bluuebunny/crossref_metadata_2025 huggingface dataset:
Topical Exposure to 2-Butoxyethanol Alters Immune Responses in Female BALB/c Mice
Authors: Singh P. , Zhao S. , Blaylock B. L.
Published: July 2002

Effects on immune parameters following topical exposure to 2-butoxyethanol (BE) in mice are reported in the present study. The objective was to determine whether subacute topical exposure to BE can modulate functional immune responses and/or nonspecific immune parameters such as lymphoid organ weight and cellularity. Female BALB/c mice were topically exposed to vehicle or BE at concentrations of 100, 500, 1000, and 1500 mg BE/kg/day for 4 consecutive days. Assessment of immune parameters began 24 hours after the final dose. No effects were observed at any of the BE concentrations on thymus cellularity or thymus to body weight ratio. A significant increase in spleen cellularity and spleen to body weight ratio was observed at 1500 mg BE/kg/day. Topical BE exposure significantly reduced the splenic T cell proliferative response to con-canavalin A (Con A) and the mixed lymphocyte response (MLR) to allogeneic antigen. No significant effect was observed in the splenic B cell proliferative response to lipopolysaccharide (LPS), nor was there an effect on the in vitro primary antibody response to sheep red blood cells (SRBCs). No significant alteration occurred in either splenocyte cytotoxic T lymphocyte (CTL) activity or natural killer (NK) cell activity following topical BE exposure. This study suggests that topical exposure to BE may suppress some aspects of T cell immunity but does not affect B cell immunity.
Evaluation of the Carcinogenic Potential of Insulin Glargine (LANTUS) in Rats and Mice
Authors: Stammberger Ingo , Bube Axel , Durchfeld-Meyer Beate , Donaubauer Hans , Troschau Gotz
Published: July 2002

Insulin glargine (LANTUS) is a new, long-acting insulin analogue with a stable profile of action. The purpose of these studies was to evaluate the carcinogenic potential of insulin glargine in rats and mice. General toxicity studies were conducted in NMRI mice (3 months' duration) and rats (Wistar rats in the 3- and 6-month studies and Sprague-Dawley rats in the 12-month study) to determine the optimal dose of insulin glargine for long-term carcinogenicity studies. Based on these results, groups of Sprague-Dawley rats or NMRI mice (50 male, 50 female) received a daily subcutaneous dose of 2, 5, or 12.5 IU/kg of insulin glargine or 12.5 (mice) or 5 IU/kg (rats) of the reference insulin (NPH insulin) in a lifetime study. Similarly treated control and vehicle-control animals received isotonic sodium chloride (Na Cl) solution or the vehicle solution, respectively. In mice, the mortality rate was comparable between all groups. In rats, the mortality rate compared with the Na Cl control was significantly increased in the following groups: males treated with the vehicle control, all insulin glargine and NPH insulin groups, and in females in the high-dose insulin glargine and NPH insulin groups. There was no difference in the incidence of mammary tumors reported in both mice and rats when comparing the insulin glargine groups with the Na Cl, vehicle-control, or the NPH insulin groups. In rats and mice, the distribution of subcutaneous malignant fibrous histiocytomas found at the injection site were not dose-dependent. These lesions are a rodent-specific event and were related to chronic tissue irritation and inflammation. In rats, neuronal necrosis of the cerebrum was attributed to persistent repeated episodes of hypoglycemia induced by high doses of insulin. In these studies, there were no neoplastic findings to indicate that insulin glargine had a systemic carcinogenic potential in mice or rats.
Endocrine Modulation and the Federal Government
Authors: Byrd Daniel M. , Roegner M. Luann
Published: July 2002

Recent events have drawn attention to the hypothesis that some xenobiotics in the environment may elicit toxicities in humans by modulating endocrine pathways. From the perspective of regulatory toxicology, pursuit of this hypothesis w ill prove difficult, because current risk assessment m ethods do not readily apply to substances with very high potencies, reversibility, transgener-ational effects, and subtle biological outcomes. Such xenobiotics typically persist in the body and bioaccumulate in the food chain. Yet the exposures are important only during critical periods of vulnerability, and no sustained bio-markers of these important exposures currently exist. This article describes some recent efforts by federal agencies to pursue the hypothesis, including research planning and screening of potential endocrine modulating xenobiotics and risk assessment of dioxin conflicts with its policy for substances that cause thyroid follicular carcinomas.
Modulation of Calmodulin and Protein Kinase C Activities by Pencillium Mycotoxins
Authors: Pala I. , Srinivasan A. , Vig P. J. S. , Desaiah D.
Published: July 2002

Calmodulin (CaM), a calcium-binding protein, is found in high concentrations in mammalian brain where it plays a pivotal role in a large number of cellular functions. Protein kinase C (PKC), a multifunctional cytosolic enzyme, in the presence of both Ca2+ and phospholipids, transduce extracellular signals into intracellu-lar events. Both CaM and PKC are partially involved in maintaining Ca2+ homeostasis in the cell. Any fluctuations in the intracel-lular Ca2+ can modulate cellular functions and may contribute to neuronal dysfunction. Hence, the present investigation was initiated to study the effects of some selected penicillium (naturally occurring tremorgenic) mycotoxins like secalonic acid, citreoviridin, and verruculogen on CaM activity, active conformation of CaM and PKC activity. Stimulation of CaM-deflcient bovine brain 3′-5′ phosphodieste rase (PDE) indicated CaM activity. The modification of CaM active conformation was studied by the binding of fluorescent probe N-phenyl-1-napthylamine (NPN) to CaM. Alterations in the fluorescence of dansyl-CaM was used to study the effect of these compounds on complex formation between CaM and PDE. Rat brain cytosolic PKC was studied using 32P-ATP as a measure of altered protein phosphorylation. The concentrations of mycotoxins used were in the range of 10 to 50 μM. All three mycotoxins inhibited CaM-stimulated PDE activity in a concentration-dependent manner. Citreoviridin and secalonic acid inhibited NPN fluorescence and Ca2+-dependent complex formation of dansyl-CaM and PDE. The IC50 values for NPN fluorescence of citreoviridin and secalonic acid were 13 μM and 19 μM respectively. However, verruculogen showed little effect on NPN fluorescence and the Ca2+-dependent complex formation of dansyl-CaM and PDE. These mycotoxins also inhibited PKC activity in a concentration-dependent manner with IC50 values of 19.8, 25.7, and 38.4 μM for secalonic acid, citreoviridin, and verruculogen, respectively. The results of our study suggest that these mycotoxins at very low concentrations are interacting with CaM and PKC. Such an effect could lead to impairment of neurotransmission and result in neurotoxicity.
Scenarios for the digital age: Convergence, personalization, exclusion
Authors: Forgacs David
Published: July 2002

The new media system that has emerged worldwide since the early 1990s is characterized by increasing use of digital technologies in every area and convergence between once distinct media. In Italy there have been a number of national variations on this global pattern: relative weakness of state regulation, a move away from the dominance of the system by ‘generalist’ television, a high rate of cellular phone use and a slower than average growth of Internet use. As far as consumption is concerned, the most common categories used to describe the emergent system—personalization of media, increased individual choice and mobility—do manage to capture some important aspects, in Italy as elsewhere, but they obscure others, notably the structural constraints limiting individual choice, the formation of new media microcommunities and, conversely, the exclusion of particular groups of citizens from full participation in the new system.
Italy in the digital age: Cinema as new technology
Authors: Brancato Sergio
Published: July 2002

Italian cinema is lagging behind in international standards, both culturally and in production terms. Starting in the mid-1970s, the Italian communication system has begun an irreversible transformation, and yet the ideologies of Italian cinema have remained impervious to the changes and they have consequently severed Italian cinema's links with the dynamic processes taking place in the other western film industries. The result is that today's Italian cinema fails to achieve either a decent production standard or essential synergies with other sectors of the cultural industry. A new political culture of communication is the only possible strategy for making Italian cinema once again a player in the global multimedia system.
Ethanol metabolism in the brain
Authors: ZIMATKIN SERGEY M. , DEITRICH RICHARD A.
Published: July 2002

Acetaldehyde is suspected of being involved in the central mechanism of central nervous system depression and addiction to ethanol, but in contrast to ethanol, it can not penetrate easily from blood into the brain because of metabolic barriers. Therefore, the possibility of ethanol metabolism and acetaldehyde formation inside the brain has been one of the crucial questions in biomedical research of alcoholism. This article reviews the recent progress in this area and summarizes the evidence on the first stage of ethanol oxidation in the brain and the specific enzyme systems involved. The brain alcohol dehydrogenase and microsomal ethanol oxidizing systems, including cytochrome P450 II E1 and catalase are considered. Their physicochemical properties, the isoform composition, substrate specificity, the regional and subcellular distribution in CNS structures, their contribution to brain ethanol metabolism, induction under ethanol administration and the role in the neurochemical mechanisms of psychopharmacological and neurotoxic effects of ethanol are discussed. In addition, the nonoxidative pathway of ethanol metabolism with the formation of fatty acid ethyl esters and phosphatidylethanol in the brain is described.
A Retrospective Clinicopathological Study of 37 Patients With Chordoma: A Danish National Series
Authors: Safwat Akmal , Nielsen Ole S. , Jurik Anne G. , Keller Johnny , Weeth Ernst R. , Lund Bjarne , Myhre-Jensen Olaf
Published: July 2002

Purpose. There are, in general, few published series on chordoma. It is a rare disease and further data are still needed.

Patients/methods. The data of 37 patients with chordoma were retrospectively analyzed. Treatment was surgical excision
in 11, radical radiotherapy in 9 and a combination of the two in 16 cases. The male to female ratio was 2.7 : 1. Median age was
59 years (range 1–89 years).

Results. The most common symptoms at diagnosis were pain (98%), neurological disturbances (42%) and incontinence
(33%). The tumours were located in the sacro‐coccygeal region in 68%, the spheno‐occipital region in 16% and the
vertebrae in 16% of the patients. Median tumour.size was 7 cm (range 1–30 cm). Local recurrence occurred in 21/36
treated cases and distant metastases developed in eight patients (23%). The median time to recurrence/progression after
primary treatment was 2 years (range 1–10 years). The actuarial 5‐year rates of overall, progression‐free and symptom‐free
survival were 40%, 31% and 20%, respectively. The corresponding 10‐year rates were 26%, 21% and 14%, respectively.
At the time of analysis, seven patients were alive, six without evidence of disease. Four of the six patients without active
disease were symptom free. A univariate analysis showed that age, sex, tumour size, histopathology, surgical safety margin,
treatment modality and radiation dose did not significantly affect overall, progression‐free or symptom‐free survival. Only
turnout site had a prognostic value with turnouts in the spheno‐occipital region carrying the worst prognosis.

Discussion. We conclude that effective treatment against chordomas is still lacking and a prospective multi‐institutional
registration study may provide more information on the optimal work‐up and treatment of this rare disease.
DRUG POLICIES, PRICES, AND WEALTH1
Authors: Braun Norman
Published: July 2002

To ensure both high prices and low prevalence rates of regular consumption of illicit substances, repressive elements dominate in the drug policies of European countries. The national drug policies differ considerably, however, to the extent in which repressive measures are taken against drug users as well. Starting from a theoretical reconstruction of the main ideas and problems of drug prohibition, this paper examines whether and how heroin and cocaine prices in European countries vary with successful supply-side drug law enforcement (i.e. seizures), national wealth levels, and the strictness of repressive orientations towards drug demand. It concludes with a brief description of experiences with an alternative policy towards heroin addicts (namely the medical prescription of narcotics in Switzerland).
NETWORKS, IDENTITY, AND (IN)ACTION
Authors: Alapuro Risto , Lonkila Markku
Published: July 2002

In analyses of the political quiescence in Russia today the emphasis is predominantly on the lack or deficit of something - above all, the weakness of civil society. In order to avoid this one-sided and arguably biased Western way to view the Russians' strategies of cooperation and challenge, a look is taken, in a comparative perspective, at people's daily practices. The data consist of the information on the personal networks of secondary school teachers in St Petersburg and Helsinki, including information on various forms of cooperation for common benefit. The majority of the Finns were engaged in the activities of formal associations; this was not true of the Russians. Typical organized activities among the St Petersburg teachers were linked to recurrent events or celebrations in the school context. Their organized action was based on the school as a community, whereas the Helsinki teachers based their organization rather on horizontal ties and group activity grounded on professional interests. A kind of community orientation in the Russian teachers' activities appears an active adaptation to the present difficult conditions in Russia. It implies that Russian civil society may be lastingly impregnated with elements of trust and solidarity not crucial to standard Western concepts of civil society.
A MARKET IN VOGUE: Fashion photography in Sweden
Authors: Aspers Patrik
Published: July 2002

The aim of this study is to understand and explain what I shall call aesthetic markets. A particular market of this kind is analysed, namely the market for fashion photography in Sweden. To understand and analyze a market from a sociological perspective, I argue, one primarily needs to perceive the construction of meaning within the market. The actors in this specific market construct what may be called ‘aesthetic values’. The market also distributes status of identities as a result of the interactions between producers and consumers. Photographers develop personal styles, and when a style intersects with status an identity is constructed in the market. The status distribution also structures the prices in the market. The conclusion of the article is that aesthetic markets can be conceptualized as status distributors of identities. The conclusion also suggests that this approach to aesthetic markets may be useful for studies of other markets as well.
Abnormal Compartmentalization of Cartilage Matrix Components in Mice Lacking Collagen X: Implications for Function
Authors: Kwan Kin Ming , Pang Michael K.M. , Zhou Sheila , Cowan Soot Keng , Kong Richard Y.C. , Pfordte Tim , Olsen Bjorn R. , Sillence David O. , Tam Patrick P.L. , Cheah Kathryn S.E.
Published: July 2002

There are conflicting views on whether collagen X is a purely structural molecule, or regulates bone mineralization during endochondral ossification. Mutations in the human collagen α1(X) gene (COL10A1) in Schmid metaphyseal chondrodysplasia (SMCD) suggest a supportive role. But mouse collagen α1(X) gene (Col10a1) null mutants were previously reported to show no obvious phenotypic change. We have generated collagen X deficient mice, which shows that deficiency does have phenotypic consequences which partly resemble SMCD, such as abnormal trabecular bone architecture. In particular, the mutant mice develop coxa vara, a phenotypic change common in human SMCD. Other consequences of the mutation are reduction in thickness of growth plate resting zone and articular cartilage, altered bone content, and atypical distribution of matrix components within growth plate cartilage. We propose that collagen X plays a role in the normal distribution of matrix vesicles and proteoglycans within the growth plate matrix. Collagen X deficiency impacts on the supporting properties of the growth plate and the mineralization process, resulting in abnormal trabecular bone. This hypothesis would accommodate the previously conflicting views of the function of collagen X and of the molecular pathogenesis of SMCD.
Dynamics of Nuclear Pore Distribution in Nucleoporin Mutant Yeast Cells
Authors: Belgareh Naïma , Doye Valérie
Published: July 2002

To follow the dynamics of nuclear pore distribution in living yeast cells, we have generated fusion proteins between the green fluorescent protein (GFP) and the yeast nucleoporins Nup49p and Nup133p. In nup133− dividing cells that display a constitutive nuclear pore clustering, in vivo analysis of GFP-Nup49p localization revealed changes in the distribution of nuclear pore complex (NPC) clusters. Furthermore, upon induction of Nup133p expression in a GAL-nup133 strain, a progressive fragmentation of the NPC aggregates was observed that in turn led to a wild-type nuclear pore distribution. To try to uncouple Nup133p- induced NPC redistribution from successive nuclear divisions and nuclear pore biogenesis, we devised an assay based on the formation of heterokaryons between nup133− mutants and cells either expressing or overexpressing Nup133p. Under these conditions, the use of GFP-Nup133p and GFP-Nup49p fusion proteins revealed that Nup133p can be rapidly targeted to the clustered nuclear pores, where its amino-terminal domain is required to promote the redistribution of preexisting NPCs.
The Chlamydomonas reinhardtii ODA3 Gene Encodes a Protein of the Outer Dynein Arm Docking Complex
Authors: Koutoulis Anthony , Pazour Gregory J. , Wilkerson Curtis G. , Inaba Kazuo , Sheng Hong , Takada Saeko , Witman George B.
Published: July 2002

We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the outer dynein arm. Among 39 insertional oda mutants characterized, two are alleles of the previously uncloned ODA3 gene, one is an allele of the uncloned ODA10 gene, and one represents a novel ODA gene (termed ODA12). ODA3 is of particular interest because it is essential for assembly of both the outer dynein arm and the outer dynein arm docking complex (ODA-DC) onto flagellar doublet microtubules (Takada, S., and R. Kamiya. 1994. J. Cell Biol. 126:737– 745). Beginning with the inserted DNA as a tag, the ODA3 gene and a full-length cDNA were cloned. The cloned gene rescues the phenotype of oda3 mutants. The cDNA sequence predicts a novel 83.4-kD protein with extensive coiled-coil domains. The ODA-DC contains three polypeptides; direct amino acid sequencing indicates that the largest of these polypeptides corresponds to ODA3. This protein is likely to have an important role in the precise positioning of the outer dynein arms on the flagellar axoneme.
Kinetochore Fiber Maturation in PtK1 Cells and Its Implications for the Mechanisms of Chromosome Congression and Anaphase Onset
Authors: McEwen Bruce F. , Heagle Amy B. , Cassels Grisel O. , Buttle Karolyn F. , Rieder Conly L.
Published: July 2002

Kinetochore microtubules (kMts) are a subset of spindle microtubules that bind directly to the kinetochore to form the kinetochore fiber (K-fiber). The K-fiber in turn interacts with the kinetochore to produce chromosome motion toward the attached spindle pole. We have examined K-fiber maturation in PtK1 cells using same-cell video light microscopy/serial section EM. During congression, the kinetochore moving away from its spindle pole (i.e., the trailing kinetochore) and its leading, poleward moving sister both have variable numbers of kMts, but the trailing kinetochore always has at least twice as many kMts as the leading kinetochore. A comparison of Mt numbers on sister kinetochores of congressing chromosomes with their direction of motion, as well as distance from their associated spindle poles, reveals that the direction of motion is not determined by kMt number or total kMt length. The same result was observed for oscillating metaphase chromosomes. These data demonstrate that the tendency of a kinetochore to move poleward is not positively correlated with the kMt number. At late prometaphase, the average number of Mts on fully congressed kinetochores is 19.7 ± 6.7 (n = 94), at late metaphase 24.3 ± 4.9 (n = 62), and at early anaphase 27.8 ± 6.3 (n = 65). Differences between these distributions are statistically significant. The increased kMt number during early anaphase, relative to late metaphase, reflects the increased kMt stability at anaphase onset. Treatment of late metaphase cells with 1 μM taxol inhibits anaphase onset, but produces the same kMt distribution as in early anaphase: 28.7 ± 7.4 (n = 54). Thus, a full complement of kMts is not sufficient to induce anaphase onset. We also measured the time course for kMt acquisition and determined an initial rate of 1.9 kMts/min. This rate accelerates up to 10-fold during the course of K-fiber maturation, suggesting an increased concentration of Mt plus ends in the vicinity of the kinetochore at late metaphase and/or cooperativity for kMt acquisition.
Capacitative Ca2+ Entry Is Closely Linked to the Filling State of Internal Ca2+ Stores: A Study Using Simultaneous Measurements of ICRAC and Intraluminal [Ca2+]
Authors: Hofer Aldebaran M. , Fasolato Cristina , Pozzan Tullio
Published: July 2002

ICRAC (the best characterized Ca2+ current activated by store depletion) was monitored concurrently for the first time with [Ca2+] changes in internal stores. To establish the quantitative and kinetic relationship between these two parameters, we have developed a novel means to clamp [Ca2+] within stores of intact cells at any level. The advantage of this approach, which is based on the membrane-permeant low-affinity Ca2+ chelator N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), is that [Ca2+] within the ER can be lowered and restored to its original level within 10–15 s without modifications of Ca2+ pumps or release channels. Using these new tools, we demonstrate here that Ca2+ release–activated Ca2+ current (ICRAC) is activated (a) solely by reduction of free [Ca2+] within the ER and (b) by any measurable decrease in [Ca2+]ER. We also demonstrate that the intrinsic kinetics of inactivation are relatively slow and possibly dependent on soluble factors that are lost during the whole-cell recording.
Occludin-deficient Embryonic Stem Cells Can Differentiate into Polarized Epithelial Cells Bearing Tight Junctions
Authors: Saitou Mitinori , Fujimoto Kazushi , Doi Yoshinori , Itoh Masahiko , Fujimoto Toyoshi , Furuse Mikio , Takano Hiroshi , Noda Tetsuo , Tsukita Shoichiro
Published: July 2002

Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.
Active MAP Kinase in Mitosis: Localization at Kinetochores and Association with the Motor Protein CENP-E
Authors: Zecevic Maja , Catling Andrew D. , Eblen Scott T. , Renzi Luigina , Hittle James C. , Yen Tim J. , Gorbsky Gary J. , Weber Michael J.
Published: July 2002

To investigate possible involvement of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 (extracellular signal-regulated kinases) in somatic cell mitosis, we have used indirect immunofluorescence with a highly specific phospho-MAP kinase antibody and found that a portion of the active MAP kinase is localized at kinetochores, asters, and the midbody during mitosis. Although the aster labeling was constant from the time of nuclear envelope breakdown, the kinetochore labeling first appeared at early prometaphase, started to fade during chromosome congression, and then disappeared at midanaphase. At telophase, active MAP kinase localized at the midbody. Based on colocalization and the presence of a MAP kinase consensus phosphorylation site, we identified the kinetochore motor protein CENP-E as a candidate mitotic substrate for MAP kinase. CENP-E was phosphorylated in vitro by MAP kinase on sites that are known to regulate its interactions with microtubules and was found to associate in vivo preferentially with the active MAP kinase during mitosis. Therefore, the presence of active MAP kinase at specific mitotic structures and its interaction with CENP-E suggest that MAP kinase could play a role in mitosis at least in part by altering the ability of CENP-E to mediate interactions between chromosomes and microtubules.
Cellular Interaction of Integrin α3β1 with Laminin 5 Promotes Gap Junctional Communication
Authors: Lampe Paul D. , Nguyen Beth P. , Gil Susana , Usui Marcia , Olerud John , Takada Yoshikazu , Carter William G.
Published: July 2002

Wounding of skin activates epidermal cell migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the individual motile cells into a synchronized colony. We found that outgrowths of human keratinocytes in wounds or epibole cultures display parallel changes in the expression of laminin 5, integrin α3β1, E-cadherin, and the gap junctional protein connexin 43. Adhesion of keratinocytes on laminin 5, collagen, and fibronectin was found to differentially regulate GJIC. When keratinocytes were adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays showed a two- to threefold increase compared with collagen and five- to eightfold over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin α3β1 with laminin 5 was sufficient to promote GJIC. Mapping of intermediate steps in the pathway linking α3β1–laminin 5 interactions to GJIC indicated that protein trafficking and Rho signaling were both required. We suggest that adhesion of epithelial cells to laminin 5 in the basement membrane via α3β1 promotes GJIC that integrates individual cells into synchronized epiboles.
Altered Cytochrome c Display Precedes Apoptotic Cell Death in Drosophila
Authors: Varkey Johnson , Chen Po , Jemmerson Ronald , Abrams John M.
Published: July 2002

Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity was necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive. Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.
Oxygen Stress: A Regulator of Apoptosis in Yeast
Authors: Madeo Frank , Fröhlich Eleonore , Ligr Martin , Grey Martin , Sigrist Stephan J. , Wolf Dieter H. , Fröhlich Kai-Uwe
Published: July 2002

Oxygen radicals are important components of metazoan apoptosis. We have found that apoptosis can be induced in the yeast Saccharomyces cerevisiae by depletion of glutathione or by low external doses of H2O2. Cycloheximide prevents apoptotic death revealing active participation of the cell. Yeast can also be triggered into apoptosis by a mutation in CDC48 or by expression of mammalian bax. In both cases, we show oxygen radicals to accumulate in the cell, whereas radical depletion or hypoxia prevents apoptosis. These results suggest that the generation of oxygen radicals is a key event in the ancestral apoptotic pathway and offer an explanation for the mechanism of bax-induced apoptosis in the absence of any established apoptotic gene in yeast.
Mitochondria Exert a Negative Feedback on the Propagation of Intracellular Ca2+ Waves in Rat Cortical Astrocytes
Authors: Boitier Eric , Rea Ruth , Duchen Michael R.
Published: July 2002

We have used digital fluorescence imaging techniques to explore the interplay between mitochondrial Ca2+ uptake and physiological Ca2+ signaling in rat cortical astrocytes. A rise in cytosolic Ca2+ ([Ca2+]cyt), resulting from mobilization of ER Ca2+ stores was followed by a rise in mitochondrial Ca2+ ([Ca2+]m, monitored using rhod-2). Whereas [Ca2+]cyt recovered within ∼1 min, the time to recovery for [Ca2+]m was ∼30 min. Dissipating the mitochondrial membrane potential (Δψm, using the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone [FCCP] with oligomycin) prevented mitochondrial Ca2+ uptake and slowed the rate of decay of [Ca2+]cyt transients, suggesting that mitochondrial Ca2+ uptake plays a significant role in the clearance of physiological [Ca2+]cyt loads in astrocytes. Ca2+ signals in these cells initiated either by receptor-mediated ER Ca2+ release or mechanical stimulation often consisted of propagating waves (measured using fluo-3). In response to either stimulus, the wave traveled at a mean speed of 22.9 ± 11.2 μm/s (n = 262). This was followed by a wave of mitochondrial depolarization (measured using tetramethylrhodamine ethyl ester [TMRE]), consistent with Ca2+ uptake into mitochondria as the Ca2+ wave traveled across the cell. Collapse of Δψm to prevent mitochondrial Ca2+ uptake significantly increased the rate of propagation of the Ca2+ waves by 50%. Taken together, these data suggest that cytosolic Ca2+ buffering by mitochondria provides a potent mechanism to regulate the localized spread of astrocytic Ca2+ signals.
Regulation of APC Activity by Phosphorylation and Regulatory Factors
Authors: Kotani Shuji , Tanaka Hirofumi , Yasuda Hideyo , Todokoro Kazuo
Published: July 2002

Ubiquitin-dependent proteolysis of Cut2/Pds1 and Cyclin B is required for sister chromatid separation and exit from mitosis, respectively. Anaphase-promoting complex/cyclosome (APC) specifically ubiquitinates Cut2/Pds1 at metaphase–anaphase transition, and ubiquitinates Cyclin B in late mitosis and G1 phase. However, the exact regulatory mechanism of substrate-specific activation of mammalian APC with the right timing remains to be elucidated. We found that not only the binding of the activators Cdc20 and Cdh1 and the inhibitor Mad2 to APC, but also the phosphorylation of Cdc20 and Cdh1 by Cdc2-Cyclin B and that of APC by Polo-like kinase and cAMP-dependent protein kinase, regulate APC activity. The cooperation of the phosphorylation/dephosphorylation and the regulatory factors in regulation of APC activity may thus control the precise progression of mitosis.
Differential Regulation of P27Kip1 Expression by Mitogenic and Hypertrophic Factors
Authors: Servant Marc J. , Coulombe Philippe , Turgeon Benjamin , Meloche Sylvain
Published: July 2002

Platelet-derived growth factor-BB (PDGF-BB) acts as a full mitogen for cultured aortic smooth muscle cells (SMC), promoting DNA synthesis and cell proliferation. In contrast, angiotensin II (Ang II) induces cellular hypertrophy as a result of increased protein synthesis, but is unable to drive cells into S phase. In an effort to understand the molecular basis for this differential growth response, we have examined the downstream effects of PDGF-BB and Ang II on regulators of the cell cycle machinery in rat aortic SMC. Both PDGF-BB and Ang II were found to stimulate the accumulation of G1 cyclins with similar kinetics. In addition, little difference was observed in the expression level of their catalytic partners, Cdk4 and Cdk2. However, while both factors increased the enzymatic activity of Cdk4, only PDGF-BB stimulated Cdk2 activity in late G1 phase. The lack of activation of Cdk2 in Ang II-treated cells was causally related to the failure of Ang II to stimulate phosphorylation of the enzyme on threonine and to downregulate p27Kip1 expression. By contrast, exposure to PDGF-BB resulted in a progressive and dramatic reduction in the level of p27Kip1 protein. The time course of p27Kip1 decline was correlated with a reduced rate of synthesis and an increased rate of degradation of the protein. Importantly, the repression of p27Kip1 synthesis by PDGF-BB was associated with a marked attenuation of Kip1 gene transcription and a corresponding decrease in Kip1 mRNA accumulation. We also show that the failure of Ang II to promote S phase entry is not related to the autocrine production of transforming growth factor-β1 by aortic SMC. These results identify p27Kip1 as an important regulator of the phenotypic response of vascular SMC to mitogenic and hypertrophic stimuli.
Dual Role of Caspase-11 in Mediating Activation of Caspase-1 and Caspase-3 under Pathological Conditions
Authors: Kang Shin-Jung , Wang Suyue , Hara Hideaki , Peterson Erin P. , Namura Shobu , Amin-Hanjani Sepideh , Huang Zhihong , Srinivasan Anu , Tomaselli Kevin J. , Thornberry Nancy A. , Moskowitz Michael A. , Yuan Junying
Published: July 2002

Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11–deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.
Tissue Plasminogen Activator–Mediated Fibrinolysis Protects against Axonal Degeneration and Demyelination after Sciatic Nerve Injury
Authors: Akassoglou Katerina , Kombrinck Keith W. , Degen Jay L. , Strickland Sidney
Published: July 2002

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin and can trigger the degradation of extracellular matrix proteins. In the nervous system, under noninflammatory conditions, tPA contributes to excitotoxic neuronal death, probably through degradation of laminin. To evaluate the contribution of extracellular proteolysis in inflammatory neuronal degeneration, we performed sciatic nerve injury in mice. Proteolytic activity was increased in the nerve after injury, and this activity was primarily because of Schwann cell–produced tPA. To identify whether tPA release after nerve damage played a beneficial or deleterious role, we crushed the sciatic nerve of mice deficient for tPA. Axonal demyelination was exacerbated in the absence of tPA or plasminogen, indicating that tPA has a protective role in nerve injury, and that this protective effect is due to its proteolytic action on plasminogen. Axonal damage was correlated with increased fibrin(ogen) deposition, suggesting that this protein might play a role in neuronal injury. Consistent with this idea, the increased axonal degeneration phenotype in tPA- or plasminogen-deficient mice was ameliorated by genetic or pharmacological depletion of fibrinogen, identifying fibrin as the plasmin substrate in the nervous system under inflammatory axonal damage. This study shows that fibrin deposition exacerbates axonal injury, and that induction of an extracellular proteolytic cascade is a beneficial response of the tissue to remove fibrin. tPA/plasmin-mediated fibrinolysis may be a widespread protective mechanism in neuroinflammatory pathologies.
Conserved Sr Protein Kinase Functions in Nuclear Import and Its Action Is Counteracted by Arginine Methylation inSaccharomyces cerevisiae
Authors: Yun Chi Y. , Fu Xiang-Dong
Published: July 2002

Mammalian serine and arginine–rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein–specific kinases (SRPKs) are conserved from humans to yeast. Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast. The yeast SR-like protein Npl3p is known to enter the nucleus through a composite nuclear localization signal (NLS) consisting of a repetitive arginine- glycine-glycine (RGG) motif and a nonrepetitive sequence. We found that the latter is the site for phosphorylation by Sky1p and that this phosphorylation regulates nuclear import of Npl3p by modulating the interaction of the RGG motif with its nuclear import receptor Mtr10p. The RGG motif is also methylated on arginine residues, but methylation does not affect the Npl3p–Mtr10p interaction in vitro. Remarkably, arginine methylation interferes with Sky1p-mediated phosphorylation, thereby indirectly influencing the Npl3p–Mtr10p interaction in vivo and negatively regulating nuclear import of Npl3p. These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells.
Absence of α-Syntrophin Leads to Structurally Aberrant Neuromuscular Synapses Deficient in Utrophin
Authors: Adams Marvin E. , Kramarcy Neal , Krall Stuart P. , Rossi Susana G. , Rotundo Richard L. , Sealock Robert , Froehner Stanley C.
Published: July 2002

The syntrophins are a family of structurally related proteins that contain multiple protein interaction motifs. Syntrophins associate directly with dystrophin, the product of the Duchenne muscular dystrophy locus, and its homologues. We have generated α-syntrophin null mice by targeted gene disruption to test the function of this association. The α-Syn−/− mice show no evidence of myopathy, despite reduced levels of α-dystrobrevin–2. Neuronal nitric oxide synthase, a component of the dystrophin protein complex, is absent from the sarcolemma of the α-Syn−/− mice, even where other syntrophin isoforms are present. α-Syn−/− neuromuscular junctions have undetectable levels of postsynaptic utrophin and reduced levels of acetylcholine receptor and acetylcholinesterase. The mutant junctions have shallow nerve gutters, abnormal distributions of acetylcholine receptors, and postjunctional folds that are generally less organized and have fewer openings to the synaptic cleft than controls. Thus, α-syntrophin has an important role in synapse formation and in the organization of utrophin, acetylcholine receptor, and acetylcholinesterase at the neuromuscular synapse.
Characterization and Reconstitution of Drosophila γ-Tubulin Ring Complex Subunits
Authors: Gunawardane Ruwanthi N. , Martin Ona C. , Cao Kan , Zhang Lijun , Dej Kimberly , Iwamatsu Akihiro , Zheng Yixian
Published: July 2002

The γ-tubulin ring complex (γTuRC) is important for microtubule nucleation from the centrosome. In addition to γ-tubulin, the Drosophila γTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small γ-tubulin complex (γTuSC) and the γTuRC, while the remaining subunits are found only in the γTuRC. To study γTuRC assembly and function, we first reconstituted γTuSC using the baculovirus expression system. Using the reconstituted γTuSC, we showed for the first time that this subcomplex of the γTuRC has microtubule binding and capping activities. Next, we characterized two new γTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known γTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with γTuSC. However, this interaction is insufficient for γTuRC assembly.
Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP
Authors: TenBroek Erica M. , Lampe Paul D. , Solan Joell L. , Reynhout James K. , Johnson Ross G.
Published: July 2002

The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.
Myelin-associated glycoprotein and myelin galactolipids stabilize developing axo-glial interactions
Authors: Marcus Jill , Dupree Jeffrey L. , Popko Brian
Published: July 2002

We have analyzed mice that lack both the myelin-associated glycoprotein (MAG) and the myelin galactolipids, two glial components implicated in mediating axo-glial interactions during the myelination process. The single-mutant mice produce abnormal myelin containing similar ultrastructural abnormalities, suggesting that these molecules may play an overlapping role in myelin formation. Furthermore, the absence of the galactolipids results in a disruption in paranodal axo-glial interactions, and we show here that similar, albeit less severe, abnormalities exist in the developing MAG mutant. In the double-mutant mice, maintenance of axo-glial adhesion is significantly more affected than in the single mutants, supporting the overlapping function hypothesis. We also show that independently of MAG, galactolipids, and paranodal junctional components, immature nodes of Ranvier form normally, but rapidly destabilize in their absence. These data indicate that distinct molecular mechanisms are responsible for the formation and maintenance of axo-glial interactions.
The Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells
Authors: Glomski Ian J. , Gedde Margaret M. , Tsang Albert W. , Swanson Joel A. , Portnoy Daniel A.
Published: July 2002

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is ∼10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.
Thymus-derived Glucocorticoids Regulate Antigen-specific Positive Selection
Authors: Vacchio Melanie S. , Ashwell Jonathan D.
Published: July 2002

While it is generally believed that the avidity of the T cell antigen receptor (TCR) for self antigen/major histocompatibility complex (MHC) determines a thymocyte's fate, how the cell discriminates between a stimulus that causes positive selection (survival) and one that causes negative selection (death) is unknown. We have previously demonstrated that glucocorticoids are produced in the thymus, and that they antagonize deletion caused by TCR cross-linking. To examine the role of glucocorticoids during MHC-dependent selection, we examined thymocyte development in organ cultures in which corticosteroid biosynthesis was inhibited. Inhibition of glucocorticoid production in thymi from α/β-TCR transgenic mice resulted in the antigen- and MHC-specific loss of thymocytes that normally recognize self antigen/MHC with sufficient avidity to result in positive selection. Furthermore, inhibition of glucocorticoid production caused an increase in apoptosis only in CD+CD8+ thymocytes bearing transgenic TCRs that recognized self antigen/MHC. These results indicate that the balance of TCR and glucocorticoid receptor signaling influences the antigen-specific thymocyte development by allowing cells with low-to-moderate avidity for self antigen/MHC to survive.