshift efficiency. A
upf3
-Δ strain demonstrates increased sensitivity to the antibiotic paromomycin and increased programmed −1 ribosomal frameshift efficiency resulting in loss of the M
1
virus. Based on these observations, we hypothesize that the Upf proteins are part of a surveillance complex that functions to monitor translational fidelity and mRNA turnover.
Direct binding of follistatin to a complex of bone-morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early
Xenopus
embryo
Authors: Iemura Shun-ichiro , Yamamoto Takamasa S. , Takagi Chiyo , Uchiyama Hideho , Natsume Tohru , Shimasaki Shunichi , Sugino Hiromu , Ueno Naoto
Published: July 2002

In early development of
Xenopus
laevis, it is known that activities of polypeptide growth factors are negatively regulated by their binding proteins. In this study, follistatin, originally known as an activin-binding protein, was shown to inhibit all aspects of bone morphogenetic protein (BMP) activity in early
Xenopus
embryos. Furthermore, using a surface plasmon resonance biosensor, we demonstrated that follistatin can directly interact with multiple BMPs at significantly high affinities. Interestingly, follistatin was found to be noncompetitive with the BMP receptor for ligand binding and to form a trimeric complex with BMP and its receptor. The results suggest that follistatin acts as an organizer factor in early amphibian embryogenesis by inhibiting BMP activities by a different mechanism from that used by chordin and noggin.