bluuebunny/crossref_metadata_2025 huggingface dataset:
β-Lactam synthetase: A new biosynthetic enzyme
Authors: Bachmann Brian O. , Li Rongfeng , Townsend Craig A.
Published: July 2002

The principal cause of bacterial resistance to penicillin and other β-lactam antibiotics is the acquisition of plasmid-encoded β-lactamases, enzymes that catalyze hydrolysis of the β-lactam bond and render these antibiotics inactive. Clavulanic acid is a potent inhibitor of β-lactamases and has proven clinically effective in combating resistant infections. Although clavulanic acid and penicillin share marked structural similarities, the biosyntheses of their bicyclic nuclei are wholly dissimilar. In contrast to the efficient iron-mediated oxidative cyclization of a tripeptide to isopenicillin N, the critical β-lactam ring of clavulanic acid is demonstrated to form by intramolecular closure catalyzed by a new type of ATP/Mg
2+
-dependent enzyme, a β-lactam synthetase (β-LS). Insertional inactivation of its encoding gene in wild-type
Streptomyces clavuligerus
resulted in complete loss of clavulanic acid production and the accumulation of N
2
-(carboxyethyl)-
l
-arginine (CEA). Chemical complementation of this blocked mutant with authentic deoxyguanidinoproclavaminic acid (DGPC), the expected product of the β-LS, restored clavulanic acid synthesis. Finally, overexpression of this gene gave the β-LS, which was shown to mediate the conversion of CEA to DGPC in the presence of ATP/Mg
2+
. Primary amino acid sequence comparisons suggest that this mode of β-lactam formation could be more widely spread in nature and mechanistically related to asparagine synthesis.
Redesigning an FKBP–ligand interface to generate chemical dimerizers with novel specificity
Authors: Clackson Tim , Yang Wu , Rozamus Leonard W. , Hatada Marcos , Amara Jane F. , Rollins Carl T. , Stevenson Lauren F. , Magari Shannon R. , Wood Susan A. , Courage Nancy L. , Lu Xiaode , Cerasoli Franklin , Gilman Michael , Holt Dennis A.
Published: July 2002

FKBP ligand homodimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP. However, use of these dimerizers
in vivo
is potentially limited by ligand binding to endogenous FKBP. We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket. A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with the modified ligand only partially filling the engineered cavity. We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor. Cells expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP.
Evolution of dendritic patterns during alloy solidification: Onset of the initial instability
Authors: Losert W. , Shi B. Q. , Cummins H. Z.
Published: July 2002

The evolution of a dendritic pattern from a planar solid–liquid
interface during directional solidification of a binary alloy was
investigated experimentally. The model alloy used was the transparent
organic crystal succinonitrile doped with the laser dye coumarin 152.
The buildup of solute ahead of the initially stable planar interface
and the subsequent instability of the planar front were measured in
detail and compared with recent theoretical calculations by Warren and
Langer [Warren, J. A. & Langer, J. S. (1993)
Phys.
Rev. E
47, 2702- 2712]. The fluorescence of coumarin 152
was used for direct observations of the evolution of the solute
concentration profile ahead of the initially planar solid–liquid
interface. UV absorption was used to produce thermal perturbations of
the sample that generated spatially periodic modulations of the planar
interface. This technique allows for measurement of both positive and
negative linear growth coefficients (determined from the growth or
decay rate of the modulation after the perturbation is switched off)
for a large range of wave vectors. Measurements of the evolution of the
concentration profile and the linear growth coefficients, and the
occurrence of the initial instability, were in good agreement with the
Warren–Langer predictions.
pRB plays an essential role in cell cycle arrest induced by DNA damage
Authors: Harrington Elizabeth A. , Bruce Jacqueline L. , Harlow Ed , Dyson Nicholas
Published: July 2002

To maintain genome stability, cells with damaged DNA must arrest to allow repair of mutations before replication. Although several key components required to elicit this arrest have been discovered, much of the pathway remains elusive. Here we report that pRB acts as a central mediator of the proliferative block induced by a diverse range of DNA damaging stimuli.
Rb
−/−
mouse embryo fibroblasts are defective in arrest after γ-irradiation, UV irradiation, and treatment with a variety of chemotherapeutic drugs. In contrast, the pRB related proteins p107 and p130 do not play an essential part in the DNA damage response. pRB is required specifically for the G
1
/S phase checkpoint induced by γ-irradiation. Despite a defect in G
1
/S phase arrest, levels of p53 and p21 are increased normally in
Rb
−/−
cells in response to γ-irradiation. These results lead us to propose a model in which pRB acts as an essential downstream target of the DNA damage-induced arrest pathway. The ability of pRB to prevent replication of damaged DNA is likely to inhibit the propagation of carcinogenic mutations and may therefore contribute to its role as a tumor suppressor. Furthermore, because many cancer therapies act by damaging DNA, these findings also have implications for the treatment of tumors in which pRB is inactivated.
Detection of 400-year-old
Yersinia pestis
DNA in human dental pulp: An approach to the diagnosis of ancient septicemia
Authors: Drancourt Michel , Aboudharam Gérard , Signoli Michel , Dutour Olivier , Raoult Didier
Published: July 2002

Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for
Yersinia pestis
, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague (“plague teeth”) and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human β-globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for
Y. pestis rpoB
(the RNA polymerase β-subunit-encoding gene) and the recognized virulence-associated
pla
(the plasminogen activator-encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific
pla
sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (
P
< 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks.
A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,
N
4
-ethenocytosine and the G/T mismatch
Authors: Hang B. , Medina M. , Fraenkel-Conrat H. , Singer B.
Published: July 2002

Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,
N
6
-ethenoadenine (ɛA) and 3,
N
4
-ethenocytosine (ɛC) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-
N
-glycosylase, which excises ɛA. The present work is directed toward identifying and purifying the human glycosylase activity releasing ɛC. HeLa cells were subjected to multiple steps of column chromatography, including two ɛC-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the ɛC activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having ɛC-DNA glycosylase activity. In addition, oligonucleotides containing ɛC⋅G or G/T(U), could compete for ɛC protein binding, further indicating that the ɛC-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for ɛC also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium,
Methanobacterium
thermoautotrophicum
THF.
Leading role of thalamic over cortical neurons during postinhibitory rebound excitation
Authors: Grenier F. , Timofeev I. , Steriade M.
Published: July 2002

The postinhibitory rebound excitation is an intrinsic property of thalamic and cortical neurons that is implicated in a variety of normal and abnormal operations of neuronal networks, such as slow or fast brain rhythms during different states of vigilance as well as seizures. We used dual simultaneous intracellular recordings of thalamocortical neurons from the ventrolateral nucleus and neurons from the motor cortex, together with thalamic and cortical field potentials, to investigate the temporal relations between thalamic and cortical events during the rebound excitation that follows prolonged periods of stimulus-induced inhibition. Invariably, the rebound spike-bursts in thalamocortical cells occurred before the rebound depolarization in cortical neurons and preceded the peak of the depth-negative, rebound field potential in cortical areas. Also, the inhibitory-rebound sequences were more pronounced and prolonged in cortical neurons when elicited by thalamic stimuli, compared with cortical stimuli. The role of thalamocortical loops in the rebound excitation of cortical neurons was shown further by the absence of rebound activity in isolated cortical slabs. However, whereas thalamocortical neurons remained hyperpolarized after rebound excitation, because of the prolonged spike-bursts in inhibitory thalamic reticular neurons, the rebound depolarization in cortical neurons was prolonged, suggesting the role of intracortical excitatory circuits in this sustained activity. The role of intrathalamic events in triggering rebound cortical activity should be taken into consideration when analyzing information processes at the cortical level; at each step, corticothalamic volleys can set into action thalamic inhibitory neurons, leading to rebound spike-bursts that are transferred back to the cortex, thus modifying cortical activities.
NMR scalar couplings across Watson–Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy
Authors: Pervushin Konstantin , Ono Akira , Fernández César , Szyperski Thomas , Kainosho Masatsune , Wüthrich Kurt
Published: July 2002

This paper describes the NMR observation of
15
N—
15
N and
1
H—
15
N scalar couplings across the hydrogen bonds in Watson–Crick base pairs in a DNA duplex,
h
J
NN
and
h
J
HN
. These couplings represent new parameters of interest for both structural studies of DNA and theoretical investigations into the nature of the hydrogen bonds. Two dimensional [
15
N,
1
H]-transverse relaxation-optimized spectroscopy (TROSY) with a
15
N-labeled 14-mer DNA duplex was used to measure
h
J
NN
, which is in the range 6–7 Hz, and the two-dimensional
h
J
NN
-correlation-[
15
N,
1
H]-TROSY experiment was used to correlate the chemical shifts of pairs of hydrogen bond-related
15
N spins and to observe, for the first time,
h
J
HN
scalar couplings, with values in the range 2–3.6 Hz. TROSY-based studies of scalar couplings across hydrogen bonds should be applicable for large molecular sizes, including protein-bound nucleic acids.
Memory fields of neurons in the primate prefrontal cortex
Authors: Rainer Gregor , Asaad Wael F. , Miller Earl K.
Published: July 2002

Many prefrontal (PF) neurons convey information about both an object’s identity (what) and its location (where). To explore how they represent conjunctions ofwhatandwhere, we explored the receptive fields of their mnemonic activity (i.e., their “memory fields”) by requiring monkeys to remember both an object and its location at many positions throughout a wide portion of central vision. Many PF neurons conveyed object information and had highly localized memory fields that emphasized the contralateral, but not necessarily foveal, visual field. These results indicate that PF neurons can simultaneously convey precise location and object information and thus may play a role in constructing a unified representation of a visual scene.
Protein component of the ribozyme ribonuclease P alters substrate recognition by directly contacting precursor tRNA
Authors: Niranjanakumari S. , Stams Travis , Crary Sharon M. , Christianson David W. , Fierke Carol A.
Published: July 2002

The protein component of ribonuclease P (RNase P) binds to the RNA subunit, forming a functional ribonucleoprotein complex
in vivo
and enhancing the affinity of the precursor tRNA (pre-tRNA) substrate. Photocrosslinking experiments with pre-tRNA bound to RNase P reconstituted with the protein component of
Bacillus subtilis
ribonuclease P (P protein) site specifically modified with a crosslinking reagent indicate that: (
i
) the central cleft of P protein directly interacts with the single-stranded 5′ leader sequence of pre-tRNA, and (
ii
) the orientation and register of the pre-tRNA leader sequence in the central cleft places the protein component in close proximity to the active site. This unique mode of interaction suggests that the catalytic active site in RNase P occurs near the interface of RNA and protein. In contrast to other ribonucleoprotein complexes where the protein mainly stabilizes the active tertiary fold of the RNA, a critical function of the protein component of RNase P is to alter substrate specificity and enhance catalytic efficiency.
Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs
Authors: Fasshauer Dirk , Sutton R. Bryan , Brunger Axel T. , Jahn Reinhard
Published: July 2002

SNARE [soluble NSF (
N
-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four α-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.
Cloned mammalian neutral sphingomyelinase: Functions in sphingolipid signaling?
Authors: Tomiuk Stefan , Hofmann Kay , Nix Michael , Zumbansen Markus , Stoffel Wilhelm
Published: July 2002

Sphingomyelin is an abundant constituent of the plasma membranes of mammalian cells. Ceramide, its primary catabolic intermediate, is released by either acid sphingomyelinase or neutral sphingomyelinase (nSMase) and has emerged as a potential lipid signaling molecule. nSMase is regarded as a key enzyme in the regulated activation of the “sphingomyelin cycle” and cell signaling. We report here the cloning, identification, and functional characterization of murine and human nSMase, a ubiquitously expressed integral membrane protein, which displays all established properties of the Mg
2+
-dependent nSMase of the plasma membrane. Stably nSMase-overexpressing U937 and human embryonic kidney cell lines have been generated for the study of the role of nSMase in signal transduction pathways. Their stimulation by tumor necrosis factor α leads only to a moderately elevated ceramide concentration. Activation of Jun kinase and NFκB and poly(ADP-ribose) polymerase cleavage are identical in mock- and nSMase-transfected cells. Tumor necrosis factor α triggers the ERK1 pathway in none of the cell lines. The cloned nSMase will facilitate further controlled experiments aiming at the definition of a possible role of ceramide as signal transduction molecule.
Separation of killing and tumorigenic effects of an alkylating agent in mice defective in two of the DNA repair genes
Authors: Kawate Hisaya , Sakumi Kunihiko , Tsuzuki Teruhisa , Nakatsuru Yoko , Ishikawa Takatoshi , Takahashi Seiichi , Takano Hiroshi , Noda Tetsuo , Sekiguchi Mutsuo
Published: July 2002

Alkylation of DNA at the
O
6
-position of guanine is one of the most critical events leading to mutation, cancer, and cell death. The enzyme
O
6
-methylguanine-DNA methyltransferase repairs
O
6
-methylguanine as well as a minor methylated base,
O
4
-methylthymine, in DNA. Mouse lines deficient in the methyltransferase (
MGMT
) gene are hypersensitive to both the killing and to the tumorigenic effects of alkylating agents. We now show that these dual effects of an alkylating agent can be dissociated by introduction of an additional defect in mismatch repair. Mice with mutations in both alleles of the
MGMT
gene and one of the mismatch repair genes,
MLH1
, are as resistant to methylnitrosourea (MNU) as are wild-type mice, in terms of survival, but do have numerous tumors after receiving MNU. In contrast to
MGMT
−/−
MLH1
+/+
mice with decrease in size of the thymus and hypocellular bone marrow after MNU administration, no conspicuous change was found in
MGMT
−/−
MLH1
−/−
mice treated in the same manner. Thus, killing and tumorigenic effects of an alkylating agent can be dissociated by preventing mismatch repair pathways.
NOEY2
(ARHI), an imprinted putative tumor suppressor gene in ovarian and breast carcinomas
Authors: Yu Yinhua , Xu Fengji , Peng Hongqi , Fang Xianjun , Zhao Shulei , Li Yang , Cuevas Bruce , Kuo Wen-Lin , Gray Joe W. , Siciliano Michael , Mills Gordon B. , Bast Robert C.
Published: July 2002

Using differential display PCR, we have identified a gene [
NOEY2
, ARHI (designation by the Human Gene Nomenclature Committee)] with high homology to ras and rap that is expressed consistently in normal ovarian and breast epithelial cells but not in ovarian and breast cancers. Reexpression of
NOEY2
through transfection suppresses clonogenic growth of breast and ovarian cancer cells. Growth suppression was associated with down-regulation of the cyclin D1 promoter activity and induction of p21
WAF1/CIP1
. In an effort to identify mechanisms leading to
NOEY2
silencing in cancer, we found that the gene is expressed monoallelically and is imprinted maternally. Loss of heterozygosity of the gene was detected in 41% of ovarian and breast cancers. In most of cancer samples with loss of heterozygosity, the nonimprinted functional allele was deleted. Thus,
NOEY2
appears to be a putative imprinted tumor suppressor gene whose function is abrogated in ovarian and breast cancers.
P/CAF associates with cyclin D1 and potentiates its activation of the estrogen receptor
Authors: McMahon Christine , Suthiphongchai Tuangporn , DiRenzo James , Ewen Mark E.
Published: July 2002

Cyclin D1 is overexpressed in a significant percentage of human breast cancers, particularly in those that also express the estrogen receptor (ER). We and others have demonstrated previously that experimentally overexpressed cyclin D1 can associate with the ER and stimulate its transcriptional functions in the absence of estrogen. This effect is separable from the established function of cyclin D1 as a regulator of cyclin-dependent kinases. Here, we demonstrate that cyclin D1 can also interact with the histone acetyltransferase, p300/CREB-binding protein-associated protein (P/CAF), thereby facilitating an association between P/CAF and the ER. Ectopic expression of P/CAF potentiates cyclin D1-stimulated ER activity in a dose-dependent manner. This effect is largely dependent on the acetyltransferase activity of P/CAF. These results suggest that cyclin D1 may trigger the activation of the ER through the recruitment of P/CAF, by providing histone acetyltransferase activity and, potentially, links to additional P/CAF-associated transcriptional coactivators.
Phosphoinositide 3-kinase lipid products regulate ATP-dependent transport by sister of P-glycoprotein and multidrug resistance associated protein 2 in bile canalicular membrane vesicles
Authors: Misra S. , Ujházy P. , Varticovski L. , Arias I. M.
Published: July 2002

Bile acid transport and secretion in hepatocytes require phosphatidylinositol (PI) 3-kinase-dependent recruitment of ATP-dependent transporters to the bile canalicular membrane and are accompanied by increased canalicular PI 3-kinase activity. We report here that the lipid products of PI 3-kinase also regulate ATP-dependent transport of taurocholate and dinitrophenyl-glutathione directly in canalicular membranes. ATP-dependent transport of taurocholate and dinitrophenyl-glutathione in isolated canalicular vesicles from rat liver was reduced 50–70% by PI 3-kinase inhibitors, wortmannin, and LY294002, at concentrations that are specific for Type I PI 3-kinase. Inhibition was reversed by addition of lipid products of PI 3-kinase (PI 3,4-bisphosphate and, to a lesser extent, PI 3-phosphate and PI 3,4,5-trisphosphate) but not by PI 4,5-bisphosphate. A membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and leads to activation of PI 3-kinase in macrophages doubled PI 3-kinase activity in canalicular membrane vesicles and enhanced taurocholate and dinitrophenyl-glutathione transport in canalicular membrane vesicles above maximal ATP-dependent transport. The effect of the peptide was blocked by wortmannin and LY294002. PI 3-kinase activity was also necessary for function of the transporters
in vivo
. ATP-dependent transport of taurocholate and PI 3-kinase activity were reduced in canalicular membrane vesicles isolated from rat liver that had been perfused with taurocholate and wortmannin. PI 3,4-bisphosphate enhanced ATP-dependent transport of taurocholate in these vesicles above control levels. Our results indicate that PI 3-kinase lipid products are necessary
in vivo
and
in vitro
for maximal ATP-dependent transport of bile acid and nonbile acid organic anions across the canalicular membrane. Our results demonstrate regulation of membrane ATP binding cassette transporters by PI 3-kinase lipid products.
Regulation of eukaryotic protein synthesis: Selective influenza viral mRNA translation is mediated by the cellular RNA-binding protein GRSF-1
Authors: Park Young Woo , Wilusz Jeffrey , Katze Michael G.
Published: July 2002

To better understand regulation of eukaryotic protein synthesis, we studied cellular and viral mRNA translation in influenza virus-infected cells. Influenza virus infection results in a dramatic shut-off of cellular protein synthesis that is concomitant with selective viral mRNA translation. Earlier work showed that these events are mediated by viral and/or cellular factors binding to the 5′ untranslated region (5′ UTR) of viral mRNAs. To identify trans-acting cellular proteins responsible for selective viral protein synthesis, we employed the yeast three-hybrid system. Using the 5′ UTR of the influenza virus nucleocapsid protein (NP) mRNA as bait, we identified the cellular RNA-recognition motif containing RNA-binding protein G-rich sequence factor 1 (GRSF-1) as a positive-acting translational regulatory factor. The
in vivo
yeast assay revealed GRSF-1 specifically bound to the NP 5′ UTR but not select NP 5′ UTR mutants or cellular RNA 5′ UTRs. These data were confirmed by gel shift assays using recombinant GRSF-1. Importantly, recombinant GRSF-1 specifically stimulated translation of a NP 5′ UTR-driven template in cell-free translation systems. Furthermore, translation efficiency of NP 5′ UTR-driven templates was reduced markedly in GRSF-1-depleted HeLa cell extracts, but restored in GRSF-1-reconstituted extracts. GRSF-1 also stimulated translation of an NP 5′ UTR-driven template in HeLa cell extracts that were depleted of essential factors by addition of RNA oligonucleotides representing the viral 5′ UTR RNA. Taken together, these data document the functional demonstration of a cellular protein binding to influenza virus RNAs and, importantly, suggest that influenza virus may recruit GRSF-1 to the 5′ UTR to ensure preferential translation of viral mRNAs in infected cells.
The neural substrate and temporal dynamics of interference effects in working memory as revealed by event-related functional MRI
Authors: D’Esposito Mark , Postle Bradley R. , Jonides John , Smith Edward E.
Published: July 2002

Research on the prefrontal cortex (PFC) of monkeys and humans indicates that this region supports a heterogeneous repertoire of mental processes that contribute to many complex behaviors, such as working memory. Anatomical evidence for some of these processes derives from functional neuroimaging experiments using blocked experimental designs, which average signal across all components of many trials and therefore cannot dissociate distinct processes with different time courses. Using event-related functional MRI, we were able to isolate temporally the neural correlates of processes contributing to the target presentation, delay, and probe portions of an item-recognition task. Two types of trials were of greatest interest: those with Recent Negative probes that matched an item from the target set of the previous, but not the present, two trials, and those with Nonrecent Negative probes that did not match a target item from either the present or the two previous trials. There was no difference between the two trial types in target presentation (i.e., encoding) or delay-period (i.e., active maintenance) PFC activation, but there was significantly greater activation for Recent Negatives than Nonrecent Negative activation associated with the probe period within left ventrolateral PFC. These findings characterize spatially and temporally a proactive interference effect that may reflect the operation of a PFC-mediated response-inhibition mechanism that contributes to working memory performance.
Structural basis of the conversion of T4 lysozyme into a transglycosidase by reengineering the active site
Authors: Kuroki Ryota , Weaver Larry H. , Matthews Brian W.
Published: July 2002

In contrast to hen egg-white lysozyme, which retains the β-configuration of the substrate in the product, T4 lysozyme (T4L) is an inverting glycosidase. The substitution Thr-26 → His, however, converts T4L from an inverting to a retaining enzyme. It is shown here that the Thr-26 → His mutant is also a transglycosidase. Indeed, the transglycosylation reaction can be more effective than hydrolysis. In contrast, wild-type T4L has no detectable transglycosidase activity. The results support the prior hypothesis that catalysis by the Thr-26 → His mutant proceeds via a covalent intermediate. Further mutations (Glu-11 → His, Asp-20 → Cys) of the T26H mutant lysozyme indicate that the catalytic mechanism of this mutant requires Glu-11 as a general acid but Asp-20 is not essential. The results help provide an overall rationalization for the activity of glycosidases, in which a highly conserved acid group (Glu-11 in T4L, Glu-35 in hen egg-white lysozyme) on the β-side of the substrate acts as a proton donor, whereas alterations in the placement and chemical identity of residues on the α-side of the substrate can lead to catalysis with or without retention of the configuration, to transglycosidase activity, or to the formation of a stable enzyme-substrate adduct.
Distributed representation of objects in the human ventral visual pathway
Authors: Ishai Alumit , Ungerleider Leslie G. , Martin Alex , Schouten Jennifer L. , Haxby James V.
Published: July 2002

Brain imaging and electrophysiological recording studies in humans have reported discrete cortical regions in posterior ventral temporal cortex that respond preferentially to faces, buildings, and letters. These findings suggest a category-specific anatomically segregated modular organization of the object vision pathway. Here we present data from a functional MRI study in which we found three distinct regions of ventral temporal cortex that responded preferentially to faces and two categories of other objects, namely houses and chairs, and had a highly consistent topological arrangement. Although the data could be interpreted as evidence for separate modules, we found that each category also evoked significant responses in the regions that responded maximally to other stimuli. Moreover, each category was associated with its own differential pattern of response across ventral temporal cortex. These results indicate that the representation of an object is not restricted to a region that responds maximally to that object, but rather is distributed across a broader expanse of cortex. We propose that the functional architecture of the ventral visual pathway is not a mosaic of category-specific modules but instead is a continuous representation of information about object form that has a highly consistent and orderly topological arrangement.
Changes in the shapes of leaves and flowers upon overexpression of cytochrome P450 in
Arabidopsis
Authors: Kim Gyung-Tae , Tsukaya Hirokazu , Saito Yoko , Uchimiya Hirofumi
Published: July 2002

In
Arabidopsis
, the two-dimensional expansion of leaves is regulated via the polarized elongation of cells. The ROTUNDIFOLIA3 (ROT3) protein, a member of the family of cytochromes P450, is involved in this process and regulates leaf length. Transgenic plants that overexpressed a wild-type
ROT3
gene had longer leaves than parent plants, without any changes in leaf width. The shapes of floral organs were also altered, but elongation of the stem, roots, and hypocotyls was unaffected. To our knowledge, no similar specific regulation of leaf length has been reported previously. Transgenic plants overexpressing the
rot3
-
2
gene had enlarged leaf blades but leaf petioles of normal length. Morphological alterations in such transgenic plants were associated with changes in shape of leaf cells. The
ROT3
gene seems to play an important role in the polar elongation of leafy organs and should be a useful tool for the biodesign of plant organs.
Selection for improved subtiligases by phage display
Authors: Atwell Shane , Wells James A.
Published: July 2002

Engineering enzyme activity has been challenging because of uncertainties in structure–function relationships and difficulties in screening a large number of mutant enzymes. A product capture strategy using phage display is presented here for the selection of improved enzymes from a large library of variants (>109independently derived mutants). Subtiligase, a double mutant of subtilisin BPN′ that catalyzes the ligation of peptides, was displayed on phage. Twenty-five active site residues were randomly mutated in groups of four or five to yield six different libraries that were independently sorted. Variants that ligated a biotin peptide onto their own extended N termini were selectively captured. Mutant subtiligases were identified that had increased ligase activity. The selection also yielded unexpected subtiligase mutants having residues known to improve the stability and oxidative resistance of wild-type subtilisin. These studies are exemplary for the use of phage to improve enzyme function when it is closely linked to a selectable catalytic event.
Protein design is a key factor for subunit–subunit association
Authors: Clementi Cecilia , Carloni Paolo , Maritan Amos
Published: July 2002

Fundamental questions about role of the quaternary structures are addressed by using a statistical mechanics off-lattice model of a dimer protein. The model, in spite of its simplicity, captures key features of the monomer–monomer interactions revealed by atomic force experiments. Force curves during association and dissociation processes are characterized by sudden jumps followed by smooth behavior and form hysteresis loops. Furthermore, the process is reversible in a finite range of temperature stabilizing the dimer, and the width of the hysteresis loop increases as the design procedure improves: i.e., stabilizes the dimer more. It is shown that, in the interface between the two monomeric subunits, the design procedure naturally favors those amino acids whose mutual interaction is stronger.
Evolutionary consequences of niche construction and their implications for ecology
Authors: Laland K. N. , Odling-Smee F. J. , Feldman M. W.
Published: July 2002

Organisms regularly modify local resource distributions, influencing both their ecosystems and the evolution of traits whose fitness depends on such alterable sources of natural selection in environments. We call these processes niche construction. We explore the evolutionary consequences of niche construction using a two-locus population genetic model, which extends earlier analyses by allowing resource distributions to be influenced both by niche construction and by independent processes of renewal and depletion. The analysis confirms that niche construction can be a potent evolutionary agent by generating selection that leads to the fixation of otherwise deleterious alleles, supporting stable polymorphisms where none are expected, eliminating what would otherwise be stable polymorphisms, and generating unusual evolutionary dynamics. Even small amounts of niche construction, or niche construction that only weakly affects resource dynamics, can significantly alter both ecological and evolutionary patterns.
Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition
Authors: Miyamoto Kazuaki , Khosrof Samer , Bursell Sven-Erik , Rohan Richard , Murata Toshinori , Clermont Allen C. , Aiello Lloyd Paul , Ogura Yuichiro , Adamis Anthony P.
Published: July 2002

Diabetic retinopathy is a leading cause of adult vision loss and blindness. Much of the retinal damage that characterizes the disease results from retinal vascular leakage and nonperfusion. This study shows that diabetic retinal vascular leakage and nonperfusion are temporally and spatially associated with retinal leukocyte stasis (leukostasis) in the rat model of streptozotocin-induced diabetes. Retinal leukostasis increases within days of developing diabetes and correlates with the increased expression of retinal intercellular adhesion molecule-1 (ICAM-1). ICAM-1 blockade with a mAb prevents diabetic retinal leukostasis and vascular leakage by 48.5% and 85.6%, respectively. These data identify the causal role of leukocytes in the pathogenesis of diabetic retinopathy and establish the potential utility of ICAM-1 inhibition as a therapeutic strategy for the prevention of diabetic retinopathy.
The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: Relationship to the structure of α-like DNA polymerases
Authors: Huang Lin , Ishii Keiko Kumura , Zuccola Harmon , Gehring Amy M. , Hwang Charles B. C. , Hogle James , Coen Donald M.
Published: July 2002

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog.
In vitro
, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates
in vivo
. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among α-like DNA polymerases. We measured
K
m
and
k
cat
values for dGTP and ACV triphosphate incorporation and
K
i
values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased
K
m
values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced
k
cat
values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered
k
m
and
k
cat
values. In most cases, the fold increase in
K
i
of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation
in vivo
; however, one mutation conferred a much greater increase in resistance than in
K
i
. The effects of mutations on enzyme kinetics could be explained by using a model of an α-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs
in vivo
, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.
A simple model for calculating the kinetics of protein folding from three-dimensional structures
Authors: Muñoz Victor , Eaton William A.
Published: July 2002

An elementary statistical mechanical model was used to calculate the folding rates for 22 proteins from their known three-dimensional structures. In this model, residues come into contact only after all of the intervening chain is in the native conformation. An additional simplifying assumption is that native structure grows from localized regions that then fuse to form the complete native molecule. The free energy function for this model contains just two contributions—conformational entropy of the backbone and the energy of the inter-residue contacts. The matrix of inter-residue interactions is obtained from the atomic coordinates of the three-dimensional structure. For the 18 proteins that exhibit two-state equilibrium and kinetic behavior, profiles of the free energy versus the number of native peptide bonds show two deep minima, corresponding to the native and denatured states. For four proteins known to exhibit intermediates in folding, the free energy profiles show additional deep minima. The calculated rates of folding the two-state proteins, obtained by solving a diffusion equation for motion on the free energy profiles, reproduce the experimentally determined values surprisingly well. The success of these calculations suggests that folding speed is largely determined by the distribution and strength of contacts in the native structure. We also calculated the effect of mutations on the folding kinetics of chymotrypsin inhibitor 2, the most intensively studied two-state protein, with some success.
Autoproteolysis in nucleoporin biogenesis
Authors: Rosenblum Jonathan S. , Blobel Günter
Published: July 2002

We have molecularly characterized a proteolytic cleavage in conserved nuclear pore complex proteins. This cleavage, previously demonstrated to be essential for the biogenesis of two nuclear pore complex proteins in mammals (Nup98 and Nup96) and yeast (Nup145-N and Nup145-C), occurs between Phe and Ser residues within a highly conserved domain in a polyprotein precursor. Here, we show that a protease is not involved in the cleavage event. By using a combination of domain mapping and site-directed mutagenesis, we demonstrate that the human nuclear pore complex protein Nup98 specifically cleaves itself between F863 and S864. A region of Nup98, amino acids 715–920, is able to cleave, whereas a smaller region, amino acids 772–920, does not cleave. In addition, we have generated a Nup98 mutant that cleaves under defined conditions
in vitro
. Further, the two cleaved fragments of Nup98 form a complex, providing a possible mechanism whereby specific, yet low-affinity, binding between Nup98 and Nup96 is responsible for the nuclear targeting of Nup96. Although apparently unrelated evolutionarily, Nup98 has converged on an autoproteolytic biogenesis mechanism similar to that of hedgehog proteins, the inteins, and the N-terminal nucleophile proteins.
Troglitazone prevents mitochondrial alterations, β cell destruction, and diabetes in obese prediabetic rats
Authors: Higa Moritake , Zhou Yan-Ting , Ravazzola Mariella , Baetens Danielle , Orci Lelio , Unger Roger H.
Published: July 2002

To determine whether the antidiabetic action of troglitazone (TGZ), heretofore attributed to insulin sensitization, also involves protection of β cells from lipoapoptosis, we treated prediabetic Zucker Diabetic Fatty rats with 200 mg/kg per day of TGZ. Their plasma-free fatty acids and triacylglycerol fell to 1.3 mM and 111 mg/dl, respectively, compared with 2.0 mM and 560 mg/dl in untreated controls. Their islet triacylglycerol content was 34% below controls. In islets of control rats, β cells were reduced by 82% and the islet architecture was disrupted; β-cell glucose transporter-2 was absent, 85% of their mitochondria were altered, and they were unresponsive to glucose. In treated rats, the loss of β cells was prevented, as were the loss of β cell glucose transporter-2, the mitochondrial alterations, and the impairment of glucose-stimulated insulin secretion. We conclude that the antidiabetic effect of TGZ in prediabetic Zucker Diabetic Fatty rats involves prevention of lipotoxicity and lipoapoptosis of β cells, as well as improvement in insulin sensitivity.
Identification and reconstitution of the origin recognition complex from
Schizosaccharomyces pombe
Authors: Moon Kyeong-Yeop , Kong Daochun , Lee Joon-Kyu , Raychaudhuri Santanu , Hurwitz Jerard
Published: July 2002

The origin recognition complex (ORC), first identified in
Saccharomyces cerevisiae
(sc), is a six-subunit protein complex that binds to DNA origins. Here, we report the identification and cloning of cDNAs encoding the six subunits of the ORC of
Schizosaccharomyces pombe
(sp). Sequence analyses revealed that spOrc1, 2, and 5 subunits are highly conserved compared with their counterparts from
S. cerevisiae
,
Xenopus
,
Drosophila
, and human. In contrast, both spOrc3 and spOrc6 subunits are poorly conserved. As reported by Chuang and Kelly [(1999)
Proc. Natl. Acad. Sci. USA
96, 2656–2661], the C-terminal region of spOrc4 is also conserved whereas the N terminus uniquely contains repeats of a sequence that binds strongly to AT-rich DNA regions. Consistent with this, extraction of
S. pombe
chromatin with 1 M NaCl, or after DNase I treatment, yielded the six-subunit ORC, whereas extraction with 0.3 M resulted in five-subunit ORC lacking spOrc4p. The spORC can be reconstituted
in vitro
with all six recombinant subunits expressed in the rabbit reticulocyte system. The association of spOrc4p with the other subunits required the removal of DNA from reaction mixture by DNase I. This suggests that a strong interaction between spOrc4p and DNA can prevent the isolation of the six-subunit ORC. The unique DNA-binding properties of the spORC may contribute to our understanding of the sequence-specific recognition required for the initiation of DNA replication in
S. pombe
.
Different population dynamics of human T cell lymphotropic virus type II in intravenous drug users compared with endemically infected tribes
Authors: Salemi Marco , Lewis Martha , Egan John Fergal , Hall William W. , Desmyter Jan , Vandamme Anne-Mieke
Published: July 2002

The phylogeny of human T cell lymphotropic virus type II (HTLV-II) was investigated by using strains isolated from Amerindian and Pygmy tribes, in which the virus is maintained primarily through mother-to-child transmission via breast-feeding, and strains from intravenous drug users (IDUs), in which spread is mainly blood-borne via needle sharing. Molecular clock analysis showed that HTLV-II has two different evolutionary rates with the molecular clock for the virus in IDUs ticking 150–350 times faster than the one in endemically infected tribes: 2.7 × 10
−4
compared with 1.71/7.31 × 10
−7
nucleotide substitutions per site per year in the long terminal repeat region. This dramatic acceleration of the evolutionary rate seems to be related with the mode of transmission. Mathematical models showed the correlation of these two molecular clocks with an endemic spread of HTLV-II in infected tribes compared with the epidemic spread in IDUs. We also noted a sharp increase in the population size of the virus among IDUs during the last decades probably caused by the worldwide increase in intravenous drug use.
Isopentenyl diphosphate biosynthesis via a mevalonate-independent pathway: Isopentenyl monophosphate kinase catalyzes the terminal enzymatic step
Authors: Lange B. Markus , Croteau Rodney
Published: July 2002

In plants, the biosynthesis of isopentenyl diphosphate, the central precursor of all isoprenoids, proceeds via two separate pathways. The cytosolic compartment harbors the mevalonate pathway, whereas the newly discovered deoxyxylulose 5-phosphate pathway, which also operates in certain eubacteria, including
Escherichia coli
, is localized to plastids. Only the first two steps of the plastidial pathway, which involve the condensation of pyruvate and glyceraldehyde 3-phosphate to deoxyxylulose 5-phosphate followed by intramolecular rearrangement and reduction to 2-
C
-methylerythritol 4-phosphate, have been established. Here we report the cloning from peppermint (
Mentha
×
piperita
) and
E. coli
, and expression, of a kinase that catalyzes the phosphorylation of isopentenyl monophosphate as the last step of this biosynthetic sequence to isopentenyl diphosphate. The plant gene defines an ORF of 1,218 bp that, when the proposed plastidial targeting sequence is excluded, corresponds to ≈308 aa with a mature size of ≈33 kDa. The
E. coli
gene (
ychB
), which is located at 27.2 min of the chromosomal map, consists of 852 nt, encoding a deduced enzyme of 283 aa with a size of 31 kDa. These enzymes represent a conserved class of the GHMP family of kinases, which includes
g
alactokinase,
h
omoserine kinase,
m
evalonate kinase, and
p
hosphomevalonate kinase, with homologues in plants and several eubacteria. Besides the preferred substrate isopentenyl monophosphate, the recombinant peppermint and
E. coli
kinases also phosphorylate isopentenol, and, much less efficiently, dimethylallyl alcohol, but dimethylallyl monophosphate does not serve as a substrate. Incubation of secretory cells isolated from peppermint glandular trichomes with isopentenyl monophosphate resulted in the rapid production of monoterpenes and sesquiterpenes, confirming that isopentenyl monophosphate is the physiologically relevant, terminal intermediate of the deoxyxylulose 5-phosphate pathway.