bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-term potentiation and dual-component quantal signaling in the dentate gyrus
Authors: Min Ming-Yuan , Asztely Fredrik , Kokaia Merab , Kullmann Dimitri M.
Published: July 2002

Long-term potentiation (LTP) of excitatory transmission is an important candidate cellular mechanism for the storage of memories in the mammalian brain. The subcellular phenomena that underlie the persistent increase in synaptic strength, however, are incompletely understood. A potentially powerful method to detect a presynaptic increase in glutamate release is to examine the effect of LTP induction on the rate at which the use-dependent blocker MK-801 attenuates successive
N
-methyl-
d
-aspartic acid (NMDA) receptor-mediated synaptic signals. This method, however, has given apparently contradictory results when applied in hippocampal CA1. The inconsistency could be explained if NMDA receptors were opened by glutamate not only released from local presynaptic terminals, but also diffusing from synapses on neighboring cells where LTP was not induced. Here we examine the effect of pairing-induced LTP on the MK-801 blocking rate in two afferent inputs to dentate granule cells. LTP in the medial perforant path is associated with a significant increase in the MK-801 blocking rate, implying a presynaptic increase in glutamate release probability. An enhanced MK-801 blocking rate is not seen, however, in the lateral perforant path. This result still could be compatible with a presynaptic contribution to LTP in the lateral perforant path if intersynaptic cross-talk occurred. In support of this hypothesis, we show that NMDA receptors consistently sense more quanta of glutamate than do α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. In the medial perforant path, in contrast, there is no significant difference in the number of quanta mediated by the two receptors. These results support a presynaptic contribution to LTP and imply that differences in intersynaptic cross-talk can complicate the interpretation of experiments designed to detect changes in transmitter release.
Activation of protein tyrosine kinase PYK2 by the m1 muscarinic acetylcholine receptor
Authors: Felsch Jason S. , Cachero Teresa G. , Peralta Ernest G.
Published: July 2002

Several G protein-coupled receptors are known to direct the tyrosine phosphorylation, and in some cases the activation, of diverse tyrosine kinases. Using a stable cell line approach, we characterize the activation of PYK2, a tyrosine kinase structurally related to focal adhesion kinase, by the G protein-coupled m1 muscarinic acetylcholine receptor. We find that PYK2 tyrosine kinase activity is critical for the m1 receptor-stimulated tyrosine phosphorylation of PYK2. Furthermore, we identify two tyrosine residues that are subject to phosphorylation in response to muscarinic signaling and show that this phosphorylation induces two cytosolic proteins, c-Src and Grb2, to bind to PYK2. This is the first demonstration of the significance played by distinct PYK2 tyrosine residues in G protein-coupled signaling to this kinase. By comparison, though m1 receptors induce the tyrosine phosphorylation of the cytoskeletal protein paxillin, the association of paxillin with PYK2 is unaffected by muscarinic signaling. We also provide evidence that PYK2 specifically phosphorylates the carboxyl-terminal cytosolic portion of the potassium channel Kv1.2 in a manner regulated by the m1 receptor. These results delineate molecular events attending the m1 muscarinic receptor stimulation of this tyrosine kinase and establish PYK2 as an effector of the m1 muscarinic receptor in the regulation of multiple cell functions.
Toward a resolution of the introns early/late debate: Only phase zero introns are correlated with the structure of ancient proteins
Authors: de Souza Sandro J. , Long Manyuan , Klein Robert J. , Roy Scott , Lin Shin , Gilbert Walter
Published: July 2002

We present evidence that a well defined subset of intron positions shows a non-random distribution in ancient genes. We analyze a database of ancient conserved regions drawn from GenBank 101 to retest two predictions of the theory that the first genes were constructed by exon shuffling. These predictions are that there should be an excess of symmetric exons (and sets of exons) flanked by introns of the same phase (positions within the codon) and that intron positions in ancient proteins should correlate with the boundaries of compact protein modules. Both these predictions are supported by the data, with considerable statistical force (
P
values < 0.0001). Intron positions correlate to modules of diameters around 21, 27, and 33 Å, and this correlation is due to phase zero introns. We suggest that 30–40% of present day intron positions in ancient genes correspond to phase zero introns originally present in the progenote, while almost all of the remaining intron positions correspond to introns added, or moved, appearing equally in all three intron phases. This proposal provides a resolution for many of the arguments of the introns-early/introns-late debate.
Cloning and characterization of a potassium-coupled amino acid transporter
Authors: Castagna Michela , Shayakul Chairat , Trotti Davide , Sacchi V. Franca , Harvey William R. , Hediger Matthias A.
Published: July 2002

Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotransporters that are driven by Na
+
or H
+
gradients. The present work extends the Na
+
and H
+
dogma by including the H
+
and K
+
paradigm. Lepidopteran insect larvae have a high K
+
and a low Na
+
content, and their midgut cells lack Na
+
/K
+
ATPase. Instead, an H
+
translocating, vacuolar-type ATPase generates a voltage of approximately −240 mV across the apical plasma membrane of so-called goblet cells, which drives H
+
back into the cells in exchange for K
+
, resulting in net K
+
secretion into the lumen. The resulting inwardly directed K
+
electrochemical gradient serves as a driving force for active amino acid uptake into adjacent columnar cells. By using expression cloning with
Xenopus laevis
oocytes, we have isolated a cDNA that encodes a K
+
-coupled amino acid transporter (KAAT1). We have cloned this protein from a larval lepidopteran midgut (
Manduca sexta
) cDNA library. KAAT1 is expressed in absorptive columnar cells of the midgut and in labial glands. When expressed in
Xenopus
oocytes, KAAT1 induced electrogenic transport of neutral amino acids but excludes α-(methylamino)isobutyric acid and charged amino acids resembling the mammalian system B. K
+
, Na
+
, and to a lesser extent Li
+
were accepted as cotransported ions, but K
+
is the principal cation, by far, in living caterpillars. Moreover, uptake was Cl
−
-dependent, and the K
+
/Na
+
selectivity increased with hyperpolarization of oocytes, reflecting the increased K
+
/Na
+
selectivity with hyperpolarization observed in midgut tissue. KAAT1 has 634 amino acid residues with 12 putative membrane spanning domains and shows a low level of identity with members of the Na
+
and Cl
−
-coupled neurotransmitter transporter family.
The crystal structure of a multifunctional protein: Phosphoglucose isomerase/autocrine motility factor/neuroleukin
Authors: Sun Yuh-Ju , Chou Chia-Cheng , Chen Wei-Shone , Wu Rong-Tsun , Meng Menghsiao , Hsiao Chwan-Deng
Published: July 2002

Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from
Bacillus stearothermophilus
at 2.3-Å resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm that PGI is neuroleukin and AMF. PGI has an open twisted α/β structural motif consisting of two globular domains and two protruding parts. Based on this substrate-free structure, together with the previously published biological, biochemical, and modeling results, we postulate a possible substrate-binding site that is located within the domains’ interface for PGI and AMF. In addition, the structure provides evidence suggesting that the top part of the large domain together with one of the protruding loops might participate in inducing the neurotrophic activity.
Fine-mapping of quantitative trait loci by identity by descent in outbred populations: Application to milk production in dairy cattle
Authors: Riquet Juliette , Coppieters Wouter , Cambisano Nadine , Arranz Juan-José , Berzi Paulette , Davis Scott K. , Grisart Bernard , Farnir Frédérick , Karim Latifa , Mni Myriam , Simon Patricia , Taylor Jeremy F. , Vanmanshoven Pascal , Wagenaar Danny , Womack James E. , Georges Michel
Published: July 2002

We previously mapped a quantitative trait locus (QTL) affecting milk production to bovine chromosome 14. To refine the map position of this QTL, we have increased the density of the genetic map of BTA14q11–16 by addition of nine microsatellites and three single nucleotide polymorphisms. Fine-mapping of the QTL was accomplished by a two-tiered approach. In the first phase, we identified seven sires heterozygous “
Qq
” for the QTL by marker-assisted segregation analysis in a Holstein-Friesian pedigree comprising 1,158 individuals. In a second phase, we genotyped the seven selected sires for the newly developed high-density marker map and searched for a shared haplotype flanking an hypothetical, identical-by-descent QTL allele with large substitution effect. The seven chromosomes increasing milk fat percentage were indeed shown to carry a common chromosome segment with an estimated size of 5 cM predicted to contain the studied QTL. The same haplotype was shown to be associated with increased fat percentage in the general population as well, providing additional support in favor of the location of the QTL within the corresponding interval.
Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle x-ray scattering
Authors: Pollack Lois , Tate Mark W. , Darnton Nicholas C. , Knight James B. , Gruner Sol M. , Eaton William A. , Austin Robert H.
Published: July 2002

Time-resolved small-angle x-ray scattering was used to measure the radius of gyration of cytochrome
c
after initiation of folding by a pH jump. Submillisecond time resolution was obtained with a microfabricated diffusional mixer and synchrotron radiation. The results show that the protein first collapses to compact denatured structures before folding very fast to the native state.
Convergence of two repressors through heterodimer formation of androgen receptor and testicular orphan receptor-4: A unique signaling pathway in the steroid receptor superfamily
Authors: Lee Yi-Fen , Shyr Chih-Rong , Thin Tin Htwe , Lin Wen-Jye , Chang Chawnshang
Published: July 2002

The androgen receptor (AR) binds to androgen response elements and regulates target genes via a mechanism involving coregulators. Here we demonstrate that the AR can interact with the testicular orphan receptor-4 (TR4) and function as a repressor to down-regulate the TR4 target genes by preventing the TR4 binding to its target DNA. Interestingly, the heterodimerization of AR and TR4 also allows TR4 to repress AR target gene expression. Simultaneous exposure to both receptors therefore could result in bidirectional suppression of their target genes. Together, these data demonstrate that the coupling of two different receptors, through the heterodimerization of AR and TR4, is a unique signaling pathway in the steroid receptor superfamily, which may facilitate further understanding of the complicated androgen action in prostate cancer or libido.
Long-term suppression of Leydig cell steroidogenesis prevents Leydig cell aging
Authors: Chen Haolin , Zirkin Barry R.
Published: July 2002

Male aging is accompanied by reduced testosterone production by the Leydig cells, the testosterone-producing cells of the testis. The mechanism by which this occurs is unknown. Based on the observations that reactive oxygen is capable of damaging components of the steroidogenic pathway and that reactive oxygen is produced during steroidogenesis itself, we hypothesized that long-term suppression of steroidogenesis might inhibit or prevent age-related deficits in Leydig cell testosterone production. To test this, we administered contraceptive doses of testosterone to groups of young (3 months old) and middle-aged (13 months old) Brown Norway rats via Silastic implants to suppress endogenous Leydig cell testosterone production. After 8 months, the implants were removed, which rapidly (days) restores the ability of the previously suppressed Leydig cells to produce testosterone. Two months after removing the implants, when the rats of the two groups were 13 and 23 months of age, respectively, the Leydig cells in both cases were found to produce testosterone at the high levels of young Leydig cells, whereas significantly lower levels were produced by the 23-month-old controls. Thus, by placing the Leydig cells in a state of steroidogenic “hibernation,” the reductions in Leydig cell testosterone production that invariably accompany aging did not occur. If hormonal contraception in the human functions the same way, the adverse consequences of reduced testosterone in later life (osteoporosis, reduced muscle mass, reduced libido, mood swings, etc.) might be delayed or prevented.
Glutamate and γ-aminobutyric acid mediate a heterosynaptic depression at mossy fiber synapses in the hippocampus
Authors: Vogt Kaspar E. , Nicoll Roger A.
Published: July 2002

Mossy fiber synapses form the major excitatory input into the autoassociative network of pyramidal cells in the CA3 area of the hippocampus. Here we demonstrate that at the mossy fiber synapses, glutamate and γ-aminobutyric acid (GABA) act as autaptic and heterosynaptic presynaptic inhibitory transmitters through metabotropic glutamate receptors (mGluRs) and GABA
B
receptors, respectively. Both GABA
B
receptors and mGluRs are activated through spillover from adjacent synapses. We demonstrate that glutamate spillover caused by brief tetanic activation of mossy fiber terminals remains intact at physiological temperatures. The activation of GABA
B
receptors increased the threshold for mossy fiber long-term potentiation (LTP), whereas activation of mGluRs did not have such an effect. We speculate that this heterosynaptic depression provides the mossy fiber synapses with a mechanism to efficiently shape input patterns into CA3, increasing the sparseness of the mossy fiber signal and enhancing the capacity and performance of the CA3 associative network. The increase in LTP threshold through activation of presynaptic inhibitory receptors imparts a piesynoptic associative nature to mossy fiber LTP.
Species realities and numbers in sexual vertebrates: Perspectives from an asexually transmitted genome
Authors: Avise John C. , Walker DeEtte
Published: July 2002

A literature review is conducted on the phylogenetic discontinuities in mtDNA sequences of 252 taxonomic species of vertebrates. About 140 of these species (56%) were subdivided clearly into two or more highly distinctive matrilineal phylogroups, the vast majority of which were localized geographically. However, only a small number (two to six) of salient phylogeographic subdivisions (those that stand out against mean within-group divergences) characterized individual species. A previous literature summary showed that vertebrate sister species and other congeners also usually have pronounced phylogenetic distinctions in mtDNA sequence. These observations, taken together, suggest that current taxonomic species often agree reasonably well in number (certainly within an order-of-magnitude) and composition with biotic entities registered in mtDNA genealogies alone. In other words, mtDNA data and traditional taxonomic assignments tend to converge on what therefore may be “real” biotic units in nature. All branches in mtDNA phylogenies are nonanastomose, connected strictly via historical genealogy. Thus, patterns of historical phylogenetic connection may be at least as important as contemporary reproductive relationshipsper sein accounting for microevolutionary unities and discontinuities in sexually reproducing vertebrates. Findings are discussed in the context of the biological and phylogenetic species concepts.
A nerve growth factor mimetic TrkA antagonist causes withdrawal of cortical cholinergic boutons in the adult rat
Authors: Debeir Thomas , Saragovi H. Uri , Cuello A. Claudio
Published: July 2002

Cholinergic neurons respond to the administration of nerve growth factor (NGF)
in vivo
with a prominent and selective increase of choline acetyl transferase activity. This suggests the possible involvement of endogenous NGF, acting through its receptor TrkA, in the maintenance of central nervous system cholinergic synapses in the adult rat brain. To test this hypothesis, a small peptide, C(92-96), that blocks NGF-TrkA interactions was delivered stereotactically into the rat cortex over a 2-week period, and its effect and potency were compared with those of an anti-NGF monoclonal antibody (mAb NGF30). Two presynaptic antigenic sites were studied by immunoreactivity, and the number of presynaptic sites was counted by using an image analysis system. Synaptophysin was used as a marker for overall cortical synapses, and the vesicular acetylcholine transporter was used as a marker for cortical cholinergic presynaptic sites. No significant variations in the number of synaptophysin-immunoreactive sites were observed. However, both mAb NGF30 and the TrkA antagonist C(92-96) provoked a significant decrease in the number and size of vesicular acetylcholine transporter–IR sites, with the losses being more marked in the C(92-96) treated rats. These observations support the notion that endogenously produced NGF acting through TrkA receptors is involved in the maintenance of the cholinergic phenotype in the normal, adult rat brain and supports the idea that NGF normally plays a role in the continual remodeling of neural circuits during adulthood. The development of neurotrophin mimetics with antagonistic and eventually agonist action may contribute to therapeutic strategies for central nervous system degeneration and trauma.
Expression of the antiproliferative gene TIS21 at the onset of neurogenesis identifies single neuroepithelial cells that switch from proliferative to neuron-generating division
Authors: Iacopetti Paola , Michelini Monica , Stuckmann Ingo , Oback Björn , Aaku-Saraste Eeva , Huttner Wieland B.
Published: July 2002

At the onset of mammalian neurogenesis, neuroepithelial (NE) cells switch from proliferative to neuron-generating divisions. Understanding the molecular basis of this switch requires the ability to distinguish between these two types of division. Here we show that in the mouse ventricular zone, expression of the mRNA of the antiproliferative gene TIS21 (PC3, BTG2) (
i
) starts at the onset of neurogenesis, (
ii
) is confined to a subpopulation of NE cells that increases in correlation with the progression of neurogenesis, and (
iii
) is not detected in newborn neurons. Expression of the TIS21 mRNA in the NE cells occurs transiently during the cell cycle, i.e., in the G
1
phase. In contrast to the TIS21 mRNA, the TIS21 protein persists through the division of NE cells and is inherited by the neurons, where it remains detectable during neuronal migration and the initial phase of differentiation. Our observations indicate that the TIS21 gene is specifically expressed in those NE cells that, at their next division, will generate postmitotic neurons, but not in proliferating NE cells. Using TIS21 as a marker, we find that the switch from proliferative to neuron-generating divisions is initiated in single NE cells rather than in synchronized neighboring cells.
Trophic cascades in a complex terrestrial community
Authors: Dyer L. A. , Letourneau D. K.
Published: July 2002

To test for direct and indirect effects of a top predator on three lower trophic levels, we conducted two multiyear predator addition experiments in a tropical wet forest. Periodic additions of a top predator (predatory clerid beetle) to a wet forest understory shrub caused a reduction in the predatory beetle’s prey (a predatory ant), increased herbivory, and reduced leaf area of the plant. These effects occurred whether beetles were added to naturally occurring shrubs or to reproductive fragments, suggesting fitness effects of top predators through three trophic levels. A correlational study showed that trophic effects of top predators also cascaded to nearby conspecifics in the forest understory. We use trends from understory plant surveys to suggest mechanisms by which these cascades could ultimately affect species diversity in the local plant community.
A critical role for eotaxin in experimental oral antigen-induced eosinophilic gastrointestinal allergy
Authors: Hogan Simon P. , Mishra Anil , Brandt Eric B. , Foster Paul S. , Rothenberg Marc E.
Published: July 2002

Despite marked advances in the understanding of allergic responses, the mechanisms regulating gastrointestinal allergy are not very well understood. We have developed a model of antigen-induced eosinophil-associated gastrointestinal allergy and characterized the role of eotaxin and IL-5. Challenge of allergen-sensitized mice with oral allergen, in the form of enteric-coated beads, resulted in marked allergen-specific IgG1and IgE, Th2-type (IL-4 and IL-5) cytokine production, and eosinophil accumulation in the blood and small intestine. In the genetic absence of eotaxin, a chemokine constitutively expressed in the gastrointestinal tract, eosinophil recruitment into the small intestine was ablated, and these mice developed enhanced eosinophil accumulation in the blood compared with wild-type mice. Interestingly, in the absence of IL-5, allergen challenge promoted partial eosinophil accumulation into the small intestine and a decline in circulating eosinophil levels. Collectively, these results establish that the accumulation of gastrointestinal eosinophils is antigen induced, can occur independent of IL-5, and provides a molecular mechanism to explain the dichotomy between peripheral blood and tissue eosinophilia. Furthermore, eotaxin is identified as a critical regulator of antigen-induced eosinophilic inflammation in the gastrointestinal tract.
Polynesian origins: Insights from the Y chromosome
Authors: Su Bing , Jin Li , Underhill Peter , Martinson Jeremy , Saha Nilmani , McGarvey Stephen T. , Shriver Mark D. , Chu Jiayou , Oefner Peter , Chakraborty Ranajit , Deka Ranjan
Published: July 2002

The question surrounding the colonization of Polynesia has remained controversial. Two hypotheses, one postulating Taiwan as the putative homeland and the other asserting a Melanesian origin of the Polynesian people, have received considerable attention. In this work, we present haplotype data based on the distribution of 19 biallelic polymorphisms on the Y chromosome in a sample of 551 male individuals from 36 populations living in Southeast Asia, Taiwan, Micronesia, Melanesia, and Polynesia. Surprisingly, nearly none of the Taiwanese Y haplotypes were found in Micronesia and Polynesia. Likewise, a Melanesian-specific haplotype was not found among the Polynesians. However, all of the Polynesian, Micronesian, and Taiwanese haplotypes are present in the extant Southeast Asian populations. Evidently, the Y-chromosome data do not lend support to either of the prevailing hypotheses. Rather, we postulate that Southeast Asia provided a genetic source for two independent migrations, one toward Taiwan and the other toward Polynesia through island Southeast Asia.
The junctional multidomain protein AF-6 is a binding partner of the Rap1A GTPase and associates with the actin cytoskeletal regulator profilin
Authors: Boettner Benjamin , Govek Eve-Ellen , Cross Justin , Van Aelst Linda
Published: July 2002

The AF-6 protein is a multidomain protein that contains two potential Ras-binding domains within its N terminus. Because of this feature, AF-6 has been isolated in both two-hybrid and biochemical approaches and is postulated to be a potential Ras-effector protein. Herein, we show that it is specifically the first Ras-binding domain of AF-6 that mediates this interaction and that the Ras-related Rap1A protein can associate with this motif even more efficiently than the oncogenic Ha-, K-, and N-Ras GTPases. We further demonstrate that both Ras and Rap1 interact with full-length AF-6
in vivo
in mammalian cells and that a fraction of Rap1 colocalizes with AF-6 at the membrane. Dominant active Rap1A, in contrast to Ras, when introduced into epithelial MDCK and MCF-7 cells, does not perturb AF-6-specific residency in cell–cell adhesion complexes. In a pursuit to gain further understanding of the role of AF-6 in junctions, we identified profilin as an AF-6-binding protein. Profilin activates monomeric actin units for subsequent polymerization steps at barbed ends of actin filaments and has been shown to participate in cortical actin assembly. To our knowledge, AF-6 is the only integral component in cell–cell junctions discovered thus far that interacts with profilin and thus could modulate actin modeling proximal to adhesion complexes.
A role for α- and β-catenins in bacterial uptake
Authors: Lecuit Marc , Hurme Reini , Pizarro-Cerdá Javier , Ohayon Hélène , Geiger Benjamin , Cossart Pascale
Published: July 2002

Interaction of internalin with E-cadherin promotes entry of
Listeria monocytogenes
into human epithelial cells. This process requires actin cytoskeleton rearrangements. Here we show, by using a series of stably transfected cell lines expressing E-cadherin variants, that the ectodomain of E-cadherin is sufficient for bacterial adherence and that the intracytoplasmic domain is required for entry. The critical cytoplasmic region was further mapped to the β-catenin binding domain. Because β-catenin is known to interact with α-catenin, which binds to actin, we generated a fusion molecule consisting of the ectodomain of E-cadherin and the actin binding site of α-catenin. Cells expressing this chimera were as permissive as E-cadherin-expressing cells. In agreement with these data, α- and β-catenins as well as E-cadherin clustered and colocalized at the entry site, where F-actin then accumulated. Taken together, these results reveal that E-cadherin, via β- and α-catenins, can trigger dynamic events of actin polymerization and membrane extensions culminating in bacterial uptake.
Enrichment for murine keratinocyte stem cells based on cell surface phenotype
Authors: Tani Hiroaki , Morris Rebecca J. , Kaur Pritinder
Published: July 2002

The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [3H]thymidine ([3H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [3H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use ofin vivocell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of α6integrin and low to undetectable expression of the transferrin receptor (CD71) termed α6briCD71dimcells, are enriched for epithelial stem cells because they represent a minor (≈8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing ≈70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed α6briCD71bri, representing the majority (≈60%) of basal keratinocytes that are actively cycling, and importantly contain ≈70% of [3H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71dimcells in the hair follicle bulge region, a well documented location for KSCs.
Risk, sexuality and economy
Authors: Adkins Lisa
Published: July 2002

From the mid 1980s onwards HIV/AIDS became a new subject of work reform, with a range of experts producing new knowledges on work and the worker in regard to HIV/AIDS and workplace organizations putting in place workplace HIV/AIDS policies and programmes. To date, much of the discussion in sociology in regard to such policies and programmes has focused on the issue of effectiveness and has been concerned with making such policy ‘better’. In this article however, and with particular reference to sexuality, I suggest that such approaches fail to register that workplace HIV/AIDS policies concern new conceptualizations of worker identities. Specifically, I suggest that such policies may be viewed as part of an assemblage of work reforms which are reworking worker identities as risk identities. Thus I argue that workplace HIV/AIDS policies and programmes are best understood as risk rationalities. Further, I consider the alignment between such rationalities and neo‐liberal modes of rule, and in particular consider the ways in which workplace HIV/AIDS policies render both HIV/AIDS and sexuality calculable and governable in terms of notions of risk, self‐responsibility and self‐management.
THE 1998 JAMES RANKIN ORATION: Reducing the toll of opioid overdose deaths in Australia
Authors: HALL WAYNE
Published: July 2002

Opioid overdose mortality among young adults in Australia has increased consistently over the past several decades. Among Australian adults aged 15–44 years, the number of these deaths has increased from six in 1964 to 600 in 1997. The rate (per million adults in this age group) increased 55‐fold, from 1.3 in 1964 to 71.5 in 1997. The proportion of all deaths in adults in this age group caused by opioid overdose increased from 0.1% in 1964 to 7.3% in 1997. The magnitude of the increase makes it unlikely to be an artefact of changes in diagnosis, especially as similar increases have also been observed in other countries. These trends are also consistent with historical information which indicates that illicit heroin use first came to police attention in Sydney and Melbourne in the late 1960s. There is an urgent need to implement and evaluate a variety of measures to reduce the unacceptable toll of opioid overdose deaths among young Australians. These include: peer education about the risks of polydrug use and overdose after resuming opioid use after periods of abstinence, and attracting more dependent users into opioid maintenance treatment. Measures are also needed to improve responses to overdose by encouraging witnesses to call ambulances, training drug users in CPR, and trialling distribution of the opiate antagonist naloxone to users at high risk of overdose.
Pharmacokinetics of ethanol: a review of the methodology
Authors: Matsumoto Hiroshi , Fukui Yuko
Published: July 2002

Abstract This paper reviews current concepts on tools for studying the pharmacokinetics of alcohol. It has been known that ethanol metabolism occurs mainly in the liver via alcohol dehydrogenase and an accessory microsomal pathway. The contribution of each pathway has been examined by administration of metabolic inhibitors. The role of gastric alcohol dehydrogenase in the first‐pass effects of ethanol has been speculative and may be relatively low. Some pharmacokinetic approaches with mathematical models have elucidated the role of gastric alcohol dehydorgenase, hepatic alcohol dehydrogenase and cytochrome P450 2E1 in ethanol elimination. The scale‐up of ethanol elimination kinetics has enabled extrapolation from animal models to human kinetics. The clarification of the pharmacokinetics of ethanol is very important for estimating the effects of ethanol on biological events.
Mildly Oxidized Glyceraldehyde‐3‐Phosphate Dehydrogenase as a Possible Regulator of Glycolysis
Authors: Danshina P. V. , Schmalhausen E. V. , Avetisyan A. V. , Muronetz V. I.
Published: July 2002

Influence of H 2 O 2 on glycolysis was investigated. A hypothesis previously formulated was tested according to which a mild oxidation of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) results in uncoupling of oxidation and phosphorylation at this step of glycolysis due to acylphosphatase activity of the oxidized enzyme. Incubation of a mixture of purified glycolytic enzymes, as well as a muscle extract, in the presence of 10‐100 μMH 2 O 2 was shown to result in an increase in the rate of glycolysis. The level of lactate accumulation in the oxidized samples increased by 80‐150% compared to the samples containing mercaptoethanol. No ATP was formed by the H 2 O 2 ‐stimulated glycolysis. Thus, H 2 O 2 really caused uncoupling of oxidation and phosphorylation in glycolysis. A role of GAPDH oxidation in regulation of glycolysis is discussed.
Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains
Authors: King Stephen J. , Dutcher Susan K.
Published: July 2002

To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning.
A Conserved Functional Domain of Drosophila Coracle Is Required for Localization at the Septate Junction and Has Membrane-organizing Activity
Authors: Ward Robert E. , Lamb Rebecca S. , Fehon Richard G.
Published: July 2002

The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members.
Microinjection of Antibody to Mad2 Protein into Mammalian Cells in Mitosis Induces Premature Anaphase
Authors: Gorbsky Gary J. , Chen Rey-Huei , Murray Andrew W.
Published: July 2002

In yeast, the Mad2 protein is required for the M phase arrest induced by microtubule inhibitors, but the protein is not essential under normal culture conditions. We tested whether the Mad2 protein participates in regulating the timing of anaphase onset in mammalian cells in the absence of microtubule drugs. When microinjected into living prophase or prometaphase PtK1 cells, anti-Mad2 antibody induced the onset of anaphase prematurely during prometaphase, before the chromosomes had assembled at the metaphase plate. Anti-Mad2 antibody-injected cells completed all aspects of anaphase including chromatid movement to the spindle poles and pole–pole separation. Identical results were obtained when primary human keratinocytes were injected with anti-Mad2 antibody. These studies suggest that Mad2 protein function is essential for the timing of anaphase onset in somatic cells at each mitosis. Thus, in mammalian somatic cells, the spindle checkpoint appears to be a component of the timing mechanism for normal mitosis, blocking anaphase onset until all chromosomes are aligned at the metaphase plate.
Signal Sequence Recognition in Cotranslational Translocation by Protein Components of the Endoplasmic Reticulum Membrane
Authors: Mothes Walther , Jungnickel Berit , Brunner Josef , Rapoport Tom A.
Published: July 2002

We have investigated the role of membrane proteins and lipids during early phases of the cotranslational insertion of secretory proteins into the translocation channel of the endoplasmic reticulum (ER) membrane. We demonstrate that all steps, including the one during which signal sequence recognition occurs, can be reproduced with purified translocation components in detergent solution, in the absence of bulk lipids or a bilayer. Photocross-linking experiments with native membranes show that upon complete insertion into the channel signal sequences are both precisely positioned with respect to the protein components of the channel and contact lipids. Together, these results indicate that signal sequences are bound to a specific binding site at the interface between the channel and the surrounding lipids, and are recognized ultimately by protein–protein interactions. Our data also suggest that at least some signal sequences reach the binding site by transfer through the interior of the channel.
A Large PEST-like Sequence Directs the Ubiquitination, Endocytosis, and Vacuolar Degradation of the Yeast a-Factor Receptor
Authors: Roth Amy F. , Sullivan Daniel M. , Davis Nicholas G.
Published: July 2002

The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode. Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G. Davis. 1996. J. Cell Biol. 134:661–674) and both depend on sequence elements within the receptor's regulatory, cytoplasmically disposed, COOH-terminal domain (CTD). Here, we concentrate on the Ste3p sequences required for constitutive endocytosis. Constitutive endocytosis is rapid. Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole where it is degraded, all with a t1/2 of 15 min. Deletion analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD that is the minimal sequence required for this rapid turnover. Deletions intruding into this interval block or severely slow the rate of endocytic turnover. Moreover, the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination. Not only necessary for ubiquitination and endocytosis, this sequence also is sufficient. When transplanted to a stable cell surface protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis and consequent vacuolar degradation. Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity—no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval. The high proportion of acidic and hydroxylated amino acid residues in this interval suggests a similarity to PEST sequences—a broad class of sequences which have been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and cytoplasmic proteins. A likely possibility, therefore, is that this sequence, responsible for both endocytosis and ubiquitination, may be first and foremost a ubiquitination signal. Finally, we present evidence suggesting that the true signal in the wild-type receptor extends beyond the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which extend another 21 residues to the COOH terminus of Ste3p. Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ubiquitination/endocytosis signal.
ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation
Authors: Sloan-Lancaster Joanne , Presley John , Ellenberg Jan , Yamazaki Tetsuo , Lippincott-Schwartz Jennifer , Samelson Lawrence E.
Published: July 2002

The nonreceptor protein tyrosine kinase ZAP-70 is a critical enzyme required for successful T lymphocyte activation. After antigenic stimulation, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the TCRζ chain expressed as a chimeric protein (TTζ and Tζζ), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tζζ fused to green fluorescent protein (ZAP-70 GFP and Tζζ–GFP, respectively). Time-lapse imaging using confocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long duration. The presence of the TCRζ chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTζ was indicated using mutant ZAP-70 GFPs and a truncated ζ chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. Tζζ– GFP is relatively immobile, while peripherally located ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided new insights into the TCRζ–ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.
The kakapo Mutation Affects Terminal Arborization and Central Dendritic Sprouting of Drosophila Motorneurons
Authors: Prokop Andreas , Uhler Jay , Roote John , Bate Michael
Published: July 2002

The lethal mutation l(2)CA4 causes specific defects in local growth of neuronal processes. We uncovered four alleles of l(2)CA4 and mapped it to bands 50A-C on the polytene chromosomes and found it to be allelic to kakapo (Prout et al. 1997. Genetics. 146:275– 285). In embryos carrying our kakapo mutant alleles, motorneurons form correct nerve branches, showing that long distance growth of neuronal processes is unaffected. However, neuromuscular junctions (NMJs) fail to form normal local arbors on their target muscles and are significantly reduced in size. In agreement with this finding, antibodies against kakapo (Gregory and Brown. 1998. J. Cell Biol. 143:1271–1282) detect a specific epitope at all or most Drosophila NMJs. Within the central nervous system of kakapo mutant embryos, neuronal dendrites of the RP3 motorneuron form at correct positions, but are significantly reduced in size. At the subcellular level we demonstrate two phenotypes potentially responsible for the defects in neuronal branching: first, transmembrane proteins, which can play important roles in neuronal growth regulation, are incorrectly localized along neuronal processes. Second, microtubules play an important role in neuronal growth, and kakapo appears to be required for their organization in certain ectodermal cells: On the one hand, kakapo mutant embryos exhibit impaired microtubule organization within epidermal cells leading to detachment of muscles from the cuticle. On the other, a specific type of sensory neuron (scolopidial neurons) shows defects in microtubule organization and detaches from its support cells.
The β4 Integrin Interactor p27BBP/eIF6 Is an Essential Nuclear Matrix Protein Involved in 60S Ribosomal Subunit Assembly
Authors: Sanvito Francesca , Piatti Simonetta , Villa Antonello , Bossi Mario , Lucchini Giovanna , Marchisio Pier Carlo , Biffo Stefano
Published: July 2002

p27BBP/eIF6 is an evolutionarily conserved protein that was originally identified as p27BBP, an interactor of the cytoplasmic domain of integrin β4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27BBP/eIF6, its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing β4 integrin, p27BBP/eIF6 is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of β4 integrin. Surprisingly, in the absence and in the presence of the β4 integrin subunit, p27BBP/eIF6 is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27BBP/eIF6 homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27BBP/eIF6 can rescue the lethal effect of the iihΔ yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27BBP/eIF6 in ribosome biogenesis or assembly rather than in translation. A further function related to the β4 integrin subunit may have evolved specifically in higher eukaryotic cells.