bluuebunny/crossref_metadata_2025 huggingface dataset:
Soldier caste-specific gene expression in the mandibular glands ofHodotermopsis japonica(Isoptera: Termopsidae)
Authors: Miura Toru , Kamikouchi Azusa , Sawata Miyuki , Takeuchi Hideaki , Natori Syunji , Kubo Takeo , Matsumoto Tadao
Published: July 2002

Although “polymorphic castes” in social insects are well known as one of the most important phenomena of polyphenism, few studies of caste-specific gene expressions have been performed in social insects. To identify genes specifically expressed in the soldier caste of the Japanese damp-wood termiteHodotermopsis japonica, we employed the differential-display method using oligo(dT) and arbitrary primers, compared mRNA from the heads of mature soldiers and pseudergates (worker caste), and identified a clone (PCR product) 329 bp in length termedSOL1. Northern blot analysis showed that theSOL1mRNA is about 1.0 kb in length and is expressed specifically in mature soldiers, but not in pseudergates, even in the presoldier induction by juvenile hormone analogue, suggesting that the product is specific for terminally differentiated soldiers. By using the method of 5′- and 3′-rapid amplification of cDNA ends, we isolated the full length ofSOL1cDNA, which contained an ORF with a putative signal peptide at the N terminus. The sequence showed no significant homology with any other known protein sequences.In situhybridization analysis showed thatSOL1is expressed specifically in the mandibular glands. These results strongly suggest that theSOL1gene encodes a secretory protein specifically synthesized in the mandibular glands of the soldiers. Histological observations revealed that the gland actually develops during the differentiation into the soldier caste.
The use of gene clusters to infer functional coupling
Authors: Overbeek Ross , Fonstein Michael , D’Souza Mark , Pusch Gordon D. , Maltsev Natalia
Published: July 2002

Previously, we presented evidence that it is possible to predict functional coupling between genes based on conservation of gene clusters between genomes. With the rapid increase in the availability of prokaryotic sequence data, it has become possible to verify and apply the technique. In this paper, we extend our characterization of the parameters that determine the utility of the approach, and we generalize the approach in a way that supports detection of common classes of functionally coupled genes (e.g., transport and signal transduction clusters). Now that the analysis includes over 30 complete or nearly complete genomes, it has become clear that this approach will play a significant role in supporting efforts to assign functionality to the remaining uncharacterized genes in sequenced genomes.
Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1
Authors: Wise Lyn M. , Veikkola Tanja , Mercer Andrew A. , Savory Loreen J. , Fleming Stephen B. , Caesar Carol , Vitali Angela , Makinen Taija , Alitalo Kari , Stacker Steven A.
Published: July 2002

Orf virus, a member of the poxvirus family, produces a pustular dermatitis in sheep, goats, and humans. The lesions induced after infection with orf virus show extensive proliferation of vascular endothelial cells, dilation of blood vessels and dermal swelling. An explanation for the nature of these lesions may lie in the discovery that orf virus encodes an apparent homolog of the mammalian vascular endothelial growth factor (VEGF) family of molecules. These molecules mediate endothelial cell proliferation, vascular permeability, angiogenesis, and lymphangiogenesis via the endothelial cell receptors VEGFR-1 (Flt1), VEGFR-2 (KDR/Flk1), and VEGFR-3 (Flt4). The VEGF-like protein of orf virus strain NZ2 (ORFV2-VEGF) is most closely related in primary structure to VEGF. In this study we examined the biological activities and receptor specificity of the ORFV2-VEGF protein. ORFV2-VEGF was found to be a disulfide-linked homodimer with a subunit of ≈25 kDa. ORFV2-VEGF showed mitogenic activity on bovine aortic and human microvascular endothelial cells and induced vascular permeability. ORFV2-VEGF was found to bind and induce autophosphorylation of VEGFR-2 and was unable to bind or activate VEGFR-1 and VEGFR-3, but bound the newly identified VEGF
165
receptor neuropilin-1. These results indicate that, from a functional viewpoint, ORFV2-VEGF is indeed a member of the VEGF family of molecules, but is unique, however, in that it utilizes only VEGFR-2 and neuropilin-1.
A mimic of HIV-1 nucleocapsid protein impairs reverse transcription and displays antiviral activity
Authors: Druillennec S. , Dong C. Z. , Escaich S. , Gresh N. , Bousseau A. , Roques B. P. , Fournié-Zaluski M. C.
Published: July 2002

Combined inhibition of HIV-1 reverse transcriptase and protease has significantly improved the treatment of AIDS. Nevertheless, resistance to these drugs occurs rapidly because of viral mutations, emphasizing the importance of identifying novel retroviral targets to develop new drug combinations. The critical role played by the nucleocapsid protein NCp7 of HIV-1 at different steps of the retrovirus life cycle makes it an attractive target for the development of new antiviral agents. NCp7 contains two highly conserved zinc fingers and is characterized by a three-dimensional structure that cannot be modified without a complete loss of infectivity of mutated viruses. Based on these structural data, we report that RB 2121, a cyclic peptide designed to mimic several essential biological determinants of NCp7, displays antiviral activity by inhibiting HIV-1 replication in CEM-4 cells infected by HIV-1.
In vitro
, RB 2121 does not interfere with HIV-1 cell entry and viral enzymes but is able to inhibit the annealing activities of NCp7 by recognizing nucleic acids. Analysis of proviral DNA synthesis by means of PCR has shown that RB 2121 acts at an early step of the retrovirus life cycle by inducing a dose-dependent reduction in transcribed DNA levels through inhibition of NCp7–reverse transcriptase interaction. Because of its original mechanism of action, RB 2121 provides an interesting lead for the rational development of new anti-HIV-1 agents that could be associated advantageously with enzyme inhibitors to counteract rapid virus mutations and resistance problems observed in tritherapies.
A small, physiological electric field orients cell division
Authors: Zhao Min , Forrester John V. , McCaig Colin D.
Published: July 2002

We report on an observation that the orientation of cell division is directed by small, applied electric fields (EFs). Cultured human corneal epithelial cells were exposed to a direct-current EF of physiological magnitude. Cells divided while attached to the culture dish, and most did so with a cleavage plane perpendicular to the EF vector. There are many instances in which cell divisions
in vivo
occur in the presence of direct-current physiological EF, for example, during embryonic morphogenesis, neuronal and epithelial differentiation, wound healing, or tumor formation. Endogenous physiological EFs may play important roles in some or all of these processes by regulating the axis of cell division and, hence, the positioning of daughter cells.
Mimicking the antenna-electron transfer properties of photosynthesis
Authors: Sykora Milan , Maxwell Kimberly A. , DeSimone Joseph M. , Meyer Thomas J.
Published: July 2002

A molecular assembly based on derivatized polystyrene is described,
which mimics both the light-harvesting and energy-conversion steps of
photosynthesis. The system is unique in that the two key parts of a
photosynthetic system are incorporated in a functional assembly
constructed from polypyridine complexes of Ru
II
. This
system is truly artificial, as none of the components used in
construction of the assembly are present in a natural photosynthetic
system. Quantitative evaluation of the energy and electron transfer
dynamics after transient irradiation by visible light offers important
insights into the mechanisms of energy transport and electron transfer
that lead to photosynthetic light-to-chemical energy conversion.
Genetic influence on the structural variations of the abnormal prion protein
Authors: Parchi Piero , Zou Wenquan , Wang Wen , Brown Paul , Capellari Sabina , Ghetti Bernardino , Kopp Nicolas , Schulz-Schaeffer Walter J. , Kretzschmar Hans A. , Head Mark W. , Ironside James W. , Gambetti Pierluigi , Chen Shu G.
Published: July 2002

Prion diseases are characterized by the presence of the abnormal prion protein PrP
Sc
, which is believed to be generated by the conversion of the α-helical structure that predominates in the normal PrP isoform into a β-sheet structure resistant to proteinase K (PK). In human prion diseases, two major types of PrP
Sc
, type 1 and 2, can be distinguished based on the difference in electrophoretic migration of the PK-resistant core fragment. In this study, protein sequencing was used to identify the PK cleavage sites of PrP
Sc
in 36 cases of prion diseases. We demonstrated two primary cleavage sites at residue 82 and residue 97 for type 1 and type 2 PrP
Sc
, respectively, and numerous secondary cleavages distributed along the region spanning residues 74–102. Accordingly, we identify three regions in PrP
Sc
: one N-terminal (residues 23–73) that is invariably PK-sensitive, one C-terminal (residues 103–231) that is invariably PK-resistant, and a third variable region (residues 74–102) where the site of the PK cleavage, likely reflecting the extent of the β-sheet structure, varies mostly as a function of the PrP genotype at codon 129.
Kinetics of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse of the mouse
Authors: Moser Tobias , Beutner Dirk
Published: July 2002

Hearing in mammals relies on the highly synchronous synaptic transfer between cochlear inner hair cells (IHCs) and the auditory nerve. We studied the presynaptic function of single mouse IHCs by monitoring membrane capacitance changes and voltage-gated Ca
2+
currents. Exocytosis initially occurred at a high rate but then slowed down within a few milliseconds, despite nearly constant Ca
2+
influx. We interpret the observed secretory depression as depletion of a readily releasable pool (RRP) of about 280 vesicles. These vesicles are probably docked close to Ca
2+
channels at the ribbon-type active zones of the IHCs. Continued depolarization evoked slower exocytosis occurring at a nearly constant rate for at least 1 s and depending on “long-distance” Ca
2+
signaling. Refilling of the RRP after depletion followed a biphasic time course and was faster than endocytosis. RRP depletion is discussed as a mechanism for fast auditory adaptation.
Subdivisions of auditory cortex and processing streams in primates
Authors: Kaas Jon H. , Hackett Troy A.
Published: July 2002

The auditory system of monkeys includes a large number of interconnected subcortical nuclei and cortical areas. At subcortical levels, the structural components of the auditory system of monkeys resemble those of nonprimates, but the organization at cortical levels is different. In monkeys, the ventral nucleus of the medial geniculate complex projects in parallel to a core of three primary-like auditory areas, AI, R, and RT, constituting the first stage of cortical processing. These areas interconnect and project to the homotopic and other locations in the opposite cerebral hemisphere and to a surrounding array of eight proposed belt areas as a second stage of cortical processing. The belt areas in turn project in overlapping patterns to a lateral parabelt region with at least rostral and caudal subdivisions as a third stage of cortical processing. The divisions of the parabelt distribute to adjoining auditory and multimodal regions of the temporal lobe and to four functionally distinct regions of the frontal lobe. Histochemically, chimpanzees and humans have an auditory core that closely resembles that of monkeys. The challenge for future researchers is to understand how this complex system in monkeys analyzes and utilizes auditory information.
Transplanted fetal striatum in Huntington's disease: Phenotypic development and lack of pathology
Authors: Freeman Thomas B. , Cicchetti Francesca , Hauser Robert A. , Deacon Terrence W. , Li Xiao-Jiang , Hersch Steven M. , Nauert G. Michael , Sanberg Paul R. , Kordower Jeffrey H. , Saporta Samuel , Isacson Ole
Published: July 2002

Neural and stem cell transplantation is emerging as a potential
treatment for neurodegenerative diseases. Transplantation of specific
committed neuroblasts (fetal neurons) to the adult brain provides such
scientific exploration of these new potential therapies. Huntington's
disease (HD) is a fatal, incurable autosomal dominant (CAG repeat
expansion of huntingtin protein) neurodegenerative disorder with
primary neuronal pathology within the caudate–putamen (striatum). In a
clinical trial of human fetal striatal tissue transplantation, one
patient died 18 months after transplantation from cardiovascular
disease, and postmortem histological analysis demonstrated surviving
transplanted cells with typical morphology of the developing striatum.
Selective markers of both striatal projection and interneurons such as
dopamine and c-AMP-related phosphoprotein, calretinin,
acetylcholinesterase, choline acetyltransferase, tyrosine hydroxylase,
calbindin, enkephalin, and substance P showed positive transplant
regions clearly innervated by host tyrosine hydroxylase fibers. There
was no histological evidence of immune rejection including microglia
and macrophages. Notably, neuronal protein aggregates of mutated
huntingtin, which is typical HD neuropathology, were not found within
the transplanted fetal tissue. Thus, although there is a genetically
predetermined process causing neuronal death within the HD striatum,
implanted fetal neural cells lacking the mutant HD gene may be able to
replace damaged host neurons and reconstitute damaged neuronal
connections. This study demonstrates that grafts derived from human
fetal striatal tissue can survive, develop, and are unaffected by the
disease process, at least for 18 months, after transplantation into a
patient with HD.
Reduced susceptibility to ischemic brain injury and
N
-methyl-
d
-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice
Authors: Iadecola Costantino , Niwa Kiyoshi , Nogawa Shigeru , Zhao Xueren , Nagayama Masao , Araki Eiichi , Morham Scott , Ross M. Elizabeth
Published: July 2002

Cyclooxygenase-2 (COX-2), a prostanoid-synthesizing enzyme that
contributes to the toxicity associated with inflammation, has recently
emerged as a promising therapeutic target for several illnesses,
ranging from osteoarthritis to Alzheimer's disease. Although COX-2 has
also been linked to ischemic stroke, its role in the mechanisms of
ischemic brain injury remains controversial. We demonstrate that
COX-2-deficient mice have a significant reduction in the brain injury
produced by occlusion of the middle cerebral artery. The protection can
be attributed to attenuation of glutamate neurotoxicity, a
critical factor in the initiation of ischemic brain injury, and to
abrogation of the deleterious effects of postischemic inflammation, a
process contributing to the secondary progression of the damage. Thus,
COX-2 is involved in pathogenic events occurring in both the early and
late stages of cerebral ischemia and may be a valuable therapeutic
target for treatment of human stroke.
Using New Media to Teach East European History
Authors: Kelly T. Mills
Published: July 2002

For decades the residents of Taos, New Mexico have been afflicted by a low frequency humming—sometimes louder, sometimes almost inaudible—but never completely absent. On almost every college campus in North America, the buzz about using technology in teaching can be almost as annoying—and with each passing year, it gets louder. Although recent events in the American stock market have taken a good deal of the shine off of the idea that the Internet will fundamentally transform the economy, in higher education there seems to be no corresponding waning of enthusiasm for the infusion of new media into the educational experiences of students.
The development of aggressive behaviour during childhood: What have we learned in the past century?
Authors: Tremblay Richard E.
Published: July 2002

Research on human aggression has been a flourishing industry in the 20th century. As the attention shifted from an instinctual paradigm to a drive paradigm and a social learning paradigm, what have we learned on the development of aggressive behaviour during childhood? Are children born with an aggressive instinct or do they have to learn to aggress?This question has deep philosophical roots, but it also has important practical implications. Should interventions prevent children from learning to aggress or should they help children learn to inhibit aggressive reactions? Since most of the 20th century work on the development of aggression was concentrated on adolescents and elementary school age children, there appeared to be an implicit assumption that aggression is learned during these developmental periods. It is argued that to understand the origins of aggressive behaviour and prevent chronic cases of physical aggression we will need to focus on the development of aggressive behaviour during the first few years after birth, and differentiate among forms of aggressive behaviour. The form of agressive behaviour that is generally considered more “serious” or “socially unacceptable” (physical aggression) is clearly ontogenetically antecedent to less “serious” forms of aggressive behaviour, such as verbal aggression or indirect aggression. Furthermore, as a rule the frequency of physical aggression appears to decrease with age. However, infants’ physical aggression has generally not been considered developmentally significant. This is probably because of “the weakness of their limbs” and the apparent lack of “intentionality”. To have a relatively complete description of the life-span developmental trajectories of human aggressive behaviour by the end of the 21st century, we will need to start recruiting pregnant women very soon.
Overview of Male Reproductive Pathology
Authors: Foley George L.
Published: July 2002

An understanding of form and function is important for examination of the male reproductive tract. A basic understanding of spermatogenesis and hormonal function in the reproductive tract is essential for the pathologists in this evaluation. Gross and histologic reproductive changes need to be distinguished from normal variation and correlated with the reproductive status of the animal. This is especially important when correlating histologic changes with organ weight and other reproductive parameters, such as seminal analysis data. Sexual maturity of animals and tissue handing can impact interpretation. Sexual immaturity of preclinical safety animals can present challenges for accurate identification of compound-related changes. Likewise, proper handling of unfixed reproductive tissues and appropriate selection of a fi xation protocol are important in avoiding artifacts that may interfere with the microscopic evaluation. The histopathology technician needs to recognize testicular landmarks that allow for correct orientation at trimming so the pathologist can assess not only the morphology of seminiferous tubules but also the outfl ow tract. For the most effective evaluation of the male reproductive tract, the testes and epididymides should be examined concurrently. Although the term ``staging'' is often used inappropriately, the pathologist should review testicular tissues in a ``stage-aware'' manner. This article reviews gross and histologic changes of the male reproductive tract as well as tissue orientation and fixation to assist in accurate interpretation of potential treatment-related changes in male reproduction.
Skeletal Changes in Rats Given Daily Subcutaneous Injections of Recombinant Human Parathyroid Hormone (1-34) for 2 Years and Relevance to Human Safety
Authors: Vahle John L. , Sato Masahiko , Long Gerald G. , Young Jamie K. , Francis Paul C. , Engelhardt Jeffery A. , Westmore Michael S. , Ma Yanfei Linda , Nold James B.
Published: July 2002

Fischer 344 rats (60/sex/group) were given daily subcutaneou s injections of recombinant human parathyroi d hormone (PTH)(1-34) for 2 years at doses of 0, 5, 30, or 75 μg/kg. Treatment caused substantial increases in bone mass consistent with the known pharmacologic effects of once-daily administration. As determined by quantitative computed tomography (QCT) and histomorphometry, bone mass was markedly increased. Substantial new bone formation resulted in a large decrease in marrow space accompanied by altered bone architecture. Bone proliferative lesions were observed in all PTH(1-34)-treated groups. Osteosarcoma occurred in 3, 21, and 31 male rats and in 4, 12, and 23 female rats in the 5-, 30-, and 75- μg/kg treatment groups, respectively. Focal osteoblast hyperplasia, osteoma, and osteoblastoma were much less frequent. Although the specific cellular or molecular mechanisms responsible for the rat bone tumors have not been fully elucidated, the data suggest that these lesions resulted from the long duration of treatment and the exaggerated pharmacologic response of the rat skeleton to daily treatment with PTH(1-34). Important differences between the rat study and clinical use in adult humans suggest that the increased incidence of bone neoplasia in rats treated for 2 years is likely not predictive of an increased risk of bone cancer in skeletally mature adult humans being given PTH(1-34) for a limited period of time in the treatment of osteoporosis.
Unilateral Free Trade Versus Reciprocity inThe Wealth of Nations
Authors: West Edwin G.
Published: July 2002

In Chapter 2, Book IV, ofThe Wealth of Nations, Adam Smith assesses the appropriateness of the policy of tariff retaliation or reciprocity. His deliberations are prefaced by a discussion of two rather innocuous cases that turn out to have very little to do with the main topic. In the first case it is advantageous “to lay some burden upon foreign, for the encouragement of domestic industry” (WN, p. 463) when that particular industry is necessary for the defence of the country. Smith's second case refers to the situation when some domestic tax is being imposed on a good produced at home. In this situation Smith believed it reasonable that an equal tax should be imposed on the same good when it was imported (WN, p. 465).
Modulation of the decrease in the seizure threshold of pentylenetetrazole in diazepam withdrawn mice by the neurosteroid 5αpregnan‐3α,21‐diol‐20‐one (alloTHDOC)
Authors: TSUDA MAKOTO , SUZUKI TSUTOMU , MISAWA MIWA
Published: July 2002

The effect of the neurosteroid 5α‐pregnan‐3α,21‐diol‐20‐one (alloTHDOC) on pentylenetetrazole (PTZ)induced diazepam‐withdrawal seizure was examined in mice. The threshold for PTZ‐induced seizure was markedly decreased by discontinuation of chronic diazepam treatment. The decrease in the seizure threshold of PTZ during diazepam withdrawal was significantly attenuated by pretreatment with alloTHDOC (10 and 20 mug/mouse, i.c.v.), which did not affect the seizure threshold of PTZ in chronically vehicle‐treated mice. However, the loss of the righting reflex (LRR) induced by other GABAA receptor activators (pentobarbital and propofol) did not differ between control and diazepam‐withdrawn mice. These findings provide the first demonstration that alloTHDOC may be able to suppress diazepam withdrawal signs, and that the sensitivity to the pharmacological effect of alloTHDOC via GABAA receptor may be enhanced in diazepam‐with‐ drawn mice.
Sleep, alcohol and alcohol abuse
Authors: VITIELLO MICHAEL V.
Published: July 2002

Abstract Use and abuse of alcohol is extremely common in many countries. Sometimes alcohol may be used to self‐treat insomnia. However, alcohol consumption for any reason will affect an individual's sleep quality. The effects of alcohol use and abuse on sleep are complex and interactive. Anyone who consumes alcohol is likely to observe them, although the particular pattern and severity of the sleep impairment will differ with both the amount and duration of alcohol consumption. The fact that alcohol is a sedative and can induce rapid onset of sleep may contribute to its use, while the resultant disturbance of night‐time sleep quality and of respiration may possibly result in post‐consumptive daytime impairment. The persistent sleep disturbance that accompanies both acute and chronic alcohol abstinence syndromes may contribute to the continued craving and urge to resume drinking seen among chronic alcoholics. This review broadly examines the interaction of alcohol consumption and sleep disturbance. Beginning with a brief overview of normal sleep it examines the effects of modest acute alcohol consumption on sleep, sleepiness, night‐time respiration and cognitive function; examines the sleep patterns of chronic alcoholics both while drinking and during acute and chronic alcohol withdrawal; reviews what is known of the relationship between alcohol use and night‐time respiratory dysfunction; and concludes with some reflections on the implications of this information for drinking practices.
A Chlamydomonas Homologue of the Putative Murine t Complex Distorter Tctex-2 Is an Outer Arm Dynein Light Chain
Authors: Patel-King Ramila S. , Benashski Sharon E. , Harrison Alistair , King Stephen M.
Published: July 2002

Molecular analysis of a 19,000-Mr protein from the Chlamydomonas flagellum reveals that it is homologous to the t complex–encoded protein Tctex-2, which is a candidate for one of the distorter products that cause the extreme transmission ratio distortion (meiotic drive) of the murine t complex. The 19,000-Mr protein is extracted from the axoneme with 0.6 M NaCl and comigrates with the outer dynein arm in sucrose density gradients. This protein also is specifically missing in axonemes prepared from a mutant that does not assemble the outer arm. These data raise the possibility that Tctex-2 is a sperm flagellar dynein component. Combined with the recent identification of Tctex-1 (another distorter candidate) as a light chain of cytoplasmic dynein, these results lead to a biochemical model for how differential defects in spermiogenesis that result in the phenomenon of meiotic drive might be generated in wild-type vs t-bearing sperm.
Large Plasma Membrane Disruptions Are Rapidly Resealed by Ca2+-dependent Vesicle–Vesicle Fusion Events
Authors: Terasaki Mark , Miyake Katsuya , McNeil Paul L.
Published: July 2002

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390–393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an ∼40 by 10 μm surface patch was torn off, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (∼5–10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was ∼3 mM Ca2+ (SW is ∼10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet–rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle–vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events.
The Pathway of Membrane Fusion Catalyzed by Influenza Hemagglutinin: Restriction of Lipids, Hemifusion, and Lipidic Fusion Pore Formation
Authors: Chernomordik Leonid V. , Frolov Vadim A. , Leikina Eugenia , Bronk Peter , Zimmerberg Joshua
Published: July 2002

The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell–cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes were merged and continuous in this intermediate, but HA restricted lipid mixing. Depending on the surface density of HA and the membrane lipid composition, this restricted hemifusion intermediate either transformed into a fusion pore or expanded into an unrestricted hemifusion, without pores but with unrestricted lipid mixing. Our results suggest that restriction of lipid flux by a ring of activated HA is necessary for successful fusion, during which a lipidic fusion pore develops in a local and transient hemifusion diaphragm.
Activity-dependent Regulation of Dendritic BC1 RNA in Hippocampal Neurons in Culture
Authors: Muslimov Ilham A. , Banker Gary , Brosius Jürgen , Tiedge Henri
Published: July 2002

Several neuronal RNAs have been identified in dendrites, and it has been suggested that the dendritic location of these RNAs may be relevant to the spatiotemporal regulation of mosaic postsynaptic protein repertoires through transsynaptic activity. Such regulation would require that dendritic RNAs themselves, or at least some of them, be subject to physiological control. We have therefore examined the functional regulation of somatodendritic expression levels of dendritic BC1 RNA in hippocampal neurons in culture. BC1 RNA, an RNA polymerase III transcript that is a component of a ribonucleoprotein particle, became first detectable in somatodendritic domains of developing hippocampal neurons at times of initial synapse formation. BC1 RNA was identified only in such neurons that had established synapses on cell bodies and/or developing dendritic arbors. When synaptic contact formation was initiated later in low-density cultures, BC1 expression was coordinately delayed. Inhibition of neuronal activity in hippocampal neurons resulted in a substantial but reversible reduction of somatodendritic BC1 expression. We conclude that expression of BC1 RNA in somatic and dendritic domains of hippocampal neurons is regulated in development, and is dependent upon neuronal activity. These results establish (for the first time to our knowledge) that an RNA polymerase III transcript can be subject to control through physiological activity in nerve cells.
Dimerization of P-Selectin Glycoprotein Ligand-1 (PSGL-1) Required for Optimal Recognition of P-Selectin
Authors: Snapp Karen R. , Craig Ron , Herron Michael , Nelson Robert D. , Stoolman Lloyd M. , Kansas Geoffrey S.
Published: July 2002

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with α1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.
Role of a New Mammalian Gene Family in the Biosynthesis of Very Long Chain Fatty Acids and Sphingolipids
Authors: Tvrdik Petr , Westerberg Rolf , Silve Sandra , Asadi Abolfazl , Jakobsson Andreas , Cannon Barbara , Loison Gerard , Jacobsson Anders
Published: July 2002

Whereas the physiological significance of microsomal fatty acid elongation is generally appreciated, its molecular nature is poorly understood. Here, we describe tissue-specific regulation of a novel mouse gene family encoding components implicated in the synthesis of very long chain fatty acids. The Ssc1 gene appears to be ubiquitously expressed, whereas Ssc2 and Cig30 show a restricted expression pattern. Their translation products are all integral membrane proteins with five putative transmembrane domains. By complementing the homologous yeast mutants, we found that Ssc1 could rescue normal sphingolipid synthesis in the sur4/elo3 mutant lacking the ability to synthesize cerotic acid (C26:0). Similarly, Cig30 reverted the phenotype of the fen1/elo2 mutant that has reduced levels of fatty acids in the C20–C24 range. Further, we show that Ssc1 mRNA levels were markedly decreased in the brains of myelin-deficient mouse mutants known to have very low fatty acid chain elongation activity. Conversely, the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity. Our results strongly implicate this new mammalian gene family in tissue-specific synthesis of very long chain fatty acids and sphingolipids.
Formation of a Complex between Nucleolin and Replication Protein a after Cell Stress Prevents Initiation of DNA Replication
Authors: Daniely Yaron , Borowiec James A.
Published: July 2002

We used a biochemical screen to identify nucleolin, a key factor in ribosome biogenesis, as a high-affinity binding partner for the heterotrimeric human replication protein A (hRPA). Binding studies in vitro demonstrated that the two proteins physically interact, with nucleolin using an unusual contact with the small hRPA subunit. Nucleolin significantly inhibited both simian virus 40 (SV-40) origin unwinding and SV-40 DNA replication in vitro, likely by nucleolin preventing hRPA from productive interaction with the SV-40 initiation complex. In vivo, use of epifluorescence and confocal microscopy showed that heat shock caused a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm. Nucleolin relocalization was concomitant with a tenfold increase in nucleolin–hRPA complex formation. The relocalized nucleolin significantly overlapped with the position of hRPA, but only poorly with sites of ongoing DNA synthesis. We suggest that the induced nucleolin–hRPA interaction signifies a novel mechanism that represses chromosomal replication after cell stress.
Fission Yeast Myosin-I, Myo1p, Stimulates Actin Assembly by Arp2/3 Complex and Shares Functions with Wasp
Authors: Lee Wei-Lih , Bezanilla Magdalena , Pollard Thomas D.
Published: July 2002

Fission yeast myo1+ encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is. Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is. Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly. First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division. Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton. Second, although deletion of myo1+ is not lethal, Δmyo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects. Third, additional disruption of wsp1+ is synthetically lethal, suggesting that these genes may share functions. In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all Δmyo1 defects. However, it fails to rescue the Δmyo1 Δwsp1 lethality. Additional tail domains, TH2 and TH3, are required to complement the double mutant. Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.
A Novel Neural Wiskott-Aldrich Syndrome Protein (N-Wasp) Binding Protein, Wish, Induces Arp2/3 Complex Activation Independent of Cdc42
Authors: Fukuoka Maiko , Suetsugu Shiro , Miki Hiroaki , Fukami Kiyoko , Endo Takeshi , Takenawa Tadaomi
Published: July 2002

We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP–induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract–induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP–depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP–dependent and –independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.
The adenomatous polyposis coli protein unambiguously localizes to microtubule plus ends and is involved in establishing parallel arrays of microtubule bundles in highly polarized epithelial cells
Authors: Mogensen Mette M. , Tucker John B. , Mackie John B. , Prescott Alan R. , Näthke Inke S.
Published: July 2002

Loss of full-length adenomatous polyposis coli (APC) protein correlates with the development of colon cancers in familial and sporadic cases. In addition to its role in regulating β-catenin levels in the Wnt signaling pathway, the APC protein is implicated in regulating cytoskeletal organization. APC stabilizes microtubules in vivo and in vitro, and this may play a role in cell migration (Näthke, I.S., C.L. Adams, P. Polakis, J.H. Sellin, and W.J. Nelson. 1996. J. Cell Biol. 134:165–179; Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000. J. Cell Biol. 148:505–517; Zumbrunn, J., K. Inoshita, A.A. Hyman, and I.S. Näthke. 2001. Curr. Biol. 11:44–49) and in the attachment of microtubules to kinetochores during mitosis (Fodde, R., J. Kuipers, C. Rosenberg, R. Smits, M. Kielman, C. Gaspar, J.H. van Es, C. Breukel, J. Wiegant, R.H. Giles, and H. Clevers. 2001. Nat. Cell Biol. 3:433–438; Kaplan, K.B., A. Burds, J.R. Swedlow, S.S. Bekir, P.K. Sorger, and I.S. Näthke. 2001. Nat. Cell Biol. 3:429–432). The localization of endogenous APC protein is complex: actin- and microtubule-dependent pools of APC have been identified in cultured cells (Näthke et al., 1996; Mimori-Kiyosue et al., 2000; Reinacher-Schick, A., and B.M. Gumbiner. 2001. J. Cell Biol. 152:491–502; Rosin-Arbesfeld, R., G. Ihrke, and M. Bienz. 2001. EMBO J. 20:5929–5939). However, the localization of APC in tissues has not been identified at high resolution. Here, we show that in fully polarized epithelial cells from the inner ear, endogenous APC protein associates with the plus ends of microtubules located at the basal plasma membrane. Consistent with a role for APC in supporting the cytoskeletal organization of epithelial cells in vivo, the number of microtubules is significantly reduced in apico-basal arrays of microtubule bundles isolated from mice heterozygous for APC.
Characterization of the galU Gene of Streptococcus pneumoniae Encoding a Uridine Diphosphoglucose Pyrophosphorylase: A Gene Essential for Capsular Polysaccharide Biosynthesis
Authors: Mollerach Marta , López Rubens , García Ernesto
Published: July 2002

The galU gene of Streptococcus pneumoniae has been cloned and sequenced. Escherichia coli cells harboring the recombinant plasmid pMMG2 (galU) overproduced a protein that has been shown to correspond to a uridine 5′-triphosphate:glucose-1-phosphate uridylyltransferase (uridine diphosphoglucose [UDP-Glc] pyrophosphorylase) responsible for the synthesis of UDP-Glc, a key compound in the biosynthesis of polysaccharides. A gene very similar to the S. pneumoniae galU has been found in a partial nucleotide sequence of the Streptococcus pyogenes genome. Knockout galU mutants of type 1 pneumococci are unable to synthesize a detectable capsule. An identical result was found in type 3 S. pneumoniae cells in spite of the fact that these bacteria contain a type-specific gene (cap3C) that also encodes a UDP-Glc pyrophosphorylase. Since eukaryotic UDP-Glc pyrophosphorylases appear to be completely unrelated to their prokaryotic counterparts, we postulate that GalU may be an appropriate target for the search of new drugs to control the pathogenicity of bacteria like pneumococcus and S. pyogenes.
Functional Redundancy of the Nuclear Factor κB Inhibitors IκBα and IκBβ
Authors: Cheng Janet D. , Ryseck Rolf-Peter , Attar Ricardo M. , Dambach Donna , Bravo Rodrigo
Published: July 2002

The transcription factor NF-κB is sequestered in the cytoplasm by the inhibitor proteins of the IκB family. Each member of the IκB exhibits structural and biochemical similarities as well as differences. In an effort to address the functional redundancy of two closely related IκB molecules, IκBα and IκBβ, we generated knock-in mice by replacing the IκBα gene with the IκBβ gene. The knock-in mice do not express IκBα, but express a T7-tagged IκBβ under the promoter and regulatory sequence of ikba. Unlike the IκBα-deficient mice, which display severe postnatal developmental defects and die by postnatal day 8, homozygous knock-in mice survive to adulthood, are fertile, and exhibit no apparent abnormalities. Furthermore, thymocytes and embryonic fibroblasts from the knock-in animals exhibit an inducible NF-κB response similar to that of wild-type animals. These results indicate that IκBα and IκBβ share significant similarities in their biochemical activity, and that they acquired their different functions from divergent expression patterns during evolution.
Biochemical Identification of a Mutated Human Melanoma Antigen Recognized by CD4+ T Cells
Authors: Pieper Rembert , Christian Robert E. , Gonzales Monica I. , Nishimura Michael I. , Gupta Gaorav , Settlage Robert E. , Shabanowitz Jeffrey , Rosenberg Steven A. , Hunt Donald F. , Topalian Suzanne L.
Published: July 2002

CD4+ T cells play a critical role in generating and maintaining immune responses against pathogens and alloantigens, and evidence suggests an important role for them in antitumor immunity as well. Although major histocompatibility complex class II–restricted human CD4+ T cells with specific antitumor reactivities have been described, no standard method exists for cloning the recognized tumor-associated antigen (Ag). In this study, biochemical protein purification methods were used in conjunction with novel mass spectrometry sequencing techniques and molecular cloning to isolate a unique melanoma Ag recognized by a CD4+ tumor-infiltrating lymphocyte (TIL) line. The HLA-DRβ1*0101–restricted Ag was determined to be a mutated glycolytic enzyme, triosephosphate isomerase (TPI). A C to T mutation identified by cDNA sequencing caused a Thr to Ile conversion in TPI, which could be detected in a tryptic digest of tumor-derived TPI by mass spectrometry. The Thr to Ile conversion created a neoepitope whose T cell stimulatory activity was enhanced at least 5 logs compared with the wild-type peptide. Analysis of T cell recognition of serially truncated peptides suggested that the mutated amino acid residue was a T cell receptor contact. Defining human tumor Ag recognized by T helper cells may provide important clues to designing more effective immunotherapies for cancer.
Secretory Leukocyte Protease Inhibitor Suppresses the Inflammation and Joint Damage of Bacterial Cell Wall–Induced Arthritis
Authors: Song Xiao-yu , Zeng Li , Jin Wenwen , Thompson John , Mizel Diane E. , Lei Ke-jian , Billinghurst R.C. , Poole A. Robin , Wahl Sharon M.
Published: July 2002

Disruption of the balance between proteases and protease inhibitors is often associated with pathologic tissue destruction. To explore the therapeutic potential of secretory leukocyte protease inhibitor (SLPI) in erosive joint diseases, we cloned, sequenced, and expressed active rat SLPI, which shares the protease-reactive site found in human SLPI. In a rat streptococcal cell wall (SCW)-induced model of inflammatory erosive polyarthritis, endogenous SLPI was unexpectedly upregulated at both mRNA and protein levels in inflamed joint tissues. Systemic delivery of purified recombinant rat SLPI inhibited joint inflammation and cartilage and bone destruction. Inflammatory pathways as reflected by circulating tumor necrosis factor α and nuclear factor κB activation and cartilage resorption detected by circulating levels of type II collagen collagenase-generated cleavage products were all diminished by SLPI treatment in acute and chronic arthritis, indicating that the action of SLPI may extend beyond inhibition of serine proteases.
Natural Interferon α/β–Producing Cells Link Innate and Adaptive Immunity
Authors: Kadowaki Norimitsu , Antonenko Svetlana , Lau Johnson Yiu-Nam , Liu Yong-Jun
Published: July 2002

Innate immune responses to pathogens critically impact the development of adaptive immune responses. However, it is not completely understood how innate immunity controls the initiation of adaptive immunities or how it determines which type of adaptive immunity will be induced to eliminate a given pathogen. Here we show that viral stimulation not only triggers natural interferon (IFN)-α/β–producing cells (IPCs) to produce vast amounts of antiviral IFN-α/β but also induces these cells to differentiate into dendritic cells (DCs). IFN-α/β and tumor necrosis factor α produced by virus-activated IPCs act as autocrine survival and DC differentiation factors, respectively. The virus-induced DCs stimulate naive CD4+ T cells to produce IFN-γ and interleukin (IL)-10, in contrast to IL-3–induced DCs, which stimulate naive CD4+ T cells to produce T helper type 2 cytokines IL-4, IL-5, and IL-10. Thus, IPCs may play two master roles in antiviral immune responses: directly inhibiting viral replication by producing large amounts of IFN-α/β, and subsequently triggering adaptive T cell–mediated immunity by differentiating into DCs. IPCs constitute a critical link between innate and adaptive immunity.