bluuebunny/crossref_metadata_2025 huggingface dataset:
Local haemorrhage induced by Bothrops jararaca venom: relationship to neurogenic inflammation
Authors: Gonçalves Luis Roberto C. , Mariano Mario
Published: July 2002

We investigated morphological alterations induced by s.c. injection of 2.5 μg of Bothrops jararaca venom in rats. Intense disorganisation of collagen fibres was observed 1 min after the venom injection, particularly at regions near vessels and nerves. Mast cells were degranulated, and erythrocytes were seen leaving venules throughout the endothelial junctions. At this time, damaged endothelial cells were not observed. In rats envenomed as above, but immediately after cardiorespiratory failure induced by deep ether anaesthesia, alterations in the connective tissue structures, as previously described, were not observed. The mediation of this haemorrhage was investigated by injecting the venom into the foot pad of mice and compared to the mediation of oedema. Local haemorrhage was significantly reduced in mice pre‐treated with capsaicin or guanethidine or submitted to a surgical section of sciatic and saphenous nerves. In these animals, oedema was not affected. Groups treated with methysergide or morphine showed both haemorrhage and oedema significantly reduced. Indomethacin or dexamethasone pre‐treatments significantly reduced the oedema, but not the haemorrhage. Moreover, in animals treated with promethazine or mepyramine, oedema and haemorrhage were not affected. These data suggest that local haemorrhage induced by Bothrops jararaca venom is partially controlled by serotonin and neurohumoral mediators. Furthermore, results indicate that haemorrhage and oedema are mediated by different pharmacological systems.
Age of Onset as a Discriminator Between Alcoholic Subtypes in a Treatment‐Seeking Outpatient Population
Authors: Johnson Bankole A. , Cloninger C. Robert , Roache John D. , Bordnick Patrick S. , Ruiz Pedro
Published: July 2002

Subtyping alcoholics may provide a more accurate guide as to the course and character of the disease. Classifications of different ages of onset of problem drinking have so far resulted in categorical inconsistencies. In the past, hospital‐based alcoholics have over‐represented those most severely ill, and comprehensive evaluations of psychopathology for discriminating between alcoholic subtypes have been infrequent. In a heterogeneous treatment‐seeking, outpatient, alcoholic population, we tested the hypothesis that age of onset represents a continuum of disease, and that greater severity of psychopathology is associated with lower ages of onset.

Using a standard questionnaire, 253 male and female treatment‐seeking alcoholics were stratified according to specific ages of onset: a) <20 years; b) 20–25 years, and c) >25 years. These age of onset groups were compared on alcohol severity and craving, family history, childhood behavior, personality, hostility, overt aggression, mood, and social functioning.

Symptom severity and age of onset were negatively correlated, and the 20–25‐year onset group usually had intermediate scores. The <20 year onset group was characterized by greater severity of alcohol‐related problems, family history, childhood behavioral problems, craving, hostility, antisocial traits, mood disturbance, and poor social functioning.

Alcoholics with an earlier age of onset have relatively greater psychopathology than those of later onset. While the preponderance of psychopathology among those in the <20‐year onset group could be conceptualized as a clinical “subtype,” such a characterization would not define an entirely homogenous category. Yet, this clinical characterization would be clinically important if specific age of onset levels were found to be differentially sensitive to pharmacological and/or psychological treatments.
In Vitro Antioxidant and Photo-oxidant Properties of Dipyridamole
Authors: Vargas Franklin , Cheng Alejandro Tse , Velutini Guillermo , Marcano Emildo , Sánchez Ysbelia , Fraile Germán , Velásquez Mait
Published: July 2002

The in vitro antioxidant and photo-oxidant activity of dipyridamole was studied by its effect on superoxide-and singlet oxygen-mediated photohemolysis and viability of neutrophils. Dipyridamole was found to be phototoxic when examined by the photohemolysis on human erythrocytes and on linoleic acid as lipid peroxidation model at concentrations above 3.0 × 10–5 M. On the contrary, when lower concentrations (1.0 × 10–5 to 1.0 × 10–6 M) were used, dipyridamole showed a protector action against singlet oxygen-mediated photohemolysis by other phototoxic compounds like triamterene. This antioxidant property is proposed to result from quenching of triamterene mediated by fluorescence energy transfer. Auto-oxidation and fluorescence-energy transfer is clearly an important mechanism for protection for this drug.
Hexamethyldisiloxane: A 13-Week Subchronic Whole-Body Vapor Inhalation Toxicity Study in Fischer 344 Rats
Authors: Cassidy Simon L. , Dotti Antonio , Kolesar Gary B. , Dochterman L. Wayne , Meeks Robert G. , Chevalier H. J.
Published: July 2002

Hexamethyldisiloxane (HMDS) is a volatile linear siloxane dimer used in many applications, including precision cleaning, active ingredient carrier, and as a manufacturing intermediate. The purpose of this study was to characterize the subchronic vapor inhalation toxicity of the material as part of a comprehensive toxicology program. Groups of 20 male and 20 female Fischer 344 (F344) rats were exposed to nominal (and mean actual) vapor concentrations of 0, 50 (50), 200 (194), 600 (593), 1500 (1509) and 5000 (5012) ppm of HMDS, 6 hours per day, 5 days per week, for 13 weeks. No treatment-related signs of clinical toxicity or mortality, statistically significant effects upon body weight gain or food consumption, ophthahnoscopic changes, gross macroscopic necropsy findings, or organ weight changes were noted. Minor hematological, clinical biochemical, and urinalysis changes were seen but were not considered to be of toxicological relevance. Histological lesions in the kidney apparently consistent with male rat-specific α-2-urinary globulin nephropathy were observed in male rats exposed to 593, 1509, and 5012 ppm of HMDS, accompanied by slightly increased plasma urea and creatinine concentrations. No other treatment-related histological changes were seen in HMDS-exposed rats.
An Automated Method for the Analysis of Methemoglobin
Authors: Olson C. T. , Menton R. G. , Blank J. A. , Hobson D. W.
Published: July 2002

Production of methemoglobin (met Hb) is effective in treating or preventing cyanide intoxication due to the affinity of cyanide for met Hb. This paper describes an automated method for measuring met Hb in mouse blood utilizing the Roche COB AS FARA centrifugal analyzer and a modified Evelyn–Malloy procedure. Blood samples were spiked with various quantities of potassium ferricyanide to produce met Hb, and automated and manual analyses of these samples were compared. Following validation of the COB AS method, two known met Hb inducers, sodium nitrite and p-aminopropiophenone (PAPP), and two sulfur donor compounds, sodium thiosulfate and ICD #1021, not known to produce met Hb were tested in vivo. Five mice per compound were injected IP and blood samples were analyzed periodically over a time period of approximately 2 h. The automated technique requires only a drop of blood, which allows repeated sampling from the same animal, and can be adapted for blood from different species. A nalyses are rapid and sensitive, and results are reproducible.
The cannabinoid receptor gene (CNR1) is not affected in German i.v. drug users
Authors: Heller D. , Schneider U. , Seifert J. , Cimander K. F. , Stuhrmann M.
Published: July 2002

The aim of the study was to investigate a possible contribution of the cannabinoid receptor gene (CNR1) to the development of i.v. drug addiction. Allele and genotype frequencies of a previously associated flanking triplet repeat polymorphism were compared between patients and controls, and the whole coding region of the CNR1 gene of all patients were screened for presence of mutations. The study took place at the Addiction Treatment Unit of the Medical School Hannover, and two outpatients departments in Hannover, Germany. Forty German unrelated opioid addicts (27 males and 13 females; mean age 37.9 years; range 16–53 years), took part, all of them satisfying ICD‐10 and DSM‐IV diagnostic criteria for opioid dependence and 81 age‐ and sex‐matched controls (German blood donors). Measurements used were lengths of alleles, genotyping and single strand conformation polymorphism (SSCP) analysis. Neither the ≥ 5 alleles of the extragenic triplet repeat (AAT) marker nor the alleles of an intragenic biallelic CNR1 polymorphism (1359G/A) were associated with i.v. drug use in our study group. In addition, we did not detect any sequence variation within the CNR1 gene which could confer susceptibility to i.v. drug abuse. In contrast to previous investigations, we found no evidence for an involvement of the CNR1 gene in i.v. drug addiction.
Group Training In Communication Skills For Carers Of Adults With Aphasia
Authors: Booth Susan , Swabey Donna
Published: July 2002

This study describes a communication skills group programme for four carers of adults with aphasia that ran once a week for 6 consecutive weeks. The content of the group was based on an approach previously not described in the literature in any detail. Conversation analysis (CA) was used to guide individualized advice that was incorporated into the group by the use of written advice sheets. Intervention was motivated by the results of a newly developed assessment tool‐the Conversation Analysis Profile for People with Aphasia (CAPPA)‐and a quantitative and qualitative analysis of collaborative repair. The CAPPA utilizes the methodology of conversation analysis (CA) as a means of both characterizing and comparing the relationship between the carers' perception of the aphasia and what is occurring in natural conversation. During the group, accurate perceptions and strategies that minimized the disruption to the conversation were reinforced, while inaccurate perceptions and strategies that appeared to impede interaction were discouraged. The use of the CAPPA results and a quantitative/qualitative analysis of repair management to measure change pre‐ and post‐group was explored. The post‐intervention analyses examined three questions in particular: (1) did the carers demonstrate more accurate perceptions of their relatives' aphasia?; (2) did the carers report a decrease in the problem severity of the aphasia?; and (3) was there a change in the time taken to repair a trouble source and was this attributable to a change in the management of repair by the carer? The study was essentially an investigation of whether this type of approach was beneficial to the carers involved. The results suggested that focusing on individualized advice and targeting conversation management in the group setting was a useful way of providing advice to carers. Furthermore, the CAPPA and a quantitative/qualitative analysis of repair management seem to have the potential for motivating the individualized advice and measuring the effectiveness of an intervention.
Significance of the Grb2 and Son of Sevenless (Sos) Proteins in Human Bladder Cancer Cell Lines
Authors: Watanabe Takafumi , Shinohara Nobuo , Moriya Kimihiko , Sazawa Ataru , Kobayashi Yoshiyasu , Ogiso Yoshifumi , Takiguchi Mitsuyoshi , Yasuda Jun , Koyanagi Tomohiko , Kuzumaki Noboru , Hashimoto Akira
Published: July 2002

The epidermal growth factor (EGF) receptor has been suggested to have an important role in tumor initiation and progression of human bladder cancers. Grb2 protein, which is the downstream effector of the EGF receptor, acts as an adaptor protein between the EGF receptor and the Ras guanine‐nucleotide exchange factor, son of sevenless (Sos) protein. Sos protein regulates the action of Ras protein by promoting the exchange of GDP for GTP . However, the significance of Grb2 and Sos proteins, which is related to EGF‐triggered Ras activation, has not been elucidated in human bladder cancer. The aim of the present study is to clarify the significance of these proteins in human bladder cancer cell lines. In the present study, we used four human bladder cancer cell lines (T24, KU‐7, UMUC‐2, UMUC‐6) and two kinds of cultured normal urothelial cells (HMKU‐1, HMKU‐2) isolated from patients with no malignancy. We examined the expression of EGF receptor, Grb2, and Sos proteins in these cells by Western blot analysis. Furthermore, the bladder cancer cell lines were subjected to sequence analysis to identify a point mutation in the c‐H‐ras gene at codon 12. There was no marked difference in the expression of the EGF receptor between human bladder cancer cell lines and cultured normal urothelial cells. On the other hand, expression of Grb2 and Sos proteins was substantially increased in all human bladder cancer cell lines examined in comparison with cultured normal urothelial cells, whether codon 12 of H‐ras was mutated or not. These results suggest that the amplification of both Grb2 and SOS proteins plays an important role in the carcinogenesis of human bladder cancer.
In Vivo Dynamics of Nuclear Pore Complexes in Yeast
Authors: Bucci Mirella , Wente Susan R.
Published: July 2002

While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP– Nup49p into existing recipient NPCs. Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures. This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.
Physiological Regulation of Membrane Protein Sorting Late in the Secretory Pathway of Saccharomyces cerevisiae
Authors: Roberg Kevin J. , Rowley Neil , Kaiser Chris A.
Published: July 2002

In mammalian cells, extracellular signals can regulate the delivery of particular proteins to the plasma membrane. We have discovered a novel example of regulated protein sorting in the late secretory pathway of Saccharomyces cerevisiae. In yeast cells grown on either ammonia or urea medium, the general amino acid permease (Gap1p) is transported from the Golgi complex to the plasma membrane, whereas, in cells grown on glutamate medium, Gap1p is transported from the Golgi to the vacuole. We have also found that sorting of Gap1p in the Golgi is controlled by SEC13, a gene previously shown to encode a component of the COPII vesicle coat. In sec13 mutants grown on ammonia, Gap1p is transported from the Golgi to the vacuole, instead of to the plasma membrane. Deletion of PEP12, a gene required for vesicular transport from the Golgi to the prevacuolar compartment, counteracts the effect of the sec13 mutation and partially restores Gap1p transport to the plasma membrane. Together, these studies demonstrate that both a nitrogen-sensing mechanism and Sec13p control Gap1p transport from the Golgi to the plasma membrane.
Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes
Authors: Gu Feng , Aniento Fernando , Parton Robert G. , Gruenberg Jean
Published: July 2002

In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in ε coatomer protein (εCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although εCOP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of εCOP, we find that a subcomplex of β, β′, and ζCOP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPγS and sensitivity to the endosomal pH. Previous studies showed that γ and δCOP are not found on endosomes. However, αCOP, which is normally present on endosomes, is no longer recruited when εCOP is missing. In contrast, all COP subunits, except obviously εCOP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas β, β′, and ζCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of α and/or εCOP.
The Sleep-inducing Lipid Oleamide Deconvolutes Gap Junction Communication and Calcium Wave Transmission in Glial Cells
Authors: Guan Xiaojun , Cravatt Benjamin F. , Ehring George R. , Hall James E. , Boger Dale L. , Lerner Richard A. , Gilula Norton B.
Published: July 2002

Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction–mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18β-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction.
Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons
Authors: Nakata Takao , Terada Sumio , Hirokawa Nobutaka
Published: July 2002

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.
Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human Cells
Authors: Jackson Dean A. , Pombo Ana
Published: July 2002

In proliferating cells, DNA synthesis must be performed with extreme precision. We show that groups of replicons, labeled together as replicon clusters, form stable units of chromosome structure. HeLa cells were labeled with 5-bromodeoxyuridine (BrdU) at different times of S phase. At the onset of S phase, clusters of replicons were activated in each of ∼750 replication sites. The majority of these replication “foci” were shown to be individual replicon clusters that remained together, as stable cohorts, throughout the following 15 cell cycles. In individual cells, the same replication foci were labeled with BrdU and 5-iododeoxyuridine at the beginning of different cell cycles. In DNA fibers, 95% of replicons in replicon clusters that were labeled at the beginning of one S phase were also labeled at the beginning of the next. This shows that a subset of origins are activated both reliably and efficiently in different cycles.

The majority of replication forks activated at the onset of S phase terminated 45–60 min later. During this interval, secondary replicon clusters became active. However, while the activation of early replicons is synchronized at the onset of S phase, different secondary clusters were activated at different times. Nevertheless, replication foci pulse labeled during any short interval of S phase were stable for many cell cycles. We propose that the coordinated replication of related groups of replicons, that form stable replicon clusters, contributes to the efficient activation and propagation of S phase in mammalian cells.
Yeast Nuclei Display Prominent Centromere Clustering That Is Reduced in Nondividing Cells and in Meiotic Prophase
Authors: Jin Quan-wen , Trelles-Sticken Edgar , Scherthan Harry , Loidl Josef
Published: July 2002

Chromosome arrangement in spread nuclei of the budding yeast, Saccharomyces cerevisiae was studied by fluorescence in situ hybridization with probes to centromeres and telomeric chromosome regions. We found that during interphase centromeres are tightly clustered in a peripheral region of the nucleus, whereas telomeres tend to occupy the area outside the centromeric domain. In vigorously growing cultures, centromere clustering occurred in ∼90% of cells and it appeared to be maintained throughout interphase. It was reduced when cells were kept under stationary conditions for an extended period. In meiosis, centromere clusters disintegrated before the emergence of the earliest precursors of the synaptonemal complex. Evidence for the contribution of centromere clustering to other aspects of suprachromosomal nuclear order, in particular the vegetative association of homologous chromosomes, is provided, and a possible supporting role in meiotic homology searching is discussed.
Role of P-Selectin Cytoplasmic Domain in Granular Targeting In Vivo and in Early Inflammatory Responses
Authors: Hartwell Daqing W. , Mayadas Tanya N. , Berger Gaëtan , Frenette Paul S. , Rayburn Helen , Hynes Richard O. , Wagner Denisa D.
Published: July 2002

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (ΔCT mice). The deletion did not affect the sorting of P-selectin into α-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The ΔCT–P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the ΔCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.
Fission Yeast α-Glucan Synthase Mok1 Requires the Actin Cytoskeleton to Localize the Sites of Growth and Plays an Essential Role in Cell Morphogenesis Downstream of Protein Kinase C Function
Authors: Katayama Satoshi , Hirata Dai , Arellano Manuel , Pérez Pilar , Toda Takashi
Published: July 2002

In fission yeast protein kinase C homologues (Pck1 and Pck2) are essential for cell morphogenesis. We have isolated mok1+ in a genetic screen to identify downstream effectors for Pck1/2. mok1+ is essential for viability and encodes a protein that has several membrane-spanning domains and regions homologous to glucan metabolic enzymes. mok1 mutant shows abnormal cell shape, randomization of F-actin and weak cell wall. Biochemical analysis shows that Mok1 appears to have α-glucan synthase activity. Mok1 localization undergoes dramatic alteration during the cell cycle. It localizes to the growing tips in interphase, the medial ring upon mitosis, a double ring before and dense dot during cytokinesis. Double immunofluorescence staining shows that Mok1 exists in close proximity to actin. The subcellular localization of Mok1 is dependent upon the integrity of the F-actin cytoskeleton. Conversely, overexpression of mok1+ blocks the translocation of cortical actin from one end of the cell to the other. pck2 mutant is synthetically lethal with mok1 mutant, delocalizes Mok1 and shows a lower level of α-glucan. These results indicate that Mok1 plays a crucial role in cell morphogenesis interdependently of the actin cytoskeleton and works as one of downstream effectors for Pck1/2.
A PDZ-containing Scaffold Related to the Dystrophin Complex at the Basolateral Membrane of Epithelial Cells
Authors: Kachinsky Amy M. , Froehner Stanley C. , Milgram Sharon L.
Published: July 2002

Membrane scaffolding complexes are key features of many cell types, serving as specialized links between the extracellular matrix and the actin cytoskeleton. An important scaffold in skeletal muscle is the dystrophin-associated protein complex. One of the proteins bound directly to dystrophin is syntrophin, a modular protein comprised entirely of interaction motifs, including PDZ (protein domain named for PSD-95, discs large, ZO-1) and pleckstrin homology (PH) domains. In skeletal muscle, the syntrophin PDZ domain recruits sodium channels and signaling molecules, such as neuronal nitric oxide synthase, to the dystrophin complex. In epithelia, we identified a variation of the dystrophin complex, in which syntrophin, and the dystrophin homologues, utrophin and dystrobrevin, are restricted to the basolateral membrane. We used exogenously expressed green fluorescent protein (GFP)-tagged fusion proteins to determine which domains of syntrophin are responsible for its polarized localization. GFP-tagged full-length syntrophin targeted to the basolateral membrane, but individual domains remained in the cytoplasm. In contrast, the second PH domain tandemly linked to a highly conserved, COOH-terminal region was sufficient for basolateral membrane targeting and association with utrophin. The results suggest an interaction between syntrophin and utrophin that leaves the PDZ domain of syntrophin available to recruit additional proteins to the epithelial basolateral membrane. The assembly of multiprotein signaling complexes at sites of membrane specialization may be a widespread function of dystrophin-related protein complexes.
Monocytes Induce Reversible Focal Changes in Vascular Endothelial Cadherin Complex during Transendothelial Migration under Flow
Authors: Allport Jennifer R. , Muller William A. , Luscinskas Francis W.
Published: July 2002

The vascular endothelial cell cadherin complex (VE-cadherin, α-, β-, and γ-catenin, and p120/p100) localizes to adherens junctions surrounding vascular endothelial cells and may play a critical role in the transendothelial migration of circulating blood leukocytes. Previously, we have reported that neutrophil adhesion to human umbilical vein endothelial cell (HUVEC) monolayers, under static conditions, results in a dramatic loss of the VE-cadherin complex. Subsequent studies by us and others (Moll, T., E. Dejana, and D. Vestweber. 1998. J. Cell Biol. 140:403–407) suggested that this phenomenon might reflect degradation by neutrophil proteases released during specimen preparation. We postulated that some form of disruption of the VE-cadherin complex might, nonetheless, be a physiological process during leukocyte transmigration. In the present study, the findings demonstrate a specific, localized effect of migrating leukocytes on the VE-cadherin complex in cytokine-activated HUVEC monolayers. Monocytes and in vitro differentiated U937 cells induce focal loss in the staining of VE-cadherin, α-catenin, β-catenin, and plakoglobin during transendothelial migration under physiological flow conditions. These events are inhibited by antibodies that prevent transendothelial migration and are reversed following transmigration. Together, these data suggest that an endothelial-dependent step of transient and focal disruption of the VE-cadherin complex occurs during leukocyte transmigration.
Spontaneous Release of Cytosolic Proteins from Posttranslational Substrates before Their Transport into the Endoplasmic Reticulum
Authors: Plath Kathrin , Rapoport Tom A.
Published: July 2002

In posttranslational translocation in yeast, completed protein substrates are transported across the endoplasmic reticulum membrane through a translocation channel formed by the Sec complex. We have used photo-cross-linking to investigate interactions of cytosolic proteins with a substrate synthesized in a reticulocyte lysate system, before its posttranslational translocation through the channel in the yeast membrane. Upon termination of translation, the signal recognition particle (SRP) and the nascent polypeptide–associated complex (NAC) are released from the polypeptide chain, and the full-length substrate interacts with several different cytosolic proteins. At least two distinct complexes exist that contain among other proteins either 70-kD heat shock protein (Hsp70) or tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1 (TRiC/CCT), which keep the substrate competent for translocation. None of the cytosolic factors appear to interact specifically with the signal sequence. Dissociation of the cytosolic proteins from the substrate is accelerated to the same extent by the Sec complex and an unspecific GroEL trap, indicating that release occurs spontaneously without the Sec complex playing an active role. Once bound to the Sec complex, the substrate is stripped of all cytosolic proteins, allowing it to subsequently be transported through the membrane channel without the interference of cytosolic binding partners.
Dynein Is a Transient Kinetochore Component Whose Binding Is Regulated by Microtubule Attachment, Not Tension
Authors: King Jennifer M. , Hays Tom S. , Nicklas R. Bruce
Published: July 2002

Cytoplasmic dynein is the only known kinetochore protein capable of driving chromosome movement toward spindle poles. In grasshopper spermatocytes, dynein immunofluorescence staining is bright at prometaphase kinetochores and dimmer at metaphase kinetochores. We have determined that these differences in staining intensity reflect differences in amounts of dynein associated with the kinetochore. Metaphase kinetochores regain bright dynein staining if they are detached from spindle microtubules by micromanipulation and kept detached for 10 min. We show that this increase in dynein staining is not caused by the retraction or unmasking of dynein upon detachment. Thus, dynein genuinely is a transient component of spermatocyte kinetochores.

We further show that microtubule attachment, not tension, regulates dynein localization at kinetochores. Dynein binding is extremely sensitive to the presence of microtubules: fewer than half the normal number of kinetochore microtubules leads to the loss of most kinetochoric dynein. As a result, the bulk of the dynein leaves the kinetochore very early in mitosis, soon after the kinetochores begin to attach to microtubules. The possible functions of this dynein fraction are therefore limited to the initial attachment and movement of chromosomes and/or to a role in the mitotic checkpoint.
Reversal of the Ras-Induced Transformed Phenotype by Hr12, a Novel Ras Farnesylation Inhibitor, Is Mediated by the Mek/ERK Pathway
Authors: Reuveni Hadas , Geiger Tamar , Geiger Benjamin , Levitzki Alexander
Published: July 2002

We have used the selective farnesylation inhibitor HR12 [cysteine-N(methyl)valine-N(cyclohexyl) glycine-methionine-O-methyl-ester] to study the role of oncogenic Ras in cytoskeletal reorganization in Ha-rasV12-transformed Rat1 cells (Rat1/ras). Application of HR12 resulted in complete restoration of the cytoskeleton and associated cell adhesions disrupted by oncogenic Ras. This included an increase in the number and size of focal adhesions, accompanied by massive stress fiber formation and enhanced tyrosine phosphorylation. Furthermore, HR12 induced assembly of adherens junctions and dramatically elevated the level of the junctional components, cadherin and β-catenin. HR12 was unable to restore the nontransformed phenotype in cells expressing farnesylation-independent, myristylated Ras. Examination of the main Ras-regulated signaling pathways revealed that HR12 induced a dose- and time-dependent decline in Erk1&2 activation (t1/2 ∼ 6 h), which correlated with the accumulation of nonfarnesylated oncogenic-Ras. Inhibition of the Mek/Erk pathway in Rat1/ras cells, using the Mek inhibitor, PD98059, resulted in complete cytoskeletal recovery, indistinguishable from that induced by HR12. Moreover, a constitutively active Mek mimicked the effect of ras transformation in Rat1 cells, and prevented HR12-induced cytoskeletal effects in Rat1/ras cells. No such effects were observed after treatment of Rat1/ras cells with the phosphatidylinositol 3-kinase inhibitor LY294002. These findings establish the Mek/Erk pathway as the dominant pathway involved in conferring the cytoskeletal and junctional manifestations of the Ras-induced transformed phenotype.
Cyclin a Is Destroyed in Prometaphase and Can Delay Chromosome Alignment and Anaphase
Authors: den Elzen Nicole , Pines Jonathon
Published: July 2002

Mitosis is controlled by the specific and timely degradation of key regulatory proteins, notably the mitotic cyclins that bind and activate the cyclin-dependent kinases (Cdks). In animal cells, cyclin A is always degraded before cyclin B, but the exact timing and the mechanism underlying this are not known. Here we use live cell imaging to show that cyclin A begins to be degraded just after nuclear envelope breakdown. This degradation requires the 26S proteasome, but is not affected by the spindle checkpoint. Neither deletion of its destruction box nor disrupting Cdk binding prevents cyclin A proteolysis, but Cdk binding is necessary for degradation at the correct time. We also show that increasing the levels of cyclin A delays chromosome alignment and sister chromatid segregation. This delay depends on the proteolysis of cyclin A and is not caused by a lag in the bipolar attachment of chromosomes to the mitotic spindle, nor is it mediated via the spindle checkpoint. Thus, proteolysis that is not under the control of the spindle checkpoint is required for chromosome alignment and anaphase.
Nuclear Import of Histone H2a and H2b Is Mediated by a Network of Karyopherins
Authors: Mosammaparast Nima , Jackson Kelley R. , Guo Yurong , Brame Cynthia J. , Shabanowitz Jeffrey , Hunt Donald F. , Pemberton Lucy F.
Published: July 2002

The first step in the assembly of new chromatin is the cell cycle–regulated synthesis and nuclear import of core histones. The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers. We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family. An abundant complex of H2A, H2B, and Kap114p was detected in cytosol. In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B. Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B. We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail. Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains. In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo. Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association. These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role.
Osp/Claudin-11 Forms a Complex with a Novel Member of the Tetraspanin Super Family and β1 Integrin and Regulates Proliferation and Migration of Oligodendrocytes
Authors: Tiwari-Woodruff Seema K. , Buznikov Alex G. , Vu Trung Q. , Micevych Paul E. , Chen Kendall , Kornblum Harley I. , Bronstein Jeff M.
Published: July 2002

Oligodendrocyte-specific protein (OSP)/claudin-11 is a major component of central nervous system myelin and forms tight junctions (TJs) within myelin sheaths. TJs are essential for forming a paracellular barrier and have been implicated in the regulation of growth and differentiation via signal transduction pathways. We have identified an OSP/claudin-11–associated protein (OAP)1, using a yeast two-hybrid screen. OAP-1 is a novel member of the tetraspanin superfamily, and it is widely expressed in several cell types, including oligodendrocytes. OAP-1, OSP/claudin-11, and β1 integrin form a complex as indicated by coimmunoprecipitation and confocal immunocytochemistry. Overexpression of OSP/claudin-11 or OAP-1 induced proliferation in an oligodendrocyte cell line. Anti–OAP-1, anti–OSP/claudin-11, and anti–β1 integrin antibodies inhibited migration of primary oligodendrocytes, and migration was impaired in OSP/claudin-11–deficient primary oligodendrocytes. These data suggest a role for OSP/claudin-11, OAP-1, and β1 integrin complex in regulating proliferation and migration of oligodendrocytes, a process essential for normal myelination and repair.
In vivo requirement of the α-syntrophin PDZ domain for the sarcolemmal localization of nNOS and aquaporin-4
Authors: Adams Marvin E. , Mueller Heather A. , Froehner Stanley C.
Published: July 2002

α-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of α-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of α-syntrophin in vivo by generating transgenic mouse lines expressing full-length α-syntrophin or a mutated α-syntrophin lacking the PDZ domain (ΔPDZ). The ΔPDZ α-syntrophin displaced endogenous α- and β1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the α-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an α-syntrophin–null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length α-syntrophin, not the ΔPDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound α-syntrophin PDZ domain.
Polyploids require Bik1 for kinetochore–microtubule attachment
Authors: Lin Haijiang , de Carvalho Pedro , Kho David , Tai Chin-Yin , Pierre Philippe , Fink Gerald R. , Pellman David
Published: July 2002

The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells. Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface. Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids. The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton. In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids. The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics. The finding that a protein involved in kinetochore–MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.
Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation
Authors: Lindsten Kristina , de Vrij Femke M.S. , Verhoef Lisette G.G.C. , Fischer David F. , van Leeuwen Fred W. , Hol Elly M. , Masucci Maria G. , Dantuma Nico P.
Published: July 2002

Loss of neurons in neurodegenerative diseases is usually preceded by the accumulation of protein deposits that contain components of the ubiquitin/proteasome system. Affected neurons in Alzheimer's disease often accumulate UBB+1, a mutant ubiquitin carrying a 19–amino acid C-terminal extension generated by a transcriptional dinucleotide deletion. Here we show that UBB+1 is a potent inhibitor of ubiquitin-dependent proteolysis in neuronal cells, and that this inhibitory activity correlates with induction of cell cycle arrest. Surprisingly, UBB+1 is recognized as a ubiquitin fusion degradation (UFD) proteasome substrate and ubiquitinated at Lys29 and Lys48. Full blockade of proteolysis requires both ubiquitination sites. Moreover, the inhibitory effect was enhanced by the introduction of multiple UFD signals. Our findings suggest that the inhibitory activity of UBB+1 may be an important determinant of neurotoxicity and contribute to an environment that favors the accumulation of misfolded proteins.
The NH2-terminal peptide of α–smooth muscle actin inhibits force generation by the myofibroblast in vitro and in vivo
Authors: Hinz Boris , Gabbiani Giulio , Chaponnier Christine
Published: July 2002

Myofibroblasts are specialized fibroblasts responsible for granulation tissue contraction and the soft tissue retractions occurring during fibrocontractive diseases. The marker of fibroblast-myofibroblast modulation is the neo expression of α–smooth muscle actin (α-SMA), the actin isoform typical of vascular smooth muscle cells that has been suggested to play an important role in myofibroblast force generation. Actin isoforms differ slightly in their NH2-terminal sequences; these conserved differences suggest different functions. When the NH2-terminal sequence of α-SMA Ac-EEED is delivered to cultured myofibroblast in the form of a fusion peptide (FP) with a cell penetrating sequence, it inhibits their contractile activity; moreover, upon topical administration in vivo it inhibits the contraction of rat wound granulation tissue. The NH2-terminal peptide of α–skeletal actin has no effect on myofibroblasts, whereas the NH2-terminal peptide of β–cytoplasmic actin abolishes the immunofluorescence staining for this isoform without influencing α-SMA distribution and cell contraction. The FPs represent a new tool to better understand the specific functions of actin isoforms. Our findings support the crucial role of α-SMA in wound contraction. The α-SMA–FP will be useful for the understanding of the mechanisms of connective tissue remodeling; moreover, it furnishes the basis for a cytoskeleton-dependent preventive and/or therapeutic strategy for fibrocontractive pathological situations.
Proteins Phosphorylated during Stress-induced Apoptosis Are Common Targets for Autoantibody Production in Patients with Systemic Lupus Erythematosus
Authors: Utz Paul J. , Hottelet Maria , Schur Peter H. , Anderson Paul
Published: July 2002

Proteins cleaved by interleukin-1β converting enzyme family proteases during apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus (SLE). We have tested the possibility that proteins phosphorylated in cells undergoing apoptosis are also targets for autoantibody production in patients with autoimmune disease. Sera from 9/12 patients containing antinuclear antibodies (10/12 meeting diagnostic criteria for SLE or a lupus overlap syndrome), precipitated new phosphoproteins from lysates derived from Jurkat T cells treated with apoptotic stimuli (i.e., Fas-ligation, gamma irradiation, ultraviolet irradiation), but not with an activation (i.e., CD3-ligation) stimulus. Sera derived from individual patients precipitated different combinations of seven distinct serine-phosphorylated proteins. None of these phosphoproteins were included in precipitates prepared using sera from patients with diseases that are not associated with autoantibody production or using serum from rheumatoid arthritis patients. Protein phosphorylation precedes, or is coincident with, the induction of DNA fragmentation, and is not observed when apoptosis is inhibited by overexpression of bcl-2. Serum from four patients precipitated a serine/threonine kinase from apoptotic cell lysates that phosphorylates proteins of 23-, 34-, and 46-kD in in vitro kinase assays. Our results suggest that proteins phosphorylated during apoptosis may be preferred targets for autoantibody production in patients with SLE.