bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppressive Activity of a Macrolide Antibiotic, Roxithromycin, on Pro‐Inflammatory Cytokine Production in Vitro and in Vivo
Authors: Suzaki H. , Asano K. , Ohki S. , Kanai K. , Mizutani T. , Hisamitsu T.
Published: July 2002

This study was designed to examine the influence of a macrolide antibiotic, roxithromycin (RXM), on the production of pro‐inflammatory cytokines, interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α. In the first experiments, we examined the effect of RXM on in vitro cytokine production from lipopolysaccharide (LPS)‐stimulated human peripheral blood monocytes. The monocytes were cultured in the presence of various doses of the agent. After 24 h, the culture supernatants were obtained and assayed for IL‐1β and TNF‐α contents by enzyme‐linked immunosorbent assay. RXM suppressed the in vitro production of IL‐1β and TNF‐α in response to LPS stimulation. This was dose dependent and first noted at a concentration of as little as 0.05 μg/ml, which is much lower than therapeutic blood levels. In the second part of the experiments, we examined the influence of RXM on the appearance of IL‐1β and TNF‐α in mouse lung extract induced by LPS inhalation. RXM was administered orally into BALB/c mice at a single dose of 2.5 mg/kg once a day for 5‐12 weeks. These mice were then instilled with LPS into the trachea and examined for the presence of cytokines in aqueous lung extracts. Pretreatment of mice with RXM for 5 weeks did not influence of the appearance of both IL‐1β and TNF‐α in aqueous lung extracts. However, pretreatment for more than 7 weeks dramatically suppressed the cytokine appearance in the extracts.
Inhibitors of Complement Activity in Human Breast‐Milk: A Proposed Hypothesis of Their Physiological Significance
Authors: Ogundele Michael Oladipo
Published: July 2002

Several natural components abundant in the fluid phase of human breast‐milk have been shown to be inhibitors of complement activation in vitro, particularly the classical pathway. These include lysozyme, lactoferrin, lactalbumin alpha and other ligand chelators, complement regulator proteins and other specific soluble inhibitors of complement activation. Their physiological significance probably resides in their ability to restrict in vivo complement activation to specialized (compartmentalized) sites on the cellular membrane structures in human milk, represented by the abundant surface area of the milk fat globule membranes. This would serve to prevent inflammatory‐induced tissue damage of the delicate immature gastrointestinal tract of the newborn as well as the mammary gland itself. A number of recognized and potential inhibitors of complement activity in human milk and other biological fluids are hereby reviewed, with a proposal of their physiological significance.

Abbreviations: HBM, human breast‐milk; APC, alternative complement activation pathway; MAC, membrane attack complex (C5b‐9); MFGM, milk fat globule membrane
Testing Guidelines for Evaluation of Foodadditives Effects on Male Reproduction
Authors: Sprando Robert L. , Collins Thomas F. X.
Published: July 2002

The revised FDA Redbook guide lines presented in this article reflect current thinking concerning male reproductive toxicology. A number of different end-points to assess m ale repro ductive function have been included in the present draft of the FDA Redbook II guidelines to better assess potential effects of direct food additives and color additives on male reproductive function. The guidelines reflect a harmony with the development of the guidelines of regulatory agencies in this country and around the world.
An Overview of Endocrine Modulators
Authors: Byrd Daniel M. , Benson William H. , Solomon Keith R. , Thomas John A. , Waddell William J.
Published: July 2002

During the American College of Toxicology's Symposium on Endocrine Modulators (Valley Forge, PA, November 1996), the speakers evaluated relevant scientific information and discussed three general ideas, as follows: (1) The available epidem iological data are inconsistent with significant increases in observed human health effects that might relate to the modulation of the endocrine system. (2) Environm ental contarn in ants have produced toxic effects in wildlife at specific locations. In select instances, the adverse effects observed may involve modulation of endocrine pathways. (3) Additional research focused on endocrine modulation is desirable in order to address this issue within a sound, scientificframework.
Dose-Dependent Modulation of Cell Death: Apoptosis Versus Necrosis in Thioacetamide Hepatotoxicity
Authors: Mangipudy Raja S. , Rao Prathibha S. , Andrews Annette , Bucci Thomas J. , Witzmann Frank A. , Mehendale Harihara M.
Published: July 2002

Apoptosis is programmed cell death, morphologically and biochemically distinct from necrosis. The objective of the present study was to examine thioacetamide-induced apoptosis over an early time course of 0 to 8 h after administration of a 12-fold dose range (50, 150, 300, and 600 mg/kg, ip) of thioacetamide (TA). Male Sprague-Daw ley rats (200-225 g) were used for the study. The incidence of apoptosis was determined by in situ end labeling, transmission electron microscopy, and charge modification of heat shock protein 70 (Hsp 70). Light microscopic examination of liver sections revealed apoptotic bodies (ABs)as early as 2 h after TA administration. A dose-dependent increase in the incidence of ABs was seen with all doses until 4 h. Thereafter, the incidence of ABs continued to increase in a temporal manner with 50 and 150 mg/kg, while it decreased in the rats treated with 300 and 600 mg/kg. Between 4 and 8 h, while necrosis as assessed by serum alanine aminotransferase (ALT) and histopathology declined in the sixfold dose range (50, 150, and 300 mg TA/kg), it increased in a temporal manner with 600 mg TA/kg. Preliminary studies indicate an inverse relation between Hsp 70 abundance and the incidence of apoptosis. Hsp 70 expression was significantly higher in the 600 mg TA/kg group compared to the lower doses. Lowest abundance was recorded in the groups receiving 50 and 150 mg TA/kg, where maximum apoptosis was noted. These findings collectively suggest that although the processes of apoptosis and necrosis are initiated simultaneously, the proportion of cells dying via either mechanism seems to be regulated by the dose of TA. Lower doses seem to favor cell death via apoptosis, while higher doses favor cell death via necrosis. Additionally, the inverse relation between Hsp 70 and apoptosis at lower doses suggests a regulatory role for Hsp 70.
Language repair via metalinguistic means
Authors: Hirschman Mary
Published: July 2002

Children with specific language impairment (SLI) have been shown to be deficient in the use of complex sentences. In an attempt to remediate this area of weakness, two groups of such children (mean ages 9;4 and 10;6) received about 55 half‐hour sessions of metalinguistic training spread over 12 months. Results showed that the use of complex sentences increased to at least normal levels in the experimental groups, and were significantly improved, at both the written and oral levels, as compared with SLI control groups, which evidenced little change over the same period. Further analysis of the data revealed the striking finding that those children who had the poorest complex sentence usage tended to benefit the most from metalinguistic training. These results are interpreted as support for the hypothesis that metalinguistic training helps to overcome the presumed neurological deficit of the language disordered child by making linguistic rules conscious, and thereby providing a A'metalinguistic bridgeA' via which information can bypass the damaged area.
Tense and finiteness in the speech of children with specific language impairment acquiring Hebrew
Authors: B. Leonard Laurence , Dromi Esther , Adam Galit , Zadunaisky‐Ehrlich Sara
Published: July 2002

Verb morphology is often an area of extraordinary difficulty for children with specific language impairment (SLI). However, in Hebrew, this difficulty appears to be more circumscribed than in other languages. In a recent study by Dromi et al, the limitations exhibited by a group of Hebrew‐speaking children with SLI were confined primarily to the use of agreement inflections within past tense. This difficulty was interpreted as being due to the fact that the agreement paradigm within past tense is rather complex (involving person, number, and gender, along with tense). This study explores another possibility ‐ that these inflections were difficult because they required past tense in particular. Also determined was whether the children's frequent use of the morphologically simplest forms (such as past third person masculine singular) in place of the correct forms could be interpreted as their selection of a non‐finite default form. A group of Hebrew‐speaking children with SLI (age 4;2 to 6;1) participated, along with a group of age controls, and a group of younger normally developing children matched for mean length of utterance (MLU). The children listened to stories that were accompanied by pictures. During each story, the children completed the experimenter's incomplete sentences using appropriate verbs. To complete the sentences accurately, the children had to alter the tense or finiteness of the verb used by the experimenter in the preceding sentence. The results indicated that the children with SLI had more difficulty than both comparison groups in the production of basic present and past forms and infinitive forms of verbs that required use of one particular phonological template or ‘binyan’. However, for the verbs requiring the remaining three phonological templates, the children with SLI were as capable as MLU controls in their command of past as well as present tense, and in their use of infinitive forms. It is concluded that tense and finiteness probably do not form the core of the problem faced by Hebrew‐speaking children with SLI in the area of verb morphology.
Non‐Word Repetition In Children With Language Impairment‐Pitfalls And Possibilities
Authors: Sahlen Birgitta , Reuterskiold‐Wagner Christina , Nettelbladt Ulrika , Radeborg Karl
Published: July 2002

Specific language impairment has, although not without controversy, been considered as a consequence of a phonological memory deficit. Non‐word repetition has been proposed as a reliable index of phonological memory and also as predictive of lexical and grammatical development in normally developing and language‐impaired children. The main aim was to study the relationship between repetition of words and non‐words and expressive language skills (phonology and grammar) in 27 5‐year‐old children with language impairment. The authors also wanted to explore the influence of lexical stress on repetition skills. The results showed that words were significantly easier to repeat than non‐words and that non‐word repetition skills were significantly correlated to phonological and grammatical development. The most important predictor of non‐word repetition skills was output phonology. The conclusion is that nonword repetition is not a single, reliable index of phonological memory in preschool children with language impairment. Also, the influence of prosodic variables on segmental aspects of speech production should not be overlooked in non‐word construction, since it was found that unstressed syllables were omitted six times more often in prestressed than in post‐stressed positions of the words and non‐words.
Nitric Oxide: The “Second Messenger” of Insulin
Authors: Kahn Nighat N. , Acharya K. , Bhattacharya S. , Acharya R. , Mazumder S. , Bauman William A. , Sinha Asru K.
Published: July 2002

Incubation of various tissues, including heart, liver, kidney, muscle, and intestine from mice and erythrocytes or their membrane fractions from humans, with physiologic concentration of insulin resulted in the activation of a membrane‐bound nitric oxide synthase (NOS). Activation of NOS and synthesis of NO were stimulated by the binding of insulin to specific receptors on the cell surface. A Lineweaver‐Burk plot of the enzymatic activity demonstrated that the stimulation of NOS by insulin was related to the decrease in the Km for L‐arginine, the substrate for NOS, with a simultaneous increase of Vmax. Addition of NG‐nitro‐L‐arginine methyl ester (LNAME), a competitive inhibitor of NOS, to the reaction mixture completely inhibited the hormone‐stimulated NO synthesis in all tissues. Furthermore, NO had an insulin‐like effect in stimulating glucose transport and glucose oxidation in muscle, a major site for insulin action. Addition of NAME to the reaction mixture completely blocked the stimulatory effect of insulin by inhibiting both NO production and glucose metabolism, without affecting the hormone‐stimulated tyrosine or phosphatidylinositol 3‐kinases of the membrane preparation. Injection of NO in alloxan‐induced diabetic mice mimicked the effect of insulin in the control of hyperglycemia (i.e., lowered the glucose content in plasma). However, injection of NAME before the administration of insulin to diabetic‐induced and nondiabetic mice inhibited not only the insulin‐stimulated increase of NO in plasma but also the glucose‐lowering effect of insulin.
Evolutionary Aspects of Calmodulin
Authors: Friedberg Felix , Rhoads Allen R.
Published: July 2002

Calmodulin (CaM) is a major cellular sensor of calcium signaling, interacts with numerous proteins associated with cellular second messenger systems (e.g., cyclic AMP, nitric oxide), and is associated with neurosecretory activity. An identical CaM protein consisting of four helix‐loop‐helix regions that arose by gene duplication is encoded by three nonallelic mammalian genes that are some of the most highly conserved genes known. Differential tissue and cellular expression of each CaM suggest unique functions that promote strong selective preservation of these replicate, yet distinct, CaM genes in mammals. Each gene displays the same exon‐intron arrangement but is characterized by distinct promoter elements and by unique 5'‐ and 3'‐untranslated regions that are highly conserved among human, rat, and mouse. These distinct untranslated regions may permit regulation of CaM levels at discrete cellular sites during differentiation and in highly specialized cell types such as neurons.
Two Peptides Derived from the Nerve Growth Factor Precursor Are Biologically Active
Authors: Dicou Eleni , Pflug Beth , Magazin Marilyn , Lehy Thérèse , Djakiew Daniel , Ferrara Pascual , Nerrière Véronique , Harvie Douglas
Published: July 2002

This report provides evidence that the proregion of the NGF precursor protein contains two novel bioactive peptides. The presence of pairs of basic amino acid (aa) residues in the NGF proregion suggests that two or three peptides other than NGF may be generated by proteolytic cleavage. Synthetic peptides of 29 aa (LIP1) and 38aa (LIP2) corresponding to the sequences −71 to −43 and −40 to −3 of the proNGF, respectively, were used in this study. ELISA specific for these two peptides revealed their presence in the rat intestine. LIP1 was localized by immunohistochemistry in endocrine cells of the intestinal epithelium, and LIP2 was immunoprecipitated from an intestinal extract. We also provide evidence for the presence of specific receptors for LIP2 in several cell lines. Scatchard analysis indicated the presence of a low affinity binding site with a Kd of ∼10−7 M and a high affinity binding site of 10−9 M. Cross-linking studies revealed receptor forms of about 140 kD and 93 kD in a prostatic adenocarcinoma cell line.

LIP1 and LIP2 induced rapid F-actin redistribution in PC12 cells within 2 min of incubation, which suggests a role of LIP1 and LIP2 in the process of neurite outgrowth. Furthermore, both propeptides induced rapid tyrosine phosphorylation of the Trk protein in both prostatic adenocarcinoma cells and PC12 cells, thus implicating trk in their mechanism of action. These results support our hypothesis that two peptides within the NGF precursor protein are biologically active.
Role of Actin Polymerization and Adhesion to Extracellular Matrix in Rac- and Rho-induced Cytoskeletal Reorganization
Authors: Machesky Laura M. , Hall Alan
Published: July 2002

Most animal cells use a combination of actin-myosin–based contraction and actin polymerization– based protrusion to control their shape and motility. The small GTPase Rho triggers the formation of contractile stress fibers and focal adhesion complexes (Ridley, A.J., and A. Hall. 1992. Cell. 70:389–399) while a close relative, Rac, induces lamellipodial protrusions and focal complexes in the lamellipodium (Nobes, C.D., and A. Hall. 1995. Cell. 81:53–62; Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. Cell. 70:401–410); the Rho family of small GTPases may thus play an important role in regulating cell movement. Here we explore the roles of actin polymerization and extracellular matrix in Rho- and Rac-stimulated cytoskeletal changes. To examine the underlying mechanisms through which these GTPases control F-actin assembly, fluorescently labeled monomeric actin, Cy3-actin, was introduced into serum-starved Swiss 3T3 fibroblasts. Incorporation of Cy3- actin into lamellipodial protrusions is concomitant with F-actin assembly after activation of Rac, but Cy3-actin is not incorporated into stress fibers formed immediately after Rho activation. We conclude that Rac induces rapid actin polymerization in ruffles near the plasma membrane, whereas Rho induces stress fiber assembly primarily by the bundling of actin filaments. Activation of Rho or Rac also leads to the formation of integrin adhesion complexes. Integrin clustering is not required for the Rho-induced assembly of actin-myosin filament bundles, or for vinculin association with actin bundles, but is required for stress fiber formation. Integrin-dependent focal complex assembly is not required for the Rac-induced formation of lamellipodia or membrane ruffles. It appears, therefore, that the assembly of large integrin complexes is not required for most of the actin reorganization or cell morphology changes induced by Rac or Rho activation in Swiss 3T3 fibroblasts.
Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo
Authors: Cardoso M. Cristina , Joseph Cuthbert , Rahn Hans-Peter , Reusch Regina , Nadal-Ginard Bernardo , Leonhardt Heinrich
Published: July 2002

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed “functional organization” of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1–28 and 111–179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH2-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH2 or the COOH termini of heterologous proteins.

We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.
Human Autoantibodies Reveal Titin as a Chromosomal Protein
Authors: Machado Cristina , Sunkel Claudio E. , Andrew Deborah J.
Published: July 2002

Assembly of the higher-order structure of mitotic chromosomes is a prerequisite for proper chromosome condensation, segregation and integrity. Understanding the details of this process has been limited because very few proteins involved in the assembly of chromosome structure have been discovered. Using a human autoimmune scleroderma serum that identifies a chromosomal protein in human cells and Drosophila embryos, we cloned the corresponding Drosophila gene that encodes the homologue of vertebrate titin based on protein size, sequence similarity, developmental expression and subcellular localization. Titin is a giant sarcomeric protein responsible for the elasticity of striated muscle that may also function as a molecular scaffold for myofibrillar assembly. Molecular analysis and immunostaining with antibodies to multiple titin epitopes indicates that the chromosomal and muscle forms of titin may vary in their NH2 termini. The identification of titin as a chromosomal component provides a molecular basis for chromosome structure and elasticity.
A Giant Ubiquitin-conjugating Enzyme Related to IAP Apoptosis Inhibitors
Authors: Hauser Hans-Peter , Bardroff Michael , Pyrowolakis George , Jentsch Stefan
Published: July 2002

Ubiquitin-conjugating enzymes (UBC) catalyze the covalent attachment of ubiquitin to target proteins and are distinguished by the presence of a UBC domain required for catalysis. Previously identified members of this enzyme family are small proteins and function primarily in selective proteolysis pathways. Here we describe BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), a giant (528-kD) ubiquitin-conjugating enzyme from mice. BRUCE is membrane associated and localizes to the Golgi compartment and the vesicular system. Remarkably, in addition to being an active ubiquitin-conjugating enzyme, BRUCE bears a baculovirus inhibitor of apoptosis repeat (BIR) motif, which to this date has been exclusively found in apoptosis inhibitors of the IAP-related protein family. The BIR motifs of IAP proteins are indispensable for their anti–cell death activity and are thought to function through protein–protein interaction. This suggests that BRUCE may combine properties of IAP-like proteins and ubiquitin-conjugating enzymes and indicates that the family of IAP-like proteins is structurally and functionally more diverse than previously expected.
The Dynamin-related GTPase, Dnm1p, Controls Mitochondrial Morphology in Yeast
Authors: Otsuga Denichiro , Keegan Brian R. , Brisch Ellen , Thatcher John W. , Hermann Greg J. , Bleazard William , Shaw Janet M.
Published: July 2002

The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.
In Vitro Repression of Brca1-associated RING Domain Gene, Bard1, Induces Phenotypic Changes in Mammary Epithelial Cells
Authors: Irminger-Finger Irmgard , Soriano Jesus V. , Vaudan Geneviève , Montesano Roberto , Sappino André-Pascal
Published: July 2002

BRCA1-associated RING domain (BARD1) was identified as a protein interacting with the breast cancer gene product BRCA1. The identification of tumorigenic missense mutations within BRCA1 that impair the formation of BARD1–BRCA1 complexes, and of BARD1 mutations in breast carcinomas, sustain the view that BARD1 is involved in BRCA1-mediated tumor suppression. We have cloned the murine Bard1 gene and determined that its expression in different tissues correlates with the expression profile of Brca1. To investigate the function of Bard1, we have reduced Bard1 gene expression in TAC-2 cells, a murine mammary epithelial cell line that retains morphogenetic properties characteristic of normal breast epithelium. Partial repression of Bard1, achieved by the transfection of TAC-2 cells with plasmids constitutively expressing ribozymes or antisense RNAs, resulted in marked phenotypic changes, consisting of altered cell shape, increased cell size, high frequency of multinucleated cells, and aberrant cell cycle progression. Furthermore, Bard1-repressed cell clones overcame contact inhibition of cell proliferation when grown in monolayer cultures and lost the capacity to form luminal structures in three-dimensional collagen gels. These results demonstrate that Bard1 repression induces complex changes in mammary epithelial cell properties which are suggestive of a premalignant phenotype.
Cell Damage-induced Conformational Changes of the Pro-Apoptotic Protein Bak In Vivo Precede the Onset of Apoptosis
Authors: Griffiths Gareth J. , Dubrez Laurence , Morgan Clive P. , Jones Neil A. , Whitehouse Jenna , Corfe Bernard M. , Dive Caroline , Hickman John A.
Published: July 2002

Investigation of events committing cells to death revealed that a concealed NH2-terminal epitope of the pro-apoptotic protein Bak became exposed in vivo before apoptosis. This occurred after treatment of human Jurkat or CEM-C7A T-lymphoma cells with the mechanistically disparate agents staurosporine, etoposide or dexamethasone. The rapid, up to 10-fold increase in Bak-associated immunofluorescence was measured with epitope-specific monoclonal antibodies using flow cytometry and microscopy. In contrast, using a polyclonal antibody to Bak, immunofluorescence was detected both before and after treatment. There were no differences in Bak protein content nor in subcellular location before or after treatment. Immunofluorescence showed Bcl-xL and Bak were largely associated with mitochondria and in untreated cells they coimmunoprecipitated in the presence of nonioinic detergent. This association was significantly decreased after cell perturbation suggesting that Bcl-xL dissociation from Bak occurred on exposure of Bak's NH2 terminus. Multiple forms of Bak protein were observed by two dimensional electrophoresis but these were unchanged by inducers of apoptosis. This indicated that integration of cellular damage signals did not take place directly on the Bak protein. Release of proteins, including Bcl-xL, from Bak is suggested to be an important event in commitment to death.
Neuropilin-1 Mediates Collapsin-1/Semaphorin III Inhibition of Endothelial Cell Motility
Authors: Miao Hua-Quan , Soker Shay , Feiner Leonard , Alonso José Luis , Raper Jonathan A. , Klagsbrun Michael
Published: July 2002

Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65–75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.
A Novel Virus–Host Cell Membrane Interaction
Authors: Poranen Minna M. , Daugelavičius Rimantas , Ojala Päivi M. , Hess Michael W. , Bamford Dennis H.
Published: July 2002

Studies on the virus–cell interactions have proven valuable in elucidating vital cellular processes. Interestingly, certain virus–host membrane interactions found in eukaryotic systems seem also to operate in prokaryotes (Bamford, D.H., M. Romantschuk, and P.J. Somerharju, 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1467–1473; Romantschuk, M., V.M. Olkkonen, and D.H. Bamford. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1821–1829). φ6 is an enveloped double-stranded RNA virus infecting a gram-negative bacterium. The viral entry is initiated by fusion between the virus membrane and host outer membrane, followed by delivery of the viral nucleocapsid (RNA polymerase complex covered with a protein shell) into the host cytosol via an endocytic-like route. In this study, we analyze the interaction of the nucleocapsid with the host plasma membrane and demonstrate a novel approach for dissecting the early events of the nucleocapsid entry process. The initial binding of the nucleocapsid to the plasma membrane is independent of membrane voltage (ΔΨ) and the K+ and H+ gradients. However, the following internalization is dependent on plasma membrane voltage (ΔΨ), but does not require a high ATP level or K+ and H+ gradients. Moreover, the nucleocapsid shell protein, P8, is the viral component mediating the membrane–nucleocapsid interaction.
Signaling by Fibroblast Growth Factors (Fgf) and Fibroblast Growth Factor Receptor 2 (Fgfr2)–Activating Mutations Blocks Mineralization and Induces Apoptosis in Osteoblasts
Authors: Mansukhani Alka , Bellosta Paola , Sahni Malika , Basilico Claudio
Published: July 2002

Fibroblast growth factors (FGF) play a critical role in bone growth and development affecting both chondrogenesis and osteogenesis. During the process of intramembranous ossification, which leads to the formation of the flat bones of the skull, unregulated FGF signaling can produce premature suture closure or craniosynostosis and other craniofacial deformities. Indeed, many human craniosynostosis disorders have been linked to activating mutations in FGF receptors (FGFR) 1 and 2, but the precise effects of FGF on the proliferation, maturation and differentiation of the target osteoblastic cells are still unclear. In this report, we studied the effects of FGF treatment on primary murine calvarial osteoblast, and on OB1, a newly established osteoblastic cell line. We show that FGF signaling has a dual effect on osteoblast proliferation and differentiation. FGFs activate the endogenous FGFRs leading to the formation of a Grb2/FRS2/Shp2 complex and activation of MAP kinase. However, immature osteoblasts respond to FGF treatment with increased proliferation, whereas in differentiating cells FGF does not induce DNA synthesis but causes apoptosis. When either primary or OB1 osteoblasts are induced to differentiate, FGF signaling inhibits expression of alkaline phosphatase, and blocks mineralization. To study the effect of craniosynostosis-linked mutations in osteoblasts, we introduced FGFR2 carrying either the C342Y (Crouzon syndrome) or the S252W (Apert syndrome) mutation in OB1 cells. Both mutations inhibited differentiation, while dramatically inducing apoptosis. Furthermore, we could also show that overexpression of FGF2 in transgenic mice leads to increased apoptosis in their calvaria. These data provide the first biochemical analysis of FGF signaling in osteoblasts, and show that FGF can act as a cell death inducer with distinct effects in proliferating and differentiating osteoblasts.
Phosphorylation of Connexin43 on Serine368 by Protein Kinase C Regulates Gap Junctional Communication
Authors: Lampe Paul D. , TenBroek Erica M. , Burt Janis M. , Kurata Wendy E. , Johnson Ross G. , Lau Alan F.
Published: July 2002

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell–cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with 32Pi yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43−/− mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of ∼50-pS channel events and a concomitant loss of ∼100-pS channel events. This change to ∼50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.
Pitx2 Regulates Procollagen Lysyl Hydroxylase (Plod) Gene Expression
Authors: Hjalt Tord A. , Amendt Brad A. , Murray Jeffrey C.
Published: July 2002

The Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1–luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI.
Cyclin E Uses Cdc6 as a Chromatin-Associated Receptor Required for DNA Replication
Authors: Furstenthal Laura , Kaiser Brett K. , Swanson Craig , Jackson Peter K.
Published: July 2002

Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E–Cdk2 to DNA. We find that cyclin E binds the NH2-terminal region of Cdc6 containing Cy–Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E–Cdk2 for chromatin binding, and fail to rescue replication in cyclin E–depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E–Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E–Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E–Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B–Cdc2, but not the polo-like kinase 1, remove cyclin E–Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E–Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase–directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E–Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.
Regulation of limb patterning by extracellular microfibrils
Authors: Arteaga-Solis Emilio , Gayraud Barbara , Lee Sui Y. , Shum Lillian , Sakai Lynn , Ramirez Francesco
Published: July 2002

To elucidate the contribution of the extracellular microfibril–elastic fiber network to vertebrate organogenesis, we generated fibrillin 2 (Fbn2)–null mice by gene targeting and identified a limb-patterning defect in the form of bilateral syndactyly. Digit fusion involves both soft and hard tissues, and is associated with reduced apoptosis at affected sites. Two lines of evidence suggest that syndactily is primarily due to defective mesenchyme differentiation, rather than reduced apoptosis of interdigital tissue. First, fusion occurs before appearance of interdigital cell death; second, interdigital tissues having incomplete separation fail to respond to apoptotic clues from implanted BMP-4 beads. Syndactyly is associated with a disorganized matrix, but with normal BMP gene expression. On the other hand, mice double heterozygous for null Fbn2 and Bmp7 alleles display the combined digit phenotype of both nullizygotes. Together, these results imply functional interaction between Fbn2-rich microfibrils and BMP-7 signaling. As such, they uncover an unexpected relationship between the insoluble matrix and soluble factors during limb patterning. We also demonstrate that the Fbn2- null mutation is allelic to the recessive shaker-with-syndactyly (sy) locus on chromosome 18.
Human VPS34 is required for internal vesicle formation within multivesicular endosomes
Authors: Futter C.E. , Collinson L.M. , Backer J.M. , Hopkins C.R.
Published: July 2002

After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3′-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3′-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3′-kinase activity, whereas vesicle formation requires PI 3′-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.
Inhibition of Phagolysosomal Biogenesis by the Leishmania Lipophosphoglycan
Authors: Desjardins Michel , Descoteaux Albert
Published: July 2002

Whereas amastigotes of the protozoan parasite Leishmania proliferate inside acidic phagolysosomal vacuoles of the macrophage, vacuoles induced by Leishmania donovani promastigotes during initiation of infection are poorly characterized. Here, evidence is presented that interaction of these parasitophorous vacuoles with endocytic organelles is very limited. In contrast, vacuoles formed around L. donovani mutants lacking the cell surface lipophosphoglycan (LPG) fuse extensively with endosomes and lysosomes. The role of LPG repeating units in the inhibition of phagosome–endosome fusion was demonstrated using two different approaches. First, genetic complementation of the LPG-defective C3PO mutant restored its ability to inhibit phagosome–endosome fusion to a degree similar to that of wild-type promastigotes. Second, opsonization of C3PO mutant cells with purified L. donovani LPG also conferred to this mutant the ability to inhibit phagosome–endosome fusion. Inasmuch as LPG is essential for infecting macrophages, these results suggest that inhibition of phagolysosomal biogenesis by LPG repeating units represents an intramacrophage survival strategy used by promastigotes to establish infection.
Murine Salmonellosis Studied by Confocal Microscopy: Salmonella typhimurium Resides Intracellularly Inside Macrophages and Exerts a Cytotoxic Effect on Phagocytes In Vivo
Authors: Richter-Dahlfors Agneta , Buchan Alison M.J. , Finlay B. Brett
Published: July 2002

Salmonella typhimurium is considered a facultative intracellular pathogen, but its intracellular location in vivo has not been demonstrated conclusively. Here we describe the development of a new method to study the course of the histopathological processes associated with murine salmonellosis using confocal laser scanning microscopy of immunostained sections of mouse liver. Confocal microscopy of 30-μm-thick sections was used to detect bacteria after injection of ∼100 CFU of S. typhimurium SL1344 intravenously into BALB/c mice, allowing salmonellosis to be studied in the murine model using more realistic small infectious doses. The appearance of bacteria in the mouse liver coincided in time and location with the infiltration of neutrophils in inflammatory foci. At later stages of disease the bacteria colocalized with macrophages and resided intracellularly inside these macrophages. Bacteria were cytotoxic for phagocytic cells, and apoptotic nuclei were detected immunofluorescently, whether phagocytes harbored intracellular bacteria or not. These data argue that Salmonella resides intracellularly inside macrophages in the liver and triggers cell death of phagocytes, processes which are involved in disease. This method is also applicable to other virulence models to examine infections at a cellular and subcellular level in vivo.
Oxazolone Colitis: A Murine Model of  T Helper Cell Type 2 Colitis Treatable with Antibodies to Interleukin 4
Authors: Boirivant Monica , Fuss Ivan J. , Chu Alan , Strober Warren
Published: July 2002

In this study we describe oxazolone colitis, a new form of experimental colitis. This model is induced in SJL/J mice by the rectal instillation of the haptenating agent, oxazolone, and is characterized by a rapidly developing colitis confined to the distal half of the colon; it consists of a mixed neutrophil/lymphocyte infiltration limited to the superficial layer of the mucosa which is associated with ulceration. Oxazolone colitis is a T helper cell type 2 (Th2)-mediated process since stimulated T cells from lesional tissue produce markedly increased amounts of interleukin (IL)-4 and IL-5; in addition, anti–IL-4 administration leads to a striking amelioration of disease, whereas anti–IL-12 administration either has no effect or exacerbates disease. Finally, this proinflammatory Th2 cytokine response is counterbalanced by a massive transforming growth factor-β (TGF-β) response which limits both the extent and duration of disease: lesional (distal) T cells manifest a 20–30-fold increase in TGF-β production, whereas nonlesional (proximal) T cells manifest an even greater 40–50-fold increase. In addition, anti–TGF-β administration leads to more severe inflammation which now involves the entire colon. The histologic features and distribution of oxazolone colitis have characteristics that resemble ulcerative colitis (UC) and thus sharply distinguish this model from most other models, which usually resemble Crohn's disease. This feature of oxazolone colitis as well as its cytokine profile have important implications to the pathogenesis and treatment of UC.
Different Protein Tyrosine Kinases Are Required for B Cell Antigen Receptor–mediated Activation of Extracellular Signal–Regulated kinase, c-Jun NH2-terminal Kinase 1, and p38 Mitogen-activated Protein Kinase
Authors: Jiang Aimin , Craxton Andrew , Kurosaki Tomohiro , Clark Edward A.
Published: July 2002

B cell antigen receptor (BCR) cross-linking activates three distinct families of nonreceptor protein tyrosine kinases (PTKs): src-family kinases, Syk, and Btk; these PTKs are responsible for initiating downstream events. BCR cross-linking in the chicken DT40 B cell line also activates three distinct mitogen-activated protein kinases (MAPKs): extracellular signal–regulated kinase (ERK)2, c-jun NH2-terminal kinase (JNK)1, and p38 MAPK. To dissect the functional roles of these PTKs in MAPK signaling, activation of MAPKs was examined in various PTK-deficient DT40 cells. BCR-mediated activation of ERK2, although maintained in Lyn-deficient cells, was abolished in Syk-deficient cells and partially inhibited in Btk-deficient cells, indicating that BCR-mediated ERK2 activation requires Syk and that sustained ERK2 activation requires Btk. BCR-mediated JNK1 activation was maintained in Lyn-deficient cells but abolished in both Syk- and Btk-deficient cells, suggesting that JNK1 is activated via a Syk- and Btk-dependent pathway. Consistent with this, BCR-mediated JNK1 activation was dependent on intracellular calcium and phorbol myristate acetate–sensitive protein kinase Cs. In contrast, BCR-mediated p38 MAPK activation was detected in all three PTK-deficient cells, suggesting that no single PTK is essential. However, BCR-mediated p38 MAPK activation was abolished in Lyn/Syk double deficient cells, demonstrating that either Lyn or Syk alone may be sufficient to activate p38 MAPK. Our data show that BCR-mediated MAPK activation is regulated at the level of the PTKs.
A Novel Mouse with B Cells but Lacking Serum Antibody Reveals an Antibody-independent Role for B Cells in Murine Lupus
Authors: Chan Owen T.M. , Hannum Lynn G. , Haberman Ann M. , Madaio Michael P. , Shlomchik Mark J.
Published: July 2002

The precise role of B cells in systemic autoimmunity is incompletely understood. Although B cells are necessary for expression of disease (Chan, O., and M.J. Shlomchik. 1998. J. Immunol. 160:51–59, and Shlomchik, M.J., M.P. Madaio, D. Ni, M. Trounstine, and D. Huszar. 1994. J. Exp. Med. 180:1295–1306), it is unclear whether autoantibody production, antigen presentation, and/or other B cell functions are required for the complete pathologic phenotype. To address this issue, two experimental approaches were used. In the first, the individual contributions of circulating antibodies and B cells were analyzed using MRL/MpJ-Faslpr (MRL/lpr) mice that expressed a mutant transgene encoding surface immunoglobulin (Ig), but which did not permit the secretion of circulating Ig. These mice developed nephritis, characterized by cellular infiltration within the kidney, indicating that B cells themselves, without soluble autoantibody production, exert a pathogenic role. The results indicate that, independent of serum autoantibody, functional B cells expressing surface Ig are essential for disease expression, either by serving as antigen-presenting cells for antigen-specific, autoreactive T cells, or by contributing directly to local inflammation.