bluuebunny/crossref_metadata_2025 huggingface dataset:
Zinc plays a key role in human and bacterial GTP cyclohydrolase I
Authors: Auerbach Günter , Herrmann Anja , Bracher Andreas , Bader Gerd , Gütlich Markus , Fischer Markus , Neukamm Martin , Garrido-Franco Marta , Richardson John , Nar Herbert , Huber Robert , Bacher Adelbert
Published: July 2002

The crystal structure of recombinant human GTP cyclohydrolase
I was solved by Patterson search methods by using the coordinates of
the
Escherichia coli
enzyme as a model. The human as
well as bacterial enzyme were shown to contain an essential zinc ion
coordinated to a His side chain and two thiol groups in each active
site of the homodecameric enzymes that had escaped detection during
earlier studies of the
E. coli
enzyme. The zinc ion is
proposed to generate a hydroxyl nucleophile for attack of imidazole
ring carbon atom eight of the substrate, GTP. It may also be involved
in the hydrolytic release of formate from the intermediate,
2-amino-5-formylamino-6-ribosylamino-4(3
H
)-pyrimidinone
5′-triphosphate, and in the consecutive Amadori rearrangement of the
ribosyl moiety.
Induction of platelet formation from megakaryocytoid cells by nitric oxide
Authors: Battinelli Elisabeth , Willoughby Scott R. , Foxall Thomas , Valeri C. Robert , Loscalzo Joseph
Published: July 2002

Although the growth factors that regulate
megakaryocytopoiesis are well known, the molecular determinants of
platelet formation from mature megakaryocytes remain poorly understood.
Morphological changes in megakaryocytes associated with platelet
formation and removal of senescent megakaryocytes are suggestive of an
apoptotic process. Previously, we have established that nitric
oxide (NO) can induce apoptosis in megakaryocytoid cell lines.
To determine whether there is an association between NO-induced
apoptosis and platelet production, we exposed Meg-01 cells to
S
-nitrosoglutathione (GSNO) with or without
thrombopoeitin (TPO) pretreatment and used flow cytometry and electron
microscopy to assess platelet-sized particle formation. Meg-01 cells
treated with TPO alone produced few platelet-sized particles (<3% of
total counts), whereas treatment with GSNO alone produced a significant
percentage of platelet-sized particles (22 ± 4% of total
counts); when combined with TPO pretreatment, however, GSNO led to a
marked increase in platelet-sized particle production (48 ± 3%
of total counts). Electron microscopy confirmed that Meg-01 cells
treated with TPO and GSNO yielded platelet-sized particles with
morphological features specific for platelet forms. The platelet-sized
particle population appears to be functional, because addition of
calcium, fibrinogen, and thrombin receptor-activating peptide led to
aggregation. These results demonstrate that NO facilitates platelet
production, thereby establishing the essential role of NO in
megakaryocyte development and thrombopoiesis.
Parathyroid hormone regulates 25-hydroxyvitamin D
3
-24-hydroxylase mRNA by altering its stability
Authors: Zierold Claudia , Mings Jamie A. , DeLuca Hector F.
Published: July 2002

The up-regulation of the 25-hydroxyvitamin D
3
-24-hydroxylase by 1,25-dihydroxyvitamin D
3
[1,25(OH)
2
D
3
] is well established and occurs at the transcriptional level through two vitamin D response elements in the promoter of the gene. However, the mechanism of down-regulation of the 24-hydroxylase by parathyroid hormone (PTH) has not yet been elucidated. To study the mechanism of PTH action, we used AOK-B50 cells, a porcine kidney-cell line with stably transfected opossum PTH receptor in which both the 24-hydroxylase mRNA and activity are down-regulated by PTH. Cells dosed with 1,25(OH)
2
D
3
at 0 h, and subsequently at 0, 1, 2, or 4 h with 100 nM of PTH, showed levels of 24-hydroxylase mRNA equivalent to 72.6, 65.3, 57.2, and 37.1%, respectively, of the levels found in cells dosed with 1,25(OH)
2
D
3
only. All cells were collected 7 h after the initial 1,25(OH)
2
D
3
dose. This pattern of expression indicated that PTH does not act by repressing transcription but rather by making the mRNA for 24-hydroxylase susceptible to degradation. At least 1 h is required for PTH to act. Further RNA and protein syntheses are required for PTH to act. However, the sites and mechanism whereby PTH causes 24-hydroxylase mRNA degradation are unknown. Because the untranslated regions of genes can determine the stability of its transcripts, we studied the 5′ untranslated region and the 3′ untranslated region of the rat 24-hydroxylase gene by using reporter-gene strategy to identify possible PTH sites of action. None was found, suggesting that the destabilization site is elsewhere in the coding region.
31P NMR magnetization transfer study of the control of ATP turnover in Saccharomyces cerevisiae.
Authors: Sheldon J G , Williams S P , Fulton A M , Brindle K M
Published: July 2002

31P NMR magnetization transfer measurements have been used to measure the steady state flux between Pi and ATP in yeast cells genetically modified to overexpress an adenine nucleotide translocase isoform. An increase in Pi -> ATP flux and apparent ratio of moles of ATP synthesized/atoms of oxygen consumed (P:O ratio), when these cells were incubated with glucose, demonstrated that the reactions catalyzed by the translocase and F1F0 ATP synthase were readily reversible in vivo. However, when the same cells were incubated with ethanol alone, translocase overexpression had no effect on the measured Pi -> ATP flux or apparent P:O ratio, suggesting that the synthase was now operating irreversibly. This change was accompanied by an increase in the intracellular ADP concentration. These observations are consistent with a model proposed for the kinetic control of mitochondrial ATP synthesis, which was based on isotope exchange measurements with isolated mammalian mitochondria [LaNoue, K. F., Jeffries, F. M. H. & Radda, G. K. (1986) Biochemistry 25, 7667-7675].
GABAergic feedforward projections from the inferior colliculus to the medial geniculate body.
Authors: Winer J A , Saint Marie R L , Larue D T , Oliver D L
Published: July 2002

A novel and robust projection from gamma-aminobutyric acid-containing (GABAergic) inferior colliculus neurons to the media] geniculate body (MGB) was discovered in the cat using axoplasmic transport methods combined with immunocytochemistry. This input travels with the classical inferior colliculus projection to the MGB, and it is a direct ascending GABAergic pathway to the sensory thalamus that may be inhibitory. This bilateral projection constitutes 10-30% of the neurons in the auditory tectothalamic system. Studies by others have shown that comparable input to the corresponding thalamic visual or somesthetic nuclei is absent. This suggests that monosynaptic inhibition or disinhibition is a prominent feature in the MGB and that differences in neural circuitry distinguish it from its thalamic visual and somesthetic counterparts.
Human cytomegalovirus inhibits antigen presentation by a sequential multistep process.
Authors: Ahn K , Angulo A , Ghazal P , Peterson P A , Yang Y , Früh K
Published: July 2002

The human cytomegalovirus (HCMV) genomic unique short (US) region encodes a family of homologous genes essential for the inhibition of major histocompatibility complex (MHC) class I-mediated antigen presentation during viral infection. Here we show that US3, the only immediate early (IE) gene within the US region, encodes an endoplasmic reticulum-resident glycoprotein that prevents intracellular transport of MHC class I molecules. In contrast to the rapid degradation of newly synthesized MHC class I heavy chains mediated by the early gene product US11, we found that US3 retains stable MHC class I heterodimers in the endoplasmic reticulum that are loaded with peptides while retained in the ER. Consistent with the expression pattern of US3 and US11, MHC class I molecules are retained but not degraded during the IE period of infection. Our data identify the first nonregulatory role of an IE protein of HCMV and suggest that HCMV uses different T-cell escape strategies at different times during the infectious cycle.
Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation.
Authors: Wang Y , Roman R , Lidofsky S D , Fitz J G
Published: July 2002

Recovery of cell volume in response to osmotic stress is mediated in part by increases in the Cl- permeability of the plasma membrane. These studies evaluate the hypothesis that ATP release and autocrine stimulation of purinergic (P2) receptors couple increases in cell volume to opening of Cl- channels. In HTC rat hepatoma cells, swelling induced by hypotonic exposure increased membrane Cl- current density to 44.8 +/- 7.1 pA/pF at -80 mV. Both the rate of volume recovery and the increase in Cl- permeability were inhibited in the presence of the ATP hydrolase apyrase (3 units/ml) or by exposure to the P2 receptor blockers suramin and Reactive Blue 2 (10-100 microM). Cell swelling also stimulated release of ATP. Hypotonic exposure increased the concentration of ATP in the effluent of perfused cells by 170 +/- 36 nM in the presence of a nucleotidase inhibitor (P < 0.01). In whole-cell recordings with ATP as the charge carrier, cell swelling increased membrane current density approximately 30-fold to 16.5 +/- 10.4 pA/pF. These findings indicate that increases in cell volume lead to efflux of ATP through opening of a conductive pathway consistent with a channel, and that extracellular ATP is required for recovery from swelling. ATP may function as an autocrine factor that couples increases in cell volume to opening of Cl- channels through stimulation of P2 receptors.
Long-term change in the organization of inventive activity
Authors: Lamoreaux Naomi R. , Sokoloff Kenneth L.
Published: July 2002

Relying on a quantitative analysis of the patenting and assignment behavior of inventors, we highlight the evolution of institutions that encouraged trade in technology and a growing division of labor between those who invented new technologies and those who exploited them commercially over the nineteenth and early-twentieth centuries. At the heart of this change in the organization of inventive activity was a set of familiar developments which had significant consequences for the supply and demand of inventions. On the supply side, the growing complexity and capital intensity of technology raised the amount of human and physical capital required for effective invention, making it increasingly desirable for individuals involved in this activity to specialize. On the demand side, the growing competitiveness of product markets induced firms to purchase or otherwise obtain the rights to technologies developed by others. These increasing incentives to differentiate the task of invention from that of commercializing new technologies depended for their realization upon the development of markets and other types of organizational supports for trade in technology. The evidence suggests that the necessary institutions evolved first in those regions of the country where early patenting activity had already been concentrated. A self-reinforcing process whereby high rates of inventive activity encouraged the evolution of a market for technology, which in turn encouraged greater specialization and productivity at invention as individuals found it increasingly feasible to sell and license their discoveries, appears to have been operating. This market trade in technological information was an important contributor to the achievement of a high level of specialization at invention well before the rise of large-scale research laboratories in the twentieth century.
Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR
Authors: Walter Nils G. , Schwille Petra , Eigen Manfred
Published: July 2002

A sensitive, labor-saving, and easily automatable
nonradioactive procedure named APEX-FCS (amplified probe extension
detected by fluorescence correlation spectroscopy) has been established
to detect specific
in vitro
amplification of pathogen
genomic sequences. As an example,
Mycobacterium
tuberculosis
genomic DNA was subjected to PCR amplification
with the Stoffel fragment of
Thermus aquaticus
DNA
polymerase in the presence of nanomolar concentrations of a
rhodamine-labeled probe (third primer), binding to the target in
between the micromolar amplification primers. The probe becomes
extended only when specific amplification occurs. Its low concentration
avoids false-positives due to unspecific hybridization under PCR
conditions. With increasing portion of extended probe molecules, the
probe’s average translational diffusion properties gradually change
over the course of the reaction, reflecting amplification kinetics.
Following PCR, this change from a stage of high to a stage of low
mobility can directly be monitored during a 30-s measurement using a
fluorescence correlation spectroscopy device. Quantitation down to 10
target molecules in a background of 2.5 μg unspecific DNA without
post-PCR probe manipulations could be achieved with different
primer/probe combinations. The assay holds the promise to
concurrently perform amplification, probe hybridization, and specific
detection without opening the reaction chamber, if sealable foils are
used.
Coexpression of
Drosophila
TRP and TRP-like proteins in
Xenopus
oocytes reconstitutes capacitative Ca
2+
entry
Authors: Gillo B. , Chorna I. , Cohen H. , Cook B. , Manistersky I. , Chorev M. , Arnon A. , Pollock J. A. , Selinger Z. , Minke B.
Published: July 2002

Capacitative Ca
2+
entry is a component of the
inositol-lipid signaling in which depletion of inositol
1,4,5-trisphosphate (Ins
P
3
)-sensitive
Ca
2+
stores activates Ca
2+
influx by a
mechanism that is still unknown. This pathway plays a central role in
cellular signaling, which is mediated by many hormones,
neurotransmitters, and growth factors. Studies of
Drosophila
photoreceptors provided the first putative
capacitative Ca
2+
entry mutant designated transient
receptor potential (
trp
) and a
Drosophila
gene encoding TRP-like protein (
trpl
). It is not clear
how the Ca
2+
store depletion signal is relayed to the
plasma membrane and whether both TRP and TRPL participate in this
process. We report here that coexpressing
Drosophila
TRP
and TRPL in
Xenopus
oocytes synergistically enhances the
endogenous Ca
2+
-activated Cl
−
current and produces a divalent inward current. Both of these currents
are activated by Ca
2+
store depletion. In the absence of
Ca
2+
, Mg
2+
is the main charge carrier of the
divalent current. This current is characterized by lanthanum
sensitivity and a voltage-dependent blocking effect of
Mg
2+
, which is relieved at both hyperpolarizing (inward
rectification) and depolarizing (outward rectification) potentials. The
store-operated divalent current is neither observed in native oocytes
nor in oocytes expressing either TRP or TRPL alone. The production of
this current implicates a cooperative action of TRP and TRPL in the
depletion-activated current.
Lipidic cubic phases: A novel concept for the crystallization of membrane proteins
Authors: Landau Ehud M. , Rosenbusch Jürg P.
Published: July 2002

Understanding the mechanisms of action of membrane proteins
requires the elucidation of their structures to high resolution. The
critical step in accomplishing this by x-ray crystallography is the
routine availability of well-ordered three-dimensional crystals. We
have devised a novel, rational approach to meet this goal using
quasisolid lipidic cubic phases. This membrane system, consisting of
lipid, water, and protein in appropriate proportions, forms a
structured, transparent, and complex three-dimensional lipidic array,
which is pervaded by an intercommunicating aqueous channel system. Such
matrices provide nucleation sites (“seeding”) and support growth
by lateral diffusion of protein molecules in the membrane
(“feeding”). Bacteriorhodopsin crystals were
obtained from bicontinuous cubic phases, but not from micellar systems,
implying a critical role of the continuity of the diffusion space (the
bilayer) on crystal growth. Hexagonal
bacteriorhodopsin crystals diffracted to 3.7
Å resolution, with a space group P6
3
, and unit cell
dimensions of
a
=
b
= 62 Å,
c
=
108 Å; α = β = 90° and γ = 120°.
Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: Relevance to diseases of the nervous system
Authors: Kahlem P. , Terré C. , Green H. , Djian P.
Published: July 2002

Many proteins contain reiterated glutamine residues, but
polyglutamine of excessive length may result in human disease by
conferring new properties on the protein containing it. One established
property of a glutamine residue, depending on the nature of the
flanking residues, is its ability to act as an amine acceptor in a
transglutaminase-catalyzed reaction and to make a glutamyl–lysine
cross-link with a neighboring polypeptide. To learn whether glutamine
repeats can act as amine acceptors, we have made peptides with variable
lengths of polyglutamine flanked by the adjacent amino acid residues in
the proteins associated with spinocerebellar ataxia type 1 (SCA1),
Machado–Joseph disease (SCA3), or dentato-rubral
pallido-luysian atrophy (DRPLA) or those residues adjacent to the
preferred cross-linking site of involucrin, or solely by arginine
residues. The polyglutamine was found to confer excellent substrate
properties on any soluble peptide; under optimal conditions, virtually
all the glutamine residues acted as amine acceptors in the reaction
with glycine ethyl-ester, and lengthening the sequence of polyglutamine
increased the reactivity of each glutamine residue. In the presence of
transglutaminase, peptides containing polyglutamine formed insoluble
aggregates with the proteins of brain extracts and these aggregates
contained glutamyl–lysine cross-links. Repeated glutamine residues
exposed on the surface of a neuronal protein should form cross-linked
aggregates in the presence of any transglutaminase activated by the
presence of Ca
2+
.
Functional reorganization in thalamocortical networks: Transition between spindling and delta sleep rhythms
Authors: Terman D. , Bose A. , Kopell N.
Published: July 2002

Thalamic reticularis, thalamocortical, and cortical cells
participate in the 7–14-hz spindling rhythm of early sleep and the
slower delta rhythms of deeper sleep, with different firing patterns.
In this case study, showing the interactions of intrinsic and synaptic
properties, a change in the conductance of one kind of cell effectively
rewires the thalamocortical circuit, leading to the transition from the
spindling to the delta rhythm. The two rhythms make different uses of
the fast (GABA
A
) and slow (GABA
B
) inhibition
generated by the thalamic reticularis cells.
Molecular cloning and characterization of SCaMPER, a sphingolipid Ca2+ release-mediating protein from endoplasmic reticulum.
Authors: Mao C , Kim S H , Almenoff J S , Rudner X L , Kearney D M , Kindman L A
Published: July 2002

Release of Ca2+ stored in endoplasmic reticulum is a ubiquitous mechanism involved in cellular signal transduction, proliferation, and apoptosis. Recently, sphingolipid metabolites have been recognized as mediators of intracellular Ca2+ release, through their action at a previously undescribed intracellular Ca2+ channel. Here we describe the molecular cloning and characterization of a protein that causes the expression of sphingosyl-phosphocholine-mediated Ca2+ release when its complementary RNA is injected into Xenopus oocytes. SCaMPER (for sphingolipid Ca2+ release-mediating protein of endoplasmic reticulum) is an 181 amino acid protein with two putative membrane-spanning domains. SCaMPER is incorporated into microsomes upon expression in SO cells or after translation in vitro. It mediates Ca2+ release at 4 degrees C as well as 22 degrees C, consistent with having ion channel function. The EC50 for Ca2+ release from Xenopus oocytes is 40 microM, similar to sphingosyl-phosphocholine-mediated Ca2+ release from permeabilized mammalian cells. Because Ca2+ release is not blocked by ryanodine or La3+, the activity described here is distinct from the Ca2+ release activity of the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. The properties of SCaMPER are identical to those of the sphingolipid-gated Ca2+ channel that we have previously described. These findings suggest that SCaMPER is a sphingolipid-gated Ca2+-permeable channel and support its role as a mediator of this pathway for intracellular Ca2+ signal transduction.
Multiplex genotype determination at a large number of gene loci.
Authors: Lin Z , Cui X , Li H
Published: July 2002

To facilitate large-scale genotype analysis, an efficient PCR-based multiplex approach has been developed. For simultaneously amplifying the target sequences at a large number of genetic loci, locus-specific primers containing 5' universal tails are used. Attaching the universal tails to the target sequences in the initial PCR steps allows replacement of all specific primers with a pair of primers identical to the universal tails and converts the multiplex amplification into "uniplex." Simultaneous amplification of 26 genetic loci with this approach is described. The multiplex amplification can be coupled with genotype determination. By incorporating a single-base mismatch between a primer and the template into the target sequences, a polymorphic site can be converted into a desirable restriction fragment length polymorphism when it is necessary. In this way, the allelic PCR products for the polymorphic loci can be discriminated by gel electrophoresis after restriction enzyme digestion. In this study, 32 loci were typed in such a multiplex way.
Increased mRNA levels for components of the lysosomal, Ca2+-activated, and ATP-ubiquitin-dependent proteolytic pathways in skeletal muscle from head trauma patients.
Authors: Mansoor O , Beaufrere B , Boirie Y , Ralliere C , Taillandier D , Aurousseau E , Schoeffler P , Arnal M , Attaix D
Published: July 2002

The cellular mechanisms responsible for enhanced muscle protein breakdown in hospitalized patients, which frequently results in lean body wasting, are unknown. To determine whether the lysosomal, Ca2+-activated, and ubiquitin-proteasome proteolytic pathways are activated, we measured mRNA levels for components of these processes in muscle biopsies from severe head trauma patients. These patients exhibited negative nitrogen balance and increased rates of whole-body protein breakdown (assessed by [13C]leucine infusion) and of myofibrillar protein breakdown (assessed by 3-methylhistidine urinary excretion). Increased muscle mRNA levels for cathepsin D, m-calpain, and critical components of the ubiquitin proteolytic pathway (i.e., ubiquitin, the 14-kDa ubiquitin-conjugating enzyme E2, and proteasome subunits) paralleled these metabolic adaptations. The data clearly support a role for multiple proteolytic processes in increased muscle proteolysis. The ubiquitin proteolytic pathway could be activated by altered glucocorticoid production and/or increased circulating levels of interleukin 1beta and interleukin 6 observed in head trauma patients and account for the breakdown of myofibrillar proteins, as was recently reported in animal studies.
Macrophage inflammatory protein-2: Chromosomal regulation in rat small intestinal epithelial cells
Authors: Ohno Yasuhiro , Lee Jenny , Fusunyan Robert D. , MacDermott Richard P. , Sanderson Ian R.
Published: July 2002

Nonpathogenic, resident bacteria participate in the pathogenesis of inflammation in the small intestine, but the molecular messages produced by such bacteria are unknown. Inflammatory responses involve the recruitment of specific leukocyte subsets. We, therefore, hypothesized that butyrate, a normal bacterial metabolite, may modulate chemokine secretion by epithelial cells, by amplifying their response to proinflammatory signals. We studied the expression of the chemokine, macrophage inflammatory protein-2 (MIP-2) by the rat small intestinal epithelial cell line, IEC-6. Cells were stimulated with lipopolysaccharide or with interleukin 1β (IL-1β) and incubated with sodium butyrate. Acetylation of histones was examined in Triton X acetic acid–urea gels by PAGE. Unstimulated IEC-6 cells did not secrete MIP-2. However, lipopolysaccharide and IL-1β induced MIP-2 expression. Butyrate enhanced MIP-2 secretion both in lipopolysaccharide-stimulated and IL-1β-stimulated enterocytes; but butyrate alone did not induce MIP-2 expression. Butyrate increased the acetylation of histones extracted from the nuclei of IEC-6 cells. Furthermore, acetylation of histones (induced by trichostatin A, a specific inhibitor of histone deacetylase) enhanced MIP-2 expression by cells stimulated with IL-1β. In conclusion, trichostatin A reproduced the effects of butyrate on MIP-2 secretion. Butyrate, therefore, increases MIP-2 secretion in stimulated cells by increasing histone acetylation. We speculate that butyrate carries information from bacteria to epithelial cells. Epithelial cells transduce this signal through histone deacetylase, modulating the secretion of chemokines.
The selectivity of the hair cell’s mechanoelectrical-transduction channel promotes Ca
2+
flux at low Ca
2+
concentrations
Authors: Lumpkin Ellen A. , Marquis Robert E. , Hudspeth A. J.
Published: July 2002

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca
2+
entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel’s open probability, an understanding of adaptation requires estimation of the magnitude of Ca
2+
influx. To determine the Ca
2+
current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K
+
, Na
+
, or Ca
2+
. For modest concentrations of a single permeant cation, Ca
2+
carried much more receptor current than did either K
+
or Na
+
. For higher cation concentrations, however, the flux of Na
+
or K
+
through the transduction channel exceeded that of Ca
2+
. For mixtures of Ca
2+
and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel’s pore. These results demonstrate not only that the hair cell’s transduction channel is selective for Ca
2+
over monovalent cations but also that Ca
2+
carries substantial current even at low Ca
2+
concentrations. At physiological cation concentrations, Ca
2+
flux through transduction channels can change the local Ca
2+
concentration in stereocilia in a range relevant for the control of adaptation.
Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases
Authors: Varghese Joseph N. , Colman Peter M. , van Donkelaar Albertus , Blick Tony J. , Sahasrabudhe Anjali , McKimm-Breschkin Jennifer L.
Published: July 2002

The x-ray structure of a complex of sialic acid (Neu5Ac) with neuraminidase N9 subtype from A/tern/Australia/G70C/75 influenza virus at 4°C has revealed the location of a second Neu5Ac binding site on the surface of the enzyme. At 18°C, only the enzyme active site contains bound Neu5Ac. Neu5Ac binds in the second site in the chair conformation in a similar way to which it binds to hemagglutinin. The residues that interact with Neu5Ac at this second site are mostly conserved in avian strains, but not in human and swine strains, indicating that it has some as-yet-unknown biological function in birds.
Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing
Authors: Yang Yi , Yang Yan , Smith Harold C.
Published: July 2002

Apolipoprotein B (apoB) mRNA editing catalyzed by
apoB
mRNA
e
diting
c
atalytic subunit
1
(APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin-(HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunofluorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1–56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS/NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.
Recovery of pituitary function after treatment with a targeted cytotoxic analog of luteinizing hormone-releasing hormone
Authors: Kovacs Magdolna , Schally Andrew V. , Nagy Attila , Koppan Miklos , Groot Kate
Published: July 2002

Recently, we developed a targeted cytotoxic analog AN-207 of luteinizing hormone-releasing hormone (LH-RH), consisting of an intensely potent derivative of doxorubicin, 2-pyrrolinodoxorubicin (AN-201) conjugated to carrier agonist [
d
-Lys
6
]LH-RH. In this study, we investigated the effects of cytotoxic analog AN-207, designed for targeted chemotherapy and radical AN-201 on pituitary function in rats. A selective damage to the pituitary gonadotroph cells was found at 1 week after a single i.v. injection of 150 nmol/kg AN-207, as evidenced by a 63% decrease in the LH-RH-stimulated release of LH
in vitro
. The release of growth hormone (GH) and thyrotropin (TSH), stimulated by GH-releasing hormone (GH-RH) and TSH-releasing hormone (TRH), respectively, was reduced by only 11–12%. In contrast, even a smaller dose of 75 nmol/kg of AN-201 nonselectively damaged pituitary function, reducing the stimulated release of LH, GH, and TSH by 57%, 74%, and 67%, respectively. Two weeks after administration, the LH-RH-stimulated LH release
in vivo
entirely normalized in the AN-207-treated rats, and only a 13% decrease in the LH response was found in the group given AN-201. GH and TSH responses to receptor-mediated stimuli with GH-RH and TRH were normal at 2 weeks in both treated groups. Neither cytotoxic compound caused changes in the concentration of pituitary LH, GH, or TSH, as determined by RIA at 1 week and 7 weeks after treatment. This study demonstrates that the cytotoxic LH-RH analog AN-207 exerts highly selective effects on the gonadotroph cells containing LH-RH receptors and is less toxic for other cells. Conversely, its cytotoxic radical AN-201 nonselectively damages the pituitary cells. The damaging effect of both cytotoxic compounds on pituitary functions is reversible. In view of its high selectivity and reduced toxicity, AN-207 could be a potential therapeutic agent for the treatment of tumors that possess receptors for LH-RH such as prostatic, mammary, ovarian, and endometrial cancers.
Feedback inhibition of Ca
2+
channels by Ca
2+
depends on a short sequence of the C terminus that does not include the Ca
2+
-binding function of a motif with similarity to Ca
2+
-binding domains
Authors: Zhou Jianming , Olcese Riccardo , Qin Ning , Noceti Francesca , Birnbaumer Lutz , Stefani Enrico
Published: July 2002

α
1C
- and α
1E
-based Ca
2+
channels differ in that the former are inhibited by Ca
2+
entering through its pore, while the latter are not. It has been proposed on the basis of analysis of α
1E
/α
1C
chimeras that the molecular determinants responsible for Ca
2+
inhibition involve both a conserved Ca
2+
-binding motif (EF hand) plus additional sequences located C-terminal to the EF hand. Through construction of similar α
1E
/α
1C
chimeras, we transferred Ca
2+
inhibition from α
1C
to α
1E
by replacing a 134-aa segment of α
1E
with the homologous 142-aa segment of α
1C
. This segment is located immediately after the proposed Ca
2+
-binding EF hand motif. Replacement of the α
1C
EF hand with the corresponding EF hand of α
1E
did not interfere with inhibition of α
1C
by Ca
2+
, and a triple mutant of α
1C
, α
1C
[D1535A,E1537A,D1546A], that disrupts the potential Ca
2+
-coordinating ability of the EF hand continued to be inhibited by Ca
2+
. These results indicate that a small portion of the α
1C
C terminus is essential for inhibition by Ca
2+
and place the Ca
2+
-binding site anywhere in α
1C
, with the exception of its EF hand-like motif.
UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y
2
receptor
Authors: Lazarowski Eduardo R. , Paradiso Anthony M. , Watt William C. , Harden T. Kendall , Boucher Richard C.
Published: July 2002

The presence of the P2Y
2
(P
2U
-purinergic) receptor on the apical surface of airway tissue raises the possibility that aerosolized UTP might be used therapeutically to induce Cl
−
secretion in individuals with cystic fibrosis. However, the duration of the effects of UTP may be limited by enzymatic degradation. We therefore have analyzed the metabolism of UTP and its metabolite UDP on polarized human nasal epithelium (HNE), and have compared the pharmacological activities of these two uridine nucleotides. HPLC analysis of medium bathing the mucosal surface of HNE cells revealed the presence of an ecto-nucleotidase(s) that hydrolyzed [
3
H]UTP and [
3
H]UDP with
t
½
values (at 1 μM nucleotide) of 14 and 27 min, respectively. An ecto-nucleoside diphosphokinase activity also was observed, which promoted conversion of [
3
H]UDP into [
3
H]UTP in the presence of ATP. The effects of UDP on [
3
H]inositol phosphate accumulation, intracellular calcium levels ([Ca
2+
]
i
), and Cl
−
secretory rates (I

Cl
−

) were quantitated in HNE cells in the presence of hexokinase and glucose to ensure that no UTP (or ATP) contaminated UDP solutions during the assays. Although UDP does not activate the human P2Y
2
receptor, mucosal addition of UDP promoted [
3
H]inositol phosphate accumulation with an EC
50
of 190 nM. Mucosal addition of UTP stimulated [
3
H]inositol phosphate accumulation with an EC
50
of 280 nM. The maximal effects of mucosal UDP on [
3
H]inositol phosphate, [Ca
2+
]
i
, and I

Cl
−

responses were approximately one-half of those observed with mucosal UTP. Serosal application of UTP promoted a 50% greater [
3
H]inositol phosphate and calcium response than did mucosal application of UTP. In contrast, UDP had no effect when added to the serosal medium. Repetitive mucosal applications of UDP to HNE cells resulted in a progressive loss, i.e., desensitization, of the [Ca
2+
]
i
and I

Cl
−

response to UDP, whereas the corresponding responses to UTP remained unchanged. Our results provide evidence for the existence of a UDP receptor on HNE cells that is pharmacologically distinct from the P2Y
2
receptor. The relative stability of UDP on the airway surface and the apparent predominant mucosal expression of this putative UDP receptor make it a potential target for cystic fibrosis treatment.
Mutations of γ-aminobutyric acid and glycine receptors change alcohol cutoff: Evidence for an alcohol receptor?
Authors: Wick Marilee J. , Mihic S. John , Ueno Susumu , Mascia Maria Paola , Trudell James R. , Brozowski Susan J. , Ye Qing , Harrison Neil L. , Harris R. Adron
Published: July 2002

Alcohols in the homologous series of
n
-alcohols increase in central nervous system depressant potency with increasing chain length until a “cutoff” is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the α subunit. We now demonstrate that these residues in the glycine α1 and the γ-aminobutyric acid ρ1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine α1 receptor, whereas mutation of Ile-307 and/or Trp-328 in the γ-aminobutyric acid ρ1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.
The role of transmembrane domain 2 in cation transport by the Na–K–Cl cotransporter
Authors: Isenring Paul , Jacoby Steven C. , Forbush Bliss
Published: July 2002

The human and shark Na–K–Cl cotransporters (NKCC), although 74% identical in amino acid sequence, exhibit marked differences in ion transport and bumetanide binding. We have utilized shark–human chimeras of NKCC1 to search for regions that confer the kinetic differences. Two chimeras (hs3.1 and its reverse sh3.1) with a junction point located at the beginning of the third transmembrane domain were examined after stable transfection in HEK-293 cells. Each carried out bumetanide-sensitive
86
Rb influx with cation affinities intermediate between shark and human cotransporters. In conjunction with the previous finding that the N and C termini are not responsible for differences in ion transport, the current observations identify the second transmembrane domain as playing an important role. Site-specific mutagenesis of two pairs of residues in this domain revealed that one pair is indeed involved in the difference in Na affinity, and a second pair is involved in the difference in Rb affinity. Substitution of the same residues with corresponding residues from NKCC2 or the Na-Cl cotransporter resulted in cation affinity changes, consistent with the hypothesis that alternative splicing of transmembrane domain 2 endows different versions of NKCC2 with unique kinetic behaviors. None of the changes in transmembrane domain 2 was found to substantially affect
K
m(Cl)
, demonstrating that the affinity difference for Cl is specified by the region beyond predicted transmembrane domain 3. Finally, unlike Cl, bumetanide binding was strongly affected by shark–human replacement of transmembrane domain 2, indicating that the bumetanide-binding site is not the same as the Cl-binding site.
Arachidonic acid mediates angiotensin II effects on p21ras in renal proximal tubular cells via the tyrosine kinase-Shc-Grb2-Sos pathway
Authors: Jiao Huaiyuan , Cui Xiao-Lan , Torti Mauro , Chang Chung-Ho , Alexander Larry D. , Lapetina Eduardo G. , Douglas Janice G.
Published: July 2002

In kidney epithelial cells, an angiotensin II (Ang II) type 2 receptor subtype (AT
2
) is linked to a membrane-associated phospholipase A
2
(PLA
2
) and the mitogen-activated protein kinase (MAPK) superfamily. However, the intervening steps in this linkage have not been determined. The aim of this study was to determine whether arachidonic acid mediates Ang II’s effect on p21ras and if so, to ascertain the signaling mechanism(s). We observed that Ang II activated p21ras and that mepacrine, a phospholipase A
2
inhibitor, blocked this effect. This activation was also inhibited by PD123319, an AT
2
receptor antagonist but not by losartan, an AT
1
receptor antagonist. Furthermore, Ang II caused rapid tyrosine phosphorylation of Shc and its association with Grb2. Arachidonic acid and linoleic acid mimicked Ang II-induced tyrosine phosphorylation of Shc and activation of p21ras. Moreover, Ang II and arachidonic acid induced an association between p21ras and Shc. We demonstrate that arachidonic acid mediates linkage of a G protein-coupled receptor to p21ras via Shc tyrosine phosphorylation and association with Grb2/Sos. These observations have important implications for other G protein-coupled receptors linked to a variety of phospholipases.
Nerve growth factor inhibits sympathetic neurons’ response to an injury cytokine
Authors: Shadiack Annette M. , Vaccariello Stacey A. , Sun Yi , Zigmond Richard E.
Published: July 2002

Axonal damage to adult peripheral neurons causes changes in neuronal gene expression. For example, axotomized sympathetic, sensory, and motor neurons begin to express galanin mRNA and protein, and recent evidence suggests that galanin plays a role in peripheral nerve regeneration. Previous studies in sympathetic and sensory neurons have established that galanin expression is triggered by two consequences of nerve transection: the induction of leukemia inhibitory factor (LIF) and the reduction in the availability of the target-derived factor, nerve growth factor. It is shown in the present study that no stimulation of galanin expression occurs following direct application of LIF to intact neurons in the superior cervical sympathetic ganglion. Injection of animals with an antiserum to nerve growth factor concomitant with the application of LIF, on the other hand, does stimulate galanin expression. The data suggest that the response of neurons to an injury factor, LIF, is affected by whether the neurons still receive trophic signals from their targets.
Cardiac G
sα
overexpression enhances L-type calcium channels through an adenylyl cyclase independent pathway
Authors: Lader Alan S. , Xiao Yong-Fu , Ishikawa Yoshihiro , Cui Yanning , Vatner Dorothy E. , Vatner Stephen F. , Homcy Charles J. , Cantiello Horacio F.
Published: July 2002

The α subunit of the stimulatory heterotrimeric G protein (G
sα
) is critical for the β-adrenergic receptor activation of the cAMP messenger system. The role of G
sα
in regulating cardiac Ca
2+
channel activity, however, remains controversial. Cultured neonatal cardiac myocytes from transgenic mice overexpressing cardiac G
sα
were used to assess the role of G
sα
on the whole-cell Ca
2+
currents (I
Ca
). Cardiac myocytes from transgenic mice had a 490% higher peak I
Ca
compared with those of either wild-type controls or G
sα
-nonexpressing littermates. The effect of G
sα
overexpression was mimicked by intracellular dialysis of wild-type cardiac myocytes with GTPγS-activated G
sα
. This effect was not mediated by protein kinase A activation as intracellular perfusion with a protein kinase A inhibitor rendered the same degree of activation in either transgenic or wild-type myocytes also dialyzed with activated G
sα
. The data indicate that G
sα
overexpression is associated with a constitutive enhancement of I
Ca
which is independent of the cAMP pathway and activation of endogenous adenylyl cyclase.
A unique pattern of photoreceptor degeneration in
cyclin D1
mutant mice
Authors: Ma Chaoyong , Papermaster David , Cepko Constance L.
Published: July 2002

Cyclin D1
-deficient mice have small eyes with thin retinas. We observed that there was a lower level of retinal cell proliferation and a unique pattern of photoreceptor cell death. Death was first observed in scattered clusters of cells in the retina. It then appeared to spread from these few cells to nearby photoreceptors, eventually producing extensive holes in the photoreceptor layer. These holes appeared to be filled with interneurons from the inner nuclear layer. The death mainly occurred during the second to fourth postnatal weeks. Other models of photoreceptor degeneration in rodents differ in that they occur more uniformly across the retina, with death proceeding over a longer period of time until all, or nearly all, of the photoreceptors degenerate. We also tested whether expression of a
bcl-2
transgene could prevent the death and found that it could not.
HIV type I envelope determinants for use of the CCR2b, CCR3, STRL33, and APJ coreceptors
Authors: Hoffman Trevor L. , Stephens Edward B. , Narayan Opendra , Doms Robert W.
Published: July 2002

The envelope (Env) proteins of primate lentiviruses interact sequentially with CD4 and a coreceptor to infect cells. Changes in coreceptor use strongly influence viral tropism and pathogenesis. We followed the evolution of coreceptor use in pig-tailed macaques that developed severe CD4 T-cell loss during the derivation of a pathogenic simian HIV (SHIV) that contained thetat,rev,vpu, andenvgenes of the HXBc2 strain of HIV-1 in a genetic background of SIVmac239. The Env from the parental virus as well as one derived from the first macaque to develop AIDS exclusively used CXCR4 as a coreceptor, indicating that CXCR4 can function as a coreceptor in macaques even though it is rarely used by simian immunodeficiency viruses. One Env (Pnb5), obtained from a macrophage-tropic virus isolated from the cerebral spinal fluid, did not use CCR5 or CXCR4. Instead, it used CCR2b and to a lesser extent CCR3, STRL33, and APJ to infect cells. Chimeras between Pnb5 and the parental X4 Env indicated that the V3 loop is the major determinant of CXCR4 use, with other regions of Env influencing the efficiency with which this coreceptor was used. In contrast, the Pnb5 V1/2 and V3 regions in combination were both necessary and sufficient to confer full use of CCR2b, CCR3, STRL33, and APJ to the parental X4 Env protein. These results are consistent with a single, conserved binding site in Env that interacts with multiple coreceptors in conjunction with the V1/2 and V3 loops, and suggest that the V1/2 region plays a more important role in governing the use of CCR2b, CCR3, STRL33, and APJ than for CXCR4.
Molecular cloning of a human cDNA encoding β-1,4-galactosyltransferase with 37% identity to mammalian UDP-Gal:GlcNAc β-1,4-galactosyltransferase
Authors: Sato Takeshi , Furukawa Kiyoshi , Bakker Hans , Van den Eijnden Dirk H. , Van Die Irma
Published: July 2002

A cDNA encoding a β-1,4-galactosyltransferase named β-1,4-GalT II was cloned from a cDNA library of the human breast tumor cell line, MRK-nu-1. Initially, a 860-bp PCR fragment was obtained from MRK-nu-1 mRNA by 3′-rapid amplification of cDNA ends by using two nested degenerate oligonucleotide primers based on a highly conserved amino acid sequence found in the catalytic domain of mammalian β-1,4-galactosyltransferases and
Lymnaea stagnalis
β-1,4-
N
-acetylglucosaminyltransferase (β-1,4-GlcNAcT), both of which utilize the same sugar acceptor. This subsequently was used as a probe to isolate a 4.7-kb cDNA that contained an ORF of 1,164 bp predicting a polypeptide of 388 aa. Its deduced amino acid sequence shows an identity of 37% with that of the previously characterized human β-1,4-galactosyltransferase (referred to as β-1,4-GalT I) and of 28% with that of
L. stagnalis
β-1,4-GlcNAcT. Study of the properties of the β-1,4-GalT II fused to protein A expressed as a soluble form in COS-7 cells revealed that it is a genuine β-1,4-GalT but has no lactose synthetase activity in the presence of α-lactalbumin. Northern blot analysis of 24 human tissues showed that they all express the β-1,4-GalT II transcript, although the levels varied. These results indicate that human cells contain another β-1,4-GalT.