bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the c-Jun N-terminal kinase pathway by a novel protein kinase related to human germinal center kinase
Authors: Diener Katrina , Wang Xuhong Sunny , Chen Cecil , Meyer Christian F. , Keesler George , Zukowski Mark , Tan Tse-Hua , Yao Zhengbin
Published: July 2002

The c-Jun N-terminal kinase (JNK), or stress-activated protein kinase plays a crucial role in cellular responses stimulated by environmental stress and proinflammatory cytokines. However, the mechanisms that lead to the activation of the JNK pathway have not been elucidated. We have isolated a cDNA encoding a novel protein kinase that has significant sequence similarities to human germinal center kinase (GCK) and human hematopoietic progenitor kinase 1. The novel GCK-like kinase (GLK) has a nucleotide sequence that encodes an ORF of 885 amino acids with 11 kinase subdomains. Endogenous GLK could be activated by UV radiation and proinflammatory cytokine tumor necrosis factor α. When transiently expressed in 293 cells, GLK specifically activated the JNK, but not the p42/44
MAPK
/extracellular signal-regulated kinase or p38 kinase signaling pathways. Interestingly, deletion of amino acids 353–835 in the putative C-terminal regulatory region, or mutation of Lys-35 in the putative ATP-binding domain, markedly reduced the ability of GLK to activate JNK. This result indicates that both kinase activity and the C-terminal region of GLK are required for maximal activation of JNK. Furthermore, GLK-induced JNK activation could be inhibited by a dominant-negative mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1) or mitogen-activated protein kinase kinase 4/SAPK/ERK kinase 1 (SEK1), suggesting that GLK may function upstream of MEKK1 in the JNK signaling pathway.
DHFR/MSH3
amplification in methotrexate-resistant cells alters the hMutSα/hMutSβ ratio and reduces the efficiency of base–base mismatch repair
Authors: Drummond James T. , Genschel Jochen , Wolf Elisabeth , Modrich Paul
Published: July 2002

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (
DHFR
) and
MSH3
genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSα (hMSH2⋅hMSH6) and hMutSβ (hMSH2⋅hMSH3). In HL-60 extracts the hMutSα to hMutSβ ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base–base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (
HPRT
) locus. Purified hMutSα and hMutSβ display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion–deletion heterology, but only hMutSα restores base–base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.
Panhandle PCR strategy to amplify
MLL
genomic breakpoints in treatment-related leukemias
Authors: Megonigal Maureen D. , Rappaport Eric F. , Jones Douglas H. , Kim Caroline S. , Nowell Peter C. , Lange Beverly J. , Felix Carolyn A.
Published: July 2002

Panhandle PCR amplifies genomic DNA with known 5′ and unknown 3′ sequences from a template with an intrastrand loop schematically shaped like a pan with a handle. We used panhandle PCR to clone
MLL
genomic breakpoints in two pediatric treatment-related leukemias. The karyotype in a case of treatment-related acute lymphoblastic leukemia showed the t(4;11)(q21;q23). Panhandle PCR amplified the translocation breakpoint at position 2158 in intron 6 in the 5′
MLL
breakpoint cluster region (bcr). The karyotype in a case of treatment-related acute myeloid leukemia was normal, but Southern blot analysis showed a single
MLL
gene rearrangement. Panhandle PCR amplified the breakpoint at position 1493 in
MLL
intron 6. Screening of somatic cell hybrid and radiation hybrid DNAs by PCR and reverse transcriptase-PCR analysis of the leukemic cells indicated that panhandle PCR identified a fusion of
MLL
intron 6 with a previously uncharacterized sequence in
MLL
intron 1, consistent with a partial duplication. In both cases, the breakpoints in the
MLL
bcr were in
Alu
repeats, and there were
Alu
repeats in proximity to the breakpoints in the partner DNAs, suggesting that
Alu
sequences were relevant to these rearrangements. This study shows that panhandle PCR is an effective method for cloning
MLL
genomic breakpoints in treatment-related leukemias. Analysis of additional pediatric cases will determine whether breakpoint distribution deviates from the predilection for 3′ distribution in the bcr that has been found in adult cases.
Localization of neuropeptide Y Y1 receptors in cerebral blood vessels
Authors: Bao Lan , Kopp Jutta , Zhang Xu , Xu Zhi-Qing David , Zhang Li-Fan , Wong Helen , Walsh John , Hökfelt Tomas
Published: July 2002

The localization of neuropeptide Y (NPY) Y1 receptor (R) -like immunoreactivity (LI) has been studied in cerebral arteries and arterioles of the rat by immunohistochemistry using fluorescence, confocal, and electron microscopy. High levels of Y1-R-LI were observed in smooth muscle cells (SMCs) in the small arterioles of the pial arterial network, especially on the basal surface of the brain, and low levels in the major basal cerebral arteries. The levels of Y1-R-LI varied strongly between adjacent SMCs. Y1-R-LI was associated with small endocytosis vesicles, mainly on the outer surface of the SMCs, but also on their endothelial side and often laterally at the interface between two SMCs. NPY-immunoreactive (Ir) nerve fibers could not be detected in association with the Y1-R-rich small arterioles but only around arteries with low Y1-R levels. A dense network of central NPY-Ir nerve fibers in the superficial layers of the brain was lying close to the strongly Y1-R-Ir small arterioles. The results indicate that NPY has a profound effect on small arterioles of the brain acting on Y1-Rs, both on the peripheral and luminal side of the SMCs. However, the source of the endogenous ligand, NPY, remains unclear. NPY released from central neurons may play a role, in addition to blood-borne NPY.
KSR stimulates Raf-1 activity in a kinase-independent manner
Authors: Michaud Neil R. , Therrien Marc , Cacace Angela , Edsall Lisa C. , Spiegel Sarah , Rubin Gerald M. , Morrison Deborah K.
Published: July 2002

Kinase suppressor of Ras (KSR) is an evolutionarily conserved component of Ras-dependent signaling pathways. Here, we find that murine KSR (mKSR1) translocates from the cytoplasm to the plasma membrane in the presence of activated Ras. At the membrane, mKSR1 modulates Ras signaling by enhancing Raf-1 activity in a kinase-independent manner. The activation of Raf-1 is mediated by the mKSR1 cysteine-rich CA3 domain and involves a detergent labile cofactor that is not ceramide. These findings reveal another point of regulation for Ras-mediated signal transduction and further define a noncatalytic role for mKSR1 in the multistep process of Raf-1 activation.
GIRK1 immunoreactivity is present predominantly in dendrites, dendritic spines, and somata in the CA1 region of the hippocampus
Authors: Drake Carrie T. , Bausch Suzanne B. , Milner Teresa A. , Chavkin Charles
Published: July 2002

Electron microscopic analysis of the CA1 region of the rat hippocampus revealed that specific immunoreactivity (IR) for a G protein-gated, inwardly rectifying potassium channel (GIRK1) was present exclusively in neurons and predominantly located in spiny dendrites of pyramidal cells. Within stratum lacunosum-moleculare and the superficial stratum radiatum, GIRK1-IR was often present immediately adjacent to asymmetric (excitatory-type) postsynaptic densities in dendritic spines. The subcellular localization of GIRK1-IR in the Golgi apparatus of pyramidal cell somata and in the plasma membrane of dendrites and dendritic spines confirms the hypothesis that GIRK1 is synthesized by pyramidal cells and transported to the more distal dendritic processes. G protein-coupled receptor activation of a dendritic potassium conductance would attenuate the propagation of excitatory synaptic inputs and thereby produce postsynaptic inhibition. Thus, these results show that the GIRK family of channels joins the list of voltage-sensitive channels now known to be expressed in dendritic spines.
Suckling and sucrose ingestion suppress persistent hyperalgesia and spinal Fos expression after forepaw inflammation in infant rats
Authors: Ren K. , Blass E. M. , Zhou Q.-q. , Dubner R.
Published: July 2002

Sweet taste and nonnutritive suckling produce analgesia to transient noxious stimuli in infant rats and humans. The present study evaluated the pain-modulating effects of sucrose and suckling in a rat model of persistent pain and hyperalgesia that mimics the response to tissue injury in humans. Fore- and hindpaw withdrawal latencies from a 30° or 48°C brass stylus were determined in 10-day-old rats following paw inflammation induced by complete Freund’s adjuvant (CFA; 1:1 injected s.c. in a 0.01 ml volume). CFA markedly decreased escape latencies to both 48° and 30°C stimulation, thereby demonstrating thermal hyperalgesia and mechanical allodynia. The combination of nonnutritive suckling and sucrose (7.5%, 0.01–0.06 ml/min) infusion markedly increased escape latencies to forepaw stimulation in both CFA-treated and control rats. In contrast, intraoral sucrose and suckling did not increase hindpaw withdrawal latencies in either control or CFA-inflamed rats. The effect was specific to sweet taste because neither water nor isotonic saline infusion affected forepaw escape latencies. Parallel findings were obtained for CFA-induced Fos-like immunoreactivity (Fos-LI), a marker of neuronal activation. Fos-LI was selectively induced in cervical and lumbar regions ipsilateral to forepaw and hindpaw inflammation, respectively. Suckling-sucrose treatment significantly reduced Fos-LI at the cervical but not at the lumbar regions. These findings demonstrate: (
i
) the development of persistent pain and hyperalgesia in 10-day-old rats that can be attenuated by endogenous pain-modulating systems activated by taste and nonnutritive suckling; (
ii
) the mediation of the sucrose-suckling analgesia and antihyperalgesia at the spinal level; and (
iii
) a differential rostrocaudal maturation of descending pain-modulating systems to the spinal cord of 10-day-old rats. These findings may provide new clinical approaches for engaging endogenous analgesic mechanisms in infants following tissue injury and inflammation.
Importance of acidic, proline/serine/threonine-rich, and GTPbinding regions in the major histocompatibility complex class II transactivator: Generation of transdominant-negative mutants
Authors: Chin Keh-Chuang , Li George G.-X. , Ting Jenny P.-Y.
Published: July 2002

The class II transactivator (CIITA) is a master transcription regulator of gene products involved in the exogenous antigen presentation pathway, including major histocompatibility complex (MHC) class II, invariant chain, and DM. An extensive analysis of the putative functional domains of CIITA is undertaken here to explore the action of CIITA. Antibodies to CIITA protein were produced to verify that these mutant proteins are expressed. Both acidic and proline/serine/threonine-rich domains are essential for class II MHC promoter activation. In addition, three guanine nucleotide-binding motifs are essential for CIITA activity. Of these mutants, two exhibited strong transdominant-negative functions. These two mutants provide a plausible approach to manipulate MHC class II expression and immune responses.
Age-dependent decrease of synaptic plasticity in the neocortex of αCaMKII mutant mice
Authors: Kirkwood Alfredo , Silva Alcino , Bear Mark F.
Published: July 2002

Synaptic long-term potentiation (LTP) and long-term depression (LTD) were studied in the visual cortex of mutant mice lacking α-calcium/calmodulin-dependent protein kinase II (αCaMKII). In adult mutants, little LTD or LTP could be elicited using standard conditioning protocols. However, substantial LTD and LTP were induced in 4- to 5-week-old mutants. Thus, the reduction in cortical plasticity in αCaMKII (−/−) mice is conditional, with the relevant condition being postnatal age.
Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates
Authors: Shah Kavita , Liu Yi , Deirmengian Carl , Shokat Kevan M.
Published: July 2002

Protein phosphorylation plays a central role in controlling many diverse signal transduction pathways in all cells. Novel protein kinases are identified at a rapid rate using homology cloning methods and genetic screens or selections; however identification of the direct substrates of kinases has proven elusive to genetic methods because of the tremendous redundancy and overlapping of substrate specificities among protein kinases. We describe the development of a protein engineering-based method to identify the direct substrates of the prototypical protein tyrosine kinase v-Src, which controls fibroblast transformation by the Rous sarcoma virus. To differentiate the substrates of v-Src from all other kinase substrates, we mutated the ATP binding site of v-Src such that the engineered v-Src uniquely accepted an ATP analog. We show that the engineered v-Src kinase displayed catalytic efficiency with the ATP analog,
N
6
-(cyclopentyl) ATP, which is similar to the wild-type kinase catalytic efficiency with ATP itself. However, the
N
6
-(cyclopentyl) ATP analog was not accepted by the wild-type kinase. Furthermore, the engineered v-Src exhibited the same protein target specificity as wild-type v-Src despite the proximity of the reengineered nucleotide binding site to the phosphoacceptor binding site. The successful engineering of v-Src’s active site to accept a unique nucleotide analog provides a unique handle by which the direct substrates of one kinase (v-Src) can be traced in the presence of any number of cellular kinases.
Fast events in protein folding: Relaxation dynamics of secondary and tertiary structure in native apomyoglobin
Authors: Gilmanshin Rudolf , Williams Skip , Callender Robert H. , Woodruff William H. , Dyer R. Brian
Published: July 2002

We report the fast relaxation dynamics of “native” apomyoglobin (pH 5.3) following a 10-ns, laser-induced temperature jump. The structural dynamics are probed using time-resolved infrared spectroscopy. The infrared kinetics monitored within the amide I absorbance of the polypeptide backbone exhibit two distinct relaxation phases which have different spectral signatures and occur on very different time scales (ν = 1633 cm
−1
, τ = 48 ns; ν = 1650 cm
−1
, τ = 132 μs). We assign these two spectral components to discrete substructures in the protein: helical structure that is solvated (1633 cm
−1
) and native helix that is protected from solvation by interhelix tertiary interactions (1650 cm
−1
). Folding rate coefficients inferred from the observed relaxations at 60°C are
k
f(solvated)
= (7 to 20) × 10
6
s
−1
and
k
f(native)
= 3.6 × 10
3
s
−1
, respectively. The faster rate is interpreted as the intrinsic rate of solvated helix formation, whereas the slower rate is interpreted as the rate of formation of tertiary contacts that determine a native helix. Thus, at 60°C helix formation precedes the formation of tertiary structure by over three orders of magnitude in this protein. Furthermore, the distinct thermodynamics and kinetics observed for the apomyoglobin substructures suggest that they fold independently, or quasi-independently. The observation of inhomogeneous folding for apomyoglobin is remarkable, given the relatively small size and structural simplicity of this protein.
Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening
Authors: Zhang Ji-Hu , Dawes Glenn , Stemmer Willem P. C.
Published: July 2002

An efficient β-fucosidase was evolved by DNA shuffling from theEscherichia coli lacZβ-galactosidase. Seven rounds of DNA shuffling and colony screening on chromogenic fucose substrates were performed, using 10,000 colonies per round. Compared with native β-galactosidase, the evolved enzyme purified from cells from the final round showed a 1,000-fold increased substrate specificity foro-nitrophenyl fucopyranoside versuso-nitrophenyl galactopyranoside and a 300-fold increased substrate specificity forp-nitrophenyl fucopyranoside versusp-nitrophenyl galactopyranoside. The evolved cell line showed a 66-fold increase inp-nitrophenyl fucosidase specific activity. The evolved fucosidase has a 10- to 20-fold increasedkcat/Kmfor the fucose substrates compared with the native enzyme. The DNA sequence of the evolved fucosidase gene showed 13 base changes, resulting in six amino acid changes from the native enzyme. This effort shows that the library size that is required to obtain significant enhancements in specificity and activity by reiterative DNA shuffling and screening, even for an enzyme of 109 kDa, is within range of existing high-throughput technology. Reiterative generation of libraries and stepwise accumulation of improvements based on addition of beneficial mutations appears to be a promising alternative to rational design.
Nitric oxide and thiol redox regulation of Janus kinase activity
Authors: Duhé Roy J. , Evans Gerald A. , Erwin Rebecca A. , Kirken Robert A. , Cox George W. , Farrar William L.
Published: July 2002

The activation of Janus kinases (JAKs) is crucial for propagation of the proliferative response initiated by many cytokines. The proliferation of various cell lines, particularly those of hematopoietic origin, is also modulated by mediators of oxidative stress such as nitric oxide and thiol redox reagents. Herein we demonstrate that nitric oxide and other thiol oxidants can inhibit the autokinase activity of rat JAK2in vitro, presumably through oxidation of crucial dithiols to disulfides within JAK2. The reduced form of JAK2 is the most active form, and the oxidized JAK2 form is inactive. Nitric oxide pretreatment of quiescent Ba/F3 cells also inhibits the interleukin 3-triggeredin vivoactivation of JAK2, a phenomenon that correlates with inhibited proliferation. Furthermore, we observed that the autokinase activity of JAK3 responds in a similar fashion to thiol redox reagentsin vitroand to nitric oxide donorsin vivo. We suggest that the thiol redox regulation of JAKs may partially explain the generally immunosuppressive effects of nitric oxide and of other thiol oxidants.
Electrostatic interactions between rotor and stator in the bacterial flagellar motor
Authors: Zhou Jiadong , Lloyd Scott A. , Blair David F.
Published: July 2002

Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor–stator interface.
Nucleolar localization of the Werner syndrome protein in human cells
Authors: Marciniak Robert A. , Lombard David B. , Johnson F. Bradley , Guarente Leonard
Published: July 2002

Werner Syndrome (WS) is a human genetic disorder with many features of premature aging. The gene defective in WS (
WRN
) has been cloned and encodes a protein homologous to several helicases, including
Escherichia coli
RecQ, the human Bloom syndrome protein (BLM), and
Saccharomyces cerevisiae
Sgs1p. To better define the function of WRN protein we have determined its subcellular localization. Indirect immunofluorescence using polyclonal anti-human WRN shows a predominant nucleolar localization. Studies of
WRN
mutant cells lines confirmed the specificity of antibody recognition. No difference was seen in the subcellular localization of the WRN protein in a variety of normal and transformed human cell lines, including both carcinomas and sarcomas. The nucleolar localization of human WRN protein was supported by the finding that upon biochemical subcellular fractionation, WRN protein is present in an increased concentration in a subnuclear fraction enriched for nucleolar proteins. We have also determined the subcellular localization of the mouse WRN homologue (mWRN). In contrast to human WRN protein, mWRN protein is present diffusely throughout the nucleus. Understanding the function of WRN in these organisms of vastly differing lifespan may yield new insights into the mechanisms of lifespan determination.
Epitopes close to the apolipoprotein B low density lipoprotein receptor-binding site are modified by advanced glycation end products
Authors: Wang Xingyu , Bucala Richard , Milne Ross
Published: July 2002

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease of patients with diabetes and renal failure, in part by preventing apolipoprotein B (apoB)-mediated cellular uptake of low density lipoproteins (LDL) by LDL receptors (LDLr). It has been proposed that AGE modification at one site in apoB, almost 1,800 residues from the putative apoB LDLr-binding domain, may be sufficient to induce an apoB conformational change that prevents binding to the LDLr. To further explore this hypothesis, we used 29 anti-human apoB mAbs to identify other potential sites on apoB that may be modified by
in vitro
advanced glycation of LDL. Glycation of LDL caused a time-dependent decrease in its ability to bind to the LDLr and in the immunoreactivity of six distinct apoB epitopes, including two that flank the apoB LDLr-binding domain. ApoB appears to be modified at multiple sites by these criteria, as the loss of glycation-sensitive epitopes was detected on both native glycated LDL and denatured, delipidated glycated apoB. Moreover, residues directly within the putative apoB LDLr-binding site are not apparently modified in glycated LDL. We propose that the inability of LDL modified by AGEs to bind to the LDLr is caused by modification of residues adjacent to the putative LDLr-binding site that were undetected by previous immunochemical studies. AGE modification either eliminates the direct participation of the residues in LDLr binding or indirectly alters the conformation of the apoB LDLr-binding site.
A novel mechanism for the acquisition of virulence by a human influenza A virus
Authors: Goto Hideo , Kawaoka Yoshihiro
Published: July 2002

Cleavage of the hemagglutinin (HA) molecule by proteases is a prerequisite for the infectivity of influenza A viruses. Here, we describe a novel mechanism of HA cleavage for a descendant of the 1918 pandemic strain of human influenza virus. We demonstrate that neuraminidase, the second major protein on the virion surface, binds and sequesters plasminogen, leading to higher local concentrations of this ubiquitous protease precursor and thus to increased cleavage of the HA. The structural basis of this unusual function of the neuraminidase molecule appears to be the presence of a carboxyl-terminal lysine and the absence of an oligosaccharide side chain at position 146 (N2 numbering). These findings suggest a means by which influenza A viruses, and perhaps other viruses as well, could become highly pathogenic in humans.
Carbohydrate utilization during exercise after high-altitude acclimation: A new perspective
Authors: McClelland Grant B. , Hochachka Peter W. , Weber Jean-Michel
Published: July 2002

At high altitude (HA), carbohydrate (CHO) is thought to be the preferred fuel because of its higher yield of ATP per mole of O
2
. We used indirect calorimetry and
d
-[6-
3
H]glucose infusions to determine total CHO and circulatory glucose utilization during exercise in HA-acclimated and sea level (SL) rats. We hypothesized that the percent contribution of CHO to total metabolism (V̇

O
2

) is determined by exercise intensity relative to an aerobic maximum (% V̇

O
2

max
). HA rats run under hypoxia (F
I
O
2
= 0.12) showed a decrease in V̇

O
2

max
compared with SL (67.55 ± 1.26 vs. 89.30 ± 1.23 ml kg
−1
min
−1
). When exercised at 60% of their respective V̇

O
2

max
, both groups showed the same relative use of CHO (38 ± 3% and 38 ± 5% of V̇

O
2

, at the beginning of exercise, in HA and SL, respectively). In both HA and SL, circulatory glucose accounted for ≈20% of V̇

O
2

, the balance was provided by muscle glycogen (≈18% of V̇

O
2

). After 20 min at a higher intensity of 80% V̇

O
2

max
, 54 ± 5% (HA) and 59 ± 4% (SL) of V̇

O
2

was accounted for by CHO. We conclude the following: (
i
) the relative contributions of total CHO, circulatory glucose, and muscle glycogen do not increase after HA acclimation because the O
2
-saving advantage of CHO is outweighed by limited CHO stores; and (
ii
) relative exercise intensity is the major determinant of metabolic fuel selection at HA, as well as at SL.
The structural distribution of cooperative interactions in proteins: Analysis of the native state ensemble
Authors: Hilser Vincent J. , Dowdy David , Oas Terrence G. , Freire Ernesto
Published: July 2002

Cooperative interactions link the behavior of different amino acid residues within a protein molecule. As a result, the effects of chemical or physical perturbations to any given residue are propagated to other residues by an intricate network of interactions. Very often, amino acids “sense” the effects of perturbations occurring at very distant locations in the protein molecule. In these studies, we have investigated by computer simulation the structural distribution of those interactions. We show here that cooperative interactions are not intrinsically bi-directional and that different residues play different roles within the intricate network of interactions existing in a protein. The effect of a perturbation to residue j on residue k is not necessarily equal to the effect of the same perturbation to residue k on residue j. In this paper, we introduce a computer algorithm aimed at mapping the network of cooperative interactions within a protein. This algorithm exhaustively performs single site thermodynamic mutations to each residue in the protein and examines the effects of those mutations on the distribution of conformational states. The algorithm has been applied to three different proteins (λ repressor fragment 6–85, chymotrypsin inhibitor 2, and barnase). This algorithm accounts well for the observed behavior of these proteins.
A putative vacuolar cargo receptor partially colocalizes with AtPEP12p on a prevacuolar compartment in
Arabidopsis
roots
Authors: Sanderfoot Anton A. , Ahmed Sharif U. , Marty-Mazars Danièle , Rapoport Iris , Kirchhausen Tomas , Marty Francis , Raikhel Natasha V.
Published: July 2002

Targeting of protein cargo to the vacuole/lysosome is a multistep process that appears to have conserved features between mammalian, yeast, and plant cells. In each case, some soluble vacuolar/lysosomal proteins are believed to be bound by transmembrane cargo receptors in the trans-Golgi network (TGN) that redirect these proteins into clathrin-coated vesicles. These vesicles then appear to be transported to the prevacuole/endosome by a trafficking machinery that requires components identified in other vesicle-targeting steps such as
N
-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment protein (SNAP), SNAP receptors (SNAREs), rab-type GTPases, and Sec1p homologs. Two likely members of this trafficking machinery have been characterized from
Arabidopsis thaliana
: AtPEP12p, a t-SNARE that resides on a what we now call a prevacuolar compartment, and AtELP, a protein that shares many common features with mammalian and yeast transmembrane cargo receptors. Here, we have further investigated the intracellular distribution of AtELP. We have found that AtELP is located at the trans-Golgi of
Arabidopsis
root cells, and that its C terminus can preferentially interact
in vitro
with the mammalian TGN-specific AP-1 clathrin–adapter complex, suggesting a likely role in clathrin-coated, vesicle-directed trafficking at the TGN. Further, consistent with a role in trafficking of vacuolar cargo, we have found that AtELP partially colocalizes with AtPEP12p on a prevacuolar compartment.
Natal dispersal and senescence
Authors: Ronce Ophélie , Clobert Jean , Massot Manuel
Published: July 2002

The potential existence of natal dispersal strategies depending on parental age has been suggested by Hamilton and May [Hamilton, W. D. & May, R. M. (1977)
Nature
269, 578–581] for organisms whose survival rates decline with age. When competition between parent and offspring is strong, any individual should disperse a smaller fraction of its offspring when it ages. Here, we verify their verbal prediction. First, we determine the evolutionarily stable dispersal strategy conditional on parental age, associated with a particular senescence curve. We show that such a conditional dispersal strategy should evolve independently from the genotype controlling the offspring dispersal behavior. Second, studying a population of common lizards, we provide empirical evidence of a relation between dispersal of female offspring and maternal senescence, in agreement with our theoretical predictions.
Molecular determinant of ion selectivity of a (Na
+
+ K
+
)-coupled rat brain glutamate transporter
Authors: Zhang Yumin , Bendahan Annie , Zarbiv Ruth , Kavanaugh Michael P. , Kanner Baruch I.
Published: July 2002

Glutamate transporters remove this neurotransmitter from the synaptic cleft by a two-stage electrogenic process, in which glutamate is first cotransported with three sodium ions and a proton. Subsequently, the cycle is completed by translocation of a potassium ion in the opposite direction. Recently, we have identified an amino acid residue of the glutamate transporter GLT-1 (Glu-404) that influences potassium coupling. We have now analyzed the effect of seven other amino acid residues in the highly conserved region surrounding this site. One of these residues, Tyr-403, also proved important for potassium coupling, because mutation to Phe (Y403F) resulted in an electroneutral obligate exchange mode of glutamate transport. This mutation in the transporter also caused an approximately 8-fold increase in the apparent sodium affinity, with no change in the apparent affinity for
l
-glutamate or
d
-aspartate. Strikingly, although exchange catalyzed by the wild-type transporter is strictly dependent on sodium, the selectivity of Y403F mutant transporters is altered so that sodium can be replaced by other alkaline metal cations including lithium and cesium. These results indicate the presence of interacting sites in or near the transporter pore that control selectivity for sodium and potassium.
Unmasking the functions of the chromaffin cell α
7
nicotinic receptor by using short pulses of acetylcholine and selective blockers
Authors: López Manuela G. , Montiel Carmen , Herrero Carlos J. , García-Palomero Esther , Mayorgas Inés , Hernández-Guijo Jesús M. , Villarroya M. , Olivares Román , Gandía Luis , McIntosh J. Michael , Olivera Baldomero M. , García Antonio G.
Published: July 2002

Methyllycaconitine (MLA), α-conotoxin ImI, and α-bungarotoxin inhibited the release of catecholamines triggered by brief pulses of acetylcholine (ACh) (100 μM, 5 s) applied to fast-superfused bovine adrenal chromaffin cells, with IC
50
s of 100 nM for MLA and 300 nM for α-conotoxin ImI and α-bungarotoxin. MLA (100 nM), α-conotoxin ImI (1 μM), and α-bungarotoxin (1 μM) halved the entry of
45
Ca
2+
stimulated by 5-s pulses of 300 μM ACh applied to incubated cells. These supramaximal concentrations of α
7
nicotinic receptor blockers depressed by 30% (MLA), 25% (α-bungarotoxin), and 50% (α-conotoxin ImI) the inward current generated by 1-s pulses of 100 μM ACh, applied to voltage-clamped chromaffin cells. In
Xenopus
oocytes expressing rat brain α
7
neuronal nicotinic receptor for acetylcholine nAChR, the current generated by 1-s pulses of ACh was blocked by MLA, α-conotoxin ImI, and α-bungarotoxin with IC
50
s of 0.1 nM, 100 nM, and 1.6 nM, respectively; the current through α
3
β
4
nAChR was unaffected by α-conotoxin ImI and α-bungarotoxin, and weakly blocked by MLA (IC
50
= 1 μM). The functions of controlling the electrical activity, the entry of Ca
2+
, and the ensuing exocytotic response of chromaffin cells were until now exclusively attributed to α
3
β
4
nAChR; the present results constitute the first evidence to support a prominent role of α
7
nAChR in controlling such functions, specially under the more physiological conditions used here to stimulate chromaffin cells with brief pulses of ACh.
Verb generation in patients with focal frontal lesions: A neuropsychological test of neuroimaging findings
Authors: Thompson-Schill Sharon L. , Swick Diane , Farah Martha J. , D’Esposito Mark , Kan Irene P. , Knight Robert T.
Published: July 2002

What are the neural bases of semantic memory? Traditional beliefs that the temporal lobes subserve the retrieval of semantic knowledge, arising from lesion studies, have been recently called into question by functional neuroimaging studies finding correlations between semantic retrieval and activity in left prefrontal cortex. Has neuroimaging taught us something new about the neural bases of cognition that older methods could not reveal or has it merely identified brain activity that is correlated with but not causally related to the process of semantic retrieval? We examined the ability of patients with focal frontal lesions to perform a task commonly used in neuroimaging experiments, the generation of semantically appropriate action words for concrete nouns, and found evidence of the necessity of the left inferior frontal gyrus for certain components of the verb generation task. Notably, these components did not include semantic retrieval
per se
.
Positive Darwinian selection after gene duplication in primate ribonuclease genes
Authors: Zhang Jianzhi , Rosenberg Helene F. , Nei Masatoshi
Published: July 2002

Evolutionary mechanisms of origins of new gene function have been a subject of long-standing debate. Here we report a convincing case in which positive Darwinian selection operated at the molecular level during the evolution of novel function by gene duplication. The genes for eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) in primates belong to the ribonuclease gene family, and the ECP gene, whose product has an anti-pathogen function not displayed by EDN, was generated by duplication of the EDN gene about 31 million years ago. Using inferred nucleotide sequences of ancestral organisms, we showed that the rate of nonsynonymous nucleotide substitution was significantly higher than that of synonymous substitution for the ECP gene. This strongly suggests that positive Darwinian selection operated in the early stage of evolution of the ECP gene. It was also found that the number of arginine residues increased substantially in a short period of evolutionary time after gene duplication, and these amino acid changes probably produced the novel anti-pathogen function of ECP.
Runaway ornament diversity caused by Fisherian sexual selection
Authors: Pomiankowski Andrew , Iwasa Yoh
Published: July 2002

Fisher’s runaway process of sexual selection is potentially an
important force generating character divergence between closely related
populations. We investigated the evolution of multiple female
preferences by Fisher’s runaway process. There are two outcomes of
runaway. The first is the evolution of mate preference to a stable
equilibrium. This evolution occurs if the benefits of mate choice are
sufficiently large relative to the cost of choice. Alternatively, mate
preferences evolve cyclically. The rate and pattern of cyclic evolution
depends primarily on the individual cost of choice and epistasis in the
joint cost of choice. If there are small differences in natural
selection (e.g., predation risk) between populations, cyclic evolution
quickly leads to divergence in mate preferences and sexual ornaments
and so to sexual isolation.
Mutation R120G in αB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function
Authors: Bova Michael P. , Yaron Orna , Huang Qingling , Ding Linlin , Haley Dana A. , Stewart Phoebe L. , Horwitz Joseph
Published: July 2002

αB-crystallin, a member of the small heat shock protein family, possesses chaperone-like function. Recently, it has been shown that a missense mutation in αB-crystallin, R120G, is genetically linked to a desmin-related myopathy as well as to cataracts [Vicart, P., Caron, A., Guicheney, P., Li, A., Prevost, M.-C., Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.-M.,et al. (1998)Nat. Genet.20, 92–95]. By using α-lactalbumin, alcohol dehydrogenase, and insulin as target proteins,in vitroassays indicated that R120G αB-crystallin had reduced or completely lost chaperone-like function. The addition of R120G αB-crystallin to unfolding α-lactalbumin enhanced the kinetics and extent of its aggregation. R120G αB-crystallin became entangled with unfolding α-lactalbumin and was a major portion of the resulting insoluble pellet. Similarly, incubation of R120G αB-crystallin with alcohol dehydrogenase and insulin also resulted in the presence of R120G αB-crystallin in the insoluble pellets. Far and near UV CD indicate that R120G αB-crystallin has decreased β-sheet secondary structure and an altered aromatic residue environment compared with wild-type αB-crystallin. The apparent molecular mass of R120G αB-crystallin, as determined by gel filtration chromatography, is 1.4 MDa, which is more than twice the molecular mass of wild-type αB-crystallin (650 kDa). Images obtained from cryoelectron microscopy indicate that R120G αB-crystallin possesses an irregular quaternary structure with an absence of a clear central cavity. The results of this study show, through biochemical analysis, that an altered structure and defective chaperone-like function of αB-crystallin are associated with a point mutation that leads to a desmin-related myopathy and cataracts.
Reprogramming of intestinal differentiation and intercalary regeneration inCdx2mutant mice
Authors: Beck Felix , Chawengsaksophak Kallayanee , Waring Paul , Playford Raymond J. , Furness John B.
Published: July 2002

The homeobox geneCdx2, a homologue of theDrosophilagenecaudal, has been implicated in the control of cell differentiation in the intestinal epithelium. Recently, we showed that mice in which one allele of theCdx2gene had been inactivated by homologous recombination developed multiple intestinal polyp-like lesions that did not expressCdx2and that contained areas of squamous metaplasia in the form of keratinizing stratified squamous epithelium, similar to that occurring in the mouse esophagus and forestomach. We have now examined colonic lesions from 98Cdx2+/− mice and report that the lesions are composed of heterotopic stomach and small intestinal mucosa. We conclude thatCdx2directs endodermal differentiation toward a caudal phenotype and that haploinsufficient levels of expression in the developing distal intestine lead to homeotic transformation to a more rostral endodermal phenotype, such as forestomach epithelium that does not expressCdx2during normal development. Intercalary growth (epimorphic regeneration), which previously has never been described in mammals, then occurs, resulting in the ordered “filling in” of tissue types at the discontinuity between the gastric and colonic epithelia. This intercalary growth in a restricted space results in the formation of the polypoid lesions observed.
Mechanism for elimination of a tumor suppressor: Aberrant splicing of a brain-specific exon causes loss of function of Bin1 in melanoma
Authors: Ge Kai , DuHadaway James , Du Wei , Herlyn Meenhard , Rodeck Ulrich , Prendergast George C.
Published: July 2002

Loss of tumor suppressors that restrain important oncoproteins such as c-Myc may contribute to malignant progression. Bin1 is an adapter protein with features of a tumor suppressor that was identified through its interaction with and inhibition of the oncogenic properties of c-Myc. In this study, we analyzed the patterns of Bin1 expression in normal melanocytes and melanoma cells at different stages of tumor progression. Evidence is provided that Bin1 function is abrogated in melanoma cells by a mechanism based on aberrant splicing of a tissue-specific exon. Specifically, most melanoma cells inappropriately expressed exon 12A, which is spliced alternately into Bin1 isoforms found in brain but not into isoforms found in melanocytes and many other nonneuronal cells. Exon 12A sequences abolished the ability of Bin1 to inhibit malignant transformation by c-Myc or adenovirus E1A. Similarly, these sequences abolished the ability of Bin1 to induce programmed cell death in melanoma cells that endogenously expressed exon 12A. Our findings suggest that aberrant splicing of Bin1 may contribute to melanoma progression, and they define a mechanism by which the activity of a tumor suppressor can be eliminated in cells.
Systematic changes in gene expression patterns following adaptive evolution in yeast
Authors: Ferea Tracy L. , Botstein David , Brown Patrick O. , Rosenzweig R. Frank
Published: July 2002

Culturing a population of
Saccharomyces cerevisiae
for many generations under conditions to which it is not optimally adapted selects for fitter genetic variants. This simple experimental design provides a tractable model of adaptive evolution under natural selection. Beginning with a clonal, founding population, independently evolved strains were obtained from three independent cultures after continuous aerobic growth in glucose-limited chemostats for more than 250 generations. DNA microarrays were used to compare genome-wide patterns of gene expression in the evolved strains and the parental strain. Several hundred genes were found to have significantly altered expression in the evolved strains. Many of these genes showed similar alterations in their expression in all three evolved strains. Genes with altered expression in the three evolved strains included genes involved in glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, and metabolite transport. These results are consistent with physiological observations and indicate that increased fitness is acquired by altering regulation of central metabolism such that less glucose is fermented and more glucose is completely oxidized.
Determinants of aminoglycoside-binding specificity for rRNA by using mass spectrometry
Authors: Griffey Richard H. , Hofstadler Steven A. , Sannes-Lowery Kristin A. , Ecker David J. , Crooke Stanley T.
Published: July 2002

We have developed methods for studying the interactions between small molecules and RNA and have applied them to characterize the binding of three classes of aminoglycoside antibiotics to ribosomal RNA subdomains. High-resolution MS was used to quantitatively identify the noncovalent binding interactions between mixtures of aminoglycosides and multiple RNA targets simultaneously. Signal overlap among RNA targets was avoided by the addition of neutral mass tags that direct each RNA target to a unique region of the spectrum. In addition to determining binding affinities, the locations of the binding sites on the RNAs were identified from a protection pattern generated by fragmenting the aminoglycoside/RNA complex. Specific complexes were observed for the prokaryotic rRNA A-site subdomain with ribostamycin, paromomycin, and lividomycin, whereas apramycin preferentially formed a complex with the eukaryotic subdomain. We show that differences in binding between paromomycin and ribostamycin can be probed by using an MS–MS protection assay. We have introduced specific base substitutions in the RNA models and have measured their impact on binding affinity and selectivity. The binding of apramycin to the prokaryotic subdomain strongly depends on the identity of position 1408, as evidenced by the selective increase in affinity for an A1408G mutant. An A1409–G1491 mismatch pair in the prokaryotic subdomain enhanced the binding of tobramycin and bekanamycin. These observations demonstrate the power of MS-based methods to provide molecular insights into small molecule/RNA interactions useful in the design of selective new antimicrobial drugs.
Molecular cloning of an apoptosis-inducing protein, pierisin, from cabbage butterfly: Possible involvement of ADP-ribosylation in its activity
Authors: Watanabe Masahiko , Kono Takuo , Matsushima-Hibiya Yuko , Kanazawa Takashi , Nishisaka Nobuyasu , Kishimoto Taketoshi , Koyama Kotaro , Sugimura Takashi , Wakabayashi Keiji
Published: July 2002

We have previously reported that the cabbage butterfly,
Pieris rapae
, contains a 98-kDa protein, named pierisin, that induces apoptosis in a variety of human cancer cell lines. In the present study, sequencing and cloning of a cDNA encoding pierisin was accomplished. PCR-direct sequencing showed that the gene encodes an 850-amino acid protein with a calculated molecular weight of 98,081. An intact clone at the amino acid level encompassing the entire coding region was obtained by recombination of two independent clones, and the molecular mass of its
in vitro
expressed protein was about 100 kDa on SDS/PAGE, the same as that of purified native pierisin. The expressed protein induced apoptosis in human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, like the native protein, indicating functional activity. The deduced amino acid sequence of pierisin showed 32% homology with a 100-kDa mosquitocidal toxin from
Bacillus sphaericus
SSII-1. In addition, pierisin showed regional sequence similarities with ADP-ribosylating toxins, such as the A subunit of cholera toxin. A glutamic acid residue at the putative NAD-binding site, conserved in all ADP-ribosylating toxins, was also found in pierisin. Substitution of another amino acid for glutamic acid 165 resulted in a great decrease in cytotoxicity and induction of apoptosis. Moreover, inhibitors of ADP-ribosylating enzymes reduced pierisin-induced apoptosis. These results suggest that the apoptosis-inducing protein pierisin might possess ADP-ribosylation activity that leads to apoptosis of the cells.
Equilibria and kinetics of folding of gelsolin domain 2 and mutants involved in familial amyloidosis–Finnish type
Authors: Isaacson Rivka L. , Weeds Alan G. , Fersht Alan R.
Published: July 2002

Mutations D187N and D187Y in domain 2 of the actin-regulating protein gelsolin cause familial amyloidosis–Finnish type (FAF). We have constructed and expressed a recombinant version of gelsolin domain 2 that is sufficiently stable for kinetic and equilibrium measurements. The wild-type domain and the two amyloidogenic mutants fold via simple two-state kinetics without the accumulation of an intermediate. Unfolding kinetics exhibits significant curvature with increasing urea concentration, indicating that the transition state for unfolding becomes more native-like under increasingly denaturing conditions in accordance with the Hammond postulate. Mutations D187N and D187Y destabilize gelsolin domain 2 by 1.22 and 2.16 kcal⋅mol
−1
(1 kcal = 4.18 kJ) respectively. The mutations do not prevent disulfide bond formation despite their direct contiguity with a cysteine residue involved in disulfide linkage. The destabilization conferred on gelsolin domain 2 by the FAF mutations is sufficient to predict that an appreciable fraction is unfolded and, therefore, extremely susceptible to proteolysis at body temperature.