bluuebunny/crossref_metadata_2025 huggingface dataset:
d
-serine and serine racemase are present in the vertebrate retina and contribute to the physiological activation of NMDA receptors
Authors: Stevens Eric R. , Esguerra Manuel , Kim Paul M. , Newman Eric A. , Snyder Solomon H. , Zahs Kathleen R. , Miller Robert F.
Published: May 2003

d
-serine has been proposed as an endogenous modulator of
N-
methyl-
d
-aspartate (NMDA) receptors in many brain regions, but its presence and function in the vertebrate retina have not been characterized. We have detected
d
-serine and its synthesizing enzyme, serine racemase, in the retinas of several vertebrate species, including salamanders, rats, and mice and have localized both constituents to Müller cells and astrocytes, the two major glial cell types in the retina. Physiological studies in rats and salamanders demonstrated that, in retinal ganglion cells,
d
-serine can enhance excitatory currents elicited by the application of NMDA, as well as the NMDA receptor component of light-evoked synaptic responses. Application of
d
-amino acid oxidase, which degrades
d
-serine, reduced the magnitude of NMDA receptor-mediated currents, raising the possibility that endogenous
d
-serine serves as a ligand for setting the sensitivity of NMDA receptors under physiological conditions. These observations raise exciting new questions about the role of glial cells in regulating the excitability of neurons through release of
d
-serine.
Spherical aggregates of β-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase-3β
Authors: Hoshi Minako , Sato Michio , Matsumoto Shinichiro , Noguchi Akihiko , Yasutake Kaori , Yoshida Natsuko , Sato Kazuki
Published: May 2003

β-Amyloid (Aβ) acquires toxicity by self-aggregation. To identify and characterize the toxic form(s) of Aβ aggregates, we examined
in vitro
aggregation conditions by using large quantities of homogenous, chemically synthesized Aβ
1–40
peptide. We found that slow rotation of Aβ
1–40
solution reproducibly gave self-aggregated Aβ
1–40
containing a stable and highly toxic moiety. Examination of the aggregates purified by glycerol-gradient centrifugation by atomic force microscopy and transmission electron microscopy revealed that the toxic moiety is a perfect sphere, which we call amylospheroid (ASPD). Other Aβ
1–40
aggregates, including fibrils, were nontoxic. Correlation studies between toxicity and sphere size indicate that 10- to 15-nm ASPD was highly toxic, whereas ASPD <10 nm was nontoxic. A positive correlation between the toxicity and ASPD >10 nm also appeared to exist when Aβ
1–42
formed ASPD by slow rotation. However, Aβ
1–42
-ASPD formed more rapidly, killed neurons at lower concentrations, and showed ≈100-fold-higher toxicity than Aβ
1–40
-ASPD. The toxic ASPD was associated with SDS-resistant oligomeric bands in immunoblotting, which were absent in nontoxic ASPD. Because the formation of ASPD was not disturbed by pentapeptides that break β-sheet interactions, Aβ may form ASPD through a pathway that is at least partly distinct from that of fibril formation. Inhibition experiments with lithium suggest the involvement of tau protein kinase I/glycogen synthase kinase-3β in the early stages of ASPD-induced neurodegeneration. Here we describe the identification and characterization of ASPD and discuss its possible role in the neurodegeneration in Alzheimer's disease.
Keratin 8 protection of placental barrier function
Authors: Jaquemar Daniel , Kupriyanov Sergey , Wankell Miriam , Avis Jacqueline , Benirschke Kurt , Baribault Hélène , Oshima Robert G.
Published: May 2003

The intermediate filament protein keratin 8 (K8) is critical for the development of most mouse embryos beyond midgestation. We find that 68% of K8−/− embryos, in a sensitive genetic background, are rescued from placental bleeding and subsequent death by cellular complementation with wild-type tetraploid extraembryonic cells. This indicates that the primary defect responsible for K8−/− lethality is trophoblast giant cell layer failure. Furthermore, the genetic absence of maternal but not paternal TNF doubles the number of viable K8−/− embryos. Finally, we show that K8−/− concepti are more sensitive to a TNF-dependent epithelial apoptosis induced by the administration of concanavalin A (ConA) to pregnant mothers. The ConA-induced failure of the trophoblast giant cell barrier results in hematoma formation between the trophoblast giant cell layer and the embryonic yolk sac in a phenocopy of dying K8-deficient concepti in a sensitive genetic background. We conclude the lethality of K8−/− embryos is due to a TNF-sensitive failure of trophoblast giant cell barrier function. The keratin-dependent protection of trophoblast giant cells from a maternal TNF-dependent apoptotic challenge may be a key function of simple epithelial keratins.
Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins
Authors: Tomita Susumu , Chen Lu , Kawasaki Yoshimi , Petralia Ronald S. , Wenthold Robert J. , Nicoll Roger A. , Bredt David S.
Published: May 2003

Functional expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in cerebellar granule cells requires stargazin, a member of a large family of four-pass transmembrane proteins. Here, we define a family of transmembrane AMPA receptor regulatory proteins (TARPs), which comprise stargazin, γ-3, γ-4, and γ-8, but not related proteins, that mediate surface expression of AMPA receptors. TARPs exhibit discrete and complementary patterns of expression in both neurons and glia in the developing and mature central nervous system. In brain regions that express multiple isoforms, such as cerebral cortex, TARP–AMPA receptor complexes are strictly segregated, suggesting distinct roles for TARP isoforms. TARPs interact with AMPA receptors at the postsynaptic density, and surface expression of mature AMPA receptors requires a TARP. These studies indicate a general role for TARPs in controlling synaptic AMPA receptors throughout the central nervous system.
αvβ3 integrin expression up-regulates cdc2, which modulates cell migration
Authors: Manes Thomas , Zheng Duo-Qi , Tognin Simona , Woodard Amy S. , Marchisio Pier Carlo , Languino Lucia R.
Published: May 2003

The αvβ3 integrin has been shown to promote cell migration through activation of intracellular signaling pathways. We describe here a novel pathway that modulates cell migration and that is activated by αvβ3 and, as downstream effector, by cdc2 (cdk1). We report that αvβ3 expression in LNCaP (β3-LNCaP) prostate cancer cells causes increased cdc2 mRNA levels as evaluated by gene expression analysis, and increased cdc2 protein and kinase activity levels. We provide three lines of evidence that increased levels of cdc2 contribute to a motile phenotype on integrin ligands in different cell types. First, increased levels of cdc2 correlate with more motile phenotypes of cancer cells. Second, ectopic expression of cdc2 increases cell migration, whereas expression of dominant-negative cdc2 inhibits migration. Third, cdc2 inhibitors reduce cell migration without affecting cell adhesion. We also show that cdc2 increases cell migration via specific association with cyclin B2, and we unravel a novel pathway of cell motility that involves, downstream of cdc2, caldesmon. cdc2 and caldesmon are shown here to localize in membrane ruffles in motile cells. These results show that cdc2 is a downstream effector of the αvβ3 integrin, and that it promotes cell migration.
Is HP1 an RNA detector that functions both in repression and activation?
Authors: Kellum Rebecca
Published: May 2003

Heterochromatin is defined as regions of compact chromatin that persist throughout the cell cycle (Heitz, 1928). The earliest cytological observations of heterochromatin were followed by ribonucleotide labeling experiments that showed it to be transcriptionally inert relative to the more typical euchromatic regions that decondense during interphase. Genetic studies of rearrangements that place euchromatic genes next to blocks of heterochromatin also pointed out the repressive nature of heterochromatin (Grigliatti, 1991; and references therein). The discovery of the heterochromatin-enriched protein heterochromatin protein 1 (HP1)**Abbreviation used in this paper: HP1, heterochromatin protein 1. by Elgin and co-workers in the mid-1980s suggested that the distinct cytological features of this chromatin may be related to its unique nucleoprotein composition (James and Elgin, 1986; James et al., 1989). HP1 immunostaining on polytene chromosomes from Drosophila larval salivary glands was used to show enrichment of the protein in pericentric heterochromatin. Since that initial discovery, HP1 homologues have been found in species ranging from fission yeast to humans where it is associated with gene silencing (Eissenberg and Elgin, 2000; and references therein). A number of euchromatic sites of localization were also reported in this original study. It has been generally assumed that these sites might constitute euchromatic sites of transcriptional repression by HP1. Indeed, several genes located at one of these sites (cytological region 31) have increased transcript levels in mutants for HP1 (Hwang et al., 2001).
A Cysteine Scan of the Inner Vestibule of Cyclic Nucleotide–gated Channels Reveals Architecture and Rearrangement of the Pore
Authors: Flynn Galen E. , Zagotta William N.
Published: May 2003

Cyclic nucleotide–gated (CNG) channels belong to the P-loop–containing family of ion channels that also includes KcsA, MthK, and Shaker channels. In this study, we investigated the structure and rearrangement of the CNGA1 channel pore using cysteine mutations and cysteine-specific modification. We constructed 16 mutant channels, each one containing a cysteine mutation at one of the positions between 384 and 399 in the S6 region of the pore. By measuring currents activated by saturating concentrations of the full agonist cGMP and the partial agonists cIMP and cAMP, we show that mutating S6 residues to cysteine caused both favorable and unfavorable changes in the free energy of channel opening. The time course of cysteine modification with 2-aminoethylmethane thiosulfonate hydrochloride (MTSEA) was complex. For many positions we observed decreases in current activated by cGMP and concomitant increases in current activated by cIMP and cAMP. A model where modification affected both gating and permeation successfully reproduced the complex time course of modification for most of the mutant channels. From the model fits to the time course of modification for each mutant channel, we quantified the following: (a) the bimolecular rate constant of modification in the open state, (b) the change in conductance, and (c) the change in the free energy of channel opening for modification of each cysteine. At many S6 cysteines, modification by MTSEA caused a decrease in conductance and a favorable change in the free energy of channel opening. Our results are interpreted within the structural framework of the known structures of KcsA and MthK. We conclude that: (a) MTSEA modification affects both gating and permeation, (b) the open configuration of the pore of CNGA1 channels is consistent with the structure of MthK, and (c) the modification of S6 residues disrupts the helical packing of the closed channel, making it easier for channels to open.
Mating Kleinian groups isomorphic to 𝐶₂∗𝐶₅ with quadratic polynomials
Authors: Freiberger Marianne
Published: May 2003

Given a quadratic polynomial

q
:

C

^

→

C

^

q:\hat {\mathbb {C}}\rightarrow \hat {\mathbb {C}}

and a representation

G
:

C

^

→

C

^

G:\hat {\mathbb {C}} \rightarrow \hat {\mathbb {C}}

of

C
2

∗

C
5

C_2\ast C_5

in

P
S
L
(
2
,

C

)

PSL(2,\mathbb {C})

satisfying certain conditions, we will construct a

4
:
4

4:4

holomorphic correspondence on the sphere (given by a polynomial relation

p
(
z
,
w
)

p(z,w)

) that mates the two actions: The sphere will be partitioned into two completely invariant sets

Ω
\Omega

and

Λ
\Lambda

. The set

Λ
\Lambda

consists of the disjoint union of two sets,

Λ
+

\Lambda _+

and

Λ
−

\Lambda _-

, each of which is conformally homeomorphic to the filled Julia set of a degree 4 polynomial

P
P

. This filled Julia set contains infinitely many copies of the filled Julia set of

q
q

. Suitable restrictions of the correspondence are conformally conjugate to

P
P

on each of

Λ
+

\Lambda _+

and

Λ
−

\Lambda _-

. The set

Λ
\Lambda

will not be connected, but it can be joined up using a family

C

\mathcal {C}

of completely invariant curves. The action of the correspondence on the complement of

Λ
∪

C

\Lambda \cup \mathcal {C}

will then be conformally conjugate to the action of

G
G

on a simply connected subset of its regular set.
Regulation of hyaluronan secretion into rabbit synovial joints in vivo by protein kinase C
Authors: Anggiansah C. L. , Scott D. , Poli A. , Coleman P. J. , Badrick E. , Mason R. M. , Levick J. R.
Published: May 2003

Hyaluronan (HA) is important for joint cavitation, lubrication, volume regulation and synovial fluid drainage but little is known about the regulation of joint HA synthesis/secretion in vivo. We investigated whether HA secretion into joints in vivo can be regulated by protein kinase C (PKC). Secretion into the knee joint cavity of anaesthetised rabbits was measured over 6 h by washout and chromatography. Joints received intra‐articular injections of Ringer vehicle (control) or an activator of classical PKC isoforms, phorbol‐12‐myristate‐13‐acetate (PMA), at 20–2000 ng ml−1. The effects of PKC inhibition by bisindolylmaleimide (BIM) and protein synthesis inhibition by cycloheximide (CX) on basal and stimulated HA secretion were also studied. The endogenous HA mass, 181 ± 8 μg (n= 26, mean ±s.e.m.), and basal secretion rate, 4.4 ± 0.4 μg h−1, indicated a turnover time of 41 h. Secretion rate showed a dose‐dependent response to PMA (n= 30), rising 5‐fold to 21.7 ± 5.0 μg h−1 (n= 5) at 2000 ng ml−1 PMA (P < 0.0001, one‐way ANOVA). PMA‐induced stimulation was partially suppressed by CX (HA secretion: 5.8 ± 1.7 μg h−1, n= 8, P < 0.01) and totally blocked by BIM (HA secretion: 3.2 ± 0.6 μg h−1, n= 9, P < 0.001). Basal HA secretion was unaffected by CX over 6 h (4.2 ± 0.7 μg h−1, n= 8) but was reduced by 29 % by BIM (3.1 ± 0.6 μg h−1, n= 10, P= 0.03). It is concluded that: (1) PKC can stimulate HA secretion into joints in vivo through mechanisms involving protein synthesis de novo as well as phosphorylation; (2) basal HA secretion is only partially PKC dependent; and (3) hyaluronan synthase turnover time is > 6 h in vivo, which is slower than in vitro (< 2–3 h).
Static and dynamic changes in carotid artery diameter in humans during and after strenuous exercise
Authors: Studinger Péter , Lénárd Zsuzsanna , Kováts Zsuzsanna , Kocsis László , Kollai Mark
Published: May 2003

Arterial baroreflex function is altered by dynamic exercise, but it is not clear to what extent baroreflex changes are due to altered transduction of pressure into deformation of the barosensory vessel wall. In this study we measured changes in mean common carotid artery diameter and the pulsatile pressure: diameter ratio (PDR) during and after dynamic exercise. Ten young, healthy subjects performed a graded exercise protocol to exhaustion on a bicycle ergometer. Carotid dimensions were measured with an ultrasound wall‐tracking system; central arterial pressure was measured with the use of radial tonometry and the generalized transfer function; baroreflex sensitivity (BRS) was assessed in the post‐exercise period by spectral analysis and the sequence method. Data are given as means ±s.e.m. Mean carotid artery diameter increased during exercise as compared with control levels, but carotid distension amplitude did not change. PDR was reduced from 27.3 ± 2.7 to 13.7 ± 1.0 μm mmHg−1. Immediately after stopping exercise, the carotid artery constricted and PDR remained reduced. At 60 min post‐exercise, the carotid artery dilated and the PDR increased above control levels (33.9 ± 1.4 μm mmHg−1). The post‐exercise changes in PDR were closely paralleled by those in BRS (0.74 ≤r≤ 0.83,P< 0.05). These changes in mean carotid diameter and PDR suggest that the mean baroreceptor activity level increases during exercise, with reduced dynamic sensitivity; at the end of exercise baroreceptors are suddenly unloaded, then at 1 h post‐exercise, baroreceptor activity increases again with increasing dynamic sensitivity. The close correlation between PDR and BRS observed at post‐exercise underlies the significance of mechanical factors in arterial baroreflex control.
Chylomicron components activate duodenal vagal afferents via a cholecystokinin A receptor‐mediated pathway to inhibit gastric motor function in the rat
Authors: Glatzle Jörg , Wang Yuhua , Adelson David W. , Kalogeris Theodore J. , Zittel Tilman T. , Tso Patrick , Wei Jen‐Yu , Raybould Helen E.
Published: May 2003

Nutrients in the intestine initiate changes in secretory and motor function of the gastrointestinal (GI) tract. The nature of the ‘sensors’ in the intestinal wall is not well characterized. Intestinal lipid stimulates the release of cholecystokinin (CCK) from mucosal entero‐endocrine cells, and it is proposed that CCK activates CCK A receptors on vagal afferent nerve terminals. There is evidence that chylomicron components are involved in this lipid transduction pathway. The aim of the present study was to determine (1) the pathway mediating reflex inhibition of gastric motility and (2) activation of duodenal vagal afferents in response to chylomicrons. Mesenteric lymph was obtained from awake rats fitted with lymph fistulas during intestinal perfusion of lipid (Intralipid, 170 μmol h−1, chylous lymph) or a dextrose and/or electrolyte solution (control lymph). Inhibition of gastric motility was measured manometrically in urethane‐anaesthetized recipient rats in response to intra‐arterial injection of lymph close to the upper GI tract. Chylous lymph was significantly more potent than control lymph in inhibiting gastric motility. Functional vagal deafferentation by perineural capsaicin or CCK A receptor antagonist (devazepide, 1 mg kg−1, I.V.) significantly reduced chylous lymph‐induced inhibition of gastric motility. The discharge of duodenal vagal afferent fibres was recorded from the dorsal abdominal vagus nerve in an in vitro preparation of the duodenum. Duodenal vagal afferent nerve fibre discharge was significantly increased by close‐arterial injection of CCK (1‐100 pmol) in 43 of 83 units tested. The discharge of 88 % of CCK‐responsive fibres was increased by close‐arterial injection of chylous lymph; devazepide (100 μg, I.A.) abolished the afferent response to chylous lymph in 83 % of these units. These data suggest that in the intestinal mucosa, chylomicrons or their products release endogenous CCK which activates CCK A receptors on vagal afferent nerve fibre terminals, which in turn initiate a vago‐vagal reflex inhibition of gastric motor function.
Pyridine nucleotide regulation of the KATP channel Kir6.2/SUR1 expressed in Xenopus oocytes
Authors: Dabrowski Michael , Trapp Stefan , Ashcroft Frances M.
Published: May 2003

The pancreatic β‐cell type of ATP‐sensitive potassium (KATP) channel (Kir6.2/SUR1) is inhibited by intracellular ATP and ADP, which bind to the Kir6.2 subunit, and is activated by Mg‐nucleotide interaction with the regulatory sulphonylurea receptor subunits (SUR1). The nicotinamide adenine dinucleotides NAD and NADP consist of an ADP molecule with a ribose group and a nicotinamide moiety attached to the terminal phosphate. Both these molecules block native KATP channels in pancreatic β‐cells at concentrations above 500 μM, and activate them at lower concentrations. We therefore investigated whether NAD and NADP interact with both Kir6.2 and SUR1 subunits of the KATP channel by comparing the potency of these agents on recombinant Kir6.2ΔC and Kir6.2/SUR1 channels expressed in Xenopus oocytes. Our results show that, at physiological concentrations, NAD and NADP interact with the nucleotide inhibitory site of Kir6.2 to inhibit Kir6.2/SUR1 currents. They may therefore contribute to the resting level of channel inhibition in the intact cell. Importantly, our data also reveal that this interaction is dependent on the presence of SUR1, which may act by increasing the width of the nucleotide‐binding pocket of Kir6.2.
Effect of charge on interstitial distribution of albumin in rat dermis in vitro
Authors: Wiig Helge , Kolmannskog Odd , Tenstad Olav , Bert Joel L.
Published: May 2003

At physiological pH, negatively charged glycosaminoglycans in the extracellular matrix may influence distribution volume of a probe. We hypothesized that by varying the probe charge we would be able to observe a graded response of available volume fraction. Human serum albumin (HSA) (isoelectric point (pI) 5.0) was made more positive by cationization. Using reaction times of 10, 45 and 60 min, cationized HSA (cHSA) with respective pIs of 6.5, 7.3 and 8.0 were made. After eight days of equilibration in a buffer containing labelled native HSA and cHSA, the distribution volumes were calculated relative to that of 51Cr‐EDTA, an extracellular tracer. The available volume in fully swollen dermis for native albumin relative to that of the extracellular tracer averaged 0.485 ± 0.008 (n= 49), with corresponding volumes for cHSA‐10 min, cHSA‐45 min and cHSA‐60 min of 0.554 ± 0.012 (n= 17), 0.647 ± 0.026 (n= 17) and 0.718 ± 0.021 (n= 12), respectively. Increasing the ionic strength of the bathing solution to 1 M NaCl, thereby screening the fixed charges of tissue elements and probes alike, resulted in similar available and thereby excluded volumes of native HSA and neutral cHSA‐45 min. These experiments suggest that fixed negative charges, most likely glycosaminoglycans, contribute significantly to interstitial exclusion of charged macromolecules, a phenomenon of importance for hydration of the interstitial fluid phase and therefore for body fluid balance. Moreover, the data indicate that previous findings of similar excluded volumes for the two differently sized major plasma proteins albumin (molecular mass 66 kDa) and IgG (molecular mass 160 kDa) may be explained by a more pronounced electrostatic repulsion of the former by the extracellular matrix.
Activation‐Dependent Properties of Pregnenolone Sulfate Inhibition of GABAA Receptor‐Mediated Current
Authors: Eisenman Lawrence N. , He Yejun , Fields Christopher , Zorumski Charles F. , Mennerick Steven
Published: May 2003

Sulfated steroids like pregnenolone sulfate (PS) are found endogenously in the central nervous system where they may modulate GABAA receptors. Understanding the mechanism of steroid inhibition is important for understanding the conditions under which endogenous steroids modulate GABAA receptor function, assessing their potential clinical utility, and for evaluating sulfated steroids as probes of receptor behaviour. Some previous studies suggest that sulfated steroid inhibition exhibits activation dependence, whilst other studies suggest only slow, time‐dependent inhibition, perhaps reflecting slow PS association with receptors. We tested activation dependence in several ways. Steroid potency increased 2‐ to 3‐fold with ≈10‐fold change in GABA concentration. PS inhibition of saturating partial agonist responses suggested that the level of channel activation, rather than receptor occupancy by agonist, is important for PS inhibition. Inhibition by sulfated steroids exhibited weak or no voltage dependence. Responses to rapid applications of exogenous GABA differed little whether PS was pre‐applied or simply co‐applied with GABA, consistent with the hypothesis that the actions of PS are facilitated by receptor activation. PS applied during steady‐state GABA responses exhibited slow onset and offset rate constants. The offset, rather than onset, was significantly slowed by elevated GABA concentration. At hippocampal synapses, large, multiquantal IPSCs were inhibited more effectively by a fixed concentration of PS than small quantal content IPSCs, consistent with known ‘pooling’ of transmitter following multiquantal release. Picrotoxinin, although superficially similar to PS in its activation dependence, was dissimilar from PS in a number of details. In summary, PS inhibition exhibits activation dependence that may be explained by activation‐dependent binding and altered desensitization.
Structural Basis for Distinctions between Substrate and Inhibitor Specificities for Feline Immunodeficiency Virus and Human Immunodeficiency Virus Proteases
Authors: Lin Ying-Chuan , Beck Zachary , Morris Garrett M. , Olson Arthur J. , Elder John H.
Published: May 2003

We used feline immunodeficiency virus (FIV) protease (PR) as a mutational framework to define determinants for the observed substrate and inhibitor specificity distinctions between FIV and human immunodeficiency virus (HIV) PRs. Multiple-substitution mutants were constructed by replacing the residues in and around the active site of FIV PR with the structurally equivalent residues of HIV-1 PR. Mutants included combinations of three critical regions (FIV numbering, with equivalent HIV numbering in superscript): I3732V in the active core region; N5546M, M5647I, and V5950I in the flap region; and L9780T, I9881P, Q9982V, P10083N, and L10184I in the 90s loop region. Significant alterations in specificity were observed, consistent with the involvement of these residues in determining the substrate-inhibitor specificity distinctions between FIV and HIV PRs. Two previously identified residues, I35 and I57 of FIV PR, were intolerant to substitution and yielded inactive PRs. Therefore, we attempted to recover the activity by introducing secondary mutations. The addition of G6253F and K6354I, located at the top of the flap and outside the active site, compensated for the activity lost in the I5748G substitution mutants. An additional two substitutions, D10588N and N8874T, facilitated recovery of activity in mutants that included the I3530D substitution. Determination ofKivalues of potent HIV-1 PR inhibitors against these mutants showed that inhibitor specificity paralleled that of HIV-1 PR. The findings indicate that maintenance of both substrate and inhibitor specificity is a function of interactions between residues both inside and outside the active site. Thus, mutations apparently peripheral to the active site can have a dramatic influence on inhibitor efficacy.
Genome Replication, Virion Secretion, and e Antigen Expression of Naturally Occurring Hepatitis B Virus Core Promoter Mutants
Authors: Parekh Sameer , Zoulim Fabien , Ahn Sang Hoon , Tsai Adrienne , Li Jisu , Kawai Shigenobu , Khan Nasser , Trépo Christian , Wands Jack , Tong Shuping
Published: May 2003

The core promoter mutants of hepatitis B virus (HBV) emerge as the dominant viral population at the late HBeAg and the anti-HBe stages of HBV infection, with the A1762T/G1764A substitutions as the hotspot mutations. The double core promoter mutations were found by many investigators to moderately enhance viral genome replication and reduce hepatitis B e antigen (HBeAg) expression. A much higher replication capacity was reported for a naturally occurring core promoter mutant implicated in the outbreak of fulminant hepatitis, which was caused by the neighboring C1766T/T1768A mutations instead. To systemically study the biological properties of naturally occurring core promoter mutants, we amplified full-length HBV genomes by PCR from sera of HBeAg
+
individuals infected with genotype A. All 12 HBV genomes derived from highly viremic sera (5 × 10
9
to 5.7 × 10
9
copies of viral genome/ml) harbored wild-type core promoter sequence, whereas 37 of 43 clones from low-viremia samples (0.2 × 10
7
to 4.6 × 10
7
copies/ml) were core promoter mutants. Of the 11 wild-type genomes and 14 core promoter mutants analyzed by transfection experiments in human hepatoma cell lines, 6 core promoter mutants but none of the wild-type genomes replicated at high levels. All had 1762/1764 mutations and an additional substitution at position 1753 (T to C), at position 1766 (C to T), or both. Moreover, these HBV clones varied greatly in their ability to secrete enveloped viral particles irrespective of the presence of core promoter mutations. High-replication clones with 1762/1764/1766 or 1753/1762/1764/1766 mutations expressed very low levels of HBeAg, whereas high-replication clones with 1753/1762/1764 triple mutations expressed high levels of HBeAg. Experiments with site-directed mutants revealed that both 1762/1764/1766 and 1753/1762/1764/1766 mutations conferred significantly higher viral replication and lower HBeAg expression than 1762/1764 mutations alone, whereas the 1753/1762/1764 triple mutant displayed only mild reduction in HBeAg expression similar to the 1762/1764 mutant. Thus, core promoter mutations other than those at positions 1762 and 1764 can have major impact on viral DNA replication and HBeAg expression.
Complete Sequence and Genomic Analysis of Rhesus Cytomegalovirus
Authors: Hansen Scott G. , Strelow Lisa I. , Franchi David C. , Anders David G. , Wong Scott W.
Published: May 2003

The complete DNA sequence of rhesus cytomegalovirus (RhCMV) strain 68-1 was determined with the whole-genome shotgun approach on virion DNA. The RhCMV genome is 221,459 bp in length and possesses a 49% G+C base composition. The genome contains 230 potential open reading frames (ORFs) of 100 or more codons that are arranged colinearly with counterparts of previously sequenced betaherpesviruses such as human cytomegalovirus (HCMV). Of the 230 RhCMV ORFs, 138 (60%) are homologous to known HCMV proteins. The conserved ORFs include the structural, replicative, and transcriptional regulatory proteins, immune evasion elements, G protein-coupled receptors, and immunoglobulin homologues. Interestingly, the RhCMV genome also contains sequences with homology to cyclooxygenase-2, an enzyme associated with inflammatory processes. Closer examination identified a series of candidate exons with the capacity to encode a full-length cyclooxygenase-2 protein. Counterparts of cyclooxygenase-2 have not been found in other sequenced herpesviruses. The availability of the complete RhCMV sequence along with the ability to grow RhCMV in vitro will facilitate the construction of recombinant viral strains for identifying viral determinants of CMV pathogenicity in the experimentally infected rhesus macaque and to the development of CMV as a vaccine vector.
Cytolysis by CCR5-Using Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Is Dependent on Membrane Fusion and Can Be Inhibited by High Levels of CD4 Expression
Authors: LaBonte Jason A. , Madani Navid , Sodroski Joseph
Published: May 2003

T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4+CXCR4+T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5+target cells. As is the case for CXCR4+target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4+CCR5+cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4+T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4+T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.
Varicella-Zoster Virus Gene 66 Transcription and Translation in Latently Infected Human Ganglia
Authors: Cohrs Randall J. , Gilden Donald H. , Kinchington Paul R. , Grinfeld Esther , Kennedy Peter G. E.
Published: May 2003

Latent infection with varicella-zoster virus (VZV) is characterized by restricted virus gene expression and the absence of virus production. Of the ∼70 predicted VZV genes, only five (genes 4, 21, 29, 62, and 63) have been shown by multiple techniques to be transcribed during latency. IE62, the protein product of VZV gene 62, is the major immediate-early (IE) virus-encoded transactivator of viral gene transcription and plays a pivotal role in transactivating viral genes during lytic infection. The protein kinase (66-pk) encoded by VZV gene 66 phosphorylates IE62, resulting in cytoplasmic accumulation of IE62 that mitigates nuclear IE62-induced gene activation. Analysis of latently infected human trigeminal ganglia for 66-pk expression by reverse transcriptase-dependent nested PCR, including DNA sequence analysis, in situ hybridization, and immunohistochemistry, revealed VZV open reading frame 66 to be a previously unrecognized latently expressed virus gene and suggests that prevention of IE62 import to the nucleus by VZV 66-pk phosphorylation is one possible mechanism by which VZV latency is maintained.
Replicating Parvoviruses That Target Colon Cancer Cells
Authors: Malerba M. , Daeffler L. , Rommelaere J. , Iggo R. D.
Published: May 2003

The wnt signaling pathway is constitutively activated in colon tumors by mutations in the adenomatous polyposis coli and β-catenin genes. We have modified the minute virus of mice (MVM) P4 promoter to make it responsive to wnt signaling by inserting binding sites for the heterodimeric β-catenin/Tcf transcription factor. In luciferase assays we can see up to 20-fold selectivity of Tcf mutant P4 promoters for cells with activated wnt signaling. Hybrid MVM/H-1 viruses containing Tcf mutant promoters were tested for NS1 expression, viral DNA replication, virus replication, and cytopathic effect on colon, lung, kidney, and cervical cancer cell lines. Activation of the wnt pathway by expression of ΔN-β-catenin increased NS1 expression and viral burst size in 293T and H1299 lung cancer cells, showing that the Tcf mutant P4 promoter can respond to wnt signals in the context of the virus. Compared to the parental virus, the burst size of the Tcf mutant viruses was reduced at least 1,000-fold in H1299, 293T, NB324K, and HeLa cells, which have inactive wnt signaling pathways. The burst size and cytopathic effect of the Tcf viruses was near wild-type levels in SW480 and Isreco1 colon cancer cell lines, which have high Tcf activity. The high specificity of these viruses should permit the development of H-1 virus-based vectors which combine high safety and greater efficacy in cancer therapy.
Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4+Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism
Authors: Yang Yida , Tikhonov Ilia , Ruckwardt Tracy J. , Djavani Mahmoud , Zapata Juan Carlos , Pauza C. David , Salvato Maria S.
Published: May 2003

The human immunodeficiency virus (HIV) Tat protein has a critical role in viral transcription, but this study focuses on its additional role as an extracellular effector of lymphocyte cell death. It is well known that Tat induces tumor necrosis factor-related apoptosis-induced ligand (TRAIL) in peripheral blood mononuclear cells (PBMC), and we show that the majority of TRAIL is produced by the monocyte subset of PBMC. Human monocytes and U937 monoblastoid cells did not take up soluble HIV Tat-86, as T cells did, yet produced more TRAIL than did T cells. TRAIL secretion was induced by Tat and by a cysteine-rich peptide of Tat but not by sulfhydryl-modified Tat toxoid. Although there was only a slight increase in cell surface expression of TRAIL on monocytes, sufficient TRAIL was secreted to be toxic for T cells. The cytotoxicity of Tat-stimulated monocyte medium could be blocked by a TRAIL-neutralizing antibody. T cells treated with Tat did not secrete enough TRAIL to mediate cell death in our assay. Remarkably, uninfected T cells are more susceptible to TRAIL than are HIV-infected T cells. The production of TRAIL by Tat-stimulated monocytes provides a mechanism by which HIV infection can destroy uninfected bystander cells.
Stem-Loop III in the 5′ Untranslated Region Is a
cis
-Acting Element in Bovine Coronavirus Defective Interfering RNA Replication
Authors: Raman Sharmila , Bouma Peter , Williams Gwyn D. , Brian David A.
Published: May 2003

Higher-order structures in the 5′ untranslated region (UTR) of plus-strand RNA viruses are known in many cases to function as
cis
-acting elements in RNA translation, replication, or transcription. Here we describe evidence supporting the structure and a
cis
-acting function in defective interfering (DI) RNA replication of stem-loop III, the third of four predicted higher-order structures mapping within the 210-nucleotide (nt) 5′ UTR of the 32-kb bovine coronavirus (BCoV) genome. Stem-loop III maps at nt 97 through 116, has a calculated free energy of −9.1 kcal/mol in the positive strand and −3.0 kcal/mol in the negative strand, and has associated with it beginning at nt 100 an open reading frame (ORF) potentially encoding an 8-amino-acid peptide. Stem-loop III is presumed to function in the positive strand, but its strand of action has not been established. Stem-loop III (i) shows phylogenetic conservation among group 2 coronaviruses and appears to have a homolog in coronavirus groups 1 and 3, (ii) has in all coronaviruses for which sequence is known a closely associated short, AUG-initiated intra-5′ UTR ORF, (iii) is supported by enzyme structure-probing evidence in BCoV RNA, (iv) must maintain stem integrity for DI RNA replication in BCoV DI RNA, and (v) shows a positive correlation between maintenance of the short ORF and maximal DI RNA accumulation in BCoV DI RNA. These results indicate that stem-loop III in the BCoV 5′ UTR is a
cis
-acting element for DI RNA replication and that its associated intra-5′ UTR ORF may function to enhance replication. It is postulated that these two elements function similarly in the virus genome.
Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL: Clues from gDgH Chimeras
Authors: Cairns Tina M. , Milne Richard S. B. , Ponce-de-Leon Manuel , Tobin Deanna K. , Cohen Gary H. , Eisenberg Roselyn J.
Published: May 2003

In alphaherpesviruses, glycoprotein B (gB), gD, gH, and gL are essential for virus entry. A replication-competent gL-null pseudorabies virus (PrV) (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999) was shown to express a gDgH hybrid protein that could replace gD, gH, and gL in cell-cell fusion and null virus complementation assays. To study this phenomenon in herpes simplex virus type 1 (HSV-1), we constructed four gDgH chimeras, joining the first 308 gD amino acids to various gH N-terminal truncations. The chimeras were named for the first amino acid of gH at which each was truncated: 22, 259, 388, and 432. All chimeras were immunoprecipitated with both gD and gH antibodies to conformational epitopes. Normally, transport of gH to the cell surface requires gH-gL complex formation. Chimera 22 contains full-length gH fused to gD308. Unlike PrV gDgH, chimera 22 required gL for transport to the surface of transfected Vero cells. Interestingly, although chimera 259 failed to reach the cell surface, chimeras 388 and 432 exhibited gL-independent transport. To examine gD and gH domain function, each chimera was tested in cell-cell fusion and null virus complementation assays. Unlike PrV gDgH, none of the HSV-1 chimeras substituted for gL for fusion. Only chimera 22 was able to replace gH for fusion and could also replace either gH or gD in the complementation assay. Surprisingly, this chimera performed very poorly as a substitute for gD in the fusion assay despite its ability to complement gD-null virus and bind HSV entry receptors (HveA and nectin-1). Chimeras 388 and 432, which contain the same portion of gD as that in chimera 22, substituted for gD for fusion at 25 to 50% of wild-type levels. However, these chimeras functioned poorly in gD-null virus complementation assays. The results highlight the fact that these two functional assays are measuring two related but distinct processes.
Dual Pressure from Antiretroviral Therapy and Cell-Mediated Immune Response on the Human Immunodeficiency Virus Type 1 Protease Gene
Authors: Karlsson Annika C. , Deeks Steven G. , Barbour Jason D. , Heiken Brandon D. , Younger Sophie R. , Hoh Rebecca , Lane Meghan , Sällberg Matti , Ortiz Gabriel M. , Demarest James F. , Liegler Teri , Grant Robert M. , Martin Jeffrey N. , Nixon Douglas F.
Published: May 2003

Human immunodeficiency virus (HIV)-specific CD8
+
T-lymphocyte pressure can lead to the development of viral escape mutants, with consequent loss of immune control. Antiretroviral drugs also exert selection pressures on HIV, leading to the emergence of drug resistance mutations and increased levels of viral replication. We have determined a minimal epitope of HIV protease, amino acids 76 to 84, towards which a CD8
+
T-lymphocyte response is directed. This epitope, which is HLA-A2 restricted, includes two amino acids that commonly mutate (V82A and I84V) in the face of protease inhibitor therapy. Among 29 HIV-infected patients who were treated with protease inhibitors and who had developed resistance to these drugs, we show that the wild-type PR82V
76-84
epitope is commonly recognized by cytotoxic T lymphocytes (CTL) in HLA-A2-positive patients and that the CTL directed to this epitope are of high avidity. In contrast, the mutant PR82A
76-84
epitope is generally not recognized by wild-type-specific CTL, or when recognized it is of low to moderate avidity, suggesting that the protease inhibitor-selected V82A mutation acts both as a CTL and protease inhibitor escape mutant. Paradoxically, the absence of a mutation at position 82 was associated with the presence of a high-avidity CD8
+
T-cell response to the wild-type virus sequence. Our results indicate that both HIV type 1-specific CD8
+
T cells and antiretroviral drugs provide complex pressures on the same amino acid sequence of the HIV protease gene and, thus, can influence viral sequence evolution.
Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Contains Hypoxia Response Elements: Relevance to Lytic Induction by Hypoxia
Authors: Haque Muzammel , Davis David A. , Wang Victoria , Widmer Isabelle , Yarchoan Robert
Published: May 2003

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also known as human herpesvirus 8, is an etiologic agent of KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease. We recently demonstrated that hypoxia can induce lytic replication of KSHV in PEL cell lines. Hypoxia induces the accumulation of hypoxia-inducible factors (HIF), and we hypothesized that the KSHV genome may respond to hypoxia through functional hypoxia response elements (HREs). Here, we demonstrate the presence of at least two promoters within the KSHV genome that are activated by hypoxia or hypoxia mimics. One is in the promoter region of the gene for Rta, the main lytic switch gene, and the other is within the promoter region of ORF34, a lytic gene of unknown function. The ORF34 promoter contains three putative consensus HREs oriented in the direction of the gene. Dissection and site-directed mutagenesis studies confirmed that one of the HREs of the ORF34 promoter is functional. Under conditions of hypoxia, the ORF34 promoter was strongly upregulated by HIF-1α and HIF-2α. By contrast, the promoter of the gene for Rta appeared to be preferentially upregulated by HIF-2α. Reverse transcription-PCR analysis revealed that specific messages for ORF34 and ORF50 are upregulated in BCBL-1 cells exposed to hypoxia. An HIF-1 binding and competition assay demonstrated that the HRE sequence from the ORF34 promoter can compete for HIF-1α binding to an erythropoietin HRE oligonucleotide while a mutant sequence cannot. Thus, we demonstrated that a viral gene can be activated by hypoxia through activation of a functional viral HRE. To our knowledge, this is the first example of a functional HRE in a viral promoter.
A Transcriptionally Active Subgenomic Promoter Supports Homologous Crossovers in a Plus-Strand RNA Virus
Authors: Wierzchoslawski Rafal , Dzianott Aleksandra , Kunimalayan Selvi , Bujarski Jozef J.
Published: May 2003

Genetic RNA recombination plays an important role in viral evolution, but its molecular mechanism is not well understood. In this work we describe homologous RNA recombination activity that is supported by a subgenomic promoter (sgp) region in the RNA3 segment of brome mosaic bromovirus (BMV), a tripartite plus-strand RNA virus. The crossover frequencies were determined by coinoculations with pairs of BMV RNA3 variants that carried a duplicated sgp region flanked by marker restriction sites. A region composed of the sgp core, a poly(A) tract, and an upstream enhancer supported homologous exchanges in 25% of the analyzed RNA3 progeny. However, mutations in the sgp core stopped both the transcription of the sgp RNA and homologous recombination. These data provide evidence for an association of RNA recombination with transcription.
CAF-Mediated Human Immunodeficiency Virus (HIV) Type 1 Transcriptional Inhibition Is Distinct from α-Defensin-1 HIV Inhibition
Authors: Chang Theresa Li-Yun , François Fleur , Mosoian Arevik , Klotman Mary E.
Published: May 2003

CD8+T lymphocytes can inhibit human immunodeficiency virus type 1 (HIV-1) replication by secreting a soluble factor(s) known as CD8+T-lymphocyte antiviral factor (CAF). One site of CAF action is inhibition of HIV-1 RNA transcription, particularly at the step of long terminal repeat (LTR)-driven gene expression. The inhibitory effect of CAF on HIV-1 LTR activation is mediated through STAT1 activation. A recent study reports that α-defensins 1 to 3 account for CAF activity against HIV-1. Here, we address whether α-defensins, particularly α-defensin-1, contribute to CAF-mediated inhibition of HIV-1 transcription. Both recombinant α-defensin-1 and CAF derived from herpesvirus saimiri (HVS)-transformed CD8+cells inhibited HIV-1 infection and gene expression. For both factors, the inhibition of HIV-1 infection did not occur at the level of viral entry. Pretreatment of cells with α-defensin-1 followed by a washing out prior to infection blocked infection by HIV-1, indicating that direct inactivation of virions was not required for its inhibitory effect. In contrast to CAF, α-defensin-1 did not inhibit phorbol myristate acetate- or Tat-mediated HIV-1 LTR activation in a transient transfection system, nor did it activate STAT1 tyrosine phosphorylation. Furthermore, α-defensins 1 to 3 were below the level of detection in a panel of HVS-transformed CD8+cells with potent HIV-1 inhibitory activity and a neutralizing antibody against α-defensins 1 to 3 did not reverse the inhibitory effect of CAF on HIV-1 gene expression in infected cells and on HIV-1 LTR activation in transfected cells. Taken together, our results suggest that α-defensin-1 inhibits HIV-1 infection following viral entry but that α-defensins 1 to 3 are not responsible for the HIV-1 transcriptional inhibition by CAF.