bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolution of a unique
Plasmodium falciparum
chloroquine-resistance phenotype in association with
pfcrt
polymorphism in Papua New Guinea and South America
Authors: Mehlotra Rajeev K. , Fujioka Hisashi , Roepe Paul D. , Janneh Omar , Ursos Lyann M. B. , Jacobs-Lorena Vanessa , McNamara David T. , Bockarie Moses J. , Kazura James W. , Kyle Dennis E. , Fidock David A. , Zimmerman Peter A.
Published: July 2002

The mechanistic basis for chloroquine resistance (CQR) in
Plasmodium falciparum
recently has been linked to the polymorphic gene
pfcrt
. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76.
P. falciparum
CQR strains of African and Southeast Asian origin carry
pfcrt
alleles encoding an amino acid haplotype of CVIET (residues 72–76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of
pfcrt
alleles in Papua New Guinean (PNG)
P. falciparum
we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of
P. falciparum
isolates carrying
pfcrt
SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET
pfcrt
-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the
P. falciparum
CQR mechanism. Our findings also suggest that
P. falciparum
CQR has arisen through multiple evolutionary pathways associated with
pfcrt
K76T.
Potentiation of pathogen-specific defense mechanisms in
Arabidopsis
by β-aminobutyric acid
Authors: Zimmerli Laurent , Jakab Gabor , Métraux Jean-Pierre , Mauch-Mani Brigitte
Published: July 2002

The nonprotein amino acids γ-aminobutyric acid (GABA) and
β-aminobutyric acid (BABA) have known biological effects in animals
and plants. Their mode of action has been the object of thorough
research in animals but remains unclear in plants. Our objective was to
study the mode of action of BABA in the protection of
Arabidopis
plants against virulent pathogens. BABA
protected
Arabidopsis
against the oomycete pathogen
Peronospora parasitica
through activation of natural
defense mechanisms of the plant such as callose deposition, the
hypersensitive response, and the formation of trailing necroses. BABA
was still fully protective against
P. parasitica
in
transgenic plants or mutants impaired in the salicylic acid, jasmonic
acid, and ethylene signaling pathways. Treatment with BABA did not
induce the accumulation of mRNA of the systemic acquired resistance
(SAR)-associated
PR-1
and the ethylene- and jasmonic
acid-dependent
PDF1.2
genes. However, BABA potentiated
the accumulation of
PR-1
mRNA after attack by virulent
pathogenic bacteria. As a result, BABA-treated
Arabidopsis
plants were less diseased compared with the
untreated control. In the case of bacteria, BABA protected mutants
insensitive to jasmonic acid and ethylene but was not active in plants
impaired in the SAR transduction pathway. Thus, BABA protects
Arabidopsis
against different virulent pathogens by
potentiating pathogen-specific plant resistance mechanisms. In
addition, we provide evidence that BABA-mediated papilla formation
after
P. parasitica
infection is independent of the SAR
signaling pathway.
Frameshift-mutation-derived peptides as tumor-specific antigens in inherited and spontaneous colorectal cancer
Authors: Sæterdal Ingvil , Bjørheim Jens , Lislerud Kari , Gjertsen Marianne K. , Bukholm Ida K. , Olsen Ole Christian , Nesland Jahn M. , Eriksen Jon Amund , Møller Mona , Lindblom Annika , Gaudernack Gustav
Published: July 2002

The functional role and specificity of tumor infiltrating lymphocytes (TIL) is generally not well characterized. Prominent lymphocyte infiltration is the hallmark of the most common form of hereditary colon cancer, hereditary nonpolyposis colon cancer (HNPCC) and the corresponding spontaneous colon cancers with the microsatellite instability (MSI) phenotype. These cancers are caused by inherited or acquired defects in the DNA mismatch–repair machinery. The molecular mechanism behind the MSI phenotype provides a clue to understanding the lymphocyte reaction by allowing reliable prediction of potential T cell epitopes created by frameshift mutations in candidate genes carrying nucleotide repeat sequences, such as
TGFβRII
and
BAX
. These tumors therefore represent an interesting human system for studying TIL and characterizing tumor-specific T cells. We here describe T cell reactivity against several T helper cell epitopes, representing a common frameshift mutation in
TGFβRII
, in TIL and peripheral blood lymphocytes from patients with MSI
+
tumors. The peptide SLVRLSSCVPVALMSAMTTSSSQ was recognized by T cells from two of three patients with spontaneous MSI
+
colon cancers and from all three patients with HNPCC. Because such mutations are present in 90% of cancers within this patient group, these newly characterized epitopes provide attractive targets for cancer vaccines, including a prophylactic vaccine for individuals carrying a genetic disposition for developing HNPCC.
A human nuclease specific for G4 DNA
Authors: Sun Hui , Yabuki Akiko , Maizels Nancy
Published: July 2002

We have identified a human nuclease that specifically cleaves four-stranded DNA stabilized by G quartets (G4 DNA). This nuclease, GQN1 (G quartet nuclease 1), cuts within the single-stranded region 5′ of the barrel formed by stacked G quartets. GQN1 does not cleave duplex or single-stranded DNA, Holliday junctions, or G4 RNA. Cleavage depends on DNA structure and not on flanking sequence. Activity is elevated in but not restricted to B cells, making GQN1 a strong candidate for function in immunoglobulin heavy chain class switch recombination. Identification of a mammalian nuclease that specifically cleaves G4 DNA provides further support for the notion that DNA structures stabilized by G quartets formin vivoand function in regulated recombination and genomic evolution.
YC-1 activation of human soluble guanylyl cyclase has both heme-dependent and heme-independent components
Authors: Martin Emil , Lee Yu-Chen , Murad Ferid
Published: July 2002

YC-1 [3-(5′-hydroxymethyl-2′furyl)-1-benzyl indazole] is an allosteric activator of soluble guanylyl cyclase (sGC). YC-1 increases the catalytic rate of the enzyme and sensitizes the enzyme toward its gaseous activators nitric oxide or carbon monoxide. In other studies the administration of YC-1 to experimental animals resulted in the inhibition of the platelet-rich thrombosis and a decrease of the mean arterial pressure, which correlated with increased cGMP levels. However, details of YC-1 interaction with sGC and enzyme activation are incomplete. Although evidence in the literature indicates that YC-1 activation of sGC is strictly heme-dependent, this report presents evidence for both heme-dependent and heme-independent activation of sGC by YC-1. The oxidation of the sGC heme by 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one completely inhibited the response to NO, but only partially attenuated activation by YC-1. We also observed activation by YC-1 of a mutant sGC, which lacks heme. These findings indicate that YC-1 activation of sGC can occur independently of heme, but that activation is substantially increased when the heme moiety is present in the enzyme.
A quorum sensing-associated virulence gene of
Pseudomonas aeruginosa
encodes a LysR-like transcription regulator with a unique self-regulatory mechanism
Authors: Cao Hui , Krishnan Gomathi , Goumnerov Boyan , Tsongalis John , Tompkins Ronald , Rahme Laurence G.
Published: July 2002

The human opportunistic pathogen
Pseudomonas
aeruginosa
strain PA14 infects both plants and animals.
Previously, using plants to screen directly for
P.
aeruginosa
virulence-attenuated mutants, we identified a locus,
pho34B12
, relevant in mammalian pathogenesis. Here,
nonsense point mutations in the two opposing ORFs identified in
the
pho34B12
locus revealed that one of them,
mvfR
(
m
ultiple
v
irulence
f
actor
R
egulator), is able to control all
of the phenotypes that mutant
phoA34B12
displays. Both
genetic and biochemical evidence demonstrate that the
mvfR
gene encodes a LysR-like transcriptional factor
that positively regulates the production of elastase, phospholipase,
and of the autoinducers, 3oxo-dodecanoyl homoserine lactone (PAI
I) and 2-heptyl-3-hydroxy-4-quinolone (PQS), as well as the expression
of the
phnAB
operon, involved in phenazine biosynthesis.
We demonstrate that the MvfR protein is membrane-associated and acts as
a transcriptional activator until cells reach stationary phase, when a
unique negative feedback mechanism is activated to signal the
down-regulation of the MvfR protein. This work reveals an unprecedented
virulence mechanism of
P. aeruginosa
and identifies a
unique indispensable player in the
P. aeruginosa
quorum-sensing cascade.
Mortality in 7B2 null mice can be rescued by adrenalectomy: Involvement of dopamine in ACTH hypersecretion
Authors: Laurent V. , Kimble A. , Peng B. , Zhu P. , Pintar J. E. , Steiner D. F. , Lindberg I.
Published: July 2002

The serine protease prohormone convertase 2 (PC2), principally involved in the processing of polypeptide hormone precursors in neuroendocrine tissues, requires interaction with the neuroendocrine protein 7B2 to generate an enzymatically active form. 7B2 null mice express no PC2 activity and release large quantities of uncleaved ACTH, resulting in a lethal endocrine condition that resembles pituitary Cushing's (Westphal, C. H., Muller, L., Zhou, A., Bonner-Weir, S., Schambelan, M., Steiner, D. F., Lindberg, I. & Leder, P. (1999)
Cell
96, 689). Here, we have compared the 7B2 and PC2 null mouse models to determine why the 7B2 null, but not the PC2 null, exhibits a lethal disease state. Both 7B2 and PC2 nulls contained highly elevated pituitary adrenocorticotropic hormone (ACTH); the neurointermediate lobe content of ACTH in 7B2 nulls was 13-fold higher than in WT mice; that of the PC2 null was 65-fold higher. However, circulating ACTH levels were much higher in the 7B2 null than in the PC2 null. Because hypothalamic inhibitory dopaminergic control represents the major influence on intermediate lobe proopiomelanocortin-derived peptide secretion, dopamine levels were measured, and they revealed that 7B2 null pituitaries contained only one-fourth of WT pituitary dopamine. Adrenalectomized 7B2 null animals survived past the usual time of death at 5 weeks; a month after adrenalectomy, they exhibited normal levels of pituitary dopamine, circulating ACTH, and corticosterone. Elevated corticosterone, therefore, seems to play a central role in the lethal phenotype of the 7B2 null, whereas a 7B2-mediated dopaminergic deficiency state may be involved in the actual ACTH hypersecretion phenomenon. Interestingly, adrenalectomized 7B2 nulls also developed unexpectedly severe obesity.
A two-subunit type I DNA topoisomerase (reverse gyrase) from an extreme hyperthermophile.
Authors: Krah R , Kozyavkin S A , Slesarev A I , Gellert M
Published: July 2002

A recently described reverse gyrase from the hyperthermophilic methanogen Methanopyrus kandleri is the only known example of a heterodimeric type I topoisomerase. The enzyme is made up of a 42-kDa subunit which covalently interacts with DNA (RgyA) and a 138-kDa subunit which binds ATP (RgyB). We have now cloned and sequenced the genes for both subunits of this enzyme. Surprisingly, the universally conserved type I topoisomerase domain [Lima, C. D., Wang, J. C. & Mondragon, A. (1994) Nature (London) 367, 138-146] which has been found as a contiguous polypeptide in the prokaryotes and eukaryotes is shared between the protomers. The subdomain with the active-site tyrosine is entirely within RgyA, whereas the subdomain implicated in noncovalent binding of the cleaved DNA strand is contained entirely in RgyB. The appearance of this unique structure in a highly conserved enzyme family supports the hypothesis that the methanogens branched from other prokaryotes and eukaryotes very early in evolution.
Structure-activity relationships derived by machine learning: the use of atoms and their bond connectivities to predict mutagenicity by inductive logic programming.
Authors: King R D , Muggleton S H , Srinivasan A , Sternberg M J
Published: July 2002

We present a general approach to forming structure-activity relationships (SARs). This approach is based on representing chemical structure by atoms and their bond connectivities in combination with the inductive logic programming (ILP) algorithm PROGOL. Existing SAR methods describe chemical structure by using attributes which are general properties of an object. It is not possible to map chemical structure directly to attribute-based descriptions, as such descriptions have no internal organization. A more natural and general way to describe chemical structure is to use a relational description, where the internal construction of the description maps that of the object described. Our atom and bond connectivities representation is a relational description. ILP algorithms can form SARs with relational descriptions. We have tested the relational approach by investigating the SARs of 230 aromatic and heteroaromatic nitro compounds. These compounds had been split previously into two subsets, 188 compounds that were amenable to regression and 42 that were not. For the 188 compounds, a SAR was found that was as accurate as the best statistical or neural network-generated SARs. The PROGOL SAR has the advantages that it did not need the use of any indicator variables handcrafted by an expert, and the generated rules were easily comprehensible. For the 42 compounds, PROGOL formed a SAR that was significantly (P < 0.025) more accurate than linear regression, quadratic regression, and back-propagation. This SAR is based on an automatically generated structural alert for mutagenicity.
Vitamin E prevents oxidative modification of brain and lymphocyte band 3 proteins during aging.
Authors: Poulin J E , Cover C , Gustafson M R , Kay M B
Published: July 2002

Antioxidants may play an important role in preventing free radical damage associated with aging by interfering directly in the generation of radicals or by scavenging them. We investigated the effects of a high vitamin E and/or a high beta-carotene diet on aging of the anion transporter, band 3, in lymphocytes and brain. The band 3 proteins function as anion transporters, acid base regulators, C02 transporters, and structural proteins that provide a framework for membrane lipids and that link the plasma membrane to the cytoskeleton. Senescent cell antigen (SCA), which terminates the life of cells, is a degradation product of band 3. This study was conducted as a double-blind study in which eight groups of middle-aged or old mice received either high levels of beta-carotene and/or vitamin E or standard levels of these supplements in their diets. Anion transport kinetic assays were performed on isolated splenic lymphocytes. Immunoreactivity of an antibody that recognizes aging changes in old band 3 preceding generation of SCA was used to quantitate aged band 3 in brain tissue. Results indicate that vitamin E prevented the observed age-related decline in anion transport by lymphocytes and the generation of aged band 3 leading to SCA formation. beta-Carotene had no significant effect on the results of either assay. Since increased aged band 3 and decreased anion transport are initial steps in band 3 aging, which culminates in the generation of SCA and cellular removal, vitamin E prevents or delays aging of band 3-related proteins in lymphocytes and brain.
Two genes abrogate the inhibition of murine hepatocarcinogenesis by ovarian hormones.
Authors: Poole T M , Drinkwater N R
Published: July 2002

Hormonal and genetic factors strongly influence the susceptibility of inbred mice to hepatocarcinogenesis. Female C57BR/cdJ (BR) mice are extremely susceptible to liver tumor induction relative to other strains because they are genetically insensitive to the inhibition of hepatocarcinogenesis by ovarian hormones. To determine the genetic basis for the sensitivity of BR mice relative to resistant C57BL/6J (B6) mice, we treated 12-day-old B6BRF1 x B6 and B6BRF1 x B6BRF1 (F2) animals with N,N-diethylnitrosamine (0.1 micromol/g of body weight) and enumerated liver tumors at 32 weeks of age in males and at 50 weeks in females. Genomic DNA samples from backcross and F2 mice were analyzed for 70 informative simple sequence length polymorphism markers. Genetic markers on chromosome 17 (D17Mit21) and chromosome 1 (D1Mit33) cosegregated with high tumor multiplicity in both sexes. Together, these loci [designated Hcf1 and Hcf2 (Hepatocarcinogenesis in females), respectively] account for virtually all of the difference in sensitivity between BR and B6 mice. The Hcf1 locus accounts for a majority of the higher susceptibility of BR mice of both sexes. Backcross female mice heterozygous at both loci (33 +/- 23 tumors per mouse) and at Hcf1 only (17 +/- 18) were 15- and 8-fold more sensitive, respectively, than mice homozygous for the B6 alleles at Hcf1 and Hcf2 (2.2 +/- 3.9). In backcross male mice, the double heterozygotes (35 +/- 22) and Hcf1 heterozygotes (28 +/- 12) were 5.4- and 4.3-fold more sensitive than mice homozygous for B6 alleles at both loci (6.5 +/- 5.4).
Genomic cloning of methylthioadenosine phosphorylase: a purine metabolic enzyme deficient in multiple different cancers.
Authors: Nobori T , Takabayashi K , Tran P , Orvis L , Batova A , Yu A L , Carson D A
Published: July 2002

5'-Deoxy-5'-methylthioadenosine phosphorylase (methylthioadeno-sine: ortho-phosphate methylthioribosyltransferase, EC 24.2.28; MTAP) plays a role in purine and polyamine metabolism and in the regulation of transmethylation reactions. MTAP is abundant in normal cells but is deficient in many cancers. Recently, the genes for the cyclin-dependent kinase inhibitors p16 and p15 have been localized to the short arm of human chromosome 9 at band p21, where MTAP and interferon alpha genes (IFNA) also map. Homozygous deletions of p16 and p15 are frequent malignant cell lines. However, the order of the MTAP, p16, p15, and IFNA genes on chromosome 9p is uncertain, and the molecular basis for MTAP deficiency in cancer is unknown. We have cloned the MTAP gene, and have constructed a topologic map of the 9p21 region using yeast artificial chromosome clones, pulse-field gel electrophoresis, and sequence-tagged-site PCR. The MTAP gene consists of eight exons and seven introns. Of 23 malignant cell lines deficient in MTAP protein, all but one had complete or partial deletions. Partial or total deletions of the MTAP gene were found in primary T-cell acute lymphoblastic leukemias (T-ALL). A deletion breakpoint of partial deletions found in cell lines and primary T-ALL was in intron 4. Starting from the centromeric end, the gene order on chromosome 9p2l is p15, p16, MTAP, IFNA, and interferon beta gene (IFNB). These results indicate that MTAP deficiency in cancer is primarily due to codeletion of the MTAP and p16 genes.
Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling.
Authors: García-Cardeña G , Oh P , Liu J , Schnitzer J E , Sessa W C
Published: July 2002

The membrane association of endothelial nitric oxide synthase (eNOS) plays an important role in the biosynthesis of nitric oxide (NO) in vascular endothelium. Previously, we have shown that in cultured endothelial cells and in intact blood vessels, eNOS is found primarily in the perinuclear region of the cells and in discrete regions of the plasma membrane, suggesting trafficking of the protein from the Golgi to specialized plasma membrane structures. Here, we show that eNOS is found in Triton X-100-insoluble membranes prepared from cultured bovine aortic endothelial cells and colocalizes with caveolin, a coat protein of caveolae, in cultured bovine lung microvascular endothelial cells as determined by confocal microscopy. To examine if eNOS is indeed in caveolae, we purified luminal endothelial cell plasma membranes and their caveolae directly from intact, perfused rat lungs. eNOS is found in the luminal plasma membranes and is markedly enriched in the purified caveolae. Because palmitoylation of eNOS does not significantly influence its membrane association, we next examined whether this modification can affect eNOS targeting to caveolae. Wild-type eNOS, but not the palmitoylation mutant form of the enzyme, colocalizes with caveolin on the cell surface in transfected NIH 3T3 cells, demonstrating that palmitoylation of eNOS is necessary for its targeting into caveolae. These data suggest that the subcellular targeting of eNOS to caveolae can restrict NO signaling to specific targets within a limited microenvironment at the cell surface and may influence signal transduction through caveolae.
Molecular characterization of a FKBP-type immunophilin from higher plants.
Authors: Luan S , Kudla J , Gruissem W , Schreiber S L
Published: July 2002

Immunophilins are intracellular receptors for the immunosuppressants cyclosporin A, FK506, and rapamycin. In addition to their use in organ transplantation, these natural products have been used to investigate signaling pathways in yeast, plant, and mammalian cells. We have recently described the identification of an immunosuppressant-sensitive signaling pathway in and the purification of several immunophilins from Vicia faba plants. We now report the molecular characterization of a 15 kDa FK506- and rapamycin-binding protein from V. faba (VfFKBP15). The amino acid sequence deduced from the cDNA starts with a signal peptide of 22 hydrophobic amino acids. The core region of VfFKBP15 is most similar to yeast and mammalian FKBP13 localized in the endoplasmic reticulum (ER). In addition, VfFKBP15 has a carboxyl-terminal sequence that is ended with SSEL, a putative ER retention signal. These findings suggest that VfFKBP15 is a functional homolog of FKBP13 from other organisms. Interestingly, two distinct cDNAs corresponding to two isoforms of FKBP15 have been cloned from Arabidopsis and also identified from rice data base, suggesting that pFKBP15 (plant FKBP15) is encoded by a small gene family in plants. This adds to the diversity of plant FKBP members even with the same subcellular localization and is in contrast with the situation in mammalian and yeast systems in which only one FKBP13 gene has been found. Like the mammalian and yeast FKBP13, the recombinant VfFKBP15 protein has rotamase activity that is inhibited by both FK506 and rapamycin with a Ki value of 30 nM and 0.9 nM, respectively, illustrating that VfFKBP15 binds rapamycin in preference over FK506. The mRNA of VfFKBP15 is ubiquitously expressed in various plant tissues including leaves, stems, and roots, consistent with the ER localization of the protein. Levels of VfFKBP15 mRNA are elevated by heat shock, suggesting a possible role for this FKBP member under stress conditions.
Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.
Authors: Crabb B S , Cowman A F
Published: July 2002

Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest.
Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex.
Authors: Chen W R , Lee S , Kato K , Spencer D D , Shepherd G M , Williamson A
Published: July 2002

The primate temporal cortex has been demonstrated to play an important role in visual memory and pattern recognition. It is of particular interest to investigate whether activity-dependent modification of synaptic efficacy, a presumptive mechanism for learning and memory, is present in this cortical region. Here we address this issue by examining the induction of synaptic plasticity in surgically resected human inferior and middle temporal cortex. The results show that synaptic strength in the human temporal cortex could undergo bidirectional modifications, depending on the pattern of conditioning stimulation. High frequency stimulation (100 or 40 Hz) in layer IV induced long-term potentiation (LTP) of both intracellular excitatory postsynaptic potentials and evoked field potentials in layers II/III. The LTP induced by 100 Hz tetanus was blocked by 50-100 microM DL-2-amino-5-phosphonovaleric acid, suggesting that N-methyl-D-aspartate receptors were responsible for its induction. Long-term depression (LTD) was elicited by prolonged low frequency stimulation (1 Hz, 15 min). It was reduced, but not completely blocked, by DL-2-amino-5-phosphonovaleric acid, implying that some other mechanisms in addition to N-methyl-DL-aspartate receptors were involved in LTD induction. LTD was input-specific, i.e., low frequency stimulation of one pathway produced LTD of synaptic transmission in that pathway only. Finally, the LTP and LTD could reverse each other, suggesting that they can act cooperatively to modify the functional state of cortical network. These results suggest that LTP and LTD are possible mechanisms for the visual memory and pattern recognition functions performed in the human temporal cortex.
Class 3 Hox genes in insects and the origin of zen.
Authors: Falciani F , Hausdorf B , Schröder R , Akam M , Tautz D , Denell R , Brown S
Published: July 2002

We have cloned, from a beetle and a locust, genes that are homologous to the class 3 Hox genes of vertebrates. Outside the homeobox they share sequence motifs with the Drosophila zerknüllt (zen) and z2 genes, and like zen, are expressed only in extraembryonic membranes. We conclude that the zen genes of Drosophila derive from a Hox class 3 sequence that formed part of the common ancestral Hox cluster, but that in insects this (Hox) gene has lost its role in patterning the anterio-posterior axis of the embryo, and acquired a new function. In the lineage leading to Drosophila, the zen genes have diverged particularly rapidly.
Circulating plasma xanthine oxidase contributes to vascular dysfunction in hypercholesterolemic rabbits.
Authors: White C R , Darley-Usmar V , Berrington W R , McAdams M , Gore J Z , Thompson J A , Parks D A , Tarpey M M , Freeman B A
Published: July 2002

Reactive oxygen species play a central role in vascular inflammation and atherogenesis, with enhanced superoxide (O2.-) production contributing significantly to impairment of nitric oxide (.NO)-dependent relaxation of vessels from cholesterol-fed rabbits. We investigated potential sources of O2.- production, which contribute to this loss of endothelium-dependent vascular responses. The vasorelaxation elicited by acetylcholine (ACh) in phenylephrine-contracted, aortic ring segments was impaired by cholesterol feeding. Pretreatment of aortic vessels with either heparin, which competes with xanthine oxidase (XO) for binding to sulfated glycosaminoglycans, or the XO inhibitor allopurinol resulted in a partial restoration (36-40% at 1 muM ACh) of ACh-dependent relaxation. Furthermore, O2.(-)-dependent lucigenin chemiluminescence, measured in intact ring segments from hypercholesterolemic rabbits, was decreased by addition of heparin, allopurinol or a chimeric, heparin-binding superoxide dismutase. XO activity was elevated more than two-fold in plasma of hypercholesterolemic rabbits. Incubation of vascular rings from rabbits on a normal diet with purified XO (10 milliunits/ml) also impaired .NO-dependent relaxation but only in the presence of purine substrate. As with vessels from hypercholesterolemic rabbits, this effect was prevented by heparin and allopurinol treatment. We hypothesize that increases in plasma cholesterol induce the release of XO into the circulation, where it binds to endothelial cell glycosaminoglycans. Only in hypercholesterolemic vessels is sufficient substrate available to sustain the production of O2.- and impair NO-dependent vasorelaxation. Chronically, the continued production of peroxynitrite, (ONOO-) which the simultaneous generation of NO and O2.- implies, may irreversibly impair vessel function.
TnrA, a transcription factor required for global nitrogen regulation in Bacillus subtilis.
Authors: Wray L V , Ferson A E , Rohrer K , Fisher S H
Published: July 2002

Expression of the Bacillus subtilis nrgAB operon is derepressed during nitrogen-limited growth. We have identified a gene, tnrA, that is required for the activation of nrgAB expression under these growth conditions. Analysis of the DNA sequence of the tnrA gene revealed that it encodes a protein with sequence similarity to GlnR, the repressor of the B. subtilis glutamine synthetase operon. The tnrA mutant has a pleiotropic phenotype. Compared with wild-type cells, the tnrA mutant is impaired in its ability to utilize allantoin, gamma-aminobutyrate, isoleucine, nitrate, urea, and valine as nitrogen sources. During nitrogen-limited growth, transcription of the nrgAB, nasB, gabP, and ure genes is significantly reduced in the tnrA mutant compared with the levels seen in wild-type cells. In contrast, the level of glnRA expression is 4-fold higher in the, tnrA mutant than in wild-type cells during nitrogen restriction. The phenotype of the tnrA mutant indicates that a global nitrogen regulatory system is present in B. subtilis and that this system is distinct from the Ntr regulatory system found in enteric bacteria.
The ocular albinism type 1 gene product is a membrane glycoprotein localized to melanosomes.
Authors: Schiaffino M V , Baschirotto C , Pellegrini G , Montalti S , Tacchetti C , De Luca M , Ballabio A
Published: July 2002

Ocular albinism type 1 (OA1) is an inherited disorder characterized by severe reduction of visual acuity, photophobia, and retinal hypopigmentation. Ultrastructural examination of skin melanocytes and of the retinal pigment epithelium reveals the presence of macromelanosomes, suggesting a defect in melanosome biogenesis. The gene responsible for OA1 is exclusively expressed in pigment cells and encodes a predicted protein of 404 aa displaying several putative transmembrane domains and sharing no similarities with previously identified molecules. Using polyclonal antibodies we have identified the endogenous OA1 protein in retinal pigment epithelial cells, in normal human melanocytes and in various melanoma cell lines. Two forms of the OA1 protein were identified by Western analysis, a 60-kDa glycoprotein and a doublet of 48 and 45 kDa probably corresponding to unglycosylated precursor polypeptides. Upon subcellular fractionation and phase separation with the nonionic detergent Triton X-114, the OA1 protein segregated into the melanosome-rich fraction and behaved as an authentic integral membrane protein. Immunofluorescence and immunogold analyses on normal human melanocytes confirmed the melanosomal membrane localization of the endogenous OA1 protein, consistent with its possible involvement in melanosome biogenesis. The identification of a novel melanosomal membrane protein involved in a human disease will provide insights into the mechanisms that control the cell-specific pathways of subcellular morphogenesis.
Interleukin 7 independent development of human B cells.
Authors: Prieyl J A , LeBien T W
Published: July 2002

Mammalian hematopoietic stem cell (HSC) commitment and differentiation into lymphoid lineage cells proceed through a series of developmentally restricted progenitor compartments. A complete understanding of this process, and how it differs from HSC commitment and differentiation into cells of the myeloid/erythroid lineages, requires the development of model systems that support HSC commitment to the lymphoid lineages. We now describe a human bone marrow stromal cell culture that preferentially supports commitment and differentiation of human HSC to CD19+ B-lineage cells. Fluorescence activated cell sorterpurified CD34++/lineage-cells were isolated from fetal bone marrow and cultured on human fetal bone marrow stromal cells in serum-free conditions containing no exogenous cytokines. Over a period of 3 weeks, CD34++/lineage- cells underwent commitment, differentiation, and expansion into the B lineage. Progressive changes included: loss of CD34, acquisition of and graded increases in the level of cell surface CD19, and appearance of immature B cells expressing mu/kappa or mu/lambda cell surface Ig receptors. The tempo and phenotype of B-cell development was not influenced by the addition of IL-7 (10 ng/ml), or by the addition of goat anti-IL-7 neutralizing antibody. These results indicate a profound difference between mouse and human in the requirement for IL-7 in normal B-cell development, and provide an experimental system to identify and characterize human bone marrow stromal cell-derived molecules crucial for human B lymphopoiesis.
Rational interleukin 2 therapy for HIV positive individuals: daily low doses enhance immune function without toxicity.
Authors: Jacobson E L , Pilaro F , Smith K A
Published: July 2002

When administered in high doses to HIV positive (HIV+) individuals, interleukin 2 (IL-2) causes extreme toxicity and markedly increases plasma HIV levels. Integration of the information from the structure-activity relationships of the IL-2 receptor interaction, the cellular distribution of the different classes of IL-2 receptors, and the pharmacokinetics of IL-2 provides for the rationale that low IL-2 doses should circumvent toxicity. Therefore, to identify a nontoxic, but effective and safe IL-2 treatment regimen that does not stimulate viral replication, doses of IL-2 from 62,500 to 250,000 IU/m2/day were administered subcutaneously for 6 months to 16 HIV+ individuals with 200-500 CD4+ T cells/mm3. IL-2 was already detectable in the plasma of most HIV+ individuals even before therapy. Peak plasma IL-2 levels were near saturating for high affinity IL-2 receptors in 10 individuals who received the maximum nontoxic dose, which ranged from 187,500 to 250,000 IU/m2/day. During the 6 months of treatment at this dose range, plasma levels of proinflammatory cytokines remained undetectable, and plasma HIV RNA levels did not change significantly. However, delayed type hypersensitivity responses to common recall antigens were markedly augmented, and there were IL-2 dose-dependent increases in circulating Natural Killer cells, eosinophils, monocytes, and CD4+ T cells. Expanded clinical trials of low dose IL-2 are now warranted, especially in combination with effective antivirals to test for the prevention of immunodeficiency and the emergence of drug-resistant mutants and for the eradication of residual virions.
Measurement of genetic structure within populations using Moran's spatial autocorrelation statistics.
Authors: Epperson B K , Li T
Published: July 2002

Spatial structure of genetic variation within populations, an important interacting influence on evolutionary and ecological processes, can be analyzed in detail by using spatial autocorrelation statistics. This paper characterizes the statistical properties of spatial autocorrelation statistics in this context and develops estimators of gene dispersal based on data on standing patterns of genetic variation. Large numbers of Monte Carlo simulations and a wide variety of sampling strategies are utilized. The results show that spatial autocorrelation statistics are highly predictable and informative. Thus, strong hypothesis tests for neutral theory can be formulated. Most strikingly, robust estimators of gene dispersal can be obtained with practical sample sizes. Details about optimal sampling strategies are also described.
CDP/cut is the DNA-binding subunit of histone gene transcription factor HiNF-D: a mechanism for gene regulation at the G1/S phase cell cycle transition point independent of transcription factor E2F.
Authors: van Wijnen A J , van Gurp M F , de Ridder M C , Tufarelli C , Last T J , Birnbaum M , Vaughan P S , Giordano A , Krek W , Neufeld E J , Stein J L , Stein G S
Published: July 2002

Transcription of the genes for the human histone proteins H4, H3, H2A, H2B, and H1 is activated at the G1/S phase transition of the cell cycle. We have previously shown that the promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors cyclin A, CDC2, and a retinoblastoma (pRB) protein-related protein. However, an intrinsic DNA-binding subunit for HiNF-D was not identified. Many genes that are up-regulated at the G1/S phase boundary are controlled by E2F, a transcription factor that associates with cyclin-, cyclin-dependent kinase-, and pRB-related proteins. Using gel-shift immunoassays, DNase I protection, and oligonucleotide competition analyses, we show that the homeodomain protein CDP/cut, not E2F, is the DNA-binding subunit of the HiNF-D complex. The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and pRB but not p107, whereas the CDP/cut complex with a nonhistone promoter (gp91-phox) reacts only with CDP and p107 antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct pRB-related proteins. Transient coexpression assays show that CDP/cut modulates H4 promoter activity via the HiNF-D-binding site. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclin A/CDC2/ RB-related proteins.
Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.
Authors: Hintermann E , Grieder N C , Amherd R , Brodbeck D , Meyer U A
Published: July 2002

In insects, neurotransmitter catabolism, melatonin precursor formation, and sclerotization involve arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) activity. It is not known if one or multiple aaNAT enzymes are responsible for these activities. We recently have purified an aaNAT from Drosophila melanogaster. Here, we report the cloning of the corresponding aaNAT cDNA (aaNAT1) that upon COS cell expression acetylates dopamine, tryptamine, and the immediate melatonin precursor serotonin. aaNAT1 represents a novel gene family unrelated to known acetyl-transferases, except in two weakly conserved amino acid motifs. In situ hybridization studies of aaNAT1 mRNA in embryos reveal hybridization signals in the brain, the ventral cord, the gut, and probably in oenocytes, indicating a broad tissue distribution of aaNAT1 transcripts. Moreover, in day/ night studies we demonstrate a diurnal rhythm of melatonin concentration without a clear-cut change in aaNAT1 mRNA levels. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes.
Elevated blood pressures in mice lacking endothelial nitric oxide synthase
Authors: Shesely Edward G. , Maeda Nobuyo , Kim H.-S. , Desai Kaushik M. , Krege John H. , Laubach Victor E. , Sherman Paula A. , Sessa William C. , Smithies Oliver
Published: July 2002

Nitric oxide produced in endothelial cells affects vascular tone.
To investigate the role of endothelial nitric oxide synthase (eNOS) in
blood pressure regulation, we have generated mice heterozygous (+/−)
or homozygous (−/−) for disruption of the eNOS gene.
Immunohistochemical staining with anti-eNOS antibodies showed reduced
amounts of eNOS protein in +/− mice and absence of eNOS protein in
−/− mutant mice. Male or female mice of all three eNOS genotypes
were indistinguishable in general appearance and histology, except that
−/− mice had lower body weights than +/+ or +/− mice. Blood
pressures tended to be increased (by approximately 4 mmHg) in +/−
mice compared with +/+, while −/− mice had a significant increase
in pressure compared with +/+ mice (≈18 mmHg) or +/− mice (≈14
mmHg). Plasma renin concentration in the −/− mice was nearly twice
that of +/+ mice, although kidney renin mRNA was modestly decreased
in the −/− mice. Heart rates in the −/− mice were significantly
lower than in +/− or +/+ mice. Appropriate genetic controls show
that these phenotypes in F
2
mice are due to the eNOS
mutation and are not due to sequences that might differ between the two
parental strains (129 and C57BL/6J) and are linked either to the eNOS
locus or to an unlinked chromosomal region containing the renin locus.
Thus eNOS is essential for maintenance of normal blood pressures and
heart rates. Comparisons between the current eNOS mutant mice and
previously generated inducible nitric oxide synthase mutants showed
that homozygous mutants for the latter differ in having unaltered blood
pressures and heart rates; both are susceptible to
lipopolysaccharide-induced death.
Effect of genetic background on the contribution of New Zealand Black loci to autoimmune lupus nephritis
Authors: Rozzo Stephen J. , Vyse Timothy J. , Drake Charles G. , Kotzin Brian L.
Published: July 2002

Autoimmune diseases such as systemic lupus erythematosus are complex genetic traits with contributions from major histocompatibility complex (MHC) genes and multiple unknown non-MHC genes. Studies of animal models of lupus have provided important insight into the immunopathogenesis of disease, and genetic analyses of these models overcome certain obstacles encountered when studying human patients. Genome-wide scans of different genetic crosses have been used to map several disease-linked loci in New Zealand hybrid mice. Although some consensus exists among studies mapping the New Zealand Black (NZB) and New Zealand White (NZW) loci that contribute to lupus-like disease, considerable variability is also apparent. A variable in these studies is the genetic background of the non-autoimmune strain, which could influence genetic contributions from the affected strain. A direct examination of this question was undertaken in the present study by mapping NZB nephritis-linked loci in backcrosses involving different non-autoimmune backgrounds. In a backcross with MHC-congenic C57BL/6J mice,H2zappeared to be the strongest genetic determinant of severe lupus nephritis, whereas in a backcross with congenic BALB/cJ mice,H2zshowed no influence on disease expression. NZB loci on chromosomes 1, 4, 11, and 14 appeared to segregate with disease in the BALB/cJ cross, but only the influence of the chromosome 1 locus spanned both crosses and showed linkage with disease when all mice were considered. Thus, the results indicate that contributions from disease-susceptibility loci, including MHC, may vary markedly depending on the non-autoimmune strain used in a backcross analysis. These studies provide insight into variables that affect genetic heterogeneity and add an important dimension of complexity for linkage analyses of human autoimmune disease.
Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene.
Authors: Hoshimaru M , Ray J , Sah D W , Gage F H
Published: July 2002

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.
Cyp1a2(-/-) null mutant mice develop normally but show deficient drug metabolism.
Authors: Liang H C , Li H , McKinnon R A , Duffy J J , Potter S S , Puga A , Nebert D W
Published: July 2002

Cytochrome P450 1A2 (CYP1A2) is a predominantly hepatic enzyme known to be important in the metabolism of numerous foreign chemicals of pharmacologic, toxicologic, and carcinogenic significance. CYP1A2 substrates include aflatoxin B1, acetaminophen, and a variety of environmental arylamines. To define better the developmental and metabolic functions of this enzyme, we developed a CYP1A2-deficient mouse line by homologous recombination in embryonic stem cells. Mice homozygous for the targeted Cyp1a2 gene, designated Cyp1a2(-/-), are completely viable and fertile; histologic examination of 15-day embryos, newborn pups, and 3-week-old mice revealed no abnormalities. No CYP1A2 mRNA was detected by Northern blot analysis. Moreover, mRNA levels of Cyp1a1, the other gene in the same subfamily, appear unaffected by loss of the Cyp1a2 gene. Because the muscle relaxant zoxazolamine is a known substrate for CYP1A2, we studied the Cyp1a2(-/-) genotype by using the zoxazolamine paralysis test: the Cyp1a2(-/-) mice exhibited dramatically lengthened paralysis times relative to the Cyp1a2(+/+) wild-type animals, and the Cyp1a2(+/-) heterozygotes showed an intermediate effect. Availability of a viable and fertile CYP1A2-deficient mouse line will provide a valuable tool for researchers wishing to define the precise role of CYP1A2 in numerous metabolic and pharmacokinetic processes.
Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.
Authors: Radford N B , Fina M , Benjamin I J , Moreadith R W , Graves K H , Zhao P , Gavva S , Wiethoff A , Sherry A D , Malloy C R , Williams R S
Published: July 2002

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.