bluuebunny/crossref_metadata_2025 huggingface dataset:
cDNA cloning of FRIL, a lectin from
Dolichos lablab
, that preserves hematopoietic progenitors in suspension culture
Authors: Colucci Gabriella , Moore Jeffrey G. , Feldman Michael , Chrispeels Maarten J.
Published: July 2002

Ex vivo
culture of hematopoietic stem cells is limited by the inability of cytokines to maintain primitive cells without inducing proliferation, differentiation, and subsequent loss of repopulating capacity. We identified recently in extracts of kidney bean and hyacinth bean a mannose-binding lectin, called FRIL, and provide here evidence that this protein appears to satisfy properties of a stem cell preservation factor. FRIL was first identified based on its ability to stimulate NIH 3T3 cells transfected with Flt3, a tyrosine kinase receptor central to regulation of stem cells. Molecular characterization from polypeptide sequencing and identification of the cDNA of hyacinth bean FRIL shows 78% amino acid identity with a mannose-binding lectin of hyacinth beans. Treatment of primitive hematopoietic progenitors in suspension culture with purified hyacinth FRIL alone is able to preserve cells for 1 month without medium changes.
In vitro
progenitor assays for human hematopoietic cells cultured 3 weeks in FRIL displayed small blast-like colonies that were capable of serial replating and persisted even in the presence of cytokines known to induce differentiation. These results suggest that FRIL is capable of preserving primitive progenitors in suspension culture for prolonged periods. FRIL’s clinical utility involving procedures for stem cell transplantation, tumor cell purging before autologous transplantation, and
ex vivo
cultures used for expansion and stem cell gene therapy currently are being explored.
Flanking nuclear matrix attachment regions synergize with the T cell receptor δ enhancer to promote V(D)J recombination
Authors: Zhong Xiao-Ping , Carabaña Juan , Krangel Michael S.
Published: July 2002

Previous studies have identified nuclear matrix attachment regions (MARs) that are closely associated with transcriptional enhancers in the IgH, Igκ, and T cell receptor (TCR) β loci, but have yielded conflicting information regarding their functional significance. In this report, a combination ofin vitroandin situmapping approaches was used to localize three MARs associated with the human TCR δ gene. Two of these are located within the Jδ3–Cδ intron, flanking the core TCR δ enhancer (Eδ) both 5′ and 3′ in a fashion reminiscent of the Ig heavy chain intronic enhancer-associated MARs. The third is located about 20 kb upstream, tightly linked to Dδ1 and Dδ2. We have previously used a transgenic minilocus V(D)J recombination reporter to establish that Eδ functions as a developmental regulator of V(D)J recombination, and that it does so by modulating substrate accessibility to the V(D)J recombinase. We show here that the Eδ-associated MARs function synergistically with the core Eδ to promote V(D)J recombination in this system, as they are required for enhancer-dependent transgene rearrangement in single-copy transgene integrants.
Dynamics of spatial summation in primary visual cortex of alert monkeys
Authors: Kapadia Mitesh K. , Westheimer Gerald , Gilbert Charles D.
Published: July 2002

One of the fundamental tasks of the visual cortex is to integrate input from different parts of the retina, parsing an image into contours and surfaces, and then assembling these features into coherent representations of objects. To examine the role of the primary visual cortex in the integration of visual information, we measured the response properties of neurons under different stimulus conditions. Surprisingly, we found that even the most conventional measures of receptive field (RF) size were not fixed, but could vary depending on stimulus contrast and foreground–background relationships. On average, the length of the excitatory RF was 4-fold greater for a low-contrast stimulus than for a stimulus at high contrast. Embedding a high-contrast stimulus in a textured background tended to suppress neuronal responses and produced an enlargement in RF size similar to that observed by decreasing the contrast of an isolated stimulus. The results show that RF dimensions are regulated in a dynamic manner that depends both on local stimulus characteristics, such as contrast, and on global relationships between a stimulus and its surroundings.
Tumor burden and clonality in multiple intestinal neoplasia mouse/normal mouse aggregation chimeras
Authors: Novelli Marco R. , Wasan Harpreet , Rosewell Ian , Bee Julie , Tomlinson Ian P. , Wright Nicholas A. , Bodmer Walter F.
Published: July 2002

Aggregation chimeras were formed between C57BL/6 mice heterozygous for the

Apc
min

(
Min
) mutation and wild-type SWR mice, that differ in their
Pla2g2a
status, a modifier of

Apc
min

, and also in their resistance to intestinal polyp formation. Variation in the dolichos biflorus agglutinin-staining patterns of the intestines of these mouse strains was used to determine the chimeric composition of the intestine in individual mice and to examine the clonal composition of adenomas. Macroscopic adenoma numbers in chimeric mice were compared with the expected adenoma numbers based on the percentage of C57BL/6J-
Apc

min
/+

epithelium in individual mice. These results unexpectedly show that there was no apparent inhibitory effect of the SWR-derived (

Pla2g2a

wild-type) tissue on adenoma formation in the C57BL/6J-
Apc

min
/+

epithelium. This suggests that the main genetic modifiers of the
Min
phenotype act at a cellular or crypt-restricted level with no discernable systemic effect. All adenomas were seen to contain C57BL/6J-
Apc

min
/+

-derived epithelium, confirming that the germ-line mutation of the
mApc
gene is necessary to initiate tumorigenesis in this model system, and that the
mApc
gene acts in a cell autonomous fashion.
Equilibrium distributions of topological states in circular DNA: Interplay of supercoiling and knotting
Authors: Podtelezhnikov Alexei A. , Cozzarelli Nicholas R. , Vologodskii Alexander V.
Published: July 2002

Two variables define the topological state of closed double-stranded DNA: the knot type,
K
, and Δ
Lk
, the linking number difference from relaxed DNA. The equilibrium distribution of probabilities of these states,
P
(Δ
Lk
,
K
), is related to two conditional distributions:
P
(Δ
Lk
|
K
), the distribution of Δ
Lk
for a particular
K
, and
P
(
K
|Δ
Lk
) and also to two simple distributions:
P
(Δ
Lk
), the distribution of Δ
Lk
irrespective of
K
, and
P
(
K
). We explored the relationships between these distributions.
P
(Δ
Lk
,
K
),
P
(Δ
Lk
), and
P
(
K
|Δ
Lk
) were calculated from the simulated distributions of
P
(Δ
Lk
|
K
) and of
P
(
K
). The calculated distributions agreed with previous experimental and theoretical results and greatly advanced on them. Our major focus was on
P
(
K
|Δ
Lk
), the distribution of knot types for a particular value of Δ
Lk
, which had not been evaluated previously. We found that unknotted circular DNA is not the most probable state beyond small values of Δ
Lk
. Highly chiral knotted DNA has a lower free energy because it has less torsional deformation. Surprisingly, even at |Δ
Lk
| > 12, only one or two knot types dominate the
P
(
K
|Δ
Lk
) distribution despite the huge number of knots of comparable complexity. A large fraction of the knots found belong to the small family of torus knots. The relationship between supercoiling and knotting
in vivo
is discussed.
Visualization of α9 acetylcholine receptor expression in hair cells of transgenic mice containing a modified bacterial artificial chromosome
Authors: Zuo Jian , Treadaway Jason , Buckner Tyler W. , Fritzsch Bernd
Published: July 2002

The α9 acetylcholine receptor (α9 AChR) is specifically expressed in hair cells of the inner ear and is believed to be involved in synaptic transmission between efferent nerves and hair cells. Using a recently developed method, we modified a bacterial artificial chromosome containing the mouse α9 AChR gene with a reporter gene encoding green fluorescent protein (GFP) to generate transgenic mice. GFP expression in transgenic mice recapitulated the known temporal and spatial expression of α9 AChR. However, we observed previously unidentified dynamic changes in α9 AChR expression in cochlear and vestibular sensory epithelia during neonatal development. In the cochlea, inner hair cells persistently expressed high levels of α9 AChR in both the apical and middle turns, whereas both outer and inner hair cells displayed dynamic changes of α9 AChR expression in the basal turn. In the utricle, we observed high levels of α9 AChR expression in the striolar region during early neonatal development and high levels of α9 AChR in the extrastriolar region in adult mice. Further, simultaneous visualization of efferent innervation and α9 AChR expression showed that dynamic expression of α9 AChR in developing hair cells was independent of efferent contacts. We propose that α9 AChR expression in developing auditory and vestibular sensory epithelia correlates with maturation of hair cells and is hair-cell autonomous.
Molecular dynamics and free-energy calculations applied to affinity maturation in antibody 48G7
Authors: Chong Lillian T. , Duan Yong , Wang Lu , Massova Irina , Kollman Peter A.
Published: July 2002

We investigated the relative free energies of hapten binding to the germ line and mature forms of the 48G7 antibody Fab fragments by applying a continuum model to structures sampled from molecular dynamics simulations in explicit solvent. Reasonable absolute and very good relative free energies were obtained. As a result of nine somatic mutations that do not contact the hapten, the affinity-matured antibody binds the hapten >10
4
tighter than the germ line antibody. Energetic analysis reveals that van der Waals interactions and nonpolar contributions to solvation are similar and drive the formations of both the germ line and mature antibody–hapten complexes. Affinity maturation of the 48G7 antibody therefore appears to occur through reorganization of the combining site geometry in a manner that optimizes the balance of gaining favorable electrostatic interactions with the hapten and losing those with solvent during the binding process. As reflected by lower rms fluctuations in the antibody–hapten complex, the mature complex undergoes more restricted fluctuations than the germ line complex. The dramatically increased affinity of the 48G7 antibody over its germ line precursor is thus made possible by electrostatic optimization.
Constitutive tyrosine phosphorylation of the inhibitory paired Ig-like receptor PIR-B
Authors: Ho Le Hong , Uehara Takahiro , Chen Ching-Cheng , Kubagawa Hiromi , Cooper Max D.
Published: July 2002

PIR-A and PIR-B are activating and inhibitory Ig-like receptors on murine B lymphocytes, dendritic cells, and myeloid-lineage cells. The inhibitory function of PIR-B is mediated via its cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, whereas PIR-A pairs with the Fc receptor common γ chain to form an activating receptor complex. In these studies, we observed constitutive tyrosine phosphorylation of PIR-B molecules on macrophages and B lymphocytes, irrespective of the cell activation status. Splenocyte PIR-B molecules were constitutively associated with the SHP-1 protein tyrosine phosphatase and Lyn protein tyrosine kinase. In Lyn-deficient mice, PIR-B tyrosine phosphorylation was greatly reduced. Unexpectedly, tyrosine phosphorylation of PIR-B was not observed in most myeloid and B cell lines but could be induced by ligation of the PIR molecules. Finally, the phosphorylation status of PIR-B was significantly reduced in MHC class I-deficient mice, although not in mice deficient in TAP1 or MHC class II expression. These findings suggest a physiological inhibitory role for PIR-B that is regulated by endogenous MHC class I-like ligands.
GLI
3
mutations in human disorders mimic
Drosophila
Cubitus interruptus protein functions and localization
Authors: Shin Sang Hoon , Kogerman Priit , Lindström Erika , Toftgárd Rune , Biesecker Leslie G.
Published: July 2002

Truncation mutations of the
GLI3
zinc finger transcription factor can cause Greig cephalopolysyndactyly syndrome (GCPS), Pallister–Hall syndrome (PHS), and postaxial polydactyly type A (PAP-A). GLI3 is homologous to
Drosophila
Cubitus interruptus (Ci), which regulates the
patched
(
ptc
),
gooseberry
(
gsb
), and
decapentaplegic
(
dpp
) genes. Ci is sequestered in the cytoplasm and is subject to posttranslational processing whereby the full-length transcriptional activator form (Ci
155
) can be cleaved to a repressor form (Ci
75
). Under hedgehog signaling, the Ci
155
form translocates to the nucleus whereas in the absence of hedgehog, the Ci
75
form translocates to the nucleus. Based on the correlation of GLI3 truncation mutations and the human phenotypes, we hypothesized that GLI3 shows transcriptional activation or repression activity and subcellular localization similar to Ci. Here we show that full-length GLI3 localizes to the cytoplasm and activates
PTCH1
expression, which is similar to full-length Ci
155
. PHS mutant protein (GLI3-PHS) localizes to the nucleus and represses GLI3-activated
PTCH1
expression, which is similar to Ci
75
. The GCPS mutant protein has no effect on GLI3-activated
PTCH1
transcription, consistent with the role of haploinsufficiency in this disorder. The PAP-A mutant protein (GLI3-PAP-A) showed less specific subcellular localization but still inhibited GLI3-activated
PTCH1
transcription, suggesting it may be a weaker allele than the GLI3-PHS mutation. These data show that
GLI3
mutations in humans mimic functional effects of the
Drosophila ci
gene and correlate with the distinct effects of these mutations on human development.
Raft association of SNAP receptors acting in apical trafficking in Madin–Darby canine kidney cells
Authors: Lafont Frank , Verkade Paul , Galli Thierry , Wimmer Christian , Louvard Daniel , Simons Kai
Published: July 2002

We have investigated the relationships between the apical sorting mechanism using lipid rafts and the soluble
N
-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin– Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP protein stimulates the apical transport whereas a SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.
Effects of mutant p53 expression on human 15-lipoxygenase-promoter activity and murine 12/15-lipoxygenase gene expression: Evidence that 15-lipoxygenase is a mutator gene
Authors: Kelavkar Uddhav P. , Badr Kamal F.
Published: July 2002

Human 15-lipoxygenase (
h15-LO
) is present on chromosome 17p13.3 in close proximity to the tumor-suppressor gene, p53.
15-LO
is implicated in antiinflammation, membrane remodeling, and cancer development/metastasis. The murine BALB/c embryo fibroblast cell line, (10)1val, expresses p53 in mutant (mt) conformation when grown at 39°C and in wild-type conformation when grown at 32°C. Transfection of
h15-LO
promoter constructs (driving luciferase reporter) into (10)1val cells and into p53-deficient (10)1 cells resulted in a marked increase in
h15-LO
promoter activity in (10)1val cells at 39°C, but not at 32°C, or as compared with (10)1 cells. Transfection of
h15-LO
promoter deletion constructs, however, resulted in total loss of activity in both cell types at 32°C and 39°C. Cotransfection of (10)1 cells with
h15-LO
promoter (driving luciferase reporter) along with increasing levels of a mt p53 expression vector demonstrated dose-dependent capacity of mt p53 to induce
15-LO
promoter activity. No effect was observed with wild-type p53. In contrast to
h15-LO
promoter activity, (10)1val cells had significantly lower levels of endogenous (murine)
12/15-LO
(mouse analog of
h15-LO
) mRNA and protein when grown at 39°C compared with cells grown at 32°C. Our data support the hypothesis that loss of a tumor-suppressor gene (p53), or “gain-of-function activities” resulting from the expression of its mutant forms, regulates
15-LO
promoter activity in man and in mouse, albeit in directionally opposite manners. The studies define a direct link between
15-LO
activity and an established tumor-suppressor gene located in close chromosomal proximity.
Patterning of the mammalian cochlea
Authors: Cantos Raquel , Cole Laura K. , Acampora Dario , Simeone Antonio , Wu Doris K.
Published: July 2002

The mammalian cochlea is sophisticated in its function and
highly organized in its structure. Although the anatomy of this sense
organ has been well documented, the molecular mechanisms underlying its
development have remained elusive. Information generated from mutant
and knockout mice in recent years has increased our understanding of
cochlear development and physiology. This article discusses factors
important for the development of the inner ear and summarizes cochlear
phenotypes of mutant and knockout mice, particularly
Otx
and
Otx2
. We also present data on gross development of
the mouse cochlea.
Electric field effect on cholesterol–phospholipid complexes
Authors: Radhakrishnan Arun , McConnell Harden M.
Published: July 2002

Monolayer mixtures of dihydrocholesterol and phospholipids at the
air–water interface are used to model membranes containing cholesterol
and phospholipids. Specific, stoichiometric interactions between
cholesterol and some but not all phospholipids have been proposed to
lead to the formation of condensed complexes. It is reported here that
an externally applied electric field of the appropriate sign can
destabilize these complexes, resulting in their dissociation. This is
demonstrated through the application of an electric field gradient that
leads to phase separations in otherwise homogeneous monolayers. This is
observed only when the monolayer composition is close to the
stoichiometry of the complex. The electric field effect is analyzed
with the same mean field thermodynamic model as that used previously to
account for pairs of upper miscibility critical points in these
mixtures. The concentrations of dihydrocholesterol, phospholipid, and
complex vary strongly and sometimes discontinuously in the monolayer
membrane in the field gradient. The model is an approximation to a
two-dimensional liquid in which molecules freely exchange between free
and complexed form so that the chemical potentials are constant
throughout the membrane. The calculations are illustrated for a complex
of about 15 molecules, composed of 5 cholesterol molecules and 10
phospholipid molecules.
Type 2 iodothyronine deiodinase expression in the cochlea before the onset of hearing
Authors: Campos-Barros Angel , Amma Lori L. , Faris Jonathan S. , Shailam Ranu , Kelley Matthew W. , Forrest Douglas
Published: July 2002

Thyroid hormone signaling during a postnatal period in the mouse is essential for cochlear development and the subsequent onset of hearing. To study the control of this temporal dependency, we investigated the role of iodothyronine deiodinases, which in target tissues convert the prohormone thyroxine into triiodothyronine (T3), the active ligand for the thyroid hormone receptor (TR). Type 2 5′-deiodinase (D2) activity rose dramatically in the mouse cochlea to peak around postnatal day 7 (P7), after which activity declined by P10. This activity peak a few days before the onset of hearing suggests a role for D2 in amplifying local T3 levels at a critical stage of cochlear development. A mouse cochlear D2 cDNA was isolated and demonstrated near identity to rat D2.
In situ
hybridization localized D2 mRNA in periosteal connective tissue in the modiolus, the cochlear outer capsule and the septal divisions between the turns of the cochlea. Surprisingly, D2 expression in these regions that give rise to the bony labyrinth was complementary to TR expression in the sensory epithelium. Thus, the connective tissue may control deiodination of thyroxine and release of T3 to confer a paracrine-like control of TR activation. These results suggest that temporal and spatial control of ligand availability conferred by D2 provides an unexpectedly important level of regulation of the TR pathways required for cochlear maturation.
Dynamics of site juxtaposition in supercoiled DNA
Authors: Huang Jing , Schlick Tamar , Vologodskii Alexander
Published: July 2002

Juxtaposition kinetics between specific sites in supercoiled DNA is
investigated at close to physiological ionic conditions by Brownian
dynamics simulations. At such conditions, supercoiled DNA is
interwound, and the probability of spatial site juxtaposition is much
higher than in relaxed DNA. We find, however, that supercoiling does
not correspondingly increase the rate of juxtaposition at these
physiological conditions. An explanation to this unexpected finding
emerges on analysis of the juxtaposition dynamics. We note that
although a particular site
i
1
in supercoiled
DNA is often in close proximity (juxtaposed) to another site
i
2
, the change of
i
2
occurs very slowly and depends largely on
internal slithering of opposite segments of the DNA superhelix. Such
slithering results in long correlations between successive values of
i
2
; these correlations increase the average
time of juxtaposition between two DNA sites. Random collisions between
sites located on different superhelix branches—although increasing in
importance with DNA size—contribute less substantially to site
juxtaposition at high salt than slithering for DNA up to 6 kb in
length.
Methadone syrup‐related deaths in New South Wales, Australia, 1990–95
Authors: SUNJIC SANDRA , ZADOR DEBORAH
Published: July 2002

The aim of this study was to determine the number, and to describe the circumstances, of accidental methadone syrup‐related deaths (MSRDs) in New South Wales (NSW), Australia from 1990 to 1995. Coronial files of all cases positive for methadone at autopsy between July 1990 and December 1995 in NSW were reviewed to determine methadone syrup‐related cases. One hundred and thirty‐four cases of MSRD were identified, of which 72 (54%) were registered in methadone maintenance treatment (MMT) at time of death. Diverted sources were implicated in the remaining 62 cases (46%) not registered in MMT. The majority of all cases were single (60%), unemployed (80%) males (75%), with a mean age of 31 years. Intravenous injection of methadone prior to death was evident in 35% of cases not in MMT, and in 10% in MMT. Polydrug use was detected in 89% of cases, most commonly benzodiazepines (52%), followed by morphine (37%) and alcohol (28%). However, forensic authorities did not implicate other drugs detected at autopsy in the cause of death in 34% of cases. The large proportion of deaths involving diverted sources of methadone may be indicative of high‐risk behaviour among some injecting drug users, who abuse methadone in combination with other drugs to enhance the effects of intoxication. Alternatively, heroin users who use illicit methadone may be doing so to medicate opioid withdrawal symptoms, which could suggest a high unmet demand for MMT. Failure to implicate other drugs detected at autopsy in the cause of death may result in over‐reporting of methadone‐detected cases as methadone‐caused deaths. The findings from this study support an urgent review of the current classification and reporting mechanisms for methadone‐related deaths. [Sunjic S, Zador D. Methadone syrup‐related deaths in New South Wales, Australia, 1990–95. Drug Alcohol Rev 1999:18:409–415]
Carbohydrate‐Deficient Transferrin: An Aid to Early Recognition of Alcohol Relapse
Authors: Allen John P. , Litten Raye Z. , Fertig Joanne B. , Sillanaukee Pekka
Published: July 2002

Although the primary use of biochemical markers of heavy drinking is to assist in screening for alcohol problems, laboratory tests may also aid in early identification of relapse. This report reviews research findings on a new marker, carbohydrate deficient transferrin (CDT), in alcoholics receiving treatment or in follow‐up. It also offers recommendations on how CDT may be employed by clinicians monitoring drinking status.
Chronic alcohol administration in the rat pup: effects upon later consumption of alcohol and other palatable solutions
Authors: LÓPEZ MARCELO FERNANDO , MOLINA JUAN CARLOS
Published: July 2002

Previous studies indicate that in rats, chronic alcohol exposure during the first weeks of life markedly affect subsequent ethanol consumption patterns. The present study examined the impact of different alcohol doses (0.5–3.0 g/kg), administered between postnatal days 6–12, upon subsequent infantile consumption of an ethanol solution as well as upon intake of various non‐ethanol solutions (water, sucrose, quinine or sucrose mixed with quinine). Alcohol administration did not strongly affect consumption scores of water, sucrose or quinine. In contrast, 15‐day‐old pups pretreated with 2.0 and 3.0 g/kg alcohol doses showed significant increases in terms of alcohol consumption when compared to saline controls. Furthermore, a positive significant correlation was observed between alcohol intake patterns and alcohol dose administered during early ontogeny. Intake of sucrose mixed with quinine was also significantly and positively correlated with pretreatment ethanol dosage. Interestingly, this taste configuration has been shown to mimic psychophysical properties of ethanol in the rat. The results appear not to be explained by teratological effects of the drug upon sensory processing of distinctive tastants. It appears that chronic alcohol exposure during early ontogeny provides specific sensoryrelated alcohol information that later modulates alcohol intake patterns.
Catalase as a regulator of the propensity to ingest alcohol in genetically determined acatalasemic individuals from Israel
Authors: AMIT ZALMAN , SMITH BRIAN R. , WEISS SHOSHANA
Published: July 2002

Direct descendants of an individual residing in Israel, who was diagnosed in the 1960s with a genetic deficiency in catalase, were examined for their propensity to consume alcohol.These individuals were found to possess a lower level of catalase activity compared to that of a group of matched controls with the same ethnic background.While no differences were observed in the propensity to drink alcohol between the two groups, the catalase deficient individuals did show a significant positive correlation between catalase activity and alcohol drinking behaviour as measured by Q‐value. No such relationship was observed in the matched controls.The findings suggest that the apparent acatalasemia in the experimental subjects may act as a limiting factor for these individuals and that catalase may play an important role in regulating alcohol drinking behaviour. These results are consistent with previous animal and human studies which suggest that catalase, via its ability to produce acetaldehyde through the metabolism of ethanol, may have a regulatory role in the propensity to drink alcohol.
An Early Stage of Membrane Fusion Mediated by the Low pH Conformation of Influenza Hemagglutinin Depends upon Membrane Lipids
Authors: Chernomordik Leonid V. , Leikina Eugenia , Frolov Vadim , Bronk Peter , Zimmerberg Joshua
Published: July 2002

While the specificity and timing of membrane fusion in diverse physiological reactions, including virus–cell fusion, is determined by proteins, fusion always involves the merger of membrane lipid bilayers. We have isolated a lipid-dependent stage of cell–cell fusion mediated by influenza hemagglutinin and triggered by cell exposure to mildly acidic pH. This stage preceded actual membrane merger and fusion pore formation but was subsequent to a low pH–induced change in hemagglutinin conformation that is required for fusion. A low pH conformation of hemagglutinin was required to achieve this lipid-dependent stage and also, downstream of it, to drive fusion to completion. The lower the pH of the medium applied to trigger fusion and, thus, the more hemagglutinin molecules activated, the less profound was the dependence of fusion on lipids. Membrane-incorporated lipids affected fusion in a manner that correlated with their dynamic molecular shape, a characteristic that determines a lipid monolayer's propensity to bend in different directions. The lipid sensitivity of this stage, i.e., inhibition of fusion by inverted cone–shaped lysophosphatidylcholine and promotion by cone-shaped oleic acid, was consistent with the stalk hypothesis of fusion, suggesting that fusion proteins begin membrane merger by promoting the formation of a bent, lipid-involving, stalk intermediate.
Kinetochore Fibers Are Not Involved in the Formation of the First Meiotic Spindle in Mouse Oocytes, but Control the Exit from the First Meiotic M Phase
Authors: Brunet Stéphane , Maria Angélica Santa , Guillaud Philippe , Dujardin Denis , Kubiak Jacek Z. , Maro Bernard
Published: July 2002

During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes.

We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore– microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore–microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion.
Structural Transitions at Microtubule Ends Correlate with Their Dynamic Properties in Xenopus Egg Extracts
Authors: Arnal Isabelle , Karsenti Eric , Hyman Anthony A.
Published: July 2002

Microtubules are dynamically unstable polymers that interconvert stochastically between growing and shrinking states by the addition and loss of subunits from their ends. However, there is little experimental data on the relationship between microtubule end structure and the regulation of dynamic instability. To investigate this relationship, we have modulated dynamic instability in Xenopus egg extracts by adding a catastrophe-promoting factor, Op18/stathmin. Using electron cryomicroscopy, we find that microtubules in cytoplasmic extracts grow by the extension of a two- dimensional sheet of protofilaments, which later closes into a tube. Increasing the catastrophe frequency by the addition of Op18/stathmin decreases both the length and frequency of the occurrence of sheets and increases the number of frayed ends. Interestingly, we also find that more dynamic populations contain more blunt ends, suggesting that these are a metastable intermediate between shrinking and growing microtubules. Our results demonstrate for the first time that microtubule assembly in physiological conditions is a two-dimensional process, and they suggest that the two-dimensional sheets stabilize microtubules against catastrophes. We present a model in which the frequency of catastrophes is directly correlated with the structural state of microtubule ends.
Close Is Not Enough
Authors: McNew James A. , Weber Thomas , Parlati Francesco , Johnston Robert J. , Melia Thomas J. , Söllner Thomas H. , Rothman James E.
Published: July 2002

Is membrane fusion an essentially passive or an active process? It could be that fusion proteins simply need to pin two bilayers together long enough, and the bilayers could do the rest spontaneously. Or, it could be that the fusion proteins play an active role after pinning two bilayers, exerting force in the bilayer in one or another way to direct the fusion process. To distinguish these alternatives, we replaced one or both of the peptidic membrane anchors of exocytic vesicle (v)- and target membrane (t)-SNAREs (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor) with covalently attached lipids. Replacing either anchor with a phospholipid prevented fusion of liposomes by the isolated SNAREs, but still allowed assembly of trans-SNARE complexes docking vesicles. This result implies an active mechanism; if fusion occurred passively, simply holding the bilayers together long enough would have been sufficient. Studies using polyisoprenoid anchors ranging from 15–55 carbons and multiple phospholipid-containing anchors reveal distinct requirements for anchors of v- and t-SNAREs to function: v-SNAREs require anchors capable of spanning both leaflets, whereas t-SNAREs do not, so long as the anchor is sufficiently hydrophobic. These data, together with previous results showing fusion is inhibited as the length of the linker connecting the helical bundle-containing rod of the SNARE complex to the anchors is increased (McNew, J.A., T. Weber, D.M. Engelman, T.H. Sollner, and J.E. Rothman, 1999. Mol. Cell. 4:415–421), suggests a model in which one activity of the SNARE complex promoting fusion is to exert force on the anchors by pulling on the linkers. This motion would lead to the simultaneous inward movement of lipids from both bilayers, and in the case of the v-SNARE, from both leaflets.
Two Pathways through Cdc42 Couple the N-Formyl Receptor to Actin Nucleation in Permeabilized Human Neutrophils
Authors: Glogauer M. , Hartwig J. , Stossel T.
Published: July 2002

We developed a permeabilization method that retains coupling between N-formyl-methionyl-leucyl-phenylalanine tripeptide (FMLP) receptor stimulation, shape changes, and barbed-end actin nucleation in human neutrophils. Using GTP analogues, phosphoinositides, a phosphoinositide-binding peptide, constitutively active or inactive Rho GTPase mutants, and activating or inhibitory peptides derived from neural Wiskott-Aldrich syndrome family proteins (N-WASP), we identified signaling pathways leading from the FMLP receptor to actin nucleation that require Cdc42, but then diverge. One branch traverses the actin nucleation pathway involving N-WASP and the Arp2/3 complex, whereas the other operates through active Rac to promote actin nucleation. Both pathways depend on phosphoinositide expression. Since maximal inhibition of the Arp2/3 pathway leaves an N17Rac inhibitable alternate pathway intact, we conclude that this alternate involves phosphoinositide-mediated uncapping of actin filament barbed ends.
Selective Induction of Apoptosis in Mature T Lymphocytes by Variant T Cell Receptor Ligands
Authors: Combadière Behazine , e Sousa Caetano Reis , Germain Ronald N. , Lenardo Michael J.
Published: July 2002

Activation, anergy, and apoptosis are all possible outcomes of T cell receptor (TCR) engagement. The first leads to proliferation and effector function, whereas the others can lead to partial or complete immunological tolerance. Structural variants of immunizing peptide–major histocompatibility complex molecule ligands that induce selective lymphokine secretion or anergy in mature T cells in association with altered intracellular signaling events have been described. Here we describe altered ligands for mature mouse CD4+ T helper 1 cells that lead to T cell apoptosis by the selective expression of Fas ligand (FasL) and tumor necrosis factor (TNF) without concomitant IL-2, IL-3, or interferon γ production. All ligands that stimulated cell death were found to induce FasL and TNF mRNA expression and TCR aggregation (“capping”) at the cell surface, but did not elicit a common pattern of tyrosine phosphorylation of the TCR-associated signal transduction chains. Thus, TCR ligands that uniquely trigger T cell apoptosis without inducing cytokines that are normally associated with activation can be identified.
Role of the S3-S4 Linker in Shaker Potassium Channel Activation
Authors: Mathur Rajesh , Zheng Jie , Yan Yangyang , Sigworth Fred J.
Published: July 2002

Structural models of voltage-gated channels suggest that flexibility of the S3-S4 linker region may be important in allowing the S4 region to undergo large conformational changes in its putative voltage-sensing function. We report here the initial characterization of 18 mutations in the S3-S4 linker of the Shaker channel, including deletions, insertions, charge changes, substitution of prolines, and chimeras replacing the 25-residue Shaker linker with 7- or 9-residue sequences from Shab, Shaw, or Shal. As measured in Xenopus oocytes with a two-microelectrode voltage clamp, each mutant construct yielded robust currents. Changes in the voltage dependence of activation were small, with activation voltage shifts of 13 mV or less. Substitution of linkers from the slowly activating Shab and Shaw channels resulted in a three- to fourfold slowing of activation and deactivation. It is concluded that the S3-S4 linker is unlikely to participate in a large conformational change during channel activation. The linker, which in some channel subfamilies has highly conserved sequences, may however be a determinant of activation kinetics in potassium channels, as previously has been suggested in the case of calcium channels.
Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation
Authors: Chen Fred S.P. , Steele David , Fedida David
Published: July 2002

K+ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K+ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K+ or Cs+, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K+ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs+ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at ECs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K+ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.
Kinetics of Recovery of the Dark-adapted Salamander Rod Photoresponse
Authors: Nikonov S. , Engheta N. , Pugh E.N.
Published: July 2002

The kinetics of the dark-adapted salamander rod photocurrent response to flashes producing from 10 to 105 photoisomerizations (Φ) were investigated in normal Ringer's solution, and in a choline solution that clamps calcium near its resting level. For saturating intensities ranging from ∼102 to 104 Φ, the recovery phases of the responses in choline were nearly invariant in form. Responses in Ringer's were similarly invariant for saturating intensities from ∼103 to 104 Φ. In both solutions, recoveries to flashes in these intensity ranges translated on the time axis a constant amount (τc) per e-fold increment in flash intensity, and exhibited exponentially decaying “tail phases” with time constant τc. The difference in recovery half-times for responses in choline and Ringer's to the same saturating flash was 5–7 s. Above ∼104 Φ, recoveries in both solutions were systematically slower, and translation invariance broke down. Theoretical analysis of the translation-invariant responses established that τc must represent the time constant of inactivation of the disc-associated cascade intermediate (R*, G*, or PDE*) having the longest lifetime, and that the cGMP hydrolysis and cGMP-channel activation reactions are such as to conserve this time constant. Theoretical analysis also demonstrated that the 5–7-s shift in recovery half-times between responses in Ringer's and in choline is largely (4–6 s) accounted for by the calcium-dependent activation of guanylyl cyclase, with the residual (1–2 s) likely caused by an effect of calcium on an intermediate with a nondominant time constant. Analytical expressions for the dim-flash response in calcium clamp and Ringer's are derived, and it is shown that the difference in the responses under the two conditions can be accounted for quantitatively by cyclase activation. Application of these expressions yields an estimate of the calcium buffering capacity of the rod at rest of ∼20, much lower than previous estimates.
Time-irreversible Subconductance Gating Associated with Ba2+ Block of Large Conductance Ca2+-activated K+ Channels
Authors: Bello Ricardo A. , Magleby Karl L.
Published: July 2002

Ba2+ block of large conductance Ca2+-activated K+ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba2+ ion would dissociate to the external solution (150 mM N-methyl-d-glucamine+o, 500 mM K+i, 10 μM Ba2+i, +30 mV, and 100 μM Ca2+i to fully activate the channel), Ba2+ blocks with a mean duration of ∼2 s occurred, on average, once every ∼100 ms of channel open time. Of these Ba2+ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at ∼0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in ∼10,000 Ba2+ blocks. Thus, the preopenings represent Ba2+-induced time-irreversible subconductance gating. The fraction of Ba2+ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba2+]i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba2+ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba2+o and no added Ba2+i. Adding K+, Rb+, or Na+ to the external solution decreased the fraction of Ba2+ blocks with preopenings, with K+ and Rb+ being more effective than Na+. These results are consistent with models in which the blocking Ba2+ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba2+ blocking site and acts directly as the preopening gate.
Coupled Ion Movement Underlies Rectification in an Inward-Rectifier K+ Channel
Authors: Spassova Maria , Lu Zhe
Published: July 2002

We studied block of the internal pore of the ROMK1 inward-rectifier K+ channel by Mg2+ and five quaternary ammoniums (tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and tetrapentylammonium). The apparent affinity of these blockers varied as a function of membrane voltage. As a consequence, the channel conducted K+ current more efficiently in the inward than the outward direction; i.e., inward rectification. Although the size of some monovalent quaternary ammoniums is rather large, the zδ values (which measure voltage dependence of their binding to the pore) were near unity in symmetric 100 mM K+. Furthermore, we observed that not only the apparent affinities of the blockers themselves, but also their dependence on membrane voltage (or zδ), varied as a function of the concentration of extracellular K+. These results suggest that there is energetic coupling between the binding of blocking and permeating (K+) ions, and that the voltage dependence of channel blockade results, at least in part, from the movement of K+ ions in the electrical field. A further quantitative analysis of the results explains why the complex phenomenon of inward rectification depends on both membrane voltage and the equilibrium potential for K+.
An Intermediate State of the γ-Aminobutyric Acid Transporter Gat1 Revealed by Simultaneous Voltage Clamp and Fluorescence
Authors: Li Ming , Farley Robert A. , Lester Henry A.
Published: July 2002

The rat γ-aminobutyric acid transporter GAT1 expressed in Xenopus oocytes was labeled at Cys74, and at one or more other sites, by tetramethylrhodamine-5-maleimide, without significantly altering GAT1 function. Voltage-jump relaxation analysis showed that fluorescence increased slightly and monotonically with hyperpolarization; the fluorescence at −140 mV was ∼0.8% greater than at +60 mV. The time course of the fluorescence relaxations was mostly described by a single exponential with voltage-dependent but history-independent time constants ranging from ∼20 ms at +60 mV to ∼150 ms at −140 mV. The fluorescence did not saturate at the most negative potentials tested, and the midpoint of the fluorescence–voltage relation was at least 50 mV more negative than the midpoint of the charge–voltage relation previously identified with Na+ binding to GAT1. The presence of γ-aminobutyric acid did not noticeably affect the fluorescence waveforms. The fluorescence signal depended on Na+ concentration with a Hill coefficient approaching 2. Increasing Cl− concentration modestly increased and accelerated the fluorescence relaxations for hyperpolarizing jumps. The fluorescence change was blocked by the GAT1 inhibitor, NO-711. For the W68L mutant of GAT1, the fluorescence relaxations occurred only during jumps to high positive potentials, in agreement with previous suggestions that this mutant is trapped in one conformational state except at these potentials. These observations suggest that the fluorescence signals monitor a novel state of GAT1, intermediate between the E*out and Eout states of Hilgemann, D.W., and C.-C. Lu (1999. J. Gen. Physiol. 114:459–476). Therefore, the study provides verification that conformational changes occur during GAT1 function.