bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurements of the concentration and composition of nuclei for cirrus formation
Authors: DeMott P. J. , Cziczo D. J. , Prenni A. J. , Murphy D. M. , Kreidenweis S. M. , Thomson D. S. , Borys R. , Rogers D. C.
Published: December 2003

This article addresses the need for new data on indirect effects of natural and anthropogenic aerosol particles on atmospheric ice clouds. Simultaneous measurements of the concentration and composition of tropospheric aerosol particles capable of initiating ice in cold (cirrus) clouds are reported. Measurements support that cirrus formation occurs both by heterogeneous nucleation by insoluble particles and homogeneous (spontaneous) freezing of particles containing solutions. Heterogeneous ice nuclei concentrations in the cirrus regime depend on temperature, relative humidity, and the concentrations and physical and chemical properties of aerosol particles. The cirrus-active concentrations of heterogeneous nuclei measured in November over the western U.S. were <0.03 cm–3. Considering previous modeling studies, this result suggests a predominant potential impact of these nuclei on cirrus formed by slow, large-scale lifting or small cooling rates, including subvisual cirrus. The most common heterogeneous ice nuclei were identified as relatively pure mineral dusts and metallic particles, some of which may have origin through anthropogenic processes. Homogeneous freezing of large numbers of particles was detected above a critical relative humidity along with a simultaneous transition in nuclei composition toward that of the sulfate-dominated total aerosol population. The temperature and humidity conditions of the homogeneous nucleation transition were reasonably consistent with expectations based on previous theoretical and laboratory studies but were highly variable. The strong presence of certain organic pollutants was particularly noted to be associated with impedance of homogeneous freezing.
Mitotic partitioning and selective reorganization of tissue-specific transcription factors in progeny cells
Authors: Zaidi Sayyed K. , Young Daniel W. , Pockwinse Shirwin M. , Javed Amjad , Lian Jane B. , Stein Janet L. , van Wijnen Andre J. , Stein Gary S.
Published: December 2003

Postmitotic gene expression requires restoration of nuclear organization and assembly of regulatory complexes. The hematopoietic and osteogenic Runx (Cbfa/AML) transcription factors are punctately organized in the interphase nucleus and provide a model for understanding the subnuclear organization of tissue-specific regulatory proteins after mitosis. Here we have used quantitative
in situ
immunofluorescence microscopy and quantitative image analysis to show that Runx factors undergo progressive changes in cellular localization during mitosis while retaining a punctate distribution. In comparison, the acetyl transferase p300 and acetylated histone H4 remain localized with DNA throughout mitosis while the RNA processing factor SC35 is excluded from mitotic chromatin. Subnuclear organization of Runx foci is completely restored in telophase, and Runx proteins are equally partitioned into progeny nuclei. In contrast, subnuclear organization of SC35 is restored subsequent to telophase. Our results show a sequential reorganization of Runx and its coregulatory proteins that precedes restoration of RNA processing speckles. Thus, mitotic partitioning and spatiotemporal reorganization of regulatory proteins together render progeny cells equivalently competent to support phenotypic gene expression.
CD24 is a genetic modifier for risk and progression of multiple sclerosis
Authors: Zhou Qunmin , Rammohan Kottil , Lin Shili , Robinson Nikki , Li Ou , Liu Xingluo , Bai Xue-feng , Yin Lijie , Scarberry Bruce , Du Peishuang , You Ming , Guan Kunliang , Zheng Pan , Liu Yang
Published: December 2003

Multiple sclerosis (MS) is a chronic neurological disease of unknown etiology, but a genetic basis for the disease is undisputed. We have reported that CD24 is required for the pathogenicity of autoreactive T cells in experimental autoimmune encephalomyelitis, the mouse model of MS. Here we investigate the contribution of CD24 to MS by studying single-nucleotide polymorphism in the ORF among 242 MS patients and 207 population controls. This single-nucleotide polymorphism results in replacement of alanine (CD24a) with valine (CD24v) in the mature protein. We found that theCD24v/vrenders a >2-fold increase in the relative risk of MS in the general population (P= 0.023). Among familial MS, theCD24vallele is preferentially transmitted into affected individuals (P= 0.017). Furthermore, 50% ofCD24v/vpatients with expanded disability status scale 6.0 reached the milestone in 5 years, whereas theCD24a/v(P= 0.00037) andCD24a/a(P= 0.0016) patients did so in 16 and 13 years, respectively. Moreover, our data suggest that theCD24v/vpatients expressed higher levels of CD24 on peripheral blood T cells than did theCD24a/apatients. Transfection withCD24aandCD24vcDNA demonstrated that theCD24vallele can be expressed at higher efficiency than theCD24aalleles. Thus, CD24 polymorphism is a genetic modifier for susceptibility and progression of MS in the central Ohio cohort that we studied, perhaps by affecting the efficiency of CD24 expression on the cell surface.
Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase
Authors: Lee Sungsoo , Lin Xiaofeng , Nam Nguyen Hai , Parang Keykavous , Sun Gongqin
Published: December 2003

Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTK–substrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substrate-docking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC
50
= 21 μM) Csk phosphorylation of Src but only moderately inhibited (IC
50
= 422 μM) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.
Segmented spiral waves in a reaction-diffusion system
Authors: Vanag Vladimir K. , Epstein Irving R.
Published: December 2003

Pattern formation in reaction-diffusion systems is often invoked as a mechanism for biological morphogenesis. Patterns in chemical systems typically occur either as propagating waves or as stationary, spatially periodic, Turing structures. The spiral and concentric (target) waves found to date in spatially extended chemical or physical systems are smooth and continuous; only living systems, such as seashells, lichens, pine cones, or flowers, have been shown to demonstrate segmentation of these patterns. Here, we report observations of segmented spiral and target waves in the Belousov–Zhabotinsky reaction dispersed in water nanodroplets of a water-in-oil microemulsion. These highly ordered chemical patterns, consisting of short wave segments regularly separated by gaps, form a link between Turing and trigger wave patterns and narrow the disparity between chemistry and biology. They exhibit aspects of such fundamental biological behavior as self-replication of structural elements and preservation of morphology during evolutionary development from a simpler precursor to a more complex structure.
The linear process of somatic evolution
Authors: Nowak Martin A. , Michor Franziska , Iwasa Yoh
Published: December 2003

Cancer is the consequence of an unwanted evolutionary process. Cells receive mutations that alter their phenotype. Especially dangerous are those mutations that increase the net reproductive rate of cells, thereby leading to neoplasia and later to cancer. The standard models of evolutionary dynamics consider well mixed populations of individuals in symmetric positions. Here we introduce a spatially explicit, asymmetric stochastic process that captures the essential architecture of evolutionary dynamics operating within tissues of multicellular organisms. The “linear process” has the property of canceling out selective differences among cells yet retaining the protective function of apoptosis. This design can slow down the rate of somatic evolution dramatically and therefore delay the onset of cancer.
Expression of conditional fear with and without awareness
Authors: Knight David C. , Nguyen Hanh T. , Bandettini Peter A.
Published: December 2003

Conditional responding during simple Pavlovian conditioning is often characterized as a form of implicit memory. The extent to which this type of associative learning is independent of awareness is an issue of continuing debate. Previous studies have demonstrated conditioning in the absence of awareness. However, their results have been questioned based on methodological concerns with postexperimental questionnaires. In the present study, skin conductance response (SCR) and unconditioned stimulus (UCS) expectancy were measured concurrently as participants were exposed to a differential delay fear conditioning procedure in which one tone (CS+) predicted a loud white noise, whereas a second tone (CS-) was presented alone. UCS predictability was varied on a trial-by-trial basis by presenting conditioned stimuli (CSs) at volumes just above or below the perceptual threshold. Differential UCS expectancy (awareness) was observed only on perceived trials, whereas differential SCR developed on both perceived and unperceived trials. Although perceived stimuli elicited larger SCRs, the magnitude of conditioning, indexed by differential conditioned response expression (conditioned SCR to CS+ minus the SCR to CS-), was not influenced by stimulus perception. These data indicate that conditional fear can be expressed when individuals are unaware of fear-eliciting stimuli and suggest that the degree of conditioning is independent of awareness during differential Pavlovian fear conditioning.
IL-27 regulates IL-12 responsiveness of naïve CD4
+
T cells through Stat1-dependent and -independent mechanisms
Authors: Lucas Sophie , Ghilardi Nico , Li Ji , de Sauvage Frédéric J.
Published: December 2003

IL-27, a novel heterodimeric cytokine produced by antigen-presenting cells, signals through the T cell cytokine receptor (TCCR)
/WSX-1
expressed on naïve CD4
+
T cells and natural killer cells. TCCR
/WSX-1
deficiency results in delayed T helper type 1 (T
H
1) development through an unresolved mechanism. We report here that IL-27 stimulation in developing murine T helper cells potently induces the expression of the major T
H
1-specific transcription factor T-bet and its downstream target IL-12R β2, independently of IFNγ. In addition, IL-27 suppresses basal expression of GATA-3, the critical T
H
2-specific transcription factor that inhibits T
H
1 development by down-regulating signal transducer and activator of transcription (Stat) 4. IL-27 signaling through TCCR
/WSX-1
induces phosphorylation of Stat1, Stat3, Stat4, and Stat5. Stat1 is required for suppression of GATA-3, but T-bet induction by IL-27 can also be mediated through a Stat1-independent pathway. Despite its T
H
1-like signaling profile, IL-27 is not sufficient to drive the differentiation of CD4
+
T cells into IFNγ-producing cells. Similarly, IL-27 induces T-bet expression in primary natural killer cells, but this does not result in an increase of IFNγ production or cytotoxic activity. Therefore, although IL-27 is unable to drive IFNγ production on its own, it plays an important role in the early steps of T
H
1 commitment by contributing in a paracrine manner to the control of IL-12 responsiveness.
CatSper1 required for evoked Ca
2+
entry and control of flagellar function in sperm
Authors: Carlson Anne E. , Westenbroek Ruth E. , Quill Timothy , Ren Dejian , Clapham David E. , Hille Bertil , Garbers David L. , Babcock Donner F.
Published: December 2003

CatSper family proteins are putative ion channels expressed exclusively in membranes of the sperm flagellum and required for male fertility. Here, we show that mouse CatSper1 is essential for depolarization-evoked Ca
2+
entry and for hyperactivated movement, a key flagellar function. CatSper1 is not needed for other developmental landmarks, including regional distributions of Ca
V
1.2, Ca
V
2.2, and Ca
V
2.3 ion channel proteins, the cAMP-mediated activation of motility by

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, and the protein phosphorylation cascade of sperm capacitation. We propose that CatSper1 functions as a voltage-gated Ca
2+
channel that controls Ca
2+
entry to mediate the hyperactivated motility needed late in the preparation of sperm for fertilization.
Interleukin 7 Regulates the Survival and Generation of Memory CD4 Cells
Authors: Kondrack Robyn M. , Harbertson Judith , Tan Joyce T. , McBreen Meghan E. , Surh Charles D. , Bradley Linda M.
Published: December 2003

Cytokines, particularly those of the common γ chain receptor family, provide extrinsic signals that regulate naive CD4 cell survival. Whether these cytokines are required for the maintenance of memory CD4 cells has not been rigorously assessed. In this paper, we examined the contribution of interleukin (IL) 7, a constitutively produced common γ chain receptor cytokine, to the survival of resting T cell receptor transgenic memory CD4 cells that were generated in vivo. IL-7 mediated the survival and up-regulation of Bcl-2 by resting memory CD4 cells in vitro in the absence of proliferation. Memory CD4 cells persisted for extended periods upon adoptive transfer into intact or lymphopenic recipients, but not in IL-7− mice or in recipients that were rendered deficient in IL-7 by antibody blocking. Both central (CD62L+) and effector (CD62L−) memory phenotype CD4 cells required IL-7 for survival and, in vivo, memory cells were comparable to naive CD4 cells in this regard. Although the generation of primary effector cells from naive CD4 cells and their dissemination to nonlymphoid tissues were not affected by IL-7 deficiency, memory cells failed to subsequently develop in either the lymphoid or nonlymphoid compartments. The results demonstrate that IL-7 can have previously unrecognized roles in the maintenance of memory in the CD4 cell population and in the survival of CD4 cells with a capacity to become memory cells.
Divergent Pro- and Antiinflammatory Roles for IL-23 and IL-12 in Joint Autoimmune Inflammation
Authors: Murphy Craig A. , Langrish Claire L. , Chen Yi , Blumenschein Wendy , McClanahan Terrill , Kastelein Robert A. , Sedgwick Jonathon D. , Cua Daniel J.
Published: December 2003

Interleukin (IL) 23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 affects memory T cell and inflammatory macrophage function through engagement of a novel receptor (IL-23R) on these cells. Recent analysis of the contribution of IL-12 and IL-23 to central nervous system autoimmune inflammation demonstrated that IL-23 rather than IL-12 was the essential cytokine. Using gene-targeted mice lacking only IL-12 (p35−/−) or IL-23 (p19−/−), we show that the specific absence of IL-23 is protective, whereas loss of IL-12 exacerbates collagen-induced arthritis. IL-23 gene-targeted mice did not develop clinical signs of disease and were completely resistant to the development of joint and bone pathology. Resistance correlated with an absence of IL-17–producing CD4+ T cells despite normal induction of collagen-specific, interferon-γ–producing T helper 1 cells. In contrast, IL-12–deficient p35−/− mice developed more IL-17–producing CD4+ T cells, as well as elevated mRNA expression of proinflammatory tumor necrosis factor, IL-1β, IL-6, and IL-17 in affected tissues of diseased mice. The data presented here indicate that IL-23 is an essential promoter of end-stage joint autoimmune inflammation, whereas IL-12 paradoxically mediates protection from autoimmune inflammation.
A Critical Role for Induced IgM in the Protection against West Nile Virus Infection
Authors: Diamond Michael S. , Sitati Elizabeth M. , Friend Lindzy D. , Higgs Stephen , Shrestha Bimmi , Engle Michael
Published: December 2003

In humans, the elderly and immunocompromised are at greatest risk for disseminated West Nile virus (WNV) infection, yet the immunologic basis for this remains unclear. We demonstrated previously that B cells and IgG contributed to the defense against disseminated WNV infection (Diamond, M.S., B. Shrestha, A. Marri, D. Mahan, and M. Engle. 2003. J. Virol. 77:2578–2586). In this paper, we addressed the function of IgM in controlling WNV infection. C57BL/6J mice (sIgM−/−) that were deficient in the production of secreted IgM but capable of expressing surface IgM and secreting other immunoglobulin isotypes were vulnerable to lethal infection, even after inoculation with low doses of WNV. Within 96 h, markedly higher levels of infectious virus were detected in the serum of sIgM−/− mice compared with wild-type mice. The enhanced viremia correlated with higher WNV burdens in the central nervous system, and was also associated with a blunted anti-WNV IgG response. Passive transfer of polyclonal anti-WNV IgM or IgG protected sIgM−/− mice against mortality, although administration of comparable amounts of a nonneutralizing monoclonal anti-WNV IgM provided no protection. In a prospective analysis, a low titer of anti-WNV IgM antibodies at day 4 uniformly predicted mortality in wild-type mice. Thus, the induction of a specific, neutralizing IgM response early in the course of WNV infection limits viremia and dissemination into the central nervous system, and protects against lethal infection.
Functional Heterogeneity of Marginal Zone B Cells Revealed by Their Ability to Generate Both Early Antibody-forming Cells and Germinal Centers with Hypermutation and Memory in Response to a T-dependent Antigen
Authors: Song Haifeng , Cerny Jan
Published: December 2003

Marginal zone (MZ) B cells play a major role in the first-line responses against blood-born T-independent bacterial antigens (TI), but the full scope of their immune functions is not known. Here we compare the responses of MZ and follicular (FO) B cells to a T-dependent antigen (TD), hapten–(4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken γ-globulin, in a cell transfer system. Consistent with the conventional paradigm, MZ B cells but not FO B cells rapidly generated the early burst of NP-specific antibody-forming cells (AFC), high levels of IgM Ab, and early IgG with relatively high affinity to NP. However, MZ B cells were also capable of forming germinal centers (GCs) albeit with a delay, compared with FO B cells. The early AFCs and the GCs originated from different MZ precursors, but the MZ- and FO-derived GCs were similar in VH gene repertoire, somatic mutation, and production of late AFC and IgG Ab. Surprisingly, the MZ but not the FO memory response included IgM Ab. We conclude that MZ B cells are heterogeneous, comprising cells for both early AFC response and GC/memory pathway against TD antigens.
Exchangeable Chaperone Modules Contribute to Specification of Type I and Type II Hsp40 Cellular Function
Authors: Fan Chun-Yang , Lee Soojin , Ren Hong-Yu , Cyr Douglas M.
Published: December 2003

Hsp40 family members regulate Hsp70s ability to bind nonnative polypeptides and thereby play an essential role in cell physiology. Type I and type II Hsp40s, such as yeast Ydj1 and Sis1, form chaperone pairs with cytosolic Hsp70 Ssa1 that fold proteins with different efficiencies and carry out specific cellular functions. The mechanism by which Ydj1 and Sis1 specify Hsp70 functions is not clear. Ydj1 and Sis1 share a high degree of sequence identity in their amino and carboxyl terminal ends, but each contains a structurally unique and centrally located protein module that is implicated in chaperone function. To test whether the chaperone modules of Ydj1 and Sis1 function in the specification of Hsp70 action, we constructed a set of chimeric Hsp40s in which the chaperone domains of Ydj1 and Sis1 were swapped to form YSY and SYS. Purified SYS and YSY exhibited protein-folding activity and substrate specificity that mimicked that of Ydj1 and Sis1, respectively. In in vivo studies, YSY exhibited a gain of function and, unlike Ydj1, could complement the lethal phenotype of sis1Δ and facilitate maintenance of the prion [RNQ+]. Ydj1 and Sis1 contain exchangeable chaperone modules that assist in specification of Hsp70 function.
The Zinc Transporter ZnT3 Interacts with AP-3 and It Is Preferentially Targeted to a Distinct Synaptic Vesicle Subpopulation
Authors: Salazar Gloria , Love Rachal , Werner Erica , Doucette Michele M. , Cheng Su , Levey Allan , Faundez Victor
Published: December 2003

Synaptic vesicles (SV) are generated by two different mechanisms, one AP-2 dependent and one AP-3 dependent. It has been uncertain, however, whether these mechanisms generate SV that differ in molecular composition. We explored this hypothesis by analyzing the targeting of ZnT3 and synaptophysin both to PC12 synaptic-like microvesicles (SLMV) as well as SV isolated from wild-type and AP-3-deficient mocha brains. ZnT3 cytosolic tail interacted selectively with AP-3 in cell-free assays. Accordingly, pharmacological disruption of either AP-2- or AP-3-dependent SLMV biogenesis preferentially reduced synaptophysin or ZnT3 targeting, respectively; suggesting that these antigens were concentrated in different vesicles. As predicted, immuno-isolated SLMV revealed that ZnT3 and synaptophysin were enriched in different vesicle populations. Likewise, morphological and biochemical analyses in hippocampal neurons indicated that these two antigens were also present in distinct but overlapping domains. ZnT3 SV content was reduced in AP-3-deficient neurons, but synaptophysin was not altered in the AP-3 null background. Our evidence indicates that neuroendocrine cells assemble molecularly heterogeneous SV and suggests that this diversity could contribute to the functional variety of synapses.
Gene Expression in the Normal Adult Human Kidney Assessed by Complementary DNA Microarray
Authors: Higgins John P.T. , Wang Lingli , Kambham Neeraja , Montgomery Kelli , Mason Veronica , Vogelmann Stefanie U. , Lemley Kevin V. , Brown Patrick O. , Brooks James D. , van de Rijn Matt
Published: December 2003

The kidney is a highly specialized organ with a complex, stereotyped architecture and a great diversity of functions and cell types. Because the microscopic organization of the nephron, the functional unit of the kidney, has a consistent relationship to the macroscopic anatomy of the kidney, knowledge of the characteristic patterns of gene expression in different compartments of the kidney could provide insight into the functions and functional organization of the normal nephron. We studied gene expression in dissected renal lobes of five adult human kidneys using cDNA microarrays representing ∼30,000 different human genes. Total RNA was isolated from sections of the inner and outer cortex, inner and outer medulla, papillary tips, and renal pelvis and from glomeruli isolated by sieving. The results revealed unique and highly distinctive patterns of gene expression for glomeruli, cortex, medulla, papillary tips, and pelvic samples. Immunohistochemical staining using selected antisera confirmed differential expression of several cognate proteins and provided histological localization of expression within the nephron. The distinctive patterns of gene expression in discrete portions of the kidney may serve as a resource for further understanding of renal physiology and the molecular and cellular organization of the nephron.
Multivesicular Body Sorting: Ubiquitin Ligase Rsp5 Is Required for the Modification and Sorting of Carboxypeptidase S
Authors: Katzmann David J. , Sarkar Srimonti , Chu Tony , Audhya Anjon , Emr Scott D.
Published: December 2003

The multivesicular body (MVB) sorting pathway provides a mechanism for delivering transmembrane proteins into the lumen of the lysosome/vacuole. Recent studies demonstrated that ubiquitin modification acts in cis as a signal for the sorting of cargoes into this pathway. Here, we present results from a genetic selection designed to identify mutants that missort MVB cargoes. This selection identified a point mutation in ubiquitin ligase Rsp5 (Rsp5-326). At the permissive temperature, this mutant is specifically defective for ubiquitination and sorting of the ubiquitin-dependent MVB cargo precursor carboxypeptidase S (pCPS), but not ligand-induced ubiquitination of Ste2. A previous study implicated Tul1 as the ubiquitin ligase responsible for MVB sorting of pCPS. However, we detected no defect in either the sorting or ubiquitination of pCPS in tul1 mutants. We had previously shown that Fab1 phosphatidylinositol 3-phosphate 5-kinase is also required for MVB sorting of pCPS, but not Ste2. However, our analyses reveal that fab1 mutants do not exhibit a defect in ubiquitination of pCPS. Thus, both Rsp5 and Fab1 play distinct and essential roles in the targeting of biosynthetic MVB cargoes. However, whereas Rsp5 seems to be responsible for cargo ubiquitination, the precise role for Fab1 remains to be elucidated.
The EF-Hand Ca2+-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca2+-binding Requirements
Authors: Andrade Josefa , Zhao Hu , Titus Brian , Timm Pearce Sandra , Barroso Margarida
Published: December 2003

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.
Clustering Induces a Lateral Redistribution of α2β1 Integrin from Membrane Rafts to Caveolae and Subsequent Protein Kinase C-dependent Internalization
Authors: Upla Paula , Marjomäki Varpu , Kankaanpää Pasi , Ivaska Johanna , Hyypiä Timo , van der Goot F. Gisou , Heino Jyrki
Published: December 2003

Integrin α2β1 mediates the binding of several epithelial and mesenchymal cell types to collagen. The composition of the surrounding plasma membrane, especially caveolin-1- and cholesterol-containing membrane structures called caveolae, may be important to integrin signaling. On cell surface α2β1 integrin was located in the raft like membrane domain, rich in GPI-anchored proteins, rather than in caveolae. However, when antibodies were used to generate clusters of α2β1 integrin, they started to move laterally on cell surface along actin filaments. During the lateral movement small clusters fused together. Finally α2β1 integrin was found inside caveolae and subsequently internalized into caveosome-like perinuclear structures. The internalization process, unlike cluster formation or lateral redistribution, was dependent on protein kinase Cα activity. Caveolae are known to be highly immobile structures and α2β1 integrin clusters represent a previously unknown mechanism to activate endocytic trafficking via caveolae. The process was specific to α2β1 integrin, because the antibody-mediated formation of αV integrin clusters activated their internalization in coated vesicles and early endosomes. In addition to natural ligands human echovirus-1 (EV1) gains entry into the cell by binding to α2β1 and taking advantage of α2β1 internalization via caveolae.
Translocation of FGF-1 and FGF-2 across Vesicular Membranes Occurs during G1-Phase by a Common Mechanism
Authors: Małecki Jedrzej , Wesche Jørgen , Skjerpen Camilla Skiple , Wiedłocha Antoni , Olsnes Sjur
Published: December 2003

The entry of exogenous fibroblast growth factor 2 (FGF-2) to the cytosolic/nuclear compartment was studied and compared with the translocation mechanism used by FGF-1. To differentiate between external and endogenous growth factor, we used FGF-2 modified to contain a farnesylation signal, a CaaX-box. Because farnesylation occurs only in the cytosol and nucleoplasm, farnesylation of exogenous FGF-2-CaaX was taken as evidence that the growth factor had translocated across cellular membranes. We found that FGF-2 translocation occurred in endothelial cells and fibroblasts, which express FGF receptors, and that the efficiency of translocation was increased in the presence of heparin. Concomitantly with translocation, the 18-kDa FGF-2 was N-terminally cleaved to yield a 16-kDa form. Translocation of FGF-2 required PI3-kinase activity but not transport through the Golgi apparatus. Inhibition of endosomal acidification did not prevent translocation, whereas dissipation of the vesicular membrane potential completely blocked it. The data indicate that translocation occurs from intracellular vesicles containing proton pumps and that an electrical potential across the vesicle membrane is required. Translocation of both FGF-1 and FGF-2 occurred during most of G1but decreased shortly before the G1→S transition. A common mechanism for FGF-1 and FGF-2 translocation into cells is postulated.
p130Cas-associated Protein (p140Cap) as a New Tyrosine-phosphorylated Protein Involved in Cell Spreading
Authors: Di Stefano Paola , Cabodi Sara , Erba Elisabetta Boeri , Margaria Valentina , Bergatto Elena , Giuffrida Maria Gabriella , Silengo Lorenzo , Tarone Guido , Turco Emilia , Defilippi Paola
Published: December 2003

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.
Differentiation-induced Colocalization of the KH-type Splicing Regulatory Protein with Polypyrimidine Tract Binding Protein and the c-srcPre-mRNA
Authors: Hall Megan P. , Huang Sui , Black Douglas L.
Published: December 2003

We have examined the subcellular localization of the KH-type splicing regulatory protein (KSRP). KSRP is a multidomain RNA-binding protein implicated in a variety of cellular processes, including splicing in the nucleus and mRNA localization in the cytoplasm. We find that KSRP is primarily nuclear with a localization pattern that most closely resembles that of polypyrimidine tract binding protein (PTB). Colocalization experiments of KSRP with PTB in a mouse neuroblastoma cell line determined that both proteins are present in the perinucleolar compartment (PNC), as well as in other nuclear enrichments. In contrast, HeLa cells do not show prominent KSRP staining in the PNC, even though PTB labeling identified the PNC in these cells. Because both PTB and KSRP interact with the c-src transcript to affect N1 exon splicing, we examined the localization of the c-src pre-mRNA by fluorescence in situ hybridization. The src transcript is present in specific foci within the nucleus that are presumably sites of src transcription but are not generally perinucleolar. In normally cultured neuroblastoma cells, these src RNA foci contain PTB, but little KSRP. However, upon induced neuronal differentiation of these cells, KSRP occurs in the same foci with src RNA. PTB localization remains unaffected. This differentiation-induced localization of KSRP with src RNA correlates with an increase in src exon N1 inclusion. These results indicate that PTB and KSRP do indeed interact with the c-src transcript in vivo, and that these associations change with the differentiated state of the cell.
RNA Interference and mRNA Silencing, 2004: How Far Will They Reach?
Authors: Pederson Thoru
Published: December 2003

The discoveries of RNA interference and RNA-mediated posttranscriptional gene silencing have opened an unanticipated new window on the regulation of gene expression as well as a facile and highly effective tool for knocking down gene expression in many organisms and cells. In addition, RNA interference and RNA silencing may conceivably be exploited for human therapeutics sometime in the future, possibly bringing greater clinical impact than have the so far disappointing antisense endeavors. This essay summarizes recent developments and offers some personalized perspectives, with emphasis on what we do not yet know.
Nasal Glioma and Encephalocele: Diagnosis and Management
Authors: Rahbar Reza , Resto Vicente A. , Robson Caroline D. , Perez‐Atayde Antonio R. , Goumnerova Liliana C. , McGill Trevor J. , Healy Gerald B.
Published: December 2003

Objective To review the biology of nasal glioma and encephalocele and to present an algorithm for preoperative evaluation and surgical management.

Design Retrospective review and analysis.

Setting Tertiary care medical center: 1970 to 2002.

Patient Sixteen patients with glioma (n = 10) and encephalocele (n = 6).

Outcome Age at the time of presentation, sex, signs and symptoms, imaging findings, surgical approach, pathology, complications, rate of recurrence, and follow‐up were recorded.

Results Ten patients presented with nasal glioma with a mean age of 9 months. All patients underwent surgical excision. No complication was encountered with a mean follow‐up of 3.5 years. Six patients presented with encephaloceles with a mean age of 15.5 months. All patients underwent surgical excision. Complications included cerebrospinal fluid leak (n = 1) and epiphora (n = 1). Follow‐up was 1 to 14 years (mean, 4 years).

Conclusion Nasal glioma and encephalocele are rare, benign, congenital lesions with a potential for intracranial extension. Evaluation should include a complete rhinologic and neurologic examination. Preoperative imaging with a thin‐cut axial and coronal computed tomography scan and/or multiplanar magnetic resonance imaging is essential. Surgical intervention should be performed soon after diagnosis to alleviate the increased risk of meningitis. A frontal craniotomy approach is recommended if intracranial extension is identified based on preoperative evaluation, followed by an extracranial resection. If there is no evidence of intracranial extension, a conservative extracranial approach is recommended.
Vascularized Radial Forearm Free Tissue Transfer for Lining in Nasal Reconstruction
Authors: Moore Eric J. , Strome Scott A. , Kasperbauer Jan L. , Sherris David A. , Manning Lance A.
Published: December 2003

Objectives/Hypothesis Total and near‐total nasal reconstruction requires the surgeon to replace external nasal cover, skeletal support, and internal nasal lining. The successful result must re‐create the form and function of the original nose. In large defects, traditional sources of internal lining may be unavailable. The study describes the recreation of nasal lining in three patients with extensive nasal defects with vascularized radial forearm tissue.

Study Design Retrospective review of three patients who underwent nasal reconstruction with radial forearm for internal lining, costal cartilage grafts for skeletal support, and paramedian forehead flap for cover.

Methods The charts of three patients who underwent total nasal reconstruction with radial forearm tissue transfer for lining were retrospectively reviewed. The technique was evaluated. The authors present a method for internal lining reconstruction in extensive nasal defects. They discuss the advantages and disadvantages of this method.

Results Three patients underwent successful total nasal reconstruction with radial forearm tissue transfer for lining, costal cartilage for skeletal support, and paramedian forehead flap. None of the patients had adequate septal mucosa or nasal subunit skin to support mucosal rotation grafts or epithelial turn‐in flaps. All patients have completed reconstruction and have satisfactory nasal form and function. Loss of the lining flap or graft loss did not occur.

Conclusion Vascularized radial forearm tissue supplies ample quantities of skin to recreate nasal lining. The tissue provides excellent support of graft material, and it prevents contracture of the covering flap. Because of its bulk and donor site morbidity, free forearm tissue transfer should not be considered for reconstruction of smaller defects when another lining flap will suffice.
Short‐Term Functional Donor Site Morbidity After Radial Forearm Fasciocutaneous Free Flap Harvest
Authors: Skoner Judith M. , Bascom Daphne A. , Cohen James I. , Andersen Peter E. , Wax Mark K.
Published: December 2003

Objectives/Hypothesis The existing literature on postoperative donor extremity function describes a spectrum of morbidity in the long term (>3 mo after surgery). However, the consensus is that there is minimal to no impact of flap harvest on patients' activities of daily living. No previous reports have examined functional donor site morbidity in the early postoperative period; such may affect patients' overall perioperative progress, especially with respect to donor extremity dominance. The authors' objective was to quantify functional morbidity of the donor site in radial forearm fasciocutaneous free flaps during the early postoperative period.

Study Design Retrospective case series review.

Methods Patient data were obtained from hospital records of 12 consecutive patients who underwent head and neck reconstruction with radial forearm fasciocutaneous free tissue transfer over a 6‐month period at a tertiary academic medical center. Functional results of each patient's donor extremity obtained preoperatively and at 5 to 8 days after surgery were determined by quantifying forearm supination and pronation, wrist flexion and extension, and sharp and dull hand sensations in radial, median, and ulnar nerve distributions.

Results Mean patient age was 57 years (age range, 42–71 y). The nondominant extremity was the donor site in 9 of 12 patients. Using the paired two‐tailed t test, statistically significant differences were demonstrated in preoperative versus postoperative forearm supination (P < .032), pronation (P < .006), wrist flexion (P < .000), and wrist extension (P < .000). Three of 12 patients demonstrated diminished sharp sensation in the “anatomical snuffbox” distribution.

Conclusion The authors describe statistically significant functional forearm and wrist range‐of‐motion morbidity associated with the harvest of a radial forearm fasciocutaneous free flap in the early postoperative period.
Amyloidosis of the Upper Aerodigestive Tract
Authors: Pribitkin Edmund , Friedman Oren , O'Hara Brian , Cunnane Mary F. , Levi David , Rosen Marc , Keane William M. , Sataloff Robert T.
Published: December 2003

Objectives/Hypothesis To delineate the clinical and pathologic characteristics of upper aerodigestive tract amyloidosis with particular attention to laryngeal amyloidosis.

Study Design Retrospective chart review of patients with amyloidosis of Thomas Jefferson University and its affiliated hospitals.

Material and Methods The charts of 16 patients with upper aerodigestive tract amyloidosis identified from the databases of the Thomas Jefferson University pathology department were reviewed and included in the study.

Results Sixteen patients (9 male and 7 female, with an average age of 49.8 years) with upper aerodigestive tract amyloidosis were identified. The most common site of amyloid involvement was the larynx. Consequently, patients most commonly presented with hoarseness (14 of 16). All patients underwent surgical removal of the amyloid deposits. Fourteen patients had primary localized amyloidosis. Two experienced systemic involvement. Seven of the 16 patients developed recurrences requiring further treatment.

Conclusions Amyloidosis of the upper aerodigestive tract generally behaves as a benign, localized condition treatable by surgical resection. Regular follow‐up with laryngoscopy is indicated for early diagnosis of recurrence, and multiple surgical procedures may be required to control symptoms.
Actinomycosis Abscess of the Thyroid Gland
Authors: Cevera John J. , Butehorn Henry F. , Shapiro Joel , Setzen Gavin
Published: December 2003

Objectives To present an unusual case of actinomycosis abscess of the thyroid gland as well as review the history, etiology, pathogenicity and treatment of actinomycosis infections of the head and neck.

Study Design Case study.

Methods A report of a 39 year‐old female status post tooth extraction that developed an actinomycosis abscess of the thyroid.

Results After a thyroid actinomycosis abscess was suggested by physical exam, ultrasound, CT scan and needle aspiration, an otolaryngology consult was obtained. The patient successfully was managed with thyroidectomy and intravenous ceftriaxone.

Conclusions Although Actinomycosis soft tissue infections of the head and neck are relatively uncommon, the head and neck surgeon must include it in the differential diagnosis when clinical presentation raises suspicion. Early biopsy is necessary for appropriate identification of the organism with the appearance of sulfur granules lendign a clue to the diagnosis. Debridement and/or excision are often necessary for antibiotics to be used successfully. Antimicrobial therapy should be used for six to twelve months to completely eradicate the disease and prevent recurrence.
Canal Wall Reconstruction with Mimix Hydroxyapatite Cement: Results in an Animal Model and Case Study
Authors: Dornhoffer John , Simmons Oliver
Published: December 2003

Objective/Hypothesis To assess Mimix hydroxyapatite cement for its applicability in canal wall reconstruction using the gerbil as a canal wall model. A case is presented to illustrate a novel technique of canal wall reconstruction using Mimix on the basis of the findings of our animal research.

Study Design This was a preclinical study.

Methods Ten Mongolian gerbils were implanted with Mimix, with the left side used to simulate mastoid obliteration and the right side used to simulate canal wall reconstruction. Pre‐ and postsurgery auditory‐evoked brainstem responses were used to assess ototoxicity, and hematoxylin‐eosin staining of histologic sections was used to assess inflammatory and foreign‐body response and new bone formation.

Results Rapid wound healing was achieved with each of the nine animals evaluated, with no erythema, edema, or drainage. Inspection of the ear canal at the time of sacrifice revealed no signs of otitis media and no middle ear effusions. Microscopic examination showed no inflammatory response or foreign‐body reaction, good mucosalization on the side of the implant facing the bulla, and minimal fibrosis adjacent to the skin. Eight of nine specimens showed new woven bone ingrowth at the bone implant interface, with active osteoblasts and viable lacunae cells. There were no apparent fractures in the implanted material.

Conclusions Mimix hydroxyapatite cement is biocompatible and suitable for canal wall reconstruction in the animal model. The characteristics of this cement, namely its ability to set quickly in a moist environment, offer advantages over previously used cements for canal wall reconstruction.
The Effect of GJB2 Allele Variants on Performance After Cochlear Implantation
Authors: Bauer Paul W. , Geers Ann E. , Brenner Christine , Moog Jean S. , H. Smith Richard J.
Published: December 2003

Objectives/Hypothesis It has been hypothesized that etiology of hearing loss may serve as an independent variable in performance after cochlear implantation. To test this hypothesis, the authors identified pediatric cochlear implant recipients with gap junction protein β2 (GJB2)–related deafness. The study examines performance outcomes associated with GJB2 deafness‐causing allele variants.

Study Design Pediatric cochlear implant patients were screened for GJB2 allele variants; statistical comparisons were made with prospectively obtained performance measures.

Methods From 181 children who participated in a nationwide cochlear implant research program, 122 children were identified with congenital nonsyndromic sensorineural hearing loss and invited to participate. Screening for GJB2 allele variants was completed for 55 children. The children were homogeneous with respect to age (8 or 9 y) and age at implant (before age 5 y). All patients have previously undergone a prospective regimented battery of performance measures.

Results Performance measures were compared between 22 children with and 33 children without mutations to determine whether GJB2 status was a significant predictor of cochlear implant outcomes. Reading and cognitive outcomes were significantly dependent on connexin status. The group of children who tested positive for GJB2‐related deafness scored significantly higher on a nonverbal cognitive measure, Block Design, and on a measure of reading comprehension.

Conclusion The isolated insult to the cochlea created by GJB2 allele variants allows for preservation of central cognitive function. Better reading performance is seen in children with GJB2‐related deafness.