bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of cyclosporin‐A on the blood–retinal barrier permeability in streptozotocin‐induced diabetes
Authors: Carmo Anália , Cunha-Vaz José G. , Carvalho Arsélio P. , Lopes Maria Celeste
Published: July 2002

Background: Our previous results showed that in retinas from streptozotocin (STZ)‐induced diabetic rats there is an increased level of interleukin‐1β (IL‐1β ). This cytokine may be involved in the expression of the inducible isoform of the nitric oxide synthase (iNOS), with consequent synthesis of large amounts of NO and
blood–retinal barrier (BRB) breakdown.

Aims: The aim of this work was to examine whether the administration of cyclosporin‐A (Cs‐A) to STZinduced diabetic rats inhibits the synthesis of IL‐1β and the expression of the inducible proteins, iNOS and cyclo‐oxygenase‐2 (COX‐2) in retinal cells, and whether the activity of these proteins contribute to BRB breakdown.

Methods: The level of IL‐1β was evaluated by ELISA and the NO production by
L‐{3H}‐citrulline formation. Expression of iNOS and COX‐2 proteins was determined by two methods, western blot and immunohistochemistry. The permeability of the BRB was assessed by quantification of the vitreous protein.

Results and discussion: Our results indicated that the levels of IL‐1β and NO in retinas from Cs‐A‐treated diabetic rats are significantly reduced, as compared to that in non‐treated diabetic rats. The treatment of diabetic rats with Cs‐A also significantly inhibited the expression of the inducible proteins, iNOS and COX2. The evaluation of the vitreous protein content revealed that Cs‐A also reduces the BRB permeability. Taken together, these results suggest that the increased production of the inflammatory mediators, IL‐1β and NO, in diabetes may affect the BRB permeability and therefore contribute to the development of diabetic retinopathy.
Role of histamine in the inhibitory effects of phycocyanin in experimental models of allergic inflammatory response
Authors: Remirez D. , Ledón N. , González R.
Published: July 2002

It has recently been reported that phycocyanin, a biliprotein found in the blue‐green microalgae Spirulina, exerts anti‐inflammatory effects in some animal models of inflammation. Taking into account these findings, we decided to elucidate whether phycocyanin might exert also inhibitory effects in the induced allergic inflammatory response and on histamine release from isolated rat mast cells. In in vivo experiments, phycocyanin (100, 200 and 300 mg/kg post‐orally (p.o.)) was administered 1 h before the challenge with 1 μg of ovalbumin (OA) in the ear of mice previously sensitized with OA. One hour later, myeloperoxidase activity and ear edema were assessed. Phycocyanin significantly reduced both parameters. In separate experiments, phycocyanin (100 and 200 mg/kg p.o.) also reduced the blue spot area induced by intradermal injections of histamine, and the histamine releaser compound 48/80 in rat skin. In concordance with the former results, phyco‐cyanin also significantly reduced histamine release induced by compound 48/80 from isolated peritoneal rat mast cells. The inhibitory effects of phycocyanin were dose dependent. Taken together, our results suggest that inhibition of allergic inflammatory response by phycocyanin is mediated, at least in part, by inhibition of histamine release from mast cells.
Antitumor effect of Bothrops jararaca venom
Authors: da Silva Reinaldo J. , da Silva Márcia G. , Vilela Lízia C. , Fecchio Denise
Published: July 2002

Many experimental studies have been carried out using snake venoms for the treatment of animal tumors, with controversial results. While some authors have reported an antitumor effect of treatment with specific snake venom fractions, others have reported no effects after this treatment. The aim of this study was to evaluate the effect of Bothrops jararaca venom (BjV) on Ehrlich ascites tumor (EAT) cells in vivo and in vitro. In the in vivo study, Swiss mice were inoculated with EAT cells by the intraperitoneal (i.p.) route and treated with BjV venom (0.4 mg/kg, i.p.), on the 1st, 4th, 7th, 10th, and 13th days. Mice were evaluated for total and differential cells number on the 2nd, 5th, 8th, 11th and 14th days. The survival time was also evaluated after 60 days of tumor growth. In the in vitro study, EAT and normal peritoneal cells were cultivated in the presence of different BjV concentrations (2.5, 5.0, 10.0, 20.0, 40.0, and 80 μg) and viability was verified after 3, 6, 12 and 24 h of cultivation. Results were analyzed statistically by the Kruskal‐Wallis and Tukey tests at the 5% level of significance. It was observed that in vivo treatment with BjV induced tumor growth inhibition, increased animal survival time, decreased mortality, increased the influx of polymorphonuclear leukocytes on the early stages of tumor growth, and did not affect the mononuclear cells number. In vitro treatment with BjV produced a dose‐dependent toxic effect on EAT and peritoneal cells, with higher effects against peritoneal cells. Taken together, our results demonstrate that BjV has an important antitumor effect. This is the first report showing this in vivo effect for this venom.
Pharmacokinetics, biological effects, and distribution of (1→3)‐β‐D‐glucan in blood and organs in rabbits
Authors: Yoshida Minoru , Roth Robert I. , Grunfeld Carl , Feingold Kenneth R. , Levin Jack
Published: July 2002

The pharmacokinetics, biological effects and distribution in blood and organs of 125I‐labeled (1→3)‐β‐D‐glucan purified from Candida albicans were analyzed in rabbits during the 24‐h period following an intravenous administration.The intravascular half‐life of (1→3)‐β‐ D‐glucan was 1.8 min in the low‐dose group (9.3 μg/kg) and 1.4 min in the high‐dose group (222 μg/kg), and the mean (±SD) total body clearance was 1.12 ± 0.30 and 1.17 ± 0.16 ml/min, respectively. The rabbits remained well and (1→3)‐β‐D‐glucan failed to alter blood cell counts. Less than 3% of the 125I‐(1→3)‐β‐D‐glucan was initially associated with the cellular compartment, and this value decreased further during the 2‐h period following administration
(P = 0.0001). Over 97% of 125I‐(1→3)‐β‐D‐glucan was associated with cell‐free plasma, and the majority in plasma appeared to be present in the unbound form (not associated with lipoproteins or plasma proteins). The liver contained more than 80% of the 125I‐(1→3)‐β‐D‐glucan detected in the six major organs analyzed.
Inhibitors of nuclear factor kappa B cause apoptosis in cultured macrophages
Authors: Mannick E. E. , Mishra J. , Marque J. , Clavell M. , Miller M. J. S. , Oliver P. D.
Published: July 2002

The precise role of the transcription factor nuclear factor kappa B (NF‐ κB) in the regulation of cell survival and cell death is still unresolved and may depend on cell type and position in the cell cycle. The aim of this study was to determine if three pharmacologic inhibitors of NF‐κB, pyrrolidine dithiocarbamate, N‐tosyl‐L‐lysl chloromethyl ketone and calpain I inhibitor, induce apoptosis in a murine macrophage cell line (RAW 264.7) at doses similar to those required for NF‐κB inhibition. We found that each of the three inhibitors resulted in a dose‐ and time‐dependent increase in morphologic indices of apoptosis in unstimulated, LPS‐stimulated and TNF‐stimulated cells. Lethal doses were consistent with those required for NF‐ κB inhibition. We conclude that nuclear NF‐κB activation may represent an important survival mechanism in macrophages.
A novel murine model of late‐phase reaction of immediate hypersensitivity
Authors: Facincone S. , De Siqueira A. L. Pereira , Jancar S. , Russo M. , Barbuto J. A. Marzagão , Mariano M.
Published: July 2002

We describe here a novel experimental model of late‐phase reaction of immediate hypersensitivity developed in mice. It consists of introducing small fragments of heat‐coagulated hen egg white into the subcutaneous tissue of mice. After 14 days, animals challenged with purified ovalbumin into the footpad presented an immediate swelling of the paw peaking at 30 min, followed by two peaks of swelling at 6 and 24 h. Histological examination of the paws showed a massive eosinophil infiltration (more than 800 cells/5 microscopic fields). This intense infiltration persisted for more than 14 days after the challenge. Furthermore, in mice immunized with coagulated egg white the delayed swelling of the paws and eosinophilic infiltration were significantly higher than in mice immunized with the classical protocol of ovalbumin in alumen adjuvant. Transfer of lymph node cells obtained from mice implanted with heat‐coagulated hen egg white induced footpad swelling and eosinophil infiltration in response to ovalbumin. High levels of ovalbuminspecific IgG1 but not of IgE were detected in the serum of these animals. The advantages of this model for the experimental study of late‐phase reaction per se and its relevance to the study of allergic diseases such as asthma are discussed.
Inhibitory effect of esculentoside A on prostaglandin E2 production from murine peritoneal macrophages and rabbit synovial cells in vitro
Authors: Wang H-B. , Fang J. , Zheng Q-Y.
Published: July 2002

Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. Previous experiments have shown that it has strong antiinflammatory effects. To investigate the mechanism of anti‐inflammatory effects of esculentoside A (EsA),[3H] arachidonic acid (AA) prelabelled murine macrophage and radioimmunoassay were used to test the effect of EsA on the total release of AA and prostaglandin E2 in culture supernatants. The results showed that EsA had no significant effect on the total release of AA from murine macrophages. EsA (2.5‐10 μmol/l), from unstimulated murine peritoneal macrophages and rabbit synovial cells, could decrease the production of
prostaglandin E2. In A23187 and LPS‐treated macrophages and synovial cells, EsA (10 μmol/l) could significantly decrease the prostaglandin E2 production. These results confirmed that EsA exerted an inhibitory effect on prostaglandin E2 production from murine macrophages and rabbit synovial cells.
Basal Secretion of Lysozyme from Human Airways in Vitro
Authors: Roger Patricia , Gascard Jean-Pierre , de Montpreville Vincent Thomas , Brink Charles
Published: July 2002

The aim of this study was to examine the basal release of lysozyme from isolated human lung tissues. Measurements of lysozyme in the fluids derived from lung preparations were performed using a rate‐of‐lysis assay subsequent to acidification of the biological samples. Lysozyme released from bronchial preparations into fluids was greater than that observed for parenchymal tissues. The lysozyme quantities detected in bronchial fluids were not modified by removal of the surface epithelium. Furthermore, the quantities of lysozyme in bronchial fluids was correlated with the size of the bronchial preparations. These results suggest that the lysozyme was principally secreted by the human bronchi (submucosal layer) rather than by parenchyma tissues and that a greater release was observed in the proximal airways.
Chronic Mild Prenatal Stress Exacerbates the Allergen‐Induced Airway Inflammation in Rats
Authors: Nogueira Paulo J. , Ferreira Heloisa H. A. , Antunes Edson , Teixeira Nancy A.
Published: July 2002

The effects of chronic mild prenatal stress on leukocyte infiltration into the airways was investigated in rat offspring. The chronic prenatal stress consisted of transitory and variable changes in the rat′s living conditions. Offspring at adult age were actively sensitized (day 0) and intratracheally challenged (day 14) with ovalbumin. Bronchoalveolar lavage was performed in the offspring at 48 h after intratracheal challenge with ovalbumin. A significant increase in total leukocyte infiltration was observed in the nonstressed offspring group and this was associated with a marked recruitment of eosinophils without a significant effect on the influx of neutrophils and mononuclear cells. In the prenatal stressed offspring, the counts of both total leukocyte and eosinophils, as well as mononuclear cells, was increased by 50% compared to the non‐stressed offspring. We provide here the first experimental evidence that chronic mild unpredictable prenatal stress produces a marked increase in the allergen‐induced airway inflammation in the rat offspring.
Soft Tissue Sarcoma: The Predominant Primary Malignancy in the Retroperitoneum
Authors: Dalen Thijs van , Coebergh Jan-Willem W. , Casparie Mariel K. , Gimbrère Charles H. F. , Hoekstra Harald J. , Van Geel Bert N. , Coevorden Frits Van , Hennipman Adriaan
Published: July 2002

Purpose. In the clinical work‐up of a retroperitoneal mass, the diagnosis of soft tissue sarcoma is often not considered. Incidence
rates of various malignant and benign retroperitoneal tumours were studied to determine the incidence of soft tissue
sarcoma in comparison with other neoplasms in the retroperitoneal space.

Method. Nation‐wide data on retroperitoneal tumours, collected prospectively over a 5‐year period (1 January 1989– 1 January
1994), were supplied by the Netherlands Cancer Registry and The Dutch Network and National Database for Pathology.

Results. Seven hundred and six patients with a primary retroperitoneal neoplasm were identified; 566 patients had a malignant
tumour (80%). A soft tissue sarcoma (STS) was the most frequently diagnosed malignant tumour (n = 192), The agestandardised
incidence of retroperitoneal STS was 2.5 per million person‐years. The male/female ratio for STS was 0.73. In
females, STS comprised 41%of all malignant retroperitoneal tumours, carcinoma of unknown primary tumour site (CUP)
comprised 31%, and malignant lymphomas (ML) comprised 22%, whereas in males these values were 28% (STS), 30%
(CUP), and 32% (ML), respectively.

Discussion. Soft tissue sarcomas, albeit rare, are relatively common primary tumours in the retroperitoneum, especially in
women.
Growth in the Lower Limb Following Chemotherapy for a Malignant Primary Bone Tumour: A Straight‐Line Graph
Authors: Cool Paul , Davies Mark , Grimer Rob J. , Carter Simon R. , Tillman Roger M.
Published: July 2002

Purpose. The aim of this paper was to assess the growth in the unaffected lower limb of children who had received
chemotherapy for a malignant primary bone tumour around the knee.

Subjects/methods. Following diagnosis, all children (45, of which 32 were boys and 13 were girls) were staged. If
limb‐salvage surgery was thought appropriate, measured radiographs of both legs was performed, the bone age was
estimated and the expected growth in the femur and tibia was calculated according to Tupman. These procedures were
repeated at follow‐up and the data plotted. Regression and correlation coefficients were also calculated.

Results. The observed regression line in boys was almost identical to Tupman′s curve. However, the observed growth in
girls was larger than the expected growth.

Discussion. It is recommended that the regression lines presented here are used in the calculation of the expected growth
in the lower limb of children who have received chemotherapy for a malignant primary bone tumour, especially in girls.
Disseminating Adamantinoma of the Tibia
Authors: Van Geel Albert N. , Hazelbag Hans M. , Slingerland Rob , Vermeulen Margit I.
Published: July 2002

Patient. This report describes a patient with a primary long bone adamantinoma. The lesion was
initially wrongly diagnosed as fibrous dysplasia and the patient was treated by curettage. At second local recurrence, the tumour
had progressed from an osteofibrous dysplasia‐like to a full‐blown classic adamantinoma, with metastatic potential to the lungs
19 years after the initial treatment. Lung metastasectomy by sternotomy was carried out twice in a period of over years. The patient is currently alive without evidence of other metastatic disease.

Discussion. From the files of the Netherlands Committee on Bone Tumors, another five patients with
lung metastaseswere studied. All types of adamantinoma should be treated by complete en bloc resection.
For patients with metastatic spread to the lungs, close radiological follow‐up and excision of tumour nodules seems
to be the only logic treatment modality.
Primary Liposarcoma of the Mediastinum
Authors: Greif Joel , Marmor Silvia , Merimsky Ofer , Kovner Felix , Inbar Moshe
Published: July 2002

Patient. A 62‐year‐old man presented with effort dyspnea, non‐productive cough and weakness of 4 month duration. He had no findings on physical examination.

Discussion. Chest X‐ray revealed a large mass in the left anterior mediastinum. Computerized tomography of the chest showed a well‐delineated homogeneous mediastinal mass with fat‐equivalent density and a small pleural effusion. Fiberoptic bronchoscopy revealed narrowing of the left main bronchus, secondary to external compression. The bronchial mucosa was normal and brush cytology was negative. A CT‐guided fine needle aspiration (FNA) of the mass yielded fragments of cells embedded in myxoid background material and closely packed atypical lipoblasts, compatible with liposarcoma. The patient underwent a left lateral thoracotomy and margibnal resection of the mass. The histopathological examination confirmed the diagnosis of mixed‐type liposarcoma, consisted of myxoid and pleomorphic liposarcoma. Postoperative two‐field radiation therapy was delivered to the mediastinum for a total midplane dose of 40 Gy. After a disease‐free interval of 8 months the disease recurred in the mediastinum and pleura. Palliative chemotherapy achieved a short duration partial response but the patient succumbed to local recurrence 2 years after the diagnosis.
Surgical Treatment for Skeletal Metastases From Soft Tissue Sarcomas: Experience With 23 Lesions in 20 Patients
Authors: Yoshikawa Hideki , Ueda Takafumi , Kudawara Ikuo , Araki Nobuhito , Yonenobu Kazuo , Ochi Takahiro , Uchida Atsumasa
Published: July 2002

Purpose. This paper reports the procedures and the clinical results of a series of surgical treatments for skeletal metastases from soft tissue sarcomas.

Subjects and methods. Surgical treatment of metastatic bony lesions from soft tissue sarcomas has been carried out over a 20 year period (1975–1996). Thirty‐two patients developed skeletal metastases from soft tissue sarcomas, and 20 of these cases received surgical treatment. The 23 metastatic bony lesions in these 20 patients were treated using the following
surgical approaches: wide resection with prosthetic replacement in five lesions, wide or marginal resection without reconstruction in four lesions, intramedullarly nailing with curettage and methylmethacrylate cementation in four lesions, marginal resection of vertebral body with replacement by a ceramic prosthesis in three lesions, laminectomy in three lesions, intramedullarly nailing in two lesions, and curettage in two lesions.

Results. Relief of pain was achieved in 17 of the 20 patients. The ambulatory status of the patients with metastasis in the lower extremity or periacetabular region was significantly improved in nine of 10 cases. Seventeen patients died of disease, with a mean survival period of 17.9 months after surgery for metastasis.

Discussion. Although surgical treatment for skeletal metastases from soft tissue sarcomas cannot save the life of the patient, it can be of value in improving their well‐being and overall quality of life. In these cases, surgical intervention may be more frequently indicated than in tumors with an osteoblastic or mixed pattern.
Concurrent Hypofractionated Radiotherapy and 5‐Fluorouracil for Advanced Sarcomas of the Bone
Authors: Zambatis Charalambos , Skarlatos John , Koukourakis Michael , Kosma Lambrini , Giatromanolaki Alexandra , Beroukas Kostantinos , Yannakakis Dimitrios
Published: July 2002

Purpose. 5‐Fluorouracil (5‐FU) has shown radiosensitizing properties in vitro. This paper reports the effects of radiotherapy and concomitant intravenous 5‐FU radiosensitization in the treatment of advanced bone sarcomas.

Subjects/methods. Four patients with large inoperable bone sarcomas (three chondrosarcomas and one fibrosarcoma) were treated with hypofractionated radiotherapy and concomitant 5‐FU bolus injection (300 mg m−2) before each fraction of radiotherapy. A radiation fraction of 5 Gy was given twice a week to a normalized total dose (α/β=4 Gy) of 75 Gy.

Results. The regimen was well tolerated, the main toxicity being grade I/II diarrhoea in two cases with pelvic irradiation. Treatment interruption for 1 week was necessary in two cases with pelvic disease but not in two patients treated for sarcoma of the extremities. A complete symptomatic relief was obtained in all cases immediately after the third to the fifth fraction and the median duration was 10 months. Computed tomography scan documented a partial response in 2/4 cases.

Discussion. Hypofractionated radiotherapy combined with potential lethal damage inhibitors for bone sarcomas requires further investigation.
A Phase II Study of Docetaxel in Chemotherapy‐Naïve Patients With Recurrent or Metastatic Adult Soft Tissue Sarcoma
Authors: Bramwell Vivien , Blackstein Martin , Belanger Karl , Verma Shail , Beare Sandra , Eisenhauer Elizabeth
Published: July 2002

Purpose: To determine the efficacy and toxicity of docetaxel as first‐line chemotherapy in adult patients with locally advanced and/or metastatic soft tissue sarcoma (STS).

Patients/methods. Thirty eligible patients, with histologically proven STS, Eastern Cooperative Oncology Group (ECOG) performance status 0–2 and bidimensionally measurable disease, entered this study. None had received previous chemotherapy. Docetaxel 100 mg m−2 was given as a 1‐h intravenous infusion every 3 weeks. Patients were evaluable for
response, evaluated by WHO criteria, after one cycle of chemotherapy and toxicity was graded by NCIC‐CTG common toxicity criteria.

Results. One hundred and thirty two cycles were aldministered, with a range per patient of 1–9. The median delivered dose intensity was 32.2 mg m−2 week−1 (planned 33.3 mg m−2 week−1 ) and 67% of patients received ≥90% planned dose intensity. There were three partial responses (10.7%; 95% confidence interval 2.3–28.2) with a median duration of 7 months (range 6.4–8.3 months). Thirty patients were evaluable for non‐haematological toxicity and 28 for haematological toxicity (repeat counts were not available in two patients). Haematological toxicity was moderately severe, with 18 (64%) patients experiencing at least one episode of grade 4 neutropenia, and 7 (25%) patients experiencing febrile neutropenia.

Conclusions. In this study, activity of docetaxel in adult chemotherapy‐naïve patients with advanced STS was modest
Pleckstrin Associates with Plasma Membranes and Induces the Formation of Membrane Projections: Requirements for Phosphorylation and the NH2-terminal PH Domain
Authors: Ma Alice D. , Brass Lawrence F. , Abrams Charles S.
Published: July 2002

Pleckstrin homology (PH) domains are sequences of ∼100 amino acids that form “modules” that have been proposed to facilitate protein/protein or protein/lipid interactions. Pleckstrin, first described as a substrate for protein kinase C in platelets and leukocytes, is composed of two PH domains, one at each end of the molecule, flanking an intervening sequence of 147 residues. Evidence is accumulating to support the hypothesis that PH domains are structural motifs that target molecules to membranes, perhaps through interactions with Gβγ or phosphatidylinositol 4,5-bisphosphate (PIP2), two putative PH domain ligands. In the present studies, we show that pleckstrin associates with membranes in human platelets. We further demonstrate that, in transfected Cos-1 cells, pleckstrin associates with peripheral membrane ruffles and dorsal membrane projections. This association depends on phosphorylation of pleckstrin and requires the presence of its NH2-terminal, but not its COOH-terminal, PH domain. Moreover, PH domains from other molecules cannot effectively substitute for pleckstrin's NH2terminal PH domain in directing membrane localization. Lastly, we show that wild-type pleckstrin actually promotes the formation of membrane projections from the dorsal surface of transfected cells, and that this morphologic change is similarly PH domain dependent. Since we have shown previously that pleckstrin-mediated inhibition of PIP2 metabolism by phospholipase C or phosphatidylinositol 3-kinase also requires pleckstrin phosphorylation and an intact NH2-terminal PH domain, these results suggest that: (a) pleckstrin's NH2terminal PH domain may regulate pleckstrin's activity by targeting it to specific areas within the cell membrane; and (b) pleckstrin may affect membrane structure, perhaps via interactions with PIP2 and/or other membrane-bound ligands.
Extracellular Matrix Survival Signals Transduced by Focal Adhesion Kinase Suppress p53-mediated Apoptosis
Authors: Ilić Duško , Almeida Eduardo A.C. , Schlaepfer David D. , Dazin Paul , Aizawa Shinichi , Damsky Caroline H.
Published: July 2002

In many malignant cells, both the anchorage requirement for survival and the function of the p53 tumor suppressor gene are subverted. These effects are consistent with the hypothesis that survival signals from extracellular matrix (ECM) suppress a p53-regulated cell death pathway. We report that survival signals from fibronectin are transduced by the focal adhesion kinase (FAK). If FAK or the correct ECM is absent, cells enter apoptosis through a p53-dependent pathway activated by protein kinase C λ/ι and cytosolic phospholipase A2. This pathway is suppressible by dominant-negative p53 and Bcl2 but not CrmA. Upon inactivation of p53, cells survive even if they lack matrix signals or FAK. This is the first report that p53 monitors survival signals from ECM/FAK in anchorage- dependent cells.
Export of a Cysteine-Free Misfolded Secretory Protein from the Endoplasmic Reticulum for Degradation Requires Interaction with Protein Disulfide Isomerase
Authors: Gillece Pauline , Luz José Manuel , Lennarz William J. , de la Cruz Francisco Javier , Römisch Karin
Published: July 2002

Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.
The Interaction of the Chaperonin Tailless Complex Polypeptide 1 (Tcp1) Ring Complex (Tric) with Ribosome-Bound Nascent Chains Examined Using Photo-Cross-Linking
Authors: McCallum Christine D. , Do Hung , Johnson Arthur E. , Frydman Judith
Published: July 2002

The eukaryotic chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) (also called chaperonin containing TCP1 [CCT]) is a hetero-oligomeric complex that facilitates the proper folding of many cellular proteins. To better understand the manner in which TRiC interacts with newly translated polypeptides, we examined its association with nascent chains using a photo-cross-linking approach. To this end, a series of ribosome-bound nascent chains of defined lengths was prepared using truncated mRNAs. Photoactivatable probes were incorporated into these 35S- labeled nascent chains during translation. Upon photolysis, TRiC was cross-linked to ribosome-bound polypeptides exposing at least 50–90 amino acids outside the ribosomal exit channel, indicating that the chaperonin associates with much shorter nascent chains than indicated by previous studies. Cross-links were observed for nascent chains of the cytosolic proteins actin, luciferase, and enolase, but not to ribosome-bound preprolactin. The pattern of cross-links became more complex as the nascent chain increased in length. These results suggest a chain length–dependent increase in the number of TRiC subunits involved in the interaction that is consistent with the idea that the substrate participates in subunit-specific contacts with the chaperonin. Both ribosome isolation by centrifugation through sucrose cushions and immunoprecipitation with anti-puromycin antibodies demonstrated that the photoadducts form on ribosome-bound polypeptides. Our results indicate that TRiC/CCT associates with the translating polypeptide shortly after it emerges from the ribosome and suggest a close association between the chaperonin and the translational apparatus.
Inhibition of Apoptotic Signaling Cascades Causes Loss of Trophic Factor Dependence during Neuronal Maturation
Authors: Putcha Girish V. , Deshmukh Mohanish , Johnson Eugene M.
Published: July 2002

During development, neurons are acutely dependent on target-derived trophic factors for survival. This dependence on trophic support decreases dramatically with maturation in several neuronal populations, including sympathetic neurons. Analyses of nerve growth factor deprivation in immature and mature sympathetic neurons indicate that maturation aborts the cell death pathway at a point that is mechanistically indistinguishable from Bax deletion. However, neither the mRNA nor protein level of BAX changes with neuronal maturation. Therefore, BAX must be regulated posttranslationally in mature neurons.

Nerve growth factor deprivation in immature sympathetic neurons induces two parallel processes: (a) a protein synthesis–dependent, caspase-independent translocation of BAX from the cytosol to mitochondria, followed by mitochondrial membrane integration and loss of cytochrome c; and (b) the development of competence-to-die, which requires neither macromolecular synthesis nor BAX expression. Activation of both signaling pathways is required for caspase activation and apoptosis in immature sympathetic neurons. In contrast, nerve growth factor withdrawal in mature sympathetic neurons did not induce the translocation of either BAX or cytochrome c. Moreover, mature neurons did not develop competence-to-die with cytoplasmic accumulation of cytochrome c. Therefore, inhibition of both BAX-dependent cytochrome c release and the development of competence-to-die contributed to the loss of trophic factor dependence associated with neuronal maturation.
Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export
Authors: Vasu Sanjay , Shah Sundeep , Orjalo Arturo , Park Minkyu , Fischer Wolfgang H. , Forbes Douglass J.
Published: July 2002

RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.
Searching for the middle ground
Authors: Kapoor Tarun M. , Compton Duane A.
Published: July 2002

The contributions of key molecules predicted to align chromosomes at the center of the mitotic spindle have been recently examined. New results dictate that models for how chromosomes align during the early stages of mitosis must be revised to integrate properties of microtubule-based motor proteins as well as microtubule dynamics.
Involvement of Bruton's Tyrosine Kinase in FcεRI-dependent Mast Cell Degranulation and Cytokine Production
Authors: Hata Daisuke , Kawakami Yuko , Inagaki Naoki , Lantz Chris S. , Kitamura Toshio , Khan Wasif N. , Maeda-Yamamoto Mari , Miura Toru , Han Wei , Hartman Stephen E. , Yao Libo , Nagai Hiroichi , Goldfeld Anne E. , Alt Frederick W. , Galli Stephen J. , Witte Owen N. , Kawakami Toshiaki
Published: July 2002

We investigated the role of Bruton's tyrosine kinase (Btk) in FcεRI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell development is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in FcεRI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon FcεRI cross-linking. Moreover, the transcriptional activities of these cytokine genes were severely reduced in FcεRI-stimulated btk mutant mast cells. The specificity of these effects of btk mutations was confirmed by the improvement in the ability of btk mutant mast cells to degranulate and to secrete cytokines after the retroviral transfer of wild-type btk cDNA, but not of vector or kinase-dead btk cDNA. Retroviral transfer of Emt (= Itk/Tsk), Btk's closest relative, also partially improved the ability of btk mutant mast cells to secrete mediators. Taken together, these results demonstrate an important role for Btk in the full expression of FcεRI signal transduction in mast cells.
The Role of Apoptosis in the Regulation of Hematopoietic Stem Cells
Authors: Domen Jos , Cheshier Samuel H. , Weissman Irving L.
Published: July 2002

Hematopoietic stem cells (HSC) give rise to cells of all hematopoietic lineages, many of which are short lived. HSC face developmental choices: self-renewal (remain an HSC with long-term multilineage repopulating potential) or differentiation (become an HSC with short-term multilineage repopulating potential and, eventually, a mature cell). There is a large overcapacity of differentiating hematopoietic cells and apoptosis plays a role in regulating their numbers. It is not clear whether apoptosis plays a direct role in regulating HSC numbers. To address this, we have employed a transgenic mouse model that overexpresses BCL-2 in all hematopoietic cells, including HSC: H2K-BCL-2. Cells from H2K-BCL-2 mice have been shown to be protected against a wide variety of apoptosis-inducing challenges. This block in apoptosis affects their HSC compartment. H2K-BCL-2–transgenic mice have increased numbers of HSC in bone marrow (2.4× wild type), but fewer of these cells are in the S/G2/M phases of the cell cycle (0.6× wild type). Their HSC have an increased plating efficiency in vitro, engraft at least as well as wild-type HSC in vivo, and have an advantage following competitive reconstitution with wild-type HSC.
Lineage-Restricted Function of Nuclear Factor κB–Inducing Kinase (Nik) in Transducing Signals via Cd40
Authors: Garceau Norman , Kosaka Yoko , Masters Sally , Hambor John , Shinkura Reiko , Honjo Tasuku , Noelle Randolph J.
Published: July 2002

CD40 signaling in B cells and dendritic cells (DCs) is critical for the development of humoral and cell-mediated immunity, respectively. Nuclear factor κB (NF-κB)–inducing kinase (NIK) has been implicated as a central transducing kinase in CD40-dependent activation. Here, we show that although NIK is essential for B cell activation, it is dispensable for activation of DCs. Such data provide compelling evidence that different intermediary kinases are used by different cellular lineages to trigger NF-κB activation via CD40.
Mechanism of Allosteric Modulation of Rod Cyclic Nucleotide–gated Channels
Authors: Sunderman Elizabeth R. , Zagotta William N.
Published: July 2002

The cyclic nucleotide–gated (CNG) channel of retinal rod photoreceptor cells is an allosteric protein whose activation is coupled to a conformational change in the ligand-binding site. The bovine rod CNG channel can be activated by a number of different agonists, including cGMP, cIMP, and cAMP. These agonists span three orders of magnitude in their equilibrium constants for the allosteric transition. We recorded single-channel currents at saturating cyclic nucleotide concentrations from the bovine rod CNG channel expressed in Xenopus oocytes as homomultimers of α subunits. The median open probability was 0.93 for cGMP, 0.47 for cIMP, and 0.01 for cAMP. The channels opened to a single conductance level of 26–30 pS at +80 mV. Using signal processing methods based on hidden Markov models, we determined that two closed and one open states are required to explain the gating at saturating ligand concentrations. We determined the maximum likelihood rate constants for two gating schemes containing two closed (denoted C) and one open (denoted O) states. For the C ↔ C ↔ O scheme, all rate constants were dependent on cyclic nucleotide. For the C ↔ O ↔ C scheme, the rate constants for only one of the transitions were cyclic nucleotide dependent. The opening rate constant was fastest for cGMP, intermediate for cIMP, and slowest for cAMP, while the closing rate constant was fastest for cAMP, intermediate for cIMP, and slowest for cGMP. We propose that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition and serve to reduce the transition state energy and stabilize the activated conformation of the channel. When 1 μM Ni2+ was applied in addition to cyclic nucleotide, the open time increased markedly, and the closed time decreased slightly. The interactions between H420 and Ni2+ occur primarily after the transition state for the allosteric transition.
Effects of Aldosterone on Biosynthesis, Traffic, and Functional Expression of Epithelial Sodium Channels in A6 Cells
Authors: de la Rosa Diego Alvarez , Li Hui , Canessa Cecilia M.
Published: July 2002

The collecting duct regulates Na+ transport by adjusting the abundance/activity of epithelial Na+ channels (ENaC). In this study we have investigated the synthesis, degradation, endocytosis, and activity of ENaC and the effects of aldosterone on these processes using endogenous channels expressed in the A6 cell line. Biochemical studies were performed with a newly raised set of specific antibodies against each of the three subunits of the amphibian ENaC. Our results indicate simultaneous transcription and translation of α, β, and γ subunits and enhancement of both processes by aldosterone: two- and fourfold increase, respectively. The biosynthesis of new channels can be followed by acquisition of endoglycosidase H–resistant oligosacharides in α and β subunits and, in the case of α, by the appearance of a form resistant to reducing agents. The half-life of the total pool of subunits (t1/2 40–70 min) is longer than the fraction of channels in the apical membrane (t1/2 12–17 min). Aldosterone induces a fourfold increase in the abundance of the three subunits in the apical membrane without significant changes in the open probability, kinetics of single channels, or in the rate of degradation of ENaC subunits. Accordingly, the aldosterone response could be accounted by an increase in the abundance of apical channels due, at least in part, to de novo synthesis of subunits.
Extremal characterizations of asplund spaces
Authors: Mordukhovich Boris , Shao Yongheng
Published: July 2002

We prove new characterizations of Asplund spaces through certain extremal principles in nonsmooth analysis and optimization. The latter principles provide necessary conditions for extremal points of set systems in terms of Fréchet normals and

ε
\varepsilon

-normals.
RanBP3 influences interactions between CRM1 and its nuclear protein export substrates
Authors: Englmeier Ludwig , Fornerod Maarten , Bischoff F Ralf , Petosa Carlo , Mattaj Iain W , Kutay Ulrike
Published: July 2002

We investigated the role of RanBP3, a nuclear member of the Ran‐binding protein 1 family, in CRM1‐mediated protein export in higher eukaryotes. RanBP3 interacts directly with CRM1 and also forms a trimeric complex with CRM1 and RanGTP. However, RanBP3 does not bind to CRM1 like an export substrate. Instead, it can stabilize CRM1–export substrate interaction. Nuclear RanBP3 stimulates CRM1‐dependent protein export in permeabilized cells. These data indicate that RanBP3 functions by a novel mechanism as a cofactor in recognition and export of certain CRM1 substrates. In vitro, RanBP3 binding to CRM1 affects the relative affinity of CRM1 for different substrates.
Going the distance with auxin: unravelling the molecular basis of auxin transport
Authors: Bennett Malcolm J. , Marchant Alan , May Sean T. , Swarup Ranjan
Published: July 2002

Auxin represents one of the most important classes of signalling molecules described in plants. Auxins regulate several fundamental cellular processes including division, elongation and differentiation. Indole–3–acetic acid (IAA), the principal form of auxin in higher plants, is first synthesized within young apical tissues, then conveyed to its basal target tissues by a specialized delivery system termed polar auxin transport. The polarity of IAA movement represents one of the most novel aspect of auxin signalling. IAA transport has been demonstrated to involve auxin influx and efflux carrier activities. The adoption of a mutational approach in the model plantArabidopsis thalianahas led to the identification of a number of genes which encode components for, or regulate the activity of, the auxin transport machinery. This paper will review the advances being made in identifying and characterizing these auxin transport–related gene products and discuss their importance within the context ofArabidopsisdevelopment.
Virulence gene regulation inside and outside
Authors: DiRita Victor J. , Cary Engleberg N. , Heath Andrew , Miller Alita , Adam Crawford J. , Yu Rosa
Published: July 2002

Much knowledge about microbial gene regulation and virulence is derived from genetic and biochemical studies done outside of hosts. The aim of this review is to correlate observations made
in vitro
and in vivo with two different bacterial pathogens in which the nature of regulated gene expression leading to virulence is quite different. The first is
Vibrio cholerae
, in which the concerted action of a complicated regulatory cascade involving several transcription activators leads ultimately to expression of cholera toxin and the toxin–coregulated pilus. The regulatory cascade is active in vivo and is also required for maintenance of
V . cholerae
in the intestinal tract during experimental infection. Nevertheless, specific signals predicted to be generated in vivo , such as bile and a temperature of 37°C, have a severe downmodulating effect on activation of toxin and pilus expression. Another unusual aspect of gene regulation in this system is the role played by inner membrane proteins that activate transcription. Although the topology of these proteins suggests an appealing model for signal transduction leading to virulence gene expression, experimental evidence suggests that such a model may be simplistic. In Streptococcus pyogenes , capsule production is critical for virulence in an animal model of necrotizing skin infection. Yet capsule is apparently produced to high levels only from mutation in a two–component regulatory system, CsrR and CsrS. Thus it seems that in
V . cholerae
a complex regulatory pathway has evolved to control virulence by induction of gene expression in vivo , whereas in
S. pyogenes
at least one mode of pathogenicity is potentiated by the absence of regulation.
RNA interference: advances and questions
Authors: Ullu Elisabetta , Djikeng Appolinaire , Shi Huafang , Tschudi Christian
Published: July 2002

In animals and protozoa gene–specific double–stranded RNA triggers the degradation of homologous cellular RNAs, the phenomenon of RNA interference (RNAi). RNAi has been shown to represent a novel paradigm in eukaryotic biology and a powerful method for studying gene function. Here we discuss RNAi in terms of its mechanism, its relationship to other post–transcriptional gene silencing phenomena in plants and fungi, its connection to retroposon silencing and possibly to translation, and its biological role. Among the organisms where RNAi has been demonstrated the protozoan parasiteTrypanosoma bruceirepresents the most ancient branch of the eukaryotic lineage. We provide a synopsis of what is currently known about RNAi inT. bruceiand outline the recent advances that make RNAi the method of choice to disrupt gene function in these organisms.
Multiple states in river and lake ecosystems
Authors: Dent C. Lisa , Cumming Graeme S. , Carpenter Stephen R.
Published: July 2002

Nonlinear models of ecosystem dynamics that incorporate positive feedbacks and multiple, internally reinforced states have considerable explanatory power. However, linear models may be adequate, particularly if ecosystem behaviour is primarily controlled by external processes. In lake ecosystems, internal (mainly biotic) processes are thought to have major impacts on system behaviour, whereas in rivers, external (mainly physical) factors have traditionally been emphasized. We consider the hypothesis that models that exhibit multiple states are useful for understanding the behaviour of lake ecosystems, but not as useful for understanding stream ecosystems. Some of the best–known examples of multiple states come from lake ecosystems. We review some of these examples, and we also describe examples of multiple states in rivers. We conclude that the hypothesis is an oversimplification; the importance of physical forcing in rivers does not eliminate the possibility of internal feedbacks that create multiple states, although in rivers these feedbacks are likely to include physical as well as biotic processes. Nonlinear behaviour in aquatic ecosystems may be more common than current theory indicates.