bluuebunny/crossref_metadata_2025 huggingface dataset:
Accurate and rapid docking of protein–protein complexes on the basis of intermolecular nuclear Overhauser enhancement data and dipolar couplings by rigid body minimization
Authors: Clore G. Marius
Published: July 2002

A simple and rapid method is presented for solving the three-dimensional structures of protein–protein complexes in solution on the basis of experimental NMR restraints that provide the requisite translational (i.e., intermolecular nuclear Overhauser enhancement, NOE, data) and orientational (i.e., backbone
1
H-
15
N dipolar couplings and intermolecular NOEs) information. Providing high-resolution structures of the proteins in the unbound state are available and no significant backbone conformational changes occur upon complexation (which can readily be assessed by analysis of dipolar couplings measured on the complex), accurate and rapid docking of the two proteins can be achieved. The method, which is demonstrated for the 40-kDa complex of enzyme I and the histidine phosphocarrier protein, involves the application of rigid body minimization using a target function comprising only three terms, namely experimental NOE-derived intermolecular interproton distance and dipolar coupling restraints, and a simple intermolecular van der Waals repulsion potential. This approach promises to dramatically reduce the amount of time and effort required to solve the structures of protein–protein complexes by NMR, and to extend the capabilities of NMR to larger protein–protein complexes, possibly up to molecular masses of 100 kDa or more.
The importance of reactant positioning in enzyme catalysis: A hybrid quantum mechanics/molecular mechanics study of a haloalkane dehalogenase
Authors: Lau Edmond Y. , Kahn Kalju , Bash Paul A. , Bruice Thomas C.
Published: July 2002

Hybrid quantum mechanics/molecular mechanics calculations using Austin Model 1 system-specific parameters were performed to study the S
N
2 displacement reaction of chloride from 1,2-dichloroethane (DCE) by nucleophilic attack of the carboxylate of acetate in the gas phase and by Asp-124 in the active site of haloalkane dehalogenase from
Xanthobacter autotrophicus
GJ10. The activation barrier for nucleophilic attack of acetate on DCE depends greatly on the reactants having a geometry resembling that in the enzyme or an optimized gas-phase structure. It was found in the gas-phase calculations that the activation barrier is 9 kcal/mol lower when dihedral constraints are used to restrict the carboxylate nucleophile geometry to that in the enzyme relative to the geometries for the reactants without dihedral constraints. The calculated quantum mechanics/molecular mechanics activation barriers for the enzymatic reaction are 16.2 and 19.4 kcal/mol when the geometry of the reactants is in a near attack conformer from molecular dynamics and in a conformer similar to the crystal structure (DCE is gauche), respectively. This haloalkane dehalogenase lowers the activation barrier for dehalogenation of DCE by 2–4 kcal/mol relative to the single point energies of the enzyme's quantum mechanics atoms in the gas phase. S
N
2 displacements of this sort in water are infinitely slower than in the gas phase. The modest lowering of the activation barrier by the enzyme relative to the reaction in the gas phase is consistent with mutation experiments.
Mechanism of the single-headed processivity: Diffusional anchoring between the K-loop of kinesin and the C terminus of tubulin
Authors: Okada Yasushi , Hirokawa Nobutaka
Published: July 2002

A motor-domain construct of KIF1A, a single-headed kinesin superfamily protein, was demonstrated to take more than 600 steps before detaching from a microtubule. However, its molecular mechanism remained unclear. Here we demonstrate the nucleotide-dependent binding between the lysine-rich, highly positively charged loop 12 of the KIF1A motor domain (K-loop) and the glutamate-rich, highly negatively charged C-terminal region of tubulin (E-hook). This binding did not contribute in the strong binding state but only in the weak binding state. This binding was demonstrated to be essential for the single-headed processivity by functioning as the anchor for the one-dimensional simple Brownian movement in the weak binding state. This Brownian movement will allow the small KIF1A motor domain to span the distance between the binding sites on microtubule and also will give the diffusive nature to the movement of single KIF1A molecules. These observations quantitatively fitted well to the predictions made from our Brownian motor model on the mechanism of the single-headed processive movement.
Coordinated plant defense responses inArabidopsisrevealed by microarray analysis
Authors: Schenk Peer M. , Kazan Kemal , Wilson Iain , Anderson Jonathan P. , Richmond Todd , Somerville Shauna C. , Manners John M.
Published: July 2002

Disease resistance is associated with a plant defense response that involves an integrated set of signal transduction pathways. Changes in the expression patterns of 2,375 selected genes were examined simultaneously by cDNA microarray analysis inArabidopsis thalianaafter inoculation with an incompatible fungal pathogenAlternaria brassicicolaor treatment with the defense-related signaling molecules salicylic acid (SA), methyl jasmonate (MJ), or ethylene. Substantial changes (up- and down-regulation) in the steady-state abundance of 705 mRNAs were observed in response to one or more of the treatments, including known and putative defense-related genes and 106 genes with no previously described function or homology. In leaf tissue inoculated withA. brassicicola,the abundance of 168 mRNAs was increased more than 2.5-fold, whereas that of 39 mRNAs was reduced. Similarly, the abundance of 192, 221, and 55 mRNAs was highly (>2.5-fold) increased after treatment with SA, MJ, and ethylene, respectively. Data analysis revealed a surprising level of coordinated defense responses, including 169 mRNAs regulated by multiple treatments/defense pathways. The largest number of genes coinduced (one of four induced genes) and corepressed was found after treatments with SA and MJ. In addition, 50% of the genes induced by ethylene treatment were also induced by MJ treatment. These results indicated the existence of a substantial network of regulatory interactions and coordination occurring during plant defense among the different defense signaling pathways, notably between the salicylate and jasmonate pathways that were previously thought to act in an antagonistic fashion.
A nucleolar protein related to ribosomal protein L7 is required for an early step in large ribosomal subunit biogenesis
Authors: Dunbar David A. , Dragon François , Lee Sarah J. , Baserga Susan J.
Published: July 2002

The
Saccharomyces cerevisiae
Rlp7 protein has
extensive identity and similarity to the large ribosomal subunit L7
proteins and shares an RNA-binding domain with them. Rlp7p is not a
ribosomal protein; however, it is encoded by an essential gene and
therefore must perform a function essential for cell growth. In this
report, we show that Rlp7p is a nucleolar protein that plays a critical
role in processing of precursors to the large ribosomal subunit RNAs.
Pulse–chase labeling experiments with Rlp7p-depleted cells reveal that
neither 5.8S
S
, 5.8S
L
, nor 25S is produced,
indicating that both the major and minor processing pathways are
affected. Analysis of processing intermediates by primer extension
indicates that Rlp7p-depleted cells accumulate the 27SA
3
precursor RNA, which is normally the major substrate (85%) used to
produce the 5.8S and 25S rRNAs, and the ratio of 27SB
L
to
27SB
S
precursors changes from approximately 1:8 to 8:1
(depleted cells). Because 27SA
3
is the direct precursor to
27SB
S
, we conclude that Rlp7p is specifically required for
the 5′ to 3′ exonucleolytic trimming of the 27SA
3
into the
27SB
S
precursor. As it is essential for processing in both
the major and minor pathways, we propose that Rlp7p may act as a
specificity factor that binds precursor rRNAs and tethers the enzymes
that carry out the early 5′ to 3′ exonucleolytic reactions that
generate the mature rRNAs. Rlp7p may also be required for the
endonucleolytic cleavage in internal transcribed spacer 2 that
separates the 5.8S rRNA from the 25S rRNA.
Nitrogen regulatory protein C-controlled genes of
Escherichia coli
: Scavenging as a defense against nitrogen limitation
Authors: Zimmer Daniel P. , Soupene Eric , Lee Haidy L. , Wendisch Volker F. , Khodursky Arkady B. , Peter Brian J. , Bender Robert A. , Kustu Sydney
Published: July 2002

Nitrogen regulatory protein C (NtrC) of enteric bacteria activates
transcription of genes/operons whose products minimize the slowing of
growth under nitrogen-limiting conditions. To reveal the NtrC regulon
of
Escherichia coli
we compared mRNA levels in a mutant
strain that overexpresses NtrC-activated genes
[
glnL
(Up)] to those in a strain with an
ntrC
(
glnG
) null allele by using DNA
microarrays. Both strains could be grown under conditions of nitrogen
excess. Thus, we could avoid differences in gene expression caused by
slow growth or nitrogen limitation
per se
. Rearranging
the spot images from microarrays in genome order allowed us to detect
all of the operons known to be under NtrC control and facilitated
detection of a number of new ones. Many of these operons encode
transport systems for nitrogen-containing compounds, including
compounds recycled during cell-wall synthesis, and hence scavenging
appears to be a primary response to nitrogen limitation. In all, ≈2%
of the
E. coli
genome appears to be under NtrC control,
although transcription of some operons depends on the nitrogen
assimilation control protein, which serves as an adapter between NtrC
and σ
70
-dependent promoters.
Targeted inhibition of transcription elongation in cells mediated by triplex-forming oligonucleotides
Authors: Faria M. , Wood C. D. , Perrouault L. , Nelson J. S. , Winter A. , White M. R. H. , Hélène C. , Giovannangeli C.
Published: July 2002

Triple-helix-forming oligonucleotides (TFOs) bind in the major groove of double-stranded DNA at oligopyrimidine⋅oligopurine sequences and therefore are candidate molecules for artificial gene regulation,
in vitro
and
in vivo
. We recently have described oligonucleotide analogues containing N3′-P5′ phosphoramidate (np) linkages that exhibited efficient inhibition of transcription elongation
in vitro
. In the present work we provide conclusive evidence that np-modified TFOs targeted to the HIV-1 polypurine tract (PPT) sequence can inhibit transcriptional elongation in cells, either in transient or stable expression systems. The same constructs were used in transient expression assays (target sequence on transfected plasmid) and in the generation of stable cell lines (target sequence integrated into cellular chromosomes). In both cases the only distinguishable feature between the cellular systems is the presence of an insert containing the wild-type PPT/HIV-1 sequence, a mutated version with two mismatches, or the absence of the insert altogether. The inhibitory action induced by np-TFOs was restricted to the cellular systems containing the complementary wild-type PPT/HIV-1 target, and consequently can be attributed only to a triple-helix-mediated mechanism. As a part of this study we also have applied an imaging technique to quantitatively investigate the dynamics of TFO-mediated specific gene silencing in single cells.
Point mutations in the murine fumarylacetoacetate hydrolase gene: Animal models for the human genetic disorder hereditary tyrosinemia type 1
Authors: Aponte Jennifer L. , Sega Gary A. , Hauser Loren J. , Dhar Madhu S. , Withrow Catherine M. , Carpenter Donald A. , Rinchik Eugene M. , Culiat Cymbeline T. , Johnson Dabney K.
Published: July 2002

Hereditary tyrosinemia type 1 (HT1) is a severe autosomal recessive
metabolic disease associated with point mutations in the human
fumarylacetoacetate hydrolase (
FAH
) gene that disrupt
tyrosine catabolism. An acute form of HT1 results in death during the
first months of life because of hepatic failure, whereas a chronic form
leads to gradual development of liver disease often accompanied by
renal dysfunction, childhood rickets, neurological crisis, and
hepatocellular carcinoma. Mice homozygous for certain chromosome 7
deletions of the albino
Tyr
;
c
locus that
also include
Fah
die perinatally as a result of liver
dysfunction and exhibit a complex syndrome characterized by structural
abnormalities and alterations in gene expression in the liver and
kidney. Here we report that two independent, postnatally lethal
mutations induced by
N
-ethyl-
N
-nitrosourea and mapped near
Tyr
are alleles of
Fah
. The
Fah
6287SB
allele is a missense mutation in
exon 6, and
Fah
5961SB
is a splice mutation
causing loss of exon 7, a subsequent frameshift in the resulting mRNA,
and a severe reduction of
Fah
mRNA levels. Increased
levels of the diagnostic metabolite succinylacetone in the urine of the
Fah
6287SB
and
Fah
5961SB
mutants indicate that these
mutations cause a decrease in Fah enzymatic activity. Thus, the
neonatal phenotype present in both mutants is due to a deficiency in
Fah caused by a point mutation, and we propose
Fah
5961SB
and
Fah
6287SB
as mouse models for acute and
chronic forms of human HT1, respectively.
Ribosome shunt is essential for infectivity of cauliflower mosaic virus
Authors: Pooggin Mikhail M. , Fütterer Johannes , Skryabin Konstantin G. , Hohn Thomas
Published: July 2002

Cauliflower mosaic virus (CaMV) is a DNA-containing pararetrovirus replicating by means of reverse transcription of a terminally redundant pregenomic 35S RNA that is also used as a polycistronic mRNA. The leader of 35S RNA is long, highly structured, and contains multiple short ORFs (sORFs), which strongly interfere with the ribosome scanning process. Translation of this RNA is initiated by a ribosome shunt mechanism, in which ribosomes translate the most 5′-proximal short ORF (sORF A), then skip a large region of the leader containing a putative RNA encapsidation signal and reinitiate translation at the first long viral ORF. Here, we demonstrate that the efficiency of the sORF A-mediated ribosome shunt is an important determinant of viral infectivity. Point mutations in sORF A, which reduced the basal level of shunt-dependent expression and the degree of shunt enhancement by a CaMV-encoded translation transactivator (TAV), consequently reduced infectivity of the virus in turnip plants. First- or second-site reversions appeared in the viral progeny. The second-site reversions restored shuntdependent expression to an extent correlating with their relative abundance in the progeny. Mutations that abolished both the basal and TAV-activated components of shunting proved to be lethal. Finally, by using an artificial stem structure that blocks scanning, we obtained direct evidence that ribosome shunt operates during CaMV infection.
Expression of scavenger receptor class B type 1 (SR-BI) promotes microvillar channel formation and selective cholesteryl ester transport in a heterologous reconstituted system
Authors: Reaven Eve , Leers-Sucheta Susan , Nomoto Ann , Azhar Salman
Published: July 2002

In the “selective” cholesteryl ester (CE) uptake process,
surface-associated lipoproteins [high density lipoprotein (HDL) and
low density lipoprotein] are trapped in the space formed between
closely apposed surface microvilli (microvillar channels) in
hormone-stimulated steroidogenic cells. This is the same location where
an HDL receptor (SR-BI) is found. In the current study, we sought to
understand the relationship between SR-BI and selective CE uptake in a
heterologous insect cell system.
Sf
9 (
Spodoptera
frugiperda
) cells overexpressing recombinant SR-BI were
examined for (
i
) SR-BI protein by Western blot analysis
and light or electron immunomicroscopy, and (
ii
)
selective lipoprotein CE uptake by the use of radiolabeled or
fluorescent (BODIPY-CE)-labeled HDL. Noninfected or infected control
Sf
9 cells do not express SR-BI, show microvillar
channels, or internalize CEs. An unexpected finding was the induction
of a complex channel system in
Sf
9 cells expressing
SR-BI. SR-BI-expressing cells showed many cell surface double-membraned
channels, immunogold SR-BI, apolipoprotein (HDL) labeling of the
channels, and high levels of selective HDL-CE uptake. Thus,
double-membraned channels can be induced by expression of recombinant
SR-BI in a heterologous system, and these specialized structures
facilitate both the binding of HDL and selective HDL-CE uptake.
Effects of Labelled Gender on Vocal Communication of Young Women with 4‐month‐old Infants
Authors: Pomerleau Andrée , Malcuit Gérard , Turgeon Lyse , Cossette Louise
Published: July 2002

Most of the previous studies analyzing the effect of gender label on adults' interactive behaviours with infants concluded that gender stereotypes affect adults' behaviours more than the actual behaviours or characteristics of the infants. These stereotypes and their ensuing behaviours would contribute to the differential socialization experiences of infants according to their gender. The objective of the present study was to investigate further the effect of gender label on adults' vocal communication with infants. Therefore, the prosodic and content features of the language addressed by young women to infants presented as girls and as boys were examined. Sixteen women were observed during two 5‐minute sessions of face‐to‐face interaction with 3‐to‐4‐month‐old infants, one introduced as a girl, the other as a boy, in a counterbalanced order. Six girls and four boys served as stimuli. The number of utterances addressed to the infants, their duration and fundamental frequency, as well as the prosodic contour and the content of each vocalization were measured. Results indicated only one significant gender label effect: Women referred more frequently to infant's global motor activity when the infant was presented as a boy. This observation is congruent with other data. However, our results do not demonstrate a consistent pattern of gender label effect when women are talking to unfamiliar infants in such a context.
Globalisation as Reterritorialisation: The Re-scaling of Urban Governance in the European Union
Authors: Brenner Neil
Published: July 2002

In the rapidly growing literatures on globalisation, many authors have emphasised the apparent disembedding of social relations from their local-territorial pre-conditions. However, such arguments neglect the relatively fixed and immobile forms of territorial organisation upon which the current round of globalisation is premised, such as urban-regional agglomerations and territorial states. This article argues that processes of reterritorialisation—the reconfiguration and re-scaling of forms of territorial organisation such as cities and states—constitute an intrinsic moment of the current round of globalisation. Globalisation is conceived here as a reterritorialisation of both socioeconomic and political-institutional spaces that unfolds simultaneously upon multiple, superimposed geographical scales. The territorial organisation of contemporary urban spaces and state institutions must be viewed at once as a presupposition, a medium and an outcome of this highly conflictual dynamic of global spatial restructuring. On this basis, various dimensions of urban governance in contemporary Europe are analysed as expressions of a politics of scale that is emerging at the geographical interface between processes of urban restructuring and state territorial restructuring.
‘Intense, but it's all worth it in the end’: The colearner's experience of the research process
Authors: Taber Keith S.
Published: July 2002

Abstract Detailed enquiry into the learning process requires in‐depth case studies of individual learners. Such research involves the informants in considerable time commitments, as well as the risk of exposing their personal limitations as learners. This can be informative for the researcher, but could also be threatening and demoralising for the ‘subject’. Such issues are especially significant for practitioner‐researchers, where there is the potential for conflicts of interest due to the dual teacher‐researcher role. This article discusses one study where a simple ethical framework was explicitly employed to protect the interests of the learners. The informants were conceptualised as ‘colearners’ who should feel they benefited from their involvement, and opportunities were taken to collect simple feedback on their perceptions of research activities. The value and limitations of such data are considered. The wider significance of the colearners' comments, and the potential methodological repercussions of prioritising the ‘ethical imperative’ are discussed.
Injecting drug use and blood‐borne viruses: a comparison of rural and urban Victoria, 1990–95
Authors: AITKEN CAMPBELL , BROUGH RODGER , CROFTS NICK
Published: July 2002

Most studies of injecting drug users (IDUs) have included only urban IDUs; information about rural IDUs is scarce. Recognizing this, the Victorian Injecting Drug Users Cohort Study (VICS) recruited IDUs from rural Victoria as well as Melbourne (population 3.2 million in 1995), capital city of the state of Victoria, Australia. Examination of data collected from IDUs living in Melbourne and the Western District (largest city: Warrnambool, pop. 27 734) revealed numerous differences in behaviour and serology. The primary drug for most Western District IDUs was amphetamines while Melbourne IDUs preferred heroin. Injecting and sharing frequencies were much lower in the rural sample. Hepatitis C antibody prevalence at first test was significantly higher in Melbourne IDUs, although this was clearly related to the difference in primary drugs; conversely, incidence among Western District amphetamine injectors was 16.2 per 100 person‐years (py) (during 18.5 py), yet no conversions occurred in metropolitan amphetamine injectors in 29.8 py. Western District IDUs were less educated and more likely to be unemployed and of aboriginal descent than metropolitan IDUs. Variation between populations of Victorian IDUs has implications for the delivery of IDU‐related health services; of particular concern is the possibility that hepatitis C has been spreading relatively rapidly among rural IDUs.
Survival and Metabolic Function of Freshly Isolated Rat Hepatocytes Exposed First to a Heat Shock and Then to an Oxidative Stress
Authors: Latour Isabelle , Buc-Calderon Pedro
Published: July 2002

The formation of heat shock proteins (hsp) leading to thermotolerance has been extensively reported in many cell types. In freshly isolated rat hepatocytes, hsp were synthesized after 60 minutes of incubation at 42°C. Cell survival was not modified by such a treatment, but protein synthesis, secretion of triglycerides as lipoproteins, and the maintenance of both ATP and glycogen levels were significantly impaired. When exposed to an oxidative stress, heat-shocked hepatocytes were not more resistant than cells always kept at 37°C. Conversely, the addition of tert-butyl hydroperoxide (tBOOH) resulted, in general, in an increased lactate dehydrogenase leakage. The metabolism of tBOOH, as estimated by the reduced glutathione (GSH) content and GSH peroxidase activity, was similar in both control and heat-shocked hepatocytes. Despite the synthesis of hsp in rat hepatocytes, the lack of resistance to a subsequent oxidant injury may be due to the metabolic impairment caused by the heat shock.
Sociocultural aspects of areca nut use
Authors: Williams S. , Malik A. , Chowdhury S. , Chauhan S.
Published: July 2002

Abstract This paper aims to describe the sociocultural context of areca nut use through exploring people's norms and values and the meaning underlying a wide range of different practices. A historical review is followed by an examination of more recent evidence, including quantitative and qualitative research conducted in the United Kingdom. It is concluded that some reports involving the various uses of areca nut and the conclusions drawn are confusing and that future studies need to be more explicit. While a sound scientific background is required, important sociocultural and religious issues around areca nut use must also be understood if health promotion initiatives are to be considered.
Quality of life, sleep, mood and alcohol consumption: a complex interaction
Authors: Foster J. H. , Peters T. J. , Kind P.
Published: July 2002

Abstract Socio‐demographic information was collected from 52 (45 men, seven women) currently drinking moderately dependent alcohol misusers attending an outpatient clinic in South London for medical assessment and treatment. Assessments at baseline and 12‐week follow‐up included: (i) Severity of Alcohol Dependence (SADQ) (baseline only), (ii) Hospital Anxiety and Depression Scale (HADS); (iii) Nottingham Health Profile sleep subscale (NHP) and (iv) the EuroQoL (EQ‐5D). The main outcome measure was drinking at a “sensible level” at 12‐week follow‐up as recommended by the Royal College of Psychiatrists. There were significant interactions between sleeping badly, lying awake at night and HADS depression scores. The Health‐related Quality of Life (HrQoL) of this group was poor compared to general population norms. Lower EQ‐5D index scores were associated with poorer educational attainment and lower EQ‐5D Visual Analogue Scale (VAS) patient ratings with greater baseline alcohol consumption. Clinician ratings on the EQ‐5D VAS were consistently lower than the patient ratings. The correlations between patient self‐assessment and clinician ratings (EQ‐5D VAS) were not significant. The forty‐seven subjects (90%) who were successfully followed‐up showed a significant reduction in the total amount of alcohol consumed. Ten (21%) subjects returned to sensible drinking levels at 12 weeks but there was not a corresponding improvement in HrQoL, sleep, or affective status scores or biochemical measures in these subjects.
Gastritis in the alcoholic: relationship to gastric alcohol metabolism and Helicobacter pylori
Authors: Lieber Charles S.
Published: July 2002

Chronic gastritis is common in the alcoholic. It is characterized by histological inflammation of the gastric mucosa and is associated with variable symptomatology. Its etiology is still the subject of debate. Recently, a new alcohol dehydrogenase isoenzyme, called sigma ADH, absent from the liver but predominant in the upper GI tract, has been fully characterized, its gene cloned, and it appears to play a major role in gastric ethanol metabolism. Indeed, it has now been established, both in vivo in experimental animals and in vitro in cultured human gastric cells, that alcohol is metabolized in the gastric mucosa, resulting in the production of acetaldehyde, a toxic metabolite. In addition, Helicobacter pylori infection is common in the alcoholic, resulting in the breakdown of urea to ammonia, another toxic product. A number of studies carried out over the last 40 years revealed that antibiotic treatment eradicates ammonia production and results in histological and symptomatic improvement in the majority of patients with alcoholic gastritis. Non‐invasive tests for the detection of H. pylori are now available which will facilitate the large scale studies needed to confirm whether, in H. pylori ‐positive patients, antibiotics should become routine treatment for alcoholic gastritis.
Non‐word repetition and grammatical morphology: normative data for children in their final year of primary school
Authors: Simkin Zoë , Conti‐Ramsden Gina
Published: July 2002

It is likely that tests of phonological short‐term memory (non‐word repetition) and grammatical morphology (past tense, third‐person singular) are clinical markers for specific language impairment (SLI). The study provides normative data on three specific tests covering the aforementioned areas for 100 children attending the final year of primary schooling (10–11 year olds). Data from typically developing age controls can be used to provide a benchmark against which to compare the ability of language‐impaired children. This is particularly important given that the transition from primary to secondary schooling is a key stage in a child's education.
Spectroscopic Contributions to the Understanding of Hemoglobin Function: Implications for Structural Biology
Authors: Shulman Robert G.
Published: July 2002

Structural biology is based on the assumption that structural determinations will explain macromolecular function. To examine the basis of these proposals, the structure/function connections in hemoglobin have been examined. Presently the Monod, Wyman, Changeux (MWC) model of hemoglobin function has great validity. In this model, ligand‐binding affinities are linked to quaternary structure, and it has been shown that the model describes the function accurately to a high first approximation. To see how this understanding developed, we review two sets of experimental studies in 1970‐71 that supported the applicability of MWC to hemoglobin oxygen binding. One set of data from NMR and ligand binding kinetics supported the quaternary‐linked nature of binding required by the MWC model. The other approach, by Perutz, proposed a structural basis for MWC, by suggesting that in one quaternary structure the binding of oxygen broke a salt bridge that caused a lowered quaternary‐linked affinity. However, experiments since that time, mostly by X‐ray crystallography of deoxygenated hemoglobin, have failed to show salt bridges breaking upon ligation, whereas affinities have remained low. This pattern of results shows that the small energies responsible for ligand‐binding affinities and reaction rates have not been identified by discrete structural features. Rather, thermodynamic and kinetic data from a variety of spectroscopic studies have played the central role in establishing the MWC model for hemoglobin.
Signaling by the Respiratory Burst in Macrophages
Authors: Forman Henry Jay , Torres Martine
Published: July 2002

During phagocytosis or stimulation with a wide variety of agents, macrophages and other phagocytic cells produce reactive oxygen species (ROS) through activation of a multicomponent NADPH oxidase. ROS production through related NADPH oxidases has recently been demonstrated in several other cell types. Furthermore, the physiological generation of ROS production has now been clearly implicated in activating signaling pathways resulting in a broad array of physiological responses from cell proliferation to gene expression and apoptosis. This brief review suggests that: 1) hydrogen peroxide and superoxide, but not the hydroxyl radical, function as second messengers; 2) antioxidant enzymes function in the “turn‐off” phase of signal transduction; 3) the chemistry of thiols is critical in redox signaling; and 4) the primary physiological role of the respiratory burst in macrophages may be in redox signaling rather than microbicidal activity.
A Chromatin-associated Kinesin-related Protein Required for Normal Mitotic Chromosome Segregation in Drosophila
Authors: Molina Isabel , Baars Sigrid , Brill Julie A. , Hales Karen G. , Fuller Margaret T. , Ripoll Pedro
Published: July 2002

The tiovivo (tio) gene of Drosophila encodes a kinesin-related protein, KLP38B, that colocalizes with condensed chromatin during cell division. Wild-type function of the tio gene product KLP38B is required for normal chromosome segregation during mitosis. Mitotic cells in tio larval brains displayed circular mitotic figures, increased ploidy, and abnormal anaphase figures. KLP38B mRNA is maternally provided and expressed in cells about to undergo division. We propose that KLP38B, perhaps redundantly with other chromosome-associated microtubule motor proteins, contributes to interactions between chromosome arms and microtubules important for establishing bipolar attachment of chromosomes and assembly of stable bipolar spindles.
Signaling and Adhesion Activities of Mammalian β-Catenin and Plakoglobin in Drosophila
Authors: White Phoebe , Aberle Hermann , Vincent Jean-Paul
Published: July 2002

The armadillo protein of Drosophila and its vertebrate homologues, β-catenin and plakoglobin, are implicated in cell adhesion and wnt signaling. Here, we examine the conservation of these two functions by assaying the activities of mammalian β-catenin and plakoglobin in Drosophila. We show that, in the female germ line, both mammalian β-catenin and plakoglobin complement an armadillo mutation. We also show that shotgun mutant germ cells (which lack Drosophila E-cadherin) have a phenotype identical to that of armadillo mutant germ cells. It therefore appears that armadillo's role in the germ line is solely in a complex with Drosophila E-cadherin (possibly an adhesion complex), and both β-catenin and plakoglobin can function in Drosophila cadherin complexes. In embryonic signaling assays, we find that plakoglobin has no detectable activity whereas β-catenin's activity is weak. Surprisingly, when overexpressed, either in embryos or in wing imaginal disks, both β-catenin and plakoglobin have dominant negative activity on signaling, an effect also obtained with COOH-terminally truncated armadillo. We suggest that the signaling complex, which has been shown by others to comprise armadillo and a member of the lymphocyte enhancer binding factor-1/T cell factor–family, may contain an additional factor that normally binds to the COOH-terminal region of armadillo.
Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity
Authors: Qiao Hui , Pelletier Sandra L. , Hoffman Lucas , Hacker Jill , Armstrong R. Todd , White Judith M.
Published: July 2002

We tested the role of the “spring-loaded” conformational change in the fusion mechanism of the influenza hemagglutinin (HA) by assessing the effects of 10 point mutants in the region of high coiled-coil propensity, HA2 54–81. The mutants included proline substitutions at HA2 55, 71, and 80, as well as a double proline substitution at residues 55 and 71. Mutants were expressed in COS or 293T cells and assayed for cell surface expression and structural features as well as for their ability to change conformation and induce fusion at low pH. We found the following: Specific mutations affected the precise carbohydrate structure and folding of the HA trimer. All of the mutants, however, formed trimers that could be expressed at the cell surface in a form that could be proteolytically cleaved from the precursor, HA0, to the fusion-permissive form, HA1-S-S-HA2. All mutants reacted with an antibody against the major antigenic site and bound red blood cells. Seven out of ten mutants displayed a wild-type (wt) or moderately elevated pH dependence for the conformational change. V55P displayed a substantial reduction (∼60– 80%) in the initial rate of lipid mixing. The other single mutants displayed efficient fusion with the same pH dependence as wt-HA. The double proline mutant V55P/ S71P displayed no fusion activity despite being well expressed at the cell surface as a proteolytically cleaved trimer that could bind red blood cells and change conformation at low pH. The impairment in fusion for both V55P and V55P/S71P was at the level of outer leaflet lipid mixing. We interpret our results in support of the hypothesis that the spring-loaded conformational change is required for fusion. An alternate model is discussed.
Requirement of Heavy Neurofilament Subunit in the Development of Axons with Large Calibers
Authors: Elder Gregory A. , Friedrich Victor L. , Kang Chulho , Bosco Paolo , Gourov Andrei , Tu Pang-Hsien , Zhang Bin , Lee Virginia M.-Y. , Lazzarini Robert A.
Published: July 2002

Neurofilaments (NFs) are prominent components of large myelinated axons. Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber. We created NF-H knockout mice to assess the contribution of NF-H to the development of axon size as well as its effect on the amounts of low and mid-sized NF subunits (NF-L and NF-M respectively). Surprisingly, we found that NF-L levels were reduced only slightly whereas NF-M and tubulin proteins were unchanged in NF-H–null mice. However, the calibers of both large and small diameter myelinated axons were diminished in NF-H–null mice despite the fact that these mice showed only a slight decrease in NF density and that filaments in the mutant were most frequently spaced at the same interfilament distance found in control. Significantly, large diameter axons failed to develop in both the central and peripheral nervous systems. These results demonstrate directly that unlike losing the NF-L or NF-M subunits, loss of NF-H has only a slight effect on NF number in axons. Yet NF-H plays a major role in the development of large diameter axons.
Role of Polo Kinase and Mid1p in Determining the Site of Cell Division in Fission Yeast
Authors: Bähler Jürg , Steever Alexander B. , Wheatley Sally , Wang Yu-li , Pringle John R. , Gould Kathleen L. , McCollum Dannel
Published: July 2002

The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin– based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.
Calreticulin Is Essential for Cardiac Development
Authors: Mesaeli Nasrin , Nakamura Kimitoshi , Zvaritch Elena , Dickie Peter , Dziak Ewa , Krause Karl-Heinz , Opas Michal , MacLennan David H. , Michalak Marek
Published: July 2002

Calreticulin is a ubiquitous Ca2+ binding protein, located in the endoplasmic reticulum lumen, which has been implicated in many diverse functions including: regulation of intracellular Ca2+ homeostasis, chaperone activity, steroid-mediated gene regulation, and cell adhesion. To understand the physiological function of calreticulin we used gene targeting to create a knockout mouse for calreticulin. Mice homozygous for the calreticulin gene disruption developed omphalocele (failure of absorption of the umbilical hernia) and showed a marked decrease in ventricular wall thickness and deep intertrabecular recesses in the ventricular walls. Transgenic mice expressing a green fluorescent protein reporter gene under the control of the calreticulin promoter were used to show that the calreticulin gene is highly activated in the cardiovascular system during the early stages of cardiac development. Calreticulin protein is also highly expressed in the developing heart, but it is only a minor component of the mature heart. Bradykinin-induced Ca2+ release by the InsP3-dependent pathway was inhibited in crt−/− cells, suggesting that calreticulin plays a role in Ca2+ homeostasis. Calreticulin-deficient cells also exhibited impaired nuclear import of nuclear factor of activated T cell (NF-AT3) transcription factor indicating that calreticulin plays a role in cardiac development as a component of the Ca2+/calcineurin/NF-AT/GATA-4 transcription pathway.
Targeted Disruption of the LAMA3 Gene in Mice Reveals Abnormalities in Survival and Late Stage Differentiation of Epithelial Cells
Authors: Ryan Maureen C. , Lee Keesook , Miyashita Yuko , Carter William G.
Published: July 2002

Laminin 5 regulates anchorage and motility of epithelial cells through integrins α6β4 and α3β1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the α3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all α3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin α6β4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin α3β1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin α3β1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin α6β4, suggesting that signaling through β1 or β4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.
The Developmental Role of warthog, the Notch Modifier Encoding Drab6
Authors: Purcell Karen , Artavanis-Tsakonas Spyros
Published: July 2002

The warthog (wrt) gene, recovered as a modifier for Notch signaling, was found to encode the Drosophila homologue of rab6, Drab6. Vertebrate and yeast homologues of this protein have been shown to regulate Golgi network to TGN trafficking. To study the function of this protein in the development of a multicellular organism, we analyzed three different warthog mutants. The first was an R62C point mutation, the second a genomic null, and the third was an engineered GTP-bound form. Our studies show, contrary to yeast, that the Drosophila homologue of rab6 is an essential gene. However, it has limited effects on development beyond the larval stage. Only the mechanosensory bristles on the head, notum, and scutellum are affected by warthog mutations. We present models for the modifying effect of Drab6 on Notch signaling.
Lst1p and Sec24p Cooperate in Sorting of the Plasma Membrane Atpase into Copii Vesicles in Saccharomyces cerevisiae
Authors: Shimoni Yuval , Kurihara Tatsuo , Ravazzola Mariella , Amherdt Mylène , Orci Lelio , Schekman Randy
Published: July 2002

Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer. We found that cytosol from an lst1-null strain supported the packaging of α-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase. Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles. When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles. Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diameter). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles. These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles.
Coordinate Control of Muscle Cell Survival by Distinct Insulin-like Growth Factor Activated Signaling Pathways
Authors: Lawlor Margaret A. , Rotwein Peter
Published: July 2002

Peptide growth factors control diverse cellular functions by regulating distinct signal transduction pathways. In cultured myoblasts, insulin-like growth factors (IGFs) stimulate differentiation and promote hypertrophy. IGFs also maintain muscle cell viability. We previously described C2 skeletal muscle lines lacking expression of IGF-II. These cells did not differentiate, but underwent progressive apoptotic death when incubated in differentiation medium. Viability could be sustained and differentiation enabled by IGF analogues that activated the IGF-I receptor; survival was dependent on stimulation of phosphatidylinositol 3-kinase (PI3-kinase). We now find that IGF action promotes myoblast survival through two distinguishable PI3-kinase–regulated pathways that culminate in expression of the cyclin-dependent kinase inhibitor, p21. Incubation with IGF-I or transfection with active PI3-kinase led to rapid induction of MyoD and p21, and forced expression of either protein maintained viability in the absence of growth factors. Ectopic expression of MyoD induced p21, and inhibition of p21 blocked MyoD-mediated survival, thus defining one PI3-kinase–dependent pathway as leading first to MyoD, and then to p21 and survival. Unexpectedly, loss of MyoD expression did not impede IGF-mediated survival, revealing a second pathway involving activation by PI3-kinase of Akt, and subsequent induction of p21. Since inhibition of p21 caused death even in the presence of IGF-I, these results establish a central role for p21 as a survival factor for muscle cells. Our observations also define a MyoD-independent pathway for regulating p21 in muscle, and demonstrate that distinct mechanisms help ensure appropriate expression of this key protein during differentiation.