bluuebunny/crossref_metadata_2025 huggingface dataset:
The nature of the active suppression of responses associated with experimental autoimmune myasthenia gravis by a dual altered peptide ligand administered by different routes
Authors: Paas-Rozner Miri , Sela Michael , Mozes Edna
Published: July 2002

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T-cell regulated, antibody-mediated diseases. Peptides p195–212 and p259–271 of the human acetylcholine receptor (AChR) α-subunit, were previously shown to be immunodominant T cell epitopes in MG patients as well as in SJL and BALB/c mice, respectively. A dual altered peptide ligand (APL) composed of the two single amino acid analogs of the myasthenogenic peptides was shown to inhibit,in vitroandin vivo, MG-associated autoimmune responses. Furthermore, the dual APL was shown to down-regulate the clinical manifestations of an established EAMG in C57BL/6 mice injected with Torpedo AChR (TAChR). In the present study we attempted the elucidation of the mechanism(s) by which the dual APL down-regulates EAMG-associated responses. It is shown here that the dual APL acts by actively suppressing, in a specific manner, myasthenogenic T cell responses. The active suppression is mediated, at least partially, by the up-regulation of the secretion of TGF-β following administration of the dual APL. The up-regulated secretion of TGF-β is accompanied by down-regulation of IFN-γ and IL-2 [T helper (Th) 1-type cytokine] secretion and by an up-regulation of IL-10 secretion (Th2-type cytokine). Furthermore, the inhibitory effect of the dual APL could be adoptively transferred to p195–212 or TAChR-immunized mice. The down-regulation of IL-2 secretion and the ability of recombinant IL-2 to rescue lymph node cells of mice treated with the dual APL from a state of unresponsiveness suggests that the dual APL acts also, at least partially, by causing the cells to undergo anergy.
The Drosophila melanogaster dodo (dod) gene, conserved in humans, is functionally interchangeable with the ESS1 cell division gene of Saccharomyces cerevisiae.
Authors: Maleszka R , Hanes S D , Hackett R L , de Couet H G , Miklos G L
Published: July 2002

We have sequenced the region of DNA adjacent to and including the flightless (fli) gene of Drosophila melanogaster and molecularly characterized four transcription units within it, which we have named tweety (twe), flightless (fli), dodo (dod), and penguin (pen). We have performed deletion and transgenic analysis to determine the consequences of the quadruple gene removal. Only the flightless gene is vital to the organism; the simultaneous absence of the other three allows the overriding majority of individuals to develop to adulthood and to fly normally. These gene deletion results are evaluated in the context of the redundancy and degeneracy inherent in many genetic networks. Our cDNA analyses and data-base searches reveal that the predicted dodo protein has homologs in other eukaryotes and that it is made up of two different domains. The first, designated WW, is involved in protein-protein interactions and is found in functionally diverse proteins including human dystrophin. The second is involved in accelerating protein folding and unfolding and is found in Escherichia coli in a new family of peptidylprolyl cis-trans isomerases (PPIases; EC 5.2.1.8). In eukaryotes, PPIases occur in the nucleus and the cytoplasm and can form stable associations with transcription factors, receptors, and kinases. Given this particular combination of domains, the dodo protein may well participate in a multisubunit complex involved in the folding and activation of signaling molecules. When we expressed the dodo gene product in Saccharomyces cerevisiae, it rescued the lethal phenotype of the ESS1 cell division gene.
Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants.
Authors: Salem N , Wegher B , Mena P , Uauy R
Published: July 2002

It is becoming clear that an adequate level of long-chain highly unsaturated fatty acids in the nervous system is required for optimal function and development; however, the ability of infants to biosynthesize long-chain fatty acids is unknown. This study explores the capacity of human infants to convert 18-carbon essential fatty acids to their elongated and desaturated forms, in vivo. A newly developed gas chromatography/negative chemical ionization/mass spectrometry method employing 2H-labeled essential fatty acids allowed assessment of this in vivo conversion with very high sensitivity and selectivity. Our results demonstrate that human infants have the capacity to convert dietary essential fatty acids administered enterally as 2H-labeled ethyl esters to their longer-chain derivatives, transport them to plasma, and incorporate them into membrane lipids. The in vivo conversion of linoleic acid (18:2n6) to arachidonic acid (20:4n6) is demonstrated in human beings. All elongases/desaturases necessary for the conversion of linolenic acid (18:3n3) to docosahexaenoic acid (22:6n3) are also active in the first week after birth. Although the absolute amounts of n-3 fatty acid metabolites accumulated in plasma are greater than those of the n-6 family, estimates of the endogenous pools of 18:2n6 and 18:3n3 indicate that n-6 fatty acid conversion rates are greater than those of the n-3 family. While these data clearly demonstrate the capability of infants to biosynthesize 22:6n3, a lipid that is required for optimal neural development, the amounts produced in vivo from 18:3n3 may be inadequate to support the 22:6n3 level observed in breast-fed infants.
4-Oxoretinol, a new natural ligand and transactivator of the retinoic acid receptors.
Authors: Achkar C C , Derguini F , Blumberg B , Langston A , Levin A A , Speck J , Evans R M , Bolado J , Nakanishi K , Buck J , Gudas L J
Published: July 2002

All-trans-retinoic acid (at-RA) induces cell differentiation in a wide variety of cell types, including F9 embryonic teratocarcinoma cells, and can influence axial pattern formation during embryonic development. We now identify a novel retinoid synthetic pathway in differentiating F9 cells that results in the intracellular production of 4-oxoretinol (4-oxo-ROL) from retinol (vitamin A). Approximately 10-15% of the total retinol in the culture is metabolized to 4-hydroxyretinol and 4-oxo-ROL by the at-RA-treated, differentiating F9 cells over an 18-hr period, but no detectable metabolism of all-trans-retinol to at-RA or 9-cis-retinoic acid is observed in these cells. Remarkably, we show that 4-oxo-ROL can bind and activate transcription of the retinoic acid receptors whereas all-trans-retinol shows neither activity. Low doses of 4-oxo-ROL (e.g., 10(-9) or 10(-10 M) can activate the retinoic acid receptors even though, unlike at-RA, 4-oxo-ROL does not contain an acid moiety at the carbon 15 position. 4-oxo-ROL does not bind or transcriptionally activate the retinoid X receptors. Treatment of F9 cells with 4-oxo-ROL induces differentiation without conversion to the acid and 4-oxo-ROL is active in causing axial truncation when administered to Xenopus embryos at the blastula stage. Thus, 4-oxo-ROL is a natural, biologically active retinoid that is present in differentiated F9 cells. Our data suggest that 4-oxo-ROL may be a novel signaling molecule and regulator of cell differentiation.
An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene.
Authors: Banerjee C , Hiebert S W , Stein J L , Lian J B , Stein G S
Published: July 2002

Tissue and cell-type specific expression of the rat osteocalcin (rOC) gene involves the interplay of multiple transcriptional regulatory factors. In this report we demonstrate that AML-1 (acute myeloid leukemia-1), a DNA-binding protein whose genes are disrupted by chromosomal translocations in several human leukemias, interacts with a sequence essential for enhancing tissue-restricted expression of the rOC gene. Deletion analysis of rOC promoter-chloramphenicol acetyltransferase constructs demonstrates that an AML-1-binding sequence within the proximal promoter (-138 to -130 nt) contributes to 75% of the level of osteocalcin gene expression. The activation potential of the AML-1-binding sequence has been established by overexpressing AML-1 in osteoblastic as well as in nonosseous cell lines. Overexpression not only enhances rOC promoter activity in osteoblasts but also mediates OC promoter activity in a nonosseous human fibroblastic cell line. A probe containing this site forms a sequence specific protein-DNA complex with nuclear extracts from osteoblastic cells but not from nonosseous cells. Antisera supershift experiments indicate the presence of AML-1 and its partner protein core-binding factor beta in this osteoblast-restricted complex. Mutations of the critical AML-1-binding nucleotides abrogate formation of the complex and strongly diminish promoter activity. These results indicate that an AML-1 related protein is functional in cells of the osteoblastic lineage and that the AML-1-binding site is a regulatory element important for osteoblast-specific transcriptional activation of the rOC gene.
Deregulation of PAX-5 by translocation of the Emu enhancer of the IgH locus adjacent to two alternative PAX-5 promoters in a diffuse large-cell lymphoma.
Authors: Busslinger M , Klix N , Pfeffer P , Graninger P G , Kozmik Z
Published: July 2002

Analyses of the human PAX-5 locus and of the 5' region of the mouse Pax-5 gene revealed that transcription from two distinct promoters results in splicing of two alternative 5' exons to the common coding sequences of exons 2-10. Transcription from the upstream promoter initiates downstream of a TATA box and occurs predominantly in B-lymphocytes, whereas the TATA-less downstream promoter is active in all Pax-5-expressing tissues. The human PAX-5 gene is located on chromosome 9 in region p13, which is involved in t(9;14)(pl3;q32) translocations recurring in small lymphocytic lymphomas of the plasmacytoid subtype and in derived large-cell lymphomas. A previous molecular analysis of a t(9;14) breakpoint from a diffuse large-cell lymphoma (KIS-1) demonstrated that the immunoglobulin heavy-chain (IgH) locus on 14q32 was juxtaposed to chromosome 9p13 sequences of unknown function [Ohno, H., Furukawa, T., Fukuhara, S., Zong, S. Q., Kamesaki, H., Shows, T. B., Le Beau, M. M., McKeithan, T. W., Kawakami, T. & Honjo, T. (1990) Proc. Natl. Acad. Sci. USA 87,628-632]. Here we show that the KIS-1 translocation breakpoint is located 1807 base pairs upstream of exon 1A of PAX-5, thus bringing the potent Emu enhancer of the IgH gene into close proximity of the PAX-5 promoters. These data suggest that deregulation of PAX-5 gene transcription by the t(9;14)(pl3;q32) translocation contributes to the pathogenesis of small lymphocytic lymphomas with plasmacytoid differentiation.
A human axillary odorant is carried by apolipoprotein D.
Authors: Zeng C , Spielman A I , Vowels B R , Leyden J J , Biemann K , Preti G
Published: July 2002

The characterization of the source of the odor in the human axillary region is not only of commercial interest but is also important biologically because axillary extracts can alter the length and timing of the female menstrual cycle. In males, the most abundant odor component is known to be E-3-methyl-2-hexenoic acid (E-3M2H), which is liberated from nonodorous apocrine secretions by axillary microorganisms. Recently, it was found that in the apocrine gland secretions, 3M2H is carried to the skin surface bound to two proteins, apocrine secretion odor-binding proteins 1 and 2 (ASOB1 and ASOB2) with apparent molecular masses of 45 kDa and 26 kDa, respectively. To better understand the formation of axillary odors and the structural relationship between 3M2H and its carrier protein, the amino acid sequence and glycosylation pattern of ASOB2 were determined by mass spectrometry. The ASOB2 protein was identified as apolipoprotein D (apoD), a known member of the alpha2mu-microglobulin superfamily of carrier proteins also known as lipocalins. The pattern of glycosylation for axillary apoD differs from that reported for plasma apoD, suggesting different sites of expression for the two glycoproteins. In situ hybridization of an oligonucleotide probe against apoD mRNA with axillary tissue demonstrates that the message for synthesis of this protein is specific to the apocrine glands. These results suggest a remarkable similarity between human axillary secretions and nonhuman mammalian odor sources, where lipocalins have been shown to carry the odoriferous signals used in pheromonal communication.
Alternative splicing of agrin regulates its binding to heparin alpha-dystroglycan, and the cell surface.
Authors: O'Toole J J , Deyst K A , Bowe M A , Nastuk M A , McKechnie B A , Fallon J R
Published: July 2002

Agrin is a basal lamina molecule that directs key events in postsynaptic differentiation, most notably the aggregation of acetylcholine receptors (AChRs) on the muscle cell surface. Agrin's AChR clustering activity is regulated by alternative mRNA splicing. Agrin splice forms having inserts at two sites (y and z) in the C-terminal region are highly active, but isoforms lacking these inserts are weakly active. The biochemical consequences of this alternative splicing are unknown. Here, the binding of four recombinant agrin isoforms to heparin, to alpha-dystroglycan (a component of an agrin receptor), and to myoblasts was tested. The presence of a four-amino acid insert at the y site is necessary and sufficient to confer heparin binding ability to agrin. Moreover, the binding of agrin to alpha-dystroglycan is inhibited by heparin when this insert is present. Agrin binding to the cell surface showed analogous properties: heparin inhibits the binding of only those agrin isoforms containing this four-amino acid insert. The results show that alternative splicing of agrin regulates its binding to heparin and suggest that agrin's interaction with alpha-dystroglycan may be modulated by cell surface glycosaminoglycans in an isoform-dependent manner.
Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.
Authors: Maegley K A , Admiraal S J , Herschlag D
Published: July 2002

Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach to this problem: the uncatalyzed hydrolysis of GTP is analyzed, and the understanding derived is applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, this analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl (-PO(3)2-) transfer, including kinases and phosphatases.
Regulation of herbivore growth by the balance of light and nutrients.
Authors: Urabe J , Sterner R W
Published: July 2002

Experiments using planktonic organisms revealed that the balance of radiant energy and available nutrients regulated herbivore growth rates through their effects on abundance and chemical composition of primary producers. Both algae and herbivores were energy limited at low light/nutrient ratios, but both were nutrient limited at high light/nutrient ratios. Herbivore growth increased with increasing light intensity at low values of the light/nutrient ratio due to increases in algal biomass, but growth decreased with increasing light at a high light/nutrient ratio due to decreases in algal quality. Herbivore production therefore was maximal at intermediate levels of the light/nutrient ratio. The results contribute to an understanding of mass transfer mechanisms in ecosystems and illustrate the importance of integration of energy-based and material-based currencies in ecology.
Locust adipokinetic hormones: carrier-independent transport and differential inactivation at physiological concentrations during rest and flight.
Authors: Oudejans R C , Vroemen S F , Jansen R F , Van der Horst D J
Published: July 2002

Since concomitant release of structurally related peptide hormones with apparently similar functions seems to be a general concept in endocrinology, we have studied the dynamics of the lifetime of the three known adipokinetic hormones (AKHs) of the migratory locust, which control flight-directed mobilization of carbohydrate and lipid from fat body stores. Although the structure of the first member of the AKHs has been known for 20 years, until now, reliable data on their inactivation and removal from the hemolymph are lacking, because measurement requires AKHs with high specific radioactivity. Employing tritiated AKHs with high specific radioactivity, obtained by catalytic reduction with tritium gas of the dehydroLeu2 analogues of the AKHs synthesized by the solid-phase procedure, studies with physiological doses of as low as 1.0 pmol per locust could be conducted. The AKHs appear to be transported in the hemolymph in their free forms and not associated with a carrier protein, despite their strong hydrophobicity. Application of AKHs in their free form in in vivo and in vitro studies therefore now has been justified. We have studied the degradation of the three AKHs during rest and flight. The first cleavage step by an endopeptidase is crucial, since the resulting degradation products lack any adipokinetic activity. Half-lives for AKH-I, -II and -III were 51, 40, and 5 min, respectively, for rest conditions and 35, 37, and 3 min, respectively, during flight. The rapid and differential degradation of structurally related hormones leads to changes in the ratio in which they are released and therefore will have important consequences for concerted hormone action at the level of the target organ or organs, suggesting that each of the known AKHs may play its own biological role in the overall syndrome of insect flight.
Subthreshold facilitation and suppression in primary visual cortex revealed by intrinsic signal imaging.
Authors: Toth L J , Rao S C , Kim D S , Somers D , Sur M
Published: July 2002

Neurons in primary visual cortex (area 17) respond vigorously to oriented stimuli within their receptive fields; however, stimuli presented outside the suprathreshold receptive field can also influence their responses. Here we describe a fundamental feature of the spatial interaction between suprathreshold center and subthreshold surround. By optical imaging of intrinsic signals in area 17 in response to a stimulus border, we show that a given stimulus generates activity primarily in iso-orientation domains, which extend for several millimeters across the cortical surface in a manner consistent with the architecture of long-range horizontal connections in area 17. By mapping the receptive fields of single neurons and imaging responses from the same cortex to stimuli that include or exclude the aggregate suprathreshold receptive field, we show that intrinsic signals strongly reveal the subthreshold surround contribution. Optical imaging and single-unit recording both demonstrate that the relative contrast of center and surround stimuli regulates whether surround interactions are facilitative or suppressive: the same surround stimulus facilitates responses when center contrast is low, but suppresses responses when center contrast is high. Such spatial interactions in area 17 are ideally suited to contribute to phenomena commonly regarded as part of "higher-level" visual processing, such as perceptual "popout" and "filling-in."
Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.
Authors: Poon P P , Wang X , Rotman M , Huber I , Cukierman E , Cassel D , Singer R A , Johnston G C
Published: July 2002

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.
Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum.
Authors: Bianchi D W , Zickwolf G K , Weil G J , Sylvester S , DeMaria M A
Published: July 2002

Rare nucleated fetal cells circulate within maternal blood. Noninvasive prenatal diagnosis by isolation and genetic analysis of these cells is currently being undertaken. We sought to determine if genetic evidence existed for persistent circulation of fetal cells from prior pregnancies. Venous blood samples were obtained from 32 pregnant women and 8 nonpregnant women who had given birth to males 6 months to 27 years earlier. Mononuclear cells were sorted by flow cytometry using antibodies to CD antigens 3, 4, 5, 19, 23, 34, and 38. DNA within sorted cells, amplified by PCR for Y chromosome sequences, was considered predictive of a male fetus or evidence of persistent male fetal cells. In the 32 pregnancies, male DNA was detected in 13 of 19 women carrying a male fetus. In 4 of 13 pregnancies with female fetuses, male DNA was also detected. All of the 4 women had prior pregnancies; 2 of the 4 had prior males and the other 2 had terminations of pregnancy. In 6 of the 8 nonpregnant women, male DNA was detected in CD34+CD38+ cells, even in a woman who had her last son 27 years prior to blood sampling. Our data demonstrate the continued maternal circulation of fetal CD34+ or CD34+CD38+ cells from a prior pregnancy. The prolonged persistence of fetal progenitor cells may represent a human analogue of the microchimerism described in the mouse and may have significance in development of tolerance of the fetus. Pregnancy may thus establish a long-term, low-grade chimeric state in the human female.
Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines.
Authors: Wischik C M , Edwards P C , Lai R Y , Roth M , Harrington C R
Published: July 2002

In Alzheimer disease (AD) the microtubule-associated protein tau is redistributed exponentially into paired helical filaments (PHFs) forming neurofibrillary tangles, which correlate with pyramidal cell destruction and dementia. Amorphous neuronal deposits and PHFs in AD are characterized by aggregation through the repeat domain and C-terminal truncation at Glu-391 by endogenous proteases. We show that a similar proteolytically stable complex can be generated in vitro following the self-aggregation of tau protein through a high-affinity binding site in the repeat domain. Once started, tau capture can be propagated by seeding the further accumulation of truncated tau in the presence of proteases. We have identified a nonneuroleptic phenothiazine previously used in man (methylene blue, MB), which reverses the proteolytic stability of protease-resistant PHFs by blocking the tau-tau binding interaction through the repeat domain. Although MB is inhibitory at a higher concentration than may be achieved clinically, the tau-tau binding assay was used to identify desmethyl derivatives of MB that have Ki values in the nanomolar range. Neuroleptic phenothiazines are inactive. Tau aggregation inhibitors do not affect the tau-tubulin interaction, which also occurs through the repeat domain. Our findings demonstrate that biologically selective pharmaceutical agents could be developed to facilitate the proteolytic degradation of tau aggregates and prevent the further propagation of tau capture in AD.
Illusory memories: A cognitive neuroscience analysis
Authors: Schacter Daniel L.
Published: July 2002

Memory illusions and distortions have long been of interest to
psychology researchers studying memory, but neuropsychologists and
neuroscientists have paid relatively little attention to them. This
article attempts to lay the foundation for a cognitive neuroscience
analysis of memory illusions and distortions by reviewing relevant
evidence from a patient with a right frontal lobe lesion, patients with
amnesia produced by damage to the medial temporal lobes, normal aging,
and healthy young volunteers studied with functional neuroimaging
techniques. Particular attention is paid to the contrasting roles of
prefrontal cortex and medial temporal lobe structures in accurate and
illusory remembering. Converging evidence suggests that the study of
illusory memories can provide a useful tool for delineating the brain
processes and systems involved in constructive aspects of remembering.
Intron of the mouse
Hoxa-7
gene contains conserved homeodomain binding sites that can functionas an enhancer element in
Drosophila
Authors: Haerry Theodor E. , Gehring Walter J.
Published: July 2002

The 5′ flanking sequences and the intron of the mouse
Hoxa-7
gene were searched for regulatory elements that
can function in
Drosophila
. Only the intron is able to
activate a
lacZ
fusion gene in various tissues of
Drosophila
embryos. This enhancer function requires a
cluster of three homeodomain binding sites (HB1-element) that are also
found in the introns of other
Hox
genes as well as in a
putative autoregulatory element of
Ultrabithorax
(
Ubx
), the
Drosophila
homolog of
Hoxa-7
. If a single binding site in the autoregulatory
element of
fushi tarazu
(
ftz
) is replaced
by the HB1-element of
Hoxa-7
, the expression pattern is
altered and newly controlled by the homeotic gene
caudal
(
cad
). These data suggest that HB1 is a potential target
for different homeodomain proteins of both vertebrates and
invertebrates.
Identification of a novel
selD
homolog from Eukaryotes, Bacteria, and Archaea: Is there an autoregulatory mechanism in selenocysteine metabolism?
Authors: Guimarães M. Jorge , Peterson David , Vicari Alain , Cocks Benjamin G. , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Ferrick David A. , Kastelein Robert A. , Bazan J. Fernando , Zlotnik Albert
Published: July 2002

Escherichia coli
selenophosphate synthetase (SPS, the
selD
gene product) catalyzes the production of
monoselenophosphate, the selenium donor compound required for synthesis
of selenocysteine (Sec) and seleno-tRNAs. We report the molecular
cloning of human and mouse homologs of the
selD
gene,
designated
Sps2
, which contains an in-frame TGA codon at a
site corresponding to the enzyme’s putative active site. These
sequences allow the identification of
selD
gene homologs in
the genomes of the bacterium
Haemophilus influenzae
and the archaeon
Methanococcus jannaschii
, which had been
previously misinterpreted due to their in-frame TGA codon.
Sps2
mRNA levels are elevated in organs previously
implicated in the synthesis of selenoproteins and in active sites of
blood cell development. In addition, we show that
Sps2
mRNA
is up-regulated upon activation of T lymphocytes and have mapped the
Sps2
gene to mouse chromosome 7. Using the mouse gene
isolated from the hematopoietic cell line FDCPmixA4, we devised a
construct for protein expression that results in the insertion of a
FLAG tag sequence at the N terminus of the SPS2 protein. This strategy
allowed us to document the readthrough of the in-frame TGA codon and
the incorporation of
75
Se into SPS2. These results suggest
the existence of an autoregulatory mechanism involving the
incorporation of Sec into SPS2 that might be relevant to blood cell
biology. This mechanism is likely to have been present in ancient life
forms and conserved in a variety of living organisms from all domains
of life.
Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles
Authors: Reiser Jakob , Harmison George , Kluepfel-Stahl Stephanie , Brady Roscoe O. , Karlsson Stefan , Schubert Manfred
Published: July 2002

The use of Moloney murine leukemia virus (Mo-MLV)-based vectors to deliver therapeutic genes into target cells is limited by their inability to transduce nondividing cells. To test the capacity of HIV-based vectors to deliver genes into nondividing cells, we have generated replication-defective HIV type 1 (HIV-1) reporter vectors carrying neomycin phosphotransferase or mouse heat stable antigen, replacing the HIV-1 sequences encoding gp160. These vectors also harbor inactivevpr,vpu, andnefcoding regions. Pseudotyped HIV-1 particles carrying either the ecotropic or the amphotropic Mo-MLV envelope proteins or the vesicular stomatitis virus G protein were released after single or double transfections of either human 293T or monkey COS-7 cells with titers of up to 107colony-forming units per milliliter. A simple ultrafiltration procedure resulted in an additional 10- to 20-fold concentration of the pseudotyped particles. These vectors along with Mo-MLV-based vectors were used to transduce primary human skin fibroblasts and human peripheral blood CD34+cells. The HIV-1 vector system was significantly more efficient than its Mo-MLV-based counterpart in transducing human skin fibroblasts arrested at the G0/G1stage of the cell cycle by density-dependent inhibition of growth. Human CD34+cells were transduced efficiently using HIV-1 pseudotype particles without prior stimulation with cytokines.
Midcycle administration of a progesterone synthesis inhibitor prevents ovulation in primates.
Authors: Hibbert M L , Stouffer R L , Wolf D P , Zelinski-Wooten M B
Published: July 2002

Progesterone receptors appear in granuloma cells of preovulatory follicles after the midcycle gonadotropin surge, suggesting important local actions of progesterone during ovulation in primates. Steroid reduction and replacement during the gonadotropin surge in macaques was used to evaluate the role of progesterone in the ovulatory process. Animals received gonadotropins to induce development of multiple preovulatory follicles, followed by human chorionic gonadotropin (hCG) administration (day 0) to promote oocyte (nuclear) maturation, ovulation, and follicular luteinization. On days 0-2, animals received no further treatment; a steroid synthesis inhibitor, trilostane (TRL); TRL + R5020; or TRL + dihydrotestosterone propionate (DHT). On day 3, ovulation was confirmed by counting ovulation sites and collecting oviductal oocytes. The meiotic status of oviductal and remaining follicular oocytes was evaluated. Peak serum estradiol levels, the total number of large follicles, and baseline serum progesterone levels at the time of hCG administration were similar in all animals. Ovulation sites and oviductal oocytes were routinely observed in controls. Ovulation was abolished in TRL. Progestin, but not androgen, replacement restored ovulation. Relative to controls, progesterone production was impaired for the first 6 days post-hCG in TRL, TRL + R5020, and TRL + DHT. Thereafter, progesterone remained low in TRL but recovered to control levels with progestin and androgen replacement. Similar percentages of mature (metaphase II) oocytes were collected among groups. Thus, steroid reduction during the gonadotropin surge inhibited ovulation and luteinization, but not reinitiation of oocyte meiotic maturation, in the primate follicle. The data are consistent with a local receptor-mediated role for progesterone in the ovulatory process.
Posttranslational amino acid epimerization: enzyme-catalyzed isomerization of amino acid residues in peptide chains.
Authors: Heck S D , Faraci W S , Kelbaugh P R , Saccomano N A , Thadeio P F , Volkmann R A
Published: July 2002

Since ribosomally mediated protein biosynthesis is confined to the L-amino acid pool, the presence of D-amino acids in peptides was considered for many years to be restricted to proteins of prokaryotic origin. Unicellular microorganisms have been responsible for the generation of a host of D-amino acid-containing peptide antibiotics (gramicidin, actinomycin, bacitracin, polymyxins). Recently, a series of mu and delta opioid receptor agonists [dermorphins and deltorphins] and neuroactive tetrapeptides containing a D-amino acid residue have been isolated from amphibian (frog) skin and mollusks. Amino acid sequences obtained from the cDNA libraries coincide with the observed dermorphin and deltorphin sequences, suggesting a stereospecific posttranslational amino acid isomerization of unknown mechanism. A cofactor-independent serine isomerase found in the venom of the Agelenopsis aperta spider provides the first major clue to explain how multicellular organisms are capable of incorporating single D-amino acid residues into these and other eukaryotic peptides. The enzyme is capable of isomerizing serine, cysteine, O-methylserine, and alanine residues in the middle of peptide chains, thereby providing a biochemical capability that, until now, had not been observed. Both D- and L-amino acid residues are susceptible to isomerization. The substrates share a common Leu-Xaa-Phe-Ala recognition site. Early in the reaction sequence, solvent-derived deuterium resides solely with the epimerized product (not substrate) in isomerizations carried out in 2H2O. Significant deuterium isotope effects are obtained in these reactions in addition to isomerizations of isotopically labeled substrates (2H at the epimerizeable serine alpha-carbon atom). The combined kinetic and structural data suggests a two-base mechanism in which abstraction of a proton from one face is concomitant with delivery from the opposite face by the conjugate acid of the second enzymic base.
Concerted repression of an immunoglobulin heavy-chain enhancer, 3' alpha E(hs1,2).
Authors: Singh M , Birshtein B K
Published: July 2002

The transcription factor, B-cell-specific activator protein (BSAP), represses the murine immunoglobulin heavy-chain 3' enhancer 3' alpha E(hs1,2) in B cells. Analysis of various 3'alpha E deletional constructs indicates that sequences flanking a and b BSAP-binding sites are essential for appropriate regulation of the enhancer. An octamer motif 5' of the a site and a specific G-rich motif 3' of the b site were identified by competition in electrophoretic mobility-shift assays and methylation-interference foot-printing analysis. Site-directed mutagenesis of either the octamer or G-rich sites resulted in the complete release of repression of 3' alpha E(hs1,2), implicating these two motifs in the repression of this enhancer in B cells. However, when both BSAP-binding sites were mutated, the octamer and G-rich motifs functioned as activators. Moreover, in plasma cells, when BSAP is not expressed, 3' alpha E(hs1,2) is active, and its activity depends on the presence of the other two factors. These results suggest that in B cells, 3' alpha E (hs1,2) is down-regulated by the concerted actions of BSAP, octamer, and G-rich DNA-binding proteins. Supporting this notion of concerted repression, a physical interaction between BSAP and octamer-binding proteins was demonstrated using glutathione S-transferase fusion proteins. Thus, concerted repression of 3' alpha E (hs1,2) in B cells provides a sensitive mechanism by which this enhancer, either individually or as part of a locus-controlling region, is highly responsive to any of several participating factors.
A σ
32
mutant with a single amino acid change in the highly conserved region 2.2 exhibits reduced core RNA polymerase affinity
Authors: Joo Daniel M. , Ng Norman , Calendar Richard
Published: July 2002

σ
32
, the product of the
rpoH
gene in
Escherichia coli
, provides promoter specificity by interacting with core RNAP. Amino acid sequence alignment of σ
32
with other sigma factors in the σ
70
family has revealed regions of sequence homology. We have investigated the function of the most highly conserved region, 2.2, using purified products of various
rpoH
alleles. Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant σ
32
proteins, Q80R. This reduced core interaction is exacerbated in the presence of σ
70
, which competes with σ
32
for binding of core RNAP. When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity. Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction.
Mapmodulin: A possible modulator of the interaction of microtubule-associated proteins with microtubules
Authors: Ulitzur Nirit , Humbert Martine , Pfeffer Suzanne R.
Published: July 2002

We have purified and characterized a 31-kDa protein named mapmodulin that binds to the microtubule-associated proteins (MAPs) MAP2, MAP4, and tau. Mapmodulin binds free MAPs in strong preference to microtubule-associated MAPs, and appears to do so via the MAP’s tubulin-binding domain. Mapmodulin inhibits the initial rate of MAP2 binding to microtubules, a property that may allow mapmodulin to displace MAPs from the path of organelles translocating along microtubules. In support of this possibility, mapmodulin stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact CHO cells. To our knowledge, mapmodulin represents the first example of a protein that can bind and potentially regulate multiple MAP proteins.
Overexpression of angiotensin AT
1
receptor transgene in the mouse myocardium produces a lethal phenotype associated with myocyte hyperplasia and heart block
Authors: Hein Lutz , Stevens Mary E. , Barsh Gregory S. , Pratt Richard E. , Kobilka Brian K. , Dzau Victor J.
Published: July 2002

Previous studies have suggested that angiotensin II (Ang II) modulates cardiac contractility, rhythm, metabolism, and structure. However, it is unclear whether the cardiac effects are due to direct actions of Ang II on the myocardium or if they are due to secondary effects mediated through the hemodynamic actions of Ang II. In this study, we used the α-myosin heavy chain (αMHC) promoter to generate transgenic mice overexpressing angiotensin II type 1 (AT
1a
) receptor selectively in cardiac myocytes. The specificity of transgene expression in the transgenic offspring was confirmed by radioligand binding studies and reverse transcription–PCR. The offspring displayed massive atrial enlargement with myocyte hyperplasia at birth, developed significant bradycardia with heart block, and died within the first weeks after birth. Thus, direct activation of AT
1
receptor signaling in cardiac myocytes
in vivo
is sufficient to induce cardiac myocyte growth and alter electrical conduction.
Quantitative neuropathology by high resolution magic angle spinning proton magnetic resonance spectroscopy
Authors: Cheng L. L. , Ma M. J. , Becerra L. , Ptak T. , Tracey I. , Lackner A. , González R. G.
Published: July 2002

We describe a method that directly relates tissue neuropathological analysis to medical imaging. Presently, only indirect and often tenuous relationships are made between imaging (such as MRI or x-ray computed tomography) and neuropathology. We present a biochemistry-based, quantitative neuropathological method that can help to precisely quantify information provided by
in vivo
proton magnetic resonance spectroscopy (
1
HMRS), an emerging medical imaging technique. This method, high resolution magic angle spinning (HRMAS)
1
HMRS, is rapid and requires only small amounts of unprocessed samples. Unlike chemical extraction or other forms of tissue processing, this method analyzes tissue directly, thus minimizing artifacts. We demonstrate the utility of this method by assessing neuronal damage using multiple tissue samples from differently affected brain regions in a case of Pick disease, a human neurodegenerative disorder. Among different regions, we found an excellent correlation between neuronal loss shown by traditional neurohistopathology and decrease of the neuronal marker
N
-acetylaspartate measured by HRMAS
1
HMRS. This result demonstrates for the first time, to our knowledge, a direct, quantitative link between a decrease in
N
-acetylaspartate and neuronal loss in a human neurodegenerative disease. As a quantitative method, HRMAS
1
HMRS has potential applications in experimental and clinical neuropathologic investigations. It should also provide a rational basis for the interpretation of
in vivo
1
HMRS studies of human neurological disorders.
Telomeres in the mouse have large inter-chromosomal variations in the number of T
2
AG
3
repeats
Authors: Zijlmans J. Mark J. M. , Martens Uwe M. , Poon Steven S. S. , Raap Anton K. , Tanke Hans J. , Ward Rabab K. , Lansdorp Peter M.
Published: July 2002

The ultra-long telomeres that have been observed in mice are not in accordance with the concept that critical telomere shortening is related to aging and immortalization. Here, we have used quantitative fluorescence
in situ
hybridization to estimate (T
2
AG
3
)
n
lengths of individual telomeres in various mouse strains. Telomere lengths were very heterogeneous, but specific chromosomes of bone marrow cells and skin fibroblasts from individual mice had similar telomere lengths. We estimate that the shortest telomeres are around 10 kb in length, indicating that each mouse cell has a few telomeres with (T
2
AG
3
)
n
lengths within the range of human telomeres. These short telomeres may be critical in limiting the replicative potential of murine cells.
Divergent and conserved features in the spatial expression of the
Drosophila pseudoobscura esterase-5
B
gene and the
esterase-6
gene of
Drosophila melanogaster
Authors: Tamarina Natalia A. , Ludwig Michael Z. , Richmond Rollin C.
Published: July 2002

The regulatory regions of homologous genes encoding
esterase
6
(
Est-6
) of
Drosophila melanogaster and
esterase 5B
(
Est-5B
) of
Drosophila
pseudoobscura
show very little similarity. We have undertaken a
comparative study of the pattern of expression directed by the
Est-5B
and
Est-6
5′-flanking DNA to
attempt to reveal conserved elements regulating tissue-specific
expression in adults. Esterase regulatory sequences were linked to a
lacZ
reporter gene and transformed into
D.
melanogaster
embryos.
Est-5B
, 5′ upstream
elements, give rise to a β-galactosidase expression pattern that
coincides with the wild-type expression of
Est-5B
in
D. pseudoobscura
. The expression patterns of the
Est-5B/lacZ
construct are different from those of a
fusion gene containing the upstream region of
Est-6
.
Common sites of expression for both kinds of constructs are the third
segment of antenna, the maxillary palps, and salivary glands.
In
vitro
deletion mutagenesis has shown that the two genes have a
different organization of regulatory elements controlling expression in
both the third segment of antenna and maxillary palps. The results
suggest that the conservation of the expression pattern in genes that
evolved from a common ancestor may not be accompanied by preservation
of the corresponding cis-regulatory elements.
Subtraction hybridization identifies a transformation progression-associated gene
PEG
-3 with sequence homology to a growth arrest and DNA damage-inducible gene
Authors: Su Zao-Zhong , Shi Yijie , Fisher Paul B.
Published: July 2002

Cancer is a progressive multigenic disorder characterized by defined changes in the transformed phenotype that culminates in metastatic disease. Determining the molecular basis of progression should lead to new opportunities for improved diagnostic and therapeutic modalities. Through the use of subtraction hybridization, a gene associated with transformation progression in virus- and oncogene-transformed rat embryo cells, progression elevated gene-3 (PEG-3), has been cloned.
PEG-3
shares significant nucleotide and amino acid sequence homology with the hamster growth arrest and DNA damage-inducible gene
gadd34
and a homologous murine gene,
MyD116
, that is induced during induction of terminal differentiation by interleukin-6 in murine myeloid leukemia cells.
PEG-3
expression is elevated in rodent cells displaying a progressed-transformed phenotype and in rodent cells transformed by various oncogenes, including Ha-
ras
, v-
src
, mutant type 5 adenovirus (Ad5), and human papilloma virus type 18. The
PEG-3
gene is transcriptionally activated in rodent cells, as is
gadd34
and
MyD116
, after treatment with DNA damaging agents, including methyl methanesulfonate and γ-irradiation. In contrast, only
PEG-3
is transcriptionally active in rodent cells displaying a progressed phenotype. Although transfection of
PEG-3
into normal and Ad5-transformed cells only marginally suppresses colony formation, stable overexpression of
PEG-3
in Ad5-transformed rat embryo cells elicits the progression phenotype. These results indicate that
PEG-3
is a new member of the
gadd
and
MyD
gene family with similar yet distinct properties and this gene may directly contribute to the transformation progression phenotype. Moreover, these studies support the hypothesis that constitutive expression of a DNA damage response may mediate cancer progression.
Evidence for rapid disappearance of initially expanded HIV-specific CD8+T cell clones during primary HIV infection
Authors: Pantaleo Giuseppe , Soudeyns Hugo , Demarest James F. , Vaccarezza Mauro , Graziosi Cecilia , Paolucci Stefania , Daucher MaryBeth , Cohen Oren J. , Denis François , Biddison William E. , Sekaly Rafick P. , Fauci Anthony S.
Published: July 2002

Down-regulation of the initial burst of viremia during primary HIV infection is thought to be mediated predominantly by HIV-specific cytotoxic T lymphocytes, and the appearance of this response is associated with major perturbations of the T cell receptor repertoire. Changes in the T cell receptor repertoire of virus-specific cytotoxic T lymphocytes were analyzed in patients with primary infection to understand the failure of the cellular immune response to control viral spread and replication. This analysis demonstrated that a significant number of HIV-specific T cell clones involved in the primary immune response rapidly disappeared. The disappearance was not the result of mutations in the virus epitopes recognized by these clones. Evidence is provided that phenomena such as high-dose tolerance or clonal exhaustion might be involved in the disappearance of these monoclonally expanded HIV-specific cytotoxic T cell clones. These findings should provide insights into how HIV, and possibly other viruses, elude the host immune response during primary infection.