bluuebunny/crossref_metadata_2025 huggingface dataset:
A critical role of neural-specific JNK3 for ischemic apoptosis
Authors: Kuan Chia-Yi , Whitmarsh Alan J. , Yang Derek D. , Liao Guanghong , Schloemer Aryn J. , Dong Chen , Bao Jue , Banasiak Kenneth J. , Haddad Gabriel G. , Flavell Richard A. , Davis Roger J. , Rakic Pasko
Published: December 2003

c-Jun N-terminal kinase (JNK) signaling is an important contributor to stress-induced apoptosis, but it is unclear whether JNK and its isoforms (JNK1, JNK2, and JNK3) have distinct roles in cerebral ischemia. Here we show that JNK1 is the major isoform responsible for the high level of basal JNK activity in the brain. In contrast, targeted deletion of
Jnk3
not only reduces the stress-induced JNK activity, but also protects mice from brain injury after cerebral ischemia–hypoxia. The downstream mechanism of JNK3-mediated apoptosis may include the induction of
Bim
and
Fas
and the mitochondrial release of cytochrome
c
. These results suggest that JNK3 is a potential target for neuroprotection therapies in stroke.
Analysis of an orf virus chemokine-binding protein: Shifting ligand specificities among a family of poxvirus viroceptors
Authors: Seet Bruce T. , McCaughan Catherine A. , Handel Tracy M. , Mercer Andrew , Brunetti Craig , McFadden Grant , Fleming Stephen B.
Published: December 2003

We identify a secreted chemokine inhibitor encoded by orf virus (ORFV), the prototypic poxvirus of theParapoxvirusgenus, and show that it is related to the poxvirus type II CC-chemokine-binding proteins (CBP-II) produced by members of theOrthopoxvirusandLeporipoxvirusgenera. The ORFV chemokine-binding protein (CBP) is functionally similar to the CBP-II proteins in its ability to bind and inhibit many CC-chemokines with high affinity. However, unlike CBP-II, the ORFV CBP also binds with high affinity to lymphotactin, a member of the C-chemokine family, demonstrating that the ORFV CBP possesses an altered binding specificity. Interestingly, the amino acid sequence of ORFV CBP more closely resembles the granulocyte–macrophage colony-stimulating factor/IL-2 inhibitory factor also produced by ORFV, implicating the granulocyte–macrophage colony-stimulating factor/IL-2 inhibitory factor protein as a highly diverged, but related, member of the CBP-II protein family. Notably, these findings suggest that the genes that encode these proteins derive from a common poxvirus ancestral gene that has since been modified in binding specificity during speciation of the poxvirus genera. Overall, these findings illustrate the concept of evolution of viral proteins at the biophysical and molecular interface.
Distinct and nonoverlapping roles for pRB and cyclin D:cyclin-dependent kinases 4/6 activity in melanocyte survival
Authors: Yu Benjamin D. , Becker-Hapak Michelle , Snyder Eric L. , Vooijs Marc , Denicourt Catherine , Dowdy Steven F.
Published: December 2003

Deregulation of the p16
INK4a
–cyclin D:cyclin-dependent kinases (cdk) 4/6 –retinoblastoma (pRB) pathway is a common paradigm in the oncogenic transformation of human cells and suggests that this pathway functions linearly in malignant transformation. However, it is not understood why p16
INK4a
and cyclin D:cdk4/6 mutations are disproportionately more common than the rare genetic event of
RB
inactivation in human malignancies such as melanoma. To better understand how these complexes contribute to altered tissue homeostasis, we blocked cdk4/6 activation and acutely inactivated
Rb
by conditional mutagenesis during mouse hair follicle cycling. Inhibition of cdk4/6 in the skin by subcutaneous administration of a membrane-transducible TAT-p16
INK4a
protein completely blocked hair follicle growth and differentiation. In contrast, acute disruption of
Rb
in the skin of homozygous

Rb
LoxP/LoxP

mice via subcutaneous administration of TAT-Cre recombinase failed to affect hair growth. However, loss of
Rb
resulted in severe depigmentation of hair follicles. Further analysis of follicular melanocytes
in vivo
and in primary cell culture demonstrated that pRB plays a cell-autonomous role in melanocyte survival. Moreover, functional inactivation of all three
Rb
family members (
Rb
,
p107
, and
p130
) in primary melanocytes by treatment with a transducible TAT-E1A protein did not rescue the apoptotic phenotype. These findings suggest that deregulated cyclin D:cdk4/6 complexes and pRB perform nonoverlapping functions
in vivo
and provide a cellular mechanism that accounts for the low incidence of
RB
inactivation in cancers such as melanoma.
A glycopeptide in complex with MHC class I uses the GalNAc residue as an anchor
Authors: Apostolopoulos Vasso , Yuriev Elizabeth , Ramsland Paul A. , Halton Jodie , Osinski Carla , Li Wenjun , Plebanski Magdalena , Paulsen Hans , McKenzie Ian F. C.
Published: December 2003

Peptides bind MHC class I molecules by anchoring hydrophobic side chains into pockets in the peptide binding groove. Here, we report an immunogenic (in vitroandin vivo) MUC1 glycopeptide (MUC1–8-5GalNAc) bound to H-2Kb, fully crossreactive with the nonglycosylated variant. Molecular modeling showed that the central P5-Thr-GalNAc residue points into the C pocket and forms van der Waals and hydrogen bond interactions with the MHC class I. As predicted, GalNAc, a modified peptide carrying an additional anchor in the central C anchor pocket, increased the affinity by ≈100-fold compared with the native low-affinity peptide (MUC1–8). The findings demonstrate that glycopeptides associated with MHC class I molecules can use GalNAc to anchor the peptide in the groove and enable high-affinity binding.
The tandem Src homology 2 domain of the Syk kinase: A molecular device that adapts to interphosphotyrosine distances
Authors: Kumaran Sangaralingam , Grucza Richard A. , Waksman Gabriel
Published: December 2003

Conformational flexibility is important for protein function. However, information on the range of conformations accessible to macromolecules in the unbound state is often difficult to obtain. By using the model system of the tandem Src homology 2 domain (i.e., two adjacent Src homology 2 domains) of the Syk kinase, we report a method combining calorimetric and crystallographic measurements that reveals the preexistence of a conformational equilibrium in the unbound state, and that shows that this equilibrium is crucial for function.
Viral protein U counteracts a human host cell restriction that inhibits HIV-1 particle production
Authors: Varthakavi Vasundhara , Smith Rita M. , Bour Stephan P. , Strebel Klaus , Spearman Paul
Published: December 2003

Human cells resist viral infections by a variety of mechanisms. Viruses must overcome host cell restrictions to successfully reproduce their genetic material. Here, we identify a host restriction to viral replication that acts at the stage of particle assembly. Viral protein U (Vpu) is an HIV-1 accessory protein that enhances particle assembly and release in most human cells, but not in simian cells. By using human-simian cell heterokaryons, we show that the inhibition of assembly in human cells is dominant. Vpu overcomes the block to assembly in human cells and in human-simian heterokaryons. The HIV-1
vpu
gene may have evolved to counteract an assembly restriction that is present in human cells.
The timing of phasic transmitter release is Ca
2+
-dependent and lacks a direct influence of presynaptic membrane potential
Authors: Felmy Felix , Neher Erwin , Schneggenburger Ralf
Published: December 2003

Ca
2+
influx through voltage-gated Ca
2+
channels and the resulting elevation of intracellular Ca
2+
concentration, [Ca
2+
]
i
, triggers transmitter release in nerve terminals. However, it is controversial whether in addition to the opening of Ca
2+
channels, membrane potential directly affects transmitter release. Here, we assayed the influence of membrane potential on transmitter release at the calyx of Held nerve terminals. Transmitter release was evoked by presynaptic Ca
2+
uncaging, or by presynaptic Ca
2+
uncaging paired with presynaptic voltage-clamp depolarizations to +80 mV, under pharmacological block of voltage-gated Ca
2+
channels. Such a change in membrane potential did not alter the Ca
2+
dependence of transmitter release rates or synaptic delays. We also found, by varying the amount of Ca
2+
influx during Ca
2+
tail-currents, that the time course of phasic transmitter release is not invariant to changes in release probability. Rather, the time difference between peak Ca
2+
current and peak transmitter release became progressively shorter with increasing Ca
2+
current amplitude. When this time difference was plotted as a function of the estimated local [Ca
2+
]
i
at the sites of vesicle fusion, a slope of ≈100 μs per 10 μM [Ca
2+
]
i
was found, in reasonable agreement with a model of cooperative Ca
2+
binding and vesicle fusion. Thus, the amplitude and time course of the [Ca
2+
]
i
signal at the sites of vesicle fusion controls the timing and the amount of transmitter release, both under conditions of brief periods of Ca
2+
influx, as well as during step-like elevations of [Ca
2+
]
i
produced by Ca
2+
uncaging.
CD4 T cell memory derived from young naive cells functions well into old age, but memory generated from aged naive cells functions poorly
Authors: Haynes Laura , Eaton Sheri M. , Burns Eve M. , Randall Troy D. , Swain Susan L.
Published: December 2003

Age-related declines in immune function have an impact on both primary and memory responses. In this study, we have examined the ability of naive CD4 T cells from young and aged T cell receptor transgenic mice to establish functional memory. We found that memory cells generated from young CD4 T cells responded well to antigen, even a year after generation, whereas memory cells derived from CD4 T cells from aged mice responded poorly bothex vivoandin vivo. Memory cells generated from aged naive cells proliferate less, produce reduced levels of cytokines, and exhibit reduced cognate helper function, compared with memory cells generated by using young naive cells. These results indicate that it is the age of the naive T cell when it first encounters antigen, rather than the age when it reencounters antigen, that is critical for good memory CD4 T cell function.
Interferon-stimulated transcription and innate antiviral immunity require deacetylase activity and histone deacetylase 1
Authors: Nusinzon Inna , Horvath Curt M.
Published: December 2003

The use of histone deacetylase (HDAC) inhibitors has revealed an essential role for deacetylation in transcription of IFN-responsive genes. The HDAC1 protein associates with both signal transducer and activator of transcription (STAT) 1 and STAT2, and IFN-α stimulation induces deacetylation of histone H4. Inhibition of HDAC1 by small interfering RNA (siRNA) decreases IFN-α responsiveness whereas expression of HDAC1 augments the IFN-α response, demonstrating that HDAC1 modulates IFN-α-induced transcription. Importantly, the innate antiviral response is inhibited in the absence of deacetylase activity. The requirement for deacetylase is shared by IFN-γ transcription response and may represent a general requirement for STAT-dependent gene expression.
Human Mps1 protein kinase is required for centrosome duplication and normal mitotic progression
Authors: Fisk Harold A. , Mattison Christopher P. , Winey Mark
Published: December 2003

The mitotic spindle is essential for the maintenance of genetic stability, and in budding yeast its assembly and function depend on the Mps1 protein kinase. Mps1p is required for centrosome duplication and the spindle checkpoint. Several recent reports demonstrate that vertebrate Mps1 proteins regulate the spindle checkpoint, but reports conflict regarding their role in centrosome duplication. Here we provide multiple lines of evidence that the human Mps1 protein (hMps1) is required for centrosome duplication. A recently described rabbit polyclonal antibody against hMps1 specifically recognizes centrosomes in a variety of human cell types. Overexpression of a dominant-negative version of hMps1 (hMps1KD) can prevent centrosome duplication in a variety of cell types, and active hMps1 accelerates centrosome reduplication in U2OS cells. Finally, we demonstrate that disruption of hMps1 function with pools of hMps1-specific small interfering RNAs causes a pleiotropic phenotype resulting from the combination of severe mitotic abnormalities and failures in centrosome duplication. This approach demonstrates that hMps1 is required for centrosome duplication and for the normal progression of mitosis, and suggests that the threshold level of hMps1 function required for centrosome duplication is lower than that required for hMps1 mitotic functions.
Interpulse growth hormone secretion in the episodic plasma profile causes the sex reversal of cytochrome P450s in senescent male rats
Authors: Dhir Ravindra N. , Shapiro Bernard H.
Published: December 2003

Humans as well as other mammals experience an aging-related decline in drug metabolism as well as a diminution in growth hormone secretion. In the case of rats, these events are more pronounced in senescent males, whose expression of male-specific isoforms of cytochrome P450, the major drug-metabolizing enzymes and constituting ≈60-70% of the total cytochrome P450 in male rat liver, is completely suppressed, whereas female-dependent isoforms are remarkably induced to female-like levels. Overlooked in these independently reported studies is the fact that “signals” inherent in the masculine episodic and female continuous growth hormone profiles regulate expression and/or suppression of the dozen or so sex-dependent cytochrome P450 isoforms in rat liver. Whereas previous studies identified profound reductions in the pulse amplitudes of the masculine growth hormone profile as the cause for the diminished hormone secretion during aging, pulse heights are not recognized by the cytochromes as regulatory signals. Instead, we have shown that just a nominal secretion of growth hormone during the usual growth hormone-devoid interpulse period in the masculine episodic profile can explain the complete repression of male-specific CYP2C11, CYP3A2, and CYP2A2 and induction of female-dependent CYP2C12, CYP2C6, and CYP2A1 observed in senescent male rats.
Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity
Authors: Chartrel Nicolas , Dujardin Cynthia , Anouar Youssef , Leprince Jérôme , Decker Annick , Clerens Stefan , Do-Régo Jean-Claude , Vandesande Frans , Llorens-Cortes Catherine , Costentin Jean , Beauvillain Jean-Claude , Vaudry Hubert
Published: December 2003

A neuropeptide was isolated from a frog brain extract by HPLC purification and characterized by mass spectrometry. This 26-aa neuropeptide, which belongs to the RFamide peptide family, was designated 26RFa, and its primary structure was established as VGTALGSLAEELNGYNRKKGGFSFRF-NH2. Research in databases revealed the presence of sequences homologous to frog 26RFa in the human genome and in rat ESTs. On the basis of this sequence information, the cDNAs encoding the human and rat 26RFa precursors were cloned. The two preproteins show a similar organization, with the 26RFa sequence located in the C-terminal region of the precursor. Human preprotein (prepro)-26RFa encodes an additional putative RFamide peptide that is not found in the rat precursor. The primary structures of human, rat, and frog 26RFa exhibit ≈80% identity, and the C-terminal octapeptide has been fully conserved from amphibians to mammals.In situhybridization histochemistry revealed that, in the rat brain, the 26RFa gene is exclusively expressed in the ventromedial hypothalamic nucleus and in the lateral hypothalamic area. 26RFa induced a dose-dependent stimulation in cAMP production by rat pituitary cellsin vitroand markedly increased food intake in mice. The conservation of the primary structure of 26RFa during vertebrate evolution, the discrete localization of the mRNA encoding its precursor in hypothalamic nuclei involved in the control of feeding behavior, and the observation that 26RFa possesses orexigenic properties indicate that this neuropeptide may play important biological functions.
Catalysis and rotation of F
1
motor: Cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation
Authors: Shimabukuro Katsuya , Yasuda Ryohei , Muneyuki Eiro , Hara Kiyotaka Y. , Kinosita Kazuhiko , Yoshida Masasuke
Published: December 2003

F
1
, a water-soluble portion of F
o
F
1
-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central γ-subunit rotates in the α
3
β
3
cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 80° substep rotation, interim dwell, and rapid 40° substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of F
1
subcomplex (α
3
β
3
γ) from thermophilic
Bacillus
PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F
1
and hydrolysis of a slowly hydrolyzable substrate ATPγS (adenosine 5′-[γ-thio]triphosphate) by wild-type F
1
. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATPγS hydrolysis by the mutant F
1
ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F
1
occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.
Ethanol enhances α
4
β
3
δ and α
6
β
3
δ γ-aminobutyric acid type A receptors at low concentrations known to affect humans
Authors: Wallner M. , Hanchar H. J. , Olsen R. W.
Published: December 2003

γ-Aminobutyric acid type A receptors (GABARs) have long been implicated in mediating ethanol (EtOH) actions, but so far most of the reported recombinant GABAR combinations have shown EtOH responses only at fairly high concentrations (≥60 mM). We show that GABARs containing the δ-subunit, which are highly sensitive to γ-aminobutyric acid, slowly inactivating, and thought to be located outside of synapses, are enhanced by EtOH at concentrations that are reached with moderate, social EtOH consumption. Reproducible ethanol enhancements occur at 3 mM, a concentration six times lower than the legal blood-alcohol intoxication (driving) limit in most states (0.08% wt/vol or 17.4 mM). GABARs responsive to these low EtOH concentrations require the GABAR δ-subunit, which is thought to be associated exclusively with α
4
- and α
6
-subunits
in vivo
, and the β
3
-subunit, which has recently been shown to be essential for the
in vivo
anesthetic actions of etomidate and propofol. GABARs containing β
2
-instead of β
3
-subunits in α
4
βδ- and α
6
βδ-receptor combinations are almost 10 times less sensitive to EtOH, with threshold enhancement at 30 mM. GABARs containing γ
2
-instead of δ-subunits with α
4
β and α
6
β are three times less sensitive to EtOH, with threshold responses at 100 mM, a concentration not usually reached with social EtOH consumption. These combined findings suggest that “extrasynaptic” δ-subunit-containing GABARs, but not their “synaptic” γ-subunit-containing counterparts, are primary targets for EtOH.
Requirement of p53 targets in chemosensitization of colonic carcinoma to death ligand therapy
Authors: Wang Shulin , El-Deiry Wafik S.
Published: December 2003

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibits specific tumoricidal activity and is under development for cancer therapy. Mismatch-repair-deficient colonic tumors evade TRAIL-induced apoptosis through mutational inactivation of Bax, but chemotherapeutics including Camptosar (CPT-11) restore TRAIL sensitivity. However, the signaling pathways in restoring TRAIL sensitivity remain to be elucidated. Here, we imaged p53 transcriptional activity in Bax
-/-
carcinomas by using bioluminescence,
in vivo
, and find that p53 is required for sensitization to TRAIL by CPT-11. Small interfering RNAs directed at proapoptotic p53 targets reveal TRAIL receptor KILLER/DR5 contributes significantly to TRAIL sensitization, whereas Bak plays a minor role. Caspase 8 inhibition protects both CPT-11 pretreated wild-type and Bax
-/-
HCT116 cells from TRAIL-induced apoptosis, whereas caspase 9 inhibition only rescued the wild-type HCT116 cells from death induced by TRAIL. The results suggest a conversion in the apoptotic mechanism in HCT116 colon carcinoma from a type II pathway involving Bax and the mitochondria to a type I pathway involving efficient extrinsic pathway caspase activation. In contrast to Bax
-/-
cells, Bak-deficient human cancers undergo apoptosis in response to TRAIL or CPT-11, implying that these proteins have nonoverlapping functions. Our studies elucidate a mechanism for restoration of TRAIL sensitivity in MMR-deficient Bax
-/-
human cancers through p53-dependent activation of KILLER/DR5 and reconstitution of a type I death pathway. Efforts to identify agents that up-regulate DR5 may be useful in cancer therapies restoring TRAIL sensitivity.
Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor
Authors: Yue Zhenyu , Jin Shengkan , Yang Chingwen , Levine Arnold J. , Heintz Nathaniel
Published: December 2003

The biochemical properties of beclin 1 suggest a role in two fundamentally important cell biological pathways: autophagy and apoptosis. We show here that beclin 1
-/-
mutant mice die early in embryogenesis and beclin 1
+/-
mutant mice suffer from a high incidence of spontaneous tumors. These tumors continue to express wild-type beclin 1 mRNA and protein, establishing that beclin 1 is a haploinsufficient tumor suppressor gene. Beclin 1
-/-
embryonic stem cells have a severely altered autophagic response, whereas their apoptotic response to serum withdrawal or UV light is normal. These results demonstrate that beclin 1 is a critical component of mammalian autophagy and establish a role for autophagy in tumor suppression. They both provide a biological explanation for recent evidence implicating beclin 1 in human cancer and suggest that mutations in other genes operating in this pathway may contribute to tumor formation through deregulation of autophagy.
p53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1
Authors: Asher Gad , Lotem Joseph , Tsvetkov Peter , Reiss Veronica , Sachs Leo , Shaul Yosef
Published: December 2003

Proteasomal degradation of p53 is mediated by two alternative pathways that are either dependent or independent of both Mdm2 and ubiquitin. The ubiquitin-independent pathway is regulated by NAD(P)H: quinone oxidoreductase 1 (NQO1) that stabilizes p53. The NQO1 inhibitor dicoumarol induces ubiquitin-independent p53 degradation. We now show that, like dicoumarol, several other coumarin and flavone inhibitors of NQO1 activity, which compete with NAD(P)H for binding to NQO1, induced ubiquitin-independent p53 degradation and inhibited wild-type p53-mediated apoptosis. Although wild-type p53 and several p53 mutants were sensitive to dicoumarol-induced degradation, the most frequent “hot-spot” p53 mutants in human cancer, R175H, R248H, and R273H, were resistant to dicoumarol-induced degradation, but remained sensitive to Mdm2-ubiquitin-mediated degradation. The two alternative pathways for p53 degradation thus have different p53 structural requirements. Further mutational analysis showed that arginines at positions 175 and 248 were essential for dicoumarol-induced p53 degradation. NQO1 bound to wild-type p53 and dicoumarol, which induced a conformational change in NQO1, inhibited this binding. Compared with wild-type p53, the hot-spot p53 mutants showed increased binding to NQO1, which can explain their resistance to dicoumarol-induced degradation. NQO1 thus has an important role in stabilizing hot-spot p53 mutant proteins in human cancer.
Biologically active fragment of a human tRNA synthetase inhibits fluid shear stress-activated responses of endothelial cells
Authors: Tzima E. , Reader J. S. , Irani-Tehrani M. , Ewalt K. L. , Schwartz M. A. , Schimmel P.
Published: December 2003

Human tryptophanyl-tRNA synthetase (TrpRS) is active in translation and angiogenesis. In particular, an N-terminally truncated fragment, T2-TrpRS, that is closely related to a natural splice variant is a potent antagonist of vascular endothelial growth factor-induced angiogenesis in severalin vivomodels. In contrast, full-length native TrpRS is inactive in the same models. However, vascular endothelial growth factor stimulation is only one of many physiological and pathophysiological stimuli to which the vascular endothelium responds. To investigate more broadly the role of T2-TrpRS in vascular homeostasis and pathophysiology, the effect of T2-TrpRS on well characterized endothelial cell (EC) responses to flow-induced fluid shear stress was studied. T2-TrpRS inhibited activation by flow of protein kinase B (Akt), extracellular signal-regulated kinase 1/2, and EC NO synthase and prevented transcription of several shear stress-responsive genes. In addition, T2-TrpRS interfered with the unique ability of ECs to align in the direction of fluid flow. In all of these assays, native TrpRS was inactive, demonstrating that angiogenesis-related activity requires fragment production. These results demonstrate that T2-TrpRS can regulate extracellular signal-activated protein kinase, Akt, and EC NO synthase activation pathways that are associated with angiogenesis, cytoskeletal reorganization, and shear stress-responsive gene expression. Thus, this biological fragment of TrpRS may have a role in the maintenance of vascular homeostasis.
Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation
Authors: Wang Haibin , Matsumoto Hiromichi , Guo Yong , Paria Bibhash C. , Roberts Richard L. , Dey Sudhansu K.
Published: December 2003

Mammalian fertility absolutely depends on synchronized development of the blastocyst to the stage when it is competent to implant, and the uterus to the stage when it is receptive to implantation. However, the molecular basis for the reciprocal interaction between the embryo and the uterus remains largely unexplored. One potentially important mechanism involves signaling between an evolutionarily conserved G protein-coupled protein cannabinoid receptor, CB1, that is expressed at high levels on the surface of the trophectoderm and anandamide (N-arachi-donoylethanolamine), an endocannabinoid ligand found to be produced at higher levels by the uterus before implantation and then down-regulated at the time of implantation. Using genetic, pharmacological, and physiological approaches, we show here that anandamide within a very narrow range regulates blastocyst function and implantation by differentially modulating mitogen-activated protein kinase signaling and Ca2+channel activity via CB1 receptors. Anandamide at a low concentration (7 nM) induces extracellular regulated kinase phosphorylation and nuclear translocation in trophectoderm cells without influencing Ca2+channels, and renders the blastocyst competent for implantation in the receptive uterus. In contrast, anandamide at a higher concentration (28 nM) inhibits Ca2+channel activity and blastocyst competency for implantation without influencing mitogen-activated protein kinase signaling. Besides uncovering a potentially important regulatory mechanism for synchronizing blastocyst and uterine competency to implantation, this observation has high clinical relevance, because elevated levels of anandamide induce spontaneous pregnancy loss in women.
The atherogenic effect of excess methionine intake
Authors: Troen Aron M. , Lutgens Esther , Smith Donald E. , Rosenberg Irwin H. , Selhub Jacob
Published: December 2003

Methionine is the precursor of homocysteine, a sulfur amino acid intermediate in the methylation and transsulfuration pathways. Elevated plasma homocysteine (hyperhomocysteinemia) is associated with occlusive vascular disease. Whether homocysteine
per se
or a coincident metabolic abnormality causes vascular disease is still an open question. Animals with genetic hyperhomocysteinemia have so far not displayed atheromatous lesions. However, when methionine-rich diets are used to induce hyperhomocysteinemia, vascular pathology is often observed. Such studies have not distinguished the effects of excess dietary methionine from those of hyperhomocysteinemia. We fed apolipoprotein E-deficient mice with experimental diets designed to achieve three conditions: (
i
) high methionine intake with normal blood homocysteine; (
ii
) high methionine intake with B vitamin deficiency and hyperhomocysteinemia; and (
iii
) normal methionine intake with B vitamin deficiency and hyperhomocysteinemia. Mice fed methionine-rich diets had significant atheromatous pathology in the aortic arch even with normal plasma homocysteine levels, whereas mice fed B vitamin-deficient diets developed severe hyperhomocysteinemia without any increase in vascular pathology. Our findings suggest that moderate increases in methionine intake are atherogenic in susceptible mice. Although homocysteine may contribute to the effect of methionine, high plasma homocysteine was not independently atherogenic in this model. Some product of excess methionine metabolism rather than high plasma homocysteine
per se
may underlie the association of homocysteine with vascular disease.
Atomically detailed folding simulation of the B domain of staphylococcal protein A from random structures
Authors: Vila Jorge A. , Ripoll Daniel R. , Scheraga Harold A.
Published: December 2003

The conformational space of the 10–55 fragment of the B-domain of staphylococcal protein A has been investigated by using the electrostatically driven Monte Carlo (EDMC) method. The ECEPP/3 (empirical conformational energy program for peptides) force-field plus two different continuum solvation models, namely SRFOPT (Solvent Radii Fixed with atomic solvation parameters OPTimized) and OONS (Ooi, Oobatake, Némethy, and Scheraga solvation model), were used to describe the conformational energy of the chain. After an exhaustive search, starting from two different random conformations, three of four runs led to native-like conformations. Boltzmann-averaged root-mean-square deviations (RMSD) for all of the backbone heavy atoms with respect to the native structure of 3.35 Å and 4.54 Å were obtained with SRFOPT and OONS, respectively. These results show that the protein-folding problem can be solved at the atomic detail level by anab initioprocedure, starting from random conformations, with no knowledge except the amino acid sequence. To our knowledge, the results reported here correspond to the largest protein ever folded from a random conformation by an initial-value formulation with a full atomic potential, without resort to knowledge-based information.
Activity of a type 1 picornavirus internal ribosomal entry site is determined by sequences within the 3′ nontranslated region
Authors: Dobrikova Elena , Florez Paola , Bradrick Shelton , Gromeier Matthias
Published: December 2003

We have proposed a cancer treatment modality based on poliovirus chimeras replicating under the translational control of an internal ribosomal entry site (IRES) derived from human rhinovirus type 2. Insertion of the heterologous IRES causes a neuron-specific propagation deficit and eliminates neurovirulence inherent in poliovirus without affecting viral growth in cells derived from malignant gliomas. We now report the elucidation of a molecular mechanism responsible for the cell type-specific defect mediated by the rhinovirus IRES. Rhinovirus IRES function in neuronal cell types depends on specific structural elements within the 3′ non-translated region of the viral genome. Our observations suggest long-range interactions between the IRES and the 3′ terminus that control IRES-mediated gene expression and virus propagation.
Transfer of neutralizing IgG to macaques 6 h but not 24 h after SHIV infection confers sterilizing protection: Implications for HIV-1 vaccine development
Authors: Nishimura Yoshiaki , Igarashi Tatsuhiko , Haigwood Nancy L. , Sadjadpour Reza , Donau Olivia K. , Buckler Charles , Plishka Ron J. , Buckler-White Alicia , Martin Malcolm A.
Published: December 2003

Passive transfer of high-titered antiviral neutralizing IgG, known to confer sterilizing immunity in pig-tailed monkeys, has been used to determine how soon after virus exposure neutralizing antibodies (NAbs) must be present to block a simian immunodeficiency virus (SIV)/HIV chimeric virus infection. Sterilizing protection was achieved in three of four macaques receiving neutralizing IgG 6 h after intravenous SIV/HIV chimeric virus inoculation as monitored by PCR analyses of and attempted virus isolations from plasma, peripheral blood mononuclear cell, and lymph node specimens. In the fourth animal, the production of progeny virus was suppressed for >4 weeks. A delay in transferring NAbs until 24 h after virus challenge resulted in infection in two of two monkeys. These results suggest that even if a vaccine capable of eliciting broadly reactive NAbs against primary HIV-1 were at hand, the Abs generated must remain at, or rapidly achieve, high levels within a relatively short period after exposure to virus to prevent the establishment of a primate lentivirus infection.
Detection of cancer-specific markers amid massive mass spectral data
Authors: Zhu Wei , Wang Xuena , Ma Yeming , Rao Manlong , Glimm James , Kovach John S.
Published: December 2003

We propose a comprehensive pattern recognition procedure that will achieve best discrimination between two or more sets of subjects with data in the same coordinate system. Applying the procedure to MS data of proteomic analysis of serum from ovarian cancer patients and serum from cancer-free individuals in the Food and Drug Administration/National Cancer Institute Clinical Proteomics Database, we have achieved perfect discrimination (100% sensitivity, 100% specificity) of patients with ovarian cancer, including early-stage disease, from normal controls for two independent sets of data. Our procedure identifies the best subset of proteomic biomarkers for optimal discrimination between the groups and appears to have higher discriminatory power than other methods reported to date. For large-scale screening for diseases of relatively low prevalence such as ovarian cancer, almost perfect specificity and sensitivity of the detection system is critical to avoid unmanageably high numbers of false-positive cases.
Light- and pH-dependent structural changes in the PsbS subunit of photosystem II
Authors: Bergantino Elisabetta , Segalla Anna , Brunetta Alessia , Teardo Enrico , Rigoni Fernanda , Giacometti Giorgio Mario , Szabò Ildikò
Published: December 2003

In higher plants, the PsbS subunit of photosystem II (PSII) plays a crucial role in pH- and xanthophyll-dependent nonphotochemical quenching of excess absorbed light energy, thus contributing to the defense mechanism against photoinhibition. We determined the amino acid sequence of
Zea mays
PsbS and produced an antibody that recognizes with high specificity a region of the protein located in the stroma-exposed loop between the second and third putative helices. By means of this antiserum, the thylakoid membranes of various higher plant species revealed the presence of a 42-kDa protein band, indicating the formation of a dimer of the 21-kDa PsbS protein. Crosslinking experiments and immunoblotting with other antisera seem to exclude the formation of a heterodimer with other PSII protein components. The PsbS monomer/dimer ratio in isolated thylakoid membranes was found to vary with luminal pH in a reversible manner, the monomer being the prevalent form at acidic and the dimer at alkaline pH. In intact chloroplasts and whole plants, dimer-to-monomer conversion is reversibly induced by light, known to cause luminal acidification. Sucrose-gradient centrifugation revealed a prevalent association of the PsbS monomer and dimer with light-harvesting complex and PSII core complexes, respectively. The finding of the existence of a light-induced change in the quaternary structure of the PsbS subunit may contribute to understanding the mechanism of PsbS action during nonphotochemical quenching.
Identification and molecular characterization of the Gα12–Rho guanine nucleotide exchange factor pathway in
Caenorhabditis elegans
Authors: Yau Douglas M. , Yokoyama Nobuhiko , Goshima Yoshio , Siddiqui Zeba K. , Siddiqui Shahid S. , Kozasa Tohru
Published: December 2003

Gα12/13-mediated pathways have been shown to be involved in various fundamental cellular functions in mammalian cells such as axonal guidance, apoptosis, and chemotaxis. Here, we identified a homologue of Rho-guanine nucleotide exchange factor (GEF) in
Caenorhabditis elegans
(
CeRhoGEF
), which functions downstream of
gpa-12
, the
C. elegans
homologue of Gα12/13. CeRhoGEF contains a PSD-95/Dlg/ZO-1 domain and a regulator of G protein signaling (RGS) domain upstream of the Dbl homology–pleckstrin homology region similar to mammalian RhoGEFs with RGS domains, PSD-95/Dlg/ZO-1–RhoGEF and leukemia-associated RhoGEF. It has been shown in mammalian cells that these RhoGEFs interact with activated forms of Gα12 or Gα13 through their RGS domains. We demonstrated by coimmunoprecipitation that the RGS domain of CeRhoGEF interacts with GPA-12 in an

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activation-dependent manner and confirmed that the Dbl homology–pleckstrin homology domain of CeRhoGEF was capable of Rho-dependent signaling. These results proved conservation of the Gα12–RhoGEF pathway in
C. elegans
. Expression of DsRed or GFP under the control of the promoter of
CeRhoGEF
or
gpa-12
revealed an overlap of their expression patterns in ventral cord motor neurons and several neurons in the head. RNA-mediated gene interference for
CeRhoGEF
and
gpa-12
resulted in similar phenotypes such as embryonic lethality and sensory and locomotive defects in adults. Thus, the Gα12/13–RhoGEF pathway is likely to be involved in embryonic development and neuronal function in
C. elegans
.
A class of sterol 14-demethylase inhibitors as anti-
Trypanosoma cruzi
agents
Authors: Buckner Frederick , Yokoyama Kohei , Lockman Jeffrey , Aikenhead Kendra , Ohkanda Junko , Sadilek Martin , Sebti Saïd , Van Voorhis Wesley , Hamilton Andrew , Gelb Michael H.
Published: December 2003

Chronic infection with the protozoan parasite
Trypanosoma cruzi
is a major cause of morbidity and mortality in Latin America. Drug treatments for the associated illness, Chagas disease, are toxic and frequently unsuccessful. In a screening effort against the drug target protein farnesyltransferase, we identified a series of disubstituted imidazoles with highly potent anti-
T. cruzi
activity that apparently acted through a mechanism independent of protein farnesylation. Metabolic labeling studies of
T. cruzi
suggested that sterol biosynthesis was inhibited. Combined GC/MS analysis confirmed depletion of cellular sterols and suggested that the site of action was sterol 14-demethylase, a cytochrome P450 enzyme. Spectral studies with recombinant
T. cruzi
sterol 14-demethylase demonstrated that the compounds bind directly to this enzyme. Two of the compounds were well absorbed when given orally to mice, gave sustained plasma levels, and were well tolerated. The compounds were administered orally to mice with acute
T. cruzi
infection and caused dramatic decrease in parasitemia and led to 100% survival. These disubstituted imidazole compounds can be prepared by a relatively short synthetic route and represent a structural class with potent anti-
T. cruzi
activity.
Contrasting dynamics in the same plant–herbivore interaction
Authors: Bonsall M. B. , van der Meijden E. , Crawley M. J.
Published: December 2003

Long-term studies of two-species interactions under field conditions are unusual; most long-term field studies are of single species dynamics (
1
–
6
). Concurrent long-term studies on the dynamics of the same two interacting species in different locations are very rare. This result has led to the tacit assumption that different cases of the same two-species interaction would involve essentially quantitative differences (e.g., context-specific differences in the numeric values of demographic parameters like fecundity or death rates). Here, we show that for one of the best-known two-species systems (ragwort and cinnabar moth), this finding does not hold. The interaction between the plant and its herbivore is fundamentally different in coastal dunes in The Netherlands and in grasslands in Southeast England. In the first case, the dynamics are cyclic and the interaction involves both direct and delayed density dependence; in the second case, the insect has little impact on plant dynamics and there are no time lags in density dependence. The difference is caused by differences in the importance of seedlimitation in plant recruitment in the two locations.
Hox cluster duplications and the opportunity for evolutionary novelties
Authors: Wagner Gunte P. , Amemiya Chris , Ruddle Frank
Published: December 2003

Hox genes play a key role in animal body plan development. These genes tend to occur in tightly linked clusters in the genome. Vertebrates and invertebrates differ in their Hox cluster number, with vertebrates having multiple clusters and invertebrates usually having only one. Recent evidence shows that vertebrate Hox clusters are structurally more constrained than invertebrate Hox clusters; they exclude transposable elements, do not undergo tandem duplications, and conserve their intergenic distances and gene order. These constraints are only relaxed after a cluster duplication. In contrast, invertebrate Hox clusters are structurally more plastic; tandem duplications are common, the linkage of Hox genes can change quickly, or they can lose their structural integrity completely. We propose that the constraints on vertebrate Hox cluster structure lead to an association between the retention of duplicated Hox clusters and adaptive radiations. After a duplication the constraints on Hox cluster structure are temporarily lifted, which opens a window of evolvability for the Hox clusters. If this window of evolvability coincides with an adaptive radiation, chances are that a modified Hox cluster becomes recruited in an evolutionary novelty and then both copies of duplicated Hox clusters are retained.
Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers
Authors: Cristian Lidia , Lear James D. , DeGrado William F.
Published: December 2003

Despite significant efforts and promising progress, the understanding of membrane protein folding lags behind that of soluble proteins. Insights into the energetics of membrane protein folding have been gained from biophysical studies in membrane-mimicking environments (primarily detergent micelles). However, the development of techniques for studying the thermodynamics of folding in phospholipid bilayers remains a considerable challenge. We had previously used thiol-disulfide exchange to study the thermodynamics of association of transmembrane α-helices in detergent micelles; here, we extend this methodology to phospholipid bilayers. The system for this study is the homotetrameric M2 proton channel protein from the influenza A virus. Transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. Thiol-disulfide exchange under equilibrium conditions was used to quantitatively measure the thermodynamics of this folding interaction in phospholipid bilayers. The effects of phospholipid acyl chain length and cholesterol on the peptide association were investigated. The association of the helices strongly depends on the thickness of the bilayer and cholesterol levels present in the phospholipid bilayer. The most favorable folding occurred when there was a good match between the width of the apolar region of the bilayer and the hydrophobic length of the transmembrane helix. Physiologically relevant variations in the cholesterol level are sufficient to strongly influence the association. Evaluation of the energetics of peptide association in the presence and absence of cholesterol showed a significantly tighter association upon inclusion of cholesterol in the lipid bilayers.
The molecular basis for the high photosensitivity of rhodopsin
Authors: Liu Robert S. H. , Colmenares Leticia U.
Published: December 2003

Based on structural information derived from the F NMR data of labeled rhodopsins, rhodopsin crystal structure, and excited-state properties of model polyenes, we propose a molecular mechanism that accounts specifically for the causes of the well-known enhanced photoreactivity of rhodopsin (increased rates and quantum yield of isomerization). It involves the key features of close proximity of C-187 to H-12 and chromophore bond lengthening upon light absorption. The resultant “sudden punch” to H-12 triggers dual processes of decay of the Franck–Condon-excited rhodopsin, a productive directed photoisomerization and a nonproductive decay returning to the ground state as two separate molecular pathways [based on real-time fluorescence results of Chosrowjan, H., Mataga, N., Shibata, Y., Tachibanaki, S., Kandori, H., Shichida, Y., Okada, T. & Kouyama, T. (1998)J. Am. Chem. Soc.120, 9706–9707]. The two processes are controlled by the local protein structure: an empty space provided by the intradiscal loop connecting transmembrane helices 4 and 5 and a protein wall composed of amino acid units in transmembrane 3. Suggestions, involving retinal analogs and rhodopsin mutants, to improve the unusually high photosensitivity of rhodopsin are proposed.
Mimicry of Pre–B Cell Receptor Signaling by Activation of the Tyrosine Kinase Blk
Authors: Tretter Theresa , Ross Ashley E. , Dordai Dominic I. , Desiderio Stephen
Published: December 2003

During B lymphoid ontogeny, assembly of the pre–B cell receptor (BCR) is a principal developmental checkpoint at which several Src-related kinases may play redundant roles. Here the Src-related kinase Blk is shown to effect functions associated with the pre-BCR. B lymphoid expression of an active Blk mutant caused proliferation of B progenitor cells and enhanced responsiveness of these cells to interleukin 7. In mice lacking a functional pre-BCR, active Blk supported maturation beyond the pro–B cell stage, suppressed VH to DJH rearrangement, relieved selection for productive heavy chain rearrangement, and stimulated κ rearrangement. These alterations were accompanied by tyrosine phosphorylation of immunoglobulin β and Syk, as well as changes in gene expression consistent with developmental maturation. Thus, sustained activation of Blk induces responses normally associated with the pre-BCR.