bluuebunny/crossref_metadata_2025 huggingface dataset:
Lysosomal-Storage Disorder Induced by Elmiron Following 90-Days Gavage Administration in Rats and Mice
Authors: Nyska Abraham , Nold James B. , Johnson Jerry D. , Abdo Kamal
Published: July 2002

Elmiron, a highly sulfated, semisynthetic pentose polysaccharide with properties similar to heparin, is used for the treatment of interstitial cystitis. Thirteen-week gavage studies were conducted by administering the drug in deionized water to F344/N rats and B6C3F1 mice once daily, 5 days per week for up to 13 consecutive weeks, at doses of 0, 63, 125, 250, 500, and 1,000 mg/kg body weight. No significant drug-related effects were observed in body weight, survival, clinical, and necropsy results. Significant organ weight increases were seen in the liver, lungs, and spleen of both species and the kidneys of rats, mainly in groups treated with 250 mg/kg/day and above. Hematological analysis indicated increases for both species in the white blood cell and lymphocyte counts. Sites of toxicity identified histopathologically were the rectum, liver, mesenteric and mandibular lymph nodes (both sexes), spleen (mice only), and lungs and kidneys (rats only). Lesions consisted mainly of infiltration into multiple tissues of vacuolated histiocytes, which, by histochemical investigation, indicated the presence of neutral and acidic mucins and lipidic material within the vacuoles. Transmission electron microscopy identified these vacuoles as lysosomal structures that exhibited a variety of contents. On the basis of our findings, we propose that Elmiron was absorbed through the focally disrupted rectal mucosa, was deposited in the lamina propria, accumulated within macrophages, and then was distributed by these cells or as a free chemical via the lymphatics and blood, to the various organ sites manifesting histiocytic infi ltration. The cytoplasmic membrane-bound structures within macrophages were lysosomes containing membranous material of cellular origin and, perhaps, remnants of the deposited test material, Elmiron.
Spontaneous Lesions in Control B6C3F1 Mice and Recommended Sectioning of Male Accessory Sex Organs
Authors: Suwa Takahiko , Nyska Abraham , Haseman Joseph K. , Mahler Joel F. , Maronpot Robert R.
Published: July 2002

Because sampling of the paired lobes (ventral, dorsal, lateral, and anterior) of the mouse prostate has often been inconsistent, comparisons among different investigations have lacked validity. The absence of site identifi cation for prostatic lesions has made reported incidences relatively nonspecific. We present here the lobe-specifi cincidences and degree of severity of spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands in 612 control B6C3F1 mice from 12 recent National Toxicology Program 2-year carcinogenicity and toxicity studies conducted in 1 of 4 different laboratories. Lymphocytic infi ltration, infl ammation, epithelial hyperplasia, mucinous cyst, and mucinous metaplasia were observed in the dorsolateral lobes. Lymphocytic infi ltration, infl ammation, epithelial hyperplasia, and edema were present in the ventral lobes. Lymphocytic infi ltration, acinar dilatation, infl ammation, epithelial hyperplasia, and atrophy occurred in the coagulating glands. No neoplastic lesions were observed in the prostate or coagulating gland. Lymphocytic infi ltration, acinar dilatation, inflammation, atrophy, adenoma, adenocarcinoma, and a granular cell tumor were observed in the seminal vesicles. Lymphocytic infi ltration was also present in the ampullary glands. The results of our survey indicate that the amounts of glandular tissues were not present consistently in slides from the different laboratories. Landmarks for uniform tissue trimming are needed. We therefore suggest an optimal trimming and embedding method for mouse prostate and seminal vesicles to ensure adequate, consistent sampling.
Biochemical and Morphological Events During Okadaic Acid-Induced Apoptosis of Tsc2-Null ERC-18 Cell Line
Authors: Kolb Todd M. , Chang Seung H. , Davis Myrtle A.
Published: July 2002

Several tumor suppressor genes have been shown to regulate cellular susceptibility to proliferation or apoptotic cell death. An essential first step in studies with the long-range goal of determining the effect of a tumor suppressor gene on cellular susceptibility to apoptosis is careful characterization of the cell's response to an apoptotic stimulus. The goals of this study were to characterize the apoptotic response of a tuberous sclerosis complex-2 (Tsc2) tumor suppressor gene-null cell line, to establish valid biochemical events that can be used as apoptosis markers, and to determine how these events correlate with apoptosis-specific morphologic changes. For characterization of apoptosis, we treated Tsc2-null renal epithelial tumor cells (ERC-18) with okadaic acid (OKA, 0.1—0.25 μM), and measured the biochemical and morphologic events during the apoptotic response. Electron microscopic and immunocytochemica l evaluation showed an early loss of microvilli and a loss of vinculin and talin staining from focal adhesions within 1 hour. During the first 2 hours of treatment with 0.25 μM OKA, ERC-18 cells rounded and ∼50% detached from the culture vessel with minimal membrane bleb formation. Phosphatidylserine externalization, chromatin margination and fragmentation, cytochrome C release, and caspase-3 and -7 cleavage were evident at 6 hours. Maximal membrane bleb formation occurred between 6 and 10 hours. Cells progressed to secondary oncotic necrosis between 10 and 24 hours of OKA treatment. Almost all cells had an oncotic phenotype after 24 hours, and 17.5% lost cell membrane integrity. A small subpopulation (< 5%) of OKA-treated cells underwent primary oncotic necrosis within 6 hours. Interestingly, the caspase-3 and -7 inhibitor Z-DEVD-FMK did not inhibit or delay OKA-induced apoptosis in these cells. Our results suggest a complex apoptotic model involving 2 or more potentially parallel death pathways. Although caspase-3 and -7 cleavage occurs during apoptosis in this model, this cleavage may not independently regulate cell death in ERC-18 cells. Therefore, measurement of apoptosi s in this model requires analysis of both biochemical and morphologi c events.
Association of Adrenal Pheochromocytoma and Lung Pathology in Inhalation Studies with Particulate Compounds in the Male F344 Rat—The National Toxicology Program Experience
Authors: Ozaki Keisuke , Haseman Joseph K. , Hailey James R. , Maronpot Robert R. , Nyska Abraham
Published: July 2002

Systemic hypoxemia, occurring in space-occupying lung pathologies such as inflammation and neoplasms, reduces the gas exchange area and stimulates catecholamine secretion from the adrenal medulla where chronic endocrine hyperactivity may lead to hyperplasia and neoplasia. We investigated the possible correlation between nonneoplastic chronic pulmonary lesions and adrenal pheochromocytom a in 9 recent, NTP, 2-year particulate inhalation studies in male F344 rats. Re-evaluation for chronic active inflammation, interstitial fibrosis, alveolar epithelial hyperplasia, squamous metaplasia, proteinosis, and histiocytosis revealed significant associations of pheochromocytom a only with the severity of inflammation and fibrosis. Nickel oxide, cobalt sulfate, indium phosphide, talc, and nickel subsulfide studies showed chemical-related incidences of adrenal pheochromocytom a and significant ( p < 0.01) associations with inflammation and fibrosis. Gallium arsenide, vanadium pentoxide, molybdenum trioxide, and nickel sulfate hexahydrate studies revealed an increased incidence and/or severity of nonneoplastic lung lesions, but no increased incidence of pheochromocytoma . Although gallium arsenide and molybdenum trioxide showed no dose-related increase in pheochromocytoma, a significant ( p < 0.01) correlation of the latter with the severity of fibrosis and inflammation occurred. In the vanadium pentoxide and nickel sulfate hexahydrate studies, no relationship between nonneoplastic lung lesions and pheochromocytoma was manifested. Our investigation assessed the strength of these various associations and supports the possible roles of 2 chronic pulmonary lesions—fibrosis and inflammation—and hypoxemi a in the induction of pheochromocytom a in the F344 male rat.
The Crick Report: difference, equality and racial justice
Authors: Osler Audrey
Published: July 2002

This article examines current policy on citizenship education in England, drawing on the recommendations of the 1998 Crick Report, Education for Citizenship and the Teaching of Democracy in Schools. In particular, it seeks to establish whether the proposals for citizenship education outlined in the report and draft frameworks for citizenship education, published by the Qualifications and Curriculum Authority as part of the National Curriculum review for England, have the potential to contribute towards racial equality. The report sets out to provide a framework for citizenship education which is intended to strengthen our democracy. The publication of the Macpherson Report of the Stephen Lawrence inquiry in 1999 led senior politicians to acknowledge institutional racism in British society and to pledge themselves to a programme to eradicate racism. Racism is therefore now officially recognized by those in government as one of the forces which operates to restrict the citizenship rights of minorities and undermines the principles of democracy. An understanding of racism, the ways in which it serves to undermine democracy, and skills to challenge this anti‐democratic force are therefore essential features of any education programme which seeks to promote the political literacy of citizens. This article argues that a key aim in any programme of citizenship education must be to enable young people to understand the barriers to citizenship and to equip them with the skills to challenge and overcome such barriers. It examines the images and understandings of multiculturalism in the Crick Report and considers how it deals with questions of difference, equality and justice. It further examines whether the proposals within the report are an adequate basis from which sound anti‐racist education programmes might be developed or whether the report itself may unwittingly reflect racism. It concludes by suggesting how the citizenship education project might be modified so that it promotes a vision of a multicultural society founded on principles of human rights and of schools where children are able to realize their rights on the basis of equality.
Neopterin inhibits ATP‐induced calcium release in alveolar epithelial cells in vitro
Authors: Hoffmann Georg , Gollnick Frank , Meyer Rainer
Published: July 2002

Background: Serum neopterin concentrations rise during activation of the cellular immune system. It is suggested that neopterin interacts with cellular redox mechanisms. This induces oxidative stress, which inhibits intracellular Ca2+ transients in various cell types. In type II alveolar epithelial cells, Ca2+ increase is considered involved in the exocytosis of surfactants. This exocytosis is disturbed during inflammation.

Aims: To clarify whether neopterin affects adenosine triphosphate (ATP)‐induced Ca2+ transients in an alveolar epithelial cell line (L2).

Methods: Ca2+ transients were detected as fura‐2 fluorescence by an image analysis system.

Results: Cells were exposed for 100 sec to ATP (1 μM, repeated four times). The first application of ATP induced an increase of the fluorescence ratio by approximately 100%, while the following stimulations resulted in smaller transients. In a second set of experiments, L2 cells were exposed to ATP or ATP + neopterin (100 nM), alternately. Simultaneous application of neopterin inhibited Ca2+ transients almost completely.

Conclusions: Inhibition of Ca2+ transients by neopterin may lead to suppressed exocytosis of surfactant proteins in alveolar epithelial cells. This might contribute to the deterioration of pulmonary functions in the course of inflammatory processes.
Inflammatory response in induced sputum mononuclear cells from patients with acute exacerbation of asthma
Authors: Hamzaoui A. , Ben Brahim M. , Zhioua A. , Ayed Kh. , Hamzaoui K.
Published: July 2002

Examination of sputum provides a direct method to investigate airway inflam mation non‐invasively in particular Th1 (IL‐2, IFN–γ) and Th2 (IL–4, IL–10) cytokine production.

IL–2, IL–4, IL–10 and IFN–γ cytokine were studied in induced sputum mononuclear cells of asthmatic patients.

Sputum induction was performed on 10 patients and 10 normal controls. Basal and mitogen‐stimulated cytokine production was determined in induced sputum T‐cell culture. Supernatants were collected and assayed not only with specific ELISA but also with polymerase chain reaction (PCR) techniques.

Data showed a significantly higher production of IL–10 by both the ELISA and the RT‐PCR techniques in asthmatic patients compared with sputum mononuclear cells from healthy controls. IL–4 production was detected at a low level using the ELISA method in asthmatic patients. The RT‐PCR analysis detected a significantly IL–4‐mRNA expression in all asthmatic patients, compared with controls. Results of IL–10 and IL–4 mRNA expression were reproducible. We did not find any alteration in the expression of the type 1 derived cytokines (IL–2 and IFN–γ) in asthmatic patients or in healthy controls.

Our study showed a tendency of induced sputum mononuclear cells to express a Th2‐like cytokine pattern in acute exacerbation of asthmatic patients, where IL–10 and IL–4 are synthesized in larger amounts. The combination of sputum induction as a non‐invasive tool to explore the lung and the identification of disease‐associated cytokine expression and of specific cytokine mRNA should help elucidate mechanisms of the immunologically mediated inflammatory
responses in asthma.
Involvement of different protein kinases and phospholipases A2 in phorbol ester (TPA)‐induced arachidonic acid liberation in bovine platelets
Authors: Lehr M. , Griessbach K.
Published: July 2002

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12‐O‐tetradecanoylphorbol–13‐acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+‐independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA‐induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31–82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.
Expression of Bcl‐2 in Inflammatory Sites from Patients with Active Behçet′s Disease
Authors: Hamzaoui K. , Hamzaoui A. , Zakraoui L. , Chabbou A.
Published: July 2002

Behçet′s disease (BD) is a current systemic vasculitis of unknown aetiology. Eyes, skin, joints, the oral cavity, genital system, blood vessels, central nervous system and lung are usually involved. Defective regulation of programmed cell death (apoptosis) may play a role in the development of (BD), and the protooncogene Bcl‐2 is involved in the control of apoptosis in immunocompetent cells. We therefore wished to investigate the expression of Bcl‐2 in the peripheral lymphocytes and in two inflammatory sites of patients with active BD: bronchoalveolar lavage (BAL) and cerebrospinal fluid (CSF) lymphocytes. Levels of Bcl‐2 expression in the lymphocytes of patients with BD and, for comparison, in the lymphocytes of healthy controls and non‐inflammatory neurological diseases (NIND), were studied by two‐colour cytofluorography and RNA analysis. In BD patients, a significant proportion of T cells expressed increased amounts of Bcl‐2 protein, both in peripheral blood and in inflammatory sites. Mononuclear cells of patients with BD showed increased amount of Bcl‐2 messenger RNA. The in vitro incubation of T lymphocytes with IL‐10, significantly increased the Bcl‐2 expression, specifically in T lymphocytes from inflammatory sites. In active BD, stimulation of HSV‐1 T lymphocytes slightly increased Bcl‐2 expression, not significantly different from unstimulated HSV‐1 T cells. The occurrence of circulating T lymphocytes with abnormally high Bcl‐2 expression in peripheral circulation and in inflammatory sites may be explained in part by the increased in vivo activation levels, and by aetiopathological agent(s): our findings seem to indicate an important role in the chronic inflammation in BD.
Preadolescent Children of Substance‐Dependent Fathers with Antisocial Personality Disorder: Psychiatric Disorders and Problem Behaviors
Authors: Moss Howard B. , Baron David A. , Hardie Thomas L. , Vanyukov Michael M.
Published: July 2002

We compared psychiatric disorders and problem behavior scores in pre‐adolescent children of fathers with alcohol or other drug dependence and ASP (SD+/ASP+), children whose fathers had substance dependence without ASP (SD+/ASP‐), and children whose fathers were without either disorder (SD—/ASP‐). SD+/ASP+ children showed elevated rates of major depression, conduct disorder, attention deficit hyperactivity disorder, oppositional defiant disorder, and separation anxiety disorder when compared to SD+/ASP—and SD—/ASP—children. SD+/ASP+ children had higher internalizing and externalizing problem behavior scores than the other two groups of children. The results suggest that SD+/ASP+ children are at significant risk for internalizing and externalizing psychopathology.
Ethanol induces taurine release in the amygdala: an in vivo microdialysis study
Authors: QUERTEMONT ETIENNE , DAHCHOUR ABDELKADER , WARD ROBERTA J. , DE WITTE PHILIPPE
Published: July 2002

The effect of acute IP ethanol injections on the extracellular aspartate, glutamate, taurine and GABA content of the basolateral amygdala microdialysate was investigated in relationship with total brain ethanol. Each acute intraperitoneal injection of ethanol, 0.5, 1.0, 2.0 and 3.0 g/kg body weight, induced an immediate increase in microdialysate taurine; both 0.5 and 1.0 g/kg ethanol evoked an increase during the first 20 minutes following injection which returned to baseline value by 40 minutes, despite the fact that ethanol was detectable in the brain until 60 or 120 minutes, respectively. After either 2.0 or 3.0 g/kg ethanol there was an increase in taurine of gradual intensity which gradually declined to reach baseline values by 100 minutes. In contrast, the ethanol concentration for 2.0 g/kg remained elevated at the end of the 120 minutes; approximately 25 mg ethanol/mg protein. The stimulated release of taurine within the amygdala could participate in the regulation of ethanolinduced changes in osmolarity, since taurine is postulated to act as an osmoregulator in the brain. Taurine could also mediate or interact with ethanol‐induced central nervous system effects, as it exerts a modulatory action on cell excitability and neurotransmitter processes.
Outcome and Toxicity of an Ifosfamide‐Based Soft Tissue Sarcoma Treatment Protocol in Children. The Importance of Local Therapy
Authors: Yule S. Murray , Skinner Roderick , English Martin W. , Cole Mike , Pearson Andrew D. J. , Lucraft Helen H. , Craft Alan W.
Published: July 2002

Background. Although the survival of children with soft tissue sarcoma (STS) has improved considerably, the outcome of patients with metastatic disease, and those with primary tumours of the extremities or parameningeal sites remains disappointing. We describe the clinical outcome of an ifosfamide‐based regimen with local therapy directed only to children who failed to achieve a complete response to initial chemotherapy.

Patients and Methods. Twenty‐one children with STS (16 rhabdomyosarcoma) who presented with unresectable tumours were treated with five courses of ifosfamide (9 g/m2) and etoposide (600 mg/m2). Patients who did not achieve a complete response then received local therapy. Chemotherapy with ifosfamide combined with etoposide, vincristine (1.5 mg/m2 and doxorubicin (60 mg/m2) or vincristine and actinomycin D (1.5 mg/m2) was continued for one year.

Results and Discussion. Objective responses to five courses of ifosfamide and etoposide were seen in all patients. Disease free survival (DFS) at a median follow up of 59 months was 57% (95% CI 29–75%). The DFS of children who received local therapy was 89% compared with 33% in those who received chemotherapy alone (p=0.027). Locoregional recurrences did not occur in children who received radiotherapy to the site of the primary tumour. Ifosfamide‐based chemotherapy does not reduce the incidence of loco‐regional recurrence in children who do not receive local therapy.
Gonadotrophin‐Releasing Hormone (GnRH) Analogues in the Treatment of Mixed Mullerian Tumours of the Uterus: Two Case Reports and Review
Authors: Katesmark Michael , Lawton Frank
Published: July 2002

Subjects/Discussion. Two cases of clinical and radiological response of recurrent mixed Mullerian tumours following treatment with either nasal (Buserilin) or intramuscular (Goserilin) GnRH analogues are reported and a short review of the evidence to support this treatment option presented.
The Role of Insulin‐Like Growth Factor System in Soft Tissue Sarcomas: From Physiopathology to Targeted Therapeutic Approaches
Authors: Zumkeller Walter
Published: July 2002

Purpose/Results. Although surgical, chemo‐ and radiotherapeutic treatment regimens in patients with soft tissue sarcomas have constantly been refined over the past two decades, the survival rate for these patients is rather low.

Discussion. There is a great need to investigate the mechanisms for oncogenesis and to identify the factors involved in malignant transformation in sarcomas. Among these factors, IGFs are thought to play a pivotal role as progression factors in various types of sarcomas. The dysregulation of the IGF‐II synthesis, e.g. by loss of imprinting which occurs in most
types of sarcomas, is a permissive effect through the suppression of cell death. In addition, cells that overexpress the type I IGF receptors are more susceptible to transformation by oncogenes. As TP53 suppresses the activity of IGF‐II P3 and P4, as well as the type I IGF receptor promoter, mutations of TP53 in sarcomas may alternatively lead to the activation of these factors. Finally, the phenomenon of non‐islet cell tumour hypoglycaemia that occurs in patients with sarcomas, and which is related to the secretion of IGF‐II prohormones, is discussed. Future therapeutic strategies may be based upon the application of antibodies or antisense oligonucleotides directed against the type I IGF receptors, with the common goal of inducing apoptosis in sarcoma cells. Ultimately, these and other therapeutic approaches may lead to a better outcome in patients suffering from sarcoma.
Longitudinal Growth Following Treatment for Osteosarcoma
Authors: Cool W. Paul , Grimer Robert J. , Carter Simon R. , Tillman Roger M. , Davies A. Mark
Published: July 2002

Purpose. The purpose of this study was to analyse the height at diagnosis and growth in 72 skeletally immature children who had been treated for osteosarcoma in the area of the knee.

Subjects. Of the patients, the average age at diagnosis was 10 years in girls and 12 years in boys. All children received neo‐adjuvant chemotherapy, and had limb salvage by endoprosthetic replacement.

Results and conclusion. The results of this study indicate that there is no evidence that children with osteosarcoma are taller at diagnosis than their normal counterparts. However, there was a marked retardation in growth in the year following the administration of cytotoxic chemotherapy. There were 19 children who reached skeletal maturity. The final height in
these children was not significantly different from the normal population.
Transferrin Promotes Endothelial Cell Migration and Invasion: Implication in Cartilage Neovascularization
Authors: Carlevaro Mariella F. , Albini Adriana , Ribatti Domenico , Gentili Chiara , Benelli Roberto , Cermelli Silvia , Cancedda Ranieri , Cancedda Fiorella Descalzi
Published: July 2002

During endochondral bone formation, avascular cartilage differentiates to hypertrophic cartilage that then undergoes erosion and vascularization leading to bone deposition. Resting cartilage produces inhibitors of angiogenesis, shifting to production of angiogenic stimulators in hypertrophic cartilage. A major protein synthesized by hypertrophic cartilage both in vivo and in vitro is transferrin. Here we show that transferrin is a major angiogenic molecule released by hypertrophic cartilage. Endothelial cell migration and invasion is stimulated by transferrins from a number of different sources, including hypertrophic cartilage. Checkerboard analysis demonstrates that transferrin is a chemotactic and chemokinetic molecule. Chondrocyte-conditioned media show similar properties. Polyclonal anti-transferrin antibodies completely block endothelial cell migration and invasion induced by purified transferrin and inhibit the activity produced by hypertrophic chondrocytes by 50–70% as compared with controls. Function-blocking mAbs directed against the transferrin receptor similarly reduce the endothelial migratory response. Chondrocytes differentiating in the presence of serum produce transferrin, whereas those that differentiate in the absence of serum do not. Conditioned media from differentiated chondrocytes not producing transferrin have only 30% of the endothelial cell migratory activity of parallel cultures that synthesize transferrin.

The angiogenic activity of transferrins was confirmed by in vivo assays on chicken egg chorioallantoic membrane, showing promotion of neovascularization by transferrins purified from different sources including conditioned culture medium.

Based on the above results, we suggest that transferrin is a major angiogenic molecule produced by hypertrophic chondrocytes during endochondral bone formation.
Translational Diffusion of Macromolecule-sized Solutes in Cytoplasm and Nucleus
Authors: Seksek Olivier , Biwersi Joachim , Verkman A.S.
Published: July 2002

Fluorescence recovery after photobleaching (FRAP) was used to quantify the translational diffusion of microinjected FITC-dextrans and Ficolls in the cytoplasm and nucleus of MDCK epithelial cells and Swiss 3T3 fibroblasts. Absolute diffusion coefficients (D) were measured using a microsecond-resolution FRAP apparatus and solution standards. In aqueous media (viscosity 1 cP), D for the FITC-dextrans decreased from 75 to 8.4 × 10−7 cm2/s with increasing dextran size (4–2,000 kD). D in cytoplasm relative to that in water (D/Do) was 0.26 ± 0.01 (MDCK) and 0.27 ± 0.01 (fibroblasts), and independent of FITC-dextran and Ficoll size (gyration radii [RG] 40–300 Å). The fraction of mobile FITC-dextran molecules (fmob), determined by the extent of fluorescence recovery after spot photobleaching, was >0.75 for RG < 200 Å, but decreased to <0.5 for RG > 300 Å. The independence of D/Do on FITC-dextran and Ficoll size does not support the concept of solute “sieving” (size-dependent diffusion) in cytoplasm. Photobleaching measurements using different spot diameters (1.5–4 μm) gave similar D/Do, indicating that microcompartments, if present, are of submicron size. Measurements of D/Do and fmob in concentrated dextran solutions, as well as in swollen and shrunken cells, suggested that the low fmob for very large macromolecules might be related to restrictions imposed by immobile obstacles (such as microcompartments) or to anomalous diffusion (such as percolation). In nucleus, D/Do was 0.25 ± 0.02 (MDCK) and 0.27 ± 0.03 (fibroblasts), and independent of solute size (RG 40–300 Å). Our results indicate relatively free and rapid diffusion of macromolecule-sized solutes up to approximately 500 kD in cytoplasm and nucleus.
Expression of a Truncated, Kinase-Defective TGF-β Type II Receptor in Mouse Skeletal Tissue Promotes Terminal Chondrocyte Differentiation and Osteoarthritis
Authors: Serra Rosa , Johnson Mahlon , Filvaroff Ellen H. , LaBorde James , Sheehan Daniel M. , Derynck Rik , Moses Harold L.
Published: July 2002

Members of the TGF-β superfamily are important regulators of skeletal development. TGF-βs signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-βs in the development and maintenance of the skeleton, we have generated transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid process and sternum. They also developed progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium was hyperplastic, and cartilaginous metaplasia was observed in the joint space.

We then tested the hypothesis that TGF-β is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen was increased in growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation. The data suggest that TGF-β may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-β promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans.
Rho-Kinase Phosphorylates COOH-terminal Threonines of Ezrin/Radixin/Moesin (ERM) Proteins and Regulates Their Head-to-Tail Association
Authors: Matsui Takeshi , Maeda Masato , Doi Yoshinori , Yonemura Shigenobu , Amano Mutsuki , Kaibuchi Kozo , Tsukita Sachiko , Tsukita Shoichiro
Published: July 2002

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho- associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and ∼30 and ∼100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase–dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase–dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.
Mode of Action of Interleukin-6 on Mature Osteoclasts. Novel Interactions with Extracellular Ca2+ Sensing in the Regulation of Osteoclastic Bone Resorption
Authors: Adebanjo Olugbenga A. , Moonga Baljit S. , Yamate Tomoo , Sun Li , Minkin Cedric , Abe Etsuko , Zaidi Mone
Published: July 2002

We describe a physiologically significant mechanism through which interleukin-6 (IL-6) and a rising ambient Ca2+ interact to regulate osteoclastic bone resorption. VOXEL-based confocal microscopy of nonpermeabilized osteoclasts incubated with anti– IL-6 receptor antibodies revealed intense, strictly peripheral plasma membrane fluorescence. IL-6 receptor expression in single osteoclasts was confirmed by in situ reverse transcriptase PCR histochemistry. IL-6 (5 ng/l to 10 μg/l), but not IL-11 (10 and 100 μg/l), reversed the inhibition of osteoclastic bone resorption induced by high extracellular Ca2+ (15 mM). The IL-6 effect was abrogated by excess soluble IL-6 receptor (500 μg/l). Additionally, IL-6 (5 pg/l to 10 μg/l) inhibited cytosolic Ca2+ signals triggered by high Ca2+ or Ni2+. In separate experiments, osteoclasts incubated in 10 mM Ca2+ or on bone released more IL-6 than those in 1.25 mM Ca2+. Furthermore, IL-6 mRNA histostaining was more intense in osteoclasts in 10 or 20 mM Ca2+ than cells in 1.25 mM Ca2+. Similarly, IL-6 receptor mRNA histostaining was increased in osteoclasts incubated in 5 or 10 mM Ca2+. Thus, while high Ca2+ enhances IL-6 secretion, the released IL-6 attenuates Ca2+ sensing and reverses inhibition of resorption by Ca2+. Such an autocrine–paracrine loop may sustain osteoclastic activity in the face of an inhibitory Ca2+ level generated locally during resorption.
The Localization of Myosin VI at the Golgi Complex and Leading Edge of Fibroblasts and Its Phosphorylation and Recruitment into Membrane Ruffles of A431 Cells after Growth Factor Stimulation
Authors: Buss Folma , Kendrick-Jones John , Lionne Corinne , Knight Alex E. , Côté Graham P. , Paul Luzio J.
Published: July 2002

Myosin VI is an unconventional myosin that may play a role in vesicular membrane traffic through actin rich regions of the cytoplasm in eukaryotic cells. In this study we have cloned and sequenced a cDNA encoding a chicken intestinal brush border myosin VI. Polyclonal antisera were raised to bacterially expressed fragments of this myosin VI. The affinity purified antibodies were highly specific for myosin VI by immunoblotting and immunoprecipitation and were used to study the localization of the protein by immunofluorescence and immunoelectron microscopy. It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool. In A431 cells in which cell surface ruffling was stimulated by EGF, myosin VI was phosphorylated and recruited into the newly formed ruffles along with ezrin and myosin V. In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain. These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.
The Integrin α9β1 Mediates Adhesion to Activated Endothelial Cells and Transendothelial Neutrophil Migration through Interaction with Vascular Cell Adhesion Molecule-1
Authors: Taooka Yasuyuki , Chen John , Yednock Ted , Sheppard Dean
Published: July 2002

The integrin α9β1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin α4β1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether α9β1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that α9β1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-α. Furthermore, we show that α9β1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, α9β1 and α4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for α9β1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.
Thin Filament Protein Dynamics in Fully Differentiated Adult Cardiac Myocytes: Toward A Model of Sarcomere Maintenance
Authors: Michele Daniel E. , Albayya Faris P. , Metzger Joseph M.
Published: July 2002

Sarcomere maintenance, the continual process of replacement of contractile proteins of the myofilament lattice with newly synthesized proteins, in fully differentiated contractile cells is not well understood. Adenoviral-mediated gene transfer of epitope-tagged tropomyosin (Tm) and troponin I (TnI) into adult cardiac myocytes in vitro along with confocal microscopy was used to examine the incorporation of these newly synthesized proteins into myofilaments of a fully differentiated contractile cell. The expression of epitope-tagged TnI resulted in greater replacement of the endogenous TnI than the replacement of the endogenous Tm with the expressed epitope-tagged Tm suggesting that the rates of myofilament replacement are limited by the turnover of the myofilament bound protein. Interestingly, while TnI was first detected in cardiac sarcomeres along the entire length of the thin filament, the epitope-tagged Tm preferentially replaced Tm at the pointed end of the thin filament. These results support a model for sarcomeric maintenance in fully differentiated cardiac myocytes where (a) as myofilament proteins turnover within the cell they are rapidly exchanged with newly synthesized proteins, and (b) the nature of replacement of myofilament proteins (ordered or stochastic) is protein specific, primarily affected by the structural properties of the myofilament proteins, and may have important functional consequences.
The 193-Kd Vault Protein, Vparp, Is a Novel Poly(Adp-Ribose) Polymerase
Authors: Kickhoefer Valerie A. , Siva Amara C. , Kedersha Nancy L. , Inman Elisabeth M. , Ruland Cristina , Streuli Michel , Rome Leonard H.
Published: July 2002

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of ∼350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.
Role of a Class Dhc1b Dynein in Retrograde Transport of Ift Motors and Ift Raft Particles along Cilia, but Not Dendrites, in Chemosensory Neurons of Living Caenorhabditis elegans
Authors: Signor Dawn , Wedaman Karen P. , Orozco Jose T. , Dwyer Noelle D. , Bargmann Cornelia I. , Rose Lesilee S. , Scholey Jonathan M.
Published: July 2002

The heterotrimeric motor protein, kinesin-II, and its presumptive cargo, can be observed moving anterogradely at 0.7 μm/s by intraflagellar transport (IFT) within sensory cilia of chemosensory neurons of living Caenorhabditis elegans, using a fluorescence microscope–based transport assay (Orozco, J.T., K.P. Wedaman, D. Signor, H. Brown, L. Rose, and J.M. Scholey. 1999. Nature. 398:674). Here, we report that kinesin-II, and two of its presumptive cargo molecules, OSM-1 and OSM-6, all move at ∼1.1 μm/s in the retrograde direction along cilia and dendrites, which is consistent with the hypothesis that these proteins are retrieved from the distal endings of the cilia by a retrograde transport pathway that moves them along cilia and then dendrites, back to the neuronal cell body. To test the hypothesis that the minus end–directed microtubule motor protein, cytoplasmic dynein, drives this retrograde transport pathway, we visualized movement of kinesin-II and its cargo along dendrites and cilia in a che-3 cytoplasmic dynein mutant background, and observed an inhibition of retrograde transport in cilia but not in dendrites. In contrast, anterograde IFT proceeds normally in che-3 mutants. Thus, we propose that the class DHC1b cytoplasmic dynein, CHE-3, is specifically responsible for the retrograde transport of the anterograde motor, kinesin-II, and its cargo within sensory cilia, but not within dendrites.
A Role for P21-Activated Kinase in Endothelial Cell Migration
Authors: Kiosses William B. , Daniels R. Hugh , Otey Carol , Bokoch Gary M. , Schwartz Martin Alexander
Published: July 2002

The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration of microvascular endothelial cells. We found that a dominant negative (DN) mutant of PAK significantly inhibited cell migration and in-creased stress fibers and focal adhesions. The DN effect mapped to the most NH2-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged α-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited migration equally well and also increased stress fibers and focal adhesions, but had a somewhat weaker effect on their dynamics. In contrast to their similar effects on motility, DN PAK decreased cell contractility, whereas active PAK increased contractility. Active PAK also increased myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.
Dynein-Mediated Cargo Transport in Vivo
Authors: Gross Steven P. , Welte Michael A. , Block Steven M. , Wieschaus Eric F.
Published: July 2002

Cytoplasmic dynein is a microtubule-based motor with diverse cellular roles. Here, we use mutations in the dynein heavy chain gene to impair the motor's function, and employ biophysical measurements to demonstrate that cytoplasmic dynein is responsible for the minus end motion of bidirectionally moving lipid droplets in early Drosophila embryos. This analysis yields an estimate for the force that a single cytoplasmic dynein exerts in vivo (1.1 pN). It also allows us to quantitate dynein-mediated cargo motion in vivo, providing a framework for investigating how dynein's activity is controlled. We identify three distinct travel states whose general features also characterize plus end motion. These states are preserved in different developmental stages. We had previously provided evidence that for each travel direction, single droplets are moved by multiple motors of the same type (Welte et al. 1998). Droplet travel distances (runs) are much shorter than expected for multiple motors based on in vitro estimates of cytoplasmic dynein processivity. Therefore, we propose the existence of a process that ends runs before the motors fall off the microtubules. We find that this process acts with a constant probability per unit distance, and is typically coupled to a switch in travel direction. A process with similar properties governs plus end motion, and its regulation controls the net direction of transport.
Ephrin-A5 Induces Collapse of Growth Cones by Activating Rho and Rho Kinase
Authors: Wahl Siegfried , Barth Holger , Ciossek Thomas , Aktories Klaus , Mueller Bernhard K.
Published: July 2002

The ephrins, ligands of Eph receptor tyrosine kinases, have been shown to act as repulsive guidance molecules and to induce collapse of neuronal growth cones. For the first time, we show that the ephrin-A5 collapse is mediated by activation of the small GTPase Rho and its downstream effector Rho kinase. In ephrin-A5–treated retinal ganglion cell cultures, Rho was activated and Rac was downregulated. Pretreatment of ganglion cell axons with C3-transferase, a specific inhibitor of the Rho GTPase, or with Y-27632, a specific inhibitor of the Rho kinase, strongly reduced the collapse rate of retinal growth cones. These results suggest that activation of Rho and its downstream effector Rho kinase are important elements of the ephrin-A5 signal transduction pathway.
Embryonic Lethality of Molecular Chaperone Hsp47 Knockout Mice Is Associated with Defects in Collagen Biosynthesis
Authors: Nagai Naoko , Hosokawa Masanori , Itohara Shigeyoshi , Adachi Eijiro , Matsushita Takatoshi , Hosokawa Nobuko , Nagata Kazuhiro
Published: July 2002

Triple helix formation of procollagen after the assembly of three α-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of α1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/− cells were resistant, but those of Hsp47−/− cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.