bluuebunny/crossref_metadata_2025 huggingface dataset:
Domain-specific gene disruption reveals critical regulation of neuregulin signaling by its cytoplasmic tail
Authors: Liu Xifu , Hwang Helen , Cao Linguang , Buckland Michael , Cunningham Anne , Chen Ju , Chien Kenneth R. , Graham Robert M. , Zhou Mingdong
Published: July 2002

Neuregulins are a multi-isoform family of growth factors that activate members of the erbB family of receptor tyrosine kinases. The membrane-anchored isoforms contain the receptor-activating ligand in their extracellular domain, a single membrane-spanning region, and a long cytoplasmic tail. To evaluate the potential biological role of the intracellular domain of the membrane-anchored neuregulin isoforms, we used a domain-specific gene disruption approach to produce a mouse line in which only the region of the neuregulin gene encoding almost the entire intracellular domain was disrupted. Consistent with previous reports in which all neuregulin isoforms were disrupted, the resulting homozygous neuregulin mutants died at E10.5 of circulatory failure and displayed defects in neural and cardiac development. To further understand these
in vivo
observations, we evaluated a similarly truncated neuregulin construct after transient expression in COS-7 cells. This cytoplasmic tail-deleted mutant, unlike wild-type neuregulin isoforms, was resistant to proteolytic release of its extracellular-domain ligand, a process required for erbB receptor activation. Thus, proteolytic processing of the membrane-bound neuregulin isoforms involved in cranial ganglia and heart embryogenesis is likely developmentally regulated and is critically controlled by their intracellular domain. This observation indicates that erbB receptor activation by membrane-bound neuregulins most likely involves a unique temporally and spatially regulated “inside-out” signaling process that is critical for processing and release of the extracellular-domain ligand.
Analysis of the S3 and S3′ subsite specificities of feline immunodeficiency virus (FIV) protease: Development of a broad-based protease inhibitor efficacious against FIV, SIV, and HIV
in vitro
and
ex vivo
Authors: Lee Taekyu , Laco Gary S. , Torbett Bruce E. , Fox Howard S. , Lerner Danica L. , Elder John H. , Wong Chi-Huey
Published: July 2002

The S3 and S3′ subsite binding specificities of HIV and feline immunodeficiency virus proteases (FIV) proteases (PRs) have been explored by using
C
2
-symmetric competitive inhibitors. The inhibitors evaluated contained (
1S, 2R, 3R, 4S
)-1,4-diamino-1,4-dibenzyl-2,3-diol as P1 and P1′ units, Val as P2 and P2′ residues, and a variety of amino acids at the P3 and P3′ positions. All inhibitors showed very high potency against HIV PR
in vitro
, and their
K
i
values ranged between 1.1 and 2.6 nM. In contrast to the low restriction of P3 and P3′ residues observed in HIV PR, FIV PR exhibited strong preference for small hydrophobic groups at the S3 and S3′ subsites. Within this series, the most effective inhibitor against FIV PR contained Ala at P3 and P3′. Its
K
i
of 41 nM was 415- and 170-fold lower than those of the inhibitors without the P3 and P3′ moieties or with the Phe at these positions, respectively. In addition, these compounds were tested against mutant FIV PRs, which contain amino acid substitutions corresponding to those in native HIV PR at homologous sites, and their efficacy of inhibition progressively increased up to 5-fold. The most potent FIV PR inhibitor was selected for examination of its effectiveness in tissue culture, and it was able to block nearly 100% of virus production in an acute infection at 1 μg/ml (1.1 μM) against HIV, FIV, and simian immunodeficiency virus. Furthermore, it was not toxic to cells, and even after 2 months of culture there was no sign of resistance development by virus. The findings suggest that inhibitors with small P3 residue may be efficacious against a broad range of HIV variants as well as interspecies PRs.
Depletion of Ca
2+
from the sarcoplasmic reticulum of cardiac muscle prompts phosphorylation of phospholamban to stimulate store refilling
Authors: Bhogal Moninder Singh , Colyer John
Published: July 2002

Nonmuscle cells have almost ubiquitously evolved a mechanism to detect and prevent Ca
2+
store depletion—store operated calcium entry. No such mechanism has, as yet, been reported in cardiac myocytes. However, it is conceivable that such a mechanism may play an important role in cardiac Ca
2+
homeostasis to ensure the availability of sufficient stored Ca
2+
to maintain normal excitation contraction coupling. We present data that confirms the presence of a mechanism that is able to monitor the Ca
2+
load of the SR and initiate a signaling process to accelerate Ca
2+
uptake by the SR when store depletion is detected. Depletion of SR Ca
2+
activates a protein kinase, the principal SR substrate of which is phospholamban. Phosphorylation of this SR protein promotes Ca
2+
pump activity and therefore store refilling. Furthermore, a protein kinase activity associated with the SR that is inhibited by Ca
2+
ions has been identified. We have measured lumenal [Ca
2+
] by using a fluorescent Ca
2+
indicator and found that by initiating Ca
2+
uptake and increasing Ca
2+
load, we can inhibit the protein kinase activity associated with the SR. This confirms that a protein kinase, that is regulated by lumenal [Ca
2+
], has been identified and represents part of a previously unidentified signalling cascade. This local feedback mechanism would allow the myocyte to detect and prevent SR Ca
2+
load depletion.
Neural cell adhesion molecule (N-CAM) domains and intracellular signaling pathways involved in the inhibition of astrocyte proliferation
Authors: Krushel Leslie A. , Tai Ming-Hong , Cunningham Bruce A. , Edelman Gerald M. , Crossin Kathryn L.
Published: July 2002

The neural cell adhesion molecule (N-CAM) inhibits astrocyte proliferationin vitroandin vivo, and this effect is partially reversed by the glucocorticoid antagonist RU-486. The present studies have tested the hypothesis that N-CAM-mediated inhibition of astrocyte proliferation is caused by homophilic binding and involves the activation of glucocorticoid receptors. It was observed that all N-CAM Ig domains inhibited astrocyte proliferation in parallel with their ability to influence N-CAM binding. The proliferation of other N-CAM-expressing cells also was inhibited by the addition of N-CAM. In contrast, the proliferation of astrocytes from knockout mice lacking N-CAM was not inhibited by added N-CAM. These findings support the hypothesis that it is binding of soluble N-CAM to N-CAM on the astrocyte surface that leads to decreased proliferation. Signaling pathways stimulated by growth factors include activation of mitogen-activated protein (MAP) kinase. Addition of N-CAM inhibited MAP kinase activity induced by basic fibroblast growth factor in astrocytes. In accord with previous findings that RU-486 could partially prevent the proliferative effects of N-CAM, inhibition of MAP kinase activity by N-CAM was reversed by RU-486. The ability of N-CAM to inhibit astrocyte proliferation was unaffected, however, by agents that block the ability of N-CAM to promote neurite outgrowth. Together, these findings indicate that homophilic N-CAM binding leads to inhibition of astrocyte proliferation via a pathway involving the glucocorticoid receptor and that the ability of N-CAM to influence astrocyte proliferation and neurite outgrowth involves different signal pathways.
An isoform of the rod photoreceptor cyclic nucleotide-gated channel β subunit expressed in olfactory neurons
Authors: Sautter Andrea , Zong Xiangang , Hofmann Franz , Biel Martin
Published: July 2002

Sensory transduction in olfactory neurons involves the activation of a cyclic nucleotide-gated (CNG) channel by cAMP. Previous studies identified a CNG channel α subunit (CNG2) and a β subunit (CNG5), which when heterologously expressed form a channel with properties similar but not identical to those of native olfactory neurons. We have cloned a new type of CNG channel β subunit (CNG4.3) from rat olfactory epithelium. CNG4.3 derives from the same gene as the rod photoreceptor β subunit (CNG4.1) but lacks the long, glutamic acid-rich domain found in the N terminus of CNG4.1. Northern blot and
in situ
hybridization revealed that CNG4.3 is expressed specifically in olfactory neurons. Expression of CNG4.3 in human embryonic kidney 293 cells did not lead to detectable currents. Coexpression of CNG4.3 with CNG2 induced a current with significantly increased sensitivity for cAMP whereas cGMP affinity was not altered. Additionally, CNG4.3 weakened the outward rectification of the current in the presence of extracellular Ca
2+
, decreased the relative permeability for Ca
2+
, and enhanced the sensitivity for
l
-
cis
diltiazem. Upon coexpression of CNG2, CNG4.3, and CNG5, a conductance with a cAMP sensitivity greater than that of either the CNG2/CNG4.3 or the CNG2/CNG5 channel and near that of native olfactory channel was observed. Our data suggest that CNG4.3 forms a subunit of the native olfactory CNG channel. The expression of various CNG4 isoforms in retina and olfactory epithelium indicates that the CNG4 subunit may be necessary for normal function of both photoreceptor and olfactory CNG channels.
Centrosomal control of microtubule dynamics
Authors: Rodionov Vladimir , Nadezhdina Elena , Borisy Gary
Published: July 2002

In many animal cells, minus ends of microtubules (MTs) are thought to be capped by the centrosome whereas plus ends are free and display dynamic instability. We tested the role of the centrosome by examining MT behavior in cytoplasts from which the centrosome was removed. Cells were injected with Cy3–tubulin to fluorescently label MTs and were enucleated by using a centrifugation procedure. Enucleation resulted in a mixture of cytoplasts containing or lacking the centrosome. Fibroblast (CHO-K1) and epithelial (BSC-1) cells were investigated. In fibroblast cytoplasts containing the centrosome, MTs showed dynamic instability indistinguishable from that in intact cells. In contrast, in cytoplasts lacking the centrosome, MTs treadmilled—shortened at the minus end at about 12 μm/min while growing at the plus end at the same rate. The change in behavior of the plus end from dynamic instability to persistent growth correlated with an elevated level of free tubulin subunits (78% in centrosome-free cytoplasts vs. 44% in intact cells) generated by minus-end depolymerization. In contrast to fibroblast cells, in centrosome-free cytoplasts prepared from epithelial cells, MTs displayed dynamic instability at plus ends and relative stability at minus ends presumably because of specific minus-end stability factors distributed throughout the cytoplasm. We suggest that, in fibroblast cells, a minus-end depolymerization mechanism functions to eliminate errors in MT organization and that dynamic instability of MT plus ends is a result of capping of minus ends by the centrosome.
Volume regulatory responses of basolateral membrane vesicles from
Necturus
enterocytes: Role of the cytoskeleton
Authors: Dubinsky William P. , Mayorga-Wark Otilia , Schultz Stanley G.
Published: July 2002

Previous studies from this laboratory have demonstrated that basolateral membrane vesicles isolated from
Necturus maculosus
small intestinal epithelial cells possess a K
+
channel that is inhibited by ATP. In the present studies, we demonstrate that these vesicles, which are essentially devoid of soluble cytoplasmic contaminants, exhibit volume regulatory responses that parallel those of intact epithelial cells. Thus, suspension of these vesicles in a solution that is hypotonic to the intravesicular solution increases channel activity whereas suspension in a solution that is hypertonic to the intravesicular solution decreases, and may abolish, channel activity. These volume regulatory responses appear to be mediated by the same K
ATP
channel and depend on an intact actin cytoskeletal network. The responses to both hypotonic and hypertonic challenge are abolished by cytochalasin D or by incubating the vesicles under conditions that are known to depolymerize actin. Phalloidin, which is known to stabilize actin filaments, partially prevents the action of cytochalasin D. Thus, the present results indicate that the K
ATP
channel activity of basolateral membrane vesicles from
Necturus
basolateral membranes respond to hypo- and hypertonic challenge monotonically around an isotonic “set point” and that these responses depend on an intact actin cytoskeleton.
Chemoreceptor discharges and cytochrome redox changes of the rat carotid body: Role of heme ligands
Authors: Lahiri Sukhamay , Ehleben Wilhelm , Acker Helmut
Published: July 2002

In superfused
in vitro
rat carotid body, we recorded chemoreceptor discharges and the redox state of cytochromes simultaneously to identify the primary oxygen-sensing protein controlling transmitter release and electrical activity of the carotid sinus nerve. These parameters were tested under the influence of heme ligands such as oxygen, cyanide, 4-(2-aminoethyl)-benzenesulfonyl fluoride, and CO. During stimulation, there was an initial increase in discharge frequency followed by a decline or suppression of activity. Photometric changes lagged and were maintained as nerve activity decreased. Reducing mitochondrial cytochromes by cyanide or prolonged severe hypoxia, suppressed the chemoreceptor discharge. 4-(2-Aminoethyl)-benzenesulfonyl fluoride, a specific inhibitor of the phagocytic cytochrome
b
558
, also silenced the chemoreceptors after an initial excitation. CO increased the chemoreceptor discharge under normoxia, an effect inhibited by light, when the cytochromes were not reduced. When the discharges were depressed by severe hypoxia, exposure to light excited the chemoreceptors and the cytochromes were reduced. The rapidity of the chemosensory responses to light and lack of effect on dopamine release from type I cells led us to hypothesize that carotid body type I cells and the apposed nerve endings use different mechanisms for oxygen sensing: the nerve endings generate action potentials in association with membrane heme proteins whereas cytosolic heme proteins signal the redox state, releasing modulators or transmitters from type I cells.
Fifty-million-year-old polymorphism at an immunoglobulin variable region gene locus in the rabbit evolutionary lineage
Authors: Su Chen , Nei Masatoshi
Published: July 2002

The Ig heavy chain variable region (VH) genes encode the antigen-binding regions of antibodies. The rabbit genome contains more than 100 VHgenes, but only one (VH1) is preferentially used in the VDJ gene rearrangement. Three highly divergent alleles occur at this VH1 locus in most rabbit populations. These three VHalleles are also present in snowshoe hare populations, indicating that the polymorphism of the VH1 alleles is trans-specific. Here we report the results of a phylogenetic analysis of rabbit Ig germ-line VHgenes (alleles) together with VHgenes from humans and mice. We have found that all rabbit VHgenes belong to one mammalian VHgroup (group C), which also includes various human and mouse VHgenes. Using the rate of nucleotide substitution obtained from human and mouse VHsequences, we have estimated that the VH1 polymorphism in the rabbit lineage has been maintained for about 50 million years. This extremely long persistence of VH1 polymorphism is apparently caused by overdominant selection, though the real mechanism is unclear.
Na
+
-H
+
exchange in salivary secretory cells is controlled by an intracellular Na
+
receptor
Authors: Ishibashi H. , Dinudom A. , Harvey K. F. , Kumar S. , Young J. A. , Cook D. I.
Published: July 2002

It recently has been shown that epithelial Na
+
channels are controlled by a receptor for intracellular Na
+
, a G protein (G
o
), and a ubiquitin-protein ligase (Nedd4). Furthermore, mutations in the epithelial Na
+
channel that underlie the autosomal dominant form of hypertension known as Liddle’s syndrome inhibit feedback control of Na
+
channels by intracellular Na
+
. Because all epithelia, including those such as secretory epithelia, which do not express Na
+
channels, need to maintain a stable cytosolic Na
+
concentration ([Na
+
]
i
) despite fluctuating rates of transepithelial Na
+
transport, these discoveries raise the question of whether other Na
+
transporting systems in epithelia also may be regulated by this feedback pathway. Here we show in mouse mandibular secretory (endpiece) cells that the Na
+
-H
+
exchanger, NHE1, which provides a major pathway for Na
+
transport in salivary secretory cells, is inhibited by raised [Na
+
]
i
acting via a Na
+
receptor and G
o
. This inhibition involves ubiquitination, but does not involve the ubiquitin protein ligase, Nedd4. We conclude that control of membrane transport systems by intracellular Na
+
receptors may provide a general mechanism for regulating intracellular Na
+
concentration.
Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with β-branched amino acids
Authors: Sarafianos Stefan G. , Das Kalyan , Clark Arthur D. , Ding Jianping , Boyer Paul L. , Hughes Stephen H. , Arnold Edward
Published: July 2002

An important component of triple-drug anti-AIDS therapy is 2′,3′-dideoxy-3′-thiacytidine (3TC, lamivudine). Single mutations at residue 184 of the reverse transcriptase (RT) in HIV cause high-level resistance to 3TC and contribute to the failure of anti-AIDS combination therapy. We have determined crystal structures of the 3TC-resistant mutant HIV-1 RT (M184I) in both the presence and absence of a DNA/DNA template-primer. In the absence of a DNA substrate, the wild-type and mutant structures are very similar. However, comparison of crystal structures of M184I mutant and wild-type HIV-1 RT with and without DNA reveals repositioning of the template-primer in the M184I/DNA binary complex and other smaller changes in residues in the dNTP-binding site. On the basis of these structural results, we developed a model that explains the ability of the 3TC-resistant mutant M184I to incorporate dNTPs but not the nucleotide analog 3TCTP. In this model, steric hindrance is expected for NRTIs with β- or
l
- ring configurations, as with the enantiomer of 3TC that is used in therapy. Steric conflict between the oxathiolane ring of 3TCTP and the side chain of β-branched amino acids (Val, Ile, Thr) at position 184 perturbs inhibitor binding, leading to a reduction in incorporation of the analog. The model can also explain the 3TC resistance of analogous hepatitis B polymerase mutants. Repositioning of the template-primer as observed in the binary complex (M184I/DNA) may also occur in the catalytic ternary complex (M184I/DNA/3TCTP) and contribute to 3TC resistance by interfering with the formation of a catalytically competent closed complex.
Polymerization and mechanical properties of single RecA–DNA filaments
Authors: Hegner Martin , Smith Steven B. , Bustamante Carlos
Published: July 2002

The polymerization of individual RecA–DNA filaments, containing
either single-stranded or double-stranded DNA, was followed in real
time, and their mechanical properties were characterized with
force-measuring laser tweezers. It was found that the stretch modulus
of a filament is dominated by its (central) DNA component, while its
bending rigidity is controlled by its (eccentric) protein component.
The longitudinal stiffness of DNA increases 6- to 12-fold when the DNA
is contained in the protein helix. Both the stretch modulus and the
bending rigidity of a fiber change in the presence of various
nucleotide cofactors—e.g., [γ-thio]ATP, ATP, and
ADP—indicating a substantial re-arrangement of spatial relationships
between the nucleic acid and the protein scaffold. In particular, when
complexed with ATP, a fiber becomes twice as extensible as a
[γ-thio]ATP fiber, suggesting that 32% of the DNA-binding sites
have been released in its core. Such release may enable easy rotation
of the DNA within the protein helix or slippage of the DNA through the
center of the protein helix.
Diet-dependent hypercalciuria in transgenic mice with reduced CLC5 chloride channel expression
Authors: Luyckx Valerie A. , Leclercq Baudouin , Dowland Lara K. , Yu Alan S. L.
Published: July 2002

Dent’s disease is an X-linked inherited disorder characterized by hypercalciuria, nephrocalcinosis, nephrolithiasis, low molecular weight proteinuria, Fanconi’s syndrome, and renal failure. It is caused by inactivating mutations in CLC5, a member of the CLC voltage-gated chloride channel family. CLC5 is known to be expressed in the endosomal compartment of the renal proximal tubule, where it may be required for endosomal acidification and trafficking. Although the Fanconi’s syndrome and low molecular weight proteinuria in Dent’s disease can be explained by disruption of endosomal function in this nephron segment, the pathogenesis of the hypercalciuria in this disease is unknown. We have generated transgenic mice (RZ) with reduced CLC5 expression by introduction of an antisense ribozyme targeted against CLC5. RZ mice are markedly hypercalciuric compared with nontransgenic control mice, at a time when their serum electrolytes and renal function are otherwise normal. This suggests that hypercalciuria in Dent’s disease is a direct consequence of CLC5 hypofunction and is not attributable to a gain of function by mutant CLC5, an effect of modifier genes, or a secondary result of nonspecific renal injury. Surprisingly, hypercalciuria in RZ mice is abolished by dietary calcium deprivation, suggesting that the hypercalciuria may be attributable to gastrointestinal hyperabsorption of calcium rather than a renal calcium leak.
The metabolic implications of intracellular circulation
Authors: Hochachka P. W.
Published: July 2002

Two views currently dominate research into cell function and
regulation. Model I assumes that cell behavior is quite similar to that
expected for a watery bag of enzymes and ligands. Model II assumes that
three-dimensional order and structure constrain and determine
metabolite behavior. A major problem in cell metabolism is determining
why essentially all metabolite concentrations are remarkably stable
(are homeostatic) over large changes in pathway fluxes—for
convenience, this is termed the [
s
] stability paradox.
For muscle cells, ATP and O
2
are the most perfectly
homeostatic, even though O
2
delivery and metabolic rate
correlate in a 1:1 fashion. In total, more than 60 metabolites are
known to be remarkably homeostatic in differing metabolic states.
Several explanations of [
s
] stability are usually
given by traditional model I studies—none of which apply to all
enzymes in a pathway, and all of which require diffusion as the means
for changing enzyme–substrate encounter rates. In contrast, recent
developments in our understanding of intracellular myosin, kinesin, and
dyenin motors running on actin and tubulin tracks or cables supply a
mechanistic basis for regulated intracellular circulation systems with
cytoplasmic streaming rates varying over an approximately 80-fold range
(from 1 to >80 μm × sec
−1
). These new studies
raise a model II hypothesis of intracellular perfusion or convection as
a primary means for bringing enzymes and substrates together under
variable metabolic conditions. In this view, change in intracellular
perfusion rates cause change in enzyme–substrate encounter rates and
thus change in pathway fluxes with no requirement for large
simultaneous changes in substrate concentrations. The ease with which
this hypothesis explains the [
s
] stability paradox is
one of its most compelling features.
A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2
Authors: Goldmacher Victor S. , Bartle Laura M. , Skaletskaya Anna , Dionne Cheryl A. , Kedersha Nancy L. , Vater Carol A. , Han Jia-wen , Lutz Robert J. , Watanabe Shinya , McFarland Ellen D. Cahir , Kieff Elliott D. , Mocarski Edward S. , Chittenden Thomas
Published: July 2002

Human cytomegalovirus (CMV), a herpesvirus that causes congenital disease and opportunistic infections in immunocompromised individuals, encodes functions that facilitate efficient viral propagation by altering host cell behavior. Here we show that CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis, denoted vMIA, capable of suppressing apoptosis induced by diverse stimuli. vMIA, a product of the viral UL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome
c
release, while residing in mitochondria and associating with adenine nucleotide translocator. These functional properties resemble those ascribed to Bcl-2; however, the absence of sequence similarity to Bcl-2 or any other known cell death suppressors suggests that vMIA defines a previously undescribed class of anti-apoptotic proteins.
Cardioprotection from ischemia by a brief exposure to physiological levels of ethanol: Role of epsilon protein kinase C
Authors: Chen Che-Hong , Gray Mary O. , Mochly-Rosen Daria
Published: July 2002

Recent epidemiological studies indicate beneficial effects of moderate ethanol consumption in ischemic heart disease. Most studies, however, focus on the effect of long-term consumption of ethanol. In this study, we determined whether brief exposure to ethanol immediately before ischemia also produces cardioprotection. In addition, because protein kinase C (PKC) has been shown to mediate protection of the heart from ischemia, we determined the role of specific PKC isozymes in ethanol-induced protection. We demonstrated that (
i
) brief exposure of isolated adult rat cardiac myocytes to 10–50 mM ethanol protected against damage induced by prolonged ischemia; (
ii
) an isozyme-selective ɛPKC inhibitor developed in our laboratory inhibited the cardioprotective effect of acute ethanol exposure; (
iii
) protection of isolated intact adult rat heart also occurred after incubation with 10 mM ethanol 20 min before global ischemia; and (
iv
) ethanol-induced cardioprotection depended on PKC activation because it was blocked by chelerythrine and GF109203X, two PKC inhibitors. Consumption of 1–2 alcoholic beverages in humans leads to blood alcohol levels of ≈10 mM. Therefore, our work demonstrates that exposure to physiologically attainable ethanol levels minutes before ischemia provides cardioprotection that is mediated by direct activation of ɛPKC in the cardiac myocytes. The potential clinical implications of our findings are discussed.
An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein
Authors: Yang Jin , Bogerd Hal P. , Peng Sheila , Wiegand Heather , Truant Ray , Cullen Bryan R.
Published: July 2002

The human endogenous retrovirus K (HERV-K) family of endogenous retroviruses consists of ≈50 proviral copies per haploid human genome. Herein, the HERV-Ks are shown to encode a sequence-specific nuclear RNA export factor, termed K-Rev, that is functionally analogous to the HIV-1 Rev protein. Like HIV-1 Rev, K-Rev binds to both the Crm1 nuclear export factor and to a cis-acting viral RNA target to activate nuclear export of unspliced RNAs. Surprisingly, this HERV-K RNA sequence, which is encoded within the HERV-K long terminal repeat, is also recognized by HIV-1 Rev. These data provide surprising evidence for an evolutionary link between HIV-1 and a group of endogenous retroviruses that first entered the human genome ≈30 million years ago.
Presenilin complexes with the C-terminal fragments of amyloid precursor protein at the sites of amyloid β-protein generation
Authors: Xia Weiming , Ray William J. , Ostaszewski Beth L. , Rahmati Talat , Kimberly W. Taylor , Wolfe Michael S. , Zhang Jimin , Goate Alison M. , Selkoe Dennis J.
Published: July 2002

An unusual intramembranous cleavage of the β-amyloid precursor protein (APP) by γ-secretase is the final step in the generation of amyloid β-peptide (Aβ). Two conserved aspartates in transmembrane (TM) domains 6 and 7 of presenilin (PS) 1 are required for Aβ production by γ-secretase. Here we report that the APP C-terminal fragments, C83 and C99, which are the direct substrates of γ-secretase, can be coimmunoprecipitated with both PS1 and PS2. PS/C83 complexes were detected in cells expressing endogenous levels of PS. The complexes accumulate when γ-secretase is inactivated either pharmacologically or by mutating the PS aspartates. PS1/C83 and PS1/C99 complexes were detected in Golgi-rich and trans-Golgi network-rich vesicle fractions. In contrast, complexes of PS1 with APP holoprotein, which is not the immediate substrate of γ-secretase, occurred earlier in endoplasmic reticulum-rich vesicles. The major portion of intracellular Aβ at steady state was found in the same Golgi/trans-Golgi network-rich vesicles, and Aβ levels in these fractions were markedly reduced when either PS1 TM aspartate was mutated to alanine. Furthermore,
de novo
generation of Aβ in a cell-free microsomal reaction occurred specifically in these same vesicle fractions and was markedly inhibited by mutating either TM aspartate. Thus, PSs are complexed with the γ-secretase substrates C83 and C99 in the subcellular locations where Aβ is generated, indicating that PSs are directly involved in the pathogenically critical intramembranous proteolysis of APP.
Rapid desensitization of the nitric oxide receptor, soluble guanylyl cyclase, underlies diversity of cellular cGMP responses
Authors: Bellamy Tomas C. , Wood John , Goodwin David A. , Garthwaite John
Published: July 2002

A major receptor for nitric oxide (NO) is the cGMP-synthesizing enzyme, soluble guanylyl cyclase (sGC), but it is not known how this enzyme behaves in cells. In cerebellar cells, NO (from diethylamine NONOate) increased astrocytic cGMP with a potency (EC
50
≤ 20 nM) higher than that reported for purified sGC. Deactivation of NO-stimulated sGC activity, studied by trapping free NO with hemoglobin, took place within seconds (or less) rather than the minute time scale reported for the purified enzyme. Measurement of the rates of accumulation and degradation of cGMP were used to follow the activity of sGC over time. The peak activity, occurring within seconds of adding NO, was swiftly followed by desensitization to a steady-state level 8-fold lower. The same desensitizing profile was observed when the net sGC activity was increased or decreased or when cGMP breakdown was inhibited. Recovery from desensitization was relatively slow (half-time = 1.5 min). When the cells were lysed, sGC desensitization was lost. Analysis of the transient cGMP response to NO in human platelets showed that sGC underwent a similar desensitization. The results indicate that, in its natural environment, sGC behaves much more like a neurotransmitter receptor than had been expected from previous enzymological studies, and that hitherto unknown sGC regulatory factors exist. Rapid sGC desensitization, in concert with variations in the rate of cGMP breakdown, provides a fundamental mechanism for shaping cellular cGMP responses and is likely to be important in decoding NO signals under physiological and pathophysiological conditions.
Aquaporins in
Saccharomyces
: Characterization of a second functional water channel protein
Authors: Carbrey Jennifer M. , Bonhivers Mélanie , Boeke Jef D. , Agre Peter
Published: July 2002

The
Saccharomyces cerevisiae
genome database
contains two ORFs with homology to aquaporins,
AQY1
and
AQY2
. Aqy1p has been shown to be a functional aquaporin
in some strains, such as Σ1278b.
AQY2
is disrupted by
a stop codon in most strains; however, Σ1278b has an intact ORF.
Because Σ1278b Aqy2p has an intracellular localization in
Xenopus
oocytes and in yeast, other strains of yeast
were examined. Aqy2p from
Saccharomyces chevalieri
has a
single amino acid in the third transmembrane domain (Ser-141) that
differs from Σ1278b Aqy2p (Pro-141).
S. chevalieri
Aqy2p is a functional water channel in oocytes and traffics to the
plasma membrane of yeast. The Σ1278b parental strain, the
aqy1
-
aqy2
double null yeast, and null
yeast expressing
S. chevalieri
Aqy2p were examined under
various conditions. Comparison of these strains revealed that the
aquaporin null cells were more aggregated and their surface was more
hydrophobic. As a result, the aquaporin null cells were more flocculent
and more efficient at haploid invasive growth. Despite its primary
intracellular localization, Σ1278b Aqy2p plays a role in yeast
similar to Aqy1p and
S. chevalieri
Aqy2p. In addition,
Aqy1p and Aqy2p can affect cell surface properties and may provide an
advantage by dispersing the cells during starvation or during sexual
reproduction.
Circulating Th1 and Th2 cytokines in patients with hepatitis C virus infection
Authors: Fan X. G. , Liu W. E. , Li C. Z. , Wang Z. C. , Luo L. X. , Tan D. M. , Hu G. L. , Zhang Z.
Published: July 2002

The imbalance of T‐helper (Th) lymphocyte cytokine production may play an important role in immunopathogenes is of persistent hepatitis C virus (HCV) infection. To know whether an imbalance between Th1 and Th2 cytokines is present in chronic HCV infection, serum levels of Th1 cytokines , interferon gamma (IFN‐γ ) and interleukin (IL)‐2, and Th2 cytokines, IL‐4 and IL‐10, were measured using enzyme ‐ linked immunosorbent as say in this study. Eighteen individuals with chronic HCV infection, 11 healthy subjects as normal controls and 10 chronic HBV infected patients as disease controls were observed. The results showed that the leve ls of Th2 cytokines (IL‐4 and IL‐10) were significantly increased inchronic HCV infected patients compared with normal controls (IL‐4: 30.49 ± 17.55 vs . 14.94 ± 13.73, pg/ml, p < 0.025; IL‐10: 50.30 ± 19.59 vs. 17.87 ± 9.49, pg/ml, p < 0.001). Similarly, the levels of Th1 cytokine, IL‐2, was also elevated in individuals with chronic HCV infection when compared with normal controls (IL‐2: 118.53 ± 95.23 vs . 61.57 ± 28.70, pg/ml, p < 0.05). However, Th1 cytokine IFN‐γ level was not significantly changed during HCV infection (IFN‐γ: 28.09 ± 15.65 vs . 24.10 ± 15.61, pg/ml, p > 0.05). Further more, the elevated levels of Th2 cytokines are greater than Th1 cytokines in HCV infection. Thus , the study indicates that an enhanced Th2 responses are present during chronic HCV infection, which may partly be
responsible for the persistence of HCV infection.
In Utero Phase Carcinogenicity Testing
Authors: Hattan David G.
Published: July 2002

Early experimentation with transplacental exposure (1940s) demonstrated that expression of lung tumors in mice was enhanced when urethane was given during development in utero. In 1970, a U. S. Food and Drug Administration (FDA) expert panel on the safety evaluation of food additives and pesticides met and recommended that an in utero exposure phase be added to carcinogenicity testing (U.S. FDA, 1 971). An analysis was conducted of studies in the open scientific literature, in food additive studies available in FDA files and in studies performed by the National Institute of Environmental Health Sciences (NIEHS). While exposure to rodents during only the adult phase provided qualitatively similar results, early neonatal exposure typically provided slightly higher incidences of turnors, and decreased latency to turn or onset in certain scientific studies. In a series of studies recently performed by the NIEHS with three known animal carcinogens, neonatal or adult exposure produced similar tumors in similar tissues. The food additive saccharin, which shows bladder tumors, and eugenol reliably produced tumors only with neonatal exposure. Implieations for carcinogenicity testing of food additives are discussed in light of these experi-mental findings.
The Peroxisome in Oxidative Stress
Authors: Dansen Tobias B. , Wirtz Karel W. A.
Published: July 2002

Peroxisomes are one of the main sites in the cell where oxygen free radicals are both generated and scavenged. The balance between these two processes is believed to be of great importance for proper functioning of cells and has been implicated in aging and carcinogenesis. We will give an overview of the peroxisomal processes involved in the oxygen radical homeostasis and its implications for the cell.
A Functional Interaction between the Carboxy-Terminal Domain of RNA Polymerase II and Pre-mRNA Splicing
Authors: Du Lei , Warren Stephen L.
Published: July 2002

In the preceding study we found that Sm snRNPs and SerArg (SR) family proteins co-immunoprecipitate with Pol II molecules containing a hyperphosphorylated CTD (Kim et al., 1997). The association between Pol IIo and splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged (Kim et al., 1997). The latter findings led us to hypothesize that a phosphorylated form of the CTD interacts with pre-mRNA splicing components in vivo. To test this idea, a nested set of CTD-derived proteins was assayed for the ability to alter the nuclear distribution of splicing factors, and to interfere with splicing in vivo. Proteins containing heptapeptides 1-52 (CTD52), 1-32 (CTD32), 1-26 (CTD26), 1-13 (CTD13), 1-6 (CTD6), 1-3 (CTD3), or 1 (CTD1) were expressed in mammalian cells. The CTD-derived proteins become phosphorylated in vivo, and accumulate in the nucleus even though they lack a conventional nuclear localization signal. CTD52 induces a selective reorganization of splicing factors from discrete nuclear domains to the diffuse nucleoplasm, and significantly, it blocks the accumulation of spliced, but not unspliced, human β-globin transcripts. The extent of splicing factor disruption, and the degree of inhibition of splicing, are proportional to the number of heptapeptides added to the protein. The above results indicate a functional interaction between Pol II's CTD and pre-mRNA splicing.
Drosophila Centrosomin Protein is Required for Male Meiosis and Assembly of the Flagellar Axoneme
Authors: Li Kaijun , Xu Eugene Yujun , Cecil Jeffrey K. , Rudolf Turner F. , Megraw Timothy L. , Kaufman Thomas C.
Published: July 2002

Centrosomes and microtubules play crucial roles during cell division and differentiation. Spermatogenesis is a useful system for studying centrosomal function since it involves both mitosis and meiosis, and also transformation of the centriole into the sperm basal body. Centrosomin is a protein localized to the mitotic centrosomes in Drosophila melanogaster. We have found a novel isoform of centrosomin expressed during spermatogenesis. Additionally, an anticentrosomin antibody labels both the mitotic and meiotic centrosomes as well as the basal body. Mutational analysis shows that centrosomin is required for spindle organization during meiosis and for organization of the sperm axoneme. These results suggest that centrosomin is a necessary component of the meiotic centrosomes and the spermatid basal body.
Temporal Differences in the Appearance of NEP-B78 and an LBR-like Protein during Xenopus Nuclear Envelope Reassembly Reflect the Ordered Recruitment of Functionally Discrete Vesicle Types
Authors: Drummond Sheona , Ferrigno Paul , Lyon Carol , Murphy Jackie , Goldberg Martin , Allen Terry , Smythe Carl , Hutchison Christopher J.
Published: July 2002

In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.
Hyaluronic Acid (Ha) Binding to Cd44 Activates Rac1 and Induces Lamellipodia Outgrowth
Authors: Oliferenko Snezhana , Kaverina Irina , Small J. Victor , Huber Lukas A.
Published: July 2002

Both cell adhesion protein CD44 and its main ligand hyaluronic acid (HA) are thought to be involved in several processes ultimately requiring cytoskeleton rearrangements. Here, we show that the small guanine nucleotide (GTP)-binding protein, Rac1, can be activated upon HA binding to CD44. When applied locally to a passive cell edge, HA promoted the formation of lamellipodial protrusions in the direction of the stimulus. This process was inhibited by the prior injection of cells with dominant-negative N17Rac recombinant protein or by pretreatment of cells with monoclonal anti-CD44 antibodies, interfering with HA binding, implying the direct involvement of CD44 in signaling to Rac1.
Tpx2, a Novel Xenopus Map Involved in Spindle Pole Organization
Authors: Wittmann Torsten , Wilm Matthias , Karsenti Eric , Vernos Isabelle
Published: July 2002

TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673–685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein–dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles.
Negative Regulation of the Sapk/Jnk Signaling Pathway by Presenilin 1
Authors: Kim Jin Woo , Chang Tong-Shin , Lee Ji Eun , Huh Sung-Ho , Yeon Seung Woo , Yang Wan Seok , Joe Cheol O. , Mook-Jung Inhee , Tanzi Rudolph E. , Kim Tae-Wan , Choi Eui-Ju
Published: July 2002

Presenilin 1 (PS1) plays a pivotal role in Notch signaling and the intracellular metabolism of the amyloid β-protein. To understand intracellular signaling events downstream of PS1, we investigated in this study the action of PS1 on mitogen-activated protein kinase pathways. Overexpressed PS1 suppressed the stress-induced stimulation of stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) in human embryonic kidney 293 cells. Interestingly, two functionally inactive PS1 mutants, PS1(D257A) and PS1(D385A), failed to inhibit UV-stimulated SAPK/JNK. Furthermore, H2O2- or UV-stimulated SAPK activity was higher in mouse embryonic fibroblast (MEF) cells from PS1-null mice than in MEF cells from PS+/+ mice. MEFPS1(−/−) cells were more sensitive to the H2O2-induced apoptosis than MEFPS1(+/+) cells. Ectopic expression of PS1 in MEFPS1(−/−) cells suppressed H2O2-stimulated SAPK/JNK activity and apoptotic cell death. Together, our data suggest that PS1 inhibits the stress-activated signaling by suppressing the SAPK/JNK pathway.
Head-to-tail oligomerization of calsequestrin
Authors: Gatti Giuliana , Trifari Sara , Mesaeli Nasrin , Parker J.M. Robert , Michalak Marek , Meldolesi Jacopo
Published: July 2002

Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.
An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin–polymerizing machine
Authors: Scita Giorgio , Tenca Pierluigi , Areces Liliana B. , Tocchetti Arianna , Frittoli Emanuela , Giardina Giuseppina , Ponzanelli Isabella , Sini Patrizia , Innocenti Metello , Di Fiore Pier Paolo
Published: July 2002

Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8–E3b1–Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure–function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1–dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac–specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.
Abp1p and cortactin, new “hand-holds” for actin
Authors: Olazabal Isabel M. , Machesky Laura M.
Published: July 2002

Recently, two new ligands of the Arp2/3 complex have been described that may shed light on the way cells organize complex networks of actin in response to signals. Abp1p, a yeast protein involved in endocytosis, and cortactin, a mammalian src substrate, both enhance the ability of the Arp2/3 complex to assemble branched actin filament networks.